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Coleman B, Bedi S, Hill JH, Morris J, Manthei KA, Hart RC, He Y, Shah AS, Jerome WG, Vaisar T, Bornfeldt KE, Song H, Segrest JP, Heinecke JW, Aller SG, Tesmer JJG, Davidson WS. Lecithin:cholesterol acyltransferase binds a discontinuous binding site on adjacent apolipoprotein A-I belts in HDL. J Lipid Res 2025; 66:100786. [PMID: 40147634 PMCID: PMC12049944 DOI: 10.1016/j.jlr.2025.100786] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2025] [Revised: 03/11/2025] [Accepted: 03/23/2025] [Indexed: 03/29/2025] Open
Abstract
Lecithin:cholesterol acyltransferase (LCAT) is a high-density lipoprotein (HDL) modifying protein that profoundly affects the composition and function of HDL subspecies. The cholesterol esterification activity of LCAT is dramatically increased by apolipoprotein A-I (APOA1) on HDL, but the mechanism remains unclear. Using site-directed mutagenesis, cross-linking, mass spectrometry, electron microscopy, protein engineering, and molecular docking, we identified two LCAT binding sites formed by helices 4 and 6 from two antiparallel APOA1 molecules in HDL. Although the reciprocating APOA1 "belts" form two ostensibly symmetrical binding locations, LCAT can adopt distinct orientations at each site, as shown by our 9.8 Å cryoEM envelope. In one case, LCAT membrane binding domains align with the APOA1 belts and, in the other, the HDL phospholipids. By introducing disulfide bonds between the APOA1 helical domains, we demonstrated that LCAT does not require helical separation during its reaction cycle. This indicates that LCAT, anchored to APOA1 belts, accesses substrates and deposits products through interactions with the planar lipid surface. This model of the LCAT/APOA1 interaction provides insights into how LCAT and possibly other HDL-modifying factors engage the APOA1 scaffold, offering potential strategies to enhance LCAT activity in individuals with genetic defects.
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Affiliation(s)
- Bethany Coleman
- Department of Molecular and Cellular Biosciences, University of Cincinnati, Cincinnati, OH, USA
| | - Shimpi Bedi
- Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH, USA
| | - John H Hill
- Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Jamie Morris
- Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH, USA
| | - Kelly A Manthei
- Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA
| | - Rachel C Hart
- Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA
| | - Yi He
- Department of Medicine, University of Washington School of Medicine, Seattle, WA, USA
| | - Amy S Shah
- Department of Pediatrics, Cincinnati Children's Hospital Medical Center and the University of Cincinnati, Cincinnati, OH, USA
| | - W Gray Jerome
- Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA
| | - Tomas Vaisar
- Department of Medicine, University of Washington School of Medicine, Seattle, WA, USA
| | - Karin E Bornfeldt
- Department of Medicine, University of Washington School of Medicine, Seattle, WA, USA
| | - Hyun Song
- Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
| | - Jere P Segrest
- Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
| | - Jay W Heinecke
- Department of Medicine, University of Washington School of Medicine, Seattle, WA, USA
| | - Stephen G Aller
- Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL, USA
| | - John J G Tesmer
- Departments of Biological Sciences and Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN, USA
| | - W Sean Davidson
- Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH, USA.
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Xu M, Xie P, Liu S, Gao X, Yang S, Hu Z, Zhao Y, Yi Y, Dong Q, Bruns C, Kong X, Hung MC, Ren N, Zhou C. LCAT deficiency promotes hepatocellular carcinoma progression and lenvatinib resistance by promoting triglyceride catabolism and fatty acid oxidation. Cancer Lett 2025; 612:217469. [PMID: 39842501 DOI: 10.1016/j.canlet.2025.217469] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2024] [Revised: 01/12/2025] [Accepted: 01/15/2025] [Indexed: 01/24/2025]
Abstract
Lecithin cholesterol acyltransferase (LCAT), a crucial enzyme in lipid metabolism, plays important yet poorly understood roles in tumours, especially in hepatocellular carcinoma (HCC). In this study, our investigation revealed that LCAT is a key downregulated metabolic gene and an independent risk factor for poor prognosis in patients with HCC. Functional experiments showed that LCAT inhibited HCC cell proliferation, migration and invasion. Mechanistically, LCAT interacts with caveolin-1 (CAV1) to promote the binding of CAV1 to PRKACA and inhibit its phosphorylation, thereby inhibiting triglyceride (TAG) catabolism. On the other hand, LCAT inhibits fatty acid oxidation (FAO) by interacting with CPT1A to promote its ubiquitination and degradation. These events result in an inadequate supply of raw materials and energy and inhibit the malignant behaviours of HCC cells. In addition, LCAT is a reliable predictive biomarker for the efficacy of lenvatinib treatment in HCC patients, and the inhibition of FAO can increase lenvatinib sensitivity in patients with LCATlow HCC. This study revealed that LCAT plays a critical role in the regulation of lipid metabolic reprogramming and is a reliable predictive biomarker for the efficacy of lenvatinib treatment in HCC patients.
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Affiliation(s)
- Min Xu
- Department of Liver Surgery and Transplantation, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, 200032, PR China
| | - Peiyi Xie
- Department of Liver Surgery and Transplantation, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, 200032, PR China
| | - Shaoqing Liu
- Department of Liver Surgery and Transplantation, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, 200032, PR China; Department of Breast Surgery, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450001, P.R. China
| | - Xukang Gao
- Department of Liver Surgery and Transplantation, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, 200032, PR China
| | - Shiguang Yang
- Key Laboratory of Whole-Period Monitoring and Precise Intervention of Digestive Cancer of Shanghai Municipal Health Commission, Shanghai, 201199, PR China; Institute of Fudan-Minhang Academic Health System, Minhang Hospital, Fudan University, Shanghai, 201199, PR China; Department of Hepatobiliary and Pancreatic Surgery, Minhang Hospital, Fudan University, Shanghai, 201199, PR China
| | - Zhiqiu Hu
- Key Laboratory of Whole-Period Monitoring and Precise Intervention of Digestive Cancer of Shanghai Municipal Health Commission, Shanghai, 201199, PR China; Institute of Fudan-Minhang Academic Health System, Minhang Hospital, Fudan University, Shanghai, 201199, PR China; Department of Hepatobiliary and Pancreatic Surgery, Minhang Hospital, Fudan University, Shanghai, 201199, PR China
| | - Yue Zhao
- Department of General, Visceral, Cancer and Transplantation Surgery, University Hospital of Cologne, Kerpener Straße 62, 50937, Cologne, Germany
| | - Yong Yi
- Department of Liver Surgery and Transplantation, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, 200032, PR China
| | - Qiongzhu Dong
- Key Laboratory of Whole-Period Monitoring and Precise Intervention of Digestive Cancer of Shanghai Municipal Health Commission, Shanghai, 201199, PR China; Institute of Fudan-Minhang Academic Health System, Minhang Hospital, Fudan University, Shanghai, 201199, PR China
| | - Christiane Bruns
- Department of General, Visceral, Cancer and Transplantation Surgery, University Hospital of Cologne, Kerpener Straße 62, 50937, Cologne, Germany
| | - Xiaoni Kong
- Central Laboratory, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, PR China.
| | - Mien-Chie Hung
- Graduate Institute of Biomedical Sciences, Institute of Biochemistry and Molecular Biology, Research Center for Cancer Biology, Cancer Biology and Precision Therapeutics Center, and Center for Molecular Medicine, China Medical University, Taichung, 40402, Taiwan.
| | - Ning Ren
- Department of Liver Surgery and Transplantation, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, 200032, PR China.
| | - Chenhao Zhou
- Department of Liver Surgery and Transplantation, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, 200032, PR China.
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Yan Y, Zhao N, Liu J, Zhang S, Zhang Y, Qin X, Zhai K, Du C. Ziziphi Spinosae Semen Flavonoid Ameliorates Hypothalamic Metabolism and Modulates Gut Microbiota in Chronic Restraint Stress-Induced Anxiety-like Behavior in Mice. Foods 2025; 14:828. [PMID: 40077533 PMCID: PMC11898499 DOI: 10.3390/foods14050828] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2025] [Revised: 02/15/2025] [Accepted: 02/22/2025] [Indexed: 03/14/2025] Open
Abstract
Ziziphi Spinosae Semen (ZSS), a homology of medicine and a type of seed, has been widely used to improve sleep quality. The present study aimed to assess the effects of ZSS flavonoid (ZSSF) extracted and isolated from ZSS on gut microbiota and hypothalamus metabolomic profiles in a chronic restraint stress (CRS)-induced anxiety mouse model. ZSSF was prepared using microporous resin chromatography, and seven compounds were determined by UPLC-MS. ZSSF treatment dramatically reduced anxiety-like behaviors, exerted sedative-hypnotic effects, increased hippocampal 5-HT and 5-HTP, and enhanced intestinal barrier function through inhibiting colon ZO-1, Claudin-1, and Occludin expression and reducing TNF-α, IL-6, and IL-1β levels. Compared with the CRS group, the diversity of gut microbiota in ZSSF-group mice was increased, with an increase in Bacteroidetes and a decrease in Firmicutes, and it was accompanied by an increase in fecal SCFAs. Hypothalamus metabolomics and lipidomics were performed to achieve 25 differential metabolites and 44 lipids, respectively. Serum metabolomics showed a total of 13 metabolites associated with anxiety were remarkably regulated by ZSSF. Weighted correlation network analysis (WGCNA) showed that glycerophospholipids (GPs) as well as phenylalanine, tyrosine, and L-tryptophan in peripheral and central parts were significant metabolites, which contributed to the pharmacological action of ZSSF. The mRNA expression of TPH2 and DDC key enzymes associated with tryptophan metabolism were upregulated, and PLA2G12A, LACT, and PLA2G6 key enzymes associated with GP metabolism were downregulated in ZSSF compared with CRS. Briefly, ZSSF improved tryptophan and GP metabolism and regulated the gut microbiome. This study may lay a theoretical basis for potentially developing ZSSF as a natural functional food ingredient for the improvement of anxiety and sleep disorders.
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Affiliation(s)
- Yan Yan
- Modern Research Center for Traditional Chinese Medicine, Shanxi University, Taiyuan 030006, China; (Y.Y.); (N.Z.); (J.L.); (Y.Z.); (X.Q.)
| | - Ni Zhao
- Modern Research Center for Traditional Chinese Medicine, Shanxi University, Taiyuan 030006, China; (Y.Y.); (N.Z.); (J.L.); (Y.Z.); (X.Q.)
| | - Jiaying Liu
- Modern Research Center for Traditional Chinese Medicine, Shanxi University, Taiyuan 030006, China; (Y.Y.); (N.Z.); (J.L.); (Y.Z.); (X.Q.)
| | - Shengmei Zhang
- School of Traditional Chinese Materia Medica, Shanxi University of Chinese Medicine, Taiyuan 030619, China;
| | - Yinjie Zhang
- Modern Research Center for Traditional Chinese Medicine, Shanxi University, Taiyuan 030006, China; (Y.Y.); (N.Z.); (J.L.); (Y.Z.); (X.Q.)
| | - Xuemei Qin
- Modern Research Center for Traditional Chinese Medicine, Shanxi University, Taiyuan 030006, China; (Y.Y.); (N.Z.); (J.L.); (Y.Z.); (X.Q.)
| | - Kefeng Zhai
- Modern Research Center for Traditional Chinese Medicine, Shanxi University, Taiyuan 030006, China; (Y.Y.); (N.Z.); (J.L.); (Y.Z.); (X.Q.)
- Engineering Research Center for Development and High Value Utilization of Genuine Medicinal Materials in North Anhui Province, School of Biological and Food Engineering, Suzhou University, Suzhou 234000, China
| | - Chenhui Du
- School of Traditional Chinese Materia Medica, Shanxi University of Chinese Medicine, Taiyuan 030619, China;
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Xu Z, Yang S, Cui L. Understanding the heterogeneity and dysfunction of HDL in chronic kidney disease: insights from recent reviews. BMC Nephrol 2024; 25:400. [PMID: 39511510 PMCID: PMC11542271 DOI: 10.1186/s12882-024-03808-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2024] [Accepted: 10/08/2024] [Indexed: 11/15/2024] Open
Abstract
Chronic kidney disease (CKD) is a complex disease that affects the global population's health, with dyslipidemia being one of its major complications. High density lipoprotein (HDL) is regarded as the "hero" in the bloodstream due to its role in reverse cholesterol transport, which lowers cholesterol levels in the blood and prevents atherosclerosis. However, in the complex internal environment of CKD, even this "hero" may struggle to perform its beneficial functions and could potentially become harmful. This article reviews HDL heterogeneity, HDL subclasses, functional changes in HDL during the progression of CKD, and the application of HDL in CKD treatment. This review aims to deepen understanding of lipid metabolism abnormalities in CKD patients and provide a basis for new therapeutic strategies.
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Affiliation(s)
- Zhen Xu
- Peking University Third Hospital, Beijing, China
| | - Shuo Yang
- Peking University Third Hospital, Beijing, China
| | - Liyan Cui
- Peking University Third Hospital, Beijing, China.
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Abu Dail Y, Flockerzi E, Flockerzi F, Matthaei M, Cursiefen C, Seitz B. A Novel Symptomatic Lecithin-Cholesterol Acyltransferase Gene Mutation With Corneal Amyloidosis. Cornea 2024; 43:1427-1430. [PMID: 39177400 DOI: 10.1097/ico.0000000000003671] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2023] [Accepted: 07/21/2024] [Indexed: 08/24/2024]
Abstract
PURPOSE To present ocular clinical, histological, systemic, and genetic findings of a patient with familial lecithin-cholesterol acyltransferase (LCAT) deficiency caused by a novel genetic variant of the LCAT gene associated with secondary corneal amyloidosis. METHODS Case report. RESULTS A 74-year-old woman presented with decreased visual acuity (VA), sensitivity to light, and progressive whitening of both corneas for approximately 20 years. The patient had undergone penetrating keratoplasty (PKP) on the right eye 6 years ago. Ophthalmologic examination revealed decreased VA in both eyes (OD: 0.05, OS: 0.3), and even further reduced glare VA (OD: 0.05, OS: 0.1), diffuse whitish corneal opacity involving the total thickness of the corneal stroma without crystalline deposits, and a marked peripheral diffuse arcus. Systemic examination revealed severely reduced plasma high-density lipoprotein cholesterol levels, target cells in blood smear, and chronic normochromic anemia. Clinically, LCAT deficiency was the most likely diagnosis. Further genetic analysis confirmed the diagnosis. The patient is homozygous for the novel variant c.943T>C (p.Trp315Arg) in the LCAT gene. Histologic examination of the cornea removed during the first keratoplasty revealed amyloid deposits. The cornea removed at the second keratoplasty had small vacuoles in the anterior stroma, indicating recurrence of lipid deposition. CONCLUSIONS LCAT deficiency is a rare genetic disorder that can cause corneal opacities because of lipid deposition in the cornea. Systemic manifestations may help in the differential diagnosis to other diseases associated with severe high-density lipoprotein cholesterol reduction. Genetic analysis is employed to confirm the diagnosis. Some mutations in the LCAT gene seem to be associated with secondary corneal amyloidosis. Further investigation of this association is warranted. A recurrence of corneal opacity after PKP seems to occur mainly in the anterior corneal stroma.
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Affiliation(s)
- Yaser Abu Dail
- Department of Ophthalmology, Saarland University Medical Center, Homburg/Saar, Germany
| | - Elias Flockerzi
- Department of Ophthalmology, Saarland University Medical Center, Homburg/Saar, Germany
| | - Fidelis Flockerzi
- Institute of Pathology, Saarland University Medical Center, Homburg/Saar, Germany; and
| | - Mario Matthaei
- Department of Ophthalmology, University of Cologne, Cologne, Germany
| | - Claus Cursiefen
- Department of Ophthalmology, University of Cologne, Cologne, Germany
| | - Berthold Seitz
- Department of Ophthalmology, Saarland University Medical Center, Homburg/Saar, Germany
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Manthei KA, Tremonti GE, Chang L, Niemelä A, Giorgi L, Koivuniemi A, Tesmer JJG. Rescue of Familial Lecithin:Cholesterol Acyltranferase Deficiency Mutations with an Allosteric Activator. Mol Pharmacol 2024; 106:188-197. [PMID: 39151949 PMCID: PMC11413911 DOI: 10.1124/molpharm.124.000932] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2024] [Revised: 07/08/2024] [Accepted: 08/01/2024] [Indexed: 08/19/2024] Open
Abstract
Lecithin:cholesterol acyltransferase (LCAT) deficiencies represent severe disorders characterized by aberrant cholesterol esterification in plasma, leading to life-threatening conditions. This study investigates the efficacy of Compound 2, a piperidinyl pyrazolopyridine allosteric activator that binds the membrane-binding domain of LCAT, in rescuing the activity of LCAT variants associated with disease. The variants K218N, N228K, and G230R, all located in the cap and lid domains of LCAT, demonstrated notable activity restoration in response to Compound 2. Molecular dynamics simulations and structural modeling indicate that these mutations disrupt the lid and membrane binding domain, with Compound 2 potentially dampening these structural alterations. Conversely, variants such as M252K and F382V in the cap and α/β-hydrolase domain, respectively, exhibited limited or no rescue by Compound 2. Future research should prioritize in vivo investigations that would validate the therapeutic potential of Compound 2 and related activators in familial LCAT deficiency patients with mutations in the cap and lid of the enzyme. SIGNIFICANCE STATEMENT: Lecithin:cholesterol acyltranferase (LCAT) catalyzes the first step of reverse cholesterol transport, namely the esterification of cholesterol in high density lipoprotein particles. Somatic mutations in LCAT lead to excess cholesterol in blood plasma and, in severe cases, kidney failure. In this study, we show that recently discovered small molecule activators can rescue function in LCAT-deficient variants when the mutations occur in the lid and cap domains of the enzyme.
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Affiliation(s)
- Kelly A Manthei
- Department of Molecular Pharmacology, University of Michigan, Ann Arbor, Michigan (K.A.M., G.E.T., L.C.); Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland (A.N., L.G., A.K.); and Departments of Biological Sciences and of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana (J.J.G.T.)
| | - Grace E Tremonti
- Department of Molecular Pharmacology, University of Michigan, Ann Arbor, Michigan (K.A.M., G.E.T., L.C.); Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland (A.N., L.G., A.K.); and Departments of Biological Sciences and of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana (J.J.G.T.)
| | - Louise Chang
- Department of Molecular Pharmacology, University of Michigan, Ann Arbor, Michigan (K.A.M., G.E.T., L.C.); Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland (A.N., L.G., A.K.); and Departments of Biological Sciences and of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana (J.J.G.T.)
| | - Akseli Niemelä
- Department of Molecular Pharmacology, University of Michigan, Ann Arbor, Michigan (K.A.M., G.E.T., L.C.); Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland (A.N., L.G., A.K.); and Departments of Biological Sciences and of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana (J.J.G.T.)
| | - Laura Giorgi
- Department of Molecular Pharmacology, University of Michigan, Ann Arbor, Michigan (K.A.M., G.E.T., L.C.); Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland (A.N., L.G., A.K.); and Departments of Biological Sciences and of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana (J.J.G.T.)
| | - Artturi Koivuniemi
- Department of Molecular Pharmacology, University of Michigan, Ann Arbor, Michigan (K.A.M., G.E.T., L.C.); Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland (A.N., L.G., A.K.); and Departments of Biological Sciences and of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana (J.J.G.T.)
| | - John Joseph Grubb Tesmer
- Department of Molecular Pharmacology, University of Michigan, Ann Arbor, Michigan (K.A.M., G.E.T., L.C.); Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland (A.N., L.G., A.K.); and Departments of Biological Sciences and of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana (J.J.G.T.)
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Li Y, Jiang LN, Zhao BK, Li ML, Jiang YY, Liu YS, Liu SH, Zhu L, Ye X, Zhao JM. Lecithin-cholesterol acyltransferase is a potential tumor suppressor and predictive marker for hepatocellular carcinoma metastasis. World J Gastrointest Oncol 2024; 16:3651-3671. [PMID: 39171187 PMCID: PMC11334038 DOI: 10.4251/wjgo.v16.i8.3651] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/24/2024] [Revised: 05/27/2024] [Accepted: 06/18/2024] [Indexed: 08/07/2024] Open
Abstract
BACKGROUND Hepatocellular carcinoma (HCC) is a major cause of cancer mortality worldwide, and metastasis is the main cause of early recurrence and poor prognosis. However, the mechanism of metastasis remains poorly understood. AIM To determine the possible mechanism affecting HCC metastasis and provide a possible theoretical basis for HCC treatment. METHODS The candidate molecule lecithin-cholesterol acyltransferase (LCAT) was screened by gene microarray and bioinformatics analysis. The expression levels of LCAT in clinical cohort samples was detected by quantitative real-time polymerase chain reaction and western blotting. The proliferation, migration, invasion and tumor-forming ability were measured by Cell Counting Kit-8, Transwell cell migration, invasion, and clonal formation assays, respectively. Tumor formation was detected in nude mice after LCAT gene knockdown or overexpression. The immunohistochemistry for Ki67, E-cadherin, N-cadherin, matrix metalloproteinase 9 and vascular endothelial growth factor were performed in liver tissues to assess the effect of LCAT on HCC. Gene set enrichment analysis (GSEA) on various gene signatures were analyzed with GSEA version 3.0. Three machine-learning algorithms (random forest, support vector machine, and logistic regression) were applied to predict HCC metastasis in The Cancer Genome Atlas and GEO databases. RESULTS LCAT was identified as a novel gene relating to HCC metastasis by using gene microarray in HCC tissues. LCAT was significantly downregulated in HCC tissues, which is correlated with recurrence, metastasis and poor outcome of HCC patients. Functional analysis indicated that LCAT inhibited HCC cell proliferation, migration and invasion both in vitro and in vivo. Clinicopathological data showed that LCAT was negatively associated with HCC size and metastasis (HCC size ≤ 3 cm vs 3-9 cm, P < 0.001; 3-9 cm vs > 9 cm, P < 0.01; metastatic-free HCC vs extrahepatic metastatic HCC, P < 0.05). LCAT suppressed the growth, migration and invasion of HCC cell lines via PI3K/AKT/mTOR signaling. Our results indicated that the logistic regression model based on LCAT, TNM stage and the serum level of α-fetoprotein in HCC patients could effectively predict high metastatic risk HCC patients. CONCLUSION LCAT is downregulated at translational and protein levels in HCC and might inhibit tumor metastasis via attenuating PI3K/AKT/mTOR signaling. LCAT is a prognostic marker and potential therapeutic target for HCC.
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Affiliation(s)
- Yan Li
- Department of Pathology and Hepatology, The Fifth Medical Center of Chinese PLA General Hospital, Beijing 100039, China
| | - Li-Na Jiang
- Department of Pathology and Hepatology, The Fifth Medical Center of Chinese PLA General Hospital, Beijing 100039, China
| | - Bo-Kang Zhao
- Department of Hepatology, Center of Infectious Diseases and Pathogen Biology, The First Hospital of Jilin University, Changchun 130061, Jilin Province, China
| | - Mei-Ling Li
- Department of Pathology and Hepatology, The Fifth Medical Center of Chinese PLA General Hospital, Beijing 100039, China
| | - Yi-Yun Jiang
- Department of Pathology and Hepatology, The Fifth Medical Center of Chinese PLA General Hospital, Beijing 100039, China
| | - Yi-Si Liu
- First Department of Liver Disease Center, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China
| | - Shu-Hong Liu
- Department of Pathology and Hepatology, The Fifth Medical Center of Chinese PLA General Hospital, Beijing 100039, China
| | - Li Zhu
- Department of Pathology and Hepatology, The Fifth Medical Center of Chinese PLA General Hospital, Beijing 100039, China
| | - Xin Ye
- Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
- Savaid Medical School, University of Chinese Academy of Sciences, Beijing 100190, China
| | - Jing-Min Zhao
- Department of Pathology, The Fifth Medical Center of Chinese PLA General Hospital, Beijing 100039, China
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Huang XW, Li Y, Jiang LN, Zhao BK, Liu YS, Chen C, Zhao D, Zhang XL, Li ML, Jiang YY, Liu SH, Zhu L, Zhao JM. Nomogram for preoperative estimation of microvascular invasion risk in hepatocellular carcinoma. Transl Oncol 2024; 45:101986. [PMID: 38723299 PMCID: PMC11101742 DOI: 10.1016/j.tranon.2024.101986] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2023] [Revised: 04/22/2024] [Accepted: 05/05/2024] [Indexed: 05/21/2024] Open
Abstract
Microvascular invasion (MVI) is an adverse prognostic indicator of tumor recurrence after surgery for hepatocellular carcinoma (HCC). Therefore, developing a nomogram for estimating the presence of MVI before liver resection is necessary. We retrospectively included 260 patients with pathologically confirmed HCC at the Fifth Medical Center of Chinese PLA General Hospital between January 2021 and April 2024. The patients were randomly divided into a training cohort (n = 182) for nomogram development, and a validation cohort (n = 78) to confirm the performance of the model (7:3 ratio). Significant clinical variables associated with MVI were then incorporated into the predictive nomogram using both univariate and multivariate logistic analyses. The predictive performance of the nomogram was assessed based on its discrimination, calibration, and clinical utility. Serum carnosine dipeptidase 1 ([CNDP1] OR 2.973; 95 % CI 1.167-7.575; p = 0.022), cirrhosis (OR 8.911; 95 % CI 1.922-41.318; p = 0.005), multiple tumors (OR 4.095; 95 % CI 1.374-12.205; p = 0.011), and tumor diameter ≥3 cm (OR 4.408; 95 % CI 1.780-10.919; p = 0.001) were independent predictors of MVI. Performance of the nomogram based on serum CNDP1, cirrhosis, number of tumors and tumor diameter was achieved with a concordance index of 0.833 (95 % CI 0.771-0.894) and 0.821 (95 % CI 0.720-0.922) in the training and validation cohorts, respectively. It fitted well in the calibration curves, and the decision curve analysis further confirmed its clinical usefulness. The nomogram, incorporating significant clinical variables and imaging features, successfully predicted the personalized risk of MVI in HCC preoperatively.
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Affiliation(s)
- Xiao-Wen Huang
- Medical School of Chinese PLA, Beijing, China; Department of Pathology and Hepatology, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, China
| | - Yan Li
- Department of Pathology and Hepatology, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, China
| | - Li-Na Jiang
- Department of Pathology and Hepatology, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, China
| | - Bo-Kang Zhao
- Department of Hepatology, Center of Infectious Diseases and Pathogen Biology, The First Hospital of Jilin University, Changchun, China
| | - Yi-Si Liu
- First Department of Liver Disease Center, Beijing Youan Hospital, Capital Medical University, Beijing, China
| | - Chun Chen
- Senior Department of Hepatology, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, China
| | - Dan Zhao
- Department of Pathology and Hepatology, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, China
| | - Xue-Li Zhang
- Department of Pathology and Hepatology, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, China
| | - Mei-Ling Li
- Department of Pathology and Hepatology, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, China
| | - Yi-Yun Jiang
- Department of Pathology and Hepatology, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, China
| | - Shu-Hong Liu
- Department of Pathology and Hepatology, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, China
| | - Li Zhu
- Department of Pathology and Hepatology, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, China
| | - Jing-Min Zhao
- Medical School of Chinese PLA, Beijing, China; Department of Pathology and Hepatology, The Fifth Medical Center of Chinese PLA General Hospital, Beijing, China.
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9
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Barbosa AD, Siniossoglou S. Membranes that make fat: roles of membrane lipids as acyl donors for triglyceride synthesis and organelle function. FEBS Lett 2024; 598:1226-1234. [PMID: 38140812 DOI: 10.1002/1873-3468.14793] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2023] [Revised: 12/05/2023] [Accepted: 12/15/2023] [Indexed: 12/24/2023]
Abstract
Triglycerides constitute an inert storage form for fatty acids deposited in lipid droplets and are mobilized to provide metabolic energy or membrane building blocks. The biosynthesis of triglycerides is highly conserved within eukaryotes and normally involves the sequential esterification of activated fatty acids with a glycerol backbone. Some eukaryotes, however, can also use cellular membrane lipids as direct fatty acid donors for triglyceride synthesis. The biological significance of a pathway that generates triglycerides at the expense of organelle membranes has remained elusive. Here we review current knowledge on how cells use membrane lipids as fatty acid donors for triglyceride synthesis and discuss the hypothesis that a primary function of this pathway is to regulate membrane lipid remodeling and organelle function.
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Affiliation(s)
- Antonio D Barbosa
- Cambridge Institute for Medical Research, University of Cambridge, UK
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10
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Sacher S, Mukherjee A, Ray A. Deciphering structural aspects of reverse cholesterol transport: mapping the knowns and unknowns. Biol Rev Camb Philos Soc 2023; 98:1160-1183. [PMID: 36880422 DOI: 10.1111/brv.12948] [Citation(s) in RCA: 16] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2022] [Revised: 02/03/2023] [Accepted: 02/24/2023] [Indexed: 03/08/2023]
Abstract
Atherosclerosis is a major contributor to the onset and progression of cardiovascular disease (CVD). Cholesterol-loaded foam cells play a pivotal role in forming atherosclerotic plaques. Induction of cholesterol efflux from these cells may be a promising approach in treating CVD. The reverse cholesterol transport (RCT) pathway delivers cholesteryl ester (CE) packaged in high-density lipoproteins (HDL) from non-hepatic cells to the liver, thereby minimising cholesterol load of peripheral cells. RCT takes place via a well-organised interplay amongst apolipoprotein A1 (ApoA1), lecithin cholesterol acyltransferase (LCAT), ATP binding cassette transporter A1 (ABCA1), scavenger receptor-B1 (SR-B1), and the amount of free cholesterol. Unfortunately, modulation of RCT for treating atherosclerosis has failed in clinical trials owing to our lack of understanding of the relationship between HDL function and RCT. The fate of non-hepatic CEs in HDL is dependent on their access to proteins involved in remodelling and can be regulated at the structural level. An inadequate understanding of this inhibits the design of rational strategies for therapeutic interventions. Herein we extensively review the structure-function relationships that are essential for RCT. We also focus on genetic mutations that disturb the structural stability of proteins involved in RCT, rendering them partially or completely non-functional. Further studies are necessary for understanding the structural aspects of RCT pathway completely, and this review highlights alternative theories and unanswered questions.
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Affiliation(s)
- Sukriti Sacher
- Department of Computational Biology, Indraprastha Institute of Information Technology, Okhla Phase III, New Delhi, 110019, India
| | - Abhishek Mukherjee
- Dhiti Life Sciences Pvt Ltd, B-107, Okhla Phase I, New Delhi, 110020, India
| | - Arjun Ray
- Department of Computational Biology, Indraprastha Institute of Information Technology, Okhla Phase III, New Delhi, 110019, India
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11
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Gao H, Wu J, Sun Z, Zhang F, Shi T, Lu K, Qian D, Yin Z, Zhao Y, Qin J, Xue B. Influence of lecithin cholesterol acyltransferase alteration during different pathophysiologic conditions: A 45 years bibliometrics analysis. Front Pharmacol 2022; 13:1062249. [PMID: 36588724 PMCID: PMC9795195 DOI: 10.3389/fphar.2022.1062249] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2022] [Accepted: 12/06/2022] [Indexed: 12/15/2022] Open
Abstract
Background: Lecithin cholesterol acyltransferase (LCAT) is an important enzyme responsible for free cholesterol (FC) esterification, which is critical for high density lipoprotein (HDL) maturation and the completion of the reverse cholesterol transport (RCT) process. Plasma LCAT activity and concentration showed various patterns under different physiological and pathological conditions. Research on LCAT has grown rapidly over the past 50 years, but there are no bibliometric studies summarizing this field as a whole. This study aimed to use the bibliometric analysis to demonstrate the trends in LCAT publications, thus offering a brief perspective with regard to future developments in this field. Methods: We used the Web of Science Core Collection to retrieve LCAT-related studies published from 1975 to 2020. The data were further analyzed in the number of studies, the journal which published the most LCAT-related studies, co-authorship network, co-country network, co-institute network, co-reference and the keywords burst by CiteSpace V 5.7. Results: 2584 publications contained 55,311 references were used to analyzed. The number of included articles fluctuated in each year. We found that Journal of lipid research published the most LCAT-related studies. Among all the authors who work on LCAT, they tend to collaborate with a relatively stable group of collaborators to generate several major authors clusters which Albers, J. published the most studies (n = 53). The United States of America contributed the greatest proportion (n = 1036) of LCAT-related studies. The LCAT-related studies have been focused on the vascular disease, lecithin-cholesterol acyltransferase reaction, phospholipid, cholesterol efflux, chronic kidney disease, milk fever, nephrotic syndrome, platelet-activating factor acetylhydrolase, reconstituted lpa-i, reverse cholesterol transport. Four main research frontiers in terms of burst strength for LCAT-related studies including "transgenic mice", "oxidative stress", "risk", and "cholesterol metabolism "need more attention. Conclusion: This is the first study that demonstrated the trends and future development in LCAT publications. Further studies should focus on the accurate metabolic process of LCAT dependent or independent of RCT using metabolic marker tracking techniques. It was also well worth to further studying the possibility that LCAT may qualify as a biomarker for risk prediction and clinical treatment.
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Affiliation(s)
- Hongliang Gao
- Core Laboratory, Sir Run Run Hospital, Nanjing Medical University, Nanjing, China,School of Clinical Medicine, Wannan Medical College, Wuhu, China,Collaborative Innovation Center of Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing, China
| | - Jing Wu
- Core Laboratory, Sir Run Run Hospital, Nanjing Medical University, Nanjing, China
| | - Zhenyu Sun
- School of Health Policy and Management, Center for Global Health, Nanjing Medical University, Nanjing, China
| | - Furong Zhang
- Core Laboratory, Sir Run Run Hospital, Nanjing Medical University, Nanjing, China
| | - Tianshu Shi
- State Key Laboratory of Pharmaceutical Biotechnology, Department of Sports Medicine and Adult Reconstructive Surgery, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, China
| | - Ke Lu
- Research Center for Computer-Aided Drug Discovery, Chinese Academy of Sciences, Shenzhen, China
| | - Dongfu Qian
- School of Health Policy and Management, Center for Global Health, Nanjing Medical University, Nanjing, China
| | - Zicheng Yin
- Nanjing Foreign Language School, Nanjing, China
| | - Yinjuan Zhao
- Collaborative Innovation Center of Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing, China,*Correspondence: Bin Xue, ; Jian Qin, ; Yinjuan Zhao,
| | - Jian Qin
- Core Laboratory, Sir Run Run Hospital, Nanjing Medical University, Nanjing, China,*Correspondence: Bin Xue, ; Jian Qin, ; Yinjuan Zhao,
| | - Bin Xue
- Core Laboratory, Sir Run Run Hospital, Nanjing Medical University, Nanjing, China,*Correspondence: Bin Xue, ; Jian Qin, ; Yinjuan Zhao,
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12
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Sun S, Wang J, Yao J, Guo H, Dai J. Transcriptome analysis of 3D primary mouse liver spheroids shows that long-term exposure to hexafluoropropylene oxide trimer acid disrupts hepatic bile acid metabolism. THE SCIENCE OF THE TOTAL ENVIRONMENT 2022; 812:151509. [PMID: 34762948 DOI: 10.1016/j.scitotenv.2021.151509] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/02/2021] [Revised: 10/27/2021] [Accepted: 11/03/2021] [Indexed: 06/13/2023]
Abstract
Hexafluoropropylene oxide trimer acid (HFPO-TA), an alternative to perfluorooctanoic acid (PFOA), has been detected in various environmental and human matrices. However, information regarding its toxicity remains limited. Here, we established a three-dimensional (3D) primary mouse liver spheroid model to compare the hepatotoxicity of HFPO-TA and PFOA. The 3D spheroids were repeatedly exposed to 25-, 50-, or 100-μM HFPO-TA and PFOA for 28 d. Compared with the PFOA groups, the HFPO-TA groups showed higher bioaccumulation potential, higher lactate dehydrogenase (LDH) leakage, and lower adenosine triphosphate (ATP), albumin, and urea secretion. Transcriptome analysis identified 1603 and 772 differentially expressed genes in the 100-μM HFPO-TA- and PFOA-treated groups, respectively. Bioinformatics analysis indicated that cholesterol metabolism, bile acid metabolism, and inflammatory response were significantly altered. Exposure to 100-μM HFPO-TA increased triglyceride content but decreased total cholesterol content, while no changes were observed in the 100-μM PFOA-treated group. Total bile acids in the re-polarized 3D spheroids increased significantly after 100-μM HFPO-TA and PFOA treatment, which did not affect bile acid synthesis but inhibited the expression levels of Bsep and Mrp2 related to bile acid transport. Thus, HFPO-TA exhibited more serious hepatotoxicity than PFOA in 3D primary liver spheroids and may not be a safe alternative.
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Affiliation(s)
- Sujie Sun
- State Environmental Protection Key Laboratory of Environmental Health Impact Assessment of Emerging Contaminants, School of Environmental Sciences and Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Minhang District, Shanghai 200240, China; Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Jianshe Wang
- School of Pharmacy, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Yantai University, Yantai 264005, Shandong Province, China
| | - Jingzhi Yao
- State Environmental Protection Key Laboratory of Environmental Health Impact Assessment of Emerging Contaminants, School of Environmental Sciences and Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Minhang District, Shanghai 200240, China; Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Hua Guo
- State Environmental Protection Key Laboratory of Environmental Health Impact Assessment of Emerging Contaminants, School of Environmental Sciences and Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Minhang District, Shanghai 200240, China; Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Jiayin Dai
- State Environmental Protection Key Laboratory of Environmental Health Impact Assessment of Emerging Contaminants, School of Environmental Sciences and Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Minhang District, Shanghai 200240, China; Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
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13
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Yang K, Wang J, Xiang H, Ding P, Wu T, Ji G. LCAT- targeted therapies: Progress, failures and future. Biomed Pharmacother 2022; 147:112677. [PMID: 35121343 DOI: 10.1016/j.biopha.2022.112677] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2021] [Revised: 01/21/2022] [Accepted: 01/26/2022] [Indexed: 11/22/2022] Open
Abstract
Lecithin: cholesterol acyltransferase (LCAT) is the only enzyme in plasma which is able to esterify cholesterol and boost cholesterol esterify with phospholipid-derived acyl chains. In order to better understand the progress of LCAT research, it is always inescapable that it is linked to high-density lipoprotein (HDL) metabolism and reverse cholesterol transport (RCT). Because LCAT plays a central role in HDL metabolism and RCT, many animal studies and clinical studies are currently aimed at improving plasma lipid metabolism by increasing LCAT activity in order to find better treatment options for familial LCAT deficiency (FLD), fish eye disease (FED), and cardiovascular disease. Recombinant human LCAT (rhLCAT) injections, cells and gene therapy, and small molecule activators have been carried out with promising results. Recently rhLCAT therapies have entered clinical phase II trials with good prospects. In this review, we discuss the diseases associated with LCAT and therapies that use LCAT as a target hoping to find out whether LCAT can be an effective therapeutic target for coronary heart disease and atherosclerosis. Also, probing the mechanism of action of LCAT may help better understand the heterogeneity of HDL and the action mechanism of dynamic lipoprotein particles.
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Affiliation(s)
- Kaixu Yang
- Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
| | - Junmin Wang
- Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
| | - Hongjiao Xiang
- Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
| | - Peilun Ding
- Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
| | - Tao Wu
- Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
| | - Guang Ji
- Institute of Digestive Disease, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China.
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14
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Lebeau PF, Platko K, Byun JH, Makda Y, Austin RC. The Emerging Roles of Intracellular PCSK9 and Their Implications in Endoplasmic Reticulum Stress and Metabolic Diseases. Metabolites 2022; 12:metabo12030215. [PMID: 35323658 PMCID: PMC8954296 DOI: 10.3390/metabo12030215] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2022] [Revised: 02/20/2022] [Accepted: 02/25/2022] [Indexed: 02/04/2023] Open
Abstract
The importance of the proprotein convertase subtilisin/kexin type-9 (PCSK9) gene was quickly recognized by the scientific community as the third locus for familial hypercholesterolemia. By promoting the degradation of the low-density lipoprotein receptor (LDLR), secreted PCSK9 protein plays a vital role in the regulation of circulating cholesterol levels and cardiovascular disease risk. For this reason, the majority of published works have focused on the secreted form of PCSK9 since its initial characterization in 2003. In recent years, however, PCSK9 has been shown to play roles in a variety of cellular pathways and disease contexts in LDLR-dependent and -independent manners. This article examines the current body of literature that uncovers the intracellular and LDLR-independent roles of PCSK9 and also explores the many downstream implications in metabolic diseases.
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15
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Chen GQ, Kim WN, Johnson K, Park ME, Lee KR, Kim HU. Transcriptome Analysis and Identification of Lipid Genes in Physaria lindheimeri, a Genetic Resource for Hydroxy Fatty Acids in Seed Oil. Int J Mol Sci 2021; 22:ijms22020514. [PMID: 33419225 PMCID: PMC7825617 DOI: 10.3390/ijms22020514] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2020] [Revised: 01/01/2021] [Accepted: 01/04/2021] [Indexed: 12/15/2022] Open
Abstract
Hydroxy fatty acids (HFAs) have numerous industrial applications but are absent in most vegetable oils. Physaria lindheimeri accumulating 85% HFA in its seed oil makes it a valuable resource for engineering oilseed crops for HFA production. To discover lipid genes involved in HFA synthesis in P. lindheimeri, transcripts from developing seeds at various stages, as well as leaf and flower buds, were sequenced. Ninety-seven percent clean reads from 552,614,582 raw reads were assembled to 129,633 contigs (or transcripts) which represented 85,948 unique genes. Gene Ontology analysis indicated that 60% of the contigs matched proteins involved in biological process, cellular component or molecular function, while the remaining matched unknown proteins. We identified 42 P. lindheimeri genes involved in fatty acid and seed oil biosynthesis, and 39 of them shared 78-100% nucleotide identity with Arabidopsis orthologs. We manually annotated 16 key genes and 14 of them contained full-length protein sequences, indicating high coverage of clean reads to the assembled contigs. A detailed profiling of the 16 genes revealed various spatial and temporal expression patterns. The further comparison of their protein sequences uncovered amino acids conserved among HFA-producing species, but these varied among non-HFA-producing species. Our findings provide essential information for basic and applied research on HFA biosynthesis.
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Affiliation(s)
- Grace Q. Chen
- Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, CA 94710, USA;
- Correspondence: (G.Q.C.); (H.U.K.)
| | - Won Nyeong Kim
- Department of Bioindustry and Bioresource Engineering, Sejong University, Seoul 05006, Korea;
| | - Kumiko Johnson
- Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, CA 94710, USA;
| | - Mid-Eum Park
- Department of Molecular Biology, Graduate School, Sejong University, Seoul 05006, Korea;
| | - Kyeong-Ryeol Lee
- Department of Agricultural Biotechnology, National Institute of Agricultural Sciences, Rural Development Administration, Jeonju 54974, Korea;
| | - Hyun Uk Kim
- Department of Bioindustry and Bioresource Engineering, Sejong University, Seoul 05006, Korea;
- Department of Molecular Biology, Graduate School, Sejong University, Seoul 05006, Korea;
- Correspondence: (G.Q.C.); (H.U.K.)
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16
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Laurenzi T, Parravicini C, Palazzolo L, Guerrini U, Gianazza E, Calabresi L, Eberini I. rHDL modeling and the anchoring mechanism of LCAT activation. J Lipid Res 2020; 62:100006. [PMID: 33518511 PMCID: PMC7859856 DOI: 10.1194/jlr.ra120000843] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2020] [Revised: 11/23/2020] [Accepted: 12/02/2020] [Indexed: 11/29/2022] Open
Abstract
Lecithin:cholesterol-acyl transferase (LCAT) plays a major role in cholesterol metabolism as it is the only extracellular enzyme able to esterify cholesterol. LCAT activity is required for lipoprotein remodeling and, most specifically, for the growth and maturation of HDLs. In fact, genetic alterations affecting LCAT functionality may cause a severe reduction in plasma levels of HDL-cholesterol with important clinical consequences. Although several hypotheses were formulated, the exact molecular recognition mechanism between LCAT and HDLs is still unknown. We employed a combination of structural bioinformatics procedures to deepen the insights into the HDL-LCAT interplay that promotes LCAT activation and cholesterol esterification. We have generated a data-driven model of reconstituted HDL (rHDL) and studied the dynamics of an assembled rHDL::LCAT supramolecular complex, pinpointing the conformational changes originating from the interaction between LCAT and apolipoprotein A-I (apoA-I) that are necessary for LCAT activation. Specifically, we propose a mechanism in which the anchoring of LCAT lid to apoA-I helices allows the formation of a hydrophobic hood that expands the LCAT active site and shields it from the solvent, allowing the enzyme to process large hydrophobic substrates.
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Affiliation(s)
- Tommaso Laurenzi
- Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, Italy
| | - Chiara Parravicini
- Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, Italy
| | - Luca Palazzolo
- Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, Italy
| | - Uliano Guerrini
- Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, Italy
| | - Elisabetta Gianazza
- Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, Italy
| | - Laura Calabresi
- Centro Enrica Grossi Paoletti, Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, Italy
| | - Ivano Eberini
- Dipartimento di Scienze Farmacologiche e Biomolecolari & DSRC, Università degli Studi di Milano, Milan, Italy.
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17
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CHEN L, ZHANG J, LI N, ZHANG L, XU XF. Suckling calves (Bos taurus) with pica exhibit blood metabolome alterations. THE INDIAN JOURNAL OF ANIMAL SCIENCES 2020. [DOI: 10.56093/ijans.v90i5.104613] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/05/2022]
Abstract
The aim of this study was to evaluate the changes of blood metabolomics in calves with pica, which causes serious harm to livestock. Two groups, each comprising 12 calves of approximately 3 weeks old and of similar weight were selected as subjects; Group A calves were control animals in good condition and Group B calves (pica animals) had rough hair, emaciation, flaccid forestomach, diarrhoea, and stunted development. Blood samples were collected from the tail root vein. Masslynx 4.1 software (Waters Company) was used to pre-process data, which were then analysed by principal component analysis, partial least squares discriminant analysis, and orthogonal partial least squares discriminant analysis. Twenty potential biomarkers were closely related to the occurrence of pica, viz. GDP-glucose, UDP-glucose, proline, creatine, arginine, glutamine, citrulline, urea, alanine, methionine, serine, glycerate, cysteine, spermine, spermidine, carnitine, xanthurenic acid, kynurenine, and thyroxine. Metabolic pathway analysis showed that, in calves, pica resulted in decreased antioxidant capacity; disruption of the mutual transformation between pentose and glucuronic acid; abnormal metabolism of cysteine, methionine, serine, arginine, and proline; impairment of lipid metabolism; reduced immunity; increased intestinal permeability; and elevated central nervous excitability. Calves with pica exhibit disruption of various metabolic pathways.
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18
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Falarz LJ, Xu Y, Caldo KMP, Garroway CJ, Singer SD, Chen G. Characterization of the diversification of phospholipid:diacylglycerol acyltransferases in the green lineage. THE PLANT JOURNAL : FOR CELL AND MOLECULAR BIOLOGY 2020; 103:2025-2038. [PMID: 32538516 DOI: 10.1111/tpj.14880] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/01/2020] [Revised: 05/28/2020] [Accepted: 06/02/2020] [Indexed: 05/03/2023]
Abstract
Triacylglycerols have important physiological roles in photosynthetic organisms, and are widely used as food, feed and industrial materials in our daily life. Phospholipid:diacylglycerol acyltransferase (PDAT) is the pivotal enzyme catalyzing the acyl-CoA-independent biosynthesis of triacylglycerols, which is unique in plants, algae and fungi, but not in animals, and has essential functions in plant and algal growth, development and stress responses. Currently, this enzyme has yet to be examined in an evolutionary context at the level of the green lineage. Some fundamental questions remain unanswered, such as how PDATs evolved in photosynthetic organisms and whether the evolution of terrestrial plant PDATs from a lineage of charophyte green algae diverges in enzyme function. As such, we used molecular evolutionary analysis and biochemical assays to address these questions. Our results indicated that PDAT underwent divergent evolution in the green lineage: PDATs exist in a wide range of plants and algae, but not in cyanobacteria. Although PDATs exhibit the conservation of several features, phylogenetic and selection-pressure analyses revealed that overall they evolved to be highly divergent, driven by different selection constraints. Positive selection, as one major driving force, may have resulted in enzymes with a higher functional importance in land plants than green algae. Further structural and mutagenesis analyses demonstrated that some amino acid sites under positive selection are critically important to PDAT structure and function, and may be central in lecithin:cholesterol acyltransferase family enzymes in general.
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Affiliation(s)
- Lucas J Falarz
- Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, T6G 2P5, Canada
- Department of Biological Sciences, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada
| | - Yang Xu
- Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, T6G 2P5, Canada
| | - Kristian Mark P Caldo
- Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, T6G 2P5, Canada
| | - Colin J Garroway
- Department of Biological Sciences, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada
| | - Stacy D Singer
- Agriculture and Agri-Food Canada, Lethbridge Research and Development Centre, Lethbridge, AB, T1J 4B1, Canada
| | - Guanqun Chen
- Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, T6G 2P5, Canada
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Piersimoni L, Sinz A. Cross-linking/mass spectrometry at the crossroads. Anal Bioanal Chem 2020; 412:5981-5987. [PMID: 32472143 PMCID: PMC7442761 DOI: 10.1007/s00216-020-02700-x] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2020] [Revised: 05/01/2020] [Accepted: 05/08/2020] [Indexed: 01/01/2023]
Abstract
Cross-linking/mass spectrometry (XL-MS) has come a long way. Originally, XL-MS was used to study relatively small, purified proteins. Meanwhile, it is employed to investigate protein-protein interactions on a proteome-wide level, giving snapshots of cellular processes. Currently, XL-MS is at the intersection of a multitude of workflows and the impact this technique has in addressing specific biological questions is steadily growing. This article is intended to give a bird's-eye view of the current status of XL-MS, the benefits of using MS-cleavable cross-linkers, and the challenges posed in the future development of this powerful technology. We also illustrate how XL-MS can deliver valuable structural insights into protein complexes when used in combination with other structural techniques, such as electron microscopy. Graphical abstract.
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Affiliation(s)
- Lolita Piersimoni
- Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Charles Tanford Center, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3a, 06120, Halle (Saale), Germany
| | - Andrea Sinz
- Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Charles Tanford Center, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3a, 06120, Halle (Saale), Germany.
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20
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p-Methoxycinnamic Acid Diesters Lower Dyslipidemia, Liver Oxidative Stress and Toxicity in High-Fat Diet Fed Mice and Human Peripheral Blood Lymphocytes. Nutrients 2020; 12:nu12010262. [PMID: 31968556 PMCID: PMC7019318 DOI: 10.3390/nu12010262] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2019] [Revised: 01/07/2020] [Accepted: 01/16/2020] [Indexed: 01/28/2023] Open
Abstract
The pursuit of cholesterol lowering natural products with less side effects is needed for controlling dyslipidemia and reducing the increasing toll of cardiovascular diseases that are associated with morbidity and mortality worldwide. The present study aimed at the examining effects of p-methoxycinnamic acid diesters (PCO-C) from carnauba (Copernicia prunifera)-derived wax on cytotoxic, genotoxic responses in vitro and on dyslipidemia and liver oxidative stress in vivo, utilizing high-fat diet (HFD) chronically fed Swiss mice. In addition, we evaluated the effect of PCO-C on the expression of key cholesterol metabolism-related genes, as well as the structural interactions between PCO-C and lecithin-cholesterol acyl transferase (LCAT) in silico. Oral treatment with PCO-C was able to reduce total serum cholesterol and low-density lipoprotein (LDL) levels following HFD. In addition, PCO-C reduced excessive weight gain and lipid peroxidation, and increased the gene expression of LCAT following HFD. Furthermore, the high affinity of the studied compound (ΔG: −8.78 Kcal/mol) towards the active sites of mutant LCAT owing to hydrophobic and van der Waals interactions was confirmed using bioinformatics. PCO-C showed no evidence of renal and hepatic toxicity, unlike simvastatin, that elevated aspartate aminotransferase (AST) levels, a marker of liver dysfunction. Finally, PCO-C showed no cytotoxicity or genotoxicity towards human peripheral blood lymphocytes in vitro. Our results suggest that PCO-C exerts hypocholesterolemic effects. The safety of PCO-C in the toxicological tests performed and the reports of its beneficial biological effects render this a promising compound for the development of new cholesterol-lowering therapeutics to control dyslipidemia. More work is needed for further elucidating PCO-C role on lipid metabolism to support future clinical studies.
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Structural analysis of lecithin:cholesterol acyltransferase bound to high density lipoprotein particles. Commun Biol 2020; 3:28. [PMID: 31942029 PMCID: PMC6962161 DOI: 10.1038/s42003-019-0749-z] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2019] [Accepted: 12/17/2019] [Indexed: 02/07/2023] Open
Abstract
Lecithin:cholesterol acyltransferase (LCAT) catalyzes a critical step of reverse cholesterol transport by esterifying cholesterol in high density lipoprotein (HDL) particles. LCAT is activated by apolipoprotein A-I (ApoA-I), which forms a double belt around HDL, however the manner in which LCAT engages its lipidic substrates and ApoA-I in HDL is poorly understood. Here, we used negative stain electron microscopy, crosslinking, and hydrogen-deuterium exchange studies to refine the molecular details of the LCAT-HDL complex. Our data are consistent with LCAT preferentially binding to the edge of discoidal HDL near the boundary between helix 5 and 6 of ApoA-I in a manner that creates a path from the lipid bilayer to the active site of LCAT. Our results provide not only an explanation why LCAT activity diminishes as HDL particles mature, but also direct support for the anti-parallel double belt model of HDL, with LCAT binding preferentially to the helix 4/6 region.
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22
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Lipid Profile Rather Than the LCAT Mutation Explains Renal Disease in Familial LCAT Deficiency. J Clin Med 2019; 8:jcm8111860. [PMID: 31684177 PMCID: PMC6912718 DOI: 10.3390/jcm8111860] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2019] [Revised: 10/28/2019] [Accepted: 10/30/2019] [Indexed: 12/25/2022] Open
Abstract
Renal complications are the major cause of morbidity and mortality in patients with familial lecithin–cholesterol acyltransferase (LCAT) deficiency (FLD). We report three FLD patients, two of them siblings—only one of whom developed renal disease—and the third case being a young man with early renal disease. The aim of this study was to analyze the clinical characteristics and possible mechanisms associated with renal disease in these patients. Plasma lipid levels, LCAT activity, lipoprotein particle profile by NMR and FPLC, free and esterified cholesterol, presence of lipoprotein X (LpX) and DNA sequencing in the three FLD patients have been determined. The three cases presented clinical characteristics of FLD, although only one of the siblings developed renal disease, at 45 years of age, while the other patient developed the disease in his youth. Genetic analysis revealed new missense homozygous mutations, p.(Ile202Thr) in both siblings and p.(Arg171Glu) in the other patient. Lipoprotein particle analysis showed that the two patients with renal disease presented higher numbers of small very low-density lipoprotein (VLDL) and a higher concentration of triglycerides in VLDL. This study reports three new cases of LCAT deficiency, not previously described. Renal disease is not only dependent on LCAT deficiency, and could be due to the presence of VLDL particles, which are rich in triglycerides, free cholesterol and LpX.
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23
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Tobar HE, Cataldo LR, González T, Rodríguez R, Serrano V, Arteaga A, Álvarez-Mercado A, Lagos CF, Vicuña L, Miranda JP, Pereira A, Bravo C, Aguilera CM, Eyheramendy S, Uauy R, Martínez Á, Gil Á, Francone O, Rigotti A, Santos JL. Identification and functional analysis of missense mutations in the lecithin cholesterol acyltransferase gene in a Chilean patient with hypoalphalipoproteinemia. Lipids Health Dis 2019; 18:132. [PMID: 31164121 PMCID: PMC6549291 DOI: 10.1186/s12944-019-1045-0] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2019] [Accepted: 04/03/2019] [Indexed: 12/16/2022] Open
Abstract
Background Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme that esterifies cholesterol in high- and low-density lipoproteins (HDL and LDL). Mutations in LCAT gene causes familial LCAT deficiency, which is characterized by very low plasma HDL-cholesterol levels (Hypoalphalipoproteinemia), corneal opacity and anemia, among other lipid-related traits. Our aim is to evaluate clinical/biochemical features of a Chilean family with a proband showing clinical signs of familial LCAT deficiency, as well as to identify and assess the functional effects of LCAT mutations. Methods An adult female proband with hypoalphalipoproteinemia, corneal opacity and mild anemia, as well as her first-degree relatives, were recruited for clinical, biochemical, genetic, in-silico and in-vitro LCAT analysis. Sequencing of exons and intron-exon boundaries was performed to identify mutations. Site-directed mutagenesis was carried out to generate plasmids containing cDNA with wild type or mutant sequences. Such expression vectors were transfected to HEK-239 T cells to asses the effect of LCAT variants in expression, synthesis, secretion and enzyme activity. In-silico prediction analysis and molecular modeling was also used to evaluate the effect of LCAT variants. Results LCAT sequencing identified rare p.V333 M and p.M404 V missense mutations in compound heterozygous state in the proband, as well the common synonymous p.L363 L variant. LCAT protein was detected in proband’s plasma, but with undetectable enzyme activity compared to control relatives. HEK-293 T transfected cells with vector expression plasmids containing either p.M404 V or p.V333 M cDNA showed detectable LCAT protein expression both in supernatants and lysates from cultured cells, but with much lower enzyme activity compared to cells transfected with the wild-type sequence. Bioinformatic analyses also supported a causal role of such rare variations in LCAT lack of function. Additionally, the proband carried the minor allele of the synonymous p.L363 L variant. However, this variant is unlikely to affect the clinical phenotype of the proband given its relatively high frequency in the Chilean population (4%) and its small putative effect on plasma HDL-cholesterol levels. Conclusion Genetic, biochemical, in vitro and in silico analyses indicate that the rare mutations p.M404 V and p.V333 M in LCAT gene lead to suppression of LCAT enzyme activity and cause clinical features of familial LCAT deficiency. Electronic supplementary material The online version of this article (10.1186/s12944-019-1045-0) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Hugo E Tobar
- Department of Nutrition, Diabetes and Metabolism, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Luis R Cataldo
- Department of Nutrition, Diabetes and Metabolism, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Trinidad González
- Department of Nutrition, Diabetes and Metabolism, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Ricardo Rodríguez
- Department of Nutrition, Diabetes and Metabolism, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Valentina Serrano
- Department of Nutrition, Diabetes and Metabolism, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Antonio Arteaga
- Department of Nutrition, Diabetes and Metabolism, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile
| | | | - Carlos F Lagos
- Facultad de Medicina y Ciencia, Universidad San Sebastián, Campus Los Leones, Santiago, Chile
| | - Lucas Vicuña
- Departamento de Estadísticas, Facultad de Matemáticas, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - José P Miranda
- Department of Nutrition, Diabetes and Metabolism, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile
| | | | - Carolina Bravo
- Department of Nutrition, Diabetes and Metabolism, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Concepción M Aguilera
- Department of Biochemistry and Molecular Biology II, Institute of Nutrition and Food Technology "José Mataix", Center of Biomedical Research, University of Granada, Granada, Spain
| | - Susana Eyheramendy
- Departamento de Estadísticas, Facultad de Matemáticas, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Ricardo Uauy
- INTA, Universidad de Chile, Santiago, Chile.,División de Pediatría, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile
| | | | - Ángel Gil
- INYTA, University of Granada, Granada, Spain
| | - Omar Francone
- Pfizer Global Research and Development, San Diego, USA
| | - Attilio Rigotti
- Department of Nutrition, Diabetes and Metabolism, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile.,Centro de Nutrición Molecular y Enfermedades Crónicas, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - José L Santos
- Department of Nutrition, Diabetes and Metabolism, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile.
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Bouley RA, Hinkovska-Galcheva V, Shayman JA, Tesmer JJG. Structural Basis of Lysosomal Phospholipase A 2 Inhibition by Zn 2. Biochemistry 2019; 58:1709-1717. [PMID: 30830753 PMCID: PMC6677152 DOI: 10.1021/acs.biochem.8b01124] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Lysosomal phospholipase A2 (LPLA2/PLA2G15) is a key enzyme involved in lipid homeostasis and is characterized by both phospholipase A2 and transacylase activity and by an acidic pH optimum. Divalent cations such as Ca2+ and Mg2+ have previously been shown to have little effect on the activity of LPLA2, but the discovery of a novel crystal form of LPLA2 with Zn2+ bound in the active site suggested a role for this divalent cation in regulating enzyme activity. In this complex, the cation directly coordinates the serine and histidine of the α/β-hydrolase triad and stabilizes a closed conformation. This closed conformation is characterized by an inward shift of the lid loop, which extends over the active site and effectively blocks access to one of its lipid acyl chain binding tracks. Therefore, we hypothesized that Zn2+ would inhibit LPLA2 activity at a neutral but not acidic pH because histidine would be positively charged at lower pH. Indeed, Zn2+ was found to inhibit the esterase activity of LPLA2 in a noncompetitive manner exclusively at a neutral pH (between 6.5 and 8.0). Because lysosomes are reservoirs of Zn2+ in cells, the pH optimum of LPLA2 might allow it to catalyze acyl transfer unimpeded within the organelle. We conjecture that Zn2+ inhibition of LPLA2 at higher pH maintains a lower activity of the esterase in environments where its activity is not typically required.
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Affiliation(s)
- Renee A. Bouley
- Life Sciences Institute and Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109, United States
| | - Vania Hinkovska-Galcheva
- Department of Internal Medicine, University of Michigan Medical School, University of Michigan, Ann Arbor, Michigan 48109, United States
| | - James A. Shayman
- Department of Internal Medicine, University of Michigan Medical School, University of Michigan, Ann Arbor, Michigan 48109, United States
| | - John J. G. Tesmer
- Departments of Biological Sciences and of Medicinal Chemistry & Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907, United States
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25
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Manthei KA, Yang SM, Baljinnyam B, Chang L, Glukhova A, Yuan W, Freeman LA, Maloney DJ, Schwendeman A, Remaley AT, Jadhav A, Tesmer JJ. Molecular basis for activation of lecithin:cholesterol acyltransferase by a compound that increases HDL cholesterol. eLife 2018; 7:41604. [PMID: 30479275 PMCID: PMC6277198 DOI: 10.7554/elife.41604] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2018] [Accepted: 11/06/2018] [Indexed: 01/29/2023] Open
Abstract
Lecithin:cholesterol acyltransferase (LCAT) and LCAT-activating compounds are being investigated as treatments for coronary heart disease (CHD) and familial LCAT deficiency (FLD). Herein we report the crystal structure of human LCAT in complex with a potent piperidinylpyrazolopyridine activator and an acyl intermediate-like inhibitor, revealing LCAT in an active conformation. Unlike other LCAT activators, the piperidinylpyrazolopyridine activator binds exclusively to the membrane-binding domain (MBD). Functional studies indicate that the compound does not modulate the affinity of LCAT for HDL, but instead stabilizes residues in the MBD and facilitates channeling of substrates into the active site. By demonstrating that these activators increase the activity of an FLD variant, we show that compounds targeting the MBD have therapeutic potential. Our data better define the substrate binding site of LCAT and pave the way for rational design of LCAT agonists and improved biotherapeutics for augmenting or restoring reverse cholesterol transport in CHD and FLD patients. Cholesterol is a fatty substance found throughout the body that is essential to our health. However, if too much cholesterol builds up in our blood vessels, it can cause blockages that lead to heart and kidney problems. The body removes excess cholesterol by sending out high-density lipoproteins (HDL) that capture the fatty molecules and carry them to the liver where they are eliminated. The first step in this process requires an enzyme called LCAT, which converts cholesterol into a form that HDL particles can efficiently pack and transport. The enzyme acts by interacting with HDL particles, and chemically joining cholesterol with another compound. Finding ways to make LCAT perform better and produce more HDL could improve treatments for heart disease. This could be particularly helpful to people with genetic changes that make LCAT defective. Several small molecules that ‘dial up’ the activity of LCAT have been identified, but how they act on the enzyme is not always well understood. Manthei et al. therefore set out to determine precisely how one such small activator promotes LCAT function. The experiments involved using a method known as crystallography to look at the structure of LCAT when it is attached to the small molecule. They also evaluated the activity of the enzyme and other aspects of the protein in the presence of the small molecule and HDL particles. Taken together, the results led Manthei et al. to suggest that the small molecule works by more efficiently bringing into LCAT the materials that this enzyme needs to create the transport-ready form of cholesterol. The small molecule also partially restored the activity of mutant LCAT found in human disease. This knowledge may help to design more drug-like chemicals to ‘boost’ the activity of LCAT and prevent heart and kidney disease, especially in people who carry a defective version of the enzyme.
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Affiliation(s)
- Kelly A Manthei
- Life Sciences Institute, University of Michigan, Ann Arbor, United States
| | - Shyh-Ming Yang
- National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, United States
| | - Bolormaa Baljinnyam
- National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, United States
| | - Louise Chang
- Life Sciences Institute, University of Michigan, Ann Arbor, United States
| | - Alisa Glukhova
- Life Sciences Institute, University of Michigan, Ann Arbor, United States
| | - Wenmin Yuan
- Department of Pharmaceutical Sciences and Biointerfaces Institute, University of Michigan, Ann Arbor, United States
| | - Lita A Freeman
- Lipoprotein Metabolism Section, Cardiovascular-Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, United States
| | - David J Maloney
- National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, United States
| | - Anna Schwendeman
- Department of Pharmaceutical Sciences and Biointerfaces Institute, University of Michigan, Ann Arbor, United States
| | - Alan T Remaley
- Lipoprotein Metabolism Section, Cardiovascular-Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, United States
| | - Ajit Jadhav
- National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, United States
| | - John Jg Tesmer
- Department of Biological Sciences, Purdue University, Indiana, United States
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26
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Xu Y, Caldo KMP, Pal-Nath D, Ozga J, Lemieux MJ, Weselake RJ, Chen G. Properties and Biotechnological Applications of Acyl-CoA:diacylglycerol Acyltransferase and Phospholipid:diacylglycerol Acyltransferase from Terrestrial Plants and Microalgae. Lipids 2018; 53:663-688. [PMID: 30252128 DOI: 10.1002/lipd.12081] [Citation(s) in RCA: 60] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2018] [Revised: 07/23/2018] [Accepted: 07/24/2018] [Indexed: 12/14/2022]
Abstract
Triacylglycerol (TAG) is the major storage lipid in most terrestrial plants and microalgae, and has great nutritional and industrial value. Since the demand for vegetable oil is consistently increasing, numerous studies have been focused on improving the TAG content and modifying the fatty-acid compositions of plant seed oils. In addition, there is a strong research interest in establishing plant vegetative tissues and microalgae as platforms for lipid production. In higher plants and microalgae, TAG biosynthesis occurs via acyl-CoA-dependent or acyl-CoA-independent pathways. Diacylglycerol acyltransferase (DGAT) catalyzes the last and committed step in the acyl-CoA-dependent biosynthesis of TAG, which appears to represent a bottleneck in oil accumulation in some oilseed species. Membrane-bound and soluble forms of DGAT have been identified with very different amino-acid sequences and biochemical properties. Alternatively, TAG can be formed through acyl-CoA-independent pathways via the catalytic action of membrane-bound phospholipid:diacylglycerol acyltransferase (PDAT). As the enzymes catalyzing the terminal steps of TAG formation, DGAT and PDAT play crucial roles in determining the flux of carbon into seed TAG and thus have been considered as the key targets for engineering oil production. Here, we summarize the most recent knowledge on DGAT and PDAT in higher plants and microalgae, with the emphasis on their physiological roles, structural features, and regulation. The development of various metabolic engineering strategies to enhance the TAG content and alter the fatty-acid composition of TAG is also discussed.
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Affiliation(s)
- Yang Xu
- Department of Agricultural, Food and Nutritional Science, University of Alberta, 116 Street and 85 Avenue, Edmonton, Alberta, T6G 2P5, Canada
| | - Kristian Mark P Caldo
- Department of Agricultural, Food and Nutritional Science, University of Alberta, 116 Street and 85 Avenue, Edmonton, Alberta, T6G 2P5, Canada
- Department of Biochemistry, University of Alberta, 116 Street and 85 Avenue, Edmonton, Alberta, T6G 2H7, Canada
| | - Dipasmita Pal-Nath
- French Associates Institute for Agriculture and Biotechnology of Drylands, The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Sede Boqer Campus, Midreshet Ben-Gurion, 8499000, Israel
| | - Jocelyn Ozga
- Department of Agricultural, Food and Nutritional Science, University of Alberta, 116 Street and 85 Avenue, Edmonton, Alberta, T6G 2P5, Canada
| | - M Joanne Lemieux
- Department of Biochemistry, University of Alberta, 116 Street and 85 Avenue, Edmonton, Alberta, T6G 2H7, Canada
| | - Randall J Weselake
- Department of Agricultural, Food and Nutritional Science, University of Alberta, 116 Street and 85 Avenue, Edmonton, Alberta, T6G 2P5, Canada
| | - Guanqun Chen
- Department of Agricultural, Food and Nutritional Science, University of Alberta, 116 Street and 85 Avenue, Edmonton, Alberta, T6G 2P5, Canada
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27
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Lysosomal phospholipase A2. Biochim Biophys Acta Mol Cell Biol Lipids 2018; 1864:932-940. [PMID: 30077006 DOI: 10.1016/j.bbalip.2018.07.012] [Citation(s) in RCA: 39] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2018] [Revised: 07/23/2018] [Accepted: 07/24/2018] [Indexed: 12/31/2022]
Abstract
Lysosomal phospholipase A2 (PLA2G15) is a ubiquitous enzyme uniquely characterized by a subcellular localization to the lysosome and late endosome. PLA2G15 has an acidic pH optimum, is calcium independent, and acts as a transacylase in the presence of N-acetyl-sphingosine as an acceptor. Recent studies aided by the delineation of the crystal structure of PLA2G15 have clarified further the catalytic mechanism, sn-1 versus sn-2 specificity, and the basis whereby cationic amphiphilic drugs inhibit its activity. PLA2G15 has recently been shown to hydrolyze short chain oxidized phospholipids which access the catalytic site directly based on their aqueous solubility. Studies on the PLA2G15 null mouse suggest a role for the enzyme in the catabolism of pulmonary surfactant. PLA2G15 may also have a role in host defense and in the processing of lipid antigens for presentation by CD1 proteins. This article is part of a Special Issue entitled Novel functions of phospholipase A2 Guest Editors: Makoto Murakami and Gerard Lambeau.
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28
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Afonso MS, Machado RM, Lavrador MS, Quintao ECR, Moore KJ, Lottenberg AM. Molecular Pathways Underlying Cholesterol Homeostasis. Nutrients 2018; 10:E760. [PMID: 29899250 PMCID: PMC6024674 DOI: 10.3390/nu10060760] [Citation(s) in RCA: 90] [Impact Index Per Article: 12.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2018] [Revised: 06/10/2018] [Accepted: 06/11/2018] [Indexed: 01/14/2023] Open
Abstract
Cholesterol is an essential molecule that exerts pleiotropic actions. Although its presence is vital to the cell, its excess can be harmful and, therefore, sustaining cholesterol homeostasis is crucial to maintaining proper cellular functioning. It is well documented that high plasma cholesterol concentration increases the risk of atherosclerotic heart disease. In the last decades, several studies have investigated the association of plasma cholesterol concentrations and the risk of cardiovascular diseases as well as the signaling pathways involved in cholesterol homeostasis. Here, we present an overview of several mechanisms involved in intestinal cholesterol absorption, the regulation of cholesterol synthesis and uptake. We also discuss the importance of reverse cholesterol transport and transintestinal cholesterol transport to maintain cholesterol homeostasis and prevent atherosclerosis development. Additionally, we discuss the influence of dietary cholesterol on plasma cholesterol concentration and the new recommendations for cholesterol intake in a context of a healthy dietary pattern.
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Affiliation(s)
- Milessa Silva Afonso
- Marc and Ruti Bell Vascular Biology and Disease Program, Leon H. Charney Division of Cardiology, Department of Medicine, New York University School of Medicine, New York, NY 10016, USA.
| | - Roberta Marcondes Machado
- Laboratorio de Lipides (LIM 10), Hospital das Clínicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP 05403-000, Brazil.
| | - Maria Silvia Lavrador
- Laboratorio de Lipides (LIM 10), Hospital das Clínicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP 05403-000, Brazil.
| | - Eder Carlos Rocha Quintao
- Laboratorio de Lipides (LIM 10), Hospital das Clínicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP 05403-000, Brazil.
| | - Kathryn J Moore
- Marc and Ruti Bell Vascular Biology and Disease Program, Leon H. Charney Division of Cardiology, Department of Medicine, New York University School of Medicine, New York, NY 10016, USA.
- Department of Cell Biology, New York University School of Medicine, New York, NY 10016, USA.
| | - Ana Maria Lottenberg
- Laboratorio de Lipides (LIM 10), Hospital das Clínicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP 05403-000, Brazil.
- Faculdade Israelita de Ciências da Saúde, Albert Einstein, São Paulo, SP 05403-000, Brazil.
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29
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Cooke AL, Morris J, Melchior JT, Street SE, Jerome WG, Huang R, Herr AB, Smith LE, Segrest JP, Remaley AT, Shah AS, Thompson TB, Davidson WS. A thumbwheel mechanism for APOA1 activation of LCAT activity in HDL. J Lipid Res 2018; 59:1244-1255. [PMID: 29773713 DOI: 10.1194/jlr.m085332] [Citation(s) in RCA: 50] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2018] [Revised: 05/08/2018] [Indexed: 01/28/2023] Open
Abstract
APOA1 is the most abundant protein in HDL. It modulates interactions that affect HDL's cardioprotective functions, in part via its activation of the enzyme, LCAT. On nascent discoidal HDL, APOA1 comprises 10 α-helical repeats arranged in an anti-parallel stacked-ring structure that encapsulates a lipid bilayer. Previous chemical cross-linking studies suggested that these APOA1 rings can adopt at least two different orientations, or registries, with respect to each other; however, the functional impact of these structural changes is unknown. Here, we placed cysteine residues at locations predicted to form disulfide bonds in each orientation and then measured APOA1's ability to adopt the two registries during HDL particle formation. We found that most APOA1 oriented with the fifth helix of one molecule across from fifth helix of the other (5/5 helical registry), but a fraction adopted a 5/2 registry. Engineered HDLs that were locked in 5/5 or 5/2 registries by disulfide bonds equally promoted cholesterol efflux from macrophages, indicating functional particles. However, unlike the 5/5 registry or the WT, the 5/2 registry impaired LCAT cholesteryl esterification activity (P < 0.001), despite LCAT binding equally to all particles. Chemical cross-linking studies suggest that full LCAT activity requires a hybrid epitope composed of helices 5-7 on one APOA1 molecule and helices 3-4 on the other. Thus, APOA1 may use a reciprocating thumbwheel-like mechanism to activate HDL-remodeling proteins.
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Affiliation(s)
- Allison L Cooke
- Departments of Pathology and Laboratory Medicine University of Cincinnati, Cincinnati, OH 45237
| | - Jamie Morris
- Departments of Pathology and Laboratory Medicine University of Cincinnati, Cincinnati, OH 45237
| | - John T Melchior
- Departments of Pathology and Laboratory Medicine University of Cincinnati, Cincinnati, OH 45237
| | - Scott E Street
- Departments of Pathology and Laboratory Medicine University of Cincinnati, Cincinnati, OH 45237
| | - W Gray Jerome
- Departments of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232
| | - Rong Huang
- Departments of Pathology and Laboratory Medicine University of Cincinnati, Cincinnati, OH 45237
| | - Andrew B Herr
- Division of Immunobiology and Center for Systems Immunology Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229
| | - Loren E Smith
- Anesthesiology, Vanderbilt University Medical Center, Nashville, TN 37232
| | - Jere P Segrest
- Medicine, Vanderbilt University Medical Center, Nashville, TN 37232
| | - Alan T Remaley
- Lipoprotein Metabolism Section, Cardiovascular-Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892
| | - Amy S Shah
- Division of Endocrinology, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229
| | - Thomas B Thompson
- Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, OH 45237
| | - W Sean Davidson
- Departments of Pathology and Laboratory Medicine University of Cincinnati, Cincinnati, OH 45237
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30
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Hinkovska-Galcheva V, Kelly R, Manthei KA, Bouley R, Yuan W, Schwendeman A, Tesmer JJG, Shayman JA. Determinants of pH profile and acyl chain selectivity in lysosomal phospholipase A 2. J Lipid Res 2018; 59:1205-1218. [PMID: 29724779 PMCID: PMC6027918 DOI: 10.1194/jlr.m084012] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2018] [Revised: 03/30/2018] [Indexed: 12/03/2022] Open
Abstract
Lysosomal phospholipase A2 (LPLA2) is characterized by broad substrate recognition, peak activity at acidic pH, and the transacylation of lipophilic alcohols, especially N-acetyl-sphingosine. Prior structural analysis of LPLA2 revealed the presence of an atypical acidic residue, Asp13, in the otherwise hydrophobic active site cleft. We hypothesized that Asp13 contributed to the pH profile and/or substrate preference of LPLA2 for unsaturated acyl chains. To test this hypothesis, we substituted Asp13 for alanine, cysteine, or phenylalanine; then, we monitored the formation of 1-O-acyl-N-acetylsphingosine to measure the hydrolysis of sn-1 versus sn-2 acyl groups on a variety of glycerophospholipids. Substitutions with Asp13 yielded significant enzyme activity at neutral pH (7.4) and perturbed the selectivity for mono- and double-unsaturated acyl chains. However, this position played no apparent role in selecting for either the acyl acceptor or the head group of the glycerophospholipid. Our modeling indicates that Asp13 and its substitutions contribute to the pH activity profile of LPLA2 and to acyl chain selectivity by forming part of a hydrophobic track occupied by the scissile acyl chain.
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Affiliation(s)
- Vania Hinkovska-Galcheva
- Department of Internal Medicine, University of Michigan Medical School, University of Michigan, Ann Arbor, MI
| | - Robert Kelly
- Department of Internal Medicine, University of Michigan Medical School, University of Michigan, Ann Arbor, MI
| | - Kelly A Manthei
- Life Sciences Institute and the Departments of Pharmacology and Biological Chemistry, University of Michigan, Ann Arbor, MI
| | - Renee Bouley
- Life Sciences Institute and the Departments of Pharmacology and Biological Chemistry, University of Michigan, Ann Arbor, MI
| | - Wenmin Yuan
- Department of Pharmaceutical Science, Biointerfaces Institute, University of Michigan, Ann Arbor, MI
| | - Anna Schwendeman
- Department of Pharmaceutical Science, Biointerfaces Institute, University of Michigan, Ann Arbor, MI
| | - John J G Tesmer
- Life Sciences Institute and the Departments of Pharmacology and Biological Chemistry, University of Michigan, Ann Arbor, MI.,Department of Biological Sciences, Purdue University, West Lafayette, IN
| | - James A Shayman
- Department of Internal Medicine, University of Michigan Medical School, University of Michigan, Ann Arbor, MI
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31
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Casteleijn MG, Parkkila P, Viitala T, Koivuniemi A. Interaction of lecithin:cholesterol acyltransferase with lipid surfaces and apolipoprotein A-I-derived peptides. J Lipid Res 2018; 59:670-683. [PMID: 29438987 PMCID: PMC5880497 DOI: 10.1194/jlr.m082685] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2017] [Revised: 01/22/2018] [Indexed: 12/12/2022] Open
Abstract
LCAT is an enzyme responsible for the formation of cholesteryl esters from unesterified cholesterol (UC) and phospholipid (PL) molecules in HDL particles. However, it is poorly understood how LCAT interacts with lipoproteins and how apoA-I activates it. Here we have studied the interactions between LCAT and lipids through molecular simulations. In addition, we studied the binding of LCAT to apoA-I-derived peptides, and their effect on LCAT lipid association-utilizing experiments. Results show that LCAT anchors itself to lipoprotein surfaces by utilizing nonpolar amino acids located in the membrane-binding domain and the active site tunnel opening. Meanwhile, the membrane-anchoring hydrophobic amino acids attract cholesterol molecules next to them. The results also highlight the role of the lid-loop in the lipid binding and conformation of LCAT with respect to the lipid surface. The apoA-I-derived peptides from the LCAT-activating region bind to LCAT and promote its lipid surface interactions, although some of these peptides do not bind lipids individually. The transfer free-energy of PL from the lipid bilayer into the active site is consistent with the activation energy of LCAT. Furthermore, the entry of UC molecules into the active site becomes highly favorable by the acylation of SER181.
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Affiliation(s)
- Marco G Casteleijn
- Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland
| | - Petteri Parkkila
- Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland
| | - Tapani Viitala
- Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland
| | - Artturi Koivuniemi
- Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland.
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Lepetit D, Gillet B, Hughes S, Kraaijeveld K, Varaldi J. Genome Sequencing of the Behavior Manipulating Virus LbFV Reveals a Possible New Virus Family. Genome Biol Evol 2018; 8:3718-3739. [PMID: 28173110 PMCID: PMC5381508 DOI: 10.1093/gbe/evw277] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/16/2016] [Indexed: 12/26/2022] Open
Abstract
Parasites are sometimes able to manipulate the behavior of their hosts. However, the molecular cues underlying this phenomenon are poorly documented. We previously reported that the parasitoid wasp Leptopilina boulardi which develops from Drosophila larvae is often infected by an inherited DNA virus. In addition to being maternally transmitted, the virus benefits from horizontal transmission in superparasitized larvae (Drosophila that have been parasitized several times). Interestingly, the virus forces infected females to lay eggs in already parasitized larvae, thus increasing the chance of being horizontally transmitted. In a first step towards the identification of virus genes responsible for the behavioral manipulation, we present here the genome sequence of the virus, called LbFV. The sequencing revealed that its genome contains an homologous repeat sequence (hrs) found in eight regions in the genome. The presence of this hrs may explain the genomic plasticity that we observed for this genome. The genome of LbFV encodes 108 ORFs, most of them having no homologs in public databases. The virus is however related to Hytrosaviridae, although distantly. LbFV may thus represent a member of a new virus family. Several genes of LbFV were captured from eukaryotes, including two anti-apoptotic genes. More surprisingly, we found that LbFV captured from an ancestral wasp a protein with a Jumonji domain. This gene was afterwards duplicated in the virus genome. We hypothesized that this gene may be involved in manipulating the expression of wasp genes, and possibly in manipulating its behavior.
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Affiliation(s)
- David Lepetit
- Université de Lyon, Université Lyon 1, CNRS, Laboratoire de Biométrie et Biologie Evolutive UMR 5558, France
| | - Benjamin Gillet
- Université de Lyon, CNRS, Ecole Normale Supérieure de Lyon, Université Lyon 1, Institut de Génomique Fonctionnelle de Lyon UMR 5242, France
| | - Sandrine Hughes
- Université de Lyon, CNRS, Ecole Normale Supérieure de Lyon, Université Lyon 1, Institut de Génomique Fonctionnelle de Lyon UMR 5242, France
| | - Ken Kraaijeveld
- Department of Ecological Science, Faculty of Earth and Life Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands
| | - Julien Varaldi
- Université de Lyon, Université Lyon 1, CNRS, Laboratoire de Biométrie et Biologie Evolutive UMR 5558, France
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Manthei KA, Ahn J, Glukhova A, Yuan W, Larkin C, Manett TD, Chang L, Shayman JA, Axley MJ, Schwendeman A, Tesmer JJG. A retractable lid in lecithin:cholesterol acyltransferase provides a structural mechanism for activation by apolipoprotein A-I. J Biol Chem 2017; 292:20313-20327. [PMID: 29030428 PMCID: PMC5724016 DOI: 10.1074/jbc.m117.802736] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2017] [Revised: 09/15/2017] [Indexed: 12/12/2022] Open
Abstract
Lecithin:cholesterol acyltransferase (LCAT) plays a key role in reverse cholesterol transport by transferring an acyl group from phosphatidylcholine to cholesterol, promoting the maturation of high-density lipoproteins (HDL) from discoidal to spherical particles. LCAT is activated through an unknown mechanism by apolipoprotein A-I (apoA-I) and other mimetic peptides that form a belt around HDL. Here, we report the crystal structure of LCAT with an extended lid that blocks access to the active site, consistent with an inactive conformation. Residues Thr-123 and Phe-382 in the catalytic domain form a latch-like interaction with hydrophobic residues in the lid. Because these residues are mutated in genetic disease, lid displacement was hypothesized to be an important feature of apoA-I activation. Functional studies of site-directed mutants revealed that loss of latch interactions or the entire lid enhanced activity against soluble ester substrates, and hydrogen-deuterium exchange (HDX) mass spectrometry revealed that the LCAT lid is extremely dynamic in solution. Upon addition of a covalent inhibitor that mimics one of the reaction intermediates, there is an overall decrease in HDX in the lid and adjacent regions of the protein, consistent with ordering. These data suggest a model wherein the active site of LCAT is shielded from soluble substrates by a dynamic lid until it interacts with HDL to allow transesterification to proceed.
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Affiliation(s)
- Kelly A Manthei
- Life Sciences Institute and the Departments of Pharmacology and Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109
| | - Joomi Ahn
- MedImmune, Gaithersburg, Maryland 20878
| | - Alisa Glukhova
- Life Sciences Institute and the Departments of Pharmacology and Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109
| | - Wenmin Yuan
- Department of Pharmaceutical Sciences and Biointerfaces Institute, University of Michigan, Ann Arbor, Michigan 48109
| | | | - Taylor D Manett
- Life Sciences Institute and the Departments of Pharmacology and Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109
| | - Louise Chang
- Life Sciences Institute and the Departments of Pharmacology and Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109
| | - James A Shayman
- Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109
| | | | - Anna Schwendeman
- Department of Pharmaceutical Sciences and Biointerfaces Institute, University of Michigan, Ann Arbor, Michigan 48109
| | - John J G Tesmer
- Life Sciences Institute and the Departments of Pharmacology and Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109.
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Freeman LA, Demosky SJ, Konaklieva M, Kuskovsky R, Aponte A, Ossoli AF, Gordon SM, Koby RF, Manthei KA, Shen M, Vaisman BL, Shamburek RD, Jadhav A, Calabresi L, Gucek M, Tesmer JJG, Levine RL, Remaley AT. Lecithin:Cholesterol Acyltransferase Activation by Sulfhydryl-Reactive Small Molecules: Role of Cysteine-31. J Pharmacol Exp Ther 2017; 362:306-318. [PMID: 28576974 PMCID: PMC5510151 DOI: 10.1124/jpet.117.240457] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2017] [Accepted: 04/19/2017] [Indexed: 12/13/2022] Open
Abstract
Lecithin:cholesterol acyltransferase (LCAT) catalyzes plasma cholesteryl ester formation and is defective in familial lecithin:cholesterol acyltransferase deficiency (FLD), an autosomal recessive disorder characterized by low high-density lipoprotein, anemia, and renal disease. This study aimed to investigate the mechanism by which compound A [3-(5-(ethylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile], a small heterocyclic amine, activates LCAT. The effect of compound A on LCAT was tested in human plasma and with recombinant LCAT. Mass spectrometry and nuclear magnetic resonance were used to determine compound A adduct formation with LCAT. Molecular modeling was performed to gain insight into the effects of compound A on LCAT structure and activity. Compound A increased LCAT activity in a subset (three of nine) of LCAT mutations to levels comparable to FLD heterozygotes. The site-directed mutation LCAT-Cys31Gly prevented activation by compound A. Substitution of Cys31 with charged residues (Glu, Arg, and Lys) decreased LCAT activity, whereas bulky hydrophobic groups (Trp, Leu, Phe, and Met) increased activity up to 3-fold (P < 0.005). Mass spectrometry of a tryptic digestion of LCAT incubated with compound A revealed a +103.017 m/z adduct on Cys31, consistent with the addition of a single hydrophobic cyanopyrazine ring. Molecular modeling identified potential interactions of compound A near Cys31 and structural changes correlating with enhanced activity. Functional groups important for LCAT activation by compound A were identified by testing compound A derivatives. Finally, sulfhydryl-reactive β-lactams were developed as a new class of LCAT activators. In conclusion, compound A activates LCAT, including some FLD mutations, by forming a hydrophobic adduct with Cys31, thus providing a mechanistic rationale for the design of future LCAT activators.
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Affiliation(s)
- Lita A Freeman
- Lipid Metabolism Section, Cardiovascular and Pulmonary Branch (L.A.F., S.J.D., S.M.G., B.L.V., R.D.S., A.T.R.), Systems Biology Center (A.A., M.G.), and Laboratory of Biochemistry (R.L.L.), National Institutes of Health National Heart, Lung, and Blood Institute, Bethesda, Maryland; Department of Chemistry, American University, Washington, DC (M.K., R.K.); University of Milano, Milano, Italy (A.F.O., L.C.); Department of Chemistry, Vanderbilt University, Nashville, Tennessee (R.F.K.); Departments of Pharmacology and Biological Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (K.A.M., J.J.G.T.); and National Institutes of Health National Center for Advancing Translational Sciences, Bethesda, Maryland (M.S., A.J.)
| | - Stephen J Demosky
- Lipid Metabolism Section, Cardiovascular and Pulmonary Branch (L.A.F., S.J.D., S.M.G., B.L.V., R.D.S., A.T.R.), Systems Biology Center (A.A., M.G.), and Laboratory of Biochemistry (R.L.L.), National Institutes of Health National Heart, Lung, and Blood Institute, Bethesda, Maryland; Department of Chemistry, American University, Washington, DC (M.K., R.K.); University of Milano, Milano, Italy (A.F.O., L.C.); Department of Chemistry, Vanderbilt University, Nashville, Tennessee (R.F.K.); Departments of Pharmacology and Biological Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (K.A.M., J.J.G.T.); and National Institutes of Health National Center for Advancing Translational Sciences, Bethesda, Maryland (M.S., A.J.)
| | - Monika Konaklieva
- Lipid Metabolism Section, Cardiovascular and Pulmonary Branch (L.A.F., S.J.D., S.M.G., B.L.V., R.D.S., A.T.R.), Systems Biology Center (A.A., M.G.), and Laboratory of Biochemistry (R.L.L.), National Institutes of Health National Heart, Lung, and Blood Institute, Bethesda, Maryland; Department of Chemistry, American University, Washington, DC (M.K., R.K.); University of Milano, Milano, Italy (A.F.O., L.C.); Department of Chemistry, Vanderbilt University, Nashville, Tennessee (R.F.K.); Departments of Pharmacology and Biological Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (K.A.M., J.J.G.T.); and National Institutes of Health National Center for Advancing Translational Sciences, Bethesda, Maryland (M.S., A.J.)
| | - Rostislav Kuskovsky
- Lipid Metabolism Section, Cardiovascular and Pulmonary Branch (L.A.F., S.J.D., S.M.G., B.L.V., R.D.S., A.T.R.), Systems Biology Center (A.A., M.G.), and Laboratory of Biochemistry (R.L.L.), National Institutes of Health National Heart, Lung, and Blood Institute, Bethesda, Maryland; Department of Chemistry, American University, Washington, DC (M.K., R.K.); University of Milano, Milano, Italy (A.F.O., L.C.); Department of Chemistry, Vanderbilt University, Nashville, Tennessee (R.F.K.); Departments of Pharmacology and Biological Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (K.A.M., J.J.G.T.); and National Institutes of Health National Center for Advancing Translational Sciences, Bethesda, Maryland (M.S., A.J.)
| | - Angel Aponte
- Lipid Metabolism Section, Cardiovascular and Pulmonary Branch (L.A.F., S.J.D., S.M.G., B.L.V., R.D.S., A.T.R.), Systems Biology Center (A.A., M.G.), and Laboratory of Biochemistry (R.L.L.), National Institutes of Health National Heart, Lung, and Blood Institute, Bethesda, Maryland; Department of Chemistry, American University, Washington, DC (M.K., R.K.); University of Milano, Milano, Italy (A.F.O., L.C.); Department of Chemistry, Vanderbilt University, Nashville, Tennessee (R.F.K.); Departments of Pharmacology and Biological Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (K.A.M., J.J.G.T.); and National Institutes of Health National Center for Advancing Translational Sciences, Bethesda, Maryland (M.S., A.J.)
| | - Alice F Ossoli
- Lipid Metabolism Section, Cardiovascular and Pulmonary Branch (L.A.F., S.J.D., S.M.G., B.L.V., R.D.S., A.T.R.), Systems Biology Center (A.A., M.G.), and Laboratory of Biochemistry (R.L.L.), National Institutes of Health National Heart, Lung, and Blood Institute, Bethesda, Maryland; Department of Chemistry, American University, Washington, DC (M.K., R.K.); University of Milano, Milano, Italy (A.F.O., L.C.); Department of Chemistry, Vanderbilt University, Nashville, Tennessee (R.F.K.); Departments of Pharmacology and Biological Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (K.A.M., J.J.G.T.); and National Institutes of Health National Center for Advancing Translational Sciences, Bethesda, Maryland (M.S., A.J.)
| | - Scott M Gordon
- Lipid Metabolism Section, Cardiovascular and Pulmonary Branch (L.A.F., S.J.D., S.M.G., B.L.V., R.D.S., A.T.R.), Systems Biology Center (A.A., M.G.), and Laboratory of Biochemistry (R.L.L.), National Institutes of Health National Heart, Lung, and Blood Institute, Bethesda, Maryland; Department of Chemistry, American University, Washington, DC (M.K., R.K.); University of Milano, Milano, Italy (A.F.O., L.C.); Department of Chemistry, Vanderbilt University, Nashville, Tennessee (R.F.K.); Departments of Pharmacology and Biological Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (K.A.M., J.J.G.T.); and National Institutes of Health National Center for Advancing Translational Sciences, Bethesda, Maryland (M.S., A.J.)
| | - Ross F Koby
- Lipid Metabolism Section, Cardiovascular and Pulmonary Branch (L.A.F., S.J.D., S.M.G., B.L.V., R.D.S., A.T.R.), Systems Biology Center (A.A., M.G.), and Laboratory of Biochemistry (R.L.L.), National Institutes of Health National Heart, Lung, and Blood Institute, Bethesda, Maryland; Department of Chemistry, American University, Washington, DC (M.K., R.K.); University of Milano, Milano, Italy (A.F.O., L.C.); Department of Chemistry, Vanderbilt University, Nashville, Tennessee (R.F.K.); Departments of Pharmacology and Biological Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (K.A.M., J.J.G.T.); and National Institutes of Health National Center for Advancing Translational Sciences, Bethesda, Maryland (M.S., A.J.)
| | - Kelly A Manthei
- Lipid Metabolism Section, Cardiovascular and Pulmonary Branch (L.A.F., S.J.D., S.M.G., B.L.V., R.D.S., A.T.R.), Systems Biology Center (A.A., M.G.), and Laboratory of Biochemistry (R.L.L.), National Institutes of Health National Heart, Lung, and Blood Institute, Bethesda, Maryland; Department of Chemistry, American University, Washington, DC (M.K., R.K.); University of Milano, Milano, Italy (A.F.O., L.C.); Department of Chemistry, Vanderbilt University, Nashville, Tennessee (R.F.K.); Departments of Pharmacology and Biological Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (K.A.M., J.J.G.T.); and National Institutes of Health National Center for Advancing Translational Sciences, Bethesda, Maryland (M.S., A.J.)
| | - Min Shen
- Lipid Metabolism Section, Cardiovascular and Pulmonary Branch (L.A.F., S.J.D., S.M.G., B.L.V., R.D.S., A.T.R.), Systems Biology Center (A.A., M.G.), and Laboratory of Biochemistry (R.L.L.), National Institutes of Health National Heart, Lung, and Blood Institute, Bethesda, Maryland; Department of Chemistry, American University, Washington, DC (M.K., R.K.); University of Milano, Milano, Italy (A.F.O., L.C.); Department of Chemistry, Vanderbilt University, Nashville, Tennessee (R.F.K.); Departments of Pharmacology and Biological Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (K.A.M., J.J.G.T.); and National Institutes of Health National Center for Advancing Translational Sciences, Bethesda, Maryland (M.S., A.J.)
| | - Boris L Vaisman
- Lipid Metabolism Section, Cardiovascular and Pulmonary Branch (L.A.F., S.J.D., S.M.G., B.L.V., R.D.S., A.T.R.), Systems Biology Center (A.A., M.G.), and Laboratory of Biochemistry (R.L.L.), National Institutes of Health National Heart, Lung, and Blood Institute, Bethesda, Maryland; Department of Chemistry, American University, Washington, DC (M.K., R.K.); University of Milano, Milano, Italy (A.F.O., L.C.); Department of Chemistry, Vanderbilt University, Nashville, Tennessee (R.F.K.); Departments of Pharmacology and Biological Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (K.A.M., J.J.G.T.); and National Institutes of Health National Center for Advancing Translational Sciences, Bethesda, Maryland (M.S., A.J.)
| | - Robert D Shamburek
- Lipid Metabolism Section, Cardiovascular and Pulmonary Branch (L.A.F., S.J.D., S.M.G., B.L.V., R.D.S., A.T.R.), Systems Biology Center (A.A., M.G.), and Laboratory of Biochemistry (R.L.L.), National Institutes of Health National Heart, Lung, and Blood Institute, Bethesda, Maryland; Department of Chemistry, American University, Washington, DC (M.K., R.K.); University of Milano, Milano, Italy (A.F.O., L.C.); Department of Chemistry, Vanderbilt University, Nashville, Tennessee (R.F.K.); Departments of Pharmacology and Biological Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (K.A.M., J.J.G.T.); and National Institutes of Health National Center for Advancing Translational Sciences, Bethesda, Maryland (M.S., A.J.)
| | - Ajit Jadhav
- Lipid Metabolism Section, Cardiovascular and Pulmonary Branch (L.A.F., S.J.D., S.M.G., B.L.V., R.D.S., A.T.R.), Systems Biology Center (A.A., M.G.), and Laboratory of Biochemistry (R.L.L.), National Institutes of Health National Heart, Lung, and Blood Institute, Bethesda, Maryland; Department of Chemistry, American University, Washington, DC (M.K., R.K.); University of Milano, Milano, Italy (A.F.O., L.C.); Department of Chemistry, Vanderbilt University, Nashville, Tennessee (R.F.K.); Departments of Pharmacology and Biological Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (K.A.M., J.J.G.T.); and National Institutes of Health National Center for Advancing Translational Sciences, Bethesda, Maryland (M.S., A.J.)
| | - Laura Calabresi
- Lipid Metabolism Section, Cardiovascular and Pulmonary Branch (L.A.F., S.J.D., S.M.G., B.L.V., R.D.S., A.T.R.), Systems Biology Center (A.A., M.G.), and Laboratory of Biochemistry (R.L.L.), National Institutes of Health National Heart, Lung, and Blood Institute, Bethesda, Maryland; Department of Chemistry, American University, Washington, DC (M.K., R.K.); University of Milano, Milano, Italy (A.F.O., L.C.); Department of Chemistry, Vanderbilt University, Nashville, Tennessee (R.F.K.); Departments of Pharmacology and Biological Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (K.A.M., J.J.G.T.); and National Institutes of Health National Center for Advancing Translational Sciences, Bethesda, Maryland (M.S., A.J.)
| | - Marjan Gucek
- Lipid Metabolism Section, Cardiovascular and Pulmonary Branch (L.A.F., S.J.D., S.M.G., B.L.V., R.D.S., A.T.R.), Systems Biology Center (A.A., M.G.), and Laboratory of Biochemistry (R.L.L.), National Institutes of Health National Heart, Lung, and Blood Institute, Bethesda, Maryland; Department of Chemistry, American University, Washington, DC (M.K., R.K.); University of Milano, Milano, Italy (A.F.O., L.C.); Department of Chemistry, Vanderbilt University, Nashville, Tennessee (R.F.K.); Departments of Pharmacology and Biological Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (K.A.M., J.J.G.T.); and National Institutes of Health National Center for Advancing Translational Sciences, Bethesda, Maryland (M.S., A.J.)
| | - John J G Tesmer
- Lipid Metabolism Section, Cardiovascular and Pulmonary Branch (L.A.F., S.J.D., S.M.G., B.L.V., R.D.S., A.T.R.), Systems Biology Center (A.A., M.G.), and Laboratory of Biochemistry (R.L.L.), National Institutes of Health National Heart, Lung, and Blood Institute, Bethesda, Maryland; Department of Chemistry, American University, Washington, DC (M.K., R.K.); University of Milano, Milano, Italy (A.F.O., L.C.); Department of Chemistry, Vanderbilt University, Nashville, Tennessee (R.F.K.); Departments of Pharmacology and Biological Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (K.A.M., J.J.G.T.); and National Institutes of Health National Center for Advancing Translational Sciences, Bethesda, Maryland (M.S., A.J.)
| | - Rodney L Levine
- Lipid Metabolism Section, Cardiovascular and Pulmonary Branch (L.A.F., S.J.D., S.M.G., B.L.V., R.D.S., A.T.R.), Systems Biology Center (A.A., M.G.), and Laboratory of Biochemistry (R.L.L.), National Institutes of Health National Heart, Lung, and Blood Institute, Bethesda, Maryland; Department of Chemistry, American University, Washington, DC (M.K., R.K.); University of Milano, Milano, Italy (A.F.O., L.C.); Department of Chemistry, Vanderbilt University, Nashville, Tennessee (R.F.K.); Departments of Pharmacology and Biological Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (K.A.M., J.J.G.T.); and National Institutes of Health National Center for Advancing Translational Sciences, Bethesda, Maryland (M.S., A.J.)
| | - Alan T Remaley
- Lipid Metabolism Section, Cardiovascular and Pulmonary Branch (L.A.F., S.J.D., S.M.G., B.L.V., R.D.S., A.T.R.), Systems Biology Center (A.A., M.G.), and Laboratory of Biochemistry (R.L.L.), National Institutes of Health National Heart, Lung, and Blood Institute, Bethesda, Maryland; Department of Chemistry, American University, Washington, DC (M.K., R.K.); University of Milano, Milano, Italy (A.F.O., L.C.); Department of Chemistry, Vanderbilt University, Nashville, Tennessee (R.F.K.); Departments of Pharmacology and Biological Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (K.A.M., J.J.G.T.); and National Institutes of Health National Center for Advancing Translational Sciences, Bethesda, Maryland (M.S., A.J.)
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Ma A, Wang J, Yang L, An Y, Zhu H. AMPK activation enhances the anti-atherogenic effects of high density lipoproteins in apoE -/- mice. J Lipid Res 2017; 58:1536-1547. [PMID: 28611100 PMCID: PMC5538277 DOI: 10.1194/jlr.m073270] [Citation(s) in RCA: 56] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2016] [Revised: 05/30/2017] [Indexed: 12/12/2022] Open
Abstract
HDL plays crucial roles at multiple stages of the pathogenesis of atherosclerosis. AMP-activated protein kinase (AMPK) is a therapeutic candidate for the treatment of cardiovascular disease. However, the effect of AMPK activation on HDL functionality has not been established in vivo. We assessed the effects of pharmacological AMPK activation using A-769662, AICAR, metformin, and IMM-H007 on the atheroprotective functions of HDL in apoE-deficient (apoE−/−) mice fed with a high-fat diet. After administration, there were no changes in serum lipid levels among the groups. However, mice treated with AMPK activators showed significantly enhanced reverse cholesterol transport in vivo and in vitro. AMPK activation also increased the expression of ABCA1 and ABCG1 in macrophages and scavenger receptor class B type I and LCAT in the liver. HDL from AMPK activation mice exhibited lower HDL inflammatory index and myeloperoxidase activity and higher paraoxonase 1 activity than HDL from untreated mice, implying superior antioxidant and anti-inflammatory capacities. Pharmacological AMPK activation also induced polarization of macrophages to the M2 state and reduced plasma lipid peroxidation, inflammatory cytokine production, and atherosclerotic plaque formation in apoE−/− mice. These observations suggest that pharmacological AMPK activation enhances the anti-atherogenic properties of HDL in vivo. This likely represents a key mechanism by which AMPK activation attenuates atherosclerosis.
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Affiliation(s)
- Ang Ma
- State Key Laboratory for Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study, and Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People's Republic of China
| | - Jing Wang
- State Key Laboratory for Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study, and Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People's Republic of China
| | - Liu Yang
- State Key Laboratory for Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study, and Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People's Republic of China
| | - Yuanyuan An
- State Key Laboratory for Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study, and Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People's Republic of China
| | - Haibo Zhu
- State Key Laboratory for Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study, and Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People's Republic of China.
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36
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Ogura Y, Parsons WH, Kamat SS, Cravatt BF. A calcium-dependent acyltransferase that produces N-acyl phosphatidylethanolamines. Nat Chem Biol 2016; 12:669-71. [PMID: 27399000 PMCID: PMC4990470 DOI: 10.1038/nchembio.2127] [Citation(s) in RCA: 80] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2016] [Accepted: 05/18/2016] [Indexed: 12/11/2022]
Abstract
More than 30 years ago, a calcium-dependent enzyme activity was described that generates N-acyl phosphatidylethanolamines (NAPEs), which are precursors for N-acyl ethanolamine (NAE) lipid transmitters, including the endocannabinoid anandamide. The identity of this calcium-dependent N-acyltransferase (Ca-NAT) has remained mysterious. Here, we use activity-based protein profiling to identify the poorly characterized serine hydrolase PLA2G4E as a mouse brain Ca-NAT and show that this enzyme generates NAPEs and NAEs in mammalian cells.
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Affiliation(s)
- Yuji Ogura
- Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA.,The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA
| | - William H Parsons
- Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA.,The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA
| | - Siddhesh S Kamat
- Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA.,The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA
| | - Benjamin F Cravatt
- Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA.,The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA
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37
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Ossoli A, Simonelli S, Vitali C, Franceschini G, Calabresi L. Role of LCAT in Atherosclerosis. J Atheroscler Thromb 2016; 23:119-27. [DOI: 10.5551/jat.32854] [Citation(s) in RCA: 47] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Affiliation(s)
- Alice Ossoli
- Centro Grossi Paoletti, Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano
| | - Sara Simonelli
- Centro Grossi Paoletti, Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano
| | - Cecilia Vitali
- Centro Grossi Paoletti, Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano
| | - Guido Franceschini
- Centro Grossi Paoletti, Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano
- Division of Chemical and Biomolecular Sciences - DeFENS, Università degli Studi di Milano
| | - Laura Calabresi
- Centro Grossi Paoletti, Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano
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38
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Gunawardane RN, Fordstrom P, Piper DE, Masterman S, Siu S, Liu D, Brown M, Lu M, Tang J, Zhang R, Cheng J, Gates A, Meininger D, Chan J, Carlson T, Walker N, Schwarz M, Delaney J, Zhou M. Agonistic Human Antibodies Binding to Lecithin-Cholesterol Acyltransferase Modulate High Density Lipoprotein Metabolism. J Biol Chem 2015; 291:2799-811. [PMID: 26644477 PMCID: PMC4742745 DOI: 10.1074/jbc.m115.672790] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2015] [Indexed: 11/28/2022] Open
Abstract
Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed because the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouseTM platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 Å LCAT-27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35% increase of the high density lipoprotein cholesterol that was observed up to 32 days after 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease.
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Affiliation(s)
| | | | | | - Stephanie Masterman
- Therapeutic Discovery, Amgen Inc., Burnaby, British Columbia V5A 1V7, Canada
| | - Sophia Siu
- From Therapeutic Discovery, Amgen Inc., Seattle, Washington 98119
| | | | - Mike Brown
- From Therapeutic Discovery, Amgen Inc., Seattle, Washington 98119
| | - Mei Lu
- Therapeutic Discovery, and
| | | | | | - Janet Cheng
- From Therapeutic Discovery, Amgen Inc., Seattle, Washington 98119
| | - Andrew Gates
- From Therapeutic Discovery, Amgen Inc., Seattle, Washington 98119
| | - David Meininger
- From Therapeutic Discovery, Amgen Inc., Seattle, Washington 98119
| | | | - Tim Carlson
- PKDM Department, Amgen Inc., South San Francisco, California 94080, and
| | | | | | - John Delaney
- Therapeutic Discovery, Amgen Inc., Burnaby, British Columbia V5A 1V7, Canada
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39
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Romanow WG, Piper DE, Fordstrom P, Thibault S, Zhou M, Walker NPC. BacMam production of active recombinant lecithin-cholesterol acyltransferase: Expression, purification and characterization. Protein Expr Purif 2015; 125:1-6. [PMID: 26363122 DOI: 10.1016/j.pep.2015.09.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2015] [Revised: 08/30/2015] [Accepted: 09/03/2015] [Indexed: 11/17/2022]
Abstract
Lecithin-cholesterol acyltransferase (LCAT) is a key enzyme in the esterification of cholesterol and its subsequent incorporation into the core of high density lipoprotein (HDL) particles. It is also involved in reverse cholesterol transport (RCT), the mechanism by which cholesterol is removed from peripheral cells and transported to the liver for excretion. These processes are involved in the development of atherosclerosis and coronary heart disease (CHD) and may have therapeutic implications. This work describes the use of baculovirus as a transducing vector to express LCAT in mammalian cells, expression of the recombinant protein as a high-mannose glycoform suitable for deglycosylation by Endo H and its purification to homogeneity and characterization. The importance of producing underglycosylated forms of secreted glycoproteins to obtain high-resolution crystal structures is discussed.
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Affiliation(s)
- William G Romanow
- Protein Technologies, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, United States.
| | - Derek E Piper
- Structural Biology, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, United States
| | - Preston Fordstrom
- Metabolic Disorders, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, United States
| | - Stephen Thibault
- Protein Technologies, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, United States
| | - Mingyue Zhou
- Metabolic Disorders, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, United States
| | - Nigel P C Walker
- Structural Biology, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, United States
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