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Ding H, Ding ZG, Liu S, Mao XN, Lu XS. Ras-related protein Rab24 plays a predictive role in hepatocellular carcinoma and enhanced tumor proliferation. World J Gastroenterol 2025; 31:101585. [PMID: 40062325 PMCID: PMC11886508 DOI: 10.3748/wjg.v31.i8.101585] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/19/2024] [Revised: 12/04/2024] [Accepted: 01/06/2025] [Indexed: 01/23/2025] Open
Abstract
BACKGROUND Ras-related protein Rab24, which belongs to the small GTPase family, plays a crucial role in regulating intracellular protein trafficking. Dysregulation of Rab24 has been recently identified in hepatocellular carcinoma (HCC). However, its clinical significance and tumor related effects remain to be further clarified. AIM To explore the expression pattern of Rab24 and its role in HCC progression. METHODS The expression profile of Rab24 was tested in HCC tissues together with adjacent tissues from transcriptional, mRNA, and protein levels. The prognostic role of Rab24 in HCC was assessed by univariate and multivariate analyses. Clinical outcomes were evaluated by the Kaplan-Meier analysis and log-rank test. The effect of Rab24 on cell proliferation was tested through cellular experiments and xenograft experiments. RESULTS Rab24 expression was elevated in HCC tissues compared to adjacent liver tissues. High expression of Rab24 was significantly associated with larger tumor size and advanced tumor stage. Moreover, HCC patients with high Rab24 expression showed poorer overall survival, and Rab24 was identified as an independent prognosis factor according to multivariate analysis. By using overexpression and shRNA knockdown strategies in HCC cell lines, we found that Rab24 can promote HCC proliferation. Finally, we validated that silencing Rab24 significantly attenuated xenograft growth in vivo. CONCLUSION Our study demonstrated that high expression of Rab24 was significantly correlated with poorer prognosis of HCC patients, indicating the potential of Rab24 as a novel clinical biomarker and therapeutic target.
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Affiliation(s)
- Han Ding
- Department of Endoscopy Center and Endoscopy Research Institute, Zhongshan Hospital, The Affiliated to Fudan University, Shanghai 200032, China
- Department of General Surgery, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
| | - Zhi-Guo Ding
- Department of General Surgery, The Third People’s Hospital of Yangzhou, Yangzhou 225126, Jiangsu Province, China
| | - Song Liu
- Department of General Surgery, The Fourth Affiliated Hospital of Soochow University, Suzhou 215000, Jiangsu Province, China
| | - Xu-Nan Mao
- Medical College, Yangzhou University, Yangzhou 225009, Jiangsu Province, China
| | - Xing-Sheng Lu
- Department of General Surgery, The Fourth Affiliated Hospital of Soochow University, Suzhou 215000, Jiangsu Province, China
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2
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Salazar CJ, Diaz-Balzac CA, Wang Y, Rahman M, Grant BD, Bülow HE. RABR-1, an atypical Rab-related GTPase, cell-nonautonomously restricts somatosensory dendrite branching. Genetics 2024; 228:iyae113. [PMID: 39028768 PMCID: PMC11457943 DOI: 10.1093/genetics/iyae113] [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: 11/15/2023] [Revised: 07/01/2024] [Accepted: 07/08/2024] [Indexed: 07/21/2024] Open
Abstract
Neurons are highly polarized cells with dendrites and axons. Dendrites, which receive sensory information or input from other neurons, often display elaborately branched morphologies. While mechanisms that promote dendrite branching have been widely studied, less is known about the mechanisms that restrict branching. Using the nematode Caenorhabditis elegans, we identify rabr-1 (for Rab-related gene 1) as a factor that restricts branching of the elaborately branched dendritic trees of PVD and FLP somatosensory neurons. Animals mutant for rabr-1 show excessively branched dendrites throughout development and into adulthood in areas where the dendrites overlay epidermal tissues. Phylogenetic analyses show that RABR-1 displays similarity to small GTPases of the Rab-type, although based on sequence alone, no clear vertebrate ortholog of RABR-1 can be identified. We find that rabr-1 is expressed and can function in epidermal tissues, suggesting that rabr-1 restricts dendritic branching cell-nonautonomously. Genetic experiments further indicate that for the formation of ectopic branches rabr-1 mutants require the genes of the Menorin pathway, which have been previously shown to mediate dendrite morphogenesis of somatosensory neurons. A translational reporter for RABR-1 reveals a subcellular localization to punctate, perinuclear structures, which correlates with endosomal and autophagosomal markers, but anticorrelates with lysosomal markers suggesting an amphisomal character. Point mutations in rabr-1 analogous to key residues of small GTPases suggest that rabr-1 functions in a GTP-bound form independently of GTPase activity. Taken together, rabr-1 encodes for an atypical small GTPase of the Rab-type that cell-nonautonomously restricts dendritic branching of somatosensory neurons, likely independently of GTPase activity.
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Affiliation(s)
| | - Carlos A Diaz-Balzac
- Department of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | - Yu Wang
- Department of Molecular Biology and Biochemistry, Rutgers Center for Lipid Research, Rutgers University, Piscataway, NJ 08854, USA
| | - Maisha Rahman
- Department of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA
- Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | - Barth D Grant
- Department of Molecular Biology and Biochemistry, Rutgers Center for Lipid Research, Rutgers University, Piscataway, NJ 08854, USA
| | - Hannes E Bülow
- Department of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA
- Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA
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3
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Erol ÖD, Şenocak Ş, Aerts-Kaya F. The Role of Rab GTPases in the development of genetic and malignant diseases. Mol Cell Biochem 2024; 479:255-281. [PMID: 37060515 DOI: 10.1007/s11010-023-04727-x] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2023] [Accepted: 04/01/2023] [Indexed: 04/16/2023]
Abstract
Small GTPases have been shown to play an important role in several cellular functions, including cytoskeletal remodeling, cell polarity, intracellular trafficking, cell-cycle, progression and lipid transformation. The Ras-associated binding (Rab) family of GTPases constitutes the largest family of GTPases and consists of almost 70 known members of small GTPases in humans, which are known to play an important role in the regulation of intracellular membrane trafficking, membrane identity, vesicle budding, uncoating, motility and fusion of membranes. Mutations in Rab genes can cause a wide range of inherited genetic diseases, ranging from neurodegenerative diseases, such as Parkinson's disease (PD) and Alzheimer's disease (AD) to immune dysregulation/deficiency syndromes, like Griscelli Syndrome Type II (GS-II) and hemophagocytic lymphohistiocytosis (HLH), as well as a variety of cancers. Here, we provide an extended overview of human Rabs, discussing their function and diseases related to Rabs and Rab effectors, as well as focusing on effects of (aberrant) Rab expression. We aim to underline their importance in health and the development of genetic and malignant diseases by assessing their role in cellular structure, regulation, function and biology and discuss the possible use of stem cell gene therapy, as well as targeting of Rabs in order to treat malignancies, but also to monitor recurrence of cancer and metastasis through the use of Rabs as biomarkers. Future research should shed further light on the roles of Rabs in the development of multifactorial diseases, such as diabetes and assess Rabs as a possible treatment target.
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Affiliation(s)
- Özgür Doğuş Erol
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, 06100, Ankara, Turkey
- Hacettepe University Center for Stem Cell Research and Development, 06100, Ankara, Turkey
| | - Şimal Şenocak
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, 06100, Ankara, Turkey
- Hacettepe University Center for Stem Cell Research and Development, 06100, Ankara, Turkey
| | - Fatima Aerts-Kaya
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, 06100, Ankara, Turkey.
- Hacettepe University Center for Stem Cell Research and Development, 06100, Ankara, Turkey.
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4
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Yu J, Lang Q, Zhong C, Wang S, Tian Y. Genome-Wide Identification of Autophagy Prognostic Signature in Pancreatic Cancer. Dose Response 2021; 19:15593258211023260. [PMID: 34262410 PMCID: PMC8252352 DOI: 10.1177/15593258211023260] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2020] [Revised: 05/07/2021] [Accepted: 05/19/2021] [Indexed: 01/07/2023] Open
Abstract
Background: Autophagy plays a vital role in cancer development. However, there is currently no comprehensive study regarding the effects of autophagy-related genes (ARGs) on pancreatic cancer prognosis. Thus, this study aimed to establish an autophagy-related signature for predicting the prognosis of patients with pancreatic cancer. Methods: We identified and validated differentially-expressed ARGs using data from The Cancer Genome Atlas (TCGA) database, Genotype-Tissue Expression project (GTEx) and Expression Omnibus (GEO) database. We performed Cox proportional hazards regression analysis on the differentially-expressed ARGs to develop an autophagy-related signature. We tested the expression of these genes through western blotting and verified their prognostic values through gene expression profiling and interactive analyses (GEPIA). Results: We identified a total of 21 differentially-expressed ARGs and screened 4 OS-related ARGs (TP63, RAB24, APOL1, and PTK6). Both the training and validation sets showed that the autophagy-related signature was more accurate than the Tumor Node Metastasis (TNM) staging system. Moreover, the western blotting result showed that the expression of TP63, APOL1, and PTK6 was high, whereas that of RAB24 was low in cancer tissues. Conclusion: This 4-ARG signature might potentially help in providing personalized therapy to patients with cancer.
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Affiliation(s)
- Jianfa Yu
- Department of General Surgery, Shengjing Hospital Affiliated to China Medical University, Shenyang, Liaoning, China
| | - Qi Lang
- Department of General Surgery, Shengjing Hospital Affiliated to China Medical University, Shenyang, Liaoning, China
| | - Chongli Zhong
- Department of General Surgery, Shengjing Hospital Affiliated to China Medical University, Shenyang, Liaoning, China
| | - Shuang Wang
- Key Laboratory of Higher Education of Liaoning Province, Shenyang, Liaoning, China
| | - Yu Tian
- Department of General Surgery, Shengjing Hospital Affiliated to China Medical University, Shenyang, Liaoning, China
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5
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Waschbüsch D, Khan AR. Phosphorylation of Rab GTPases in the regulation of membrane trafficking. Traffic 2020; 21:712-719. [PMID: 32969543 PMCID: PMC7756361 DOI: 10.1111/tra.12765] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2020] [Revised: 09/17/2020] [Accepted: 09/17/2020] [Indexed: 12/15/2022]
Abstract
Rab GTPases are master regulators of membrane trafficking in eukaryotic cells. Phosphorylation of Rab GTPases was characterized in the 1990s and there have been intermittent reports of its relevance to Rab functions. Phosphorylation as a regulatory mechanism has gained prominence through the identification of Rabs as physiological substrates of leucine‐rich repeat kinase 2 (LRRK2). LRRK2 is a Ser/Thr kinase that is associated with inherited and sporadic forms of Parkinson disease. In recent years, numerous kinases and their associated signaling pathways have been identified that lead to phosphorylation of Rabs. These emerging studies suggest that serine/threonine and tyrosine phosphorylation of Rabs may be a widespread and under‐appreciated mechanism for controlling their membrane trafficking functions. Here we survey current knowledge of Rab phosphorylation and discuss models for how this post‐translational mechanism exerts control of membrane trafficking.
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Affiliation(s)
- Dieter Waschbüsch
- School of Biochemistry and Immunology, Trinity College, Dublin, Ireland
| | - Amir R Khan
- School of Biochemistry and Immunology, Trinity College, Dublin, Ireland.,Division of Newborn Medicine, Boston Children's Hospital, Boston, Massachusetts, USA
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6
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Qiu D, Li S, Guo L, Yuan R, Ou X. Rab24 functions in meiotic apparatus assembly and maturational progression in mouse oocyte. Cell Cycle 2019; 18:2893-2901. [PMID: 31496367 PMCID: PMC6791699 DOI: 10.1080/15384101.2019.1660115] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2019] [Revised: 08/17/2019] [Accepted: 08/21/2019] [Indexed: 01/09/2023] Open
Abstract
Rab GTPases have multiple regulatory functions in intracellular vesicle transport. In recent years, there has been an increasing interest in the roles of Rab proteins in mammalian oocytes. In this paper, we show the specific distribution pattern of Rab24 during mouse oocyte meiosis. Furthermore, we find that Rab24 depletion results in the failure of maturational progression in mouse oocytes. Notably, the frequency of meiotic apparatus abnormality is significantly increased in Rab24-depleted oocytes relative to controls. In addition, lagging chromosomes are readily observed in anaphase/telophase oocytes with Rab24 knockdown. In support of this, the depletion of Rab24 disturbs the kinetochore-microtubule attachments in oocytes, and contributes to the production of aneuploid eggs. Taken together, the results of this study identify Rab24 as a novel factor in the modulation of meiotic apparatus assembly and meiotic progression during mouse oocyte maturation.
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Affiliation(s)
- Danhong Qiu
- Fertility Preservation Laboratory, Human Reproduction Medical Center, Guangdong Second Provincial General Hospital, Guangzhou, China
- Department of Physiology, School of Basic Medical Sciences, Shenzhen University Health Sciences Center, Shenzhen, China
| | - Sen Li
- Fertility Preservation Laboratory, Human Reproduction Medical Center, Guangdong Second Provincial General Hospital, Guangzhou, China
| | - Lei Guo
- Fertility Preservation Laboratory, Human Reproduction Medical Center, Guangdong Second Provincial General Hospital, Guangzhou, China
| | - Ruiying Yuan
- Fertility Preservation Laboratory, Human Reproduction Medical Center, Guangdong Second Provincial General Hospital, Guangzhou, China
| | - Xianghong Ou
- Fertility Preservation Laboratory, Human Reproduction Medical Center, Guangdong Second Provincial General Hospital, Guangzhou, China
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7
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Abstract
Autophagy is an evolutionarily conserved degradation pathway for cells to maintain homeostasis, produce energy, degrade misfolded proteins and damaged organelles, and fight against intracellular pathogens. The process of autophagy entails the isolation of cytoplasmic cargo into double membrane bound autophagosomes that undergo maturation by fusion with endosomes and lysosomes to obtain degradation capacity. RAB proteins regulate intracellular vesicle trafficking events including autophagy. RAB24 is an atypical RAB protein that is required for the clearance of late autophagic vacuoles under basal conditions. RAB24 has also been connected to several diseases including ataxia, cancer and tuberculosis. This review gives a short summary on autophagy and RAB proteins, and an overview on the current knowledge on the roles of RAB24 in autophagy and disease.
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Affiliation(s)
- Päivi Ylä-Anttila
- a Department of Biosciences , University of Helsinki , Helsinki , Finland
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8
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Abstract
Autophagy is an evolutionarily conserved degradation pathway for cells to maintain homeostasis, produce energy, degrade misfolded proteins and damaged organelles, and fight against intracellular pathogens. The process of autophagy entails the isolation of cytoplasmic cargo into double membrane bound autophagosomes that undergo maturation by fusion with endosomes and lysosomes to obtain degradation capacity. RAB proteins regulate intracellular vesicle trafficking events including autophagy. RAB24 is an atypical RAB protein that is required for the clearance of late autophagic vacuoles under basal conditions. RAB24 has also been connected to several diseases including ataxia, cancer and tuberculosis. This review gives a short summary on autophagy and RAB proteins, and an overview on the current knowledge on the roles of RAB24 in autophagy and disease.
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Affiliation(s)
- Päivi Ylä-Anttila
- a Department of Biosciences , University of Helsinki , Helsinki , Finland
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9
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Amaya C, Militello RD, Calligaris SD, Colombo MI. Rab24 interacts with the Rab7/Rab interacting lysosomal protein complex to regulate endosomal degradation. Traffic 2016; 17:1181-1196. [PMID: 27550070 DOI: 10.1111/tra.12431] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2015] [Revised: 08/17/2016] [Accepted: 08/17/2016] [Indexed: 12/11/2022]
Abstract
Endocytosis is a multistep process engaged in extracellular molecules internalization. Several proteins including the Rab GTPases family coordinate the endocytic pathway. The small GTPase Rab7 is present in late endosome (LE) compartments being a marker of endosome maturation. The Rab interacting lysosomal protein (RILP) is a downstream effector of Rab7 that recruits the functional dynein/dynactin motor complex to late compartments. In the present study, we have found Rab24 as a component of the endosome-lysosome degradative pathway. Rab24 is an atypical protein of the Rab GTPase family, which has been attributed a function in vesicle trafficking and autophagosome maturation. Using a model of transiently expressed proteins in K562 cells, we found that Rab24 co-localizes in vesicular structures labeled with Rab7 and LAMP1. Moreover, using a dominant negative mutant of Rab24 or a siRNA-Rab24 we showed that the distribution of Rab7 in vesicles depends on a functional Rab24 to allow DQ-BSA protein degradation. Additionally, by immunoprecipitation and pull down assays, we have demonstrated that Rab24 interacts with Rab7 and RILP. Interestingly, overexpression of the Vps41 subunit from the homotypic fusion and protein-sorting (HOPS) complex hampered the co-localization of Rab24 with RILP or with the lysosomal GTPase Arl8b, suggesting that Vps41 would affect the Rab24/RILP association. In summary, our data strongly support the hypothesis that Rab24 forms a complex with Rab7 and RILP on the membranes of late compartments. Our work provides new insights into the molecular function of Rab24 in the last steps of the endosomal degradative pathway.
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Affiliation(s)
- Celina Amaya
- Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM)-CONICET, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina
| | - Rodrigo D Militello
- Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM)-CONICET, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina
| | - Sebastián D Calligaris
- Centro de Medicina Regenerativa, Facultad de Medicina, Universidad del Desarrollo Clínica Alemana, Santiago, Chile
| | - María I Colombo
- Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM)-CONICET, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina.
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10
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Ylä-Anttila P, Mikkonen E, Happonen KE, Holland P, Ueno T, Simonsen A, Eskelinen EL. RAB24 facilitates clearance of autophagic compartments during basal conditions. Autophagy 2016; 11:1833-48. [PMID: 26325487 DOI: 10.1080/15548627.2015.1086522] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
RAB24 belongs to a family of small GTPases and has been implicated to function in autophagy. Here we confirm the intracellular localization of RAB24 to autophagic vacuoles with immuno electron microscopy and cell fractionation, and show that prenylation and guanine nucleotide binding are necessary for the targeting of RAB24 to autophagic compartments. Further, we show that RAB24 plays a role in the maturation and/or clearance of autophagic compartments under nutrient-rich conditions, but not during short amino acid starvation. Quantitative electron microscopy shows an increase in the numbers of late autophagic compartments in cells silenced for RAB24, and mRFP-GFP-LC3 probe and autophagy flux experiments indicate that this is due to a hindrance in their clearance. Formation of autophagosomes is shown to be unaffected by RAB24-silencing with siRNA. A defect in aggregate clearance in the absence of RAB24 is also shown in cells forming polyglutamine aggregates. This study places RAB24 function in the termination of the autophagic process under nutrient-rich conditions.
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Affiliation(s)
- Päivi Ylä-Anttila
- a Department of Biosciences, Division of Biochemistry and Biotechnology; University of Helsinki ; Helsinki , Finland
| | - Elisa Mikkonen
- a Department of Biosciences, Division of Biochemistry and Biotechnology; University of Helsinki ; Helsinki , Finland
| | - Kaisa E Happonen
- a Department of Biosciences, Division of Biochemistry and Biotechnology; University of Helsinki ; Helsinki , Finland
| | - Petter Holland
- b Department of Biochemistry, Institute of Basic Medical Sciences; University of Oslo ; Oslo , Norway
| | - Takashi Ueno
- c Laboratory of Proteomics and Biomolecular Science; Research Support Center; Juntendo University Graduate School of Medicine ; Tokyo , Japan
| | - Anne Simonsen
- b Department of Biochemistry, Institute of Basic Medical Sciences; University of Oslo ; Oslo , Norway
| | - Eeva-Liisa Eskelinen
- a Department of Biosciences, Division of Biochemistry and Biotechnology; University of Helsinki ; Helsinki , Finland
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Ishida M, E Oguchi M, Fukuda M. Multiple Types of Guanine Nucleotide Exchange Factors (GEFs) for Rab Small GTPases. Cell Struct Funct 2016; 41:61-79. [PMID: 27246931 DOI: 10.1247/csf.16008] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
Rab small GTPases are highly conserved master regulators of membrane traffic in all eukaryotes. The same as the activation and inactivation of other small GTPases, the activation and inactivation of Rabs are tightly controlled by specific GEFs (guanine nucleotide exchange factors) and GAPs (GTPase-activating proteins), respectively. Although almost all Rab-GAPs reported thus far have a TBC (Tre-2/Bub2/Cdc16)/Rab-GAP domain in common, recent accumulating evidence has indicated the existence of a number of structurally unrelated types of Rab-GEFs, including DENN proteins, VPS9 proteins, Sec2 proteins, TRAPP complexes, heterodimer GEFs (Mon1-Ccz1, HPS1-HPS4 (BLOC-3 complex), Ric1-Rgp1 and Rab3GAP1/2), and other GEFs (e.g., REI-1 and RPGR). In this review article we provide an up-to-date overview of the structures and functions of all putative Rab-GEFs in mammals, with a special focus on their substrate Rabs, interacting proteins, associations with genetic diseases, and intracellular localizations.
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Affiliation(s)
- Morié Ishida
- Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University
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12
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Schöppner P, Csaba G, Braun T, Daake M, Richter B, Lange OF, Zacharias M, Zimmer R, Haslbeck M. Regulatory Implications of Non-Trivial Splicing: Isoform 3 of Rab1A Shows Enhanced Basal Activity and Is Not Controlled by Accessory Proteins. J Mol Biol 2016; 428:1544-57. [PMID: 26953259 DOI: 10.1016/j.jmb.2016.02.028] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2015] [Revised: 02/19/2016] [Accepted: 02/24/2016] [Indexed: 01/04/2023]
Abstract
Alternative splicing often affects structured and highly conserved regions of proteins, generating so called non-trivial splicing variants of unknown structure and cellular function. The human small G-protein Rab1A is involved in the regulation of the vesicle transfer from the ER to Golgi. A conserved non-trivial splice variant lacks nearly 40% of the sequence of the native Rab1A, including most of the regulatory interaction sites. We show that this variant of Rab1A represents a stable and folded protein, which is still able to bind nucleotides and co-localizes with membranes. Nevertheless, it should be mentioned that compared to other wild-typeRabGTPases, the measured nucleotide binding affinities are dramatically reduced in the variant studied. Furthermore, the Rab1A variant forms hetero-dimers with wild-type Rab1A and its presence in the cell enhances the efficiency of alkaline phosphatase secretion. However, this variant shows no specificity for GXP nucleotides, a constantly enhanced GTP hydrolysis activity and is no longer controlled by GEF or GAP proteins, indicating a new regulatory mechanism for the Rab1A cycle via alternative non-trivial splicing.
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Affiliation(s)
- Patricia Schöppner
- Center for Integrated Protein Science, Department Chemie, Technische Universität München, Lichtenbergstrasse 4, 85748 Garching, Germany
| | - Gergely Csaba
- Department of Informatics, Ludwig-Maximilians-Universität München, Amalienstr. 17, 80333 München, Germany
| | - Tatjana Braun
- Center for Integrated Protein Science, Department Chemie, Technische Universität München, Lichtenbergstrasse 4, 85748 Garching, Germany
| | - Marina Daake
- Center for Integrated Protein Science, Department Chemie, Technische Universität München, Lichtenbergstrasse 4, 85748 Garching, Germany
| | - Bettina Richter
- Center for Integrated Protein Science, Department Chemie, Technische Universität München, Lichtenbergstrasse 4, 85748 Garching, Germany
| | - Oliver F Lange
- Center for Integrated Protein Science, Department Chemie, Technische Universität München, Lichtenbergstrasse 4, 85748 Garching, Germany
| | - Martin Zacharias
- Physics Department, Technische Universität München, James-Franck-Strasse 1, 85747 Garching, Germany
| | - Ralf Zimmer
- Department of Informatics, Ludwig-Maximilians-Universität München, Amalienstr. 17, 80333 München, Germany.
| | - Martin Haslbeck
- Center for Integrated Protein Science, Department Chemie, Technische Universität München, Lichtenbergstrasse 4, 85748 Garching, Germany.
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13
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Miserey-Lenkei S, Colombo MI. Small RAB GTPases Regulate Multiple Steps of Mitosis. Front Cell Dev Biol 2016; 4:2. [PMID: 26925400 PMCID: PMC4756281 DOI: 10.3389/fcell.2016.00002] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2015] [Accepted: 01/11/2016] [Indexed: 12/12/2022] Open
Abstract
GTPases of the RAB family are key regulators of multiple steps of membrane trafficking. Several members of the RAB GTPase family have been implicated in mitotic progression. In this review, we will first focus on the function of endosome-associated RAB GTPases reported in early steps of mitosis, spindle pole maturation, and during cytokinesis. Second, we will discuss the role of Golgi-associated RAB GTPases at the metaphase/anaphase transition and during cytokinesis.
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Affiliation(s)
- Stéphanie Miserey-Lenkei
- Institut Curie, PSL Research University, Molecular Mechanisms of Intracellular Transport Group, CNRS UMR 144 Paris, France
| | - María I Colombo
- Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología-CONICET, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo Mendoza, Argentina
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14
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Amaya C, Fader CM, Colombo MI. Autophagy and proteins involved in vesicular trafficking. FEBS Lett 2015; 589:3343-53. [PMID: 26450776 DOI: 10.1016/j.febslet.2015.09.021] [Citation(s) in RCA: 77] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2015] [Revised: 09/19/2015] [Accepted: 09/22/2015] [Indexed: 12/16/2022]
Abstract
Autophagy is an intracellular degradation system that, as a basic mechanism it delivers cytoplasmic components to the lysosomes in order to maintain adequate energy levels and cellular homeostasis. This complex cellular process is activated by low cellular nutrient levels and other stress situations such as low ATP levels, the accumulation of damaged proteins or organelles, or pathogen invasion. Autophagy as a multistep process involves vesicular transport events leading to tethering and fusion of autophagic vesicles with several intracellular compartments. This review summarizes our current understanding of the autophagic pathway with emphasis in the trafficking machinery (i.e. Rabs GTPases and SNAP receptors (SNAREs)) involved in specific steps of the pathway.
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Affiliation(s)
- Celina Amaya
- Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM)-CONICET, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Casilla de Correo 56, Centro Universitario, Parque General San Martín, 5500 Mendoza, Argentina
| | - Claudio Marcelo Fader
- Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM)-CONICET, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Casilla de Correo 56, Centro Universitario, Parque General San Martín, 5500 Mendoza, Argentina
| | - María Isabel Colombo
- Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM)-CONICET, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Casilla de Correo 56, Centro Universitario, Parque General San Martín, 5500 Mendoza, Argentina.
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15
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Matsuto M, Kano F, Murata M. Reconstitution of the targeting of Rab6A to the Golgi apparatus in semi-intact HeLa cells: A role of BICD2 in stabilizing Rab6A on Golgi membranes and a concerted role of Rab6A/BICD2 interactions in Golgi-to-ER retrograde transport. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2015; 1853:2592-609. [PMID: 25962623 DOI: 10.1016/j.bbamcr.2015.05.005] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/13/2014] [Revised: 05/02/2015] [Accepted: 05/05/2015] [Indexed: 12/21/2022]
Abstract
Rab is a small GTP-binding protein family that regulates various pathways of vesicular transport. Although more than 60 Rab proteins are targeted to specific organelles in mammalian cells, the mechanisms underlying the specificity of Rab proteins for the respective organelles remain unknown. In this study, we reconstituted the Golgi targeting of Rab6A in streptolysin O (SLO)-permeabilized HeLa cells in a cytosol-dependent manner and investigated the biochemical requirements of targeting. Golgi-targeting assays identified Bicaudal-D (BICD)2, which is reportedly involved in the dynein-mediated transport of mRNAs during oogenesis and embryogenesis in Drosophila, as a cytosolic factor for the Golgi targeting of Rab6A in SLO-permeabilized HeLa cells. Subsequent immunofluorescence analyses indicated decreased amounts of the GTP-bound active form of Rab6 in BICD2-knockdown cells. In addition, fluorescence recovery after photobleaching (FRAP) analyses revealed that overexpression of the C-terminal region of BICD2 decreased the exchange rate of GFP-Rab6A between the Golgi membrane and the cytosol. Collectively, these results indicated that BICD2 facilitates the binding of Rab6A to the Golgi by stabilizing its GTP-bound form. Moreover, several analyses of vesicular transport demonstrated that Rab6A and BICD2 play crucial roles in Golgi tubule fusion with the endoplasmic reticulum (ER) in brefeldin A (BFA)-treated cells, indicating that BICD2 is involved in coat protein I (COPI)-independent Golgi-to-ER retrograde vesicular transport.
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Affiliation(s)
- Mariko Matsuto
- Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan
| | - Fumi Kano
- Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan; PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan
| | - Masayuki Murata
- Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan.
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16
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Kim M, Tan YS, Cheng WC, Kingsbury TJ, Heimfeld S, Civin CI. MIR144 and MIR451 regulate human erythropoiesis via RAB14. Br J Haematol 2014; 168:583-97. [PMID: 25312678 DOI: 10.1111/bjh.13164] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2014] [Accepted: 08/15/2014] [Indexed: 12/11/2022]
Abstract
Expression levels of MIR144 and MIR451 increase during erythropoiesis, a pattern that is conserved from zebrafish to humans. As these two miRs are expressed from the same polycistronic transcript, we manipulated MIR144 and MIR451 in human erythroid cells individually and together to investigate their effects on human erythropoiesis. Inhibition of endogenous human MIR451 resulted in decreased numbers of erythroid (CD71(hi) CD235a(hi) CD34(-) ) cells, consistent with prior studies in zebrafish and mice. In addition, inhibition of MIR144 impaired human erythroid differentiation, unlike in zebrafish and mouse studies where the functional effect of MIR144 on erythropoiesis was minimal. In this study, we found RAB14 is a direct target of both MIR144 and MIR451. As MIR144 and MIR451 expression increased during human erythropoiesis, RAB14 protein expression decreased. Enforced RAB14 expression phenocopied the effect of MIR144 and/or MIR451 depletion, whereas shRNA-mediated RAB14 knockdown protected cells from MIR144 and/or MIR451 depletion-mediated erythropoietic inhibition. RAB14 knockdown increased the frequency and number of erythroid cells, increased β-haemoglobin expression, and decreased CBFA2T3 expression during human erythropoiesis. In summary, we utilized MIR144 and MIR451 to identify RAB14 as a novel physiological inhibitor of human erythropoiesis.
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Affiliation(s)
- MinJung Kim
- Departments of Physiology and Pediatrics, Center for Stem Cell Biology & Regenerative Medicine, Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD, USA
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17
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Chandra M, Mukherjee M, Srivastava VK, Saito-Nakano Y, Nozaki T, Datta S. Insights into the GTP/GDP cycle of RabX3, a novel GTPase from Entamoeba histolytica with tandem G-domains. Biochemistry 2014; 53:1191-205. [PMID: 24471929 DOI: 10.1021/bi401428f] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Members of the small GTPase Ras superfamily regulate a host of systems through their ability to catalyze the GTP/GDP cycle. All family members reported thus far possess a single GTPase domain with a P-loop containing a nucleoside triphosphate hydrolase fold. Here for the first time we report a novel member from Entamoeba histolytica, EhRabX3, which harbors two GTPase domains in tandem and exhibits unique biochemical properties. A combination of biochemical and microcalorimetric studies revealed that EhRabX3 binds to a single guanine nucleotide through its N-terminal domain. Unlike most of the members of the Ras superfamily, the dissociation of the nucleotide from EhRabX3 is independent of Mg(2+), perhaps indicating a novel mechanism of nucleotide exchange by this protein. We found that EhRabX3 is extremely sluggish in hydrolyzing GTP, and that could be attributed to its atypical nucleotide binding pocket. It harbors substitutions at two positions that confer oncogenicity to Ras because of impaired GTP hydrolysis. Engineering these residues into the conserved counterparts enhanced their GTPase activity by at least 20-fold. In contrast to most of the members of the Ras superfamily, EhRabX3 lacks the prenylation motif. Using indirect immunofluorescence and biochemical fractionation, we demonstrated that the protein is distributed all over the cytosol in amoebic trophozoites. Collectively, this unique ancient GTPase exhibits a striking evolutionary divergence from the other members of the superfamily.
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Affiliation(s)
- Mintu Chandra
- Department of Biological Sciences, Indian Institute of Science Education and Research Bhopal , Bhopal 462023, India
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18
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Agler C, Nielsen DM, Urkasemsin G, Singleton A, Tonomura N, Sigurdsson S, Tang R, Linder K, Arepalli S, Hernandez D, Lindblad-Toh K, van de Leemput J, Motsinger-Reif A, O'Brien DP, Bell J, Harris T, Steinberg S, Olby NJ. Canine hereditary ataxia in old english sheepdogs and gordon setters is associated with a defect in the autophagy gene encoding RAB24. PLoS Genet 2014; 10:e1003991. [PMID: 24516392 PMCID: PMC3916225 DOI: 10.1371/journal.pgen.1003991] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2013] [Accepted: 10/16/2013] [Indexed: 11/19/2022] Open
Abstract
Old English Sheepdogs and Gordon Setters suffer from a juvenile onset, autosomal recessive form of canine hereditary ataxia primarily affecting the Purkinje neuron of the cerebellar cortex. The clinical and histological characteristics are analogous to hereditary ataxias in humans. Linkage and genome-wide association studies on a cohort of related Old English Sheepdogs identified a region on CFA4 strongly associated with the disease phenotype. Targeted sequence capture and next generation sequencing of the region identified an A to C single nucleotide polymorphism (SNP) located at position 113 in exon 1 of an autophagy gene, RAB24, that segregated with the phenotype. Genotyping of six additional breeds of dogs affected with hereditary ataxia identified the same polymorphism in affected Gordon Setters that segregated perfectly with phenotype. The other breeds tested did not have the polymorphism. Genome-wide SNP genotyping of Gordon Setters identified a 1.9 MB region with an identical haplotype to affected Old English Sheepdogs. Histopathology, immunohistochemistry and ultrastructural evaluation of the brains of affected dogs from both breeds identified dramatic Purkinje neuron loss with axonal spheroids, accumulation of autophagosomes, ubiquitin positive inclusions and a diffuse increase in cytoplasmic neuronal ubiquitin staining. These findings recapitulate the changes reported in mice with induced neuron-specific autophagy defects. Taken together, our results suggest that a defect in RAB24, a gene associated with autophagy, is highly associated with and may contribute to canine hereditary ataxia in Old English Sheepdogs and Gordon Setters. This finding suggests that detailed investigation of autophagy pathways should be undertaken in human hereditary ataxia. Neurodegenerative diseases are one of the most important causes of decline in an aging population. An important subset of these diseases are known as the hereditary ataxias, familial neurodegenerative diseases that affect the cerebellum causing progressive gait disturbance in both humans and dogs. We identified a mutation in RAB24, a gene associated with autophagy, in Old English Sheepdogs and Gordon Setters with hereditary ataxia. Autophagy is a process by which cell proteins and organelles are removed and recycled and its critical role in maintenance of the continued health of cells is becoming clear. We evaluated the brains of affected dogs and identified accumulations of autophagosomes within the cerebellum, suggesting a defect in the autophagy pathway. Our results suggest that a defect in the autophagy pathway results in neuronal death in a naturally occurring disease in dogs. The autophagy pathway should be investigated in human hereditary ataxia and may represent a therapeutic target in neurodegenerative diseases.
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Affiliation(s)
- Caryline Agler
- Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, United States of America
| | - Dahlia M. Nielsen
- Bioinformatics Research Center, North Carolina State University, Raleigh, North Carolina, United States of America
| | - Ganokon Urkasemsin
- Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, United States of America
| | - Andrew Singleton
- Laboratory of Neurogenetics, National Institute on Aging, Bethesda, Maryland, United States of America
| | - Noriko Tonomura
- Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America
- Department of Clinical Sciences, Tufts Cummings School of Veterinary Medicine, North Grafton, Massachusetts, United States of America
| | - Snaevar Sigurdsson
- Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America
| | - Ruqi Tang
- Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America
| | - Keith Linder
- Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, United States of America
| | - Sampath Arepalli
- Laboratory of Neurogenetics, National Institute on Aging, Bethesda, Maryland, United States of America
| | - Dena Hernandez
- Laboratory of Neurogenetics, National Institute on Aging, Bethesda, Maryland, United States of America
| | - Kerstin Lindblad-Toh
- Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America
- Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden
| | - Joyce van de Leemput
- Laboratory of Neurogenetics, National Institute on Aging, Bethesda, Maryland, United States of America
| | - Alison Motsinger-Reif
- Bioinformatics Research Center, North Carolina State University, Raleigh, North Carolina, United States of America
- Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, North Carolina, United States of America
| | - Dennis P. O'Brien
- Department of Veterinary Medicine & Surgery, College of Veterinary Medicine, University of Missouri, Columbia, Missouri, United States of America
| | - Jerold Bell
- Department of Clinical Sciences, Tufts Cummings School of Veterinary Medicine, North Grafton, Massachusetts, United States of America
| | - Tonya Harris
- Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, United States of America
| | - Steven Steinberg
- VCA Veterinary Referral Associates, Gaithersbrug, Maryland, United States of America
| | - Natasha J. Olby
- Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, United States of America
- Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, North Carolina, United States of America
- * E-mail:
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19
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Abstract
Whereas most of what we know today about the Ras-related small GTPases of the Rab family stems from observations made on Golgi complex, endosome and plasma membrane trafficking, a subset of Rabs localizes in part or predominantly to the ER (endoplasmic reticulum). Here, Rabs such as Rab1, Rab2, Rab6 and Rab33 can regulate the anterograde and retrograde trafficking of vesicles between the Golgi complex, the ERGIC (ER-Golgi intermediate compartment) and the ER itself. However, among the ER-associated Rabs, some Rabs appear to perform roles not directly related to trafficking: these Rabs (e.g. Rab32 or Rab24) could aid proteins of the atlastin and reticulon families in determining the extent and direction of ER tubulation. In so doing, these Rabs regulate not only ER contacts with other organelles such as mitochondria, but also the formation of autophagosomes.
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20
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Militello RD, Munafó DB, Berón W, López LA, Monier S, Goud B, Colombo MI. Rab24 is required for normal cell division. Traffic 2013; 14:502-18. [PMID: 23387408 DOI: 10.1111/tra.12057] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2012] [Revised: 02/04/2013] [Accepted: 02/06/2013] [Indexed: 12/18/2022]
Abstract
Rab24 is an atypical member of the Rab GTPase family whose distribution in interphase cells has been characterized; however, its function remains largely unknown. In this study, we have analyzed the distribution of Rab24 throughout cell division. We have observed that Rab24 was located at the mitotic spindle in metaphase, at the midbody during telophase and in the furrow during cytokinesis. We have also observed partial co-localization of Rab24 and tubulin and demonstrated its association to microtubules. Interestingly, more than 90% of transiently transfected HeLa cells with Rab24 presented abnormal nuclear connections (i.e., chromatin bridges). Furthermore, in CHO cells stably transfected with GFP-Rab24wt, we observed a large percentage of binucleated and multinucleated cells. In addition, these cells presented an extremely large size and multiple failures in mitosis, as aberrant spindle formation (metaphase), delayed chromosomes (telophase) and multiple cytokinesis. A marked increase in binucleated, multinucleated and multilobulated nucleus formation was observed in HeLa cells depleted of Rab24. We also present evidence that a fraction of Rab24 associates with microtubules. In addition, Rab24 knock down resulted in misalignment of chromosomes and abnormal spindle formation in metaphase leading to the appearance of delayed chromosomes during late telophase and failures in cytokinesis. Our findings suggest that an adequate level of Rab24 is necessary for normal cell division. In summary, Rab24 modulates several mitotic events, including chromosome segregation and cytokinesis, perhaps through the interaction with microtubules.
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Affiliation(s)
- Rodrigo D Militello
- Laboratorio de Biología Celular y Molecular- Instituto de Histología y Embriología (IHEM), Facultad de Ciencias Médicas, Universidad Nacional de Cuyo-CONICET, Mendoza, Argentina
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21
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Gallegos ME, Balakrishnan S, Chandramouli P, Arora S, Azameera A, Babushekar A, Bargoma E, Bokhari A, Chava SK, Das P, Desai M, Decena D, Saramma SDD, Dey B, Doss AL, Gor N, Gudiputi L, Guo C, Hande S, Jensen M, Jones S, Jones N, Jorgens D, Karamchedu P, Kamrani K, Kolora LD, Kristensen L, Kwan K, Lau H, Maharaj P, Mander N, Mangipudi K, Menakuru H, Mody V, Mohanty S, Mukkamala S, Mundra SA, Nagaraju S, Narayanaswamy R, Ndungu-Case C, Noorbakhsh M, Patel J, Patel P, Pendem SV, Ponakala A, Rath M, Robles MC, Rokkam D, Roth C, Sasidharan P, Shah S, Tandon S, Suprai J, Truong TQN, Uthayaruban R, Varma A, Ved U, Wang Z, Yu Z. The C. elegans rab family: identification, classification and toolkit construction. PLoS One 2012; 7:e49387. [PMID: 23185324 PMCID: PMC3504004 DOI: 10.1371/journal.pone.0049387] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2012] [Accepted: 10/09/2012] [Indexed: 11/29/2022] Open
Abstract
Rab monomeric GTPases regulate specific aspects of vesicle transport in eukaryotes including coat recruitment, uncoating, fission, motility, target selection and fusion. Moreover, individual Rab proteins function at specific sites within the cell, for example the ER, golgi and early endosome. Importantly, the localization and function of individual Rab subfamily members are often conserved underscoring the significant contributions that model organisms such as Caenorhabditis elegans can make towards a better understanding of human disease caused by Rab and vesicle trafficking malfunction. With this in mind, a bioinformatics approach was first taken to identify and classify the complete C. elegans Rab family placing individual Rabs into specific subfamilies based on molecular phylogenetics. For genes that were difficult to classify by sequence similarity alone, we did a comparative analysis of intron position among specific subfamilies from yeast to humans. This two-pronged approach allowed the classification of 30 out of 31 C. elegans Rab proteins identified here including Rab31/Rab50, a likely member of the last eukaryotic common ancestor (LECA). Second, a molecular toolset was created to facilitate research on biological processes that involve Rab proteins. Specifically, we used Gateway-compatible C. elegans ORFeome clones as starting material to create 44 full-length, sequence-verified, dominant-negative (DN) and constitutive active (CA) rab open reading frames (ORFs). Development of this toolset provided independent research projects for students enrolled in a research-based molecular techniques course at California State University, East Bay (CSUEB).
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Affiliation(s)
- Maria E Gallegos
- Department of Biological Sciences, California State University East Bay, Hayward, CA, USA.
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22
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Delprato A. Topological and functional properties of the small GTPases protein interaction network. PLoS One 2012; 7:e44882. [PMID: 23028658 PMCID: PMC3441499 DOI: 10.1371/journal.pone.0044882] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2012] [Accepted: 08/15/2012] [Indexed: 12/31/2022] Open
Abstract
Small GTP binding proteins of the Ras superfamily (Ras, Rho, Rab, Arf, and Ran) regulate key cellular processes such as signal transduction, cell proliferation, cell motility, and vesicle transport. A great deal of experimental evidence supports the existence of signaling cascades and feedback loops within and among the small GTPase subfamilies suggesting that these proteins function in a coordinated and cooperative manner. The interplay occurs largely through association with bi-partite regulatory and effector proteins but can also occur through the active form of the small GTPases themselves. In order to understand the connectivity of the small GTPases signaling routes, a systems-level approach that analyzes data describing direct and indirect interactions was used to construct the small GTPases protein interaction network. The data were curated from the Search Tool for the Retrieval of Interacting Genes (STRING) database and include only experimentally validated interactions. The network method enables the conceptualization of the overall structure as well as the underlying organization of the protein-protein interactions. The interaction network described here is comprised of 778 nodes and 1943 edges and has a scale-free topology. Rac1, Cdc42, RhoA, and HRas are identified as the hubs. Ten sub-network motifs are also identified in this study with themes in apoptosis, cell growth/proliferation, vesicle traffic, cell adhesion/junction dynamics, the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase response, transcription regulation, receptor-mediated endocytosis, gene silencing, and growth factor signaling. Bottleneck proteins that bridge signaling paths and proteins that overlap in multiple small GTPase networks are described along with the functional annotation of all proteins in the network.
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Affiliation(s)
- Anna Delprato
- BioScience Project, Wakefield, Massachusetts, United States of America.
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23
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The GTPase TcRjl of the human pathogen Trypanosoma cruzi is involved in the cell growth and differentiation. Biochem Biophys Res Commun 2012; 419:38-42. [PMID: 22326867 DOI: 10.1016/j.bbrc.2012.01.119] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2012] [Accepted: 01/25/2012] [Indexed: 11/20/2022]
Abstract
The protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas Disease, undergoes through a complex life cycle where rounds of cell division and differentiation occur initially in the gut of triatominae vectors and, after transmission, inside of infected cells in vertebrate hosts. Members of the Ras superfamily of GTPases are molecular switches which play pivotal regulatory functions in cell growth and differentiation. We have previously described a novel GTPase in T. cruzi, TcRjl, which belongs to the RJL family of Ras-related GTP binding proteins. Here we show that most of TcRjl protein is found bound to GTP nucleotides and may be locked in this stage. In addition, we show that TcRjl is located close to the kinetoplast, in a region corresponding possibly to flagellar pocket of the parasite and the expression of a dominant-negative TcRjl construct (TcRjlS37N) displays a significative growth phenotype in reduced serum medium. Remarkably, overexpression of TcRjl inhibits differentiation of epimastigotes to trypomastigote forms and promotes the accumulation of intermediate differentiation stages. Our data suggest that TcRjl might play a role in the control of the parasite growth and differentiation.
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24
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Schardt A, Brinkmann BG, Mitkovski M, Sereda MW, Werner HB, Nave KA. The SNARE protein SNAP-29 interacts with the GTPase Rab3A: Implications for membrane trafficking in myelinating glia. J Neurosci Res 2010; 87:3465-79. [PMID: 19170188 DOI: 10.1002/jnr.22005] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
During myelin formation, vast amounts of specialized membrane proteins and lipids are trafficked toward the growing sheath in cell surface-directed transport vesicles. Soluble N-ethylmaleimide-sensitive factor (NSF) attachment proteins (SNAPs) are important components of molecular complexes required for membrane fusion. We have analyzed the expression profile and molecular interactions of SNAP-29 in the nervous system. In addition to its known enrichment in neuronal synapses, SNAP-29 is abundant in oligodendrocytes during myelination and in noncompact myelin of the peripheral nervous system. By yeast two-hybrid screen and coimmunoprecipitation, we found that the GTPases Rab3A, Rab24, and septin 4 bind to the N-terminal domain of SNAP-29. The interaction with Rab24 or septin 4 was GTP independent. In contrast, interaction between SNAP-29 and Rab3A was GTP dependent, and colocalization was extensive both in synapses and in myelinating glia. In HEK293 cells, cytoplasmic SNAP-29 pools were redistributed upon coexpression with Rab3A, and surface-directed trafficking of myelin proteolipid protein was enhanced by overexpression of SNAP-29 and Rab3A. Interestingly, the abundance of SNAP-29 in sciatic nerves was increased during remyelination and in a rat model of Charcot-Marie-Tooth disease, two pathological situations with increased myelin membrane biogenesis. We suggest that Rab3A may regulate SNAP-29-mediated membrane fusion during myelination.
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Affiliation(s)
- Anke Schardt
- Department of Neurogenetics, Max-Planck-Institute of Experimental Medicine, Göttingen, Germany
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25
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26
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Dong B, Gong D, Gu Z, Meng H. Molecular cloning and characterization of Rab6 gene in duck. ACTA ACUST UNITED AC 2007; 18:307-11. [PMID: 17541837 DOI: 10.1080/10425170701248509] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
Abstract
Rab (ras-like in rat brain) proteins are small GTP-binding proteins that belong to largest subfamily in the small G protein, which are important for molecular modulation of membrane in the vesicular trafficking pathways. We have cloned and sequenced full length cDNA of Rab6 gene in duck. The cDNA sequence consists of 761 nucleotides and contains a complete open reading frame (ORF) of 627 nucleotides; the putative protein includes 208 amino acids. The CDS of duck Rab6 gene shares 86.1-90.0% homology with house mouse, silurana tropicalis, dog, human and orangutan, which indicates the Rab6 gene is high evolutional conservation in above animals.
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Affiliation(s)
- Biao Dong
- School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, P. R. China
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27
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Montalbano J, Jin W, Sheikh MS, Huang Y. RBEL1 is a novel gene that encodes a nucleocytoplasmic Ras superfamily GTP-binding protein and is overexpressed in breast cancer. J Biol Chem 2007; 282:37640-9. [PMID: 17962191 DOI: 10.1074/jbc.m704760200] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Rab family proteins are generally known as regulators of protein transport and trafficking. A number of Rab proteins have been implicated in cancer development and/or progression. Here we report the identification of a novel Rab-like protein, which we have named RBEL1 (Rab-like protein 1) for its higher similarity to the Rab subfamily members. We have characterized two isoforms of RBEL1 including the predominant RBEL1A and the less abundant RBEL1B that results from alternative splicing. Both isoforms harbor conserved N-terminal guanine trinucleotide phosphate (GTP) binding domains and, accordingly, are capable of binding to GTP. Both isoforms contain variable C termini and exhibit differential subcellular localization patterns. Unlike known Rabs that are mostly cytosolic, RBEL1B predominantly resides in the nucleus, whereas RBEL1A is localized primarily to the cytosol. Interestingly, a point mutation affecting RBEL1B GTP binding also alters the ability of mutant protein to accumulate in the nucleus, suggesting GTP binding potential to be important for RBEL1B nuclear localization. Our results also indicate that RBEL1A is overexpressed in about 67% of primary breast tumors. Thus, RBEL1A and RBEL1B are novel Rab-like proteins that localize in the nucleus and cytosol and may play an important role in breast tumorigenesis.
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Affiliation(s)
- JoAnne Montalbano
- Department of Pharmacology, State University of New York, Upstate Medical University, Syracuse, NY 13210, USA
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28
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Kaul A, Overmeyer JH, Maltese WA. Activated Ras induces cytoplasmic vacuolation and non-apoptotic death in glioblastoma cells via novel effector pathways. Cell Signal 2006; 19:1034-43. [PMID: 17210246 PMCID: PMC1894854 DOI: 10.1016/j.cellsig.2006.11.010] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2006] [Revised: 11/22/2006] [Accepted: 11/23/2006] [Indexed: 10/23/2022]
Abstract
Expression of activated H-Ras induces a unique form of non-apoptotic cell death in human glioblastoma cells and other specific tumor cell lines. The major cytopathological features of this form of death are the accumulation of large phase-lucent, LAMP1-positive, cytoplasmic vacuoles. In this study we sought to determine if induction of cytoplasmic vacuolation a) depends on Ras farnesylation, b) is specific to H-Ras, and c) is mediated by signaling through the major known Ras effector pathways. We find that the unusual effects of activated H-Ras depend on farnesylation and membrane association of the GTPase. Both H-Ras(G12V) and K-Ras4B(G12V) stimulate vacuolation, but activated forms of Cdc42 and RhoA do not. Amino acid substitutions in the Ras effector domain, which are known to selectively impair its interactions with Raf kinase, class-I phosphatidylinositide 3-kinase (PI3K), or Ral nucleotide exchange factors, initially pointed to Raf as a possible mediator of cell vacuolation. However, the MEK inhibitor, PD98059, did not block the induction of vacuoles, and constitutively active Raf-Caax did not mimic the effects of Ras(G12V). Introduction of normal PTEN together with H-Ras(G12V) into U251 glioblastoma cells reduced the PI3K-dependent activation of Akt, but had no effect on vacuolation. Finally, co-expression of H-Ras(G12V) with a dominant-negative form of RalA did not suppress vacuolation. Taken together, the observations indicate that Ras activates non-conventional and perhaps unique effector pathways to induce cytoplasmic vacuolation in glioblastoma cells. Identification of the relevant signaling pathways may uncover specific molecular targets that can be manipulated to activate non-apoptotic cell death in this type of cancer.
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Affiliation(s)
| | | | - William A. Maltese
- *Correspondence: Dr. William A. Maltese, Department of Biochemistry & Cancer Biology, Block Health Sciences Bldg, University of Toledo College of Medicine, 3035 Arlington Ave., Toledo, Ohio, 43614 E-mail:
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Egami Y, Kiryu-Seo S, Yoshimori T, Kiyama H. Induced expressions of Rab24 GTPase and LC3 in nerve-injured motor neurons. Biochem Biophys Res Commun 2005; 337:1206-13. [PMID: 16236257 DOI: 10.1016/j.bbrc.2005.09.171] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2005] [Accepted: 09/22/2005] [Indexed: 11/30/2022]
Abstract
Rab24 is a member of the Rab GTPase family, but its function is unclear. Here, we demonstrated increase in Rab24 mRNA in nerve-injured hypoglossal motor neurons of rats. Expression of Rab24 mRNA was also induced in differentiated PC12 cells following proteasome inhibitor (MG132) treatment. MG132 treatment further induced expression of microtubule-associated protein light chain 3 (LC3), and accumulation of LC3-II, a processed form of LC3 and the most reliable marker for autophagy. Induction of LC3 mRNA and accumulation of LC3-II were also observed in nerve-injured hypoglossal motor neurons, and partial co-localization of Rab24 and LC3 was demonstrated by immunohistochemistry. The present data suggest that nerve injury promotes autophagy-like events, and this may be an important response for degradation of unnecessary and misfolded proteins to recycle limited amino acids, and synthesize new proteins that are necessary for survival and nerve regeneration responses.
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Affiliation(s)
- Youhei Egami
- Department of Anatomy and Neurobiology, Osaka City University, Graduate School of Medicine, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan
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30
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Bergbrede T, Pylypenko O, Rak A, Alexandrov K. Structure of the extremely slow GTPase Rab6A in the GTP bound form at 1.8A resolution. J Struct Biol 2005; 152:235-8. [PMID: 16332443 DOI: 10.1016/j.jsb.2005.10.001] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2005] [Revised: 09/28/2005] [Accepted: 10/13/2005] [Indexed: 11/18/2022]
Abstract
Rab/Ypt GTPases represent a>60 member large family of membrane traffic regulators in eukaryotic cells. Members of this group display intrinsic GTPase activity varying over two orders of magnitude. Here, we show that Rab6A represents the RabGTPase with the slowest spontaneous GTPase activity yet measured (5x10(-6)s(-1)). Due to the very low intrinsic hydrolysis rate we were able to crystallise and solve the structure of the Rab6A:GTP complex to 1.82A resolution. Analysis of the structure suggests that low catalytic activity of the Rab6A might be due to high flexibility of the Switch II region and a low degree of constraint of critically important for catalysis Gln 72.
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Affiliation(s)
- Tim Bergbrede
- Max-Planck-Institute for Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany
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31
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Ramos FP, Araripe JR, Urményi TP, Silva R, Cunha e Silva NL, Leite Fontes CF, da Silveira JF, Rondinelli E. Characterization of RAB-like4, the first identified RAB-like protein from Trypanosoma cruzi with GTPase activity. Biochem Biophys Res Commun 2005; 333:808-17. [PMID: 15975556 DOI: 10.1016/j.bbrc.2005.05.183] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2005] [Accepted: 05/26/2005] [Indexed: 11/25/2022]
Abstract
RAB proteins, which belong to the RAS superfamily, regulate exocytic and endocytic pathways of eukaryotic cells, controlling vesicle docking and fusion. Few RAB proteins have been identified in parasites. Molecular markers for cellular compartments are important to studies concerning about the protein traffic in Trypanosoma cruzi, the causal agent of Chagas disease. In this work, we describe the characterization of TcRABL4, the first RAB-like gene identified in T. cruzi (GenBank Accession No.: ), present as a single-copy gene. TcRABL4 contains all five consensus RAB motifs but lacks cysteine residues at the C terminus, which are essential to isoprenylation, an absolute prerequisite for membrane association of these proteins. TcRABL4 is a functional GTPase that is able to bind and hydrolyze GTP, and its gene is transcribed as a single 1.2 kb mRNA in epimastigotes. TcRABL4 appears to be differentially regulated in the three cell forms of the parasite, and the protein is not associated to membranes, unlike other RAB proteins. It is possible that TcRABL4 may be a member of a novel family of small GTPases.
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Affiliation(s)
- Fabiane Pereira Ramos
- Laboratório de Metabolismo Macromolecular Firmino Torres de Castro, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS, Cidade Universitária, Rio de Janeiro 21949-900, Brazil
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32
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Wang W, Zhi H, Chai B, Liang A. Cloning and sequence analysis of the micronuclear and macronuclear gene encoding Rab protein of Euplotes octocarinatus. Biosci Biotechnol Biochem 2005; 69:649-52. [PMID: 15785000 DOI: 10.1271/bbb.69.649] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The DNA in a micronucleus undergoes remarkable rearrangements when it develops into a macronucleus after cell mating in the hypotrichous ciliate. A Rab gene was isolated from the macronuclear plasmid mini-library of Euplotes octocarinatus. A micronuclear version of the Rab gene was amplified by polymerase chain reaction (PCR). The macronuclear DNA molecule carrying the Rab gene is 767 bp long and shows characteristics typical of macronuclear chromosomes of hypotrichous ciliates. Three of the five cysteines are encoded by the opal codon UGA. The deduced protein is a 207-amino acid (aa) with a molecular mass of 23 kDa. The protein shares 36% identity with Rab 1 protein of Plasmodium and yeast. Analysis of the sequences indicated that the micronuclear version of the Rab gene contains two internal eliminated sequences, internal eliminated sequence (IES)1 and IES2. IES1 is flanked by a pair of hepta-nucleotide 5'-AAATTTT-3' direct repeats, and IES2 is flanked by 5'-TA-3' direct repeats.
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Affiliation(s)
- Wei Wang
- Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Shanxi University, Taiyuan, P.R. China
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33
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Meijer AJ, Codogno P. Regulation and role of autophagy in mammalian cells. Int J Biochem Cell Biol 2005; 36:2445-62. [PMID: 15325584 DOI: 10.1016/j.biocel.2004.02.002] [Citation(s) in RCA: 459] [Impact Index Per Article: 23.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2003] [Revised: 01/16/2004] [Accepted: 02/05/2004] [Indexed: 01/25/2023]
Abstract
The recent period has witnessed progress in the understanding of the lysosomal autophagic pathway. The discovery of a family of genes conserved from yeast to humans, and involved in the formation of autophagosomes, has unraveled new protein-conjugation systems and has shed light on the importance of autophagy in physiology and pathophysiology. The elucidation of the molecular control of autophagy will also lead to a better understanding of the role of autophagy during cell death. As a great number of extracellular stimuli (starvation, hormonal or therapeutic treatment) as well as intracellular stimuli (accumulation of misfolded proteins, invasion of microorganisms) is able to modulate the autophagic response, it is not surprising that several signaling pathways are involved in the control of autophagy. The mammalian Target of Rapamycin (mTOR) signaling pathway plays a major role in transmitting autophagic stimuli because of its ability to sense nutrient, metabolic and hormonal signals. In addition, autophagy, which is characterized by a flux of membrane from the formation of the autophagosome to the fusion with the lysosome, is regulated by GTPases, similarly to the vesicular transport along the exocytic/endocytic pathway. The aim of the present review is to give an overview of autophagy and to discuss its regulation by activators and effectors of mTOR and GTPases.
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Affiliation(s)
- Alfred J Meijer
- Department of Biochemistry, Academic Medical Center, University of Amsterdam, Meibergreef 15, 1105 AZ Amsterdam, The Netherlands
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34
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Abstract
Tyrosine phosphorylation is a fundamental mechanism for regulating the functions of numerous proteins in eukaryotic cells. It has been known for some time that several members of the Rab GTPase family can undergo phosphorylation on serine or threonine residues, but the potential for tyrosine phosphorylation has been appreciated only recently, based on a single example-Rab24. Herein we describe a series of straightforward methods to facilitate an initial assessment of the potential for tyrosine phosphorylation of epitope-tagged Rab proteins transiently expressed in mammalian cells. The approach takes advantage of the availability of highly specific monoclonal antibodies against phosphotyrosine and specific chemical inhibitors for tyrosine kinases. We also describe the use of site-directed mutagenesis to identify tyrosine residues that may be targets for phosphorylation, and we discuss the possible relevance of this modification for regulating Rab function.
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35
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Chen Y, Vallee S, Wu J, Vu D, Sondek J, Ghosh G. Inhibition of NF-kappaB activity by IkappaBbeta in association with kappaB-Ras. Mol Cell Biol 2004; 24:3048-56. [PMID: 15024091 PMCID: PMC371134 DOI: 10.1128/mcb.24.7.3048-3056.2004] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023] Open
Abstract
IkappaBbeta, one of the major IkappaB proteins, is only partially degraded in response to most extracellular signals. However, the molecular mechanism of this event is unknown. We show here that IkappaBbeta exists in at least two different forms: one that is bound to the NF-kappaB dimer and the other bound to both NF-kappaB and kappaB-Ras, a Ras-like small G protein. Removal of cellular kappaB-Ras enhances whereas excess kappaB-Ras blocks induced IkappaBbeta degradation. Remarkably, kappaB-Ras functions in both GDP- and GTP-bound states, and mutations of the conserved guanine-binding residues of kappaB-Ras abrogate its ability to block degradation of IkappaBbeta. kappaB-Ras also directly blocks the in vitro phosphorylation of IkappaBbeta by IKKbeta. These observations suggest that IkappaBbeta in the ternary complex is resistant to degradation by most signals. We suggest that specific signals, in addition to those that activate only IKK, are essential for the complete degradation of IkappaBbeta.
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Affiliation(s)
- Yi Chen
- Department of Chemistry and Biochemistry, University of California-San Diego, La Jolla, California 92093, USA
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Ding J, Soule G, Overmeyer JH, Maltese WA. Tyrosine phosphorylation of the Rab24 GTPase in cultured mammalian cells. Biochem Biophys Res Commun 2004; 312:670-5. [PMID: 14680817 DOI: 10.1016/j.bbrc.2003.10.171] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2003] [Indexed: 01/05/2023]
Abstract
Several members of the large family of Rab GTPases have been shown to function in vesicular trafficking in mammalian cells. However, the exact role of Rab24 remains poorly defined. Rab24 differs from other Rab proteins in that it has a low intrinsic GTPase activity and is not efficiently prenylated. Here we report an additional unique property of Rab24; i.e., the protein can undergo tyrosine phosphorylation when overexpressed in cultured cells. Immunoblot analyses with specific anti-phosphotyrosine monoclonal antibodies revealed the presence of phosphotyrosine (pTyr) on myc-Rab24 in whole cell lysates and immunoprecipitated samples. No pTyr was detected on other overexpressed myc-tagged GTPases (H-Ras, Rab1b, Rab6, Rab11 or Rab13). Comparisons of myc-Rab24 in the soluble and particulate fractions from HEK293 and HEp-2 cells indicated that the cytosolic pool of Rab24 was more heavily phosphorylated than the membrane pool. Treatment of transfected cells with the broad-spectrum tyrosine kinase inhibitor, genistein, as well as the specific Src-family kinase inhibitor, PP2, eliminated the pTyr signal from Rab24. In contrast the receptor tyrosine kinase inhibitor, tyrphostin A25, had no effect. Tyrosine phosphorylation of Rab24 was reduced by alanine substitution of two unique tyrosines, one found in a strong consensus phosphorylation motif (Y [Formula: see text] ) in the hypervariable domain (Y172) and the other falling within the GXXXGK(S/T) motif known as the P-loop (Y17). The latter region is known to influence GTP hydrolysis in Rab proteins, so the phosphorylation of Y17 could contribute to the low intrinsic GTPase activity of Rab24. This is the first report of tyrosine phosphorylation in any member of the Ras superfamily and it raises the possibility that this type of modification could influence Rab24 targeting and interactions with effector protein complexes.
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Affiliation(s)
- Jane Ding
- Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, Ohio 43614, USA
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37
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Nepomuceno-Silva JL, de Melo LDB, Mendonçã SM, Paixão JC, Lopes UG. RJLs: a new family of Ras-related GTP-binding proteins. Gene 2004; 327:221-32. [PMID: 14980719 DOI: 10.1016/j.gene.2003.11.010] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2003] [Revised: 10/30/2003] [Accepted: 11/14/2003] [Indexed: 10/26/2022]
Abstract
The Ras superfamily of GTP binding proteins encompasses several gene families that regulate a plethora of events in the eukaryotic cell. Here we describe a novel branch of this superfamily which we have named RJLs. These are present in many unicellular organisms and also in deuterostomes but apparently missing in some intermediary phyla, suggesting an intriguing possibility of lateral gene transference between lower and higher eukaryotes. RJLs lack classical membrane targeting signals and the conserved glutamine residue that coordinates GTP hydrolysis in other proteins from the Ras superfamily. Interestingly, chordate orthologues are chimeras fused to "J" domains in their C-terminal, suggesting that these proteins recruit Hsc70 to specific sites in the cell. Expression analysis of RJLs from chordates suggests predominant expression in nervous tissues, possibly reflecting a role for RJLs in the development or maintenance of the sophisticated chordate nervous system.
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Affiliation(s)
- José L Nepomuceno-Silva
- Instituto de Biofísica Carlos Chagas Filho, Universidades Federal do Rio de Janeiro, Rio de Janeiro, RJ 21949-900, Brazil
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Ogier-Denis E, Codogno P. Autophagy: a barrier or an adaptive response to cancer. BIOCHIMICA ET BIOPHYSICA ACTA 2003; 1603:113-28. [PMID: 12618311 DOI: 10.1016/s0304-419x(03)00004-0] [Citation(s) in RCA: 117] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Macroautophagy or autophagy is a degradative pathway terminating in the lysosomal compartment after the formation of a cytoplasmic vacuole that engulfs macromolecules and organelles. The recent discovery of the molecular controls of autophagy that are common to eukaryotic cells from yeast to human suggests that the role of autophagy in cell functioning is far beyond its nonselective degradative capacity. The involvement of proteins with properties of tumor suppressor and oncogenic properties at different steps of the pathway implies that autophagy must be considered in tumor progression. Autophagy as a stress response mechanism protects cancer cells from low nutrient supply or therapeutic insults. Autophagy is also involved in the elimination of cancer cells by triggering a non-apoptotic cell death program, suggesting a negative role in tumor development. These two aspects of autophagy will be discussed in this review.
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Affiliation(s)
- Eric Ogier-Denis
- INSERM U504 Glycobiologie et Signalisation cellulaire, Institut André Lwoff, 16 avenue Paul-Vaillant-Couturier, 94807 Villejuif Cedex, France
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Quevillon E, Spielmann T, Brahimi K, Chattopadhyay D, Yeramian E, Langsley G. The Plasmodium falciparum family of Rab GTPases. Gene 2003; 306:13-25. [PMID: 12657463 DOI: 10.1016/s0378-1119(03)00381-0] [Citation(s) in RCA: 71] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Rab GTPases are key regulators of vesicular traffic in eukaryotic cells. Here we sought a global characterization and description of the Plasmodium falciparum family of Rab GTPases. We used a combination of bioinformatic analyses, experimental testing of predictions, structure modelling and phylogenetics. These analyses led to the identification of seven new parasite Rabs. Accordingly we estimate that the P. falciparum family is made up of 11 genes. We show that ten members of this family are transcribed in infected erythrocytes. Concerning the various members of the family, a series of specific as well as global conclusions can be drawn. Rabs predicted to be compartment-specific show different subcellular distributions. This is demonstrated for PfRab1A and PfRab11A, with the generation of specific antisera. The sequence analyses reveal several peculiarities, with possible functional implications. One of the transcribed genes, Pfrab5b, does not encode a classical C-terminus, suggestive of a novel regulatory role for this GTPase. Another, Pfrab5a, previously identified as a rab gene located on chromosome 2, possesses a 30-amino-acid insertion in its GTP-binding domain. Structural considerations suggest that this insertion could represent a novel interaction interface. We used conserved RabF and RabSF motifs to discriminate between specific parasite Rabs, and followed their predicted change in position on the structure of PfRab6, as GTP is hydrolysed to GDP. This allowed us to propose their involvement in potential interaction surfaces, that we extended to human Rab6 and the motifs known to mediate Rabkinesine-6 binding. Finally, we compared the P. falciparum Rab family to those of Saccharomyces cerevisiae and Schizosaccharomyces pombe and found that parasite Rabs segregate into possible functional clads. Such grouping into clads may give clues to parasite Rab function, and may shed light on P. falciparum secretory/endocytic pathways.
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Affiliation(s)
- Emmanuel Quevillon
- Laboratoire de Signalisation Immunoparasitaire, URA CNRS 1960, Department of Parasitology, Institut Pasteur, Paris, France
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Lee M, Kwon J, Kim SN, Kim JE, Koh WS, Kim EJ, Chung MK, Han SS, Song CW. cDNA microarray gene expression profiling of hydroxyurea, paclitaxel, and p-anisidine, genotoxic compounds with differing tumorigenicity results. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2003; 42:91-97. [PMID: 12929121 DOI: 10.1002/em.10177] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/24/2023]
Abstract
The potential application of toxicogenomics to predictive toxicology has been discussed widely, but the utility of the approach remains largely unproven. Using cDNA microarrays, we compared the gene expression profiles produced in mouse lymphoma cells by three genotoxic compounds, hydroxyurea (a carcinogen), p-anisidine (a noncarcinogen), and paclitaxel (carcinogenicity unknown). To minimize the effect of biological variability and technological limitations, quadruplicate observations were made for each compound and a subset of genes yielding reproducible induction/repression was selected for comparison. A method was applied to attach normalized expression data to genes with a low false-discovery rate (<0.1) to yield more confidence regarding differential expression. This analysis identified genotoxicity-specific gene expression. Seven genes were consistently upregulated and 12 downregulated more than 2-fold by the three genotoxic compounds. Using additional genes, the expression pattern induced by the genotoxic noncarcinogen, p-anisidine, was readily distinguished from that associated with the genotoxic carcinogen, hydroxyurea. Comparison of paclitaxel-induced expression data to data for p-anisidine and hydroxyurea suggested that paclitaxel's profile is more similar to the genotoxic noncarcinogen. With further supporting evidence it may be possible to perform large-scale monitoring of gene expression during drug and chemical development that can provide an early warning of potential toxicological responses.
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Affiliation(s)
- Michael Lee
- Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, Yusong, Daejeon, Republic of Korea.
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Petiot A, Pattingre S, Arico S, Meley D, Codogno P. Diversity of signaling controls of macroautophagy in mammalian cells. Cell Struct Funct 2002; 27:431-41. [PMID: 12576636 DOI: 10.1247/csf.27.431] [Citation(s) in RCA: 78] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
Macroautophagy is a major lysosomal catabolic process conserved from yeast to human. The formation of autophagic vacuoles is stimulated by a variety of intracellular and extracellular stress situations including amino acid starvation, aggregation of misfolded proteins, and accumulation of damaged organelles. Several signaling pathways control the formation of autophagic vacuoles. As some of them are engaged in the control of protein synthesis or cell survival this suggests that macroautophagy is intimately associated with the execution of cell proliferation and cell death programs. Whether or not these different signaling pathways converge to a unique point to trigger the formation of autophagic vacuole remains an open question.
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Affiliation(s)
- Anne Petiot
- Department of Biochemistry, University of Geneva, Science II, 30 quai Ernest Ansermet, 1211 Geneva, Switzerland
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42
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Maltese WA, Soule G, Gunning W, Calomeni E, Alexander B. Mutant Rab24 GTPase is targeted to nuclear inclusions. BMC Cell Biol 2002; 3:25. [PMID: 12323076 PMCID: PMC130051 DOI: 10.1186/1471-2121-3-25] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2002] [Accepted: 09/25/2002] [Indexed: 11/30/2022] Open
Abstract
BACKGROUND Members of the Rab GTPase family regulate intracellular protein trafficking, but the specific function of Rab24 remains unknown. Several attributes distinguish this protein from other members of the Rab family, including a low intrinsic GTPase activity. RESULTS The functions of other Rab proteins have been defined through the use of dominant-negative mutants with amino acid substitutions in the conserved N(T)KxD nucleotide binding motif. Surprisingly, when such Rab24 constructs were expressed in cultured cells, they accumulated in nuclear inclusions which disrupted the integrity of the nuclear envelope. The inclusions reacted positively with antibodies against ubiquitin and Hsp70, similar to protein aggregates observed in polyglutamine disorders. They also appeared to sequester importin-beta and GFP-coupled glucocorticoid receptor. Other Rab GTPases with similar mutations in the N(T)KxD motif were never found in inclusions, suggesting that the unusual localization of Rab24 is not related solely to misfolding of its nucleotide-free form. Studies with Rab24/Rab1B chimeras indicated that targeting of the mutant protein to inclusions requires the unique C-terminal domain of Rab24. CONCLUSION These studies demonstrate that mutations in Rab24 can trigger a cytopathic cellular response involving accumulation of nuclear inclusions. If the N(T)KxD mutants of Rab24 function as dominant suppressors, these studies may point to a unique role for Rab24 in degradation of misfolded cellular proteins or trafficking of proteins to the nuclear envelope. However, we cannot yet eliminate the possibility that these phenomena are related to unusual non-physiological protein interactions with the mutant form of Rab24.
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Affiliation(s)
- William A Maltese
- Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, OH 43614, USA
| | - Gwendolyn Soule
- Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, OH 43614, USA
| | - William Gunning
- Department of Pathology, Medical College of Ohio, Toledo, OH 43614, USA
| | - Edward Calomeni
- Department of Pathology, Medical College of Ohio, Toledo, OH 43614, USA
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Munafó DB, Colombo MI. Induction of autophagy causes dramatic changes in the subcellular distribution of GFP-Rab24. Traffic 2002; 3:472-82. [PMID: 12047555 DOI: 10.1034/j.1600-0854.2002.30704.x] [Citation(s) in RCA: 150] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Rab GTPases comprises a large family of proteins, with more than 50 gene products localized in distinct subcellular compartments. Rab24 is a member of this family whose function is not presently known. In order to elucidate the role of this protein we have generated a GFP-tagged Rab24 and studied the distribution of this chimera by fluorescence microscopy. GFP-Rab24 showed a perinuclear reticular localization that often encircled the nucleus. This reticular pattern partially overlapped with ER markers, cis-Golgi, and the ER-Golgi intermediate compartment. Surprisingly, when GFP-Rab24-transfected cells were starved to induce autophagy the distribution of the protein changed dramatically. GFP-Rab24 localized in large dots, cup-shaped structures and ring-shaped vesicles. Some of these vesicles were labeled with monodansylcadaverine, a specific autophagosome marker. In the presence of vinblastine, an agent that induces the formation of very large autophagic vesicles, GFP-Rab24 accumulated in the large vacuoles that were also labeled by monodansylcadaverine. Furthermore, Rab24 colocalized with LC3, a mammalian homolog of the yeast protein Apg8/Aut7, an essential gene for autophagy. This is the first report indicating that Rab24 localizes on autophagosomes, suggesting that this Rab protein is involved in the autophagic pathway.
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Affiliation(s)
- Daniela B Munafó
- Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, CONICET, Mendoza 5500, Argentina
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He H, Dai F, Yu L, She X, Zhao Y, Jiang J, Chen X, Zhao S. Identification and characterization of nine novel human small GTPases showing variable expressions in liver cancer tissues. Gene Expr 2002; 10:231-42. [PMID: 12450215 PMCID: PMC5977521 DOI: 10.3727/000000002783992406] [Citation(s) in RCA: 104] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/29/2002] [Indexed: 11/24/2022]
Abstract
Digestion and detoxification are the two most important functions of the liver, and liver cells always keep a high metabolism level and active vesicular traffic. The malfunction of the vesicular traffic system might be a cause of the abnormal biological behavior of cancerous liver cells. The Ras superfamily is known to regulate various steps of vesicular traffic in eukaryotic cells. It would be significant to determine the change of vesicular transport molecules such as the members of Ras superfamily in carcinogenesis of liver cells. In the present study, we have cloned nine novel genes encoding human small GTPases: RAB1B, RAB4B, RAB10, RAB22A, RAB24, RAB25 ARL5, SARA1, and SARA2, among which the former six belong to the RAB family and the latter three belong to the ARF/SAR1 family. The identification of these new genes has greatly enlarged the pool of the Ras superfamily. It is interesting to find that they are upregulated in most of the 11 hepatocellular carcinoma and 1 cholangiohepatoma cases. Furthermore, the expression in 16 normal human adult tissues, the chromosome loci, and the gene structures of the nine genes are also described. The above findings could be valuable for understanding the vesicular transport system and elucidating the molecular basis of liver cancer carcinogenesis.
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Affiliation(s)
- Hua He
- State Key Laboratory of Genetic Engineering, Group of Liver Cancer Research, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, P.R. China
| | - Fangyan Dai
- State Key Laboratory of Genetic Engineering, Group of Liver Cancer Research, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, P.R. China
| | - Long Yu
- State Key Laboratory of Genetic Engineering, Group of Liver Cancer Research, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, P.R. China
- Address correspondence to Prof. Long Yu, Institute of Genetics, Fudan University, 220 Handan Road, Shanghai 200433, P.R. China. Tel: 86-21-65642422; Fax: 86-21-65643250; E-mail:
| | - Xingyu She
- State Key Laboratory of Genetic Engineering, Group of Liver Cancer Research, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, P.R. China
| | - Yong Zhao
- State Key Laboratory of Genetic Engineering, Group of Liver Cancer Research, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, P.R. China
| | - Jianmin Jiang
- State Key Laboratory of Genetic Engineering, Group of Liver Cancer Research, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, P.R. China
| | - Xiaosong Chen
- State Key Laboratory of Genetic Engineering, Group of Liver Cancer Research, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, P.R. China
| | - Shouyuan Zhao
- State Key Laboratory of Genetic Engineering, Group of Liver Cancer Research, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, P.R. China
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Abstract
Rab proteins are small GTP-binding proteins that form the largest family within the Ras superfamily. Rab proteins regulate vesicular trafficking pathways, behaving as membrane-associated molecular switches. Here, we have identified the complete Rab families in the Caenorhabditis elegans (29 members), Drosophila melanogaster (29), Homo sapiens (60) and Arabidopsis thaliana (57), and we defined criteria for annotation of this protein family in each organism. We studied sequence conservation patterns and observed that the RabF motifs and the RabSF regions previously described in mammalian Rabs are conserved across species. This is consistent with conserved recognition mechanisms by general regulators and specific effectors. We used phylogenetic analysis and other approaches to reconstruct the multiplication of the Rab family and observed that this family shows a strict phylogeny of function as opposed to a phylogeny of species. Furthermore, we observed that Rabs co-segregating in phylogenetic trees show a pattern of similar cellular localisation and/or function. Therefore, animal and fungi Rab proteins can be grouped in "Rab functional groups" according to their segregating patterns in phylogenetic trees. These functional groups reflect similarity of sequence, localisation and/or function, and may also represent shared ancestry. Rab functional groups can help the understanding of the functional evolution of the Rab family in particular and vesicular transport in general, and may be used to predict general functions for novel Rab sequences.
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Affiliation(s)
- J B Pereira-Leal
- Cell and Molecular Biology Section, Division of Biomedical Sciences, Faculty of Medicine, Imperial College, London, SW7 2AZ, UK
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Overmeyer JH, Wilson AL, Maltese WA. Membrane targeting of a Rab GTPase that fails to associate with Rab escort protein (REP) or guanine nucleotide dissociation inhibitor (GDI). J Biol Chem 2001; 276:20379-86. [PMID: 11389151 DOI: 10.1074/jbc.m101511200] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The targeting of various Rab proteins to different subcellular compartments appears to be determined by variable amino acid sequences located upstream from geranylgeranylated cysteine residues in the C-terminal tail. All nascent Rab proteins are prenylated by geranylgeranyltransferase II, which recognizes the Rab substrate only when it is bound to Rab escort protein (REP). After prenylation, REP remains associated with the modified Rab until it is delivered to the appropriate subcellular membrane. It remains unclear whether docking of the Rab with the correct membrane is solely a function of features contained within the prenylated Rab itself (with REP serving as a "passive" carrier) or whether REP actively participates in the targeting process. To address this issue, we took advantage of a mutation in the alpha2 helix of Rab1B (i.e. Y78D) that abolishes REP and GDI interaction without disrupting nucleotide binding or hydrolysis. These studies demonstrate that replacing the C-terminal GGCC residues of Rab1B(Y78D) with a CLLL motif permits this protein to be prenylated by geranylgeranyltransferase I but not II both in cell-free enzyme assays and in transfected cells. Subcellular fractionation and immunofluorescence studies reveal that the prenylated Rab1B(Y78D)CLLL, which remains deficient in REP and GDI association is, nonetheless, delivered to the Golgi and endoplasmic reticulum (ER) membranes. When the dominant-negative S22N mutation was inserted into Rab1B-CLLL, the resulting monoprenylated construct suppressed ER --> Golgi protein transport. However, when the Y78D mutation was added to the latter construct, its inhibitory effect on protein trafficking was lost despite the fact that it was localized to the ER/Golgi membrane. Therefore, protein interactions mediated by the alpha2 helical domain of Rab1B(S22N) appear to be essential for its functional interaction with components of the ER --> Golgi transport machinery.
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Affiliation(s)
- J H Overmeyer
- Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, Ohio 43614-5804, USA
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Erdman RA, Maltese WA. Different Rab GTPases associate preferentially with alpha or beta GDP-dissociation inhibitors. Biochem Biophys Res Commun 2001; 282:4-9. [PMID: 11263962 DOI: 10.1006/bbrc.2001.4560] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
GDIs (GDP-dissociation inhibitors) bind to Rab GTPases and mediate their membrane targeting and recycling. In vitro, most Rabs can bind to either of the major isoforms of GDI, leading to the assumption that the proportion of each specific Rab/GDI complex in vivo reflects the relative abundance of the alpha versus beta forms of GDI. Here we show that when human teratocarcinoma cells (Ntera2) are induced to differentiate into postmitotic neurons (NT2N), there is a major change in the proportion of GDIalpha relative to GDIbeta. Under these conditions, certain Rab GTPases associate preferentially with either GDIalpha or GDIbeta, irrespective of the relative abundance of the GDI isoform. These findings suggest that heretofore unrecognized functional specificity may exist between the two major forms of GDI.
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Affiliation(s)
- R A Erdman
- Weis Center for Research, Penn State College of Medicine, Danville, Pennsylvania, 17822, USA
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Pereira-Leal JB, Seabra MC. The mammalian Rab family of small GTPases: definition of family and subfamily sequence motifs suggests a mechanism for functional specificity in the Ras superfamily. J Mol Biol 2000; 301:1077-87. [PMID: 10966806 DOI: 10.1006/jmbi.2000.4010] [Citation(s) in RCA: 349] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The Rab/Ypt/Sec4 family forms the largest branch of the Ras superfamily of GTPases, acting as essential regulators of vesicular transport pathways. We used the large amount of information in the databases to analyse the mammalian Rab family. We defined Rab-conserved sequences that we designate Rab family (RabF) motifs using the conserved PM and G motifs as "landmarks". The Rab-specific regions were used to identify new Rab proteins in the databases and suggest rules for nomenclature. Surprisingly, we find that RabF regions cluster in and around switch I and switch II regions, i.e. the regions that change conformation upon GDP or GTP binding. This finding suggests that specificity of Rab-effector interaction cannot be conferred solely through the switch regions as is usually inferred. Instead, we propose a model whereby an effector binds to RabF (switch) regions to discriminate between nucleotide-bound states and simultaneously to other regions that confer specificity to the interaction, possibly Rab subfamily (RabSF) specific regions that we also define here. We discuss structural and functional data that support this model and its general applicability to the Ras superfamily of proteins.
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Affiliation(s)
- J B Pereira-Leal
- Molecular Genetics Section Division of Biomedical Sciences, Imperial College School of Medicine, Sir Alexander Fleming Building, London, SW7 2AZ, UK
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Stickney JT, Buss JE. Murine guanylate-binding protein: incomplete geranylgeranyl isoprenoid modification of an interferon-gamma-inducible guanosine triphosphate-binding protein. Mol Biol Cell 2000; 11:2191-200. [PMID: 10888661 PMCID: PMC14912 DOI: 10.1091/mbc.11.7.2191] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
Farnesylation of Ras proteins is necessary for transforming activity. Although farnesyl transferase inhibitors show promise as anticancer agents, prenylation of the most commonly mutated Ras isoform, K-Ras4B, is difficult to prevent because K-Ras4B can be alternatively modified with geranylgeranyl (C20). Little is known of the mechanisms that produce incomplete or inappropriate prenylation. Among non-Ras proteins with CaaX motifs, murine guanylate-binding protein (mGBP1) was conspicuous for its unusually low incorporation of [(3)H]mevalonate. Possible problems in cellular isoprenoid metabolism or prenyl transferase activity were investigated, but none that caused this defect was identified, implying that the poor labeling actually represented incomplete prenylation of mGBP1 itself. Mutagenesis indicated that the last 18 residues of mGBP1 severely limited C20 incorporation but, surprisingly, were compatible with farnesyl modification. Features leading to the expression of mutant GBPs with partial isoprenoid modification were identified. The results demonstrate that it is possible to alter a protein's prenylation state in a living cell so that graded effects of isoprenoid on function can be studied. The C20-selective impairment in prenylation also identifies mGBP1 as an important model for the study of substrate/geranylgeranyl transferase I interactions.
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Affiliation(s)
- J T Stickney
- Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA
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