Cancer and cardiovascular diseases (CVDs) are global health challenges. In cancer patients, CVD is the second leading cause of death following disease progression. There are few specialized services for cardio-oncology patients worldwide currently. Branched-chain amino acids (BCAAs) are essential amino acids that promote protein synthesis and energy homeostasis. The disruption of BCAAs metabolism facilitates the development of cancer and CVDs while the benefit of BCAA supplement is full of controversy. In this review, we summarized BCAA-related studies in cardiometabolism, cancer and chemotherapy-induced cardiotoxicity, and provided our perspectives on the roles of BCAAs in cardio-oncology. We find that supplementation of BCAAs presents protective effects in cardiometabolic diseases, while the influence on cancer is intricate and varies across different types of cancers. Large-scale clinical studies are needed to understand the long-term effects of BCAA intake and its impact on different stages of the disease. BCAAs have potential to mitigate chemotherapy-induced cardiotoxicity. Continued research is still essential to understand the precise mechanisms, determine optimal dosage and timing, and assess the effectiveness of BCAA supplement in cardio-oncology, in particular clinical research.

The mechanistic target of rapamycin complex 1 (mTORC1) is a crucial nutrient sensor and a major regulator of cell growth and proliferation. While mTORC1 activity is frequently upregulated in cancer, the mechanisms regulating mTORC1 are not fully understood. POLR1D, a shared subunit of RNA polymerases I and III, is often upregulated in colorectal cancer (CRC) and mutated in Treacher-Collins syndrome. POLR1D, together with its binding partner POLR1C, forms a dimer that is believed to initiate the assembly of the multisubunit RNA polymerases I and III. Our data reveal an unexpected link between POLR1D and mTORC1 signalling. We found that the overproduction of POLR1D in human cells stimulates mTORC1 activity. In contrast, the downregulation of POLR1D leads to the repression of the mTORC1 pathway. Additionally, we demonstrate that a pool of POLR1D localises to the cytoplasm and interacts with the mTORC1 regulator RAGA and RAPTOR. Furthermore, POLR1D enhances the interaction between RAPTOR and RAGA and sustains mTORC1 activity under starvation conditions. We have identified a novel role for the RNA polymerase I/III subunit POLR1D in regulating mTORC1 signalling. Our findings suggest the existence of a new node in the already complex mTORC1 signalling network, where POLR1D functions to convey the cell's internal status, namely polymerase assembly, to this kinase.

Amino acids are organic compounds that serve as the building blocks of proteins and peptides. Additionally, they function as bioactive molecules that play important roles in metabolic regulation and signal transduction. The ability of cells to sense fluctuations in intracellular and extracellular amino acid levels is vital for effectively regulating protein synthesis and catabolism, maintaining homeostasis, adapting to diverse nutritional environments and influencing cell fate decision. In this review, the recent molecular insights into amino acids sensing are discussed, along with the different sensing mechanisms in distinct organisms.

Herpesviruses require membrane fusion for entry and spread, a process facilitated by the fusion glycoprotein B (gB) and the regulatory factor gH/gL. The human cytomegalovirus (HCMV) gH/gL can be modified by the accessory protein gO, or the set of proteins UL128, UL130, and UL131. While the binding of the gH/gL/gO and gH/gL/UL128-131 complexes to cellular receptors, including PDGFRα and NRP2, has been well-characterized structurally, the specific role of receptor engagements by the gH/gL/gO and gH/gL/UL128-131 in regulation of fusion has remained unclear. We describe a cell-cell fusion assay that can quantitatively measure fusion on a timescale of minutes and demonstrate that binding of gH/gL/gO to PDGFRα dramatically enhances gB-mediated cell-cell fusion. In contrast, gH/gL/pUL128-131-regulated fusion is significantly slower, and gH/gL alone cannot promote gB fusion activity within this timescale. The genetic diversity of gO influenced the observed cell-cell fusion rates, correlating with previously reported effects on HCMV infectivity. Mutations in gL that had no effect on the formation of gH/gL/gO or binding to PDGFRa dramatically reduced the cell-cell fusion rate, suggesting that gL plays a critical role in linking the gH/gL/gO-PDGFRa receptor binding to activation of gB. Several neutralizing human monoclonal antibodies were found to potently block gH/gL/gO-PDGFRa-regulated cell-cell fusion, suggesting this mechanism as a therapeutic target.

Development of vaccines and therapeutics targeting the fusion apparatus of human cytomegalovirus (HCMV) has been limited by the lack of an in vitro cell-cell fusion assay that faithfully models the receptor-dependent fusion characteristic of HCMV entry. The cell-cell fusion assay described here demonstrated that the binding of gH/gL/gO to its receptor, PDGFRα, serves to regulate the activity of the fusion protein gB, and this is specifically vulnerable to inhibition by neutralizing antibodies. Moreover, the measurement of fusion kinetics allows for mutational studies of the fusion mechanism, assessing the influence of genetic diversity among the viral glycoproteins and studying the mechanism of neutralizing antibodies.

To become infectious, assembling enveloped viruses must acquire viral glycoproteins to mediate downstream infection events. Human immunodeficiency virus-1 (HIV-1) envelope glycoproteins (Env) are well characterized to function as trimers for membrane fusion and entry; however, we sought to understand whether the trimeric structure of Env is required for incorporation into virus particles. Using superresolution live-cell imaging and biochemical assays, we demonstrate that a monomeric receptor chimera containing the Env cytoplasmic tail (Env-CT), known to regulate Env incorporation, is sufficient for lattice trapping and incorporation into virus assembly sites. We also demonstrate that these Env-CT monomers can restrict the incorporation of native Env trimers, competing for an apparently limited number of interaction sites in each assembling particle. Furthermore, this monomeric construct can restrict the incorporation of Env glycoproteins from an evolutionarily distant HIV-1 primary isolate. Our findings support a model where a monomeric Env-CT mediates Env incorporation, with this mechanism of Env incorporation being conserved between distant clades of HIV-1.IMPORTANCETo combat the prevalence of HIV-1 and antiviral resistance, new classes of antivirals are needed. An attractive target for new classes includes virus assembly because released virus particles unable to obtain Env glycoproteins are non-infectious and unable to propagate HIV-1 infection. One requisite to the development of an antiviral targeting Gag-Env coalescence is the need to define the functional units constituting this molecular interface. Although Env functions as an obligatory trimer for virus entry, we demonstrate that a monomeric Env-CT is sufficient for Env incorporation into HIV-1 particles. Monomeric Env-CT displayed saturability in viral lattices and the ability to compete with native Env trimers for particle incorporation. These results suggest a less complex Env-CT structure mediates virus incorporation and that Env-CT mimetics could yield broad competitive activity against HIV-1 infection.

Signals from the microenvironment are known to be critical for development, stem cell self-renewal and oncogenic progression. Although some niche-driven signals that promote cancer progression have been identified1-5, concerted efforts to map disease-relevant microenvironmental ligands of cancer stem cell receptors have been lacking. Here, we use temporal single-cell RNA-sequencing (scRNA-seq) to identify molecular cues from the bone marrow stromal niche that engage leukaemia stem-enriched cells (LSCs) during oncogenic progression. We integrate these data with our human LSC RNA-seq and in vivo CRISPR screen of LSC dependencies6 to identify LSC-niche interactions that are essential for leukaemogenesis. These analyses identify the taurine-taurine transporter (TAUT) axis as a critical dependency of aggressive myeloid leukaemias. We find that cysteine dioxygenase type 1 (CDO1)-driven taurine biosynthesis is restricted to osteolineage cells, and increases during myeloid disease progression. Blocking CDO1 expression in osteolineage cells impairs LSC growth and improves survival outcomes. Using TAUT genetic loss-of-function mouse models and patient-derived acute myeloid leukaemia (AML) cells, we show that TAUT inhibition significantly impairs in vivo myeloid leukaemia progression. Consistent with elevated TAUT expression in venetoclax-resistant AML, TAUT inhibition synergizes with venetoclax to block the growth of primary human AML cells. Mechanistically, our multiomic approaches indicate that the loss of taurine uptake inhibits RAG-GTP dependent mTOR activation and downstream glycolysis. Collectively, our work establishes the temporal landscape of stromal signals during leukaemia progression and identifies taurine as a key regulator of myeloid malignancies.

Alterations in mitochondrial metabolism in obesity may indicate disrupted communication between mitochondria and nucleus, and DNA methylation may influence this interplay. Here, we leverage data from the Finnish Twin Cohort study subcohort (n = 173; 86 full twin pairs, 1 singleton), including comprehensive measurements of obesity-related outcomes, mitochondrial DNA quantity and nuclear DNA methylation levels in adipose and muscle tissue, to identify one CpG at SH3BP4 significantly associated with mitochondrial DNA quantity in adipose tissue (FDR < 0.05). We also show that SH3BP4 methylation correlates with its gene expression. Additionally, we find that 14 out of the 35 obesity-related traits display significant associations with both SH3BP4 methylation and mitochondrial DNA quantity in adipose tissue. We use data from TwinsUK and the Scandinavian T2D-discordant monozygotic twin cohort, to validate the observed associations. Further analysis using ICE FALCON suggests that mitochondrial DNA quantity, insulin sensitivity and certain body fat measures are causal to SH3BP4 methylation. Examining mitochondrial DNA quantity and obesity-related traits suggests causation from mitochondrial DNA quantity to obesity, but unmeasured within-individual confounding cannot be ruled out. Our findings underscore the impact of mitochondrial DNA quantity on DNA methylation and expression of the SH3BP4 gene within adipose tissue, with potential implications for obesity.

Sestrins (SESN1-3) are a family of stress-responsive proteins that regulate cellular metabolism and redox balance, both of which are frequently disrupted in cancer. As direct targets of stress-responsive transcription factors, including tumour suppressor p53, Sestrins function as leucine-dependent inhibitors of mTORC1 and potent antioxidants. Their downregulation is widely observed across multiple cancers and is associated with increased tumour growth and poor prognosis. Despite their consistent tumour-suppressive effects through mTORC1 inhibition and promotion of p53-dependent apoptosis, Sestrins exhibit a limited role in tumour initiation, which appears to be context-dependent. Their antioxidant activity reduces oxidative damage, thereby protecting against genomic instability and other cancer-promoting events. However, in certain contexts, Sestrins may promote tumour survival and progression by stimulating pro-survival pathways, such as AKT signalling through mTORC2 activation. This review examines the molecular mechanisms underlying these dual functions, with a particular focus on mTOR signalling and oxidative stress. We also discuss Sestrin expression patterns and functional outcomes in various cancer types, including lung, liver, colon, skin, prostate, and follicular lymphomas, highlighting their potential as diagnostic markers and therapeutic targets.

Amino acid homeostasis is essential for cellular functions such as growth, metabolism, and signaling. In budding yeast Saccharomyces cerevisiae, the General Amino Acid Control (GAAC) and Target of Rapamycin Complex 1 (TORC1) pathways are utilized for intracellular amino acid sensing, while the Ssy1-Ptr3-Ssy5 (SPS) pathway is used for extracellular sensing. These pathways maintain homeostasis by responding to variations in amino acid levels to regulate amino acid biosynthesis and uptake. However, their interactions under various conditions and behavior at single-cell resolution remain insufficiently understood. We developed fluorescent transcriptional reporters to monitor amino acid biosynthesis and uptake pathways in single cells, revealing pathway engagement in response to different amino acid levels and types. Inhibition experiments demonstrated that the SPS pathway influences TORC1 and GAAC activities differently. Additionally, pathway engagement varied between liquid culture and colony environments. In colonies, some cells specialized in either amino acid synthesis or uptake. Disruption of the SPS pathway hindered this specialization and increased cell death rates in aging colonies, indicating a role for metabolic differentiation in maintaining colony viability. Collectively, this study introduces a new tool for exploring cellular amino acid homeostasis and highlights the importance of cellular differentiation in amino acid control for colony survival.

Cholesterol plays a pivotal role in modulating the activity of mechanistic target of rapamycin complex 1 (mTOR1), thereby regulating cell growth and metabolic homeostasis. LYCHOS, a lysosome-localized G-protein-coupled receptor-like protein, emerges as a cholesterol sensor and is capable of transducing the cholesterol signal to affect the mTORC1 function. However, the precise mechanism by which LYCHOS recognizes cholesterol remains unknown. Here, using cryo-electron microscopy, we determined the three-dimensional structural architecture of LYCHOS in complex with cholesterol molecules, revealing a unique arrangement of two sequential structural domains. Through a comprehensive analysis of this structure, we elucidated the specific structural features of these two domains and their collaborative role in the process of cholesterol recognition by LYCHOS.

The tuberous sclerosis complex (TSC1/TSC2/TBC1D7) primarily functions to inhibit the mechanistic target of rapamycin complex 1 (mTORC1), a crucial regulator of cell growth. Mutations in TSC1 or TSC2 cause tuberous sclerosis complex (TSC), a rare autosomal dominant genetic disorder marked by benign tumors in multiple organs that rarely progress to malignancy. Traditionally, TSC proteins are considered tumor suppressive due to their inhibition of mTORC1 and other mechanisms. However, more recent studies have shown that TSC proteins can also promote tumorigenesis in certain cancer types. In this review, we explore the composition and function of the TSC protein complex, the roles of its individual components in cancer biology, and potential future therapeutic targeting strategies.

Transplant recipients require lifelong, multimodal immunosuppression to prevent rejection by reducing alloreactive immunity. Rapamycin is known to modulate adaptive and innate immunity, but its full mechanism remains incompletely understood. We investigated the understudied effects of rapamycin on lymph node (LN) architecture, leukocyte trafficking, and gut microbiome and metabolism after 3 (early), 7 (intermediate), and 30 (late) days of rapamycin treatment. Rapamycin significantly reduced CD4+ T cells, CD8+ T cells, and Tregs in peripheral LNs, mesenteric LNs, and spleen. Rapamycin induced early proinflammation transition to protolerogenic status by modulating the LN laminin α4/α5 expression ratios (La4/La5) through LN stromal cells, laminin α5 expression, and adjustment of Treg numbers and distribution. Additionally, rapamycin shifted the Bacteroides/Firmicutes ratio and increased amino acid bioavailability in the gut lumen. These effects were evident by 7 days and became most pronounced by 30 days in naive mice, with changes as early as 3 days in allogeneic splenocyte-stimulated mice. These findings reveal what we believe to be a novel mechanism of rapamycin action through time-dependent modulation of LN architecture and gut microbiome, which orchestrates changes in immune cell trafficking, providing a framework for understanding and optimizing immunosuppressive therapies.

The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway is frequently hyperactivated in triple-negative breast cancers (TNBCs) associated with poor prognosis and is a therapeutic target in breast cancer management. Here, we describe the effects of repression of mTOR-containing complex 1 (mTORC1) through knockdown of several key mTORC1 components or with mTOR inhibitors used in cancer therapy. mTORC1 repression results in an ∼10-fold increase in extracellular matrix proteolytic degradation. Repression in several TNBC models, including in patient-derived xenografts (PDXs), induces nuclear translocation of transcription factor EB (TFEB), which drives a transcriptional program that controls endolysosome function and exocytosis. This response triggers a surge in endolysosomal recycling and the surface exposure of membrane type 1 matrix metalloproteinase (MT1-MMP) associated with invadopodia hyperfunctionality. Furthermore, repression of mTORC1 results in a basal-like breast cancer cell phenotype and disruption of ductal carcinoma in situ (DCIS)-like organization in a tumor xenograft model. Altogether, our data call for revaluation of mTOR inhibitors in breast cancer therapy.

Serendipity plays a huge role in science, and having a prepared mind that can seize upon a chance observation or occurrence can drive a project forward. This happened in my lab with a project centered on the regulation of trophoblast cell behavior at implantation. We discovered that amino acids regulate the onset of trophoblast motility through the activation of the kinase complex mTORC1, and that this acts as a checkpoint to trophoblast differentiation. This finding not only broadened our understanding of the mechanisms underlying embryo implantation, but also provided new ways of thinking about the regulation of diapause, a state of suspended embryonic development that occurs in many species. I should say that we re-discovered the fact that amino acids regulate the onset of trophoblast motility, as reading the literature showed us that others had made this same observation some 30 years previously and we were fortuitously able to build upon those findings. This project confirmed to me how valuable it is to read the literature widely, both historical papers and those in fields outside one's area of research, and to go to seminars on topics outside one's area.

Macropinocytosis is a nonselective form of endocytosis that allows cancer cells to largely take up the extracellular fluid and its contents, including nutrients, growth factors, etc. We first elaborate meticulously on the process of macropinocytosis. Only by thoroughly understanding this entire process can we devise targeted strategies against it. We then focus on the central role of the MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) in regulating macropinocytosis, highlighting its significance as a key signaling hub where various pathways converge to control nutrient uptake and metabolic processes. The article covers a comprehensive analysis of the literature on the molecular mechanisms governing macropinocytosis, including the initiation, maturation, and recycling of macropinosomes, with an emphasis on how these processes are hijacked by cancer cells to sustain their growth. Key discussions include the potential therapeutic strategies targeting macropinocytosis, such as enhancing drug delivery via this pathway, inhibiting macropinocytosis to starve cancer cells, blocking the degradation and recycling of macropinosomes, and inducing methuosis - a form of cell death triggered by excessive macropinocytosis. Targeting macropinocytosis represents a novel and innovative approach that could significantly advance the treatment of cancers that rely on this pathway for survival. Through continuous research and innovation, we look forward to developing more effective and safer anti-cancer therapies that will bring new hope to patients.Abbreviation: AMPK: AMP-activated protein kinase; ASOs: antisense oligonucleotides; CAD: carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase; DC: dendritic cell; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; ERBB2: erb-b2 receptor tyrosine kinase 2; ESCRT: endosomal sorting complex required for transport; GAP: GTPase-activating protein; GEF: guanine nucleotide exchange factor; GRB2: growth factor receptor bound protein 2; LPP: lipopolyplex; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; MTORC2: mechanistic target of rapamycin kinase complex 2; NSCLC: non-small cell lung cancer; PADC: pancreatic ductal adenocarcinoma; PDPK1: 3-phosphoinositide dependent protein kinase 1; PI3K: phosphoinositide 3-kinase; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns(3,4,5)P3: phosphatidylinositol-(3,4,5)-trisphosphate; PtdIns(4,5)P2: phosphatidylinositol-(4,5)-bisphosphate; PTT: photothermal therapies; RAC1: Rac family small GTPase 1; RPS6: ribosomal protein S6; RPS6KB1: ribosomal protein S6 kinase B1; RTKs: receptor tyrosine kinases; SREBF: sterol regulatory element binding transcription factor; TFEB: transcription factor EB; TNBC: triple-negative breast cancer; TSC2: TSC complex subunit 2; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system.

Activating mutations in STAT6 are common in Follicular Lymphoma (FL) and transformed FL and various other B cell lymphomas. Here, we report RNA-seq based gene expression data on normal human lymph node derived B lymphocytes (NBC; N = 6), and primary human FL WT (N = 11) or mutant (N = 4) for STAT6 before and after ex vivo stimulation with IL4. We found that STAT6 mutants result in broad based augmentation of IL4-induced gene expression. Unexpectedly, in FL with WT STAT6 we measured reduced baseline and IL4-induced gene expression levels when compared with NBC lymphocytes or FL with STAT6 mutations. We tracked the attenuated IL4/JAK/STAT6 response to co-existing CREBBP mutations and experimentally verified that intact CREBBP is required for the induction of many IL4-induced genes. One of the IL4-induced genes here identified is RRAGD, a small G-protein involved in lysosomal mTOR activation. We show that IL4 treatment induced RRAGD expression, that RRAGD is required for mTOR activation in lymphoma cells and that IL4-enhanced BCR signaling induced mTOR activation. The IL4 and BCR-induced mTOR activation was reduced by CREBBP mutants and augmented by mutant STAT6, establishing a link between STAT6 mutations and mTOR regulated pro-growth pathways in lymphoma.

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate that is an effective therapy for HER2-positive breast cancer; however, its efficacy is limited by drug resistance. While multiple mechanisms of resistance have been proposed, these are not yet well understood. Greater understanding of T-DM1 sensitivity and resistance could provide new combination strategies to overcome resistance or predictive biomarkers to guide therapy.

We have conducted CRISPR/Cas9 functional genomics modifier screens in HER2-positive breast cancer cell lines to allow for unbiased discovery of T-DM1 sensitivity and resistance genes. Whole-genome knockout screens were carried out in MDA-MB-361 and MDA-MB-453 cells treated with T-DM1 and its payload cytotoxin DM1. Hits were validated in secondary T-DM1 screens using a focused single-guide RNA (sgRNA) library and subsequently by individual gene knockout.

The whole-genome CRISPR screens with T-DM1 and DM1 identified 599 genes as potential modifiers of T-DM1 sensitivity and resistance. Of these, 17 genes were significantly enriched and 3 genes depleted at P < 0.001 in either or both MDA-MB-361 and MDA-MB-453 libraries in the secondary screens. Among the top hits, were known T-DM1 sensitivity genes ERBB2 and SLC46A3, in addition to negative regulators of mTOR complex 1: TSC1 and TSC2. MDA-MB-453 clones with knockout of TSC1 or partial knockout of TSC2 were more resistant to T-DM1 than wild type cells in competition growth assays and to T-DM1 and other HER2 targeting therapies (T-DXd, lapatinib and neratinib) in growth inhibition assays, and had increased internalisation of T-DM1 at 6 h. T-DM1 and the mTOR inhibitor everolimus demonstrated synergistic activity at inhibiting cell proliferation at multiple T-DM1 concentrations across four HER2-positive breast cancer cell lines.

Our CRISPR screening approach with T-DM1 in HER2-positive breast cancer cell lines identified genes not previously implicated in T-DM1 sensitivity or resistance, including TSC1 and TSC2. These genes may inform new strategies to enhance T-DM1 therapy in the clinic.

Age-related hearing loss (ARHL) is one of the most prevalent and complex disorders. Our previous study demonstrated that abnormal activation of mammalian target of rapamycin complex 1 (mTORC1) signaling in the cochlear neurosensory epithelium causes auditory hair cell (HC) damage and contributes to ARHL. However, the underlying mechanism of mTORC1 activation remains unclear. In this study, we identified tumor necrosis factor-alpha-induced protein 8-like 2 (TNFAIP8L2), an immune regulatory gene, as a potential candidate. To elucidate the effect of TNFAIP8L2 on mTORC1 signaling in the neurosensory epithelium and on hearing function, we generated a Tnfaip8l2-deficient (Tnfaip8l2-/-) mouse model. We discovered that Tnfaip8l2 deficiency led to features of oxidative stress in cochlear HCs and age-related hearing degeneration, exhibiting a similar phenotype to the mTORC1-over-activated Tsc1-cKO mice described previously. Furthermore, rapamycin, a well-known mTORC1 inhibitor, significantly mitigated the hearing dysfunction caused by Tnfaip8l2-deficiency. Mechanistically, we found that TNFAIP8L2 regulates mTORC1 signaling by simultaneously inhibiting the GTPase activity of Ras homolog enriched in brain (RHEB) and Ras-related C3 botulinum toxin substrate 1 (RAC1). Notably, both RHEB and RAC1 inhibitors alleviated the hearing phenotype observed in Tnfaip8l2-/- mice by inhibiting mTORC1 signaling. Collectively, our results provide insights into the activation of the mTORC1 pathway in aged mouse cochleae and positions TNFAIP8L2 as a valuable theoretical strategy.

Unlike protein-coding genes, most human long noncoding RNAs (lncRNAs) lack conservation based on their sequences, posing a challenge for investigating their role in a pathophysiological context for clinical translation. This study explores the hypothesis that nonconserved lncRNAs in human and mouse livers may share similar metabolic functions, giving rise to functionally conserved lncRNA metabolic regulators (fcLMRs).

We developed a sequence-independent strategy to select putative fcLMRs and performed extensive analysis to determine the functional similarities of putative human and mouse (h/m)LMR pairs.

We found that several pairs of putative fcLMRs share similar functions in regulating gene expression. We further demonstrated that a pair of fcLMRs, h/mLMR1, robustly regulated triglyceride levels by modulating the expression of a similar set of lipogenic genes. Mechanistically, h/mLMR1 binds to poly(A)-binding protein cytoplasmic 1 (PABPC1), a regulator of protein translation, via short motifs on either lncRNA with divergent sequences but similar structures. This interaction inhibits protein translation, activating an amino acid- mechanistic target of rapamycin-sterol regulatory element-binding transcription factor 1 axis to regulate lipogenic gene expression. Intriguingly, PABPC1-binding motifs on each lncRNA fully rescued the functions of their corresponding LMRs in the opposite species. Given the elevated expression of h/mLMR1 in humans and mice with hepatic steatosis, the PABPC1-binding motif on hLMR1 emerges as a potential nonconserved human drug target whose functions can be fully validated in a physiologically relevant setting before clinical studies.

Our study supports that fcLMRs represent a novel and prevalent biological phenomenon and that deep phenotyping of genetic mLMR mouse models constitutes a powerful approach to understand the pathophysiological role of lncRNAs in the human liver.

One of the biggest challenges to eukaryotic gene expression is coordinating transcription in the nucleus and protein synthesis in the cytoplasm. However, little is known about how these major steps in gene expression are connected. The Target of Rapamycin (TOR) signaling pathway is crucial in connecting these critical phases of gene expression. Highly conserved among eukaryotic cells, TOR regulates growth, metabolism, and cellular equilibrium in response to changes in nutrients, energy levels, and stress conditions. This review examines the extensive role of TOR in gene expression regulation. We highlight how TOR is involved in phosphorylation, remodeling chromatin structure, and managing the factors that facilitate transcription and translation. Furthermore, the critical functions of TOR extend to processing RNA, assembling RNA-protein complexes, and managing their export from the nucleus, demonstrating its wide-reaching impact throughout the cell. Our discussion emphasizes the integral roles of TOR in bridging the processes of transcription and translation and explores how it orchestrates these complex cellular processes.

Oleaginous microorganisms can produce polyunsaturated fatty acids beneficial to human health through adjusting the nitrogen content in the medium. The target of rapamycin complex 1 (TORC1) is important for nitrogen sensing and then regulates lipid metabolism. However, the function of Kog1, a subunit of TORC1, in TORC1-regulated lipid metabolism in oleaginous microorganisms remains unclear. In this study, the gene kog1 was knocked out to explore the mechanism of lipid accumulation in the oleaginous fungus M. circinelloides under nitrogen-limited and nitrogen-rich conditions. The results showed that the cell dry weight (CDW) of the kog1 deletion mutant was obviously decreased from 22.2 to 15.4 g/L under nitrogen-limited conditions; however, the lipid content markedly increased by 43.2% compared to the control, from 20.8% of CDW to 29.9%. A similar trend was observed under nitrogen-rich conditions; the cell growth was significantly inhibited, the CDW was decreased from 28.6 to 23.0 g/L, and the lipid content increased by 79.6% compared to the control strain, reaching 9.7% of CDW. The addition of rapamycin further enhanced lipid accumulation in the kog1 knockout mutant but not in the tor knockout mutant, indicating that Kog1 is the upstream target of rapamycin (TOR) in regulating lipid regulation. Transcriptional analysis under both nitrogen-limited and nitrogen-rich conditions notably suggested that nitrogen stress may activate Snf1/AMPK to inhibit Kog1, facilitating SREBP-1c nuclear translocation and activating fatty acid biosynthesis genes.

The ubiquitin ligase Nedd4 (Nedd4-1), comprised of C2-WW(n)-HECT domains, regulates protein trafficking. We recently described a primate-specific Nedd4-1 splice isoform with an extended N-terminus replacing the C2 domain, called Nedd4-1(NE). Here, we show that while canonical Nedd4-1 is primarily localized to the cytosol, Nedd4-1(NE) localizes to late endosomes. This localization is mediated by the NE region, is dependent on amino acid availability, is independent of mTORC1, and is inhibited by the autophagy inducer IKKβ. We further demonstrate that VPS16B, which regulates late endosome to lysosome maturation, is a unique Nedd4-1(NE) substrate that co-localizes with Nedd4-1(NE) in the presence of nutrients. Importantly, a potentially pathogenic homozygous variant identified in the NE region (E70Q) of a patient with lymphangiectasia and protein-losing enteropathy leads to reduced VPS16B ubiquitination by Nedd4-1(NE). Finally, we report that Nedd4-1(NE) inhibits autophagy, likely by disrupting late endosome to autophagosome maturation. This work identified an mTORC1-independent, IKK-driven mechanism to regulate Nedd4-1(NE) localization to late endosomes in primates in response to nutrient availability, and uncovered suppression of autophagy by this ubiquitin ligase.

Transcription Factor EB (TFEB) controls lysosomal biogenesis and autophagy in response to nutritional status and other stress factors. Although its regulation by nuclear translocation is known to involve a complex network of well-studied regulatory processes, the precise contribution of each of these mechanisms is unclear. Using microfluidics technology and real-time imaging coupled with mathematical modelling, we explored the dynamic regulation of TFEB under different conditions. We found that TFEB nuclear translocation upon nutrient deprivation happens in two phases: a fast one characterised by a transient boost in TFEB dephosphorylation dependent on transient calcium release mediated by mucolipin 1 (MCOLN1) followed by activation of the Calcineurin phosphatase, and a slower one driven by inhibition of mTORC1-dependent phosphorylation of TFEB. Upon refeeding, TFEB cytoplasmic relocalisation kinetics are determined by Exportin 1 (XPO1). Collectively, our results show how different mechanisms interact to regulate TFEB activation and the power of microfluidics and quantitative modelling to elucidate complex biological mechanisms.

Diet influences macronutrient availability to cells, and although mechanisms of sensing dietary glucose and amino acids are well characterized, less is known about sensing lipids. We defined a nutrient signaling mechanism involving fatty acid-binding protein 5 (FABP5) and mechanistic target of rapamycin complex 1 (mTORC1) that is activated by the essential polyunsaturated fatty acid (PUFA) ω-6 linoleic acid (LA). FABP5 directly bound to the regulatory-associated protein of mTOR (Raptor) to enhance formation of functional mTORC1 and substrate binding, ultimately converging on increased mTOR signaling and proliferation. The amounts of FABP5 protein were increased in tumors and serum from triple-negative compared with those from receptor-positive breast cancer patients, which highlights its potential role as a biomarker that mediates cellular responses to ω-6 LA intake in this disease subtype.

Pyruvate and branched-chain amino acid (BCAA) metabolism are pivotal pathways in tumor progression, yet the intricate interplay between them and its implications for tumor progression remain elusive. Our research reveals that dihydrolipoamide S-acetyltransferase (DLAT), a pyruvate metabolism enzyme, promotes leucine accumulation and sustains mammalian target of rapamycin (mTOR) complex activation in hepatocellular carcinoma (HCC). Mechanistically, DLAT directly acetylates the K109 residue of AU RNA-binding methylglutaconyl-coenzyme A (CoA) hydratase (AUH), a critical enzyme in leucine catabolism, inhibiting its activity and leading to leucine accumulation. Notably, DLAT upregulation correlates with poor prognosis in patients with HCC. Therefore, we developed an AUHK109R-mRNA lipid nanoparticles (LNPs) therapeutic strategy, which effectively inhibits tumor growth by restoring leucine catabolism and inhibiting mTOR activation in vivo. In summary, our findings uncover DLAT's unexpected role as an acetyltransferase for AUH, suppressing leucine catabolism. Restoring leucine catabolism with AUHK109R-mRNA LNP effectively inhibits HCC development, highlighting a novel direction for cancer research.

This article provides a comprehensive analysis of the signaling pathways implicated in breast cancer (BC), the most prevalent malignancy among women and a leading cause of cancer-related mortality globally. Special emphasis is placed on the structural dynamics of protein complexes that are integral to the regulation of these signaling cascades. Dysregulation of cellular signaling is a fundamental aspect of BC pathophysiology, with both upstream and downstream signaling cascade activation contributing to cellular process aberrations that not only drive tumor growth, but also contribute to resistance against current treatments. The review explores alterations within these pathways across different BC subtypes and highlights potential therapeutic strategies targeting these pathways. Additionally, the influence of specific mutations on therapeutic decision-making is examined, underscoring their relevance to particular BC subtypes. The article also discusses both approved therapeutic modalities and ongoing clinical trials targeting disrupted signaling pathways. However, further investigation is necessary to fully elucidate the underlying mechanisms and optimize personalized treatment approaches.

The nutrient-sensitive protein kinases AMPK and mTORC1 form a fundamental negative feedback loop that governs cell growth and proliferation. mTORC1 phosphorylates α2-S345 in the AMPK αβγ heterotrimer to suppress its activity and promote cell proliferation under nutrient stress conditions. Whether AMPK contains other functional mTORC1 substrates is unknown. Using mass spectrometry, we generated precise stoichiometry profiles of phosphorylation sites across all twelve AMPK complexes expressed in proliferating human cells and identified seven sites displaying sensitivity to pharmacological mTORC1 inhibition. These included the abundantly phosphorylated residues β1-S182 and β2-S184, which were confirmed as mTORC1 substrates on purified AMPK, and four residues in the unique γ2 N-terminal extension. β-S182/184 phosphorylation was elevated in α1-containing complexes relative to α2, an effect attributed to the α-subunit serine/threonine-rich loop. Mutation of β1-S182 to non-phosphorylatable Ala had no effect on basal and ligand-stimulated AMPK activity; however, β2-S184A mutation increased nuclear AMPK activity, enhanced cell proliferation under nutrient stress and altered expression of genes implicated in glucose metabolism and Akt signalling. Our results indicate that mTORC1 directly or indirectly phosphorylates multiple AMPK residues that may contribute to metabolic rewiring in cancerous cells.

Obesity is a major risk factor for poor breast cancer outcomes, but the impact of obesity-induced tumor microenvironment (TME) metabolites on breast cancer growth and metastasis remains unclear. Here, we performed TME metabolomic analysis in high-fat diet (HFD) mouse models and found that glutathione (GSH) levels were elevated in the TME of obesity-accelerated breast cancer. The deletion of glutamate-cysteine ligase catalytic subunit (GCLC), the rate-limiting enzyme in GSH biosynthesis, in adipocytes but not tumor cells reduced obesity-related tumor progression. Mechanistically, we identified that GSH entered tumor cells and directly bound to lysosomal integral membrane protein-2 (scavenger receptor class B, member 2 [SCARB2]), interfering with the interaction between its N and C termini. This, in turn, recruited mTORC1 to lysosomes through ARF1, leading to the activation of mTOR signaling. Overall, we demonstrated that GSH links obesity and breast cancer progression by acting as an activator of mTOR signaling. Targeting the GSH/SCARB2/mTOR axis could benefit breast cancer patients with obesity.

Brown adipose tissue (BAT) thermogenesis results from the uncoupling of mitochondrial inner membrane proton gradient mediated by uncoupling protein 1 (UCP-1), which is activated by lipolysis-derived fatty acids. Norepinephrine (NE) secreted by sympathetic innervation not only activates BAT lipolysis and UCP-1 but, uniquely in brown adipocytes, promotes "futile" metabolic cycles and enhances BAT thermogenic capacity by increasing UCP-1 content, mitochondrial biogenesis, and brown adipocyte hyperplasia. NE exerts these actions by triggering signaling in the canonical G protein-coupled β-adrenergic receptors, cAMP, and protein kinase A (PKA) pathway, which in brown adipocytes is under a complex and intricate cross talk with important growth-promoting signaling pathways such as those of mechanistic target of rapamycin (mTOR) complexes 1 (mTORC1) and 2 (mTORC2). This article reviews evidence suggesting that mTOR complexes are modulated by and participate in the thermogenic, metabolic, and growth-promoting effects elicited by NE in BAT and discusses current gaps and future directions in this field of research.

mTOR plays a pivotal role in cancer growth control upon amino acid response. Recently, CDK inhibitor P27KIP1 has been reported as a noncanonical inhibitor of mTOR signaling in MEFs, via unclear mechanisms. Here, we find that P27KIP1 degradation via E3 ligase TRIM21 is inhibited by human micropeptide hSPAR through its C-terminus (hSPAR-C), causing P27KIP1's cytoplasmic accumulation in breast cancer cells. Furthermore, hSPAR/hSPAR-C also serves as an inhibitor of glutamine transporter SLC38A2 expression and thereby decreases the cellular glutamine levels specifically in cancer cells. The resultant glutamine deprivation sequentially triggers translocation of cytoplasmic P27KIP1 to lysosomes, where P27KIP1 disrupts the Ragulator complex and suppresses mTORC1 assembly. Administration of hSPAR or hSPAR-C significantly impedes breast cancer cell proliferation and tumor growth in xenograft models. These findings define hSPAR as an intrinsic control factor for cellular glutamine levels and as a novel tumor suppressor inhibiting mTORC1 assembly.

Diabetic kidney disease2 (DKD) is a chronic complication of diabetes characterized by kidney damage due to persistent hyperglycemia. A growing number of evidence indicated that circular RNAs3 (circRNAs) play a crucial role in diabetes and associated complications. However, the function and mechanism of circRNAs in DKD remain unclear. Herein, we investigated the expression profiles of circRNAs in DKD mice compared to non-diabetic mice using RNA-seq analysis. A novel circRNA, circMRP4, derived from the circularization of Multidrug resistance-associated protein 44 (MRP4) was identified. The expression of circMRP4 was significantly increased in both kidney tissues of DKD and mouse podocytes exposed to high glucose5 (HG). In addition, knockdown of circMRP4 alleviated podocytes apoptosis and inflammation induced by HG, while circMRP4 overexpression resulted in the opposite impact. Dual-luciferase reporter, RNA immunoprecipitation and RNA pull-down assay demonstrated that circMRP4 could directly target miR-499-5p which was closely associated with podocytes apoptosis and inflammation. Furthermore, circMRP4 was found to act as a sponge for miR-499-5p, leading to the upregulation of its target RRAGB, thereby activating the mTORC1/P70S6K signaling. In summary, our findings suggested that circMRP4 mediated podocytes apoptosis and inflammation in DKD by modulating the miR-499-5p/RRAGB/mTORC1/P70S6K axis, highlighting circMRP4 as a potential therapeutic target for DKD.

Acetyl-coenzyme A is a central metabolite that participates in many cellular pathways. Evidence suggests that acetyl-CoA metabolism is highly compartmentalized in mammalian cells. Yet methods to measure acetyl-CoA in living cells are lacking. Herein, we engineered an acetyl-CoA biosensor from the bacterial protein PanZ and circularly permuted green fluorescent protein (cpGFP). The sensor, "PancACe," has a maximum change of ∼2-fold and a response range of ∼10 μM-2 mM acetyl-CoA. We demonstrated that the sensor has a greater than 7-fold selectivity over coenzyme A, butyryl-CoA, malonyl-CoA, and succinyl-CoA, and a 2.3-fold selectivity over propionyl-CoA. We expressed the sensor in E. coli and showed that it enables detection of rapid changes in acetyl-CoA levels. By localizing the sensor to either the cytoplasm, nucleus, or mitochondria in human cells, we showed that it enables subcellular detection of changes in acetyl-CoA levels, the magnitudes of which agreed with an orthogonal PicoProbe assay.