Guang Yang

Genetic variants associated with complex traits often lie in distal enhancers. While candidate enhancers have been mapped genome wide, their functional state and gene targets in specific cell types remain unclear. Here we present AstroREG, a resource of enhancer–gene interactions in human primary astrocytes, generated by combining CRISPR inhibition (CRISPRi), single-cell RNA-seq and machine learning. By functionally testing nearly 1,000 PsychENCODE enhancers, we identified more than 150 regulatory interactions, revealing enhancers that control key astrocyte functions and genes implicated in Alzheimer’s disease. The CRISPRi screen also provided valuable ground-truth data from a primary cell type for training and benchmarking prediction models of enhancer activity. We thus developed EGrf, a random forest (RF) model trained on these data, and applied it genome wide to predict regulatory interactions with high specificity. Together, our data provide a comprehensive functional map of enhancer-mediated regulation in a key glial cell type, shedding light on brain function and disease.

The glycerol-3-phosphate (G-3-P) pathway is central to the synthesis of triacylglycerols (TAGs) and glycerophospholipids, essential for membrane biogenesis and lipid storage. The first and rate-limiting step in this pathway is catalyzed by glycerol-3-phosphate acyltransferases (GPATs), with microsomal GPAT3 and GPAT4 being evolutionarily conserved and predominant in most tissues. While previous studies have implicated Calcineurin B homologous protein 1 (CHP1) as a cofactor for GPAT4, the broader role of CHP1 in regulating microsomal GPATs and TAG biosynthesis remains unclear. Here, we demonstrate that CHP1 is a critical regulator of both GPAT3 and GPAT4, essential for their stability, enzymatic activity, and lipid droplet (LD) localization. Structural modeling and mutational analyses identified key hydrophobic interfaces mediating the CHP1–GPAT interaction, which are required for optimal GPAT activity and LD growth. Loss of CHP1 impairs LD expansion and disrupts the localization of GPAT3/4 and downstream enzymes in the TAG synthesis pathway, including 1-acylglycerol-3-phosphate O-acyltransferase 3 (AGPAT3) and diacylglycerol O-acyltransferase 2 (DGAT2). Mechanistically, CHP1 helps circumvent seipin-mediated restriction of late LD-targeting enzymes, facilitating their access to mature LDs. Together, our findings reveal CHP1 as a dual-function regulator that stabilizes and activates microsomal GPATs while enabling the coordinated recruitment of TAG biosynthetic enzymes to LDs. This work uncovers a previously unrecognized mechanism for regulating LD growth and glycerolipid metabolism, with broad implications for lipid homeostasis and metabolic diseases.

Protein phosphorylation dynamically integrates environmental and cellular information to control biological processes. Identifying functional phosphorylation amongst the thousands of phosphosites regulated by a perturbation at a global scale is a major challenge. Here we introduce ‘personalized phosphoproteomics’, a combination of experimental and computational analyses to link signaling with biological function by utilizing human phenotypic variance. We measure individual subject phosphoproteome responses to interventions with corresponding phenotypes measured in parallel. Applying this approach to investigate how exercise potentiates insulin signaling in human skeletal muscle, we identify both known and previously unidentified phosphosites on proteins involved in glucose metabolism. This includes a cooperative relationship between mTOR and AMPK whereby the former directly phosphorylates the latter on S377, for which we find a role in metabolic regulation. These results establish personalized phosphoproteomics as a general approach for investigating the signal transduction underlying complex biology.

RapaLink1 enables differentiation between functions mediated by the closely related mTORC1 and mTORC2.

The PI3K/MTOR signalling network regulates a broad array of critical cellular processes, including cell growth, metabolism and autophagy. The mechanistic target of rapamycin (MTOR) kinase functions as a core catalytic subunit in two physically and functionally distinct complexes mTORC1 and mTORC2, which also share other common components including MLST8 (also known as GβL) and DEPTOR. Despite intensive research, how mTORC1 and 2 assembly and activity are coordinated, and how they are functionally linked remain to be fully characterized. This is due in part to the complex network wiring, featuring multiple feedback loops and intricate post-translational modifications. Here, we integrate predictive network modelling, in vitro experiments and -omics data analysis to elucidate the emergent dynamic behaviour of the PI3K/MTOR network. We construct new mechanistic models that encapsulate critical mechanistic details, including mTORC1/2 coordination by MLST8 (de)ubiquitination and the Akt-to-mTORC2 positive feedback loop. Model simulations validated by experimental studies revealed a previously unknown biphasic, threshold-gated dependence of mTORC1 activity on the key mTORC2 subunit SIN1, which is robust against cell-to-cell variation in protein expression. In addition, our integrative analysis demonstrates that ubiquitination of MLST8, which is reversed by OTUD7B, is regulated by IRS1/2. Our results further support the essential role of MLST8 in enabling both mTORC1 and 2’s activity and suggest MLST8 as a viable therapeutic target in breast cancer. Overall, our study reports a new mechanistic model of PI3K/MTOR signalling incorporating MLST8-mediated mTORC1/2 formation and unveils a novel regulatory linkage between mTORC1 and mTORC2.

Calcium/calmodulin-dependent protein serine kinase (CASK) is a key player in vesicle transport and release in neurons. However, its precise role, particularly in nonneuronal systems, is incompletely understood. We report that CASK functions as an important regulator of insulin secretion. CASK depletion in mouse islets/β cells substantially reduces insulin secretion and vesicle docking/fusion. CASK forms a ternary complex with Mint1 and Munc18-1, and this event is regulated by glucose stimulation in β cells. The crystal structure of the CASK/Mint1 complex demonstrates that Mint1 exhibits a unique “whip”-like structure that wraps tightly around the CASK-CaMK domain, which contains dual hydrophobic interaction sites. When triggered by CASK binding, Mint1 modulates the assembly of the complex. Further investigation revealed that CASK-Mint1 binding is critical for ternary complex formation, thereby controlling Munc18-1 membrane localization and insulin secretion. Our work illustrates the distinctive molecular basis underlying CASK/Mint1/Munc18-1 complex formation and reveals the importance of the CASK-Mint1-Munc18 signaling axis in insulin secretion.

The PI3K/mTOR signalling network critically regulates a broad array of important biological processes, including cell growth, metabolism and autophagy. Dysregulation of PI3K/mTOR signalling is associated with a variety of human diseases, including cancer and metabolic disorders. The mechanistic target of rapamycin (mTOR) is a kinase that functions as a core catalytic subunit in two physically and functionally distinct complexes termed mTOR complex 1 (mTORC1) and mTORC2, which also share other common components such as mLTS8 (also known as GβL) and DEPTOR. Despite being the subject of intensive research, a full picture of how mTORC1/2 assembly and activity are coordinated, and how they are functionally connected remain to be fully characterised. This is due primarily to the complex network wiring, featuring a growing number of intricate feedback loops and post-translational modifications, which require quantitative systems-level approaches to decipher. Here, we integrate predictive computational modelling, in vitro experiments and -omics data analysis to elucidate the dynamic and emergent features of the PI3K/mTOR network behavior. We construct new mechanistic models of the network that encapsulate novel critical mechanistic details, including mTORC1/2 coordination by mLTS8 (de)ubiquitination, and Akt-to-mTORC2 positive feedback loop. Model simulations subsequently confirmed by experimental validation revealed a previously unknown biphasic, threshold-gated dependence of mTORC1 activity on the key mTORC2 subunit Sin1, which is robust against cell-to-cell variation in protein expression. Furthermore, our results support the essential role of mLST8 in both mTORC1 and 2 activity, and suggest mLST8 could serve as a viable therapeutic target in breast cancer. Overall, our integrated analyses provide fresh systems-level insights into the dynamic behavior of PI3K/mTOR signalling and shed new light on the complexity of this important network. AUTHOR SUMMARY: Signalling networks are the key information-processing machineries that underpin the ability of living cells to respond proportionately to extra- (and intra-) cellular cues. The PI3K/mTOR signalling network is one of the most important signalling networks in human cells that regulates cellular response to hormones such as insulin, yet our understanding of the network behaviour remains far from complete. Here, we employed a highly integrative approach that combines predictive mathematical modelling, biological experimentation, and data analysis to gain novel systems-level insights into PI3K/mTOR signalling. We constructed new mathematical models of this complex network incorporating important regulatory mechanisms. In contrary to commonly held views that mTORC2 lies upstream and is a positive regulator of mTORC1, we found that their relationship is highly nonlinear and dose dependent. This finding has major implications for mTORC2-directed anti-cancer strategies as depending on the cellular contexts, blocking mTORC2 may reduce or even enhance mTORC1 activation, the latter could inadvertently blunt the effect of mTORC2 blockade. Furthermore, our results demonstrate that mLST8 is required for the assembly and activity of both mTOR complexes, and suggest mLST8 is a viable therapeutic target in breast cancer, notably breast cancer.

Protein oxidation sits at the intersection of multiple signalling pathways, yet the magnitude and extent of crosstalk between oxidation and other post-translational modifications remains unclear. Here, we delineate global changes in adipocyte signalling networks following acute oxidative stress and reveal considerable crosstalk between cysteine oxidation and phosphorylation-based signalling. Oxidation of key regulatory kinases, including Akt, mTOR and AMPK influences the fidelity rather than their absolute activation state, highlighting an unappreciated interplay between these modifications. Mechanistic analysis of the redox regulation of Akt identified two cysteine residues in the pleckstrin homology domain (C60 and C77) to be reversibly oxidized. Oxidation at these sites affected Akt recruitment to the plasma membrane by stabilizing the PIP₃binding pocket. Our data provide insights into the interplay between oxidative stress-derived redox signalling and protein phosphorylation networks and serve as a resource for understanding the contribution of cellular oxidation to a range of diseases.

The mechanistic (or mammalian) target of rapamycin complex 1 (mTORC1) controls cell growth, proliferation, and metabolism in response to diverse stimuli. Two major parallel pathways are implicated in mTORC1 regulation including a growth factor‐responsive pathway mediated via TSC2/Rheb and an amino acid‐responsive pathway mediated via the Rag GTPases. Here, we identify and characterize three highly conserved growth factor‐responsive phosphorylation sites on RagC, a component of the Rag heterodimer, implicating cross talk between amino acid and growth factor‐mediated regulation of mTORC1. We find that RagC phosphorylation is associated with destabilization of mTORC1 and is essential for both growth factor and amino acid‐induced mTORC1 activation. Functionally, RagC phosphorylation suppresses starvation‐induced autophagy, and genetic studies in Drosophila reveal that RagC phosphorylation plays an essential role in regulation of cell growth. Finally, we identify mTORC1 as the upstream kinase of RagC on S21. Our data highlight the importance of RagC phosphorylation in its function and identify a previously unappreciated auto‐regulatory mechanism of mTORC1 activity. Synopsis The mechanistic target of rapamycin complex1 (mTORC1) controls cell growth, proliferation and metabolism in response to diverse stimuli. This study identifies a new auto‐regulatory branch of mTORC1 signaling involving phosphorylation of the Rag GTPase RagC by mTORC1, which is required for full mTORC1 activation by growth factors and amino acids. Growth factors and amino acids stimulate mTORC1‐mediated phosphorylation of RagC. RagC phosphorylation is essential for both growth factor‐ and amino acid‐induced mTORC1 activity. RagC phosphorylation is required for mTORC1 regulation of cell size and autophagy in vitro and in Drosophila wing discs. RagC phosphorylation is associated with activation and concomitant destabilization of mTORC1.