Advances in biological regulation最新文献

Esophageal squamous cell carcinoma (eSCC) is an aggressive malignancy with poor prognosis and limited therapeutic options. The phosphoinositide 3-kinase (PI3K)/AKT pathway is frequently activated in eSCC, but clinical use of PI3K or AKT inhibitors is restricted by toxicity and compensatory signaling. SHIP2, an inositol 5-phosphatase encoded by INPPL1, modulates this pathway by converting PI(3,4,5)P₃ to PI(3,4)P₂, thereby regulating AKT activation. We previously identified INPPL1 amplification as recurrent in eSCC and demonstrated that SHIP2 inhibition suppresses tumor growth and synergizes with PLK1 inhibition. Here, we extend these findings and show that SHIP2-PLK1 synergy is not confined to eSCC but is also observed in multiple colorectal cancer cell lines, revealing a conserved vulnerability across tumor types. Mechanistic analyses demonstrate that this synergy depends on PI3K/AKT signaling, with SHIP2 inhibition producing stronger effects than direct PI3K blockade, suggesting additional regulatory functions beyond canonical PI3K control. Furthermore, SHIP2 inhibition enhances the cytotoxic activity of standard chemotherapies, including 5-fluorouracil and paclitaxel, in eSCC cells. Importantly, these effects occur at sub-cytotoxic drug concentrations, indicating potential therapeutic benefit with reduced toxicity. Collectively, our results identify SHIP2 as a central regulator of the PI3K/AKT axis in eSCC and colorectal cancer and highlight its value as a combinatorial target. SHIP2 inhibition represents a promising strategy to potentiate existing chemotherapies and targeted agents, opening new avenues for the treatment of refractory gastrointestinal cancers.

Class IA phosophoinositide kinases (PI3Ks) are master regulators of growth, metabolism, and immunity. The class IA PI3Ks are a heterodimer composed of a p110 catalytic subunit and one of five possible regulatory subunits (p85α, p85β, p55γ, p55α, p50α). The regulatory subunit plays critical roles in stability, inhibition, and activation of the p110 catalytic subunit. The p110α catalytic subunit frequently contains activating mutations in human cancer, with many of these mutations altering the interaction between catalytic and regulatory subunits. It has been found that different regulatory subunits play unique roles in human disease, but it is unknown how these different subunits regulate p110α. Here, using a synergy of biochemical assays and hydrogen deuterium exchange mass spectrometry (HDX-MS) we examined how the five different regulatory subunits inhibit, activate, and interact with the p110α catalytic subunit. We find that there are no significant differences in lipid kinase activity or in membrane recruitment between the different heterodimer complexes. HDX-MS in the presence and absence of an activating phosphopeptide also showed only minor conformational differences between different regulatory subunit complexes. Overall, our work reveals that the different regulatory subunits interact with and inhibit p110α in a similar fashion at a molecular level.

The unfolded protein response (UPR) is a central regulator of proteostasis, coordinating cellular adaptation to endoplasmic reticulum (ER) stress. It is comprised of three signaling branches: ATF6 (activating transcription factor 6), IRE1 (inositol-requiring enzyme 1), and PERK (protein kinase RNA-like ER kinase), which mediate transcriptional and translational reprogramming of the proteostasis network. These pathways display both functional redundancy and branch-specific activities. Dysregulated UPR signaling contributes to diverse pathologies: in cancer, UPR activation supports uncontrolled proliferation and treatment resistance, whereas in aging, proteostasis decline and diminished UPR responsiveness are hallmarks. Traditional approaches, including transcriptomics and western blotting, have been widely used to monitor UPR activity, but they offer limited insight into its regulation at the protein level. In contrast, liquid chromatography-tandem mass spectrometry (LC-MS/MS) based proteomics allows comprehensive, branch-specific profiling of UPR signaling. Recent advances, including data-independent acquisition (DIA) MS and automated sample preparation, have further improved sensitivity, reproducibility, and detection of low-abundance UPR target proteins. Proteomics thus provides a systematic and scalable framework to interrogate UPR regulation across cell types and disease models. When integrated with complementary datasets, protein-level measurements can uncover context-dependent molecular signatures of UPR activity, offering insights into disease mechanisms and guiding the rational design of targeted pharmacological interventions. Future work integrating high-resolution LC-MS/MS proteomics with tissue and single-cell analyses will further clarify the role of the UPR in health and disease.

Tepsin is an accessory protein in Adaptor Protein 4 (AP-4) coated vesicles responsible for trafficking cargo from the trans-Golgi network (TGN). AP-4 vesicles recognize and sort multiple cargoes including ATG9A, a lipid scramblase essential for autophagosome maturation. In cultured cells, tepsin loss alters ATG9A distribution and autophagosome morphology, and tepsin has been shown to contain a canonical LC3-interacting region (LIR) motif required for proper ATG9A distribution. Computational modeling in AlphaFold Multimer combined with biochemical and biophysical experiments identified three additional LC3B binding motifs within tepsin disordered regions. Structural models paired with bio-layer interferometry (BLI) uncovered and confirmed specific residues involved in each interaction and indicated all four motifs independently engage the LC3B LIR docking site (LDS). Thermodynamic and kinetic properties associated with each motif found in full-length tepsin were quantified. BLI and biochemical data reveal all four motifs in tepsin must be mutated to abrogate binding to LC3B in vitro, while stoichiometry data estimate one tepsin likely binds two LC3B at one time on a surface or membrane. Together, data suggest tepsin could respond dynamically to LC3B concentrations on membranes by leveraging multivalency to modulate binding strength.

Phosphatidic acid (PA) has emerged as a central regulator of membrane dynamics, vesicle trafficking, exocytosis, and intracellular signaling. Building on recent advances, including subspecies-specific functions of PA in neuroendocrine exocytosis, the primacy of PLD1-derived PA in vivo, and the development of natural-mimetic PA analogues, this review integrates biochemical, biophysical, and systems-level insights across eukaryotes. We contextualize the role of PA in vesicular trafficking, delineate how acyl-chain composition encodes molecular specificity, summarize enzymatic sources and sinks sculpting spatiotemporal control of PA pools within cells, and examine emerging tools used for measuring and disturbing PA in living cells to unravel its function. Given the pleiotropic roles of PA among numerous experimental contexts such as the nervous, endocrine, immune, and metabolic systems, mapping mechanistic connections to disease through mTOR and RAF/MEK/ERK signaling, autophagy, and organelle contact-site biology. Finally, we outline future directions spanning single-cell lipidomics, imaging mass spectrometry, and therapeutic lipid engineering. Together, available evidence positions PA as a conserved, tunable molecular switch that coordinates membrane mechanics with signal transduction to enable realisation of a wide range of function within cells.

The Endosomal Sorting Complex Required for Transport (ESCRT) machinery orchestrates a wide range of membrane remodeling and repair events, spanning multivesicular body biogenesis, viral budding, nuclear envelope surveillance, cytokinesis, lysosomal repair and plasma membrane resealing. These processes depend on the hierarchical assembly of ESCRT complexes to detect and remodel membranes, ultimately driving membrane scission with topological precision. A growing body of evidence indicates that phosphoinositides (PtdIns), a versatile class of phosphorylated lipids, are central determinants of ESCRT function by defining membrane identity, recruiting specific ESCRT modules and integrating lipid signaling into biological processes. This review synthesizes current understanding of how distinct phosphoinositide pools govern ESCRT recruitment and activity, with a focus on the molecular components and their interaction.

Canonically known both for structural contributions to lipid bilayers and roles in cell signaling, the sphingolipids comprise a dynamic, multifaceted class of molecules which are studied to understand cell biology and pathophysiology. All sphingolipids are downstream products of the rate-limiting and initiating enzyme in the de novo sphingolipid synthesis pathway, serine palmitoyltransferase (SPT). SPT activity is strictly regulated. This regulation is accomplished through the ORMDLs, transmembrane polypeptides embedded in the lipid bilayer of the endoplasmic reticulum, which are the regulatory subunits of the SPT complex. Recently the specific mechanism of ORMDL's regulation of SPT was established: ceramide, a downstream product of the de novo biosynthetic pathway, binds directly to a binding site of ORMDL to induce an inhibitory conformational change. Here, we validate a computational docking approach to interrogate the binding efficiency of a range of sphingolipids in the ceramide binding site. We demonstrate that docking poses predicted by this in silico approach reflect experimental data on the efficiency of sphingolipid species to accomplish ORMDL-dependent inhibition of SPT. We propose that this docking analysis will be a valuable complement to experimental tests of compounds that bind to this site to regulate sphingolipid biosynthesis.

DNA double-strand breakage is the most lethal damage to chromosomal DNA. It activates a series of cellular DNA damage response pathways, including DNA damage sensing, control of cell cycle arrest and apoptosis, and DNA repair. DNA damage response pathways are regulated by complex signaling machineries. Of the intracellular signaling cascades, diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to generate phosphatidic acid (PA). Because both DG and PA serve as second messengers, DGK activity induces a shift of signaling pathways from DG-mediated to PA-mediated cascades, thereby implicating DGK in the regulation of widely various functions. Reportedly, one member of the DGK family, DGKζ, is intimately involved in the regulation of stress responses through p53 and NF-κB. Stresses such as ischemia and infarction cause DGKζ downregulation. Experimental DGKζ depletion renders cells and mice vulnerable to various stressors such as chemotherapeutic agents and ionizing irradiation. Nevertheless, how DGKζ is involved in DNA repair, a critical event of DNA damage response for survival remains unknown. For this study, we examined how DGKζ depletion affects DNA repair mechanisms. We demonstrated that DGKζ depletion causes attenuation of Akt activation and DNA-PK protein expression upon DNA damage, which might engender downregulated BRCA1 protein synthesis and stability. Results suggest that DGKζ depletion attenuates BRCA1-mediated DNA repair machinery, thereby conferring vulnerability to DNA damage.

Despite decades of research since phorbol esters first linked protein kinase C (PKC) to tumor promotion, the biological role of this family of kinases in cancer has remained ambiguous because of isozyme-specific functions and tissue-type-dependent effects. Here, we delineate critical roles for PKC in lung cancer. We previously showed that sustained activation of PKCβ_II activates mTOR, an effect evident in lung cancer cell lines with high expression of classical PKCs (cPKCs). These findings prompted us to examine lung cancers driven by mutant EGFR (mEGFR), in which PKCα is highly expressed. We find that mEGFR-dependent activation of PKCα drives serum-deprived proliferation, anchorage-independent growth (AIG), and anchorage-independent survival (AIS). Subsequent studies revealed that the mutant receptor is impaired in ligand-independent activation and, due to altered autophosphorylation, exhibits biased activation of the PLC arm, preferentially propagating signals through a PLC-PKCα-AKT-mTORC1 axis required for AIG and AIS. In parallel, we investigated the basis of PKCα upregulation and found that elevated PKCα levels are independent of mEGFR. Bioinformatic analysis of mEGFR lung cancers highlighted basal cells, a subtype of lung cell which intrinsically express high PRKCA, as the likely cell-of-origin, suggesting that cell lineage sets a high ceiling for PKCα abundance, while mEGFR licenses the activation of the kinase. Collectively, these data define a pathway-specific role for cPKCs, particularly PKCα, as upstream effectors of mTORC1 in mEGFR systems, establishing a neomorphic dependency on the PKCα-AKT-mTORC1 signaling arm that sustains tumorigenesis via biased signaling by the mutant receptor.

The modification of nuclear, cytoplasmic, and mitochondrial proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has emerged as an essential post-translational modification in mammals. More than 5000 human proteins are subject to O-GlcNAcylation, influencing key cellular processes such as signal transduction, epigenetic regulation, transcription, translation, and bioenergetics. Dysregulation of this modification has been implicated in a wide range of diseases, including metabolic disorders, cancer, neurodegeneration, ischemic injury, and heart failure. O-GlcNAc-cycling is orchestrated by two enzymes: the O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which catalyze the addition and removal of O-GlcNAc, respectively. A central challenge in the field is understanding how this minimal enzymatic machinery achieves such broad substrate specificity. It is hypothesized that OGT's functional versatility is mediated through interactions with a diverse network of protein partners that act as adaptors, scaffolds, or substrates, thereby directing its localization, modulating its activity, and shaping its substrate selectivity. In this review, we discuss key interactors and their functional impact on OGT. We also explore how post-translational modifications and substrate availability contribute to OGT regulation and specificity.