Biology subjects

Gygi, S.

Publications and source records attributed to Gygi, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Traffickome infers directed, compartment-resolved membrane trafficking from proteomic data

Membrane trafficking determines the subcellular localization and compartment-residence time of receptors, channels, transporters and other transmembrane proteins and thereby influences where in the cell and how long these proteins function. Functional-enrichment computational approaches may predict the membrane-trafficking regulations but do not resolve the directionality of the transport between cellular compartments. We present Traffickome, a deterministic method organizing 754 trafficking proteins into 11 directed transitions across seven compartments and 1,837 signaling proteins across 22 pathways. Implemented as an interactive browser and open-source Python package, Traffickome infers compartment-to-compartment transport, cargo fate and signaling-pathway activity and enables in silico perturbation from a single proteomic comparison. Across 29 proteomic comparisons spanning 14 studies encompassing over 66,000 gene-level observations across over 10,000 genes, Traffickome ranked the expected trafficking transition first in 23 cases (79%), outperforming the best conventional enrichment methods (17 of 29, 59%). Mean accuracy fell to 18% after randomizing the protein-to-transition assignments. To illustrate the power of Traffickome, we performed time-resolved analysis of the phosphoproteome in EGF-stimulated cells with the goal to distinguish signaling by internalized versus plasma-membrane EGFR. In the resulting datasets, comprising over 18,000 phosphosite-level measurements, Traffickome revealed localization-dependent differences in EGF-induced phosphorylation and prioritized trafficking regulators for testing their effects on signaling processes.

cell biology↗

bioRxiv2026

Tirzepatide preserves hematopoietic stem and progenitor cycling while remodeling inflammatory monocytes in obese mice

Obesity expands myeloid progenitors, myelopoiesis and increases the production of monocytes. While weight loss (WL) alleviates aspects of this inflammatory dysregulation, it is not known whether GLP-1 receptor agonists or other traditional modalities of WL differentially modify hematopoietic stem/progenitor cells (HSPCs), hematopoiesis, or inflammatory cell production. To test this, we compared the hematopoietic compartment in lean, obese and weight-reduced mice from tirzepatide treatment and caloric restriction (CR) implemented to match the body weight in both groups. At equal WL, we found CR induced multilineage cytopenias, whereas tirzepatide preserved blood lineages while specifically reducing classical Ly6Chi CCR2+ monocytes. To define the mechanisms underlying these changes we performed single-cell mRNA sequencing of bone marrow HSPCs and mature mononuclear blood cells. CR-HSPCs suppressed gene sets associated with nutrient sensing, proliferation and oxidative phosphorylation (OXPHOS) and exhibited lower inferred cell cycle activity, whereas tirzepatide-HSPCs attenuated these changes. Unlike CR, we found that across progressively differentiated cells from HSPCs to mature blood monocytes, tirzepatide increasingly suppressed OXPHOS and simultaneously shifted the maturation spectrum away from classical monocytes. Following six weeks of tirzepatide withdrawal and weight regain, Ly6Chi CCR2+ monocytes rebounded to levels seen in obese mice. These findings suggest that tirzepatide uncouples WL from the broad hematopoietic suppression seen in CR by preserving progenitor activity but selectively remodeling inflammatory/classical monocytes. We demonstrate that WL modality differentially impacts hematopoietic adaptation and provide evidence that classical monocytes are an effector cell through which tirzepatide may dampen obesity-associated inflammation.

immunology↗

bioRxiv2026

Amino Acid Stress Induces Non-AUG Initiation of c-myc Translation

Initiation of translation at non-AUG start codons can generate novel isoforms of important cellular regulators, with activities or localization distinct from their AUG-initiated counterparts. Ribosomes scanning the 5UTR upstream of canonical AUG starts recognize near-cognate starts inefficiently, however, and the mechanisms by which non-canonical starts might be regulated physiologically are poorly understood. We show here that the restriction of utilization of individual amino acid induces a switch in expression of c-myc from its AUG-initiated isoform, c-myc-2, to an upstream CUG-initiated isoform, c-myc-1. This switch is enhanced in cells lacking GCN2, the protein kinase linking amino acid stress to phosphorylation and inhibition of eif2. Induction of upstream CUG start site usage in c-myc by restriction of proline utilization depends on the proximity of proline codons downstream of the canonical AUG start site. These observations support a model in which the stalling of an elongating ribosome near a canonical start leads to the accumulation of a ribosome queue that facilitates initiation at an upstream non-canonical start site. We find similar effects associated with deprivation of amino acids other than proline, and isoform changes with ATF-3 as well as c-myc. These findings suggest a broad mechanism by which amino acid stress can regulate translation initiation site use to modify the N-terminal proteome.

cell biology↗

bioRxiv2026

Analysis of Confounding Factors in Reactive Cysteine Profiling Reveals Enhanced Chromatin-Protein Association via CDK7 Inhibition by THZ1

Recent advances in activity-based proteome profiling (ABPP) have enabled global mapping of cysteine ligandability, uncovering novel biological insights and opportunities for identifying disease vulnerabilities. While both live cell-based and native lysate-based ABPP have been applied, how cysteine ligandability differs between these systems and what factors influence these measurements remain unclear. Building on our previous development of a high-throughput TMT-ABPP workflow for native lysates, here we adapt the protocol for live cells and systematically compare cysteine ligandability across both platforms. Our analysis reveals three major contributors to the discrepancies: in-cellulo cysteine accessibility, protein abundance changes, and protein relocalization. Notably, we highlight that CDK7 inhibitor THZ1 induces substantial protein relocalization and promotes chromatin binding. Together, these results provide a practical framework for ABPP experimental design and data interpretation, supporting more accurate application of ABPP in functional proteomics and drug discovery.

cell biology↗

bioRxiv2026

CDK/mTOR-dependent phosphorylation of UBE2H restrains its charging with ubiquitin and regulates CTLH-dependent degradation

The C-terminal to LisH (CTLH) complex is a modular multi-subunit E3 ligase with diverse biological functions, yet how its overall ubiquitylation activity is tuned remains unclear. Here, we identify CDK- and mTOR-dependent phosphorylation of the cognate E2 enzyme UBE2H as a key regulator of CTLH E3 catalytic capacity. Phosphorylation of two N-terminal serine residues (S3/S5) reduces UBE2H charging with ubiquitin, thereby limiting the pool of active E2 available to CTLH. Mitotic CDK activity inactivates UBE2H during mitosis, whereas mTOR restrains UBE2H charging in interphase to couple CTLH-dependent ubiquitylation to nutrient status. Preventing this phosphorylation maintains UBE2H charging, enhances CTLH-mediated substrate degradation, promotes CTLH subunit turnover, and causes proliferation and mitotic defects. Using hyperactive UBE2H, we identify two additional CTLH substrates, the mitotic kinase NEK9 and Angio-associated migratory cell protein (AAMP) and define a DR-like C-degron recognized by the CTLH subunit MKLN1. These findings reveal how regulation of an E2 enzyme by cell cycle and nutrient signaling pathways dynamically shape CTLH activity.

cell biology↗

bioRxiv2025

Ribosome remodeling drives translation adaptation during viral infection and cellular stress

The ribosome is the highly conserved molecular machine that decodes mRNAs during protein synthesis. While traditionally thought to consist of a uniform set of proteins, here we discover that ribosome composition is reprogrammed to adapt to intrinsic and external cellular perturbations. During infection by non-segmented negative-sense viruses, viral entry into cells recruits the large ribosomal subunit protein rpL40 to a noncanonical site on the small subunit of 80S ribosomes near the mRNA entry site. These specialized ribosomes preferentially bind viral mRNAs to drive enhanced viral protein synthesis that is critical for replication under host pressures. Unexpectedly, we find that viruses have co-opted this translation pathway from a previously unrecognized endogenous ribosome remodeling program in which metabolic stress alters ribosome structure to promote mRNA translation required for cell survival. Thus, ribosome remodeling is a conserved mechanism enabling dynamic protein synthesis across pathogen and cellular adaptation.

molecular biology↗

bioRxiv2025

Shifted assembly and function of mSWI/SNF family subcomplexes underlie targetable dependencies in dedifferentiated endometrial carcinomas

The mammalian SWI/SNF (mSWI/SNF) family of chromatin remodelers govern cell type-specific chromatin accessibility and gene expression and assemble as three distinct complexes: canonical BAF (cBAF), Polybromo-associated BAF (PBAF), and non-canonical BAF (ncBAF). ARID1A and ARID1B are paralog subunits that specifically nucleate the assembly of cBAF complexes and are frequently co-mutated in highly aggressive dedifferentiated/undifferentiated endometrial carcinomas (DDEC/UECs). Here, in cellular models and primary human tumors, we find that ARID1A/B deficiency-mediated cBAF loss results in increased ncBAF and PBAF biochemical abundance and chromatin-level functions to maintain the DDEC oncogenic state. Further, treatment with clinical-grade SMARCA4/2 ATPase inhibitors markedly attenuates DDEC cell proliferation and tumor growth in vivo and synergizes with carboplatin-based chemotherapy to extend survival. These findings reveal the oncogenic contributions of shifted mSWI/SNF family complex stoichiometry and resulting gene regulatory dysregulation and suggest therapeutic utility of mSWI/SNF small molecule inhibitors in DDEC/UEC and other cBAF-disrupted cancer types.

molecular biology↗

bioRxiv2025

Catalytic pocket of Clr4 (Suv39h) methyltransfer-ase serves as a substrate receptor for Cullin 4-dependent histone H3 ubiquitination

Histone H3 lysine 9 (H3K9) methylation must be regulated to prevent inappropriate heterochromatin formation. Regulation of the conserved fission yeast H3K9 methyltransferase Clr4 (Suv39h) involves an automethylation-induced conformational switch and interaction of its catalytic SET domain with mono-ubiquitinated histone H3 lysine 14 (H3K14ub), a modification catalyzed by the Cul4 subunit of the CLRC complex. Using reconstituted CLRC, we show that Clr4 catalytic pocket serves as a substrate receptor for Cul4-dependent H3K14 ubiquitination. H3K14ub activates Clr4 to catalyze cis methylation of H3K9 on the same histone tail, while Clr4 auto-methylation enables H3K14ub-bound Clr4 to methylate H3K9 on an unmodified H3 tail in trans. Crosslinking and structural modeling reveal interactions between Clr4 chromo and SET domains, and between the chromo-domain and H3K14ub, suggesting that the chromodomain reads H3K9me3 and H3K14ub to allosterically regulate Clr4 activity. H3K14 ubiquitination therefore regulates Clr4 by promoting its recruitment and by positioning H3K9 in the active site.

biochemistry↗

bioRxiv2025

A cyclin-polarity feedback network ensures healthy cell proliferation

Healthy proliferation requires the coordination of cell cycle progression with cell polarity. In budding yeast, polarity is established when G1-cyclin-Cdc28Cdk1 triggers Cdc42 activation to generate a cell pole that is used as an axis for growth and division. While polarity defects delay the cell cycle temporally, permitting error correction, it is unknown if Cdc28Cdk1 directly rectifies errant polarity. Here, we identify an adaptive response where G1-cyclin-Cdc28Cdk1 participates in error correction via the augmentation of its kinase activity towards substrates that activate Cdc42. The response involves temporal and spatial cell cycle reconfiguration via extended G1 cyclin expression, nucleocytoplasmic rerouting and signaling. However, this strategy has a cost: if the defect is irreparable, high G1-cyclin levels enforce inexorable cell cycle commitment in the absence of a daughter cell, generating multinucleate cells. G1-cyclins therefore not only trigger G1 events, but also monitor their execution, employing feedback to coordinate polarity with cell cycle progression.

cell biology↗

bioRxiv2025

Ex vivo recapitulation of intramuscular mRNA vaccination with naive and recall antigens using a human Lymphoid Follicle Chip platform

Predicting the efficacy and toxicity of intramuscular mRNA vaccines remains challenging. Here, we describe an ex vivo human cell-based model that replicates immune responses to lipid nanoparticle (LNP)-based mRNA vaccines that require intramuscular injection. Vaccines are administered into a biomimetic muscle module containing human skeletal myoblasts and antigen-presenting cells (APCs) to mimic intramuscular vaccination, followed by transfer of the APCs and soluble factors to a microfluidic human lymphoid follicle chip (LF Chip) to mimic lymphatic drainage. Non-replicating mRNA vaccines directly induce antigen expression in APCs, whereas self-amplifying mRNA vaccines require muscle cell-APC contact within the intramuscular vaccination module. Transfer of APCs and soluble factors to the LF Chip induces LF expansion, de novo antigen-specific IgG production against a naive antigen (rabies virus glycoprotein), and cytokine release, with responses varying depending on LNP type. Vaccination of LF chips against SARS- COV-2 Spike recall antigen using the Moderna Spikevax vaccine generates neutralizing antibodies and induces somatic hypermutation. This biomimetic platform offers an all-human alternative for evaluating vaccine-induced immunity, potentially obviating the need for non-human primates and accelerating vaccine development.

immunology↗

bioRxiv2025

Insights into the Absence of Lymphoma Despite Fulminant Epstein-Barr Virus Infection in Patients with XIAP Deficiency

X-linked Lymphoproliferative Syndromes (XLP), which arise from mutations in the SH2D1A or XIAP genes, are characterized by the inability to control Epstein-Barr Virus (EBV) infection. While primary EBV infection triggers severe diseases in each, lymphomas occur at high rates with XLP-1 but not with XLP-2. Why XLP-2 patients are apparently protected from EBV-driven lymphomagenesis, in contrast to all other described congenital conditions that result in heightened susceptibility to EBV, remains a key open question. To gain insights, we cross-compared newly EBV infected versus immune stimulated B-cells from XLP-2 patients or upon XIAP CRISPR knockout, relative to healthy controls. XIAP perturbation impeded outgrowth of newly EBV-infected primary human B-cells, though had no impact on proliferation of B-cells stimulated by CD40 ligand and interleukin-21 or upon established EBV-immortalized lymphoblastoid cell lines (LCLs). B-cells from XLP-2 patients or in which XIAP was depleted by CRISPR editing exhibited a markedly lower EBV transformation efficiency than healthy control B-cells. Mechanistically, nascent EBV infection activated p53-mediated apoptosis signaling, whose effects on transforming B-cell death were counteracted by XIAP. In the absence of XIAP, EBV infection triggered high rates of apoptosis, not seen with CD40L/IL-21 stimulation. Moreover, inflammatory cytokines are present at high levels in XLP-2 patient serum with fulminant EBV infection, which heightened apoptosis induction in newly EBV-infected cells. These findings highlight the crucial role of XIAP in supporting early stages of EBV-driven B-cell immortalization and provide insights into the absence of EBV+ lymphoma in XLP-2 patients. Key pointsO_LIXIAP loss-of-function markedly impairs EBV+ B-cells outgrowth over the first week post-infection, particularly in the presence of IFN-{gamma}. C_LIO_LIXIAP mutation impedes EBV-driven B-cell transformation by potentiating p53-driven caspase activation and apoptosis. C_LI

microbiology↗

bioRxiv2024

Episodic Transport of Protein Aggregates Achieves a Positive Size Selectivity in Aggresome Formation

Eukaryotic cells direct toxic misfolded proteins to various protein quality control pathways based on their chemical features and aggregation status. Aggregated proteins are targeted to selective autophagy or specifically sequestered into the "aggresome," a perinuclear inclusion at the microtubule-organizing center (MTOC). However, the mechanism for selectively sequestering protein aggregates into the aggresome remains unclear. To investigate aggresome formation, we reconstituted MTOC-directed aggregate transport in Xenopus laevis egg extract using AgDD, a chemically inducible aggregation system. High-resolution single-particle tracking revealed that dynein-mediated transport of aggregates was highly episodic, with average velocity positively correlated with aggregate size. Our mechanistic model suggests that the recurrent formation of the dynein transport complex biases larger aggregates towards the active transport state, compensating for the slowdown due to viscosity. Both episodic transport and positive size selectivity are specifically associated with aggresome-dynein adaptors. Coupling conventional dynein-activating adaptors to the aggregates perturbs aggresome formation and reverses size selectivity.

cell biology↗

bioRxiv2023

Cell growth and nutrient availability control the mitotic exit signaling network in budding yeast

Cell growth is required for cell cycle progression. The amount of growth required for cell cycle progression is reduced in poor nutrients, which leads to a reduction in cell size. In budding yeast, nutrients influence cell size by modulating the duration and extent of bud growth, which occurs predominantly in mitosis. However, the mechanisms are unknown. Here, we used mass spectrometry to identify proteins that mediate the effects of nutrients on bud growth. This led to the discovery that nutrients regulate numerous components of the Mitotic Exit Network (MEN), which controls exit from mitosis. A key component of the MEN undergoes gradual multi-site phosphorylation during bud growth that is dependent upon growth and correlated with the extent of growth. Furthermore, activation of the MEN is sufficient to over-ride a growth requirement for mitotic exit. The data suggest a model in which the MEN integrates signals regarding cell growth and nutrient availability to ensure that mitotic exit occurs only when sufficient growth has occurred.

cell biology↗

bioRxiv2023

ALS-related p97 R155H mutation disrupts lysophagy in iPSC-derived motor neurons

Mutations in the AAA+ ATPase p97 (also known as valosin containing protein, VCP) cause multisystem proteinopathy 1 (MSP-1) which includes amyotrophic-lateral sclerosis (ALS); however, the pathogenic mechanisms that contribute to motor neuron loss in familial ALS caused by p97 mutations remain obscure. Here, we use two distinct induced pluripotent stem cell models differentiated into spinal motor neurons to investigate how p97 mutations perturb the motor neuron proteome. Using multiplexed quantitative proteomics in these cells, we find that motor neurons harboring the p97 R155H mutation have deficits in lysosome quality control and the selective autophagy of lysosomes (lysophagy). p97 R155H motor neurons are unable to efficiently clear damaged lysosomes and have reduced viability. Additionally, lysosomes in mutant motor neurons have increased pH compared to their wildtype counterparts. The endo-lysosomal damage repair (ELDR) complex is required for clearance of damaged lysosomes and involves UBXD1-p97 interaction which is disrupted in mutant motor neurons. Finally, we report that inhibition of the ATPase activity of p97 R155H using the ATP competitive inhibitor CB-5083 rescues lysophagy defects in mutant motor neurons. These results add to the increasing evidence that endo-lysosomal dysfunction is a key aspect of disease pathogenesis in p97-related disorders.

neuroscience↗

bioRxiv2023

Frontotemporal Dementia Patient Neurons With Progranulin Deficiency Display Protein Dyshomeostasis

Haploinsufficiency of progranulin (PGRN) causes frontotemporal dementia (FTD), a devastating neurodegenerative disease with no effective treatment. PGRN is required for efficient proteostasis, as loss of neuronal PGRN results in dysfunctional lysosomes and impaired clearance and cytoplasmic aggregation of TDP-43, a protein involved in neurodegeneration in FTD. These and other events lead to neurodegeneration and neuroinflammation. However, the detailed mechanisms leading to protein dyshomeostasis in PGRN-deficient cells remain unclear. We report here the development of human cell models of FTD with PGRN-deficiency to explore the molecular mechanisms underlying proteostasis breakdown and TDP-43 aggregation in FTD. Neurons differentiated from FTD patient induced pluripotent stem cells (iPSCs) have reduced PGRN levels, and the neurons recapitulate key disease features, including impaired lysosomal function, defective TDP-43 turnover and accumulation, neurodegeneration, and death. Proteomic analysis revealed altered levels of proteins linked to the autophagy-lysosome pathway (ALP) and the ubiquitin-proteasome system (UPS) in FTD patient neurons, providing new mechanistic insights into the link between PGRN-deficiency and disease pathobiology.

neuroscience↗

bioRxiv2022

Membrane curvature-generating proteins crucial for autophagosome formation

Autophagy is essential for cellular homeostasis and begins with the formation of a phagophore, a cup-like membrane sheet consisting of two closely apposed lipid bilayers connected by a highly curved rim. How the membrane sheet forms, bends, and eventually generates an autophagosome that enwraps cargo remains enigmatic. Specifically, it is unclear how the high membrane curvature of the phagophore rim, an energetically unfavorable state, is stabilized. Here, we demonstrate that phagophore formation requires the conserved, membrane curvature-generating REEP1 proteins. The REEP1 family proteins (REEP1-4 in vertebrates) differ from the related endoplasmic reticulum-shaping REEPs in abundance and membrane topology. In fission yeast, the single REEP1 ortholog is involved in both bulk and selective autophagy. It is recruited at early stages of phagophore formation and required for their maturation into autophagosomes. The function of REEP1 relies on its ability to generate high membrane curvature and its localization to the phagophore rim. Mammalian REEP1 proteins also associate with phagophores upon induction of autophagy and colocalize with early autophagic markers. We propose that REEP1 proteins stabilize the phagophores highly curved rim so that the two membrane sheets are kept in close proximity to form the autophagosome. Defective autophagy may underlie the effect of curvature-compromising mutations in human REEP1 proteins linked to neurological disease.

cell biology↗

bioRxiv2022

Determination of cross-tissue and tissue-specific aging changes in murine proteomes

Maintenance of protein homeostasis degrades with age, contributing to aging-related decline and disease. Previous studies have primarily surveyed transcriptional changes with age. To define the effects of age directly at the protein level, we performed discovery-based proteomics in 10 tissues from 20 C57BL/6J mice, representing both sexes at adult and late midlife ages (8 and 18 months). Consistent with previous studies, age-related changes in protein abundance often have no corresponding transcriptional change. Aging resulted in increases in immune protein abundance across all tissues, consistent with a global pattern of immune infiltration with age. Our protein-centric data revealed tissue-specific aging changes with potential functional consequences, including altered endoplasmic reticulum and protein trafficking in the spleen. We further observed changes in the stoichiometry of protein complexes with important roles in protein homeostasis such as the CCT/TriC complex and large ribosomal subunit. These data provide a foundation for understanding how proteins contribute to systemic aging across tissues.

systems biology↗

bioRxiv2022

Genetic dissection of the pluripotent proteome through multi-omics data integration

Genetic background is a major driver of phenotypic variability in pluripotent stem cells (PSCs). Most studies of variation in PSCs have relied on transcript abundance as the primary molecular readout of cell state. However, little is known about how proteins, the primary functional units in the cell, vary across genetically diverse PSCs, how protein abundance relates to variation in other cell characteristics, and how genetic background confers these effects. Here we present a comprehensive genetic study characterizing the pluripotent proteome of 190 unique mouse embryonic stem cell lines (mESCs) derived from genetically heterogeneous Diversity Outbred (DO) mice. The quantitative proteome is highly variable across DO mESCs, and we identified differentially activated pluripotency-associated pathways in the proteomics data that were not evident in transcriptome data from the same cell lines. Comparisons of protein abundance to transcript levels and chromatin accessibility show broad co-variation across molecular layers and variable correlation across samples, with some lines showing high and others low correlation between these multi-omics datasets. Integration of these three molecular data types using multi-omics factor analysis revealed shared and unique drivers of quantitative variation in pluripotency-associated pathways. QTL mapping localized the genetic drivers of this quantitative variation to a number of genomic hotspots, and demonstrated that multi-omics data integration consolidates the influence of genetic signals shared across molecular traits to increase QTL detection power and overcome the limitations inherent in mapping individual molecular features. This study reveals transcriptional and post-transcriptional mechanisms and genetic interactions that underlie quantitative variability in the pluripotent proteome, and in so doing provides a regulatory map for mouse ESCs that can provide a rational basis for future mechanistic studies, including studies of human PSCs.

genetics↗