Biology subjects

Gygi, S. P.

Publications and source records attributed to Gygi, S. P..

At least 19 recordsLinked to original sources

bioRxiv2026

Structures of MyD88 and its oncogenic mutant reveal DNA binding with implications for cytosolic DNA sensing

MyD88 is a universal adaptor that connects to Toll-like receptors and interleukin-1 receptors through its Toll/IL-1R homology (TIR) domain and activates inflammatory signaling via its death domain (DD). However, how MyD88TIR serves as the platform for these functions and if it has additional functions remain unclear. Here, we report cryogenic electron microscopy (cryo-EM) structures of MyD88TIR and its cancer-specific mutant L252P (previously L265P) mainly in ring and spiral forms, respectively. Both assemblies are formed by antiparallel double-filaments, using canonical TIR interfaces within each strand but unique surfaces between the strands, and position MyD88DD at the same sides of the assemblies for self-oligomerization and pathway activation. Surprisingly, these WT or L252P filaments are bound to dsDNA using the concave sides of the rings and the spirals. In vitro assays and cellular studies establish that MyD88 directly binds dsDNA and promotes inflammatory cytokine expression, particularly pro-IL-1{beta}, following dsDNA transfection or viral infection. Notably, these responses are unaffected by deficiency of UNC93B1, the essential chaperone required for trafficking endosomal nucleic acid-sensing TLRs. Structure-guide MyD88 mutant that disrupts dsDNA binding specifically compromises dsDNA-induced transcriptional program while maintaining TLR signaling. RNA-seq further reveals that MyD88 plays a crucial role in inducing gene expression upon dsDNA sensing and that about half of cGAS-dependent genes are also MyD88-dependent. Together, our structural, biochemical and genetic data support the findings that MyD88 directly engages with cytosolic dsDNA and cooperates with cGAS to promote the full innate immune transcriptional response.

immunology↗

bioRxiv2026

A multivalent docking platform and Rcn1-mediated inhibition control the extent of calcineurin recruitment to the cell division site for the dephosphorylation of multiple cytokinetic proteins

Cytokinesis requires coordinated signaling to ensure the accurate physical separation of daughter cells. Calcineurin (CN), a conserved Ca2+/calmodulin-dependent phosphatase, is required for cytokinesis in organisms ranging from yeast to humans, yet how CN is regulated at the division site and the substrates through which it promotes cell division remain poorly understood. Here we use the fission yeast Schizosaccharomyces pombe, which display striking cell division defects in the absence of CN, to define how CN is anchored at the cell division site. We show that CN recruitment to the cytokinetic ring (CR) requires its PxIxIT- and LxVP-binding surfaces and is mediated by multivalent interactions with the CR components paxillin-like Pxl1 and the F-BAR protein Cdc15. Disrupting these interactions nearly eliminates CN from the CR and causes gross cytokinetic defects similar to complete loss of CN function. Cell cycle stage-specific quantitative phosphoproteomics combined with proximity labeling-based proteomics were used to identify candidate CN substrates involved in cytokinesis. Validation of a cohort of these proteins localizing to the CR, including the F-BAR protein Rga7, the actin regulator Aim21 and three protein kinases, revealed that CN targets a broad network of structural and signaling components involved in cell division. We also identify the conserved CN inhibitor Rcn1 as a CN substrate and show that Rcn1 restricts CN accumulation at the CR to provide an additional layer of spatial regulation. Thus, spatial control of CN enables proper protein dephosphorylation for successful cytokinesis.

cell biology↗

bioRxiv2026

Activation and inactivation pathways of a p53-like transcription factor govern lipid homeostasis in yeast

Membrane fluidity depends on unsaturated acyl chains that are generated in Saccharomyces cerevisiae by the desaturase Ole1, whose expression is primarily under the control of the transcription factor Mga2. Here, we show that the endoplasmic reticulum-anchored Mga2 precursor is ubiquitinated by the E3 ligase Rsp5 and then processively degraded by the proteasome until proteolysis stalls at a defined site, releasing a soluble fragment that enters the nucleus and activates Ole1 transcription. Unexpectedly, Mga2 contains a DNA-binding domain and a trans-activation-like segment structurally and functionally related to those of the human tumor suppressor p53. The mature transcription factor is degraded in the nucleus in a DNA binding-dependent manner; blocking this degradation causes unsaturated acyl chains to accumulate in lipid droplets, a detoxification response required for cell viability. These findings define the pathways that activate and inactivate Mga2, and uncover an evolutionary connection between the yeast lipid homeostasis regulator Mga2 and p53.

cell biology↗

bioRxiv2026

2-Hydroxyglutarate Redirects Fatty Acid Partitioning to Mitigate Lipotoxic Stress and Preserve Metabolic Fuel

The role of 2-hydroxyglutarate in lipid metabolism is currently unknown. Here we show that 2HG redistributes the partitioning of fatty acids into triglyceride storage and away from membrane phospholipid synthesis, mitochondrial oxidation, and lipotoxic intermediates. In primary human cardiac and vascular cells, both enantiomers, D2HG and L2HG, expanded triglyceride stores and lipid droplets while selectively depleting phosphatidylethanolamine, with L2HG acting more potently than D2HG despite lower intracellular accumulation. Mechanistically, L2HG increases DGAT-dependent triglyceride synthesis, slows triglyceride turnover, and constrains the ethanolamine branch of the Kennedy pathway. This response limits fatty acid oxidation, long-chain acylcarnitine accumulation, and lipid peroxidation independently of pseudohypoxic transcription or canonical lipid storage regulators, while also remodeling the phosphoproteome and redox proteome. L2HG accumulation induces hypertriglyceridemia in mice, redistributes the acyl chain composition of cardiac triglycerides, and limits ischemia-induced acylcarnitine accumulation in the heart, mirroring a positive association between circulating 2HG and triglycerides in humans. Thus, 2HG expands metabolic flexibility from whether fatty acids are used as fuel to how that fuel is allocated among storage, membrane synthesis, and oxidation.

cell biology↗

bioRxiv2026

Lineage specifying transcription factors determine cell function by direct control of metabolism

Execution of a complex cellular function requires the coherent expression of a set of genes that encode the requisite biochemical program. It is believed that this is typically accomplished by employing common transcription factors to activate those genes, thus ensuring coherence. However, many of these programmatic gene sets are expressed at differing levels and in distinct configurations in different cell types. This creates a unique challenge for the logic of gene regulation--precisely tuning the expression of programs across cell types while maintaining coherence. Using the glycolysis pathway as a model of co-expressed genes with highly divergent cell-specific regulation, we developed a CRISPR screening approach to identify trans-acting factors that control RNA abundance. Using this platform, we discovered that lineage-specifying transcription factors directly activate glycolytic gene expression and alter metabolism. We showed that impaired metabolism disrupts lineage-specific activities via loss of specialized biochemical pathways and functions. We hypothesize that lineage-specifying transcription factors directly regulate metabolic gene expression to ensure that cellular metabolism is tuned to the specific demands imparted by specialized cell function.

cell biology↗

bioRxiv2026

Mitochondrial carrier SLC25A34 links clock, diet, and temperature control of interorganellar lipid cycling

Adipocyte lipid metabolism is coordinated by circadian rhythms, diet, and environmental temperature. Yet how these diverse signals are molecularly integrated remains unknown. Here we show that clock, diet, and temperature cues converge on the orphan mitochondrial transporter, SLC25A34, to orchestrate thermogenic cycling of lipid synthesis and oxidation. During sleep, the clock suppresses Slc25a34 transcription through REV-ERB. Waking, lipid-rich diets, or cold exposure abolish this repression, allowing lipolytic signals to stimulate Slc25a34 expression via PPAR. SLC25A34 then imports oxaloacetate into mitochondria to accelerate the export of substrates used for acetyl-CoA production in the cytosol. This feeds into cytosolic lipid synthesis and transcriptional induction of mitochondrial biogenesis, which collectively promote mitochondrial lipid oxidation. Thus, SLC25A34 confers circadian, dietary, and environmental control of thermogenic metabolism through interorganellar lipid cycling.

molecular biology↗

bioRxiv2026

High Resolution Multi-Pass Astral Analyzer Quantification Enables Highly Multiplexed 35-Plex Tandem Mass Tag Proteomics

Tandem mass tags (TMT) allow highly multiplexed and thus high-throughput, precisely quantitative proteomic analysis. Incorporation of additional deuterated reporter channels has near-doubled the multiplexation achieved with Thermo Scientific TMTpro reagents from 18 to 35-plex but requires extremely high [~]100k analyzer resolving power at m/z 128 to differentiate and quantify reporter ion channels, far beyond any single reflection time-of-flight analyzer, and exceeding the multi-reflection Thermo Scientific Astral analyzer in its standard operation. A multi-pass mode of Astral operation has been developed for the Thermo Scientific Orbitrap Astral Zoom mass spectrometer that triples the ion path to 90 m, more than doubling resolving power for a narrow m/z range. This "TMT HR mode" has been integrated into a new method of TMT proteomic analysis that splits regular MS2 analysis of labeled peptides into paired measurements comprising wide mass range scans for peptide identification, and TMT HR mode scans for reporter ion quantification. The method has been shown to accurately quantify 32-plex labeled HeLa protein lysate and provide far greater depth of analysis as state-of-the-art Orbitrap-only methods, while analysis of 11-plex labeled yeast showed no analytical depth sacrificed vs regular Orbitrap Astral TMT analysis. Further comparative measurements of a 2-cell line 35-plex sample demonstrated greater analytical depth, and similar quantitative precision, to "gold standard" Orbitrap MS3 methods.

biochemistry↗

bioRxiv2025

Proteome Landscapes Decode Organelle Vulnerabilities in cortical and dopaminergic-like induced neurons Across Lysosomal Storage Disorders

Lysosomes maintain cellular homeostasis by degrading proteins delivered via endocytosis and autophagy, and by recycling building blocks for organelle biogenesis. Lysosomal Storage Disorders (LSDs) comprise a group of diseases affecting diverse lysosomal functions. To facilitate molecular phenotyping across diverse LSD gene classes, we are developing a library of human embryonic stem cells engineered to lack individual LSD genes as a resource for the field. Here, we report our initial stem cell toolkit lacking one of 23 LSD genes, including the majority of genes associated with sphingolipidoses and neuronal ceroid lipofuscinoses, and its use in the generation of a proteomic resource for induced cortical-like and midbrain dopaminergic-like neurons In-depth abundance and correlation profiling across organelles and sub-organelle components revealed potential vulnerabilities that reflect distinct patterns of proteome alterations across both genotypes and neuronal cell types. We characterize alterations in the mitochondrial proteome associated with GBA1 and ASAH1 deficiency and identify synaptic and mitochondrial defects in ASAH1-/- induced neurons that correlate with defects in neuronal firing rates. Moreover, we developed an informatic pipeline for proteome-wide identification of individual protein interactions and protein complexes that may be disrupted as a result of LSD gene deficiency. Finally, we visualized structural alterations of ASAH1-deficient endolysosomes in situ using cryo-electron tomography, revealing swollen organelles that were largely devoid of dense internal membranes characteristic of wild-type cells, but containing numerous intralumenal vesicle compartments. This toolkit and associated proteomic landscapes provide a resource for defining molecular signatures associated with LSD gene dysfunction and organelle vulnerability.

cell biology↗

bioRxiv2025

Lysosome-dependent nutrient scavenging underliesstress adaptation during epithelial-to-mesenchymaltransition

Metastatic cancer cells invade tissue, overcome nutrient stress, and survive transit to distant sites. Many of the mechanisms that support these processes are incompatible with proliferation. This study defines cellular transition states in breast epithelial cells undergoing epithelial-mesenchymal transition (EMT) driven by ERK2 and TGF-{beta} signaling. EMT triggers robust endolysosomal system upregulation and metabolic adaptations that balance proliferative and invasive states. Surprisingly, invasive cells rely on scavenging via lysosomes and macropinocytosis to acquire amino acids, rather than plasma membrane transport, even in nutrient-rich conditions. Macropinocytosis increases intracellular amino acid storage, promoting survival during amino acid deprivation. This metabolic shift depends on c-MYC downregulation, an early EMT event. Reintroducing c-MYC suppresses the metabolic switch, endolysosomal induction, macropinocytosis, and the proliferation-to-migration transition. These findings reveal how cells dynamically balance proliferation and invasion, offering insights into transition states difficult to capture in models of breast cancer metastasis.

cell biology↗

bioRxiv2025

Next-Generation Multiplexed Targeted Proteomics Quantifies Post-Translational Modifications, Compound-Protein Interactions, and Disease Biomarkers with High Throughput

The GoDig platform enables sensitive, multiplexed targeted pathway proteomics without manual scheduling or synthetic standards. Here we present GoDig 2.0, which increases sample multiplexing to 35-fold, improves time efficiency and reduces scan delays for higher success rates, and allows flexible spectral and elution library generation from different mass spectrometry data types. GoDig 2.0 measures 2.4x more targets than GoDig 1.0, quantifying >99% of 800 peptides in a single run. We compiled a library of 23,989 human phosphorylation sites from a phosphoproteomic dataset and used it to profile kinase signaling differences across cell lines. In human brain tissue, we established a hyperphosphorylated tau assay including pTau127, revealing potential biomarkers for Alzheimers disease. We also quantified diglycyl-lysine peptides to assess polyubiquitin branching. Finally, we built a library of 20,946 reactive cysteines and profiled covalent compound-protein interactions spanning diverse pathways. GoDig 2.0 enables high-throughput analyses of site-specific protein modifications across many biological contexts.

biochemistry↗

bioRxiv2025

Library-Free Multiplexed Targeted Proteomics Enables Quantitative Protein-Level Genotyping

Existing targeted mass spectrometry methods require synthetic standard peptides, manual scan scheduling, or data libraries to quantify target proteins. Here we present GoDig-LiF, which uses spectra and retention times predicted by the Prosit-TMT model in place of data libraries, enabling targeted proteomics with only a tandem mass tag-labeled sample, target list, and mass spectrometer. We applied GoDig-LiF to the quantification of mutated proteins in cancer cell lines, including KRAS G13D.

bioinformatics↗

bioRxiv2025

HuD controls widespread RNA stability to drive neuronal activity-dependent responses

Neuronal activity shapes brain development and refines synaptic connectivity in part through dynamic changes in gene expression. While activity-regulated transcriptional programs have been extensively characterized, the holistic effects of neuronal activity on the full RNA life cycle remain relatively unexplored. Here, we show that neuronal activity influences multiple stages of RNA metabolism in vitro and in vivo. Among these, RNA stability emerges as a previously underappreciated regulator of gene expression, exerting a stronger influence than transcription on total RNA levels for [~]15% of activity-dependent genes. We go on to profile 3'UTR mRNA motifs that are sufficient to modulate activity-dependent mRNA stability and employ machine learning to identify the neuronal-specific RNA-binding protein HuD as a key regulator of activity-dependent mRNA stabilization. We demonstrate that HuD shapes activity-dependent mRNA abundance of hundreds of transcripts in both soma and distal neuronal processes and that neuronal activity drives the reorganization of HuD-interacting proteins, thereby stabilizing HuD-bound mRNAs and directing them into translationally active granules. Finally, we find that many variants associated with autism spectrum disorder (ASD) and other neurodevelopmental disorders disrupt or promote aberrant activity-dependent changes in mRNA stability. These findings reveal mRNA stability as a widespread mechanism of stimulus-responsive gene regulation in neurons with direct implications for the understanding of neurodevelopmental disorders.

neuroscience↗

bioRxiv2025

On-target toxicity limits the efficacy of CDK11 inhibition against cancers with 1p36 deletions

The cyclin-dependent kinase CDK11 is an understudied kinase that has been the subject of conflicting reports regarding its function in cancer. Here, we combine genetic and pharmacological approaches to demonstrate that CDK11 is a critical regulator of cancer cell survival that is required for RNA splicing and the expression of homologous recombination genes. Inhibition of CDK11 disrupts genome stability, promotes the retention of intronic sequences in mature mRNAs, and induces synthetic lethality with PARP inhibitors. Through integrative analysis of functional genomics datasets, we identify heterozygous deletions of chromosome 1p36 - which encompasses CDK11 and its activating cyclin CCNL2 - as a recurrent and predictive biomarker of sensitivity to CDK11 inhibition. To assess the therapeutic potential of CDK11, we develop MEL-495R, a selective and orally bioavailable CDK11 inhibitor. Additionally, we establish a genetically-engineered mouse model that allows us to differentiate between the on-target and off-target effects of CDK11 inhibitors in vivo. Using this platform, we demonstrate that MEL-495R induces widespread on-target toxicity, revealing a narrow therapeutic index. Together, these findings define CDK11 as a core cancer dependency, uncover a chromosomal deletion that sensitizes tumors to CDK11 inhibition, and provide a generalizable strategy for deconvolving drug efficacy and toxicity in vivo for novel oncology targets.

cancer biology↗

bioRxiv2025

A SWI/SNF-specific Ig-like domain, SWIFT, is a transcription factor binding platform

Mammalian SWI/SNF (BAF) chromatin remodeling complexes modulate DNA accessibility and gene expression, however, their genomic targeting mechanisms remain incompletely understood. Here, we identify SWIFT (SWI/SNF Ig-Fold for Transcription Factor Interactions), a conserved, broad transcription factor (TF) binding platform on the SMARCD subunits. SWIFT is necessary and sufficient for direct engagement with the transactivation domain of PU.1, a single mutation in which disrupts PU.1-mSWI/SNF binding, impairs complex targeting, and attenuates oncogenic transcription and proliferation in PU.1-dependent cancer cells. Dominant expression of the SWIFT domain in isolation sequesters TFs from mSWI/SNF and poisons TF-addicted cancer cells. Finally, TFs across diverse families interact with SMARCD paralog-specific SWIFT domains. These results define a major mechanism of cell type- and disease-specific mSWI/SNF chromatin targeting and inform approaches toward therapeutic modulation.

molecular biology↗

bioRxiv2025

Baseline expression of c-Myc defines the tissue specificity of oncogenic K-Ras

KRAS is among the most frequently mutated oncogenes in cancer. Yet, mutations in KRAS are common only in tumors originating from a subset of tissues. It is critical to understand the molecular mechanisms underlying this oncogene tissue specificity. Utilizing genetically engineered mouse models carrying a conditional oncogenic allele of Kras, we expressed activated K-Ras in adult tissues to investigate its specificity. We discovered that the ability of K-RasG12D to influence the fitness of cells in a given tissue is not determined by its canonical signaling through MAPK. Instead, low baseline expression of c-Myc renders tissues non-permissive to oncogenic K-Ras, a context that can be reversed in the liver by ectopically expressing c-Myc. This functions independently of the proliferative index of the tissue or the induction of cell cycle arrest or apoptosis. Our findings reveal the importance of the basal state of the tissue-inherent signaling network for determining oncogene specificity.

cancer biology↗

bioRxiv2025

Cell-surface proteomic profiling identifies CD72 as a regulator of microglial tiling

Microglial tiling--the phenomenon of consistent cell-to-cell distances and non-overlapping processes--is regarded as a qualitative indicator of homeostasis, but mechanisms of microglial tiling are unknown. We used cell-surface proximity labeling and mass spectrometry to profile the microglial cell-surface proteome in an in vitro model of homeostatic glial physiology and used single-cell RNA sequencing and public databases to identify candidate cell-surface proteins that might modulate tiling. We designed an image-based functional assay which measures six morphological/spatial readouts to screen these proteins for modulation of tiling. CD72, a coreceptor to the B cell receptor that is expressed by microglia, disrupted tiling; we validated its effects in vitro and in situ in organotypic hippocampal brain slices. Phosphoproteomic studies revealed that CD72 modulates pathways associated with cell adhesion, repulsive receptors, microglial activation, and cytoskeletal organization. These results lay the groundwork for further investigation of the functional roles of tiling in homeostasis and disease.

neuroscience↗

bioRxiv2025

Interrupting Elmsan1 repression of nuclear Acetyl-CoA production therapeutically reprograms cancer cells

Metabolites are essential substrates for epigenetic modifications. Although nuclear acetyl-CoA constitutes a small fraction of the whole cell pool, it regulates cell fate by locally providing histone acetylation substrate. Here, we combined phenotypic chemical screen and genome-wide CRISPR screen to demonstrate a nucleus-specific acetyl-CoA regulatory mechanism that can be modulated to achieve therapeutic cancer cell reprogramming. While previously thought that nucleus-localized pyruvate dehydrogenase complex (nPDC) is constitutively active, we found that nPDC is constitutively inhibited by the nuclear protein ELMSAN1 through direct interaction. Pharmacologic inhibition of the ELMSAN1-nPDC interaction derepressed nPDC activity, enhancing nuclear acetyl-CoA generation and reprogramming cancer cells to a postmitotic state with diminished cell-of-origin signatures. Reprogramming was synergistically enhanced by histone deacetylase 1/2 inhibition, resulting in inhibited tumor growth, durably suppressed tumor-initiating ability, and improved survival in multiple cancer types in vivo, including therapy-resistant sarcoma patient-derived xenografts and carcinoma cell line xenografts. Our findings highlight the potential of targeting ELMSAN1-nPDC as epigenetic cancer therapy.

cancer biology↗

bioRxiv2025

Deazaguanylation is a nucleobase-protein conjugation required for type IV CBASS immunity

7-deazapurines are nucleobase analogs essential for nucleic acid modifications in nearly all cellular life. Here, we discover a role for 7-deazapurines in protein modification within type IV CBASS anti-phage defense and define functions for CBASS ancillary proteins Cap9 and Cap10 in nucleobase-protein conjugation. A structure of Cap10 reveals a tRNA transglycosylase-family enzyme remodeled to bind the modified N-terminus of a partner cGAS/DncV-like nucleotidyltransferase linked to a 7-amido-7-deazaguanine (NDG) nucleobase. The structure of Cap9 explains how this QueC-like enzyme co-opts a 7-deazapurine biosynthetic reaction mechanism for NDG conjugation. We demonstrate that Cap9, Cap10, and NDG conjugation are essential for host defense against phage infection. Our results define a previously unknown 7-deazapurine protein modification and explain how nucleobase biosynthetic machinery has been repurposed for antiviral immunity.

microbiology↗