Biology subjects

Gygi, S. P.

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

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Muscle-specific increased expression of JAG1 improves skeletal muscle phenotype in dystrophin-deficient mice.

Therapeutic strategies for Duchenne Muscular Dystrophy (DMD) will likely require complementary approaches. One possibility is to explore genetic modifiers that improve muscle regeneration and function. The beneficial effects of the overexpression of Jagged-1 were described in escaper golden retriever muscular dystrophy (GRMD) dogs that had a near-normal life and validated in dystrophin-deficient zebrafish (1). To clarify the underlying biology of JAG1 overexpression in dystrophic muscles, we generated a transgenic mouse (mdx5cv-JAG1) model that lacks dystrophin and overexpresses human JAG1 in striated muscles. Skeletal muscles from mdx5cv-JAG1 and mdx5cv mice were studied at one, four, and twelve-month time points. JAG1 expression in mdx5cv-JAG1 increased by three to five times compared to mdx5cv. Consequently, mdx5cv-JAG1 muscles were significantly bigger and stronger than dystrophic controls, along with an increased number of myofibers. Proteomics data show increased dysferlin in mdx5cv-JAG1 muscles and an association of Nsd1 with the phenotype. Our data supports the positive effect of JAG1 overexpression in dystrophic muscles. Significance StatementDuchenne Muscular Dystrophy (DMD) patients present a progressive decline in motor function. DMD is caused by mutations in the DMD gene that lead to the absence of dystrophin - an essential component of muscle cells. However, dystrophin-deficient dogs overexpressing JAG1 had a normal lifespan with remarkable motor function. In this study, we increased expression of human JAG1 in mouse skeletal muscles lacking dystrophin to explore mechanisms responsible for these benefits. Our observations show that overexpression of JAG1 counterbalances the lack of dystrophin by generating bigger and stronger muscles as the mouse ages. Moreover, our proteomics dataset suggests a role of dysferlin in the phenotype. Therefore, our study supports the exploration of JAG1 in pre-clinical models.

physiology↗

bioRxiv2025

Structural basis for the midnolin-proteasome pathway and its role in suppressing myeloma

The midnolin-proteasome pathway degrades many nuclear proteins without ubiquitination, but how it operates mechanistically remains unclear. Here, we present structures of the midnolin-proteasome complex, revealing how established proteasomal components are repurposed to enable a unique form of proteolysis. While the proteasomal subunit PSMD2/Rpn1 binds to ubiquitinated or ubiquitin-like proteins, we discover that it also interacts with the midnolin nuclear localization sequence, elucidating how midnolins activity is confined to the nucleus. Likewise, PSMD14/Rpn11, an enzyme that normally cleaves ubiquitin chains, surprisingly functions non-enzymatically as a receptor for the midnolin ubiquitin-like (Ubl) domain, positioning the substrate-binding Catch domain directly above the proteasomal entry site to guide substrates into the proteasome. Moreover, we demonstrate that midnolin downregulation is critical for the survival of myeloma cells by promoting the expression of its transcription factor substrate IRF4. Our findings uncover the mechanisms underlying the midnolin-proteasome pathway and midnolin downregulation as a driver of multiple myeloma.

cell biology↗

bioRxiv2024

CEBPA repression by MECOM blocks differentiation to drive aggressive leukemias

Acute myeloid leukemias (AMLs) have an overall poor prognosis with many high-risk cases co-opting stem cell gene regulatory programs, yet the mechanisms through which this occurs remain poorly understood. Increased expression of the stem cell transcription factor, MECOM, underlies one key driver mechanism in largely incurable AMLs. How MECOM results in such aggressive AML phenotypes remains unknown. To address existing experimental limitations, we engineered and applied targeted protein degradation with functional genomic readouts to demonstrate that MECOM promotes malignant stem cell-like states by directly repressing pro-differentiation gene regulatory programs. Remarkably and unexpectedly, a single node in this network, a MECOM-bound cis-regulatory element located 42 kb downstream of the myeloid differentiation regulator CEBPA, is both necessary and sufficient for maintaining MECOM-driven leukemias. Importantly, targeted activation of this regulatory element promotes differentiation of these aggressive AMLs and reduces leukemia burden in vivo, suggesting a broadly applicable differentiation-based approach for improving therapy.

cancer biology↗

bioRxiv2024

Expanding the Landscape of Aging via Orbitrap Astral Mass Spectrometry and Tandem Mass Tag (TMT) Integration

Aging results in a progressive decline in physiological function due to the deterioration of essential biological processes, such as transcription and RNA splicing, ultimately increasing mortality risk. Although proteomics is emerging as a powerful tool for elucidating the molecular mechanisms of aging, existing studies are constrained by limited proteome coverage and only observe a narrow range of lifespan. To overcome these limitations, we integrated the Orbitrap Astral Mass Spectrometer with the multiplex tandem mass tag (TMT) technology to profile the proteomes of three brain tissues (cortex, hippocampus, striatum) and kidney in the C57BL/6JN mouse model, achieving quantification of 8,954 to 9,376 proteins per tissue (cumulatively 12,749 across all tissues). Our sample population represents balanced sampling across both sexes and three age groups (3, 12, and 20 months), comprising young adulthood to early late life (approximately 20-60 years of age for human lifespan). To enhance quantitative accuracy, we developed a peptide filtering strategy based on resolution and signal-to-noise thresholds. Our analysis uncovered distinct tissue-specific patterns of protein abundance, with age and sex differences in the kidney, while brain tissues exhibit notable age changes and limited sex differences. In addition, we identified both proteomic changes that are linear with age (i.e., continuous) and that have a non-linear pattern (i.e., non-continuous), revealing complex protein dynamics over the adult lifespan. Integrating our findings with early developmental proteomic data from brain tissues highlighted further divergent age-related trajectories, particularly in synaptic proteins. This study not only provides a robust data analysis workflow for TMT datasets generated using the Orbitrap Astral mass spectrometer but also expands the proteomic landscape of aging, capturing proteins with age and sex effects with unprecedented depth.

systems biology↗

bioRxiv2024

Isobaric Tagging and Data Independent Acquisition as Complementary Strategies for Proteome Profiling on an Orbitrap Astral Mass Spectrometer

Comprehensive global proteome profiling that is amenable to high throughput processing will broaden our understanding of complex biological systems. Here, we evaluated two leading mass spectrometry techniques, Data Independent Acquisition (DIA) and Tandem Mass Tagging (TMT), for extensive protein abundance profiling. DIA provides label-free quantification with a broad dynamic range, while TMT enables multiplexed analysis using isobaric tags for efficient cross-sample comparisons. We analyzed 18 samples, including four cell lines (IHCF, HCT116, HeLa, MCF7) under standard growth conditions, in addition to IHCF treated with two H2O2 concentrations, all in triplicate. Experiments were conducted on an Orbitrap Astral mass spectrometer, employing Field Asymmetric Ion Mobility Spectrometry (FAIMS). Despite utilizing different acquisition strategies, both the DIA and TMT approaches achieved comparable proteome depth and quantitative consistency, with each method quantifying over 10,000 proteins across all samples, with slightly more protein-level precision for the TMT strategy. Relative abundance correlation analysis showed strong agreement at both peptide and protein levels. Our findings highlight the complementary strengths of DIA and TMT for high-coverage proteomic studies, providing flexibility in method selection based on specific experimental needs.

biochemistry↗

bioRxiv2024

Direct mitochondrial import of lactate supports resilient carbohydrate oxidation

Lactate is the highest turnover circulating metabolite in mammals. While traditionally viewed as a waste product, lactate is an important energy source for many organs, but first must be oxidized to pyruvate for entry into the tricarboxylic acid cycle (TCA cycle). This reaction is thought to occur in the cytosol, with pyruvate subsequently transported into mitochondria via the mitochondrial pyruvate carrier (MPC). Using 13C stable isotope tracing, we demonstrated that lactate is oxidized in the myocardial tissue of mice even when the MPC is genetically deleted. This MPC-independent lactate import and mitochondrial oxidation is dependent upon the monocarboxylate transporter 1 (MCT1/Slc16a1). Mitochondria isolated from the myocardium without MCT1 exhibit a specific defect in mitochondrial lactate, but not pyruvate, metabolism. The import and subsequent mitochondrial oxidation of lactate by mitochondrial lactate dehydrogenase (LDH) acts as an electron shuttle, generating sufficient NADH to support respiration even when the TCA cycle is disrupted. In response to diverse cardiac insults, animals with hearts lacking MCT1 undergo rapid progression to heart failure with reduced ejection fraction. Thus, the mitochondrial import and oxidation of lactate enables carbohydrate entry into the TCA cycle to sustain cardiac energetics and maintain myocardial structure and function under stress conditions.

biochemistry↗

bioRxiv2024

Potent and Selective SETDB1 Covalent Negative Allosteric Modulator Reduces Methyltransferase Activity in Cells

A promising drug target, SETDB1, is a dual Kme reader and methyltransferase, which has been implicated in cancer and neurodegenerative disease progression. To help understand the role of the triple Tudor domain (3TD) of SETDB1, its Kme reader, we first identified a low micromolar small molecule ligand, UNC6535, which occupies simultaneously both the TD2 and TD3 reader binding sites. Further optimization led to the discovery of UNC10013, the first covalent 3TD ligand targeting Cys385 of SETDB1. UNC10013 is potent with a kinact/KI of 1.0 x 106 M-1s-1 and demonstrated proteome-wide selectivity. In cells, negative allosteric modulation of SETDB1-mediated Akt methylation was observed after treatment with UNC10013. Therefore, UNC10013 is a potent, selective and cell-active covalent ligand for the 3TD of SETDB1, demonstrating negative allosteric modulator properties and making it a promising tool to study the biological role of SETDB1 in disease progression. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/615363v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@6db8e2org.highwire.dtl.DTLVardef@1852ee3org.highwire.dtl.DTLVardef@c95bb6org.highwire.dtl.DTLVardef@1a43951_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Chromosome X Dosage Modulates Development of Aneuploidy in Genetically Diverse Mouse Embryonic Stem Cells

The genetic integrity of pluripotent stem cells (PSC) is critical to their applications in research and therapy, but it is compromised by frequent development of structural chromosome variants associated with malignancy. Many cell lines exhibit remarkable genetic stability, but little is known about the basis of the known variation in genomic integrity amongst different PSC isolates. Here we identify aneuploidies using RNA-seq and proteomics data from a panel of mouse embryonic stem cell (mESC) lines derived from 170 Diversity Outbred mice. We found 62 lines with detectable aneuploid subpopulations and a subset of originally XX lines that lost one Chromosome X (XO). Strikingly, a much lower proportion of XX lines were aneuploid, compared to XY or XO lines. Two single-cell RNA-seq data sets demonstrated that aneuploid XY DO mESC also show lower Chromosome X gene expression, and a prospective study confirmed that XY mESC accumulate higher aneuploid proportions in culture than isogenic XX lines. We identify potential mechanisms for this protective effect of X chromosome dosage, including our findings that the lines with two active X Chromosomes have a higher proportion of 2-cell-like cells, a state associated with maintenance of genetic integrity of mESC, and that they show differential expression of X-linked tumor suppressor genes associated with the DNA damage response. HighlightsO_LIFirst genetic analysis of predisposition to aneuploidy in pluripotent stem cell cultures C_LIO_LIChromosomal regions duplicated in aneuploid mouse embryonic stem cells are syntenic with regions overrepresented in human pluripotent stem cell lines bearing recurrent genetic abnormalities C_LIO_LIX-Chromosome dosage strongly influences susceptibility to aneuploidy in mouse embryonic stem cells and to a lesser degree in human pluripotent stem cells C_LIO_LIXX mouse embryonic stem cell lines show a higher proportion of cells in 2 cell-like state and higher expression of tumor suppressor genes associated with DNA damage response C_LI

genetics↗

bioRxiv2024

Ergothioneine boosts mitochondrial respiration and exercise performance via direct activation of MPST

Ergothioneine (EGT) is a diet-derived, atypical amino acid that accumulates to high levels in human tissues. Reduced EGT levels have been linked to age-related disorders, including neurodegenerative and cardiovascular diseases, while EGT supplementation is protective in a broad range of disease and aging models in mice. Despite these promising data, the direct and physiologically relevant molecular target of EGT has remained elusive. Here we use a systematic approach to identify how mitochondria remodel their metabolome in response to exercise training. From this data, we find that EGT accumulates in muscle mitochondria upon exercise training. Proteome-wide thermal stability studies identify 3-mercaptopyruvate sulfurtransferase (MPST) as a direct molecular target of EGT; EGT binds to and activates MPST, thereby boosting mitochondrial respiration and exercise training performance in mice. Together, these data identify the first physiologically relevant EGT target and establish the EGT-MPST axis as a molecular mechanism for regulating mitochondrial function and exercise performance.

molecular biology↗

bioRxiv2024

Inserting Pre-Analytical Chromatographic Priming Runs Significantly Improves Targeted Pathway Proteomics With Sample Multiplexing

GoDig, a recent platform for targeted pathway proteomics without the need for manual assay scheduling or synthetic standard peptides, is a relatively flexible and easy-to-use method that uses tandem mass tags (TMT) to increase sample throughput up to 18-fold relative to label-free targeted proteomics. Though the protein quantification success rate of GoDig is generally high, the peptide-level success rate is more limited, hampering the extension of GoDig to assays of harder-to-quantify proteins and site-specific phenomena. In order to guide the optimization of GoDig assays as well as improvements to the GoDig platform, we created GoDigViewer, a new stand-alone software that provides detailed visualizations of GoDig runs. GoDigViewer guided the implementation of "priming runs," an acquisition mode with significantly higher success rates due to improved elution order calibration. In this mode, one or more chromatographic priming runs are automatically performed to determine accurate and precise target elution orders, followed by analytical runs which quantify targets. Using priming runs, peptide-level quantification success rates exceeded 97% for a list of 400 peptide targets and 95% for a list of 200 targets that are usually not quantified using untargeted mass spectrometry. We used priming runs to establish a quantitative assay of 125 macroautophagy proteins that had a >95% success rate and revealed differences in macroautophagy protein expression profiles across four human cell lines.

cell biology↗

bioRxiv2024

Highly specific intracellular ubiquitination of a small molecule

Ubiquitin is a small, highly conserved protein that acts as a posttranslational modification in eukaryotes. Ubiquitination of proteins frequently serves as a degradation signal, marking them for disposal by the proteasome. Here, we report a novel small molecule from a diversity-oriented synthesis library, BRD1732, that is directly ubiquitinated in cells, resulting in dramatic accumulation of inactive ubiquitin monomers and polyubiquitin chains causing broad inhibition of the ubiquitin-proteasome system. Ubiquitination of BRD1732 and its associated cytotoxicity are stereospecific and dependent upon two homologous E3 ubiquitin ligases, RNF19A and RNF19B. Our finding opens the possibility for indirect ubiquitination of a target through a ubiquitinated bifunctional small molecule, and more broadly raises the potential for posttranslational modification in trans.

biochemistry↗

bioRxiv2024

Large-scale characterization of drug mechanism of action using proteome-wide thermal shift assays

In response to an ever-increasing demand of new small molecules therapeutics, numerous chemical and genetic tools have been developed to interrogate compound mechanism of action. Owing to its ability to approximate compound-dependent changes in thermal stability, the proteome-wide thermal shift assay has emerged as a powerful tool in this arsenal. The most recent iterations have drastically improved the overall efficiency of these assays, providing an opportunity to screen compounds at a previously unprecedented rate. Taking advantage of this advance, we quantified more than one million thermal stability measurements in response to multiple classes of therapeutic and tool compounds (96 compounds in living cells and 70 compounds in lysates). When interrogating the dataset as a whole, approximately 80% of compounds (with quantifiable targets) caused a significant change in the thermal stability of an annotated target. There was also a wealth of evidence portending off-target engagement despite the extensive use of the compounds in the laboratory and/or clinic. Finally, the combined application of cell-and lysate-based assays, aided in the classification of primary (direct ligand binding) and secondary (indirect) changes in thermal stability. Overall, this study highlights the value of these assays in the drug development process by affording an unbiased and reliable assessment of compound mechanism of action.

biochemistry↗

bioRxiv2023

Bidirectional substrate shuttling between the 26S proteasome and the Cdc48 ATPase promotes protein degradation

Most eukaryotic proteins are degraded by the 26S proteasome after modification with a polyubiquitin chain. Substrates lacking unstructured segments cannot be degraded directly and require prior unfolding by the Cdc48 ATPase (p97 or VCP in mammals) in complex with its ubiquitin-binding partner Ufd1-Npl4 (UN). Here, we use purified yeast components to reconstitute Cdc48-dependent degradation of well-folded model substrates by the proteasome. We show that a minimal system consists of the 26S proteasome, the Cdc48-UN ATPase complex, the proteasome cofactor Rad23, and the Cdc48 cofactors Ubx5 and Shp1. Rad23 and Ubx5 stimulate polyubiquitin binding to the 26S proteasome and the Cdc48-UN complex, respectively, allowing these machines to compete for substrates before and after their unfolding. Shp1 stimulates protein unfolding by the Cdc48-UN complex, rather than substrate recruitment. In vivo experiments confirm that many proteins undergo bidirectional substrate shuttling between the 26S proteasome and Cdc48 ATPase before being degraded.

biochemistry↗

bioRxiv2023

Substrate identification and specificity profiling of deubiquitylases against endogenously-generatedubiquitin-protein conjugates

Deubiquitylating enzymes (DUBs) remove ubiquitin from proteins thereby regulating their stability or activity. Our understanding of DUB-substrate specificity is limited because DUBs are typically not compared to each other against many physiological substrates. By broadly inhibiting DUBs in Xenopus egg extract, we generated hundreds of ubiquitylated proteins and compared the ability of 30 DUBs to deubiquitylate them using quantitative proteomics. We identified five high impact DUBs (USP7, USP9X, USP36, USP15 and USP24) that each reduced ubiquitylation of over ten percent of the isolated proteins. Candidate substrates of high impact DUBs showed substantial overlap and were enriched for disordered regions, suggesting this feature may promote substrate recognition. Other DUBs showed lower impact and non-overlapping specificity, targeting distinct non-disordered proteins including complexes such as the ribosome or the proteasome. Altogether our study identifies candidate DUB substrates and defines patterns of functional redundancy and specificity, revealing substrate characteristics that may influence DUB-substrate recognition.

biochemistry↗

bioRxiv2023

The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

Precise control of protein ubiquitination is essential for brain development, and hence, disruption of ubiquitin signaling networks can lead to neurological disorders. Mutations of the deubiquitinase USP7 cause the Hao-Fountain syndrome (HAFOUS), characterized by developmental delay, intellectual disability, autism, and aggressive behavior. Here, we report that conditional deletion of USP7 in excitatory neurons in the mouse forebrain triggers diverse phenotypes including sensorimotor deficits, learning and memory impairment, and aggressive behavior, resembling clinical features of HAFOUS. USP7 deletion induces neuronal apoptosis in a manner dependent of the tumor suppressor p53. However, most behavioral abnormalities in USP7 conditional mice persist despite p53 loss. Strikingly, USP7 deletion in the brain perturbs the synaptic proteome and dendritic spine morphogenesis independently of p53. Integrated proteomics analysis reveals that the neuronal USP7 interactome is enriched for proteins implicated in neurodevelopmental disorders and specifically identifies the RNA splicing factor Ppil4 as a novel neuronal substrate of USP7. Knockdown of Ppil4 in cortical neurons impairs dendritic spine morphogenesis, phenocopying the effect of USP7 loss on dendritic spines. These findings reveal a novel USP7-Ppil4 ubiquitin signaling link that regulates neuronal connectivity in the developing brain, with implications for our understanding of the pathogenesis of HAFOUS and other neurodevelopmental disorders.

neuroscience↗

bioRxiv2023

Synaptic Activity Causes Minute-scale Changes in BAF Complex Composition and Function

Genes encoding subunits of the SWI/SNF or BAF ATP-dependent chromatin remodeling complex are among the most enriched for deleterious de novo mutations in intellectual disabilities and autism spectrum disorder, but the causative molecular pathways are not fully known1,2. Synaptic activity in neurons is critical for learning and memory and proper neural development3. Neural activity prompts calcium influx and transcription within minutes, facilitated in the nucleus by various transcription factors (TFs) and chromatin modifiers4. While BAF is required for activity-dependent developmental processes such as dendritic outgrowth5-7, the immediate molecular consequences of neural activity on BAF complexes and their functions are unknown. Here we mapped minute-scale biochemical consequences of neural activity, modeled by membrane depolarization of embryonic mouse primary cortical neurons, on BAF complexes. We used acute chemical perturbations of BAF ATPase activity and kinase signaling to define the activity-dependent effects on BAF complexes and activity-dependent BAF functions. Our studies found that BAF complexes change in subunit composition and are selectively phosphorylated within 10 minutes of depolarization. Increased levels of the core PBAF subunit Baf200/Arid2, uniquely containing an RFX-like DNA-binding domain, are concurrent with ATPase-dependent opening of chromatin at RFX/X-box motifs. Changes in BAF composition and phosphorylation lead to the regulation of chromatin accessibility for critical neurogenesis TFs. These biochemical effects are a convergent phenomenon downstream of multiple growth factor signaling pathways in mouse neurons and fibroblasts suggesting that BAF integrates signaling information from the membrane. In support of such a membrane-to-nucleus signaling cascade, we also identified a BAF-interacting kinase, Dclk2, whose inhibition attenuates BAF phosphorylation selectively. Our findings support a direct role of BAF complexes in responding to synaptic activity to regulate TF binding and transcription.

molecular biology↗

bioRxiv2023

Selective Mitochondrial Respiratory Complex I Subunit Deficiency Causes Tumor Immunogenicity

Targeting of specific metabolic pathways in tumor cells has the potential to sensitize them to immune-mediated attack. Here we provide evidence for a specific means of mitochondrial respiratory Complex I (CI) inhibition that improves tumor immunogenicity and sensitivity to immune checkpoint blockade (ICB). Targeted genetic deletion of the CI subunits Ndufs4 and Ndufs6, but not other subunits, induces an immune-dependent tumor growth attenuation in mouse melanoma models. We show that deletion of Ndufs4 induces expression of the transcription factor Nlrc5 and genes in the MHC class I antigen presentation and processing pathway. This induction of MHC-related genes is driven by an accumulation of pyruvate dehydrogenase-dependent mitochondrial acetyl-CoA downstream of CI subunit deletion. This work provides a novel functional modality by which selective CI inhibition restricts tumor growth, suggesting that specific targeting of Ndufs4, or related CI subunits, increases T-cell mediated immunity and sensitivity to ICB.

cancer biology↗

bioRxiv2023

Multifaceted proteome analysis at solubility, redox, and expression dimensions for target identification

Multifaceted interrogation of the proteome deepens the system-wide understanding of biological systems; however, mapping the redox changes in the proteome has so far been significantly more challenging than expression and solubility/stability analyses. Here, we devise the first high- throughput redox proteomics approach integrated with expression analysis (REX) and combine it with Proteome Integral Solubility Alteration (PISA) assay. The whole PISA-REX experiment with up to four biological replicates can be multiplexed into a single tandem mass tag TMTpro set. For benchmarking this compact tool, we analyzed HCT116 cells treated with auranofin, showing great improvement compared with previous such studies. Then we applied PISA-REX to study proteome remodeling upon stimulation of human monocytes by interferon . We also studied the proteome changes in plasmacytoid dendritic cells isolated from wild type vs. Ncf1- mutant mice treated with interferon , showing that NCF1 deficiency enhances the STAT1 pathway and modulates the expression, solubility and redox state of interferon-induced proteins. Providing comprehensive multifaceted information on the proteome, the compact PISA-REX has the potential to become an industry standard in proteomics and to open new windows into the biology of health and disease.

biochemistry↗