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Deubiquitinases (DUBs) antagonize protein ubiquitination by removing ubiquitin from substrates. Identifying the physiological substrates of each DUB is critical for understanding DUB function and the principles that govern the specificity of this class of enzymes. Since multiple DUBs can act on the same substrate, it can be challenging to identify substrates using inactivating a single enzyme. Here, we outline a method that enables the identification of proteins whose stability depends on DUB activity and an approach to profile DUB specificity in Xenopus egg extract. By coupling broad DUB inhibition with quantitative proteomics, we circumvent DUB redundancy to identify DUB substrates. By adding back recombinant DUBs individually to the extract, we pinpoint DUBs sufficient to counteract proteasomal degradation of these newly identified substrates. We apply this method to Xenopus egg extract but suggest that it can also be adapted to other cell lysates.

Alzheimer’s disease (AD) pathologies were discovered in the accessible neurosensory retina. However, their exact nature and topographical distribution, particularly in the early stages of functional impairment, and how they relate to disease progression in the brain remain largely unknown. To better understand the pathological features of AD in the retina, we conducted an extensive histopathological and biochemical investigation of postmortem retina and brain tissues from 86 human donors. Quantitative examination of superior and inferior temporal retinas from mild cognitive impairment (MCI) and AD patients compared to those with normal cognition (NC) revealed significant increases in amyloid β-protein (Aβ42) forms and novel intraneuronal Aβ oligomers (AβOi), which were closely associated with exacerbated retinal macrogliosis, microgliosis, and tissue atrophy. These pathologies were unevenly distributed across retinal layers and geometrical areas, with the inner layers and peripheral subregions exhibiting most pronounced accumulations in the MCI and AD versus NC retinas. While microgliosis was increased in the retina of these patients, the proportion of microglial cells engaging in Aβ uptake was reduced. Female AD patients exhibited higher levels of retinal microgliosis than males. Notably, retinal Aβ42, S100 calcium-binding protein B+ macrogliosis, and atrophy correlated with severity of brain Aβ pathology, tauopathy, and atrophy, and most retinal pathologies reflected Braak staging. All retinal biomarkers correlated with the cognitive scores, with retinal Aβ42, far-peripheral AβOi and microgliosis displaying the strongest correlations. Proteomic analysis of AD retinas revealed activation of specific inflammatory and neurodegenerative processes and inhibition of oxidative phosphorylation/mitochondrial, and photoreceptor-related pathways. This study identifies and maps retinopathy in MCI and AD patients, demonstrating the quantitative relationship with brain pathology and cognition, and may lead to reliable retinal biomarkers for noninvasive retinal screening and monitoring of AD.

Background: ALPK3 encodes α-kinase 3, a muscle-specific protein of unknown function. ALPK3 loss-of-function variants cause cardiomyopathy with distinctive clinical manifestations in both children and adults, but the molecular functions of ALPK3 remain poorly understood. Methods: We explored the putative kinase activity of ALPK3 and the consequences of damaging variants using isogenic human induced pluripotent stem cell–derived cardiomyocytes, mice, and human patient tissues. Results: Multiple sequence alignment of all human α-kinase domains and their orthologs revealed 4 conserved residues that were variant only in ALPK3, demonstrating evolutionary divergence of the ALPK3 α-kinase domain sequence. Phosphoproteomic evaluation of both ALPK3 kinase domain inhibition and overexpression failed to detect significant changes in catalytic activity, establishing ALPK3 as a pseudokinase. Investigations into alternative functions revealed that ALPK3 colocalized with myomesin proteins (MYOM1, MYOM2) at both the nuclear envelope and the sarcomere M-band. ALPK3 loss-of-function variants caused myomesin proteins to mislocalize and also dysregulated several additional M-band proteins involved in sarcomere protein turnover, which ultimately impaired cardiomyocyte structure and function. Conclusions: ALPK3 is an essential cardiac pseudokinase that inserts in the nuclear envelope and the sarcomere M-band. Loss of ALPK3 causes mislocalization of myomesins, critical force-buffering proteins in cardiomyocytes, and also dysregulates M-band proteins necessary for sarcomere protein turnover. We conclude that ALPK3 cardiomyopathy induces ventricular dilatation caused by insufficient myomesin-mediated force buffering and hypertrophy by impairment of sarcomere proteostasis.

Advanced gastrointestinal stromal tumour (GIST) is characterised by genomic perturbations of key cell cycle regulators. Oncogenic activation of CDK4/6 results in RB1 inactivation and cell cycle progression. Given that single-agent CDK4/6 inhibitor therapy failed to show clinical activity in advanced GIST, we evaluated strategies for maximising response to therapeutic CDK4/6 inhibition.Targeted next-generation sequencing and multiplexed protein imaging were used to detect cell cycle regulator aberrations in GIST clinical samples. The impact of inhibitors of CDK2, CDK4 and CDK2/4/6 was determined through cell proliferation and protein detection assays. CDK-inhibitor resistance mechanisms were characterised in GIST cell lines after long-term exposure.We identify recurrent genomic aberrations in cell cycle regulators causing co-activation of the CDK2 and CDK4/6 pathways in clinical GIST samples. Therapeutic co-targeting of CDK2 and CDK4/6 is synergistic in GIST cell lines with intact RB1, through inhibition of RB1 hyperphosphorylation and cell proliferation. Moreover, RB1 inactivation and a novel oncogenic cyclin D1 resulting from an intragenic rearrangement (CCND1::chr11.g:70025223) are mechanisms of acquired CDK-inhibitor resistance in GIST.These studies establish the biological rationale for CDK2 and CDK4/6 co-inhibition as a therapeutic strategy in patients with advanced GIST, including metastatic GIST progressing on tyrosine kinase inhibitors.

Human genomic DNA contains a number of diverse repetitive sequence motifs, often identified as fragile sites leading to genetic instability. Among them, expansion events occurring at triplet repeats have been extensively studied due to their association with neurological disorders, including Huntington’s disease (HD). In the case of HD, expanded CAG triplet repeats in the HTT gene are thought to cause the onset. The expansion of CAG triplet repeats is believed to be triggered by the emergence of stem-loops composed of CAG triplet repeats, while the underlying molecular mechanisms are largely unknown. Therefore, identifying proteins recruited on such stem loops would be useful to understand the molecular mechanisms leading to the genetic instability of CAG triplet repeats. We previously developed a plasmid DNA pull-down methodology that captures proteins specifically assembled on any sequence of interest using nuclear extracts. Analysis by Mass Spectrometry revealed that among the proteins specifically bound to a stem-loop composed of CAG triplet repeats, many turned out to belong to DNA repair pathways. We expect our data set to represent a useful entry point for the design of assays allowing the molecular mechanisms of genetic instability at CAG triplet repeats to be explored.

Peroxisomes are ubiquitous organelles that house various metabolic reactions and are essential for human health1–4. Luminal peroxisomal proteins are imported from the cytosol by mobile receptors, which then recycle back to the cytosol by a poorly understood process1–4. Recycling requires receptor modification by a membrane-embedded ubiquitin ligase complex comprising three RING finger domain-containing proteins (Pex2, Pex10 and Pex12)5,6. Here we report a cryo-electron microscopy structure of the ligase complex, which together with biochemical and in vivo experiments reveals its function as a retrotranslocation channel for peroxisomal import receptors. Each subunit of the complex contributes five transmembrane segments that co-assemble into an open channel. The three ring finger domains form a cytosolic tower, with ring finger 2 (RF2) positioned above the channel pore. We propose that the N terminus of a recycling receptor is inserted from the peroxisomal lumen into the pore and monoubiquitylated by RF2 to enable extraction into the cytosol. If recycling is compromised, receptors are polyubiquitylated by the concerted action of RF10 and RF12 and degraded. This polyubiquitylation pathway also maintains the homeostasis of other peroxisomal import factors. Our results clarify a crucial step during peroxisomal protein import and reveal why mutations in the ligase complex cause human disease.

The yeast, Saccharomyces cerevisiae, is a widely used model system for investigating conserved biological functions and pathways. Advancements in sample multiplexing have facilitated the study of the yeast proteome, yet many yeast proteins remain uncharacterized or only partially characterized. Yeast deletion strain collections are powerful resources for yeast proteome studies, uncovering the effects of gene function, genetic interactions, and cellular stresses. As complex biological systems cannot be understood by simply analyzing the individual components, a systems approach is often required in which a protein is represented as a component of large, interacting networks. Here, we evaluate the current state of yeast proteome analysis using isobaric tag-based sample multiplexing (TMTpro16) to profile the proteomes of 75 yeast deletion strains for which we measured the abundance of nearly 5000 proteins. Using statistical approaches, we highlighted covariance and regulation subnetworks and the enrichment of gene ontology classifications for covarying and coregulated proteins. This dataset presents a resource that is amenable to further data mining to study individual deletion strains, pathways, proteins, and/or interactions thereof while serving as a template for future network-based investigations using yeast deletion strain collections.

Glaucoma is an age-related neurodegenerative disorder characterized by retinal ganglion cell (RGC) degeneration and excavation of the optic nerve head (ONH). It is associated with an increase in intraocular pressure (IOP) and progressive decline in the visual field. Reduction in the retrograde axonal transport of neurotrophic factors such as brain-derived neurotrophic factor (BDNF) from the brain to the neuronal cell bodies in retina, has been suggested as one of the key mechanisms underlying selective degeneration of ganglion cells and optic nerve in glaucoma. Multiple studies have indicated that BDNF and its high affinity receptor Tropomyosin receptor kinase B (TrkB) play crucial roles in survival of RGCs and that upregulating BDNF/TrkB signalling using gene therapy can protect the ganglion cells against degeneration. This study corroborates previous findings and demonstrates that glaucoma is associated with downregulation of TrkB downstream signalling and enhanced levels of amyloid β (Aβ 1–42) accumulation in the retina. 7,8 dihydroxyflavone (7,8 DHF) is a TrkB agonist and regular administration of this compound imparted significant protection against loss of GCL density and preserved inner retinal function in experimental glaucoma models. 7,8 DHF treatment stimulated activation of TrkB intracellular signalling as well as ameliorated the increase in the levels of soluble Aβ (1–42) in the retinas of rats and mice exposed to high IOP. The protective effects of 7,8 DHF were also evident in BDNF(+/−) mice indicating that TrkB agonist mediated activation of TrkB signalling was not altered upon BDNF allelic impairment. These data support BDNF/TrkB axis as a promising therapeutic target in glaucoma and highlight that the detrimental effects of high IOP exposure can be compensated by the exogenous administration of a TrkB agonist.

Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) are histone-modifying and -binding complexes that mediate the formation of facultative heterochromatin and are required for silencing of developmental genes and maintenance of cell fate1–3. Multiple pathways of RNA decay work together to establish and maintain heterochromatin in fission yeast, including a recently identified role for a conserved RNA-degradation complex known as the rixosome or RIX1 complex4–6. Whether RNA degradation also has a role in the stability of mammalian heterochromatin remains unknown. Here we show that the rixosome contributes to silencing of many Polycomb targets in human cells. The rixosome associates with human PRC complexes and is enriched at promoters of Polycomb target genes. Depletion of either the rixosome or Polycomb results in accumulation of paused and elongating RNA polymerase at Polycomb target genes. We identify point mutations in the RING1B subunit of PRC1 that disrupt the interaction between PRC1 and the rixosome and result in diminished silencing, suggesting that direct recruitment of the rixosome to chromatin is required for silencing. Finally, we show that the RNA endonuclease and kinase activities of the rixosome and the downstream XRN2 exoribonuclease, which degrades RNAs with 5′ monophosphate groups generated by the rixosome, are required for silencing. Our findings suggest that rixosomal degradation of nascent RNA is conserved from fission yeast to human, with a primary role in RNA degradation at facultative heterochromatin in human cells.

Cancer cell energy metabolism plays an important role in dictating the efficacy of oncolysis by oncolytic viruses. To understand the role of multiple myeloma metabolism in reovirus oncolysis, we performed semi-targeted mass spectrometry-based metabolomics on 12 multiple myeloma cell lines and revealed a negative correlation between NAD+ levels and susceptibility to oncolysis. Likewise, a negative correlation was observed between the activity of the rate-limiting NAD+ synthesis enzyme NAMPT and oncolysis. Indeed, depletion of NAD+ levels by pharmacological inhibition of NAMPT using FK866 sensitized several myeloma cell lines to reovirus-induced killing. The myelomas that were most sensitive to this combination therapy expressed a functional p53 and had a metabolic and transcriptomic profile favoring mitochondrial metabolism over glycolysis, with the highest synergistic effect in KMS12 cells. Mechanistically, U

Tumor neurogenesis, a process by which new nerves invade tumors, is a growing area of interest in cancer research. Nerve presence has been linked to aggressive features of various solid tumors, including breast and prostate cancer. A recent study suggested that the tumor microenvironment may influence cancer progression through recruitment of neural progenitor cells from the central nervous system. However, the presence of neural progenitors in human breast tumors has not been reported. Here, we investigate the presence of Doublecortin (DCX) and Neurofilament-Light (NFL) co-expressing (DCX+/NFL+) cells in patient breast cancer tissue using Imaging Mass Cytometry. To map the interaction between breast cancer cells and neural progenitor cells further, we created anin vitromodel mimicking breast cancer innervation, and characterized using mass spectrometry-based proteomics on the two cell types as they co- evolved in co-culture. Our results indicate stromal presence of DCX+/NFL+ cells in breast tumor tissue from a cohort of 107 patient cases, and that neural interaction contribute to drive a more aggressive breast cancer phenotype in our co-culture models. Our results support that neural involvement plays an active role in breast cancer and warrants further studies on the interaction between nervous system and breast cancer progression.

The relaxin family peptide receptor 1 (RXFP1) is the receptor for relaxin-2, an important regulator of reproductive and cardiovascular physiology. RXFP1 is a multi-domain G protein-coupled receptor (GPCR) with an ectodomain consisting of an LDLa module and leucine-rich repeats. The mechanism of RXFP1 signal transduction is clearly distinct from that of other GPCRs, but remains very poorly understood. Here, we present the cryo-electron microscopy structure of active-state human RXFP1, bound to a single-chain version of the endogenous agonist relaxin-2 and to the heterotrimeric Gs protein. Evolutionary coupling analysis and structure-guided functional experiments reveal that RXFP1 signals through a mechanism of autoinhibition, wherein the receptor’s extracellular loop 2 occupies the orthosteric site in the active state but is inhibited by the ectodomain in the absence of relaxin-2. Our results explain how an unusual GPCR family functions, providing a path to rational drug development targeting the relaxin receptors.

GSEA analysis of spleens expressing WT or mutant K-Ras.

Proteomics data from human cell lines.

Supplementary Figures S1-S18

Photoreceptor cells are highly susceptible to oxidative-stress-induced damage due to their high metabolic rate. Oxidative stress plays a key role in driving pathological events in several different ocular diseases, which lead to retinal degeneration and ultimately blindness. A growing number of studies have been performed to understand downstream events caused by ROS induced oxidative stress in photoreceptor cells; however, the underlying mechanisms of ROS toxicity are not fully understood. To shed light on ROS induced downstream pathological events, we employed a tandem mass tag (TMT) labelling-based quantitative mass-spectrometric approach to determine proteome changes in 661W photoreceptor cells following oxidative stress induction via the application of different concentrations of H2O2 at different time points. Overall, 5920 proteins were identified and quantified, and 450 differentially expressed proteins (DEPs) were identified, which were altered in a dose and time dependent manner in all treatment groups compared to the control group. These proteins were involved in several biological pathways, including spliceosome and ribosome response, activated glutathione metabolism, decreased ECM-receptor interaction, oxidative phosphorylation, abnormally regulated lysosome, apoptosis, and ribosome biogenesis. Our results highlighted ECM receptor interaction, oxidative phosphorylation and spliceosome pathways as the major targets of oxidative stress that might mediate vascular dysfunction and cellular senescence.

Pancreatic ductal adenocarcinoma (PDAC) evolves a complex microenvironment comprised of multiple cell types, including pancreatic stellate cells (PSC). Previous studies have demonstrated that stromal supply of alanine, lipids, and nucleotides supports the metabolism, growth, and therapeutic resistance of PDAC. Here we demonstrate that alanine cross-talk between PSCs and PDAC is orchestrated by the utilization of specific transporters. PSCs utilize SLC1A4 and other transporters to rapidly exchange and maintain environmental alanine concentrations. Moreover, PDAC cells upregulate SLC38A2 to supply their increased alanine demand. Cells lacking SLC38A2 fail to concentrate intracellular alanine and undergo a profound metabolic crisis resulting in markedly impaired tumor growth. Our results demonstrate that stromal–cancer metabolic niches can form through differential transporter expression, creating unique therapeutic opportunities to target metabolic demands of cancer.Significance:This work identifies critical neutral amino acid transporters involved in channeling alanine between pancreatic stellate and PDAC cells. Targeting PDAC-specific alanine uptake results in a metabolic crisis impairing metabolism, proliferation, and tumor growth. PDAC cells specifically activate and require SLC38A2 to fuel their alanine demands that may be exploited therapeutically.This article is highlighted in the In This Issue feature, p. 890

SummaryThe role of proteostasis and organelle homeostasis dysfunction in human aging and Alzheimer’s disease (AD) remains unclear. Analyzing proteome-wide changes in human donor fibroblasts and their corresponding transdifferentiated neurons (tNeurons), we find aging and AD synergistically impair multiple proteostasis pathways, most notably lysosomal quality control (LQC). In particular, we show that ESCRT-mediated lysosomal repair defects are associated with both sporadic and PSEN1 familial AD. Aging- and AD-linked defects are detected in fibroblasts but highly exacerbated in tNeurons, leading to enhanced neuronal vulnerability, unrepaired lysosomal damage, inflammatory factor secretion and cytotoxicity. Surprisingly, tNeurons from aged and AD donors spontaneously develop amyloid-β inclusions co-localizing with LQC markers, LAMP1/2-positive lysosomes and proteostasis factors; we observe similar inclusions in brain tissue from AD patients and APP-transgenic mice. Importantly, compounds enhancing lysosomal function broadly ameliorate these AD-associated pathologies. Our findings establish cell-autonomous LQC dysfunction in neurons as a central vulnerability in aging and AD pathogenesis.

Genetic background drives phenotypic variability in pluripotent stem cells (PSCs). Most studies to date have used transcript abundance as the primary molecular readout of cell state in PSCs. We performed a comprehensive proteogenomics analysis of 190 genetically diverse mouse embryonic stem cell (mESC) lines. The quantitative proteome is highly variable across lines, and we identified pluripotency-associated pathways that were differentially activated in the proteomics data that were not evident in transcriptome data from the same lines. Integration of protein abundance to transcript levels and chromatin accessibility revealed broad co-variation across molecular layers as well as shared and unique drivers of quantitative variation in pluripotency-associated pathways. Quantitative trait locus (QTL) mapping localized the drivers of these multi-omic signatures to genomic hotspots. This study reveals post-transcriptional mechanisms and genetic interactions that underlie quantitative variability in the pluripotent proteome and provides a regulatory map for mESCs that can provide a basis for future mechanistic studies.

The intimate association between the endoplasmic reticulum (ER) and mitochondrial membranes at ER-Mitochondria contact sites (ERMCS) is a platform for critical cellular processes, particularly lipid synthesis. How contacts are remodeled and the impact of altered contacts on lipid metabolism remains poorly understood. We show that the p97 AAA-ATPase and its adaptor ubiquitin-X domain adaptor 8 (UBXD8) regulate ERMCS. The p97-UBXD8 complex localizes to contacts and its loss increases contacts in a manner that is dependent on p97 catalytic activity. Quantitative proteomics and lipidomics of ERMCS demonstrates alterations in proteins regulating lipid metabolism and a significant change in membrane lipid saturation upon UBXD8 deletion. Loss of p97-UBXD8 increased membrane lipid saturation via SREBP1 and the lipid desaturase SCD1. Aberrant contacts can be rescued by unsaturated fatty acids or overexpression of SCD1. We find that the SREBP1-SCD1 pathway is negatively impacted in the brains of mice with p97 mutations that cause neurodegeneration. We propose that contacts are exquisitely sensitive to alterations to membrane lipid composition and saturation.

Targeted proteomics enables hypothesis-driven research by measuring the cellular expression of protein cohorts related by function, disease, or class after perturbation. Here, we present a pathway-centric approach and an assay builder resource for targeting entire pathways of up to 200 proteins selected from >10,000 expressed proteins to directly measure their abundances, exploiting sample multiplexing to increase throughput by 16-fold. The strategy, termed GoDig, requires only a single-shot LC-MS analysis, ~1 µg combined peptide material, a list of up to 200 proteins, and real-time analytics to trigger simultaneous quantification of up to 16 samples for hundreds of analytes. We apply GoDig to quantify the impact of genetic variation on protein expression in mice fed a high-fat diet. We create several GoDig assays to quantify the expression of multiple protein families (kinases, lipid metabolism- and lipid droplet-associated proteins) across 480 fully-genotyped Diversity Outbred mice, revealing protein quantitative trait loci and establishing potential linkages between specific proteins and lipid homeostasis.

CD8 T cells play a central role in antiviral immunity. Type I interferons are among the earliest responders after virus exposure and can cause extensive reprogramming and antigen-independent bystander activation of CD8 T cells. Although bystander activation of pre-existing memory CD8 T cells is known to play an important role in host defense and immunopathology, its impact on naïve CD8 T cells remains underappreciated. Here we report that exposure to reovirus, both in vitro or in vivo, promotes bystander activation of naïve CD8 T cells within 24 hours and that this distinct subtype of CD8 T cell displays an innate, antiviral, type I interferon sensitized signature. The induction of bystander naïve CD8 T cells is STAT1 dependent and regulated through nicotinamide phosphoribosyl transferase (NAMPT)-mediated enzymatic actions within NAD+ salvage metabolic biosynthesis. These findings identify a novel aspect of CD8 T cell activation following virus infection with implications for human health and physiology.

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.

RNA alternative splicing (AS) expands the regulatory potential of eukaryotic genomes. The mechanisms regulating liver-specific AS profiles and their contribution to liver function are poorly understood. Here, we identify a key role for the splicing factor RNA-binding Fox protein 2 (RBFOX2) in maintaining cholesterol homeostasis in a lipogenic environment in the liver. Using enhanced individual-nucleotide-resolution ultra-violet cross-linking and immunoprecipitation, we identify physiologically relevant targets of RBFOX2 in mouse liver, including the scavenger receptor class B type I (Scarb1). RBFOX2 function is decreased in the liver in diet-induced obesity, causing a Scarb1 isoform switch and alteration of hepatocyte lipid homeostasis. Our findings demonstrate that specific AS programmes actively maintain liver physiology, and underlie the lipotoxic effects of obesogenic diets when dysregulated. Splice-switching oligonucleotides targeting this network alleviate obesity-induced inflammation in the liver and promote an anti-atherogenic lipoprotein profile in the blood, underscoring the potential of isoform-specific RNA therapeutics for treating metabolism-associated diseases.

Endoplasmic reticulum (ER) protein homeostasis (proteostasis) is essential to facilitate proper folding and maturation of proteins in the secretory pathway. Loss of ER proteostasis due to cell stress or mutations in ER proteins can lead to the accumulation of misfolded or aberrant proteins in the ER and triggers the unfolded protein response (UPR). In this study we find that the p97 adaptor UBXN1 is an important negative regulator of the UPR. Loss of UBXN1 significantly sensitizes cells to ER stress and activates canonical UPR signaling pathways. This in turn leads to widespread upregulation of the ER stress transcriptional program. Using comparative, quantitative proteomics we show that deletion of UBXN1 results in a significant enrichment of proteins belonging to ER-quality control processes including those involved in protein folding and import. Notably, we find that loss of UBXN1 does not perturb p97-dependent ER associated degradation (ERAD). Our studies indicate that loss of UBXN1 increases translation in both resting and ER-stressed cells. Surprisingly, this process is independent of p97 function. Taken together, our studies have identified a new role for UBXN1 in repressing translation and maintaining ER proteostasis in a p97 independent manner.

During nutrient stress, macroautophagy is employed to degrade cellular macromolecules, thereby providing biosynthetic building blocks while simultaneously remodeling the proteome. While the machinery responsible for initiation of macroautophagy is well characterized, our understanding of the extent to which individual proteins, protein complexes and organelles are selected for autophagic degradation, and the underlying targeting mechanisms is limited. Here, we use orthogonal proteomic strategies to provide a global molecular inventory of autophagic cargo during nutrient stress in mammalian cell lines. Through prioritization of autophagic cargo, we identify a heterodimeric pair of membrane-embedded proteins, YIPF3 and YIPF4, as receptors for Golgiphagy. During nutrient stress, YIPF4 is mobilized into ATG8-positive vesicles that traffic to lysosomes as measured via Golgiphagy flux reporters in a process that requires the VPS34 and ULK1-FIP200 arms of the autophagy system. Cells lacking YIPF3 or YIPF4 are selectively defective in elimination of Golgi membrane proteins during nutrient stress. By merging absolute protein abundance with autophagic turnover, we create a global protein census describing how autophagic degradation maps onto protein abundance and subcellular localization. Our results, available via an interactive web tool, reveal that autophagic turnover prioritizes membrane-bound organelles (principally Golgi and ER) for proteome remodeling during nutrient stress.One-Sentence SummaryDuring nutrient stress, macroautophagy uses organelle-phagy receptors to prioritize recycling of Golgi and ER membrane proteins.

The protein kinase PINK1 and ubiquitin ligase Parkin promote removal of damaged mitochondria via a feed-forward mechanism involving ubiquitin (Ub) phosphorylation (pUb), Parkin activation, and ubiquitylation of mitochondrial outer membrane proteins to support recruitment of mitophagy receptors. The ubiquitin ligase substrate receptor FBXO7/PARK15 is mutated in an early-onset parkinsonian-pyramidal syndrome. Previous studies have proposed a role for FBXO7 in promoting Parkin-dependent mitophagy. Here, we systematically examine the involvement of FBXO7 in depolarization andmtUPR-dependent mitophagy in the well-established HeLa and induced-neurons cell systems. We find that FBXO7-/-cells have no demonstrable defect in: 1) kinetics of pUb accumulation, 2) pUb puncta on mitochondria by super-resolution imaging, 3) recruitment of Parkin and autophagy machinery to damaged mitochondria, 4) mitophagic flux, and 5) mitochondrial clearance as quantified by global proteomics. Moreover, global proteomics of neurogenesis in the absence of FBXO7 reveals no obvious alterations in mitochondria or other organelles. These results argue against a general role for FBXO7 in Parkin-dependent mitophagy and point to the need for additional studies to define how FBXO7 mutations promote parkinsonian-pyramidal syndrome.

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 phagophore’s 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.

Mass-spectrometry-based phosphoproteomics has become indispensable for understanding cellular signaling in complex biological systems. Despite the central role of protein phosphorylation, the field still lacks inexpensive, regenerable, and diverse phosphopeptides with ground-truth phosphorylation positions. Here, we present Iterative Synthetically Phosphorylated Isomers (iSPI), a proteome-scale library of human-derived phosphoserine-containing phosphopeptides that is inexpensive, regenerable, and diverse, with precisely known positions of phosphorylation. We demonstrate possible uses of iSPI, including use as a phosphopeptide standard, a tool to evaluate and optimize phosphorylation-site localization algorithms, and a benchmark to compare performance across data analysis pipelines. We also present AScorePro, an updated version of the AScore algorithm specifically optimized for phosphorylation-site localization in higher energy fragmentation spectra, and the FLR viewer, a web tool for phosphorylation-site localization, to enable community use of the iSPI resource. iSPI and its associated data constitute a useful, multi-purpose resource for the phosphoproteomics community.

Alzheimer’s disease (AD) pathologies were discovered in the easily accessible neurosensory retina. Yet, their specific nature, topographical distribution, and relationship with disease status remain undefined. Here, we histologically determined burden and spatial distribution of amyloid β-protein (Aβ42), intraneuronal scFvA13+-Aβ species, macro- and microgliosis, and atrophy in superior- and inferior-temporal retinas of human donors with mild cognitive impairment (MCI) or AD versus normal cognition. AD and MCI patients had enhanced retinopathy, predominantly affecting inner layers and peripheral subregions, which quantitatively correlated with severity of cerebral amyloid, tau, and neurodegeneration, and cognitive scores. In advanced clinical stages AD retinopathy further affected central outer segments. Increased retinal macrogliosis and Aβ-phagocytosing microglia were detected in MCI and AD patients. Further, distinct proteome profiles of AD retinas were identified, displaying greater overlap with the temporal cortices than with hippocampi or cerebella. AD retinas exhibited upregulated inflammatory and neurodegenerative processes and downregulated oxidative-phosphorylation/mitochondrial, and photoreceptor-related pathways. This study identifies and maps AD retinopathy, demonstrating the quantitative relationship with brain pathology and cognition.

Summary Genetically diverse mouse populations are powerful tools for characterizing the regulation of the proteome and its relationship to whole-organism phenotypes. We used mass spectrometry to profile and quantify the abundance of 6,798 proteins in liver tissue from mice of both sexes across 58 Collaborative Cross (CC) inbred strains. We previously collected liver proteomics data from the related Diversity Outbred (DO) mice and their founder strains. We show concordance across the proteomics datasets despite being generated from separate experiments, allowing comparative analysis. We map protein abundance quantitative trait loci (pQTLs), identifying 1,087 local and 285 distal in the CC mice and 1,706 local and 414 distal in the DO mice. We find that regulatory effects on individual proteins are conserved across the mouse populations, in particular for local genetic variation and sex differences. In comparison, proteins that form complexes are often co-regulated, displaying varying genetic architectures, and overall show lower heritability and map fewer pQTLs. We have made this resource publicly available to enable quantitative analyses of the regulation of the proteome.

Haploinsufficiency of GRN causes frontotemporal dementia (FTD). The GRN locus produces progranulin (PGRN), which is cleaved to lysosomal granulin polypeptides. The function of lysosomal granulins and why their absence causes neurodegeneration are unclear. Here we discover that PGRN-deficient human cells and murine brains, as well as human frontal lobes from GRN-mutation FTD patients have increased levels of gangliosides, glycosphingolipids that contain sialic acid. In these cells and tissues, levels of lysosomal enzymes that catabolize gangliosides were normal, but levels of bis(monoacylglycero)phosphates (BMP), lipids required for ganglioside catabolism, were reduced with PGRN deficiency. Our findings indicate that granulins are required to maintain BMP levels to support ganglioside catabolism, and that PGRN deficiency in lysosomes leads to gangliosidosis. Lysosomal ganglioside accumulation may contribute to neuroinflammation and neurodegeneration susceptibility observed in FTD due to PGRN deficiency and other neurodegenerative diseases.

The conformational stability of the proteome has tremendous implications for the health of the cell and its capacity to determine longevity or susceptibility to age-associated degenerative diseases. For humans, this question of proteome conformational stability has the additional complexity from non-synonymous mutations in thousands of protein coding genes challenging the capacity of the proteostasis network to properly fold, transport, assemble and degrade proteins. Here, we quantify the proteome-wide capacity to such challenges using the isogenic organism Caenorhabditis elegans by examining the dynamics of global proteome conformational stability in animals expressing different temperature-sensitive (ts) proteins or short polyglutamine (polyQ) expansions in the context of biological aging. Using limited proteolysis of native extracts together with tandem mass tag-based quantitative proteomics, we identify proteins that become metastable under these conditions and monitor the effects on proteome solubility and abundance. Expression of different mutant proteins in the same tissue identifies hundreds to a thousand proteins that become metastable affecting multiple compartments and processes in a cell autonomous and non-autonomous manner. Comparison of the network of metastable proteins, however, reveals only a small number of common proteins. The most dramatic effects on global proteome dynamics occur in aging with one-third of the proteome undergoing conformational changes in early adulthood. These age-dependent metastable proteins overlap substantially with ts proteins and polyQ; moreover, expression of polyQ accelerates the aging phenotype. Together, these results reveal that the proteome responds to misfolding one-at-a-time to generate a metastable sub-proteome network with features of a fingerprint for which aging is the dominant determinant of proteome metastability.

Isobaric labeling has rapidly become a predominant strategy for proteome-wide abundance measurements. Coupled to mass spectrometry, sample multiplexing techniques using isobaric labeling are unparalleled for profiling proteins and posttranslational modifications across multiple samples in a single experiment. Here, I highlight aspects of isobaric labeling in the context of expanding the breadth of multiplexing, improving quantitative accuracy and proteome depth, and developing a wide range of diverse applications. I underscore two facets that enhance quantitative accuracy and reproducibility, specifically the availability of quality control standards for isobaric labeling experiments and the evolution of data acquisition methods. I also emphasize the necessity for standardized methodologies, particularly for emerging high-throughput workflows. Future developments in sample multiplexing will further strengthen the importance of isobaric labeling for comprehensive proteome profiling.

Fingolimod (FTY720) is an oral drug approved by the Food and Drug Administration (FDA) for management of multiple sclerosis (MS) symptoms, which has also shown beneficial effects against Alzheimer's (AD) and Parkinson's (PD) diseases pathologies. Although an extensive effort has been made to identify mechanisms underpinning its therapeutic effects, much remains unknown. Here, we investigated Fingolimod induced proteome changes in the cerebellum (CB) and frontal cortex (FC) regions of the brain which are known to be severely affected in MS, using a tandem mass tag (TMT) isobaric labeling-based quantitative mass-spectrometric approach to investigate the mechanism of action of Fingolimod. This study identified 6749 and 6319 proteins in CB and FC, respectively, and returned 2609 and 3086 differentially expressed proteins in mouse CB and FC, respectively, between Fingolimod treated and control groups. Subsequent bioinformatics analyses indicated a metabolic reprogramming in both brain regions of the Fingolimod treated group, where oxidative phosphorylation was upregulated while glycolysis and pentose phosphate pathway were downregulated. In addition, modulation of neuroinflammation in the Fingolimod treated group was indicated by upregulation of retrograde endocannabinoid signaling and autophagy pathways, and downregulation of neuroinflammation related pathways including neutrophil degranulation and the IL-12 mediated signaling pathway. Our findings suggest that Fingolimod may exert its protective effects on the brain by inducing metabolic reprogramming and neuroinflammation pathway modulation.

Background Phosphorylation of proteins is a key step in the regulation of many cellular processes including activation of enzymes and signaling cascades. The abundance of a phosphorylated peptide (phosphopeptide) is determined by the abundance of its parent protein and the proportion of target sites that are phosphorylated. Results We quantified phosphopeptides, proteins, and transcripts in heart, liver, and kidney tissue samples of mice from 58 strains of the Collaborative Cross strain panel. We mapped ~700 phosphorylation quantitative trait loci (phQTL) across the three tissues and applied genetic mediation analysis to identify causal drivers of phosphorylation. We identified kinases, phosphatases, cytokines, and other factors, including both known and potentially novel interactions between target proteins and genes that regulate site-specific phosphorylation. Our analysis highlights multiple targets of pyruvate dehydrogenase kinase 1 (PDK1), a regulator of mitochondrial function that shows reduced activity in the NZO/HILtJ mouse, a polygenic model of obesity and type 2 diabetes. Conclusions Together, this integrative multi-omics analysis in genetically diverse CC strains provides a powerful tool to identify regulators of protein phosphorylation. The data generated in this study provides a resource for further exploration.

SUMMARYDegradation and recycling of plasma membrane-localized proteins occurs via the endolysosomal system, wherein endosomes bud into the cytosol from the plasma membrane and subsequently mature into degradative lysosomal compartments. The endolysosomal system also participates in intracellular membrane protein trafficking, signaling, and protein degradation via autophagy. Our understanding of the endolysosomal system has been significantly advanced through the development of methods for selective capture of lysosomes (termed Lyso-IP), but methods for analogous isolation of early intermediates in the endolysosomal system are lacking. Here, we develop an approach for rapid isolation of early/sorting endosomes through affinity capture of the early endosome-associated protein EEA1 (Endo-IP) and provide proteomic and lipidomic snapshots of early endosomes in action. In addition to resident endosomal components, we identify numerous recycling, regulatory and membrane fusion complexes as well as candidate cargo, providing a proteomic landscape of early/sorting endosomes that we distinguished from lysosomes via comparative proteomics. The approach allows capture of internalized transferrin within minutes of its addition to cells, indicating the utility of the approach for dynamic analysis of early/sorting endosomes. Among the endocytic cargo identified by Endo-IP was the amyloid precursor protein (APP) genetically linked with Alzheimer’s disease. Processing of APP to amyloidogenic Aβ peptides by β- and γ-Secretases can occur within the endolysosomal system among other compartments, but methods for spatial quantification of Aβ products in individual organelles are lacking. We combined Endo- and Lyso-IP with targeted proteomics to provide a spatial digital snapshot of Aβ products. This analysis revealed that products of Aβ processing by β- and γ-Secretases, and alterations in the specificity of cleavage by small molecule γ-Secretase modulators, can be quantified in both early/sorting endosomes and lysosomes. We anticipate that the Endo-IP approach will facilitate systematic interrogation of the many processes that are coordinated on early endosomes.

Degradation and recycling of plasma membrane proteins occurs via the endolysosomal system, wherein endosomes bud into the cytosol from the plasma membrane and subsequently mature into degradative lysosomal compartments. While methods have been developed for rapid selective capture of lysosomes (Lyso-IP), analogous methods for isolation of early endosome intermediates are lacking. Here, we develop an approach for rapid isolation of early/sorting endosomes through affinity capture of the early endosome-associated protein EEA1 (Endo-IP) and provide proteomic and lipidomic snapshots of EEA1-positive endosomes in action. We identify recycling, regulatory and membrane fusion complexes, as well as candidate cargo, providing a proteomic landscape of early/sorting endosomes. To demonstrate the utility of the method, we combined Endo- and Lyso-IP with multiplexed targeted proteomics to provide a spatial digital snapshot of amyloid precursor protein (APP) processing by β and γ-Secretases, which produce amyloidogenic Aβ species, and quantify small molecule modulation of Secretase action on endosomes. We anticipate that the Endo-IP approach will facilitate systematic interrogation of processes that are coordinated on EEA1-positive endosomes.

The mammalian SWI/SNF (mSWI/SNF or BAF) ATP-dependent chromatin remodeling complexes collectively represent one of the most frequently mutated cellular entities in cancer, second only to TP53. Mutations across the 29 human genes that encode mSWI/SNF complex subunits occur in over 20% of human cancers, with mutations affecting ARID1A and ARID1B, the largest BAF subunits, being the most frequent. However, the functional contributions of these subunits, particularly their commonly mutated N-terminal intrinsically disordered regions (IDRs) and a highly conserved ARID DNA-binding domain, to BAF function remain poorly understood. Here, we demonstrate that the IDRs of ARID1A/B, coupled with the ARID domain, drive biomolecular condensate formation and BAF chromatin localization in cells. We define ARID1A/B IDRs as two-part systems, facilitating homotypic BAF complex interactions (i.e., valence generated by localized condensation) and heterotypic complex interactions, that together establish a highly specific, sequence-encoded protein interaction network within condensates. Both types of interactions are required for appropriate genome-wide targeting of BAF complexes, DNA accessibility generation, and appropriate gene expression. Replacement of the ARID1A N-terminal IDR with IDRs derived from two unrelated proteins FUS and DDX4, rescues generic condensation of BAF but not chromatin occupancy, DNA accessibility, and heterotypic interactions, highlighting the sequence-specificity embedded in the IDR of ARID1A. Taken together, these data establish a role for the largest and most frequently perturbed IDRs within a major chromatin remodeler and explain how biomolecular condensate formation enables both genomic localization and functional partner recruitment. Furthermore, these findings lay the groundwork for mapping IDR sequence specificity or “grammar”, that dictates the co-condensation network formation, and suggests that targeted disruption of these mechanisms may represent new targeted therapeutic opportunities across multiple cancers. Citation Format: Ajinkya Patil, Amy R. Strom, Clayton K. Collings, Joao A. Paulo, Tobias Wauer, Akshay Sankar, Jessica D. St.Laurent, Kasey S. Cervantes, Steven P. Gygi, Clifford P. Brangwynne, Cigall Kadoch. Intrinsically disordered regions of the ARID1A/B tumor suppressors encode an interaction network within biomolecular condensates that directs mSWI/SNF chromatin remodeler complex activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3485.

Brown adipose tissue (BAT) regulates metabolic physiology. However, nearly all mechanistic studies of BAT protein function occur in a single inbred mouse strain, which has limited the understanding of generalizable mechanisms of BAT regulation over physiology. Here, we perform deep quantitative proteomics of BAT across a cohort of 163 genetically defined diversity outbred mice, a model that parallels the genetic and phenotypic variation found in humans. We leverage this diversity to define the functional architecture of the outbred BAT proteome, comprising 10,479 proteins. We assign co-operative functions to 2,578 proteins, enabling systematic discovery of regulators of BAT. We also identify 638 proteins that correlate with protection from, or sensitivity to, at least one parameter of metabolic disease. We use these findings to uncover SFXN5, LETMD1, and ATP1A2 as modulators of BAT thermogenesis or adiposity, and provide OPABAT as a resource for understanding the conserved mechanisms of BAT regulation over metabolic physiology.

The biosynthesis of thousands of proteins requires targeting a signal sequence or transmembrane segment (TM) to the endoplasmic reticulum (ER). These hydrophobic ɑ helices must localize to the appropriate cellular membrane and integrate in the correct topology to maintain a high-fidelity proteome. Here, we show that the P5A-ATPase ATP13A1 prevents the accumulation of mislocalized and misoriented proteins, which are eliminated by different ER-associated degradation (ERAD) pathways in mammalian cells. Without ATP13A1, mitochondrial tail-anchored proteins mislocalize to the ER through the ER membrane protein complex and are cleaved by signal peptide peptidase for ERAD. ATP13A1 also facilitates the topogenesis of a subset of proteins with an N-terminal TM or signal sequence that should insert into the ER membrane with a cytosolic N terminus. Without ATP13A1, such proteins accumulate in the wrong orientation and are targeted for ERAD by distinct ubiquitin ligases. Thus, ATP13A1 prevents ERAD of diverse proteins capable of proper folding.