Polygenic scores (PGS) summarize the combined effects of common single-nucleotide polymorphisms and contribute to predictions of disease severity, but biological consequences linked to these common variants remain poorly defined. Here, we focused on polygenic liability for a measurable electrophysiologic trait (the QT interval). Prolonged QT interval, measured on patient electrocardiograms, is associated with an increased risk for cardiac arrhythmia. We investigated human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) from donors with extreme PGS (i.e., high and low) related to QT interval duration. We paired global proteomics with multiplexed affinity purification mass spectrometry (AP-MS) centered on Kv11.1 (hERG), a major determinant of QT-interval repolarization. Global proteomics indicated increased mitochondrial protein abundance in high-PGS cardiomyocytes, but this did not explain the Kv11.1 interactome. In high-PGS cells, Kv11.1 showed increased associations with myosin motor proteins and endosomal recycling machinery, consistent with altered (and potentially increased) recycling/trafficking dynamics rather than trafficking deficiency observed with most pathogenic Kv11.1 variants. This proof-of-concept study underscores a framework for linking polygenic factors to tractable biological consequences by combining patient-specific hiPSCs, proteomics and affinity-purification. Linking polygenic scores to changes in protein networks provides testable mechanisms that can be applied across many diseases.

C57BL/6J mice are widely used in biomedical research and are susceptible to insulin resistance and dyslipidemia when challenged with high fat diets relative to other inbred strains. Interestingly, C57Bl/6J mice contain a naturally occurring premature termination codon in the lipid flippase Atp10D, and previous studies have linked Atp10D to the metabolic disease-prone phenotype in mice and atherosclerotic severity in humans. In this study, we used CRISPR/Cas9 to revert the premature termination codon to the wild-type glutamine codon (Atp10D *817Q) in the C57Bl/6J mouse strain. The RNA transcripts from original and corrected alleles are dually expressed in heterozygous mice, suggesting that the mutant transcript escapes nonsense-mediated decay. Expression of two corrected Atp10D alleles restores wildtype expression levels of the transcript and protein in the liver. When challenged with a high fat diet, Atp10D-/- (original) and Atp10D+/+ (corrected) C57Bl/6J mice showed no significant difference in weight gain, glucose tolerance, or plasma levels of triglycerides, cholesterol, or free fatty acids. However, the female Atp10D+/+ mice displayed an increase in complex glycosphingolipids and a reduction in cardiolipins in the plasma. These results suggest that restoring the expression of Atp10D in C57Bl/6J mice does not reverse insulin resistance and dyslipidemia in response to high fat diet feeding.

Proteostasis, or protein homeostasis, is a tightly regulated network of cellular pathways essential for maintaining proper protein folding, trafficking, and degradation. Neurons are particularly vulnerable to proteostasis collapse due to their postmitotic and long-lived nature and thus represent a unique cell type to understand the dynamics of proteostasis throughout development and maturation. Here, we utilized a dual-species co-culture model of human excitatory neurons and mouse glia to recapitulate and investigate cell type-specific, maturation-related changes in the proteostasis network using data-independent acquisition LC-MS/MS proteomics. We quantified branch-specific unfolded protein response (UPR) activation by monitoring curated effector proteins downstream of the ATF6, IRE1/XBP1s, and PERK pathways, enabling a comprehensive, unbiased evaluation of UPR dynamics during in vitro neuronal maturation between 30 d and 60 d. Species-specific analysis revealed that mature neurons largely preserved proteostasis, although they showed some signs of collapse, primarily in endoplasmic reticulum (ER)-to-Golgi transport mechanisms. However, these changes were accompanied by upregulation of proteostasis-related machinery and activation of the ATF6 branch, as well as maintenance of the XBP1s and PERK branches of the UPR over time. In contrast, glia exhibited broad downregulation of proteostasis factors and UPR components, independent of neuronal presence. Furthermore, we quantified stimulus-specific modulation of select UPR branches in matured neurons exposed to pharmacologic ER stressors. These findings highlight distinct, cell-type-specific stress adaptations during in vitro maturation and provide a valuable proteomic resource for dissecting proteostasis and UPR regulation in human neurons.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has greatly impacted public health due to high rates of transmissibility and mutation during the COVID-19 pandemic. Macrodomain 1 (Mac1) of non-structural protein 3 remained well conserved across variants and is critical to suppression of host immune response to infection, making Mac1 a promising target for therapeutic development. Mac1 binds and cleaves the post-translational modification ADP-ribose and is hypothesized to have a downstream effect on the host interferon response, but the exact cellular targets of Mac1 are still unknown. Characterizing the substrates of Mac1 ADP-ribosyl hydrolase activity using a catalytically inactive mutant N40D can reveal critical virus-host interactions to identify protein targets of Mac1 and reveal mechanisms of host interferon suppression. Here, we performed affinity enrichment with WT Mac1 and Mac1 N40D in HEK293T and A549 cells and quantified changes in protein interactions by TMT-multiplexed tandem mass spectrometry. We identified interactions between Mac1 and ADP-ribosylated substrates involved in DNA damage response, cytoskeletal components, and cell cycle regulation. Additionally, several members of the TRiC complex involved in protein folding were selectively enriched with mutant Mac1 from A549 cells. These findings suggest a novel role of Mac1 in regulating host protein folding.

More than 1,200 variants of the cystic fibrosis transmembrane conductance regulator gene (CFTR) are associated with cystic fibrosis (CF), an autosomal recessive pulmonary disease affecting over 100,000 people. Most people with CF bear a common CFTR variant (F508del) that can be treated with therapeutics containing "correctors" that suppress the misfolding of the CFTR chloride channel. However, the pharmacological responsiveness of other rare CF variants can vary tremendously. The approval of VX-121, a VX-445 analog that serves as a key component of Alyftrek™, potentially provides a new therapeutic option for those with rare CF variants. Nevertheless, it remains unclear whether VX-121 offers superior rescue across the entire spectrum of rare CF variants. In this work, we use deep mutational scanning (DMS) to survey the impact of VX-121 on the plasma membrane expression of 232 rare CF variants. Our results show that VX-121 generally enhances CF variant expression more than VX-445 and is most potent towards variants with mutations in the first membrane spanning domain (MSD1). However, we identify one variant (Y1032C) with diminished proteostatic and functional selectivity for VX-121 relative to VX-445. Computational docking suggests that the native Y1032 side chain forms favorable interactions with VX-121 that are disrupted by this mutation in a manner that alters its coordination. Finally, using photo-crosslinking, we show that VX-121 avoids a key off-target interaction of VX-445. Together, our findings provide new insights into the similarities and differences between current approved CF therapeutics.

Spirocyclic β-lactones (SβLs) are ring-strain activated natural products produced by actinomycetota, possessing potent and selective cytotoxicity, and with an unknown pharmacological mechanism. To further the understanding of their mechanism of action and the discovery of new SβLs, we obtained a series of known and previously not isolated SβLs from Streptomyces platensis, which contained a cryptic SβL-producing gene cluster. Utilizing single cell Multiplexed Activity Metabolomics to assess a panel of regulated cell injury and cell death markers in metabolomic arrays tested against MV-4-11 cells, phosphoprotein-S6 phosphorylation inhibition was observed for several metabolomic features, prompting us to prioritize these metabolites for structure elucidation. Several compounds were isolated including one new variant, permitting correlation of a cryptic biosynthetic gene cluster entry for oxazolomycin D (OxD) family metabolites. Cytotoxicity and flow cytometric structure-activity relationships for isolated analogs were determined using an expanded phospho-flow metabolism marker panel suggesting that SβLs possess two pharmacophores: a mixed polyketide component, involved in target engagement and inhibition, and a nonribosomal peptide synthetase-derived covalent β-lactone warhead, contributing to cytotoxicity. Global cellular thermal proteome profiling analysis of OxD treated cells implicated potent interaction with and modulation of spliceosome associated proteins. Spliceosome-associated functional changes were validated by alternate splicing analysis upon induction by OxD. As aberrant RNA splicing occurs in nearly all cancer types, the spliceosome-associated cellular response profiles of OxD define an unexplored opportunity for therapeutic development in cancers dependent upon alternative splicing.

Hypothyroidism is a prevalent endocrine disorder characterized by insufficient thyroid hormone (T3T4) production. Thyroglobulin (Tg) serves as the prohormone for T3 and T4 production, with many variants of uncertain clinical significance due to genetic diversity in the Tg gene. We leveraged the large-scale All of Us biobank to investigate the disease association of prevalent yet undercharacterized Tg variants. We related variant presence to thyroid-stimulating hormone levels and levothyroxine (LT4) usage as proxies for thyroid function. This identified R152H, Q870H, A993T, P1012L, and P1494L variants linked to increased LT4 usage and decreased thyroid function, while the R320C variant was associated with decreased thyroid function. Molecular characterization in Fischer rat thyroid cells revealed decreased secretion efficiency of R152H, Q870H, and R320C variants. Affinity purification-mass spectrometry demonstrated that secretion-deficient variants showed higher engagement with the protein homeostasis network, indicating protein quality control defects as the pathophysiology mechanism. In contrast, secretion-competent A993T and P1494L variants showed elevated interactions with degradation and antigen-presentation pathways, suggesting an alternative pathophysiology possibly linked to Hashimoto’s disease, an autoimmune condition with overproduction of autoantibodies that target thyroid proteins. In support, participants carrying the A993T or P1494L variants had elevated anti-thyroidperoxidase (TPO) antibody levels. We estimate ∼115,000 US individuals currently taking levothyroxine could benefit from precision medicine targeting these variants, with ∼85,000 carrying Q870H. Our findings highlight the power of combining large public biobank data with molecular characterization to understand Tg genotype-to-phenotype relationships. Q870H represents a candidate for molecular therapies to restore secretion, offering precision medicine beyond LT4 replacement therapy.

INTRODUCTION:The advent of variant-specific disease-modifying drugs into clinical practice has provided remarkable benefits for people with cystic fibrosis (PwCF), a multi-organ life-limiting inherited disease. However, further efforts are needed to maximize therapeutic benefits as well as to increase the number of PwCF taking CFTR modulators. AREA COVERED:The authors discuss some of the key limitations of the currently available CFTR modulator therapies (e.g. adverse reactions) and strategies in development to increase the number of available therapeutics for CF. These include novel methods to accelerate theratyping and identification of novel small molecules and cellular targets representing alternative or complementary therapies for CF. EXPERT OPINION:While the CF therapy development pipeline continues to grow, there is a critical need to optimize strategies that will accelerate testing and approval of effective therapies for (ultra)rare CFTR variants as traditional assays and trials are not suitable to address such issues. Another major barrier that needs to be solved is the restricted access to currently available modulator therapies, which remains a significant burden for PwCF who are from racial and ethnic minorities and/or living in underprivileged regions.

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

Norovirus (NoV) is a leading cause of global acute gastroenteritis, particularly in young children, with no current licensed vaccine. Epidemiological studies have shown that asymptomatic cases are common, and infected patients may test positive for prolonged periods; however, the impact of these phenomena on transmission and public health measures remains unclear. A major limiting factor is our ability to measure infection, which is constrained to real-time reverse transcription polymerase chain reaction or antibody-based assays, both of which are susceptible to loss of detection by rapid NoV evolution. This review highlights the potential for proteomics to overcome current technical limitations and advance basic science discovery and clinical research. Importantly, proteomics-based protein detection can span NoV, host, and microbiome proteins and could help identify host or microbiome factors that correlate with disease outcome. Further developing proteomics tools to complement existing diagnostic technologies will improve our ability to assess NoV pathogenesis and transmission, as well as therapeutic efficacy.

Liquid handling robots have been developed to automate various steps of the bottom-up proteomics workflow, however, protocols for the generation of isobarically labeled peptides remain limited. Existing methods often require costly specialty devices and are constrained by fixed workflows. To address this, we developed a cost-effective, flexible, automated sample preparation protocol for TMT-labeled peptides using the Biomek i5 liquid handler (Beckman Coulter). Our approach leverages single-pot solid-phase-enhanced sample preparation with paramagnetic beads to streamline protein cleanup and digestion. The protocol also allows for adjustment of trypsin concentration and peptide-to-TMT ratio to increase throughput and reduce costs, respectively. We compared our automated and manual 18-plex TMT-Pro labeling workflows by monitoring select protein markers of the unfolded protein response in pharmacologically activatable, engineered cell lines. Overall, the automated protocol demonstrated equivalent performance in peptide and protein identifications, digestion and labeling efficiency, and an enhancement in the dynamic range of TMT quantifications. Compared to the manual method, the Biomek protocol significantly reduces hands-on time and minimizes sample handling errors. The 96-well format additionally allows for the number of TMT reactions to be scaled up quickly without a significant increase in user interaction. Our optimized automated workflow enhances throughput, reproducibility, and cost-effectiveness, making it a valuable tool for high-throughput proteomics studies.

Cystic fibrosis (CF) pharmacological correctors improve the cystic fibrosis transmembrane conductance regulator (CFTR) protein trafficking and function. Several side effects of these correctors and adverse drug interactions have been reported, emphasizing the need to understand off-targets. We synthesized VU439, a functionalized photoaffinity ligand (PAL) of VX-445. Chemoproteomics analysis by mass spectrometry (MS) was used to identify cross-linked proteins in CF bronchial epithelial cells expressing F508del CFTR. We identified saccharopine dehydrogenase-like oxidoreductase (SCCPDH), an uncharacterized putative oxidoreductase, as a VX-445-specific off-target. We also characterized changes in the metabolomic profiles of cells overexpressing SCCPDH to determine the consequence of binding of VX-445 to SCCPDH. These data show dysregulation of amino acid metabolism and a potential inhibitory activity of VX-445 on SCCPDH. The identified off-target may explain the exacerbation of psychological symptoms observed in the clinic, thus emphasizing the need for further optimization of correctors.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are part of a spectrum of diseases that share several causative genes, resulting in a combinatory of motor and cognitive symptoms and abnormal protein aggregation. Multiple unbiased studies have revealed that proteostasis impairment at the level of the endoplasmic reticulum (ER) is a transversal pathogenic feature of ALS/FTD. The transcription factor XBP1s is a master regulator of the unfolded protein response (UPR), the main adaptive pathway to cope with ER stress. Here, we provide evidence of suboptimal activation of the UPR in ALS/FTD models under experimental ER stress. To artificially engage the UPR, we intracerebroventricularly administrated adeno-associated viruses (AAVs) to express the active form of XBP1 (XBP1s) in the nervous system of ALS/ FTD models. XBP1s expression improved motor performance and extended lifespan of mutant SOD1 mice, associated with reduced protein aggregation. AAV-XBP1s administration also attenuated disease progression in models of TDP-43 and C9orf72 pathogenesis. Proteomic profiling of spinal cord tissue revealed that XBP1s overexpression improved proteostasis and modulated the expression of a cluster of synaptic and cell morphology proteins. Our results suggest that strategies to improve ER proteostasis may serve as a pan-therapeutic strategy to treat ALS/FTD.

Background & Aims:Collagen is the main cargo of the secretory pathway, contributing to hepatic fibrogenesis due to extensive accumulation of extracellular matrix. An excess of collagen deposition is a characteristic feature of several chronic liver diseases. Collagen overproduction imposes pressure on the secretory pathway, altering endoplasmic reticulum (ER) proteostasis. Here, we investigated the possible contribution of the unfolded protein response, the main adaptive pathway that monitors and adjusts protein production capacity at the ER, to collagen biogenesis and liver disease.Approach & Results:We used multi-mice models combined with multi-omics approaches followed by cell culture studies. In addition, we validated results using human metabolic dysfunction-associated steatohepatitis (MASH) samples. Genetic ablation of the ER stress sensor IRE1 in the liver using conditional knockout mice reduced liver damage and collagen deposition in models of fibrosis, steatosis, and acute hepatotoxicity. Proteomic profiling identified the prolyl 4-hydroxylase (P4HB, also known as PDIA1) as a major IRE1-regulated gene, a critical factor involved in collagen maturation. Cell culture studies demonstrated that IRE1 deficiency results in collagen retention at the ER, reducing its secretion, and this phenotype is rescued by P4HB/PDIA1 overexpression. Analyses of human MASH samples revealed a positive correlation between IRE1 signaling and P4HB/PDIA1 expression as well as the severity of the disease.Conclusions:Altogether, our results establish a role of the IRE1/P4HB axis in the regulation of collagen production and support its implication in the pathogenesis of liver fibrosis.

Cystic Fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). Though most people with CF have one or two copies of the ΔF508 mutation, there are hundreds of other distinct CF mutations that vary in their mechanistic effects and response to therapeutics. Endogenous chaperones are known to have divergent effects on the druggability of CF variants. Nevertheless, it remains unclear how this proteostatic modulation is related to the underlying mechanistic effects of distinct classes of CF mutations. Here, we survey the effects of a previously discovered effector (calnexin, CANX) on the expression and pharmacological rescue of 232 CF variants using deep mutational scanning. We find that CANX is generally required for robust plasma membrane expression of the CFTR protein- particularly for CF variants that perturb its second nucleotide binding domain. CANX also appears to be critical for the pharmacological rescue of CF variants with poor basal expression. Though corrector selectivity is generally dictated by the properties of mutations, we find that CANX enhances the sensitivity of CF variants within a domain swapped region of membranes spanning domain 2 to the type III corrector VX-445. Overall, mutagenic trends suggest CANX modulates the later stages of CFTR assembly and disproportionately affects variants bearing mutations within the C-terminal domains. Interestingly, we find that the loss of CANX results in widespread perturbations of CF variant interactomes and that the proteostatic effects of CANX are generally decoupled from changes in CFTR activity. Together, our findings reveal how the proteostasis machinery may shape the variant-specific effects of corrector molecules.

Coronaviruses (CoV) rewire host protein homeostasis (proteostasis) networks through interactions between viral nonstructural proteins (nsps) and host factors to promote infection. With the emergence of SARS-CoV-2, it is imperative to characterize host interactors shared across nsp homologs. Using quantitative proteomics and functional genetic screening, we identify conserved proteostasis interactors of nsp2 and nsp4 that serve pro-viral roles during infection of murine hepatitis virus – a model betacoronavirus. We uncover a glycoprotein quality control factor, Malectin (MLEC), which significantly reduces infectious titers when knocked down. During infection, nsp2 interacts with MLEC-associated proteins and the MLEC-interactome is drastically altered, stabilizing association with the Oligosaccheryltransferase (OST) complex, a crucial component of viral glycoprotein production. MLEC promotes viral protein levels and genome replication through its quality control activity. Lastly, we show MLEC promotes SARS-CoV-2 replication. Our results reveal a role for MLEC in mediating CoV infection and identify a potential target for pan-CoV antivirals.

Cystic fibrosis (CF) is a lethal genetic disorder caused by variants in CF transmembrane conductance regulator (CFTR). Many variants are treatable with correctors, which enhance the folding and trafficking of CFTR. However, approximately 3% of persons with CF harbor poorly responsive variants. Here, we used affinity purification mass spectrometry proteomics to profile the protein homeostasis (proteostasis) changes of CFTR variants during correction to assess modulated interactions with protein folding and maturation pathways. Responsive variant interactions converged on similar proteostasis pathways during correction. In contrast, poorly responsive variants subtly diverged, revealing a partial restoration of protein quality control surveillance and partial correction. Computational structural modeling showed that corrector VX-445 failed to confer enough NBD1 stability to poor responders. NBD1 secondary stabilizing mutations rescued poorly responsive variants, revealing structural vulnerabilities in NBD1 required for treating poor responders. Our study provides a framework for discerning the underlying protein quality control and structural defects of CFTR variants not reached with existing drugs to expand therapeutics to all susceptible CFTR variants.

Dynamic protein-protein interactions are key drivers of many cellular processes. Determining the relative sequence and precise timing of these interactions is crucial for elucidating the functional dynamics of biological processes. Here, we developed a time-resolved analysis of protein-protein ensembles using a destabilizing domain (TRAPPED) to study protein-protein interactions in a temporal manner. We have taken advantage of a dihydrofolate reductase-destabilizing domain (DHFR(DD)) that can be fused to a protein of interest and is constitutively degraded by the proteosome. Addition of the ligand trimethoprim (TMP) can stabilize DHFR(DD), preventing proteasomal degradation of the fusion protein and thereby inducing accumulation in cells. We synthesized and optimized TRimethoprim Analog Probes that maintain stabilization activity and contain a terminal alkyne for Click functionalization and a thiol reactive group to covalently tag DHFR(DD). Click reaction with a biotin tag and subsequent streptavidin enrichment enable time-resolved mass spectrometric identification of interacting partners. We evaluated the timing of protein interactions of SARS-CoV-2 and SARS-CoV nonstructural protein 15 (nsp15) over a 2 h period. We found interactors GEMIN5 and YBX3, known regulators of SARS-CoV-2 infection that bind viral RNA, as well as CACYBP and FHL1 that implicate nsp15 in the disruption of host ERK1/2 signaling. We further revealed that these interactions remain relatively steady from 0 to 2 h post translation of nsp15. TRAPPED methodology can be applied to determine the sequence and timing of protein-protein interactions of temporally regulated biological processes such as viral infection or signal transduction.

SARS-CoV-2 virus and its variants remain a global health threat, due to their capacity for rapid evolution. Variants throughout the COVID-19 pandemic exhibited variations in virulence, impacting vaccine protection and disease severity. Investigating nonstructural protein variants is critical to understanding viral evolution and manipulation of host protein interactions. We focus on nonstructural protein 3 (nsp3), with multiple domains with different activities, including viral polyprotein cleavage, host deubiquitylation, de-ISGylation, and double-membrane vesicle formation. Using affinity purification–mass spectrometry (AP-MS), we identify differential protein interactions in nsp3 caused by mutations found in variants identified between 2019 and 2024: Alpha 20I, Beta 20H, Delta 21I, Delta 21J, Gamma 20J, Kappa 21B, Lambda 21G, Omicron 21K, and Omicron 21L. A small set of amino acid substitutions in the N-terminal region of nsp3 (nsp3.1) could be traced to increased interactions with RNA-binding proteins, which are vital in viral replication. Meanwhile, variants of the central region of nsp3 (nsp3.2) were found to share interactions with protein quality control machinery, including ER-associated degradation. In this construct, shared trends in interactor enrichment are observed between Omicron 21K and Delta 21I. These results underscore how minor mutations reshape host interactions, emphasizing the evolutionary arms race between the host and virus. We provide a roadmap to track the interaction changes driven by SARS-CoV-2 variant evolution.