Communicating Mass Spectrometry Quality Information in mzQC with Python, R, and Java.

Mass spectrometry is a powerful technique for analyzing molecules in complex biological samples. However, inter- and intralaboratory variability and bias can affect the data due to various factors, including sample handling and preparation, instrument calibration and performance, and data acquisition and processing. To address this issue, the Quality Control (QC) working group of the Human Proteome Organization's Proteomics Standards Initiative has established the standard mzQC file format for reporting and exchanging information relating to data quality. mzQC is based on the JavaScript Object Notation (JSON) format and provides a lightweight yet versatile file format that can be easily implemented in software. Here, we present open-source software libraries to process mzQC data in three programming languages: Python, using pymzqc; R, using rmzqc; and Java, using jmzqc. The libraries follow a common data model and provide shared functionalities, including the (de)serialization and validation of mzQC files. We demonstrate use of the software libraries in a workflow for extracting, analyzing, and visualizing QC metrics from different sources. Additionally, we show how these libraries can be integrated with each other, with existing software tools, and in automated workflows for the QC of mass spectrometry data. All software libraries are available as open source under the MS-Quality-Hub organization on GitHub (https://github.com/MS-Quality-Hub).

Sexual dimorphism and the multi-omic response to exercise training in rat subcutaneous white adipose tissue.

Subcutaneous white adipose tissue (scWAT) is a dynamic storage and secretory organ that regulates systemic homeostasis, yet the impact of endurance exercise training (ExT) and sex on its molecular landscape is not fully established. Utilizing an integrative multi-omics approach, and leveraging data generated by the Molecular Transducers of Physical Activity Consortium (MoTrPAC), we show profound sexual dimorphism in the scWAT of sedentary rats and in the dynamic response of this tissue to ExT. Specifically, the scWAT of sedentary females displays -omic signatures related to insulin signaling and adipogenesis, whereas the scWAT of sedentary males is enriched in terms related to aerobic metabolism. These sex-specific -omic signatures are preserved or amplified with ExT. Integration of multi-omic analyses with phenotypic measures identifies molecular hubs predicted to drive sexually distinct responses to training. Overall, this study underscores the powerful impact of sex on adipose tissue biology and provides a rich resource to investigate the scWAT response to ExT.

The mitochondrial multi-omic response to exercise training across rat tissues.

Mitochondria have diverse functions critical to whole-body metabolic homeostasis. Endurance training alters mitochondrial activity, but systematic characterization of these adaptations is lacking. Here, the Molecular Transducers of Physical Activity Consortium mapped the temporal, multi-omic changes in mitochondrial analytes across 19 tissues in male and female rats trained for 1, 2, 4, or 8 weeks. Training elicited substantial changes in the adrenal gland, brown adipose, colon, heart, and skeletal muscle. The colon showed non-linear response dynamics, whereas mitochondrial pathways were downregulated in brown adipose and adrenal tissues. Protein acetylation increased in the liver, with a shift in lipid metabolism, whereas oxidative proteins increased in striated muscles. Exercise-upregulated networks were downregulated in human diabetes and cirrhosis. Knockdown of the central network protein 17-beta-hydroxysteroid dehydrogenase 10 (HSD17B10) elevated oxygen consumption, indicative of metabolic stress. We provide a multi-omic, multi-tissue, temporal atlas of the mitochondrial response to exercise training and identify candidates linked to mitochondrial dysfunction.

Cross-family small GTPase ubiquitination by the intracellular pathogen Legionella pneumophila.

The intracellular bacterial pathogen Legionella pneumophila (L.p.) manipulates eukaryotic host ubiquitination machinery to form its replicative vacuole. While nearly 10% of L.p.'s ∼330 secreted effector proteins are ubiquitin ligases or deubiquitinases, a comprehensive measure of temporally resolved changes in the endogenous host ubiquitinome during infection has not been undertaken. To elucidate how L.p. hijacks host cell ubiquitin signaling, we generated a proteome-wide analysis of changes in protein ubiquitination during infection. We discover that L.p. infection increases ubiquitination of host regulators of subcellular trafficking and membrane dynamics, most notably ∼40% of mammalian Ras superfamily small GTPases. We determine that these small GTPases undergo nondegradative ubiquitination at the Legionella-containing vacuole (LCV) membrane. Finally, we find that the bacterial effectors SidC/SdcA play a central role in cross-family small GTPase ubiquitination, and that these effectors function upstream of SidE family ligases in the polyubiquitination and retention of GTPases in the LCV membrane. This work highlights the extensive reconfiguration of host ubiquitin signaling by bacterial effectors during infection and establishes simultaneous ubiquitination of small GTPases across the Ras superfamily as a novel consequence of L.p. infection. Our findings position L.p. as a tool to better understand how small GTPases can be regulated by ubiquitination in uninfected contexts.

Cross-family small GTPase ubiquitination by the intracellular pathogen Legionella pneumophila.

The intracellular bacterial pathogen Legionella pneumophila (L.p.) manipulates eukaryotic host ubiquitination machinery to form its replicative vacuole. While nearly 10% of L.p.'s arsenal of ~330 secreted effector proteins have been biochemically characterized as ubiquitin ligases or deubiquitinases, a comprehensive measure of temporally resolved changes in the endogenous host ubiquitinome during infection has not been undertaken. To elucidate how L.p hijacks ubiquitin signaling within the host cell, we undertook a proteome-wide analysis of changes in protein ubiquitination during infection. We discover that L.p. infection results in increased ubiquitination of host proteins regulating subcellular trafficking and membrane dynamics, most notably 63 of ~160 mammalian Ras superfamily small GTPases. We determine that these small GTPases predominantly undergo non-degradative monoubiquitination, and link ubiquitination to recruitment to the Legionella-containing vacuole membrane. Finally, we find that the bacterial effectors SidC/SdcA play a central, but likely indirect, role in cross-family small GTPase ubiquitination. This work highlights the extensive reconfiguration of host ubiquitin signaling by bacterial effectors during infection and establishes simultaneous ubiquitination of small GTPases across the Ras superfamily as a novel consequence of L.p. infection. This work positions L.p. as a tool to better understand how small GTPases can be regulated by ubiquitination in uninfected contexts.

Quantitative Proteomic Analysis Reveals apoE4-Dependent Phosphorylation of the Actin-Regulating Protein VASP.

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer's disease. While neurons generally produce a minority of the apoE in the central nervous system, neuronal expression of apoE increases dramatically in response to stress and is sufficient to drive pathology. Currently, the molecular mechanisms of how apoE4 expression may regulate pathology are not fully understood. Here, we expand upon our previous studies measuring the impact of apoE4 on protein abundance to include the analysis of protein phosphorylation and ubiquitylation signaling in isogenic Neuro-2a cells expressing apoE3 or apoE4. ApoE4 expression resulted in a dramatic increase in vasodilator-stimulated phosphoprotein (VASP) S235 phosphorylation in a protein kinase A (PKA)-dependent manner. This phosphorylation disrupted VASP interactions with numerous actin cytoskeletal and microtubular proteins. Reduction of VASP S235 phosphorylation via PKA inhibition resulted in a significant increase in filopodia formation and neurite outgrowth in apoE4-expressing cells, exceeding levels observed in apoE3-expressing cells. Our results highlight the pronounced and diverse impact of apoE4 on multiple modes of protein regulation and identify protein targets to restore apoE4-related cytoskeletal defects.

Sexual dimorphism and the multi-omic response to exercise training in rat subcutaneous white adipose tissue.

Subcutaneous white adipose tissue (scWAT) is a dynamic storage and secretory organ that regulates systemic homeostasis, yet the impact of endurance exercise training and sex on its molecular landscape has not been fully established. Utilizing an integrative multi-omics approach with data generated by the Molecular Transducers of Physical Activity Consortium (MoTrPAC), we identified profound sexual dimorphism in the dynamic response of rat scWAT to endurance exercise training. Despite similar cardiorespiratory improvements, only male rats reduced whole-body adiposity, scWAT adipocyte size, and total scWAT triglyceride abundance with training. Multi-omic analyses of adipose tissue integrated with phenotypic measures identified sex-specific training responses including enrichment of mTOR signaling in females, while males displayed enhanced mitochondrial ribosome biogenesis and oxidative metabolism. Overall, this study reinforces our understanding that sex impacts scWAT biology and provides a rich resource to interrogate responses of scWAT to endurance training.

The mitochondrial multi-omic response to exercise training across tissues.

Mitochondria are adaptable organelles with diverse cellular functions critical to whole-body metabolic homeostasis. While chronic endurance exercise training is known to alter mitochondrial activity, these adaptations have not yet been systematically characterized. Here, the Molecular Transducers of Physical Activity Consortium (MoTrPAC) mapped the longitudinal, multi-omic changes in mitochondrial analytes across 19 tissues in male and female rats endurance trained for 1, 2, 4 or 8 weeks. Training elicited substantial changes in the adrenal gland, brown adipose, colon, heart and skeletal muscle, while we detected mild responses in the brain, lung, small intestine and testes. The colon response was characterized by non-linear dynamics that resulted in upregulation of mitochondrial function that was more prominent in females. Brown adipose and adrenal tissues were characterized by substantial downregulation of mitochondrial pathways. Training induced a previously unrecognized robust upregulation of mitochondrial protein abundance and acetylation in the liver, and a concomitant shift in lipid metabolism. The striated muscles demonstrated a highly coordinated response to increase oxidative capacity, with the majority of changes occurring in protein abundance and post-translational modifications. We identified exercise upregulated networks that are downregulated in human type 2 diabetes and liver cirrhosis. In both cases HSD17B10, a central dehydrogenase in multiple metabolic pathways and mitochondrial tRNA maturation, was the main hub. In summary, we provide a multi-omic, cross-tissue atlas of the mitochondrial response to training and identify candidates for prevention of disease-associated mitochondrial dysfunction.

Nuclear accumulation of host transcripts during Zika Virus Infection.

Zika virus (ZIKV) infects fetal neural progenitor cells (NPCs) causing severe neurodevelopmental disorders in utero. Multiple pathways involved in normal brain development are dysfunctional in infected NPCs but how ZIKV centrally reprograms these pathways remains unknown. Here we show that ZIKV infection disrupts subcellular partitioning of host transcripts critical for neurodevelopment in NPCs and functionally link this process to the up-frameshift protein 1 (UPF1). UPF1 is an RNA-binding protein known to regulate decay of cellular and viral RNAs and is less expressed in ZIKV-infected cells. Using infrared crosslinking immunoprecipitation and RNA sequencing (irCLIP-Seq), we show that a subset of mRNAs loses UPF1 binding in ZIKV-infected NPCs, consistent with UPF1's diminished expression. UPF1 target transcripts, however, are not altered in abundance but in subcellular localization, with mRNAs accumulating in the nucleus of infected or UPF1 knockdown cells. This leads to diminished protein expression of FREM2, a protein required for maintenance of NPC identity. Our results newly link UPF1 to the regulation of mRNA transport in NPCs, a process perturbed during ZIKV infection.

Murine blastocysts generated by in vitro fertilization show increased Warburg metabolism and altered lactate production.

In vitro fertilization (IVF) has resulted in the birth of over 8 million children. Although most IVF-conceived children are healthy, several studies suggest an increased risk of altered growth rate, cardiovascular dysfunction, and glucose intolerance in this population compared to naturally conceived children. However, a clear understanding of how embryonic metabolism is affected by culture condition and how embryos reprogram their metabolism is unknown. Here, we studied oxidative stress and metabolic alteration in blastocysts conceived by natural mating or by IVF and cultured in physiologic (5%) or atmospheric (20%) oxygen. We found that IVF-generated blastocysts manifest increased reactive oxygen species, oxidative damage to DNA/lipid/proteins, and reduction in glutathione. Metabolic analysis revealed IVF-generated blastocysts display decreased mitochondria respiration and increased glycolytic activity suggestive of enhanced Warburg metabolism. These findings were corroborated by altered intracellular and extracellular pH and increased intracellular lactate levels in IVF-generated embryos. Comprehensive proteomic analysis and targeted immunofluorescence showed reduction of lactate dehydrogenase-B and monocarboxylate transporter 1, enzymes involved in lactate metabolism. Importantly, these enzymes remained downregulated in the tissues of adult IVF-conceived mice, suggesting that metabolic alterations in IVF-generated embryos may result in alteration in lactate metabolism. These findings suggest that alterations in lactate metabolism are a likely mechanism involved in genomic reprogramming and could be involved in the developmental origin of health and disease.

Deconvoluting complex correlates of COVID-19 severity with a multi-omic pandemic tracking strategy.

The SARS-CoV-2 pandemic has differentially impacted populations across race and ethnicity. A multi-omic approach represents a powerful tool to examine risk across multi-ancestry genomes. We leverage a pandemic tracking strategy in which we sequence viral and host genomes and transcriptomes from nasopharyngeal swabs of 1049 individuals (736 SARS-CoV-2 positive and 313 SARS-CoV-2 negative) and integrate them with digital phenotypes from electronic health records from a diverse catchment area in Northern California. Genome-wide association disaggregated by admixture mapping reveals novel COVID-19-severity-associated regions containing previously reported markers of neurologic, pulmonary and viral disease susceptibility. Phylodynamic tracking of consensus viral genomes reveals no association with disease severity or inferred ancestry. Summary data from multiomic investigation reveals metagenomic and HLA associations with severe COVID-19. The wealth of data available from residual nasopharyngeal swabs in combination with clinical data abstracted automatically at scale highlights a powerful strategy for pandemic tracking, and reveals distinct epidemiologic, genetic, and biological associations for those at the highest risk.

Ceragenins and Antimicrobial Peptides Kill Bacteria through Distinct Mechanisms.

Ceragenins are a family of synthetic amphipathic molecules designed to mimic the properties of naturally occurring cationic antimicrobial peptides (CAMPs). Although ceragenins have potent antimicrobial activity, whether their mode of action is similar to that of CAMPs has remained elusive. Here, we reported the results of a comparative study of the bacterial responses to two well-studied CAMPs, LL37 and colistin, and two ceragenins with related structures, CSA13 and CSA131. Using transcriptomic and proteomic analyses, we found that Escherichia coli responded similarly to both CAMPs and ceragenins by inducing a Cpx envelope stress response. However, whereas E. coli exposed to CAMPs increased expression of genes involved in colanic acid biosynthesis, bacteria exposed to ceragenins specifically modulated functions related to phosphate transport, indicating distinct mechanisms of action between these two classes of molecules. Although traditional genetic approaches failed to identify genes that confer high-level resistance to ceragenins, using a Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) approach we identified E. coli essential genes that when knocked down modify sensitivity to these molecules. Comparison of the essential gene-antibiotic interactions for each of the CAMPs and ceragenins identified both overlapping and distinct dependencies for their antimicrobial activities. Overall, this study indicated that, while some bacterial responses to ceragenins overlap those induced by naturally occurring CAMPs, these synthetic molecules target the bacterial envelope using a distinctive mode of action. IMPORTANCE The development of novel antibiotics is essential because the current arsenal of antimicrobials will soon be ineffective due to the widespread occurrence of antibiotic resistance. The development of naturally occurring cationic antimicrobial peptides (CAMPs) for therapeutics to combat antibiotic resistance has been hampered by high production costs and protease sensitivity, among other factors. The ceragenins are a family of synthetic CAMP mimics that kill a broad spectrum of bacterial species but are less expensive to produce, resistant to proteolytic degradation, and seemingly resistant to the development of high-level resistance. Determining how ceragenins function may identify new essential biological pathways of bacteria that are less prone to the development of resistance and will further our understanding of the design principles for maximizing the effects of synthetic CAMPs.

Mono- and Biallelic Protein-Truncating Variants in Alpha-Actinin 2 Cause Cardiomyopathy Through Distinct Mechanisms.

BACKGROUND: ACTN2 (alpha-actinin 2) anchors actin within cardiac sarcomeres. The mechanisms linking ACTN2 mutations to myocardial disease phenotypes are unknown. Here, we characterize patients with novel ACTN2 mutations to reveal insights into the physiological function of ACTN2. METHODS: Patients harboring ACTN2 protein-truncating variants were identified using a custom mutation pipeline. In patient-derived iPSC-cardiomyocytes, we investigated transcriptional profiles using RNA sequencing, contractile properties using video-based edge detection, and cellular hypertrophy using immunohistochemistry. Structural changes were analyzed through electron microscopy. For mechanistic studies, we used co-immunoprecipitation for ACTN2, followed by mass-spectrometry to investigate protein-protein interaction, and protein tagging followed by confocal microscopy to investigate introduction of truncated ACTN2 into the sarcomeres. RESULTS: Patient-derived iPSC-cardiomyocytes were hypertrophic, displayed sarcomeric structural disarray, impaired contractility, and aberrant Ca2+-signaling. In heterozygous indel cells, the truncated protein incorporates into cardiac sarcomeres, leading to aberrant Z-disc ultrastructure. In homozygous stop-gain cells, affinity-purification mass-spectrometry reveals an intricate ACTN2 interactome with sarcomere and sarcolemma-associated proteins. Loss of the C-terminus of ACTN2 disrupts interaction with ACTN1 (alpha-actinin 1) and GJA1 (gap junction protein alpha 1), 2 sarcolemma-associated proteins, which may contribute to the clinical arrhythmic and relaxation defects. The causality of the stop-gain mutation was verified using CRISPR-Cas9 gene editing. CONCLUSIONS: Together, these data advance our understanding of the role of ACTN2 in the human heart and establish recessive inheritance of ACTN2 truncation as causative of disease.

A protein network map of head and neck cancer reveals PIK3CA mutant drug sensitivity.

We outline a framework for elucidating tumor genetic complexity through multidimensional protein-protein interaction maps and apply it to enhancing our understanding of head and neck squamous cell carcinoma. This network uncovers 771 interactions from cancer and noncancerous cell states, including WT and mutant protein isoforms. Prioritization of cancer-enriched interactions reveals a previously unidentified association of the fibroblast growth factor receptor tyrosine kinase 3 with Daple, a guanine-nucleotide exchange factor, resulting in activation of Gαi- and p21-activated protein kinase 1/2 to promote cancer cell migration. Additionally, we observe mutation-enriched interactions between the human epidermal growth factor receptor 3 (HER3) receptor tyrosine kinase and PIK3CA (the alpha catalytic subunit of phosphatidylinositol 3-kinase) that can inform the response to HER3 inhibition in vivo. We anticipate that the application of this framework will be valuable for translating genetic alterations into a molecular and clinical understanding of the underlying biology of many disease areas.

Restriction factor compendium for influenza A virus reveals a mechanism for evasion of autophagy.

The fate of influenza A virus (IAV) infection in the host cell depends on the balance between cellular defence mechanisms and viral evasion strategies. To illuminate the landscape of IAV cellular restriction, we generated and integrated global genetic loss-of-function screens with transcriptomics and proteomics data. Our multi-omics analysis revealed a subset of both IFN-dependent and independent cellular defence mechanisms that inhibit IAV replication. Amongst these, the autophagy regulator TBC1 domain family member 5 (TBC1D5), which binds Rab7 to enable fusion of autophagosomes and lysosomes, was found to control IAV replication in vitro and in vivo and to promote lysosomal targeting of IAV M2 protein. Notably, IAV M2 was observed to abrogate TBC1D5-Rab7 binding through a physical interaction with TBC1D5 via its cytoplasmic tail. Our results provide evidence for the molecular mechanism utilised by IAV M2 protein to escape lysosomal degradation and traffic to the cell membrane, where it supports IAV budding and growth.

Brown adipocyte ATF4 activation improves thermoregulation and systemic metabolism.

Cold-induced thermogenesis in endotherms demands adaptive thermogenesis fueled by mitochondrial respiration and Ucp1-mediated uncoupling in multilocular brown adipocytes (BAs). However, dietary regulation of thermogenesis in BAs isn't fully understood. Here, we describe that the deficiency of Leucine-rich pentatricopeptide repeat containing-protein (Lrpprc) in BAs reduces mtDNA-encoded ETC gene expression, causes ETC proteome imbalance, and abolishes the mitochondria-fueled thermogenesis. BA-specific Lrpprc knockout mice are cold resistant in a 4°C cold-tolerance test in the presence of food, which is accompanied by the activation of transcription factor 4 (ATF4) and proteome turnover in BAs. ATF4 activation genetically by BA-specific ATF4 overexpression or physiologically by a low-protein diet feeding can improve cold tolerance in wild-type and Ucp1 knockout mice. Furthermore, ATF4 activation in BAs improves systemic metabolism in obesogenic environment regardless of Ucp1's action. Therefore, our study reveals a diet-dependent but Ucp1-independent thermogenic mechanism in BAs that is relevant to systemic thermoregulation and energy homeostasis.

Multi-omic profiling reveals widespread dysregulation of innate immunity and hematopoiesis in COVID-19.

Our understanding of protective versus pathological immune responses to SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), is limited by inadequate profiling of patients at the extremes of the disease severity spectrum. Here, we performed multi-omic single-cell immune profiling of 64 COVID-19 patients across the full range of disease severity, from outpatients with mild disease to fatal cases. Our transcriptomic, epigenomic, and proteomic analyses revealed widespread dysfunction of peripheral innate immunity in severe and fatal COVID-19, including prominent hyperactivation signatures in neutrophils and NK cells. We also identified chromatin accessibility changes at NF-κB binding sites within cytokine gene loci as a potential mechanism for the striking lack of pro-inflammatory cytokine production observed in monocytes in severe and fatal COVID-19. We further demonstrated that emergency myelopoiesis is a prominent feature of fatal COVID-19. Collectively, our results reveal disease severity-associated immune phenotypes in COVID-19 and identify pathogenesis-associated pathways that are potential targets for therapeutic intervention.

Endosomal cAMP production broadly impacts the cellular phosphoproteome.

Endosomal signaling downstream of G-protein-coupled receptors (GPCRs) has emerged as a novel paradigm with important pharmacological and physiological implications. However, our knowledge of the functional consequences of intracellular signaling is incomplete. To begin to address this gap, we combined an optogenetic approach for site-specific generation of the prototypical second messenger generated by active GPCRs, cyclic AMP (cAMP), with unbiased mass-spectrometry-based analysis of the phosphoproteome. We identified 218 unique, high-confidence sites whose phosphorylation is either increased or decreased in response to cAMP elevation. We next determined that the same amount of cAMP produced from the endosomal membrane led to more robust changes in phosphorylation than the plasma membrane. Remarkably, this was true for the entire repertoire of 218 identified targets and irrespective of their annotated subcellular localizations (endosome, cell surface, nucleus, cytosol). Furthermore, we identified a particularly strong endosome bias for a subset of proteins that are dephosphorylated in response to cAMP. Through bioinformatics analysis, we established these targets as putative substrates for protein phosphatase 2A (PP2A), and we propose compartmentalized activation of PP2A by cAMP-responsive kinases as the likely underlying mechanism. Altogether, our study extends the concept that endosomal signaling is a significant functional contributor to cellular responsiveness to cAMP by establishing a unique role for localized cAMP production in defining categorically distinct phosphoresponses.

High-throughput SARS-CoV-2 and host genome sequencing from single nasopharyngeal swabs.

During COVID19 and other viral pandemics, rapid generation of host and pathogen genomic data is critical to tracking infection and informing therapies. There is an urgent need for efficient approaches to this data generation at scale. We have developed a scalable, high throughput approach to generate high fidelity low pass whole genome and HLA sequencing, viral genomes, and representation of human transcriptome from single nasopharyngeal swabs of COVID19 patients.

Dynamic post-translational modification profiling of Mycobacterium tuberculosis-infected primary macrophages.

Macrophages are highly plastic cells with critical roles in immunity, cancer, and tissue homeostasis, but how these distinct cellular fates are triggered by environmental cues is poorly understood. To uncover how primary murine macrophages respond to bacterial pathogens, we globally assessed changes in post-translational modifications of proteins during infection with Mycobacterium tuberculosis, a notorious intracellular pathogen. We identified hundreds of dynamically regulated phosphorylation and ubiquitylation sites, indicating that dramatic remodeling of multiple host pathways, both expected and unexpected, occurred during infection. Most of these cellular changes were not captured by mRNA profiling, and included activation of ubiquitin-mediated autophagy, an evolutionarily ancient cellular antimicrobial system. This analysis also revealed that a particular autophagy receptor, TAX1BP1, mediates clearance of ubiquitylated Mtb and targets bacteria to LC3-positive phagophores. These studies provide a new resource for understanding how macrophages shape their proteome to meet the challenge of infection.