Discovery Proteomics Analysis Determines That Driver Oncogenes Suppress Antiviral Defense Pathways Through Reduction in Interferon-ß Autocrine Stimulation.

Since the discovery of oncogenes, there has been tremendous interest to understand their mechanistic basis and to develop broadly actionable therapeutics. Some of the most frequently activated oncogenes driving diverse cancers are c-MYC, EGFR, HER2, AKT, KRAS, BRAF, and MEK. Using a reductionist approach, we explored how cellular proteomes are remodeled in isogenic cell lines engineered with or without these driver oncogenes. The most striking discovery for all oncogenic models was the systematic downregulation of scores of antiviral proteins regulated by type 1 interferon. These findings extended to cancer cell lines and patient-derived xenograft models of highly refractory pancreatic cancer and osteosarcoma driven by KRAS and MYC oncogenes. The oncogenes reduced basal expression of and autocrine stimulation by type 1 interferon causing remarkable convergence on common phenotypic and functional profiles. In particular, there was dramatically lower expression of dsRNA sensors including DDX58 (RIG-I) and OAS proteins, which resulted in attenuated functional responses when the oncogenic cells were treated with the dsRNA mimetic, polyI:C, and increased susceptibility to infection with an RNA virus shown using SARS-CoV-2. Our reductionist approach provides molecular and functional insights connected to immune evasion hallmarks in cancers and suggests therapeutic opportunities.

A deep analysis of the proteomic and phosphoproteomic alterations that occur in skeletal muscle after the onset of immobilization.

KEY POINTS: A decrease in protein synthesis plays a major role in the loss of muscle mass that occurs in response to immobilization. In mice, immobilization leads to a rapid (within 6 h) and progressive decrease in the rate of protein synthesis and this effect is mediated by a decrease in translational efficiency. Deep proteomic and phosphoproteomic analyses of mouse skeletal muscles revealed that the rapid immobilization-induced decrease in protein synthesis cannot be explained by changes in the abundance or phosphorylation state of proteins that have been implicated in the regulation of translation. ABSTRACT: The disuse of skeletal muscle, such as that which occurs during immobilization, can lead to the rapid loss of muscle mass, and a decrease in the rate of protein synthesis plays a major role in this process. Indeed, current dogma contends that the decrease in protein synthesis is mediated by changes in the activity of protein kinases (e.g. mTOR); however, the validity of this model has not been established. Therefore, to address this, we first subjected mice to 6, 24 or 72 h of unilateral immobilization and then used the SUnSET technique to measure changes in the relative rate of protein synthesis. The result of our initial experiments revealed that immobilization leads to a rapid (within 6 h) and progressive decrease in the rate of protein synthesis and that this effect is mediated by a decrease in translational efficiency. We then performed a deep mass spectrometry-based analysis to determine whether this effect could be explained by changes in the expression and/or phosphorylation state of proteins that regulate translation. From this analysis, we were able to quantify 4320 proteins and 15,020 unique phosphorylation sites, and surprisingly, the outcomes revealed that the rapid immobilization-induced decrease in protein synthesis could not be explained by changes in either the abundance, or phosphorylation state, of proteins. The results of our work not only challenge the current dogma in the field, but also provide an expansive resource of information for future studies that are aimed at defining how disuse leads to loss of muscle mass.

Mapping of the contraction-induced phosphoproteome identifies TRIM28 as a significant regulator of skeletal muscle size and function.

Mechanical signals, such as those evoked by maximal-intensity contractions (MICs), can induce an increase in muscle mass. Rapamycin-sensitive signaling events are widely implicated in the regulation of this process; however, recent studies indicate that rapamycin-insensitive signaling events are also involved. Thus, to identify these events, we generate a map of the MIC-regulated and rapamycin-sensitive phosphoproteome. In total, we quantify more than 10,000 unique phosphorylation sites and find that more than 2,000 of these sites are significantly affected by MICs, but remarkably, only 38 of the MIC-regulated events are mediated through a rapamycin-sensitive mechanism. Further interrogation of the rapamycin-insensitive phosphorylation events identifies the S473 residue on Tripartite Motif-Containing 28 (TRIM28) as one of the most robust MIC-regulated phosphorylation sites, and extensive follow-up studies suggest that TRIM28 significantly contributes to the homeostatic regulation of muscle size and function as well as the hypertrophy that occurs in response to increased mechanical loading.

Peak Filtering, Peak Annotation, and Wildcard Search for Glycoproteomics.

Glycopeptides in peptide or digested protein samples pose a number of analytical and bioinformatics challenges beyond those posed by unmodified peptides or peptides with smaller posttranslational modifications. Exact structural elucidation of glycans is generally beyond the capability of a single mass spectrometry experiment, so a reasonable level of identification for tandem mass spectrometry, taken by several glycopeptide software tools, is that of peptide sequence and glycan composition, meaning the number of monosaccharides of each distinct mass, e.g., HexNAc(2)Hex(5) rather than man5. Even at this level, however, glycopeptide analysis poses challenges: finding glycopeptide spectra when they are a tiny fraction of the total spectra; assigning spectra with unanticipated glycans, not in the initial glycan database; and finding, scoring, and labeling diagnostic peaks in tandem mass spectra. Here, we discuss recent improvements to Byonic, a glycoproteomics search program, that address these three issues. Byonic now supports filtering spectra by m/z peaks, so that the user can limit attention to spectra with diagnostic peaks, e.g., at least two out of three of 204.087 for HexNAc, 274.092 for NeuAc (with water loss), and 366.139 for HexNAc-Hex, all within a set mass tolerance, e.g., ± 0.01 Da. Also, new is glycan "wildcard" search, which allows an unspecified mass within a user-set mass range to be applied to N- or O-linked glycans and enables assignment of spectra with unanticipated glycans. Finally, the next release of Byonic supports user-specified peak annotations from user-defined posttranslational modifications. We demonstrate the utility of these new software features by finding previously unrecognized glycopeptides in publicly available data, including glycosylated neuropeptides from rat brain.

Phosphorylation controls RNA binding and transcription by the influenza virus polymerase.

The influenza virus polymerase transcribes and replicates the viral genome. The proper timing and balance of polymerase activity is important for successful replication. Genome replication is controlled in part by phosphorylation of NP that regulates assembly of the replication machinery. However, it remains unclear whether phosphorylation directly regulated polymerase activity. Here we identified polymerase phosphosites that control its function. Mutating phosphosites in the catalytic subunit PB1 altered polymerase activity and virus replication. Biochemical analyses revealed phosphorylation events that disrupted global polymerase function by blocking the NTP entry channel or preventing RNA binding. We also identified a regulatory site that split polymerase function by specifically suppressing transcription. These experiments show that host kinases phospho-regulate viral RNA synthesis directly by modulating polymerase activity and indirectly by controlling assembly of replication machinery. Further, they suggest polymerase phosphorylation may bias replication versus transcription at discrete times or locations during the infectious cycle.

Discovering How Heme Controls Genome Function Through Heme-omics.

Protein ensembles control genome function by establishing, maintaining, and deconstructing cell-type-specific chromosomal landscapes. A plethora of small molecules orchestrate cellular functions and therefore may link physiological processes with genome biology. The metabolic enzyme and hemoglobin cofactor heme induces proteolysis of a transcriptional repressor, Bach1, and regulates gene expression post-transcriptionally. However, whether heme controls genome function broadly or through prescriptive actions is unclear. Using assay for transposase-accessible chromatin sequencing (ATAC-seq), we establish a heme-dependent chromatin atlas in wild-type and mutant erythroblasts lacking enhancers that confer normal heme synthesis. Amalgamating chromatin landscapes and transcriptomes in cells with sub-physiological heme and post-heme rescue reveals parallel Bach1-dependent and Bach1-independent mechanisms that target heme-sensing chromosomal hotspots. The hotspots harbor a DNA motif demarcating heme-regulated chromatin and genes encoding proteins not known to be heme regulated, including metabolic enzymes. The heme-omics analysis establishes how an essential biochemical cofactor controls genome function and cellular physiology.

Post-Translation Regulation of Influenza Virus Replication.

Influenza virus exploits cellular factors to complete each step of viral replication. Yet, multiple host proteins actively block replication. Consequently, infection success depends on the relative speed and efficacy at which both the virus and host use their respective effectors. Post-translational modifications (PTMs) afford both the virus and the host means to readily adapt protein function without the need for new protein production. Here we use influenza virus to address concepts common to all viruses, reviewing how PTMs facilitate and thwart each step of the replication cycle. We also discuss advancements in proteomic methods that better characterize PTMs. Although some effectors and PTMs have clear pro- or antiviral functions, PTMs generally play regulatory roles to tune protein functions, levels, and localization. Synthesis of our current understanding reveals complex regulatory schemes where the effects of PTMs are time and context dependent as the virus and host battle to control infection.

Broad and thematic remodeling of the surfaceome and glycoproteome on isogenic cells transformed with driving proliferative oncogenes.

The cell surface proteome, the surfaceome, is the interface for engaging the extracellular space in normal and cancer cells. Here we apply quantitative proteomics of N-linked glycoproteins to reveal how a collection of some 700 surface proteins is dramatically remodeled in an isogenic breast epithelial cell line stably expressing any of six of the most prominent proliferative oncogenes, including the receptor tyrosine kinases, EGFR and HER2, and downstream signaling partners such as KRAS, BRAF, MEK, and AKT. We find that each oncogene has somewhat different surfaceomes, but the functions of these proteins are harmonized by common biological themes including up-regulation of nutrient transporters, down-regulation of adhesion molecules and tumor suppressing phosphatases, and alteration in immune modulators. Addition of a potent MEK inhibitor that blocks MAPK signaling brings each oncogene-induced surfaceome back to a common state reflecting the strong dependence of the oncogene on the MAPK pathway to propagate signaling. Cell surface protein capture is mediated by covalent tagging of surface glycans, yet current methods do not afford sequencing of intact glycopeptides. Thus, we complement the surfaceome data with whole cell glycoproteomics enabled by a recently developed technique called activated ion electron transfer dissociation (AI-ETD). We found massive oncogene-induced changes to the glycoproteome and differential increases in complex hybrid glycans, especially for KRAS and HER2 oncogenes. Overall, these studies provide a broad systems-level view of how specific driver oncogenes remodel the surfaceome and the glycoproteome in a cell autologous fashion, and suggest possible surface targets, and combinations thereof, for drug and biomarker discovery.

Proteomic and transcriptomic analyses of early and late-chronic Toxoplasma gondii infection shows novel and stage specific transcripts.

BACKGROUND: The protozoan pathogen Toxoplasma gondii has the unique ability to develop a chronic infection in the brain of its host by transitioning from the fast growing tachyzoite morphology to latent bradyzoite morphology. A hallmark of the bradyzoite is the development of neuronal cysts that are resilient against host immune response and current therapeutics. The bradyzoite parasites within the cyst have a carbohydrate and protein-rich wall and a slow-replication cycle, allowing them to remain hidden from the host. The intracellular, encysted lifestyle of T. gondii has made them recalcitrant to molecular analysis in vivo. RESULTS: Here, we detail the results from transcriptional and proteomic analyses of bradyzoite-enriched fractions isolated from mouse brains infected with T. gondii over a time course of 21 to 150 days. The enrichment procedure afforded consistent identification of over 2000 parasitic peptides from the mixed-organism sample, representing 366 T. gondii proteins at 28, 90, and 120 day timepoints. Deep sequencing of transcripts expressed during these three timepoints revealed that a subpopulation of genes that are transcriptionally expressed at a high level. Approximately one-third of these transcripts are more enriched during bradyzoite conditions compared to tachyzoites and approximately half are expressed at similar levels during each phase. The T. gondii transcript which increased the most over the course of chronic infection, sporoAMA1, shows stage specific isoform expression of the gene. CONCLUSIONS: We have expanded the transcriptional profile of in vivo bradyzoites to 120 days post-infection and provided the first in vivo proteomic profile of T. gondii bradyzoites. The RNA sequencing depth of in vivo bradyzoite T. gondii was over 250-fold greater than previous reports and allowed us to identify low level transcripts and a novel bradyzoite-specific isoform of sporoAMA1.

Pptc7 is an essential phosphatase for promoting mammalian mitochondrial metabolism and biogenesis.

Mitochondrial proteins are replete with phosphorylation, yet its functional relevance remains largely unclear. The presence of multiple resident mitochondrial phosphatases, however, suggests that protein dephosphorylation may be broadly important for calibrating mitochondrial activities. To explore this, we deleted the poorly characterized matrix phosphatase Pptc7 from mice using CRISPR-Cas9 technology. Strikingly, Pptc7-/- mice exhibit hypoketotic hypoglycemia, elevated acylcarnitines and serum lactate, and die soon after birth. Pptc7-/- tissues have markedly diminished mitochondrial size and protein content despite normal transcript levels, and aberrantly elevated phosphorylation on select mitochondrial proteins. Among these, we identify the protein translocase complex subunit Timm50 as a putative Pptc7 substrate whose phosphorylation reduces import activity. We further find that phosphorylation within or near the mitochondrial targeting sequences of multiple proteins could disrupt their import rates and matrix processing. Overall, our data define Pptc7 as a protein phosphatase essential for proper mitochondrial function and biogenesis during the extrauterine transition.

GATA/Heme Multi-omics Reveals a Trace Metal-Dependent Cellular Differentiation Mechanism.

By functioning as an enzyme cofactor, hemoglobin component, and gene regulator, heme is vital for life. One mode of heme-regulated transcription involves amplifying the activity of GATA-1, a key determinant of erythrocyte differentiation. To discover biological consequences of the metal cofactor-transcription factor mechanism, we merged GATA-1/heme-regulated sectors of the proteome and transcriptome. This multi-omic analysis revealed a GATA-1/heme circuit involving hemoglobin subunits, ubiquitination components, and proteins not implicated in erythrocyte biology, including the zinc exporter Slc30a1. Though GATA-1 induced expression of Slc30a1 and the zinc importer Slc39a8, Slc39a8 dominantly increased intracellular zinc, which conferred erythroblast survival. Subsequently, a zinc transporter switch, involving decreased importer and sustained exporter expression, reduced intracellular zinc during terminal differentiation. Downregulating Slc30a1 increased intracellular zinc and, strikingly, accelerated differentiation. This analysis established a conserved paradigm in which a GATA-1/heme circuit controls trace metal transport machinery and trace metal levels as a mechanism governing cellular differentiation.

Identifying Novel Signaling Pathways: An Exercise Scientists Guide to Phosphoproteomics.

We propose that phosphoproteomic-based studies will radically advance our knowledge about exercise-regulated signaling events. However, these studies use cutting-edge technologies that can be difficult for nonspecialists to understand. Hence, this review is intended to help nonspecialists 1) understand the fundamental technologies behind phosphoproteomic analysis and 2) use various bioinformatic tools that can be used to interrogate phosphoproteomic datasets.

Multi-omics Reveal Specific Targets of the RNA-Binding Protein Puf3p and Its Orchestration of Mitochondrial Biogenesis.

Coenzyme Q (CoQ) is a redox-active lipid required for mitochondrial oxidative phosphorylation (OxPhos). How CoQ biosynthesis is coordinated with the biogenesis of OxPhos protein complexes is unclear. Here, we show that the Saccharomyces cerevisiae RNA-binding protein (RBP) Puf3p regulates CoQ biosynthesis. To establish the mechanism for this regulation, we employed a multi-omic strategy to identify mRNAs that not only bind Puf3p but also are regulated by Puf3p in vivo. The CoQ biosynthesis enzyme Coq5p is a critical Puf3p target: Puf3p regulates the abundance of Coq5p and prevents its detrimental hyperaccumulation, thereby enabling efficient CoQ production. More broadly, Puf3p represses a specific set of proteins involved in mitochondrial protein import, translation, and OxPhos complex assembly (pathways essential to prime mitochondrial biogenesis). Our data reveal a mechanism for post-transcriptionally coordinating CoQ production with OxPhos biogenesis, and they demonstrate the power of multi-omics for defining genuine targets of RBPs.

Unusual Reactivity of a Potent Platinum-Acridine Hybrid Antitumor Agent.

The formation of unusual seven-membered, sterically overloaded chelates [Pt(en)(L/L´)](NO(3))(2) (4a/4b) from the corresponding potent hybrid antitumor agents [PtCl(en)(LH/L´H)](NO(3))(2) (3a/3b) is described, where en is ethane-1,2-diamine and L(H) and L´(H) are (protonated) N-(2-(acridin-9-ylamino)ethyl)-N-methylpropionimidamide and N-(2-(acridin-9-ylamino)ethyl)-N-methylacetimidamide, respectively. Compounds 3a and 3b inhibit H460 lung cancer cell proliferation with IC(50) values of 12 ± 2 nM and 2.8 ± 0.3 nM, respectively. The new derivative 3b proves to be not only the most cytotoxic platinum-acridine hybrid of this kind, but also one of the most potent platinum-based anticancer agents described to date. The chelates 4a and 4b do not undergo ligand substitution reactions with nucleobase nitrogen and cysteine sulfur and do not intercalate into DNA. Despite their inertness, the two chelates appear to maintain micromolar activity in H460 cells. The results are discussed in the context of potential DNA-mediated and DNA-independent cell kill mechanisms and the potential use of the chelates as prodrugs.

Cytosolic protein interactions of the schizophrenia susceptibility gene dysbindin.

Using immunoprecipitation, mass spectrometry, and western blot analysis we investigated cytosolic protein interactions of the schizophrenia susceptibility gene dysbindin in mammalian cells. We identified novel interactions with members of the exocyst, dynactin and chaperonin containing T-complex protein complexes, and we confirmed interactions reported previously with all members of the biogenesis of lysosome-related organelles complex-1 and the adaptor-related protein complex 3. We report interactions between dysbindin and the exocyst and dynactin complex that confirm a link between two important schizophrenia susceptibility genes: dysbindin and disrupted-in-schizophrenia-1. To expand upon this network of interacting proteins we also investigated protein interactions for members of the exocyst and dynactin complexes in mammalian cells. Our results are consistent with the notion that impairment of aspects of the synaptic vesicle life cycle may be a pathogenic mechanism in schizophrenia.

Comparison of source apportionment and sensitivity analysis in a particulate matter air duality model.

Two efficient methods to study relationships between particulate matter (PM) concentrations and emission sources are compared in the three-dimensional comprehensive air quality model with extensions (CAMx). Particulate source apportionment technology (PSAT) is a tagged species method that apportions concentrations of PM components to their respective primary precursors, e.g., sulfate is apportioned to SOx, nitrate to NOx, etc. The decoupled direct method (DDM) calculates first-order sensitivities of PM concentrations to model inputs. Both tools were applied to two month long (February and July) PM modeling episodes and evaluated against changes in PM concentrations due to various emission reductions. The results show that source contributions calculated by PSAT start to deviate from the actual model responses as indirect effects from limiting reactants or nonprimary precursor emissions become important The DDM first-order sensitivity is useful for determining source contributions only if the model response to input changes is reasonably linear. For secondary inorganic PM, the response is linear for emission reductions of 20% in all cases considered and reasonably linear for reductions of 100% inthe case of on-road mobile sources. The model response for secondary organic aerosols and primary PM remains nearly linear to 100% reductions in anthropogenic emissions.

DNA copy-number analysis in bipolar disorder and schizophrenia reveals aberrations in genes involved in glutamate signaling.

Using bacterial artificial chromosome (BAC) array comparative genome hybridization (aCGH) at approximately 1.4 Mbp resolution, we screened post-mortem brain DNA from bipolar disorder cases, schizophrenia cases and control individuals (n=35 each) for DNA copy-number aberrations. DNA copy number is a largely unexplored source of human genetic variation that may contribute risk for complex disease. We report aberrations at four loci which were seen in affected but not control individuals, and which were verified by quantitative real-time PCR. These aberrant loci contained the genes encoding EFNA5, GLUR7, CACNG2 and AKAP5; all brain-expressed proteins with known or postulated roles in neuronal function, and three of which (GLUR7, CACNG2 and AKAP5) are involved in glutamate signaling. A second cohort of psychiatric samples was also tested by quantitative PCR using the primer/probe sets for EFNA5, GLUR7, CACNG2 and AKAP5, and samples with aberrant copy number were found at three of the four loci (GLUR7, CACNG2 and AKAP5). Further scrutiny of these regions may reveal insights into the etiology and genetic risk factors for these complex psychiatric disorders.

Identification by full-coverage array CGH of human DNA copy number increases relative to chimpanzee and gorilla.

Duplication of chromosomal segments and associated genes is thought to be a primary mechanism for generating evolutionary novelty. By comparative genome hybridization using a full-coverage (tiling) human BAC array with 79-kb resolution, we have identified 63 chromosomal segments, ranging in size from 0.65 to 1.3 Mb, that have inferred copy number increases in human relative to chimpanzee. These segments span 192 Ensembl genes, including 82 gene duplicates (41 reciprocal best BLAST matches). Synonymous and nonsynonymous substitution rates across these pairs provide evidence for general conservation of the amino acid sequence, consistent with the maintenance of function of both copies, and one case of putative positive selection for an uncharacterized gene. Surprisingly, the core histone genes H2A, H2B, H3, and H4 have been duplicated in the human lineage since our split with chimpanzee. The observation of increased copy number of a human cluster of core histone genes suggests that altered dosage, even of highly constrained genes, may be an important evolutionary mechanism.

Activation/division of lymphocytes results in increased levels of cytoplasmic activation/proliferation-associated protein-1: prototype of a new family of proteins.

We purified from activated T lymphocytes a novel, highly conserved, 116-kDa, intracellular protein that occurred at high levels in the large, dividing cells of the thymus, was up-regulated when resting T or B lymphocytes or hemopoietic progenitors were activated, and was down-regulated when a monocytic leukemia, M1, was induced to differentiate. Expression of the protein was highest in the thymus and spleen and lowest in tissues with a low proportion of dividing cells such as kidney or muscle, although expression was high in the brain. The protein was localized to the cytosol and was phosphorylated, which is consistent with a previous report that the Xenopus laevis ortholog was phosphorylated by a mitotically activated kinase (1 ). The cDNA was previously mischaracterized as encoding p137, a 137-kDa GPI-linked membrane protein (2 ). We propose that the authentic protein encoded by this cDNA be called cytoplasmic activation/proliferation-associated protein-1 (caprin-1), and show that it is the prototype of a novel family of proteins characterized by two novel protein domains, termed homology regions-1 and -2 (HR-1, HR-2). Although we have found evidence for caprins only in urochordates and vertebrates, two insect proteins exhibit well-conserved HR-1 domains. The HR-1 and HR-2 domains have no known function, although the HR-1 of caprin-1 appeared necessary for formation of multimeric complexes of caprin-1. Overexpression of a fusion protein of enhanced green fluorescent protein and caprin-1 induced a specific, dose-dependent suppression of the proliferation of NIH-3T3 cells, consistent with the notion that caprin-1 plays a role in cellular activation or proliferation.

Comparison of source apportionment and source sensitivity of ozone in a three-dimensional air quality model.

The ozone source apportionment technology (OSAT) estimates the contributions of different sources to ozone concentrations using a set of tracers for NOx, total VOCs, and ozone and an indicator that ascribes instantaneous ozone production to NOx or VOCs. These source contributions were compared to first-order sensitivities obtained by the decoupled direct method (DDM) for a three-dimensional simulation of an ozone episode in the Lake Michigan region. The cut-point for the OSAT indicator between VOC- and NOx-sensitive ozone production agrees well with the DDM sensitivities to VOC and NOx. In a ranking of the most important contributors to ozone concentrations >80 ppb, the OSAT and DDM results agreed on four of the top five contributors on average. The spatial distributions of the sensitivities and source contributions are similar, and the OSAT and DDM results for ozone >80 ppb correlate well. However, the source contributions ascribe substantially less relative importance to anthropogenic emissions and greater relative importance to the boundary concentrations than do the sensitivities. In regions where NOx inhibits ozone formation and the sensitivity is negative, the source contribution is small and positive. For the same subdivision of the emissions, the OSAT is 14 times faster than the DDM, but the DDM has greater flexibility in defining which emissions to include and generates results for species other than ozone. The first-order sensitivities explain, on average, 70% of the ozone concentrations.