The Human Ganglioside Interactome in Live Cells Revealed Using Clickable Photoaffinity Ganglioside Probes.

Gangliosides, sialic acid bearing glycosphingolipids, are components of the outer leaflet of plasma membranes of all vertebrate cells. They contribute to cell regulation by interacting with proteins in their own membranes (cis) or their extracellular milieu (trans). As amphipathic membrane constituents, gangliosides present challenges for identifying their ganglioside protein interactome. To meet these challenges, we synthesized bifunctional clickable photoaffinity gangliosides, delivered them to plasma membranes of cultured cells, then captured and identified their interactomes using proteomic mass spectrometry. Installing probes on ganglioside lipid and glycan moieties, we captured cis and trans ganglioside-protein interactions. Ganglioside interactomes varied with the ganglioside structure, cell type, and site of the probe (lipid or glycan). Gene ontology revealed that gangliosides engage with transmembrane transporters and cell adhesion proteins including integrins, cadherins, and laminins. The approach developed is applicable to other gangliosides and cell types, promising to provide insights into molecular and cellular regulation by gangliosides.

Arginine methylation of RNA-binding proteins is impaired in Huntington's disease.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion in the HD gene, coding for huntingtin protein (HTT). Mechanisms of HD cellular pathogenesis remain undefined and likely involve disruptions in many cellular processes and functions presumably mediated by abnormal protein interactions of mutant HTT. We previously found HTT interaction with several protein arginine methyl-transferase (PRMT) enzymes. Protein arginine methylation mediated by PRMT enzymes is an important post-translational modification with an emerging role in neurodegeneration. We found that normal (but not mutant) HTT can facilitate the activity of PRMTs in vitro and the formation of arginine methylation complexes. These interactions appear to be disrupted in HD neurons. This suggests an additional functional role for HTT/PRMT interactions, not limited to substrate/enzyme relationship, which may result in global changes in arginine protein methylation in HD. Our quantitative analysis of striatal precursor neuron proteome indicated that arginine protein methylation is significantly altered in HD. We identified a cluster highly enriched in RNA-binding proteins with reduced arginine methylation, which is essential to their function in RNA processing and splicing. We found that several of these proteins interact with HTT, and their RNA-binding and localization are affected in HD cells likely due to a compromised arginine methylation and/or abnormal interactions with mutant HTT. These studies reveal a potential new mechanism for disruption of RNA processing in HD, involving a direct interaction of HTT with methyl-transferase enzymes and modulation of their activity and highlighting methylation of arginine as potential new therapeutic target for HD.

Differential Detection of O-GlcNAcylated proteins in the heart using antibodies.

Thousands of mammalian intracellular proteins are dynamically modified by O-linked ß-N-acetylglucosamine (O-GlcNAc). Global changes in O-GlcNAcylation have been associated with the development of cardiomyopathy, heart failure, hypertension, and neurodegenerative disease. Levels of O-GlcNAc in cells and tissues can be detected using numerous approaches; however, immunoblotting using GlcNAc-specific antibodies and lectins is commonplace. The goal of this study was to optimize the detection of O-GlcNAc in heart lysates by immunoblotting. Using a combination of tissue fractionation, immunoblotting, and galactosyltransferase labeling, as well as hearts from wild-type and O-GlcNAc transferase transgenic mice, we demonstrate that contractile proteins in the heart are differentially detected by two commercially available antibodies (CTD110.6 and RL2). As CTD110.6 displays poor reactivity toward contractile proteins, and as these proteins represent a major fraction of the heart proteome, a better assessment of cardiac O-GlcNAcylation is obtained in total tissue lysates with RL2. The data presented highlight tissue lysis approaches that should aid the assessment of the cardiac O-GlcNAcylation by immunoblotting.

The Interaction of Borrelia burgdorferi with Human Dendritic Cells: Functional Implications.

Dendritic cells bridge the innate and adaptive immune responses by serving as sensors of infection and as the primary APCs responsible for the initiation of the T cell response against invading pathogens. The naive T cell activation requires the following three key signals to be delivered from dendritic cells: engagement of the TCR by peptide Ags bound to MHC molecules (signal 1), engagement of costimulatory molecules on both cell types (signal 2), and expression of polarizing cytokines (signal 3). Initial interactions between Borrelia burgdorferi, the causative agent of Lyme disease, and dendritic cells remain largely unexplored. To address this gap in knowledge, we cultured live B. burgdorferi with monocyte-derived dendritic cells (mo-DCs) from healthy donors to examine the bacterial immunopeptidome associated with HLA-DR. In parallel, we examined changes in the expression of key costimulatory and regulatory molecules as well as profiled the cytokines released by dendritic cells when exposed to live spirochetes. RNA-sequencing studies on B. burgdorferi-pulsed dendritic cells show a unique gene expression signature associated with B. burgdorferi stimulation that differs from stimulation with lipoteichoic acid, a TLR2 agonist. These studies revealed that exposure of mo-DCs to live B. burgdorferi drives the expression of both pro- and anti-inflammatory cytokines as well as immunoregulatory molecules (e.g., PD-L1, IDO1, Tim3). Collectively, these studies indicate that the interaction of live B. burgdorferi with mo-DCs promotes a unique mature DC phenotype that likely impacts the nature of the adaptive T cell response generated in human Lyme disease.

Global Discovery and Temporal Changes of Human Albumin Modifications by Pan-Protein Adductomics: Initial Application to Air Pollution Exposure.

Assessing personal exposure to environmental toxicants is a critical challenge for predicting disease risk. Previously, using human serum albumin (HSA)-based biomonitoring, we reported dosimetric relationships between adducts at HSA Cys34 and ambient air pollutant levels (Smith et al., Chem. Res. Toxicol. 2021, 34, 1183). These results provided the foundation to explore modifications at other sites in HSA to reveal novel adducts of complex exposures. Thus, the Pan-Protein Adductomics (PPA) technology reported here is the next step toward an unbiased, comprehensive characterization of the HSA adductome. The PPA workflow requires <2 µL serum/plasma and uses nanoflow-liquid chromatography, gas-phase fractionation, and overlapping-window data-independent acquisition high-resolution tandem mass spectrometry. PPA analysis of albumin from nonsmoking women exposed to high levels of air pollution uncovered 68 unique location-specific modifications (LSMs) across 21 HSA residues. While nearly half were located at Cys34 (33 LSMs), 35 were detected on other residues, including Lys, His, Tyr, Ser, Met, and Arg. HSA adduct relative abundances spanned a ∼400 000-fold range and included putative products of exogenous (SO2, benzene, phycoerythrobilin) and endogenous (oxidation, lipid peroxidation, glycation, carbamylation) origin, as well as 24 modifications without annotations. PPA quantification revealed statistically significant changes in LSM levels across the 84 days of monitoring (∼3 HSA lifetimes) in the following putative adducts: Cys34 trioxidation, ß-methylthiolation, benzaldehyde, and benzene diol epoxide; Met329 oxidation; Arg145 dioxidation; and unannotated Cys34 and His146 adducts. Notably, the PPA workflow can be extended to any protein. Pan-Protein Adductomics is a novel and powerful strategy for untargeted global exploration of protein modifications.

De novo methylation of histone H3K23 by the methyltransferases EHMT1/GLP and EHMT2/G9a.

Epigenetic modifications to histone proteins serve an important role in regulating permissive and repressive chromatin states, but despite the identification of many histone PTMs and their perceived role, the epigenetic writers responsible for generating these chromatin signatures are not fully characterized. Here, we report that the canonical histone H3K9 methyltransferases EHMT1/GLP and EHMT2/G9a are capable of catalyzing methylation of histone H3 lysine 23 (H3K23). Our data show that while both enzymes can mono- and di-methylate H3K23, only EHMT1/GLP can tri-methylate H3K23. We also show that pharmacologic inhibition or genetic ablation of EHMT1/GLP and/or EHMT2/G9a leads to decreased H3K23 methylation in mammalian cells. Taken together, this work identifies H3K23 as a new direct methylation target of EHMT1/GLP and EHMT2/G9a, and highlights the differential activity of these enzymes on H3K23 as a substrate.

Tbx18 Orchestrates Cytostructural Transdifferentiation of Cardiomyocytes to Pacemaker Cells by Recruiting the Epithelial-Mesenchymal Transition Program.

Previously, we reported that heterologous expression of an embryonic transcription factor, Tbx18, reprograms ventricular cardiomyocytes into induced pacemaker cells (Tbx18-iPMs), though the key pathways are unknown. Here, we have used a tandem mass tag proteomic approach to characterize the impact of Tbx18 on neonatal rat ventricular myocytes. Tbx18 expression triggered vast proteome remodeling. Tbx18-iPMs exhibited increased expression of known pacemaker ion channels, including Hcn4 and Cx45 as well as upregulation of the mechanosensitive ion channels Piezo1, Trpp2 (PKD2), and TrpM7. Metabolic pathways were broadly downregulated, as were ion channels associated with ventricular excitation-contraction coupling. Tbx18-iPMs also exhibited extensive intracellular cytoskeletal and extracellular matrix remodeling, including 96 differentially expressed proteins associated with the epithelial-to-mesenchymal transition (EMT). RNAseq extended coverage of low abundance transcription factors, revealing upregulation of EMT-inducing Snai1, Snai2, Twist1, Twist2, and Zeb2. Finally, network diffusion mapping of >200 transcriptional regulators indicates EMT and heart development factors occupy adjacent network neighborhoods downstream of Tbx18 but upstream of metabolic control factors. In conclusion, transdifferentiation of cardiac myocytes into pacemaker cells entails massive electrogenic, metabolic, and cytostructural remodeling. Structural changes exhibit hallmarks of the EMT. The results aid ongoing efforts to maximize the yield and phenotypic stability of engineered biological pacemakers.

Interaction of huntingtin with PRMTs and its subsequent arginine methylation affects HTT solubility, phase transition behavior and neuronal toxicity.

Huntington's disease (HD) is an incurable neurodegenerative disorder caused by a CAG expansion in the huntingtin gene (HTT). Post-translational modifications of huntingtin protein (HTT), such as phosphorylation, acetylation and ubiquitination, have been implicated in HD pathogenesis. Arginine methylation/dimethylation is an important modification with an emerging role in neurodegeneration; however, arginine methylation of HTT remains largely unexplored. Here we report nearly two dozen novel arginine methylation/dimethylation sites on the endogenous HTT from human and mouse brain and human cells suggested by mass spectrometry with data-dependent acquisition. Targeted quantitative mass spectrometry identified differential arginine methylation at specific sites in HD patient-derived striatal precursor cell lines compared to normal controls. We found that HTT can interact with several type I protein arginine methyltransferases (PRMTs) via its N-terminal domain. Using a combination of in vitro methylation and cell-based experiments, we identified PRMT4 (CARM1) and PRMT6 as major enzymes methylating HTT at specific arginines. Alterations of these methylation sites had a profound effect on biochemical properties of HTT rendering it less soluble in cells and affected its liquid-liquid phase separation and phase transition patterns in vitro. We found that expanded HTT 1-586 fragment can form liquid-like assemblies, which converted into solid-like assemblies when the R200/205 methylation sites were altered. Methyl-null alterations increased HTT toxicity to neuronal cells, while overexpression of PRMT 4 and 6 was beneficial for neuronal survival. Thus, arginine methylation pathways that involve specific HTT-modifying PRMT enzymes and modulate HTT biochemical and toxic properties could provide targets for HD-modifying therapies.

Deleting a UBE3A substrate rescues impaired hippocampal physiology and learning in Angelman syndrome mice.

In humans, loss-of-function mutations in the UBE3A gene lead to the neurodevelopmental disorder Angelman syndrome (AS). AS patients have severe impairments in speech, learning and memory, and motor coordination, for which there is currently no treatment. In addition, UBE3A is duplicated in > 1-2% of patients with autism spectrum disorders-a further indication of the significant role it plays in brain development. Altered expression of UBE3A, an E3 ubiquitin ligase, is hypothesized to lead to impaired levels of its target proteins, but identifying the contribution of individual UBE3A targets to UBE3A-dependent deficits remains of critical importance. Ephexin5 is a putative UBE3A substrate that has restricted expression early in development, regulates synapse formation during hippocampal development, and is abnormally elevated in AS mice, modeled by maternally-derived Ube3a gene deletion. Here, we report that Ephexin5 can be directly ubiquitylated by UBE3A. Furthermore, removing Ephexin5 from AS mice specifically rescued hippocampus-dependent behaviors, CA1 physiology, and deficits in dendritic spine number. Our findings identify Ephexin5 as a key driver of hippocampal dysfunction and related behavioral deficits in AS mouse models. These results demonstrate the exciting potential of targeting Ephexin5, and possibly other UBE3A substrates, to improve symptoms of AS and other UBE3A-related developmental disorders.

Biomonitoring of Ambient Outdoor Air Pollutant Exposure in Humans Using Targeted Serum Albumin Adductomics.

Outdoor air pollution, a spatially and temporally complex mixture, is a human carcinogen. However, ambient measurements may not reflect subject-level exposures, personal monitors do not assess internal dose, and spot assessments of urinary biomarkers may not recapitulate chronic exposures. Nucleophilic sites in serum albumin-particularly the free thiol at Cys34-form adducts with electrophiles. Due to the 4-week lifetime of albumin in circulation, accumulating adducts can serve as intermediate- to long-residence biomarkers of chronic exposure and implicate potential biological effects. Employing nanoflow liquid chromatography-high-resolution mass spectrometry (nLC-HRMS) and parallel reaction monitoring (PRM), we have developed and validated a novel targeted albumin adductomics platform capable of simultaneously monitoring dozens of Cys34 adducts per sample in only 2.5 µL of serum, with on-column limits of detection in the low-femtomolar range. Using this platform, we characterized the magnitude and impact of ambient outdoor air pollution exposures with three repeated measurements over 84 days in n = 26 nonsmoking women (n = 78 total samples) from Qidong, China, an area with a rising burden of lung cancer incidence. In concordance with seasonally rising ambient concentrations of NO2, SO2, and PM10 measured at stationary monitors, we observed elevations in concentrations of Cys34 adducts of benzoquinone (p < 0.05), benzene diol epoxide (BDE; p < 0.05), crotonaldehyde (p < 0.01), and oxidation (p < 0.001). Regression analysis revealed significant elevations in oxidation and BDE adduct concentrations of 300% to nearly 700% per doubling of ambient airborne pollutant levels (p < 0.05). Notably, the ratio of irreversibly oxidized to reduced Cys34 rose more than 3-fold during the 84-day period, revealing a dramatic perturbation of serum redox balance and potentially serving as a portent of increased pollution-related mortality risk. Our targeted albumin adductomics assay represents a novel and flexible approach for sensitive and multiplexed internal dosimetry of environmental exposures, providing a new strategy for personalized biomonitoring and prevention.

Quantitative Proteomics Reveals that the OGT Interactome Is Remodeled in Response to Oxidative Stress.

The dynamic modification of specific serine and threonine residues of intracellular proteins by O-linked N-acetyl-ß-D-glucosamine (O-GlcNAc) mitigates injury and promotes cytoprotection in a variety of stress models. The O-GlcNAc transferase (OGT) and the O-GlcNAcase are the sole enzymes that add and remove O-GlcNAc, respectively, from thousands of substrates. It remains unclear how just two enzymes can be specifically controlled to affect glycosylation of target proteins and signaling pathways both basally and in response to stress. Several lines of evidence suggest that protein interactors regulate these responses by affecting OGT and O-GlcNAcase activity, localization, and substrate specificity. To provide insight into the mechanisms by which OGT function is controlled, we have used quantitative proteomics to define OGT's basal and stress-induced interactomes. OGT and its interaction partners were immunoprecipitated from OGT WT, null, and hydrogen peroxide-treated cell lysates that had been isotopically labeled with light, medium, and heavy lysine and arginine (stable isotopic labeling of amino acids in cell culture). In total, more than 130 proteins were found to interact with OGT, many of which change their association upon hydrogen peroxide stress. These proteins include the major OGT cleavage and glycosylation substrate, host cell factor 1, which demonstrated a time-dependent dissociation after stress. To validate less well-characterized interactors, such as glyceraldehyde 3-phosphate dehydrogenase and histone deacetylase 1, we turned to parallel reaction monitoring, which recapitulated our discovery-based stable isotopic labeling of amino acids in cell culture approach. Although the majority of proteins identified are novel OGT interactors, 64% of them are previously characterized glycosylation targets that contain varied domain architecture and function. Together these data demonstrate that OGT interacts with unique and specific interactors in a stress-responsive manner.

Borrelia burgdorferi-Induced Changes in the Class II Self-Immunopeptidome Displayed on HLA-DR Molecules Expressed by Dendritic Cells.

The MHC class II antigen processing and presentation pathway has evolved to derive short amino acid peptides from proteins that enter the endocytic pathway, load them onto MHC class II molecules and display them on the surface of antigen presenting cells for recognition by CD4+ T cells. Under normal circumstances, peptides bound to MHC class II molecules are derived from host (self) proteins and not recognized by T cells due to tolerance mechanisms. Pathogens induce significant changes in the biology of antigen presenting cells, including upregulation of MHC processing and presentation. We therefore hypothesized that exposure to pathogens may alter the repertoire of self-peptides bound to MHC class II molecules. To test this hypothesis, we isolated monocyte-derived dendritic cells from healthy subjects, exposed them to the TLR-2 agonist lipoteichoic acid or live Borrelia burgdorferi, the causative agent of Lyme disease, and isolated and characterized HLA-DR associated peptides using mass spectrometry. Our results show that lipoteichoic acid-stimulated, B. burgdorferi-stimulated and unstimulated monocyte-derived dendritic cells largely derive their self-peptides from similar overlapping sets of host proteins. However, lipoteichoic acid and B. burgdorferi stimulation promote the processing and presentation of new sets of HLA-DR associated self-peptides derived from unique protein sources. Examination of processes and compartments these proteins reside in, indicate that activation of monocyte-derived dendritic cells changes the range of host self-proteins available for processing and presentation on MHC class II molecules. These findings reveal that the HLA-DR-bound self-immunopeptidome presented by mo-DCs is dynamic in nature and changes with activation state reflective of cellular function. In addition, among the repertoire of self-peptides bound to HLA-DR are several epitopes known to be recognized by autoreactive T cells. These studies are relevant to our basic understanding of pathogen-induced changes in monocyte-derived dendritic cell function, and the mechanisms involved in infection-induced autoimmune illnesses such as Lyme arthritis.

Expanded Chinese hamster organ and cell line proteomics profiling reveals tissue-specific functionalities.

Chinese hamster ovary (CHO) cells are the predominant production vehicle for biotherapeutics. Quantitative proteomics data were obtained from two CHO cell lines (CHO-S and CHO DG44) and compared with seven Chinese hamster (Cricetulus griseus) tissues (brain, heart, kidney, liver, lung, ovary and spleen) by tandem mass tag (TMT) labeling followed by mass spectrometry, providing a comprehensive hamster tissue and cell line proteomics atlas. Of the 8470 unique proteins identified, high similarity was observed between CHO-S and CHO DG44 and included increases in proteins involved in DNA replication, cell cycle, RNA processing, and chromosome processing. Alternatively, gene ontology and pathway analysis in tissues indicated increased protein intensities related to important tissue functionalities. Proteins enriched in the brain included those involved in acidic amino acid metabolism, Golgi apparatus, and ion and phospholipid transport. The lung showed enrichment in proteins involved in BCAA catabolism, ROS metabolism, vesicle trafficking, and lipid synthesis while the ovary exhibited enrichments in extracellular matrix and adhesion proteins. The heart proteome included vasoconstriction, complement activation, and lipoprotein metabolism enrichments. These detailed comparisons of CHO cell lines and hamster tissues will enhance understanding of the relationship between proteins and tissue function and pinpoint potential pathways of biotechnological relevance for future cell engineering.

Definition of Naturally Processed Peptides Reveals Convergent Presentation of Autoantigenic Topoisomerase I Epitopes in Scleroderma.

OBJECTIVE: Autoimmune responses to DNA topoisomerase I (topo I) are found in a subset of scleroderma patients who are at high risk for interstitial lung disease (ILD) and mortality. Anti-topo I antibodies (ATAs) are associated with specific HLA-DRB1 alleles, and the frequency of HLA-DR-restricted topo I-specific CD4+ T cells is associated with the presence, severity, and progression of ILD. Although this strongly implicates the presentation of topo I peptides by HLA-DR in scleroderma pathogenesis, the processing and presentation of topo I has not been studied. METHODS: We developed a natural antigen processing assay (NAPA) to identify putative CD4+ T cell epitopes of topo I presented by monocyte-derived dendritic cells (mo-DCs) from 6 ATA-positive patients with scleroderma. Mo-DCs were pulsed with topo I protein, HLA-DR-peptide complexes were isolated, and eluted peptides were analyzed by mass spectrometry. We then examined the ability of these naturally presented peptides to induce CD4+ T cell activation in 11 ATA-positive and 11 ATA-negative scleroderma patients. RESULTS: We found that a common set of 10 topo I epitopes was presented by Mo-DCs from scleroderma patients with diverse HLA-DR variants. Sequence analysis revealed shared peptide-binding motifs within the HLA-DRß chains of ATA-positive patients and a subset of topo I epitopes with distinct sets of anchor residues capable of binding to multiple different HLA-DR variants. The NAPA-derived epitopes elicited robust CD4+ T cell responses in 73% of ATA-positive patients (8 of 11), and the number of epitopes recognized correlated with ILD severity (P = 0.025). CONCLUSION: These findings mechanistically implicate the presentation of a convergent set of topo I epitopes in the development of scleroderma.

Insulin-induced de novo lipid synthesis occurs mainly via mTOR-dependent regulation of proteostasis of SREBP-1c.

Insulin stimulates de novo lipid synthesis in the liver and in cultured hepatocytes via its ability to activate sterol regulatory element-binding protein 1c (SREBP-1c). Although PI3K-AKT-mTORC1-p70S6K-signaling kinases are known to drive feed-forward expression of SREBP-1c, the identity of the phosphorylated amino acid residue(s) putatively involved in insulin-stimulated de novo lipogenesis remains elusive. We obtained in silico and mass spectrometry evidence, that was combined with siRNA strategies, to discover that insulin-induced phosphorylation of serine 418, serine 419, and serine 422 in rat SREBP-1c was most likely mediated by p70S6 kinase. Here, for the first time, we show that insulin-induced phosphorylation of these 3 serine residues mainly impinged on the mechanisms of proteostasis of both full-length and mature SREBP-1c in the McArdle-RH7777 hepatoma cells. Consistent with this conclusion, nascent SREBP-1c, substituted with phosphomimetic aspartic acid residues at these 3 sites, was resistant to proteasomal degradation. As a consequence, endoplasmic reticulum to Golgi migration and proteolytic maturation of pSREBP-1c was significantly enhanced which led to increased accumulation of mature nSREBP-1c, even in the absence of insulin. Remarkably, aspartic acid substitutions at S418, S419 and S422 also protected the nascent SREBP-1c from ubiquitin-mediated proteasome degradation thus increasing its steady-state levels and transactivation potential in the nucleus. These complementary effects of p70S6K-mediated phosphorylation on proteostasis of pSREBP-1c were necessary and sufficient to account for insulin's ability to enhance transcription of genes controlling de novo lipogenesis in hepatocytes.

The structural unit of melanin in the cell wall of the fungal pathogen Cryptococcus neoformans.

Melanins are synthesized macromolecules that are found in all biological kingdoms. These pigments have a myriad of roles that range from microbial virulence to key components of the innate immune response in invertebrates. Melanins also exhibit unique properties with potential applications in physics and material sciences, ranging from electrical batteries to novel therapeutics. In the fungi, melanins, such as eumelanins, are components of the cell wall that provide protection against biotic and abiotic elements. Elucidation of the smallest fungal cell wall-associated melanin unit that serves as a building block is critical to understand the architecture of these polymers, its interaction with surrounding components, and their functional versatility. In this study, we used isopycnic gradient sedimentation, NMR, EPR, high-resolution microscopy, and proteomics to analyze the melanin in the cell wall of the human pathogenic fungus Cryptococcus neoformans We observed that melanin is assembled into the cryptococcal cell wall in spherical structures ∼200 nm in diameter, termed melanin granules, which are in turn composed of nanospheres ∼30 nm in diameter, termed fungal melanosomes. We noted that melanin granules are closely associated with proteins that may play critical roles in the fungal melanogenesis and the supramolecular structure of this polymer. Using this structural information, we propose a model for C. neoformans' melanization that is similar to the process used in animal melanization and is consistent with the phylogenetic relatedness of the fungal and animal kingdoms.

The prionlike domain of FUS is multiphosphorylated following DNA damage without altering nuclear localization.

FUS (fused in sarcoma) is an abundant, predominantly nuclear protein involved in RNA processing. Under various conditions, FUS functionally associates with RNA and other macromolecules to form distinct, reversible phase-separated liquid structures. Persistence of the phase-separated state and increased cytoplasmic localization are both hypothesized to predispose FUS to irreversible aggregation, which is a pathological hallmark of subtypes of amyotrophic lateral sclerosis and frontotemporal dementia. We previously showed that phosphorylation of FUS's prionlike domain suppressed phase separation and toxic aggregation, proportionally to the number of added phosphates. However, phosphorylation of FUS's prionlike domain was previously reported to promote its cytoplasmic localization, potentially favoring pathological behavior. Here we used mass spectrometry and human cell models to further identify phosphorylation sites within FUS's prionlike domain, specifically following DNA-damaging stress. In total, 28 putative sites have been identified, about half of which are DNA-dependent protein kinase (DNA-PK) consensus sites. Custom antibodies were developed to confirm the phosphorylation of two of these sites (Ser-26 and Ser-30). Both sites were usually phosphorylated in a subpopulation of cellular FUS following a variety of DNA-damaging stresses but not necessarily equally or simultaneously. Importantly, we found DNA-PK-dependent multiphosphorylation of FUS's prionlike domain does not cause cytoplasmic localization.

Lessons from the Hamster: Cricetulus griseus Tissue and CHO Cell Line Proteome Comparison.

Chinese hamster ovary cells represent the dominant host for therapeutic recombinant protein production. However, few large-scale data sets have been generated to characterize this host organism and derived CHO cell lines at the proteomics level. Consequently, an extensive label-free quantitative proteomics analysis of two cell lines (CHO-S and CHO DG44) and two Chinese hamster tissues (liver and ovary) was used to identify a total of 11 801 unique proteins containing at least two unique peptides. 9359 unique proteins were identified specifically in the cell lines, representing a 56% increase over previous work. Additionally, 6663 unique proteins were identified across liver and ovary tissues, providing the first Chinese hamster tissue proteome. Protein expression was more conserved within cell lines during both growth phases than across cell lines, suggesting large genetic differences across cell lines. Overall, both gene ontology and KEGG pathway analysis revealed enrichment of cell-cycle activity in cells. In contrast, upregulated molecular functions in tissue include glycosylation and lipid transporter activity. Furthermore, cellular components including Golgi apparatus are upregulated in both tissues. In conclusion, this large-scale proteomics analysis enables us to delineate specific changes between tissues and cells derived from these tissues, which can help explain specific tissue function and the adaptations cells incur for applications in biopharmaceutical productions.

Phosphorylation of the FUS low-complexity domain disrupts phase separation, aggregation, and toxicity.

Neuronal inclusions of aggregated RNA-binding protein fused in sarcoma (FUS) are hallmarks of ALS and frontotemporal dementia subtypes. Intriguingly, FUS's nearly uncharged, aggregation-prone, yeast prion-like, low sequence-complexity domain (LC) is known to be targeted for phosphorylation. Here we map in vitro and in-cell phosphorylation sites across FUS LC We show that both phosphorylation and phosphomimetic variants reduce its aggregation-prone/prion-like character, disrupting FUS phase separation in the presence of RNA or salt and reducing FUS propensity to aggregate. Nuclear magnetic resonance spectroscopy demonstrates the intrinsically disordered structure of FUS LC is preserved after phosphorylation; however, transient domain collapse and self-interaction are reduced by phosphomimetics. Moreover, we show that phosphomimetic FUS reduces aggregation in human and yeast cell models, and can ameliorate FUS-associated cytotoxicity. Hence, post-translational modification may be a mechanism by which cells control physiological assembly and prevent pathological protein aggregation, suggesting a potential treatment pathway amenable to pharmacologic modulation.

Kinetic and structural analyses reveal residues in phosphoinositide 3-kinase α that are critical for catalysis and substrate recognition.

Phosphoinositide 3-kinases (PI3Ks) are ubiquitous lipid kinases that activate signaling cascades controlling cell survival, proliferation, protein synthesis, and vesicle trafficking. PI3Ks have dual kinase specificity: a lipid kinase activity that phosphorylates the 3'-hydroxyl of phosphoinositides and a protein-kinase activity that includes autophosphorylation. Despite the wealth of biochemical and structural information on PI3Kα, little is known about the identity and roles of individual active-site residues in catalysis. To close this gap, we explored the roles of residues of the catalytic domain and the regulatory subunit of human PI3Kα in lipid and protein phosphorylation. Using site-directed mutagenesis, kinetic assays, and quantitative mass spectrometry, we precisely mapped key residues involved in substrate recognition and catalysis by PI3Kα. Our results revealed that Lys-776, located in the P-loop of PI3Kα, is essential for the recognition of lipid and ATP substrates and also plays an important role in PI3Kα autophosphorylation. Replacement of the residues His-936 and His-917 in the activation and catalytic loops, respectively, with alanine dramatically changed PI3Kα kinetics. Although H936A inactivated the lipid kinase activity without affecting autophosphorylation, H917A abolished both the lipid and protein kinase activities of PI3Kα. On the basis of these kinetic and structural analyses, we propose possible mechanistic roles of these critical residues in PI3Kα catalysis.