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The discovery of activating epidermal growth factor receptor (EGFR) mutations spurred the use of EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib, as the first-line treatment of lung cancers. We previously reported that differential degradation of TKI-sensitive (e.g. L858R) and resistant (T790M) EGFR mutants upon erlotinib treatment correlates with drug sensitivity. We also reported that SMAD ubiquitination regulatory factor 2 (SMURF2) ligase activity is important in stabilizing EGFR. However, the molecular mechanisms involved remain unclear. Here, using in vitro and in vivo ubiquitination assays, MS, and superresolution microscopy, we show SMURF2–EGFR functional interaction is important for EGFR stability and response to TKI. We demonstrate that L858R/T790M EGFR is preferentially stabilized by SMURF2-UBCH5 (an E3-E2)-mediated polyubiquitination. We identified four lysine residues as the sites of ubiquitination and showed that replacement of one of them with acetylation-mimicking glutamine increases the sensitivity of mutant EGFR to erlotinib-induced degradation. We show that SMURF2 extends membrane retention of EGF-bound EGFR, whereas SMURF2 knockdown increases receptor sorting to lysosomes. In lung cancer cell lines, SMURF2 overexpression increased EGFR levels, improving TKI tolerance, whereas SMURF2 knockdown decreased EGFR steadystate levels and sensitized lung cancer cells. Overall, we propose that SMURF2-mediated polyubiquitination of L858R/T790M EGFR competes with acetylation-mediated receptor internalization that correlates with enhanced receptor stability; therefore, disruption of the E3-E2 complex may be an attractive target to overcome TKI resistance. [ABSTRACT FROM AUTHOR]

Identification of biomarkers and therapeutic targets is a critical goal of precision medicine. N-glycoproteins are a particularly attractive class of proteins that constitute potential cancer biomarkers and therapeutic targets for smallmolecules, antibodies, and cellular therapies. Using mass spectrometry (MS), we generated a compendium of 1,091 N-glycoproteins (from 40 human primary lymphomas and cell lines). Hierarchical clustering revealed distinct subtype signatures that included several subtype-specific biomarkers. Orthogonal immunological studies in 671 primary lymphoma tissue biopsies and 32 lymphomaderived cell lines corroborated MS data. In anaplastic lymphoma kinase-positive (ALK+) anaplastic large cell lymphoma (ALCL), integration of N-glycoproteomics and transcriptome sequencing revealed an ALK-regulated cytokine/receptor signaling network, including vulnerabilities corroborated by a genome-wide clustered regularly interspaced short palindromic screen. Functional targeting of IL-31 receptor β, an ALCL-enriched and ALK-regulated N-glycoprotein in this network, abrogated ALK+ALCL growth in vitro and in vivo. Our results highlight the utility of functional proteogenomic approaches for discovery of cancer biomarkers and therapeutic targets. [ABSTRACT FROM AUTHOR]

UBE2W ubiquitinates Ntermini of proteins rather than internal lysine residues, showing a preference for substrates with intrinsically disordered N termini. The in vivo functions of this intriguing E2, however, remain unknown. We generated Ube2w germ line KO mice that proved to be susceptible to early postnatal lethality without obvious developmental abnormalities. Although the basis of early death is uncertain, several organ systems manifest changes in Ube2w KO mice. Newborn Ube2w KO mice often show altered epidermal maturation with reduced expression of differentiation markers. Mirroring higher UBE2W expression levels in testis and thymus, Ube2w KO mice showed a disproportionate decrease in weight of these two organs (~50%), suggesting a functional role for UBE2W in the immune and male reproductive systems. Indeed, Ube2w KO mice displayed sustained neutrophilia accompanied by increased G-CSF signaling and testicular vacuolation associated with decreased fertility. Proteomic analysis of a vulnerable organ, presymptomatic testis, showed a preferential accumulation of disordered proteins in the absence of UBE2W, consistent with the view that UBE2W preferentially targets disordered polypeptides. These mice further allowed us to establish that UBE2W is ubiquitously expressed as a single isoform localized to the cytoplasm and that the absence of UBE2W does not alter cell viability in response to various stressors. Our results establish that UBE2W is an important, albeit not essential, protein for early postnatal survival and normal functioning of multiple organ systems. [ABSTRACT FROM AUTHOR]

Hepatic accumulation of protoporphyrin-IX (PP-IX) in erythropoietic protoporphyria (EPP) or X-linked-dominant protoporphyria (XLP) cause liver damage. Hepatocyte nuclear lamin aggregation is a sensitive marker for PP-IX-mediated liver injury. We tested the hypothesis that extracellular or intracellular protoporphyria cause damage to different subcellular compartments, in a light-triggered manner. Three hepatoma cell lines (HepG2, Hepa-1, and Huh-7) were treated with exogenous PP-IX (mimicking XLP extrahepatic protoporphyria) or with the iron chelator deferoxamine and the porphyrin precursor 5-aminolevulinic acid (ALA) (mimicking intracellular protoporphyrin accumulation in EPP). Exogenous PP-IX accumulated predominantly in the nuclear fraction and caused nuclear shape deformation and cytoplasmic vacuoles containing electron-dense particles, whereas ALA+deferoxamine treatment resulted in higher PP-IX in the cytoplasmic fraction. Protein aggregation in the nuclear and cytoplasmic fractions paralleled PP-IX levels and, in cell culture, the effects were exclusively ambient light-mediated. PP-IX and ALA caused proteasomal inhibition, whereas endoplasmic reticulum protein aggregation was more prominent in ALA-treated cells. The enhanced ALA-related toxicity is likely due to generation of additional porphyrin intermediates including uroporphyrin and coproporphyrin, based on HPLC analysis of cell lysates and the culture medium, as well as cell-free experiments with uroporphyrin/coproporphyrin. Mouse livers from drug-induced porphyria phenocopied the in vitro findings, and mass spectrometry of liver proteins isolated in light/dark conditions showed diminished (as compared with light-harvested) but detectable aggregation under dark-harvested conditions. Therefore, PP-IX leads to endoplasmic reticulum stress and proteasome inhibition in a manner that depends on the source of porphyrin buildup and light exposure. Porphyrin-mediated selective protein aggregation provides a potential mechanism for porphyria-associated tissue injury. [ABSTRACT FROM AUTHOR]

Analysis of the full spectrum of secreted proteins in cell culture is complicated by leakage of intracellular proteins from damaged cells. To address this issue, we compared the abundance of individual proteins between the cell lysate and the conditioned medium, reasoning that secreted proteins should be relatively more abundant in the conditioned medium. Marked enrichment for signal-peptide-bearing proteins with increasing conditioned media to cell lysate ratio, as well loss of this signal following brefeldin A treatment, confirmed the sensitivity and specificity of this approach. The subset of proteins demonstrating increased conditioned media to cell lysate ratio in the presence of Brefeldin A identified candidates for unconventional secretion via a pathway independent of ER to Golgi trafficking. [Display omitted] • Intracellular proteins that leak out into conditioned media confound identification of secreted proteins. • We demonstrate that secreted proteins are relatively more abundant in the cell conditioned media relative to the cell lysate. • Calculation of a media to lysate protein abundance ratio allows high sensitivity identification of secreted proteins. • Brefeldin-A resistant proteins with high media to lysate ratios are likely unconventionally secreted via ER-Golgi independent pathways. [ABSTRACT FROM AUTHOR]

Background Burkitt lymphoma (BL) is the most common non-Hodgkin lymphoma in children worldwide and the most common paediatric malignancy in sub-Saharan Africa. The endemic (eBL) and sporadic (sBL) variants have distinct epidemiologic and virologic characteristics. Although gene expression studies have defined the transcriptional profiles of both, their proteomic signatures have not been studied. Methods We compared the proteomic expression profiles using differential mass spectrometry-based isotope tag for relative and absolute quantitation (iTRAQ) analysis of a cell line representing Epstein–Barr virus (EBV)+ eBL, EBV+ and EBV– sBL, and EBV+/– normal B cells from healthy donors. Results In total, there were 144 differentially expressed proteins with a statistically significant false discovery rate (FDR) of ⩽0.2. Results revealed over-expression of specific proteins with well-established links to lymphomagenesis such as TUBB2C (FDR 0.05), UCHL1 (FDR 0.05) and HSP90AB1 (FDR 0.1). Distinct characteristics based upon the epidemiologic and virologic subtypes of BL were also identified. In sBL, PCNA (FDR 0.05) and SLC3A2 (FDR 0.1) were significantly over-expressed. In eBL, C1QBP (FDR 0.1) and ENO1 (FDR 0.25) were significantly over-expressed. Comparison of EBV+ to EBV– BL cell lines and B cells revealed significant over-expression of DDX3X (FDR 0.1). Proteins were validated using Western blot analysis. Conclusion Our results suggest unique signal transduction pathways associated with EBV infection and epidemiological subtype of BL that may contribute to lymphomagenesis. These proteomic findings provide potential diagnostic, prognostic and therapeutic links to BL. [ABSTRACT FROM AUTHOR]

Attempts to target mutant KRAS have been unsuccessful. Here, we report the identification of Smad ubiquitination regulatory factor 2 (SMURF2) and UBCH5 as a critical E3:E2 complex maintaining KRAS protein stability. Loss of SMURF2 either by small interfering RNA/short hairpin RNA (siRNA/shRNA) or by overexpression of a catalytically inactive mutant causes KRAS degradation, whereas overexpression of wild-type SMURF2 enhances KRAS stability. Importantly, mutant KRAS is more susceptible to SMURF2 loss where protein half-life decreases from >12 hours in control siRNA-treated cells to <3 hours on Smurf2 silencing, whereas only marginal differences were noted for wild-type protein. This loss of mutant KRAS could be rescued by overexpressing a siRNA-resistant wild-type SMURF2. Our data further show that SMURF2 monoubiquitinates UBCH5 at lysine 144 to form an active complex required for efficient degradation of a RAS-family E3, β-transducing repeat containing protein 1 (β-TrCP1). Conversely, β-TrCP1 is accumulated on SMURF2 loss, leading to increased KRAS degradation. Therefore, as expected, β-TrCP1 knockdown following Smurf2 siRNA treatment rescues mutant KRAS loss. Further, we identify two conserved proline (P) residues in UBCH5 critical for SMURF2 interaction; mutation of either of these P to alanine also destabilizes KRAS. As a proof of principle, we demonstrate that Smurf2 silencing reduces the clonogenic survival in vitro and prolongs tumor latency in vivo in cancer cells including mutant KRAS-driven tumors. Taken together, we show that SMURF2:UBCH5 complex is critical in maintaining KRAS protein stability and propose that targeting such complex may be a unique strategy to degrade mutant KRAS to kill cancer cells. [ABSTRACT FROM AUTHOR]

Cardiotonic steroids (such as ouabain) signaling through Na/K-ATPase regulate sodium reabsorption in the renal proximal tubule. We report here that reactive oxygen species are required to initiate ouabain-stimulated Na/K-ATPasec-Src signaling. Pretreatment with the antioxidant N-acetyl-L-cysteine prevented ouabain-stimulated Na/K-ATPasec-Src signaling, protein carbonylation, redistribution ofNa/K-ATPase and sodium/proton exchanger isoform 3, and inhibition of active tran- sepithelial 22Na transport. Disruption of the Na/K-ATPasec-Src signaling complex attenuated ouabain-stimulated protein carbonylation. Ouabain-stimulated protein carbonylation is reversed after removal of ouabain, and this reversibility is largely independent of de novo protein synthesis and degradation by either the lysosome or the proteasome pathways. Furthermore, ouabain stimulated direct carbonylation of two amino acid residues in the actuator domain of theNa/K-ATPase1 subunit. Taken together, the data indicate that carbonylation modification of the Na/K-ATPase1 subunit is involved in a feed-forward mechanism of regulation of ouabain-mediated renal proximal tubule Na/K-ATPase signal transduction and subsequent sodium transport. [ABSTRACT FROM AUTHOR]

The mechanisms underlying the pathogenesis of the constitutively active tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressing anaplastic large cell lymphoma are not completely understood. Here we show using an integrated phospho proteomic and metabolomic strategy that NPM-ALK induces a metabolic shift toward aerobic glycolysis, increased lactate production, and biomass production. The metabolic shift is mediated through the anaplastic lymphoma kinase (ALK) phosphor ylation of the tumor-specific isoform of pyruvate kinase (PKM2) at Y105, resulting in decreased enzymatic activity. Small molecule activation of PKM2 or expression of Y105F PKM2 mutant leads to reversal of the metabolic switch with increased oxidative phosphorylation and reduced lactate production coincident with increased cell death, decreased colony formation, and reduced tumor growth in an in vivo xenograft model. This study provides comprehensive profiling of the phosphoproteomic and metabolomic consequences of NPM-ALK expression and reveals a novel role of ALK in the regulation of multiple components of cellular metabolism. Our studies show that PKM2 is a novel substrate of ALK and plays a critical role in mediating the metabolic shift toward biomass production and tumorigenesis. [ABSTRACT FROM AUTHOR]

Summary: Fragile X-associated tremor ataxia syndrome (FXTAS) results from a CGG repeat expansion in the 5′ UTR of FMR1. This repeat is thought to elicit toxicity as RNA, yet disease brains contain ubiquitin-positive neuronal inclusions, a pathologic hallmark of protein-mediated neurodegeneration. We explain this paradox by demonstrating that CGG repeats trigger repeat-associated non-AUG-initiated (RAN) translation of a cryptic polyglycine-containing protein, FMRpolyG. FMRpolyG accumulates in ubiquitin-positive inclusions in Drosophila, cell culture, mouse disease models, and FXTAS patient brains. CGG RAN translation occurs in at least two of three possible reading frames at repeat sizes ranging from normal (25) to pathogenic (90), but inclusion formation only occurs with expanded repeats. In Drosophila, CGG repeat toxicity is suppressed by eliminating RAN translation and enhanced by increased polyglycine protein production. These studies expand the growing list of nucleotide repeat disorders in which RAN translation occurs and provide evidence that RAN translation contributes to neurodegeneration. [ABSTRACT FROM AUTHOR]

Fbxw7? is a member of the F-box family of proteins, which function as the substrate-targeting subunits of SCF (Skp1/Cul1/F-box protein) ubiquitin ligase complexes. Using differential purifications and mass spectrometry, we identified p100, an inhibitor of NF-?B signalling, as an interactor of Fbxw7?. p100 is constitutively targeted in the nucleus for proteasomal degradation by Fbxw7?, which recognizes a conserved motif phosphorylated by GSK3. Efficient activation of non-canonical NF-?B signalling is dependent on the elimination of nuclear p100 through either degradation by Fbxw7? or exclusion by a newly identified nuclear export signal in the carboxy terminus of p100. Expression of a stable p100 mutant, expression of a constitutively nuclear p100 mutant, Fbxw7? silencing or inhibition of GSK3 in multiple myeloma cells with constitutive non-canonical NF-?B activity results in apoptosis both in cell systems and xenotransplant models. Thus, in multiple myeloma, Fbxw7? and GSK3 function as pro-survival factors through the control of p100 degradation. [ABSTRACT FROM AUTHOR]

In Saccharomyces cerevisae, expanded polyglutamine (polyQ) fragments are assembled into discrete cytosolic aggregates in a process regulated by the molecular chaperones Hsp26, Hsp70, Hsp90, and Hsp104. To better understand how the different chaperones might cooperate during polyQ aggregation, we used sequential immunoprecipitations and mass spectrometry to identify proteins associated with either soluble (Q25) or aggregation-prone (Q103) fragments at both early and later times after induction of their expression. We found that Hsp26, Hsp70, Hsp90, and other chaperones interact with Q103, but not Q25, within the first 2 h. Further, Hsp70 and Hsp90 appear to be partially released from Q103 prior to the maturation of the aggregates and before the recruitment of Hsp104. To test the importance of this seemingly ordered process, we used a chemical probe to artificially enhance Hsp70 binding to Q103. This treatment retained both Hsp70 and Hsp90 on the polyQ fragment and, interestingly, limited subsequent exchange for Hsp26 and Hsp104, resulting in incomplete aggregation. Together, these results suggest that partial release of Hsp70 may be an essential step in the continued processing of expanded polyQ fragments in yeast. [ABSTRACT FROM AUTHOR]

Summary: Recurrent fusions of ETS genes are considered driving mutations in a diverse array of cancers, including Ewing''s sarcoma, acute myeloid leukemia, and prostate cancer. We investigate the mechanisms by which ETS fusions mediate their effects, and find that the product of the predominant ETS gene fusion, TMPRSS2:ERG, interacts in a DNA-independent manner with the enzyme poly (ADP-ribose) polymerase 1 (PARP1) and the catalytic subunit of DNA protein kinase (DNA-PKcs). ETS gene-mediated transcription and cell invasion require PARP1 and DNA-PKcs expression and activity. Importantly, pharmacological inhibition of PARP1 inhibits ETS-positive, but not ETS-negative, prostate cancer xenograft growth. Finally, overexpression of the TMPRSS2:ERG fusion induces DNA damage, which is potentiated by PARP1 inhibition in a manner similar to that of BRCA1/2 deficiency. [Copyright &y& Elsevier]

Deubiquitinating enzymes (DUbs) play important roles in many ubiquitin-dependent pathways, yet how DUbs them-selves are regulated is not well understood. Here, we provide insight into the mechanism by which ubiquitination directly enhances the activity of ataxin-3, a DUb implicated in protein quality control and the disease protein in the polyglutamine neurodegenerative disorder, Spinocerebellar Ataxia Type 3. We identify Lys-117, which resides near the catalytic triad, as the primary site of ubiquitination in wild type and pathogenic ataxin-3. Further studies indicate that ubiquitin-dependent activation of ataxin-3 at Lys-117 is important for its ability to reduce high molecular weight ubiquitinated species in cells. Ubiquitination at Lys-117 also facilitates the ability of ataxin-3 to induce aggresome formation in cells. Finally, structure-function studies support a model of activation whereby ubiquitination at Lys-117 enhances ataxin-3 activity independent of the known ubiquitin-binding sites in ataxin-3, most likely through a direct conformational change in or near the catalytic domain. [ABSTRACT FROM AUTHOR]

Summary: MLL1 fusion proteins activate HoxA9 gene expression and cause aggressive leukemias that respond poorly to treatment, but how they recognize and stably bind to HoxA9 is not clearly understood. In a systematic analysis of MLL1 domain recruitment activity, we identified an essential MLL1 recruitment domain that includes the CXXC domain and PHD fingers and is controlled by direct interactions with the PAF elongation complex and H3K4Me2/3. MLL1 fusion proteins lack the PHD fingers and require prebinding of a wild-type MLL1 complex and CXXC domain recognition of DNA for stable HoxA9 association. Together, these results suggest that specific recruitment of MLL1 requires multiple interactions and is a precondition for stable recruitment of MLL1 fusion proteins to HoxA9 in leukemogenesis. Since wild-type MLL1 and oncogenic MLL1 fusion proteins have overlapping yet distinct recruitment mechanisms, this creates a window of opportunity that could be exploited for the development of targeted therapies. [Copyright &y& Elsevier]

Summary: MLL is involved in chromosomal rearrangements that generate fusion proteins with deregulated transcriptional activity. The mechanisms of MLL fusion protein-mediated transcriptional activation are poorly understood. Here we show MLL interacts directly with the polymerase associated factor complex (PAFc) through sequences flanking the CxxC domain. PAFc interacts with RNA polymerase II and stimulates posttranslational histone modifications. PAFc augments MLL and MLL-AF9 mediated transcriptional activation of Hoxa9. Conversely, knockdown of PAFc disrupts MLL fusion protein-mediated transcriptional activation and MLL recruitment to target loci. PAFc gene expression is downregulated during hematopoiesis and likely serves to regulate MLL function. Deletions of MLL that abolish interactions with PAFc also eliminate MLL-AF9 mediated immortalization indicating an essential function for this interaction in leukemogenesis. [Copyright &y& Elsevier]

The effect of cardiac glycosides to increase cardiac inotropy by altering Ca2+ cycling is well known but still poorly understood. The studies described in this report focus on defining the effects of ouabain signaling on sarcoplasmic reticulum Ca2+-ATPase function. Rat cardiac myocytes treated with 50 µM ouabain demonstrated substantial increases in systolic and diastolic Ca2+ concentrations. The recovery time constant for the Ca2+ transient, τCa2+, was significantly prolonged by ouabain. Exposure to 10 µM H2O2, which causes an increase in intracellular reactive oxygen species similar to that of 50 µM ouabain, caused a similar increase in τCa2+. Concurrent exposure to 10 mM N-acetylcysteine or an aqueous extract from green tea (50 mg/ml) both prevented the increases in τCa2+ as well as the changes in systolic or diastolic Ca2+ concentrations. We also observed that 50 µM ouabain induced increases in developed pressure in addition to diastolic dysfunction in the isolated perfused rat heart. Coadministration of ouabain with N-acetylcysteine prevented these increases. Analysis of sarcoplasmic reticulum Ca2+-ATPase protein revealed increases in both the oxidation and nitrotyrosine content in the ouabain-treated hearts. Liquid chromatography-mass spectrometric analysis confirmed that the sarcoplasmic reticulum Ca2+-ATPase protein from ouabain-treated hearts had modifications consistent with oxidative and nitrosative stress. These data suggest that ouabain induces oxidative changes of the sarcoplasmic reticulum Ca2+-ATPase structure and function that may, in turn, produce some of the associated changes in Ca2+ cycling and physiological function. [ABSTRACT FROM AUTHOR]

Objective: The aim of this study was to better understand how mixed lineage leukemia (MLL) fusion proteins deregulate the expression of genes critical for leukemia. Materials and Methods: The transforming domain of one of the most common MLL fusion partners, AF9, was immunopurified after expression in myeloblastic M1 cells, and associating proteins were identified by mass spectrometric analysis. Chromatin immunoprecipitation followed by quantitative polymerase chain reaction was used to determine how binding of associating proteins compare across Hoxa9 and Meis1 in cell lines with and without MLL fusion proteins and how binding is altered during gene down-regulation and differentiation. Results: Consistent with earlier purifications of ENL and AF4 from 293 cells, the 90 amino acid C−terminal domain of AF9 associates with many other MLL translocation partners including Enl, Af4, Laf4, Af5q31, Ell, and Af10. This complex, termed elongation assisting proteins (EAPs), also contains the RNA polymerase II C-terminal domain kinase Cdk9/Cyclin T1/T2 (pTEFb) and the histone H3 lysine 79 methyltransferase Dot1L. Myeloid cells transformed by MLL fusions show higher levels and a broader distribution of EAP components at genes critical for leukemia. Inhibition of EAP components pTEFb and Dot1l show that both contribute significantly to activation of Hoxa9 and Meis1 expression. EAP is dynamically associated with the Hoxa9 and Meis1 loci in hematopoietic cells and rapidly dissociates during induction of differentiation. In the presence of MLL fusion proteins, its dissociation is prevented. Conclusions: The findings suggest that MLL fusion proteins deregulate genes critical for leukemia by excessive recruitment and impaired dissociation of EAP from target loci. [Copyright &y& Elsevier]

The role of ubiquitin-mediated degradation mechanisms in the pathogenesis of diffuse large B cell (DLBCL) and follicular lymphoma (FL) is not completely understood. We show that conditional deletion of the E3 ubiquitin ligase Fbxo45 in germinal center B-cells results in B-cell lymphomagenesis in homozygous (100%) and heterozygous (48%) mice. Mechanistically, FBXO45 targets the RHO guanine exchange factor ARHGEF2/GEF-H1 for ubiquitin-mediated degradation. Double genetic ablation of Fbxo45 and Arhgef2 ameliorated lymphoma formation. Transgenic knock-in mice harboring a GEF-H1 mutant unable to bind FBXO45 develop B-cell lymphomas with ~50% penetrance. Genome sequencing in human lymphomas identified mutually-exclusive FBXO45 copy number losses and ARHGEF2 gains, with combined frequencies ranging from 26.32% in FL to 45.12% in DLBCL. Notably, FBXO45 silencing enhances sensitivity to MEK1/2 inhibition. These results identify FBXO45 and ARHGEF2 as a novel tumor-suppressor and oncogene pair involved in the pathogenesis of B-cell lymphomas with significant implications for targeted therapies.

Starvation, intermittent fasting and exercise, all of which are recommended lifestyle modifiers share a common metabolic signature, ketogenesis to generate the ketone bodies, predominantly β-hydroxybutyrate. β-hydroxybutyrate exerts beneficial effects across various contexts, preventing or mitigating disease. We hypothesized that these dynamic health benefits of β-hydroxybutyrate might stem from its ability to regulate genome architecture through chromatin remodeling via histone β-hydroxybutyrylation, thereby influencing the transcriptome. Focusing on the kidney, which is an end organ protected by β-hydroxybutyrate, we examined histone β-hydroxybutyrylation-mediated chromatin remodeling. Notably, regions of the genome associated with lipid catabolism were predominantly in an open chromatin configuration, leading to active transcription and translation. Significant β-hydroxybutyrylation was observed in the kidneys and the most highly upregulated gene actively transcribed and translated was 3-hydroxy-3-methyglutaryl CoA Synthase 2 ( Hmgcs2 ), a gene responsible for the biosynthesis of β-hydroxybutyrate in mitochondria. In contrast, regions with more compact chromatin structures were enriched with genes related to immune function such as protein tyrosine phosphatase receptor type C ( Ptprc ) and lymphocyte cytosolic protein 1 ( Lcp1 ), which exhibited reduced transcription and translation. These results reveal that renal epigenetic histone β-hydroxybutyrylation, which concurrently modulates both energy metabolism and immune function is a mechanism underlying the dynamic health effects of β-hydroxybutyrate to protect kidneys and lower hypertension.

The human genome is replete with sequences derived from foreign elements including endogenous retrovirus-like proteins of unknown function. Here we show that UBQLN2, a ubiquitin-proteasome shuttle factor implicated in neurodegenerative diseases, is regulated by the linked actions of two retrovirus-like proteins, RTL8 and PEG10. RTL8 confers on UBQLN2 the ability to complex with and regulate PEG10. PEG10, a core component of stress granules, drives the recruitment of UBQLN2 to stress granules under various stress conditions, but can only do so when RTL8 is present. Changes in PEG10 levels further remodel the kinetics of stress granule disassembly and overall composition by incorporating select extracellular vesicle proteins. Within stress granules, PEG10 forms virus-like particles, underscoring the structural heterogeneity of this class of biomolecular condensates. Together, these results reveal an unexpected link between pathways of cellular proteostasis and endogenous retrovirus-like proteins.

Plasminogen activator inhibitor-1 (PAI-1) has been previously shown to promote lung fibrosis via a mechanism that requires an intact vitronectin (VTN) binding site. In the present study, employing two distinct murine fibrosis models, we find that VTN is not required for PAI-1 to drive lung scarring. This result suggested the existence of a previously unrecognized profibrotic PAI-1-protein interaction involving the VTN-binding site for PAI-1. Using an unbiased proteomic approach, we identified sortilin related receptor 1 (SorlA) as the most highly enriched PAI-1 interactor in the fibrosing lung. We next investigated the role of SorlA in pulmonary fibrosis and found that SorlA deficiency protected against lung scarring in a murine model. We further show that, while VTN deficiency does not influence fibrogenesis in the presence or absence of PAI-1, SorlA is required for PAI-1 to promote scarring. These results, together with data showing increased SorlA levels in human IPF lung tissue, support a novel mechanism through which the potent profibrotic mediator PAI-1 drives lung fibrosis and implicate SorlA as a new therapeutic target in IPF treatment.

Proteotoxicity is a contributor to the development of type 2 diabetes (T2D), but it is unknown whether protein misfolding in T2D is generalized or has special features. Here, we report a robust accumulation of misfolded proteins within the mitochondria of human pancreatic islets in T2D and elucidate its impact on β cell viability. Surprisingly, quantitative proteomics studies of protein aggregates reveal that human islets from donors with T2D have a signature more closely resembling mitochondrial rather than ER protein misfolding. The matrix protease LonP1 and its chaperone partner mtHSP70 were among the proteins enriched in protein aggregates. Deletion of LONP1 in mice yields mitochondrial protein misfolding and reduced respiratory function, ultimately leading to β cell apoptosis and hyperglycemia. Intriguingly, LONP1 gain of function ameliorates mitochondrial protein misfolding and restores human β cell survival following glucolipotoxicity via a protease-independent effect requiring LONP1-mtHSP70 chaperone activity. Thus, LONP1 promotes β cell survival and prevents hyperglycemia by facilitating mitochondrial protein folding. These observations may open novel insights into the nature of impaired proteostasis on β cell loss in the pathogenesis of T2D that could be considered as future therapeutic targets.

Most proteins secreted into the extracellular space are first recruited from the endoplasmic reticulum into coat protein complex II (COPII)-coated vesicles or tubules that facilitate their transport to the Golgi apparatus. Although several secreted proteins have been shown to be actively recruited into COPII vesicles and tubules by the cargo receptors LMAN1 and SURF4, the full cargo repertoire of these receptors is unknown. We now report mass spectrometry analysis of conditioned media and cell lysates from HuH7 cells CRISPR targeted to inactivate the LMAN1 or SURF4 gene. We found that LMAN1 has limited clients in HuH7 cells, whereas SURF4 traffics a broad range of cargoes. Analysis of putative SURF4 cargoes suggests that cargo recognition is governed by complex mechanisms rather than interaction with a universal binding motif..

The extreme polymorphisms of HLA class I proteins result in structural variations in their peptide binding sites to achieve diversity in Ag presentation. External factors could independently constrict or alter HLA class I peptide repertoires. Such effects of the assembly factor tapasin were assessed for HLA-B*44:05 (Y116) and a close variant, HLA-B*44:02 (D116), which have low and high tapasin dependence, respectively, for their cell surface expression. Analyses of the HLA-B*44:05 peptidomes in the presence and absence of tapasin reveal that peptides with C-terminal tryptophans and higher predicted affinities are preferentially selected by tapasin, coincident with reduced frequencies of peptides with other C-terminal amino acids, including leucine. Comparisons of the HLA-B*44:05 and HLA-B*44:02 peptidomes indicate the expected structure-based alterations near the peptide C termini, but also C-terminal amino acid frequency and predicted affinity changes among the unique and shared peptide groups for B*44:02 and B*44:05. Overall, these findings indicate that the presence of tapasin and the tapasin dependence of assembly alter HLA class I peptide-binding preferences at the peptide C terminus. The particular C-terminal amino acid preferences that are altered by tapasin are expected to be determined by the intrinsic peptide-binding specificities of HLA class I allotypes. Additionally, the findings suggest that tapasin deficiency and reduced tapasin dependence expand the permissive affinities of HLA class I-bound peptides, consistent with prior findings that HLA class I allotypes with low tapasin dependence have increased breadth of CD8+ T cell epitope presentation and are more protective in HIV infections., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Conventional kinesin-1 is the primary anterograde motor in cells for transporting cellular cargo. While there is a consensus that the C-terminal tail of kinesin-1 inhibits motility, the molecular architecture of a full-length autoinhibited kinesin-1 remains unknown. Here, we combine cross-linking mass spectrometry (XL-MS), electron microscopy (EM), and AlphaFold structure prediction to determine the architecture of the full-length autoinhibited kinesin-1 homodimer [kinesin-1 heavy chain (KHC)] and kinesin-1 heterotetramer [KHC bound to kinesin light chain 1 (KLC1)]. Our integrative analysis shows that kinesin-1 forms a compact, bent conformation through a break in coiled coil 3. Moreover, our XL-MS analysis demonstrates that kinesin light chains stabilize the folded inhibited state rather than inducing a new structural state. Using our structural model, we show that disruption of multiple interactions between the motor, stalk, and tail domains is required to activate the full-length kinesin-1. Our work offers a conceptual framework for understanding how cargo adaptors and microtubule-associated proteins relieve autoinhibition to promote activation.

Aberrant enhancer activation is a key mechanism driving oncogene expression in many cancers. While much is known about the regulation of larger chromosome domains in eukaryotes, the details of enhancer-promoter interactions remain poorly understood. Recent work suggests co-activators like BRD4 and Mediator have little impact on enhancer-promoter interactions. In leukemias controlled by the MLL-AF4 fusion protein, we use the ultra-high resolution technique Micro-Capture-C (MCC) to show that MLL-AF4 binding promotes broad, high-density regions of enhancer-promoter interactions at a subset of key targets. These enhancers are enriched for transcription elongation factors like PAF1C and FACT, and the loss of these factors abolishes enhancer-promoter contact. This work not only provides an additional model for how MLL-AF4 is able to drive high levels of transcription at key genes in leukemia but also suggests a more general model linking enhancer-promoter crosstalk and transcription elongation., (© 2023. Springer Nature Limited.)

Th17 cells that produce Interleukin IL-17 are pathogenic in many human diseases, including inflammatory bowel disease, but are, paradoxically, essential for maintaining the integrity of the intestinal barrier in a non-inflammatory state. However, the intracellular mechanisms that regulate distinct transcriptional profiles and functional diversity of Th17 cells remain unclear. Here we show Raftlin1, a lipid raft protein, specifically upregulates and forms a complex with RORγt in pathogenic Th17 cells. Disruption of the RORγt-Raftlin1 complex results in the reduction of pathogenic Th17 cells in response to Citrobacter rodentium; however, there is no effect on nonpathogenic Th17 cells in response to commensal segmented filamentous bacteria. Mechanistically, we show that Raftlin1 recruits distinct phospholipids to RORγt and promotes the pathogenicity of Th17 cells. Thus, we have identified a mechanism that drives the pathogenic function of Th17 cells, which could provide a platform for advanced therapeutic strategies to dampen Th17-mediated inflammatory diseases., (© 2023. Springer Nature Limited.)

Dysregulated interleukin-17 (IL-17) expression and its downstream signaling is strongly linked to inflammatory bowel diseases (IBDs). However, the molecular mechanisms by which the function of RORγt, the transcription factor of IL-17, is regulated remains elusive. By a mass spectrometry-based approach, we identify that Pak2, a serine (S)/threonine (T) kinase, directly associates with RORγt. Pak2 recognizes a conserved KRLS motif within RORγt and phosphorylates the S-316 within this motif. Genetic deletion of Pak2 in Th17 cells reduces RORγt phosphorylation, increases IL-17 expression, and induces severe colitis upon adoptive transfer to Rag1 -/- mice. Similarly, reconstitution of RORγt-S316A mutant in Rorc -/- Th17 cells enhances IL-17 expression and colitis severity. Mechanistically, we demonstrate that Pak2-mediated phosphorylation causes a conformational change resulting in exposure of the ubiquitin ligase Itch interacting PPLY motif and degradation of RORγt. Thus, we have uncovered a mechanism by which the activity of RORγt is regulated that can be exploited therapeutically., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Repeat associated non-AUG (RAN) translation of CGG repeats in the 5'UTR of FMR1 produces toxic proteins that contribute to fragile X-associated tremor/ataxia syndrome (FXTAS) pathogenesis. The most abundant RAN product, FMRpolyG, initiates predominantly at an ACG upstream of the repeat. Accurate FMRpolyG measurements in FXTAS patients are lacking. We used data-dependent acquisition and parallel reaction monitoring (PRM) mass spectrometry coupled with stable isotope labeled standard peptides to identify signature FMRpolyG fragments in patient samples. Following immunoprecipitation, PRM detected FMRpolyG signature peptides in transfected cells, and FXTAS tissues and cells, but not in controls. We identified two amino-terminal peptides: an ACG-initiated Ac-MEAPLPGGVR and a GUG-initiated Ac-TEAPLPGGVR, as well as evidence for RAN translation initiation within the CGG repeat itself in two reading frames. Initiation at all sites increased following cellular stress, decreased following eIF1 overexpression and was eIF4A and M7G cap-dependent. These data demonstrate that FMRpolyG is quantifiable in human samples and FMR1 RAN translation initiates via similar mechanisms for near-cognate codons and within the repeat through processes dependent on available initiation factors and cellular environment., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Kir2.1, a strong inward rectifier potassium channel encoded by the KCNJ2 gene, is a key regulator of the resting membrane potential of the cardiomyocyte and plays an important role in controlling ventricular excitation and action potential duration in the human heart. Mutations in KCNJ2 result in inheritable cardiac diseases in humans, e.g. the type-1 Andersen-Tawil syndrome (ATS1). Understanding the molecular mechanisms that govern the regulation of inward rectifier potassium currents by Kir2.1 in both normal and disease contexts should help uncover novel targets for therapeutic intervention in ATS1 and other Kir2.1-associated channelopathies. The information available to date on protein-protein interactions involving Kir2.1 channels remains limited. Additional efforts are necessary to provide a comprehensive map of the Kir2.1 interactome. Here we describe the generation of a comprehensive map of the Kir2.1 interactome using the proximity-labeling approach BioID. Most of the 218 high-confidence Kir2.1 channel interactions we identified are novel and encompass various molecular mechanisms of Kir2.1 function, ranging from intracellular trafficking to cross-talk with the insulin-like growth factor receptor signaling pathway, as well as lysosomal degradation. Our map also explores the variations in the interactome profiles of Kir2.1 WT versus Kir2.1 Δ314-315 , a trafficking deficient ATS1 mutant, thus uncovering molecular mechanisms whose malfunctions may underlie ATS1 disease. Finally, using patch-clamp analysis, we validate the functional relevance of PKP4, one of our top BioID interactors, to the modulation of Kir2.1-controlled inward rectifier potassium currents. Our results validate the power of our BioID approach in identifying functionally relevant Kir2.1 interactors and underline the value of our Kir2.1 interactome as a repository for numerous novel biological hypotheses on Kir2.1 and Kir2.1-associated diseases., Competing Interests: Conflict of interest—Authors declare no competing interests., (© 2020 Park et al.)

The inflammasome NLRP6 plays a crucial role in regulating inflammation and host defense against microorganisms in the intestine. However, the molecular mechanisms by which NLRP6 function is inhibited to prevent excessive inflammation remain unclear. Here, we demonstrate that the deubiquitinase Cyld prevents excessive interleukin 18 (IL-18) production in the colonic mucosa by deubiquitinating NLRP6. We show that deubiquitination inhibited the NLRP6-ASC inflammasome complex and regulated the maturation of IL-18. Cyld deficiency in mice resulted in elevated levels of active IL-18 and severe colonic inflammation following Citrobacter rodentium infection. Further, in patients with ulcerative colitis, the concentration of active IL-18 was inversely correlated with CYLD expression. Thus, we have identified a novel regulatory mechanism that inhibits the NLRP6-IL-18 pathway in intestinal inflammation.

Metaplastic breast carcinoma (MBC) is a highly aggressive form of triple-negative cancer (TNBC), defined by the presence of metaplastic components of spindle, squamous, or sarcomatoid histology. The protein profiles underpinning the pathological subtypes and metastatic behavior of MBC are unknown. Using multiplex quantitative tandem mass tag-based proteomics we quantify 5798 proteins in MBC, TNBC, and normal breast from 27 patients. Comparing MBC and TNBC protein profiles we show MBC-specific increases related to epithelial-to-mesenchymal transition and extracellular matrix, and reduced metabolic pathways. MBC subtypes exhibit distinct upregulated profiles, including translation and ribosomal events in spindle, inflammation- and apical junction-related proteins in squamous, and extracellular matrix proteins in sarcomatoid subtypes. Comparison of the proteomes of human spindle MBC with mouse spindle (CCN6 knockout) MBC tumors reveals a shared spindle-specific signature of 17 upregulated proteins involved in translation and 19 downregulated proteins with roles in cell metabolism. These data identify potential subtype specific MBC biomarkers and therapeutic targets.

Rational new strategies are needed to treat tumors resistant to kinase inhibitors. Mechanistic studies of resistance provide fertile ground for development of new approaches. Cancer drug addiction is a paradoxical resistance phenomenon, well-described in MEK-ERK-driven solid tumors, in which drug-target overexpression promotes resistance but a toxic overdose of signaling if the inhibitor is withdrawn. This can permit prolonged control of tumors through intermittent dosing. We and others showed previously that cancer drug addiction arises also in the hematologic malignancy ALK-positive anaplastic large-cell lymphoma (ALCL) resistant to ALK-specific tyrosine kinase inhibitors (TKIs). This is driven by the overexpression of the fusion kinase NPM1-ALK, but the mechanism by which ALK overactivity drives toxicity upon TKI withdrawal remained obscure. Here we reveal the mechanism of ALK-TKI addiction in ALCL. We interrogated the well-described mechanism of MEK/ERK pathway inhibitor addiction in solid tumors and found it does not apply to ALCL. Instead, phosphoproteomics and confirmatory functional studies revealed that the STAT1 overactivation is the key mechanism of ALK-TKI addiction in ALCL. The withdrawal of TKI from addicted tumors in vitro and in vivo leads to overwhelming phospho-STAT1 activation, turning on its tumor-suppressive gene-expression program and turning off STAT3's oncogenic program. Moreover, a novel NPM1-ALK-positive ALCL PDX model showed a significant survival benefit from intermittent compared with continuous TKI dosing. In sum, we reveal for the first time the mechanism of cancer drug addiction in ALK-positive ALCL and the benefit of scheduled intermittent dosing in high-risk patient-derived tumors in vivo.

CD73, a cell-surface N -linked glycoprotein that produces extracellular adenosine, is a novel target for cancer immunotherapy. Although anti-CD73 antibodies have entered clinical development, CD73 has both protumor and antitumor functions, depending on the target cell and tumor type. The aim of this study was to characterize CD73 regulation in human hepatocellular carcinoma (HCC). We examined CD73 expression, localization, and activity using molecular, biochemical, and cellular analyses on primary HCC surgical specimens, coupled with mechanistic studies in HCC cells. We analyzed CD73 glycan signatures and global alterations in transcripts encoding other N -linked glycoproteins by using mass spectrometry glycomics and RNA sequencing (RNAseq), respectively. CD73 was expressed on tumor hepatocytes where it exhibited abnormal N -linked glycosylation, independent of HCC etiology, tumor stage, or fibrosis presence. Aberrant glycosylation of tumor-associated CD73 resulted in a 3-fold decrease in 5'-nucleotidase activity ( P <  0.0001). Biochemically, tumor-associated CD73 was deficient in hybrid and complex glycans specifically on residues N311 and N333 located in the C-terminal catalytic domain. Blocking N311/N333 glycosylation by site-directed mutagenesis produced CD73 with significantly decreased 5'-nucleotidase activity in vitro , similar to the primary tumors. Glycosylation-deficient CD73 partially colocalized with the Golgi structural protein GM130, which was strongly induced in HCC tumors. RNAseq analysis further revealed that N -linked glycoprotein-encoding genes represented the largest category of differentially expressed genes between HCC tumor and adjacent tissue. Conclusion: We provide the first detailed characterization of CD73 glycosylation in normal and tumor tissue, revealing a novel mechanism that leads to the functional suppression of CD73 in human HCC tumor cells. The present findings have translational implications for therapeutic candidate antibodies targeting cell-surface CD73 in solid tumors and small-molecule adenosine receptor agonists that are in clinical development for HCC., (© 2019 The Authors. Hepatology Communications published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.)

Background & Aims: Porphyrias are caused by porphyrin accumulation resulting from defects in the heme biosynthetic pathway that typically lead to photosensitivity and possible end-stage liver disease with an increased risk of hepatocellular carcinoma. Our aims were to study the mechanism of porphyrin-induced cell damage and protein aggregation, including liver injury, where light exposure is absent., Methods: Porphyria was induced in vivo in mice using 3,5-diethoxycarbonyl-1,4-dihydrocollidine or in vitro by exposing human liver Huh7 cells and keratinocytes, or their lysates, to protoporphyrin-IX, other porphyrins, or to δ-aminolevulinic acid plus deferoxamine. The livers, cultured cells, or porphyrin exposed purified proteins were analyzed for protein aggregation and oxidation using immunoblotting, mass spectrometry, and electron paramagnetic resonance spectroscopy. Consequences on cell-cycle progression were assessed., Results: Porphyrin-mediated protein aggregation required porphyrin-photosensitized singlet oxygen and porphyrin carboxylate side-chain deprotonation, and occurred with site-selective native protein methionine oxidation. Noncovalent interaction of protoporphyrin-IX with oxidized proteins led to protein aggregation that was reversed by incubation with acidified n-butanol or high-salt buffer. Phototoxicity and the ensuing proteotoxicity, mimicking porphyria photosensitivity conditions, were validated in cultured keratinocytes. Protoporphyrin-IX inhibited proteasome function by aggregating several proteasomal subunits, and caused cell growth arrest and aggregation of key cell proliferation proteins. Light-independent synergy of protein aggregation was observed when porphyrin was applied together with glucose oxidase as a secondary peroxide source., Conclusions: Photo-excitable porphyrins with deprotonated carboxylates mediate protein aggregation. Porphyrin-mediated proteotoxicity in the absence of light, as in the liver, requires porphyrin accumulation coupled with a second tissue oxidative injury. These findings provide a potential mechanism for internal organ damage and photosensitivity in porphyrias., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Arginase is one of the key enzymes responsible for maintaining the essential levels of nitrogen among plants, but biochemical and functional characterization of arginase among plants is limited. While screening for stable plant arginase, we found cilantro possessing an abundant and stable arginase. We purified arginase to apparent homogeneity (3300-fold purification) with a specific activity of 81,728 nmoles of urea formed/mg of protein/min and its eight-tryptic fragments had amino acid sequences identical to Arabidopsis thaliana arginase. Cilantro arginase exhibited absolute requirement for Mn 2+ (0.5 mM-1 mM). Unlike other known plant arginases, cilantro arginase did not hydrolyse d-arginine and other arginine analogues. While for sulfhydryl reagents the enzyme was sensitive, l-NOHA, an arginase inhibitor showed only moderate inhibition - a property distinct from tomato arginase. We also found arginine derived amino acids and polyamines can regulate cilantro arginase in vitro. In addition, we also noticed an increase in cilantro arginase activity to both biotic and abiotic stress. We conclude that, cilantro may be used as a model plant to study plant arginases and to delineate arginase role, beyond its classical role in nitrogen recycling and polyamine biosynthesis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cardiac myosin binding protein C (MYBPC3) is the most commonly mutated gene associated with hypertrophic cardiomyopathy (HCM). Haploinsufficiency of full-length MYBPC3 and disruption of proteostasis have both been proposed as central to HCM disease pathogenesis. Discriminating the relative contributions of these 2 mechanisms requires fundamental knowledge of how turnover of WT and mutant MYBPC3 proteins is regulated. We expressed several disease-causing mutations in MYBPC3 in primary neonatal rat ventricular cardiomyocytes. In contrast to WT MYBPC3, mutant proteins showed reduced expression and failed to localize to the sarcomere. In an unbiased coimmunoprecipitation/mass spectrometry screen, we identified HSP70-family chaperones as interactors of both WT and mutant MYBPC3. Heat shock cognate 70 kDa (HSC70) was the most abundant chaperone interactor. Knockdown of HSC70 significantly slowed degradation of both WT and mutant MYBPC3, while pharmacologic activation of HSC70 and HSP70 accelerated degradation. HSC70 was expressed in discrete striations in the sarcomere. Expression of mutant MYBPC3 did not affect HSC70 localization, nor did it induce a protein folding stress response or ubiquitin proteasome dysfunction. Together these data suggest that WT and mutant MYBPC3 proteins are clients for HSC70, and that the HSC70 chaperone system plays a major role in regulating MYBPC3 protein turnover.

Tumor: tumor microenvironment (TME) interactions are critical for tumor progression and the composition and structure of the local extracellular matrix (ECM) are key determinants of tumor metastasis. We recently reported that activation of Wnt/beta-catenin signaling in Ewing sarcoma cells induces widespread transcriptional changes that are associated with acquisition of a metastatic tumor phenotype. Significantly, ECM protein-encoding genes were found to be enriched among Wnt/beta-catenin induced transcripts, leading us to hypothesize that activation of canonical Wnt signaling might induce changes in the Ewing sarcoma secretome. To address this hypothesis, conditioned media from Ewing sarcoma cell lines cultured in the presence or absence of Wnt3a was collected for proteomic analysis. Label-free mass spectrometry was used to identify and quantify differentially secreted proteins. We then used in silico databases to identify only proteins annotated as secreted. Comparison of the secretomes of two Ewing sarcoma cell lines revealed numerous shared proteins, as well as a degree of heterogeneity, in both basal and Wnt-stimulated conditions. Gene set enrichment analysis of secreted proteins revealed that Wnt stimulation reproducibly resulted in increased secretion of proteins involved in ECM organization, ECM receptor interactions, and collagen formation. In particular, Wnt-stimulated Ewing sarcoma cells up-regulated secretion of structural collagens, as well as matricellular proteins, such as the metastasis-associated protein, tenascin C (TNC). Interrogation of published databases confirmed reproducible correlations between Wnt/beta-catenin activation and TNC and COL1A1 expression in patient tumors. In summary, this first study of the Ewing sarcoma secretome reveals that Wnt/beta-catenin activated tumor cells upregulate secretion of ECM proteins. Such Wnt/beta-catenin mediated changes are likely to impact on tumor: TME interactions that contribute to metastatic progression., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Nonalcoholic fatty liver disease (NAFLD) is becoming the major chronic liver disease in many countries. Its pathogenesis is multifactorial, but twin and familial studies indicate significant heritability, which is not fully explained by currently known genetic susceptibility loci. Notably, mutations in genes encoding nuclear lamina proteins, including lamins, cause lipodystrophy syndromes that include NAFLD. We hypothesized that variants in lamina-associated proteins predispose to NAFLD and used a candidate gene-sequencing approach to test for variants in 10 nuclear lamina-related genes in a cohort of 37 twin and sibling pairs: 21 individuals with and 53 without NAFLD. Twelve heterozygous sequence variants were identified in four lamina-related genes (ZMPSTE24, TMPO, SREBF1, SREBF2). The majority of NAFLD patients (>90%) had at least one variant compared to <40% of controls (P < 0.0001). When only insertions/deletions and changes in conserved residues were considered, the difference between the groups was similarly striking (>80% versus <25%; P < 0.0001). Presence of a lamina variant segregated with NAFLD independently of the PNPLA3 I148M polymorphism. Several variants were found in TMPO, which encodes the lamina-associated polypeptide-2 (LAP2) that has not been associated with liver disease. One of these, a frameshift insertion that generates truncated LAP2, abrogated lamin-LAP2 binding, caused LAP2 mislocalization, altered endogenous lamin distribution, increased lipid droplet accumulation after oleic acid treatment in transfected cells, and led to cytoplasmic association with the ubiquitin-binding protein p62/SQSTM1., Conclusion: Several variants in nuclear lamina-related genes were identified in a cohort of twins and siblings with NAFLD; one such variant, which results in a truncated LAP2 protein and a dramatic phenotype in cell culture, represents an association of TMPO/LAP2 variants with NAFLD and underscores the potential importance of the nuclear lamina in NAFLD. (Hepatology 2018;67:1710-1725)., (© 2017 by the American Association for the Study of Liver Diseases.)

The Polymerase Associated Factor 1 complex (PAF1c) is an epigenetic co-modifying complex that directly contacts RNA polymerase II (RNAPII) and several epigenetic regulating proteins. Mutations, overexpression and loss of expression of subunits of the PAF1c are observed in various forms of cancer suggesting proper regulation is needed for cellular development. However, the biochemical interactions with the PAF1c that allow dynamic gene regulation are unclear. We and others have shown that the PAF1c makes a direct interaction with MLL fusion proteins, which are potent oncogenic drivers of acute myeloid leukemia (AML). This interaction is critical for the maintenance of MLL translocation driven AML by targeting MLL fusion proteins to the target genes Meis1 and Hoxa9 . Here, we use a proteomics approach to identify protein-protein interactions with the PAF1c subunit CDC73 that regulate the function of the PAF1c. We identified a novel interaction with a histone H3 lysine 9 (H3K9) methyltransferase protein, SETDB1. This interaction is stabilized with a mutant CDC73 that is incapable of supporting AML cell growth. Importantly, transcription of Meis1 and Hoxa9 is reduced and promoter H3K9 trimethylation (H3K9me3) increased by overexpression of SETDB1 or stabilization of the PAF1c-SETDB1 interaction in AML cells. These findings were corroborated in human AML patients where increased SETDB1 expression was associated with reduced HOXA9 and MEIS1 . To our knowledge, this is the first proteomics approach to search for CDC73 protein-protein interactions in AML, and demonstrates that the PAF1c may play a role in H3K9me3-mediated transcriptional repression in AML., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

PKM2 (pyruvate kinase M2), a critical regulator of glycolysis, is phosphorylated by numerous growth factor receptors and oncogenic tyrosine kinases including NPM-ALK which is expressed in a subset of aggressive T-cell non-Hodgkin lymphomas known as anaplastic large cell lymphoma, ALK-positive. Our previous work demonstrated that phosphorylation of Y105-PKM2 by NPM-ALK regulates a major metabolic shift to promote lymphomagenesis. In addition to its role in metabolism, recent studies have shown that PKM2 promotes oncogenesis by phosphorylating nuclear STAT3 (signal transducer and activator of transcription 3) and regulating transcription of genes involved in cell survival and proliferation. We hypothesized that identification of novel PKM2 interactors could provide additional insights into its expanding functional role in cancer. To this end, immunocomplexes of FLAG-tagged PKM2 were isolated from NPM-ALK-positive ALCL (anaplastic large cell lymphoma) cells and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) which led to the identification of polypyrimidine tract-binding protein (PTBP1) as a novel interactor of PKM2. The interaction between PTBP1 and PKM2 was restricted to the nucleus and was dependent on NPM-ALK mediated Y105 phosphorylation of PKM2. Stable shRNA-mediated silencing of PTBP1 resulted in a marked decrease in pY105-PKM2 and pY705-STAT3 which led to decreased ALCL cell proliferation and colony formation. Overall, our data demonstrate that PTBP1 interacts with PKM2 and promotes ALCL oncogenesis by facilitating PKM2-dependent activation of STAT3 within the nucleus.

Fbxo45 is an atypical E3 ubiquitin ligase, which specifically targets proteins for ubiquitin-mediated degradation. Fbxo45 ablation results in defective neuronal differentiation and abnormal formation of neural connections; however, the mechanisms underlying these defects are poorly understood. Using an unbiased mass spectrometry-based proteomic screen, we show here that N-cadherin is a novel interactor of Fbxo45. N-cadherin specifically interacts with Fbxo45 through two consensus motifs overlapping the site of calcium-binding and dimerization of the cadherin molecule. N-cadherin interaction with Fbxo45 is significantly abrogated by calcium treatment. Surprisingly, Fbxo45 depletion by RNAi-mediated silencing results in enhanced proteolysis of N-cadherin. Conversely, ectopic expression of Fbxo45 results in decreased proteolysis of N-cadherin. Fbxo45 depletion results in dramatic reduction in N-cadherin expression, impaired neuronal differentiation, and diminished formation of neuronal processes. Our studies reveal an unanticipated role for an F-box protein that inhibits proteolysis in the regulation of a critical biological process., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Deregulation of signaling pathways controlled by protein phosphorylation underlies the pathogenesis of hematological malignancies; however, the extent to which deregulated phosphorylation may be involved in B-cell non-Hodgkin lymphoma (B-NHL) pathogenesis is largely unknown. To identify phosphorylation events important in B-NHLs, we performed mass spectrometry-based, label-free, semiquantitative phosphoproteomic profiling of 11 cell lines derived from three B-NHL categories: Burkitt lymphoma, follicular lymphoma, and mantle-cell lymphoma. In all, 6579 unique phosphopeptides, corresponding to 1701 unique phosphorylated proteins, were identified and quantified. The data are available via ProteomeXchange with identifier PXD000658. Hierarchical clustering highlighted distinct phosphoproteomic signatures associated with each lymphoma subtype. Interestingly, germinal center-derived B-NHL cell lines were characterized by phosphorylation of proteins involved in the B-cell receptor signaling. Of these proteins, phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (PAG1) was identified with the most phosphorylated tyrosine peptides in Burkitt lymphoma and follicular lymphoma. PAG1 knockdown resulted in perturbation of the tyrosine phosphosignature of B-cell receptor signaling components. Significantly, PAG1 knockdown increased cell proliferation and response to antigen stimulation of these germinal center-derived B-NHLs. These data provide a detailed annotation of phosphorylated proteins in human lymphoid cancer. Overall, our study revealed the utility of unbiased phosphoproteome interrogation in characterizing signaling networks that may provide insights into pathogenesis mechanisms in B-cell lymphomas., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Chromosomal translocations encoding chimeric fusion proteins constitute one of the most common mechanisms underlying oncogenic transformation in human cancer. Fusion peptides resulting from such oncogenic chimeric fusions, though unique to specific cancer subtypes, are unexplored as cancer biomarkers. Here we show, using an approach termed fusion peptide multiple reaction monitoring mass spectrometry, the direct identification of different cancer-specific fusion peptides arising from protein chimeras that are generated from the juxtaposition of heterologous genes fused by recurrent chromosomal translocations. Using fusion peptide multiple reaction monitoring mass spectrometry in a clinically relevant scenario, we demonstrate the specific, sensitive, and unambiguous detection of a specific diagnostic fusion peptide in clinical samples of anaplastic large cell lymphoma, but not in a diverse array of benign lymph nodes or other forms of primary malignant lymphomas and cancer-derived cell lines. Our studies highlight the utility of fusion peptides as cancer biomarkers and carry broad implications for the use of protein biomarkers in cancer detection and monitoring.

Attachment of ubiquitin to substrate is typically thought to occur via formation of an isopeptide bond between the C-terminal glycine residue of ubiquitin and a lysine residue in the substrate. In vitro, Ube2w is nonreactive with free lysine yet readily ubiquitinates substrate. Ube2w also contains novel residues within its active site that are important for its ability to ubiquitinate substrate. To identify the site of modification, we analyzed ubiquitinated substrates by mass spectrometry and found the N-terminal -NH2 group as the site of conjugation. To confirm N-terminal ubiquitination, we generated lysine-less and N-terminally blocked versions of one substrate, the polyglutamine disease protein ataxin-3, and showed that Ube2w can ubiquitinate a lysine-less, but not N-terminally blocked, ataxin-3. This was confirmed with a second substrate, the neurodegenerative disease protein Tau. Finally, we directly sequenced the N terminus of unmodified and ubiquitinated ataxin-3, demonstrating that Ube2w attaches ubiquitin to the N terminus of its substrates. Together these data demonstrate that Ube2w has novel enzymatic properties that direct ubiquitination of the N terminus of substrates.

Wild-type plasminogen activator inhibitor type 1 (PAI-1) is a fast-acting uPA and tPA inhibitor with half-life of 1-2 h. Recombinant PAI-1 with two mutations, Q197C and G355C, shows a very long half-life (VLHL). An introduced disulfide bridge holds together two central, parallel strands of beta-sheet A, preventing their separation to incorporate residues P4-P14 during the serpin's transition into latency. An active PAI-1 is usually described as a single structure with the reactive center loop (RCL) with P1-P1' (R369-M370) extended far from the bulk of the serpin's body. We have found that VLHL PAI-1 exists in several active forms that travel with different electrophoretic mobilities. Under aerobic conditions, two distinct active forms are observed. Upon reduction of cysteines, the VLHL mutant converts into the latent form, which spontaneously reactivates into a fully or partially active serpin, with yet another mobility. Utilizing electrophoresis, zymography (to check PAI-1 activity toward uPA) and theoretical calculations for molecular modeling, we have characterized active 1, 2, 3 and latent conformers of VLHL PAI-1 and their behaviors at normal and elevated temperatures, and in normal or reducing environments. VLHL PAI-1 activity is not affected, and the molecules do not polymerize unless reduced and/or heated. VLHL PAI-1 associates into dimers and bigger oligomers when -SH groups become available for oxidation and formation of intra- or intermolecular -S-S- bridges between conformers of different shapes and activities. We postulate that the active structures differ in RCL conformation and their position in relation to the gate region and the rest of the molecule.

The integrin alpha3beta1 plays important roles in development, angiogenesis, and the pathogenesis of cancer, suggesting potential therapeutic uses for antagonists of this receptor. Recently, an alpha3beta1 integrin-binding site was mapped to residues 190-201 (FQGVLQNVRFVF) of the N-terminal domain of the secreted protein thrombospondin-1 (TSP1). This sequence displays diverse biological activities in vitro and inhibits angiogenesis in vivo. Herein we describe the NMR solution conformation of this segment in both water and dodecylphosphocholine micelles. While essentially unstructured in water, a more well-defined conformation is populated in micelles, particularly in the C-terminal half of the peptide and correlated with increased biological activity of the micellar peptide. The data suggested that the residues that are critical for biological activity are contained in a structurally well-defined segment of the peptide. These data support the role of the NVR motif as a required element of full-length TSP1 for specific molecular recognition by the alpha3beta1 integrin.

Melanin is a heterogeneous biopolymer produced only by specific cells termed melanocytes, which synthesize and deposit the pigment in specialized membrane-bound organelles known as melanosomes. Although melanosomes have been suspected of being closely related to lysosomes and platelets, the total number of melanosomal proteins is still unknown. Thus far, six melanosome-specific proteins have been identified, and the challenge is to characterize the complete proteome of the melanosome to further understand its mechanism of biogenesis. In this report, we used mass spectrometry and subcellular fractionation to identify protein components of early melanosomes. Using this approach, we have identified all 6 of the known melanosome-specific proteins, 56 proteins that are shared with other organelles, and confirmed the presence of 6 novel melanosomal proteins using western blotting and by immunohistochemistry.