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Calmodulin (CaM) is a Ca2+ binding protein modulating multiple targets, several of which are associated with cardiac pathophysiology. Recently, CaM mutations were linked to heart arrhythmia. CaM is crucial for cell growth and viability, yet the effect of the arrhythmogenic CaM mutations on cell viability, as well as heart rhythm, remains unknown, and only a few targets with relevance for heart physiology have been analyzed for their response to mutant CaM. We show that the arrhythmia-associated CaM mutants support growth and viability of DT40 cells in the absence of WT CaM except for the long QT syndrome mutant CaM D129G. Of the six CaM mutants tested (N53I, F89L, D95V, N97S, D129G, and F141L), three showed a decreased activation of Ca2+/CaM-dependent kinase II, most prominently the D129G CaM mutation, which was incapable of stimulating Thr286 autophosphorylation. Furthermore, the CaM D129G mutation led to bradycardia in zebrafish and an arrhythmic phenotype in a subset of the analyzed zebrafish.

Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses (1). Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important part in innate immune responses (2). Viral-encoded core basic proteins compact viral genomes, but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones (3). Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles (4,5), it is unknown whether protein VII affects cellular chromatin. Here we show that protein VII alters cellular chromatin, leading us to hypothesize that this has an impact on antiviral responses during adenovirus infection in human cells. We find that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter the protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in the chromatin of members of the high-mobility-group protein B family (HMGB1, HMGB2 and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses (6,7). We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together, our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signalling., As viruses commandeer cellular functions to promote viral production, they induce numerous cellular changes. Manipulation of host chromatin is important for viral takeover of cellular functions (1,8-11). Although there are [...]

The epigenetic reader BRD4 plays a vital role in transcriptional regulation, cellular growth control and cell-cycle progression. Dysregulation of BRD4 function has been implicated in the pathogenesis of a wide range of cancers. However, how BRD4 is regulated to maintain its normal function in healthy cells and how alteration of this process leads to cancer remain poorly understood. In this study, we discovered that BRD4 is hyperphosphorylated in NUT midline carcinoma and identified CDK9 as a potential kinase mediating BRD4 hyperphosphorylation. Disruption of BRD4 hyperphosphorylation using both chemical and molecular inhibitors led to the repression of BRD4 downstream oncogenes and abrogation of cellular transformation. BRD4 hyperphosphorylation is also observed in other cancers displaying enhanced BRD4 oncogenic activity. Our study revealed a mechanism that may regulate BRD4 biological function through phosphorylation, which, when dysregulated, could lead to oncogenesis. Our finding points to strategies to target the aberrant BRD4 signaling specifically for cancer intervention. [ABSTRACT FROM AUTHOR]

Mass spectrometry (MS) is an essential part of the cell biologist's proteomics toolkit, allowing analyses at molecular and system-wide scales. However, proteomics still lag behind genomics in popularity and ease of use. We discuss key differences between MS-based -omics and other booming -omics technologies and highlight what we view as the future of MS and its role in our increasingly deep understanding of cell biology. [ABSTRACT FROM AUTHOR]

Posttranslational modifications (PTMs) such as phosphorylation, acetylation, and glycosylation are an essential regulatory mechanism of protein function and interaction, and they are associated with a wide range of biological processes. Since most PTMs alter the molecular mass of a protein, mass spectrometry (MS) is the ideal analytical tool for studying various PTMs. However, PTMs are often present in substoichiometric levels, and therefore their unmodified counterpart often suppresses their signal in MS. Consequently, PTM analysis by MS is a challenging task, requiring highly specialized and sensitive PTM-specific enrichment methods. Currently, several methods have been implemented for PTM enrichment, and each of them has its drawbacks and advantages as they differ in selectivity and specificity toward specific protein modifications. Unfortunately, for the vast majority of more than 400 known modifications, we have no or poor tools for selective enrichment.Here, we describe a comprehensive workflow to simultaneously study phosphorylation, acetylation, and N-linked sialylated glycosylation from the same biological sample. The protocol involves an initial titanium dioxide (TiO 2 ) step to enrich for phosphopeptides and sialylated N-linked glycopeptides followed by glycan release and post-fractionation using sequential elution from immobilized metal affinity chromatography (SIMAC) to separate mono-phosphorylated and deglycosylated peptides from multi-phosphorylated ones. The IMAC flow-through and acidic elution are subsequently subjected to a next round of TiO 2 enrichment for further separation of mono-phosphopeptides from deglycosylated peptides. Furthermore, the lysine-acetylated peptides present in the first TiO 2 flow-through fraction are enriched by immunoprecipitation (IP) after peptide cleanup. Finally, the samples are fractionated by high pH reversed phase chromatography (HpH) or hydrophilic interaction liquid chromatography (HILIC ) to reduce sample complexity and increase the coverage in the subsequent LC-MS /MS analysis. This allows the analysis of multiple types of modifications from the same highly complex biological sample without decreasing the quality of each individual PTM study.

Post-translational modifications (PTMs) such as phosphorylation, acetylation, and glycosylation are an essential regulatory mechanism of protein function and they are associated with a range of biological processes. Since most PTMs alter the molecular mass of a protein, mass spectrometry (MS) is the ideal analytical tool for studying various PTMs. However, PTMs are generally present in substoichiometric levels and therefore their unmodified counterpart often suppresses their signal in MS. Consequently, PTM analysis by MS is a challenging task requiring highly specialized and sensitive enrichment methods. Currently, several methods have been implemented for PTM enrichment and each of them has its drawbacks and advantages as they differ in selectivity and specificity toward specific protein modifications. Unfortunately, for most of the more than 300 known modifications we have none or poor tools for selective enrichment.Here, we describe a comprehensive workflow to simultaneously study phosphorylation, acetylation, and N-linked sialylated glycosylation from the same biological sample. The protocol involves an initial titanium dioxide (TiO2) step to enrich for phosphopeptides and sialylated N-linked glycopeptides followed by glycan release and post-fractionation using sequential elution from immobilized metal affinity chromatography (SIMAC) to separate mono-phosphorylated and deglycosylated peptides from multi-phosphorylated ones. The IMAC flow-through and acidic elution is subsequently subjected to a next round of TiO2 enrichment for further separation of mono-phosphopeptides from deglycosylated peptides. In addition, the acetylated peptides present in the first TiO2 flow-through are enriched by immunoprecipitation (IP). Finally, the samples are fractionated by hydrophilic interaction liquid chromatography (HILIC) to reduce sample complexity and increase the coverage during LC-MS/MS analysis. This allows the analysis of multiple types of modifications from the same highly complex biological sample without decreasing the quality of each individual PTM study.