Whole-exome sequencing uncovers frequent GNAS mutations in intraductal papillary mucinous neoplasms of the pancreas

Intraductal papillary mucinous neoplasm (IPMN) is a common pancreatic cystic neoplasm that is often invasive and metastatic, resulting in a poor prognosis. Few molecular alterations unique to IPMN are known. We performed whole-exome sequencing for a primary IPMN tissue, which uncovered somatic mutations in KCNF1, DYNC1H1, PGCP, STAB1, PTPRM, PRPF8, RNASE3, SPHKAP, MLXIPL, VPS13C, PRCC, GNAS, KRAS, RBM10, RNF43, DOCK2, and CENPF. We further analyzed GNAS mutations in archival cases of 118 IPMNs and 32 pancreatic ductal adenocarcinomas (PDAs), which revealed that 48 (40.7%) of the 118 IPMNs but none of the 32 PDAs harbored GNAS mutations. G-protein alpha-subunit encoded by GNAS and its downstream targets, phosphorylated substrates of protein kinase A, were evidently expressed in IPMN; the latter was associated with neoplastic grade. These results indicate that GNAS mutations are common and specific for IPMN, and activation of G-protein signaling appears to play a pivotal role in IPMN

Integrated Proteomics Unveils Nuclear PDE3A2 as a Regulator of Cardiac Myocyte Hypertrophy

Background: Signaling by cAMP is organized in multiple distinct subcellular nanodomains regulated by cAMP-hydrolyzing PDEs (phosphodiesterases). Cardiac β-adrenergic signaling has served as the prototypical system to elucidate cAMP compartmentalization. Although studies in cardiac myocytes have provided an understanding of the location and properties of a handful of cAMP subcellular compartments, an overall view of the cellular landscape of cAMP nanodomains is missing. Methods: Here, we combined an integrated phosphoproteomics approach that takes advantage of the unique role that individual PDEs play in the control of local cAMP, with network analysis to identify previously unrecognized cAMP nanodomains associated with β-adrenergic stimulation. We then validated the composition and function of one of these nanodomains using biochemical, pharmacological, and genetic approaches and cardiac myocytes from both rodents and humans. Results: We demonstrate the validity of the integrated phosphoproteomic strategy to pinpoint the location and provide critical cues to determine the function of previously unknown cAMP nanodomains. We characterize in detail one such compartment and demonstrate that the PDE3A2 isoform operates in a nuclear nanodomain that involves SMAD4 (SMAD family member 4) and HDAC-1 (histone deacetylase 1). Inhibition of PDE3 results in increased HDAC-1 phosphorylation, leading to inhibition of its deacetylase activity, derepression of gene transcription, and cardiac myocyte hypertrophic growth. Conclusions: We developed a strategy for detailed mapping of subcellular PDE-specific cAMP nanodomains. Our findings reveal a mechanism that explains the negative long-term clinical outcome observed in patients with heart failure treated with PDE3 inhibitors

Genome Wide Analysis of Acute Myeloid Leukemia Reveal Leukemia Specific Methylome and Subtype Specific Hypomethylation of Repeats

Methylated DNA immunoprecipitation followed by high-throughput sequencing (MeDIP-seq) has the potential to identify changes in DNA methylation important in cancer development. In order to understand the role of epigenetic modulation in the development of acute myeloid leukemia (AML) we have applied MeDIP-seq to the DNA of 12 AML patients and 4 normal bone marrows. This analysis revealed leukemia-associated differentially methylated regions that included gene promoters, gene bodies, CpG islands and CpG island shores. Two genes (SPHKAP and DPP6) with significantly methylated promoters were of interest and further analysis of their expression showed them to be repressed in AML. We also demonstrated considerable cytogenetic subtype specificity in the methylomes affecting different genomic features. Significantly distinct patterns of hypomethylation of certain interspersed repeat elements were associated with cytogenetic subtypes. The methylation patterns of members of the SINE family tightly clustered all leukemic patients with an enrichment of Alu repeats with a high CpG density (P<0.0001). We were able to demonstrate significant inverse correlation between intragenic interspersed repeat sequence methylation and gene expression with SINEs showing the strongest inverse correlation (R2 = 0.7). We conclude that the alterations in DNA methylation that accompany the development of AML affect not only the promoters, but also the non-promoter genomic features, with significant demethylation of certain interspersed repeat DNA elements being associated with AML cytogenetic subtypes. MeDIP-seq data were validated using bisulfite pyrosequencing and the Infinium array

Studying protein–protein affinity and immobilized ligand–protein affinity interactions using MS-based methods

This review discusses the most important current methods employing mass spectrometry (MS) analysis for the study of protein affinity interactions. The methods are discussed in depth with particular reference to MS-based approaches for analyzing protein–protein and protein–immobilized ligand interactions, analyzed either directly or indirectly. First, we introduce MS methods for the study of intact protein complexes in the gas phase. Next, pull-down methods for affinity-based analysis of protein–protein and protein–immobilized ligand interactions are discussed. Presently, this field of research is often called interactomics or interaction proteomics. A slightly different approach that will be discussed, chemical proteomics, allows one to analyze selectivity profiles of ligands for multiple drug targets and off-targets. Additionally, of particular interest is the use of surface plasmon resonance technologies coupled with MS for the study of protein interactions. The review addresses the principle of each of the methods with a focus on recent developments and the applicability to lead compound generation in drug discovery as well as the elucidation of protein interactions involved in cellular processes. The review focuses on the analysis of bioaffinity interactions of proteins with other proteins and with ligands, where the proteins are considered as the bioactives analyzed by MS

Спроби розв’язання Радою Народних Міністрів Української Народної Республіки земельного питання у березні-квітні 1918 року

У статті розглядається діяльність РНМ УНР із реформування земельних відносин в Україні, реалізації тимчасового земельного закону, вирішення питання засіву землі весною 1918 р.В статье рассматривается деятельность СНМ УНР по реформированию земельных отношений в Украине, реализации временного земельного закона, решение вопроса засева земли весной 1918 г.In the article activity of CFМ of UNR is examined from reformation of the landed relations in Ukraine, realization of the temporal landed law, decision of sowing of the land in spring 1918

A Proteomics Investigation of Anchored PKA-RI Signaling

Compartmentalization of kinases and phosphatases plays an important role in the specificity of second messenger mediated signaling events. Localization of the cAMP-dependent protein kinase is mediated by interaction of its regulatory subunit (PKA-R) with the versatile family of A-kinase anchoring proteins (AKAPs). Most AKAPs bind avidly to PKA-RII, while some have dual specificity for both PKA-RI and PKA-RII, however, no mammalian PKA-RI specific AKAPs have thus far been assigned. This was mainly attributed to the observation that PKA-RI is more cytosolic, as compared to the more heavily compartmentalized PKA-RII. Chemical proteomics screens of the cAMP interactome in mammalian heart tissue recently identified sphingosine kinase type 1-interacting protein (SKIP, SPHKAP) as a putative novel AKAP. Biochemical characterization now shows that SPHKAP can be considered as the first mammalian AKAP that preferentially binds to PKA-RIα. Recombinant human SPHKAP functions as an RI-specific AKAP that utilizes the for AKAPs characteristic amphipathic helix for interaction. Further chemical proteomic screening utilizing differential binding characteristics of specific cAMP-resins confirms SPHKAPs endogenous specificity for PKA-RI directly in mammalian heart and spleen tissue. Alignment of SPHKAPs amphipathic helix with peptide models of PKA-RI or PKA-RII specific anchoring domains shows that it has largely only PKA-RIα characteristics. To further investigate the role of the protein, we combined subcellular fractionation, confocal microscopy and LC-MS/MS analysis to study its subcellular localization and exact molecular environment. We found that SPHKAP localizes to the cytoplasm as well as to the mitochondrial inner membrane space and matrix. Here, it colocalizes with PKA-RIα and associates with a mitochondrial inner membrane organizing system (MINOS) complex that is responsible for maintaining crista structure. Additionally, SPHKAP associates with apoptosis-inducing factor (AIF) and the phosphatase PGAM5. We found that the protein N-terminus is required for its mitochondrial localization and overexpression of SPHKAP results in abnormal mitochondria. We show that SPHKAP is a substrate of PKA and that phosphorylation regulates the interaction between SPHKAP and AIF upon cAMP and apoptosis stimulation. Sequence analysis revealed that SPHKAP contains a 13-amino acid motif similar to the BH3 domain present in BCL-2 family proteins. As with other BH3-only proteins, co-immunoprecipitation and deletion of BH3 core amino acids demonstrated that SPHKAP interacts with the anti-apoptotic BCL-2 and BCL-xL through its BH3 domain. Taken together, The data suggest a putative role for the mitochondrial SPHKAP-PKA type I complex in regulating mitochondrial crista structure and mitochondrial-mediated apoptosi

Applications of stable isotope dimethyl labeling in quantitative proteomics.

Mass spectrometry has proven to be an indispensable tool for protein identification, characterization, and quantification. Among the possible methods in quantitative proteomics, stable isotope labeling by using reductive dimethylation has emerged as a cost-effective, simple, but powerful method able to compete at any level with the present alternatives. In this review, we briefly introduce experimental and software methods for proteome analysis using dimethyl labeling and provide a comprehensive overview of reported applications in the analysis of (1) differential protein expression, (2) posttranslational modifications, and (3) protein interactions