Disentangling thermal stress responses in a reef-calcifier and its photosymbionts by shotgun proteomics

This project was funded by the Leibniz Association (SAW-2014-ISAS-2) awarded to AS and HW and supported by the Ministerium für Kultur und Wissenschaft des Landes Nordrhein-Westfalen, the Regierende Bürgermeister von Berlin - inkl. Wissenschaft und Forschung, and the Bundesministerium für Bildung und Forschung. Sampling was conducted under the Research Permit No. FKNMS-2015–026, issued to Pamela Hallock who is warmly acknowledged for her general support and assistance during fieldwork.Peer reviewedPublisher PD

Regulatory Function of Sympathetic Innervation on the Endo/Lysosomal Trafficking of Acetylcholine Receptor

Recent studies have demonstrated that neuromuscular junctions are co-innervated by sympathetic neurons. This co-innervation has been shown to be crucial for neuromuscular junction morphology and functional maintenance. To improve our understanding of how sympathetic innervation affects nerve–muscle synapse homeostasis, we here used in vivo imaging, proteomic, biochemical, and microscopic approaches to compare normal and sympathectomized mouse hindlimb muscles. Live confocal microscopy revealed reduced fiber diameters, enhanced acetylcholine receptor turnover, and increased amounts of endo/lysosomal acetylcholine-receptor-bearing vesicles. Proteomics analysis of sympathectomized skeletal muscles showed that besides massive changes in mitochondrial, sarcomeric, and ribosomal proteins, the relative abundance of vesicular trafficking markers was affected by sympathectomy. Immunofluorescence and Western blot approaches corroborated these findings and, in addition, suggested local upregulation and enrichment of endo/lysosomal progression and autophagy markers, Rab 7 and p62, at the sarcomeric regions of muscle fibers and neuromuscular junctions. In summary, these data give novel insights into the relevance of sympathetic innervation for the homeostasis of muscle and neuromuscular junctions. They are consistent with an upregulation of endocytic and autophagic trafficking at the whole muscle level and at the neuromuscular junction

Deciphering the role of autophagy and exosomes in human lymphoma

The orchestrated homeostatic action of autophagy and exosome biogenesis provides a potential causal relation with lymphomagenesis. This study aims to characterize the differently expressed proteins associated with both above-mentioned pathways, as well as to explore their potential implication in therapeutic response controlling lymphomagenesis via N3a-induced re-activated p53 in different lymphoma subtypes

The FERM protein EPB41L5 regulates actomyosin contractility and focal adhesion formation to maintain the kidney filtration barrier

Podocytes form the outer part of the glomerular filter, where they have to withstand enormous transcapillary filtration forces driving glomerular filtration. Detachment of podocytes from the glomerular basement membrane precedes most glomerular diseases. However, little is known about the regulation of podocyte adhesion in vivo. Thus, we systematically screened for podocyte-specific focal adhesome (FA) components, using genetic reporter models in combination with iTRAQ-based mass spectrometry. This approach led to the identification of FERM domain protein EPB41L5 as a highly enriched podocyte-specific FA component in vivo. Genetic deletion of Epb41l5 resulted in severe proteinuria, detachment of podocytes, and development of focal segmental glomerulosclerosis. Remarkably, by binding and recruiting the RhoGEF ARGHEF18 to the leading edge, EPB41L5 directly controls actomyosin contractility and subsequent maturation of focal adhesions, cell spreading, and migration. Furthermore, EPB41L5 controls matrix-dependent outside-in signaling by regulating the focal adhesome composition. Thus, by linking extracellular matrix sensing and signaling, focal adhesion maturation, and actomyosin activation EPB41L5 ensures the mechanical stability required for podocytes at the kidney filtration barrier. Finally, a diminution of EPB41L5-dependent signaling programs appears to be a common theme of podocyte disease, and therefore offers unexpected interventional therapeutic strategies to prevent podocyte loss and kidney disease progression

Proteomic insights into non-small cell lung cancer: New ideas for cancer diagnosis and therapy from a functional viewpoint

We recently characterized SEC62 as an oncogene in non-small-cell lung cancer (NSCLC). Here we aimed to gain further insight into the molecular mechanisms of the cancer-related functions of this oncogene. We performed 2D-DIGE proteome analysis of tumor material from patients with NSCLC and of HEK293 cells stably overexpressing plasmid-encoded SEC62, combined with investigation of the Sec62 interactome. Furthermore, we analyzed the proteomic effects of siRNA-mediated depletion of the Sec62-interacting protein Sec63. We identified a comprehensive list of differentially regulated proteins, providing new insights into the molecular mechanisms of the cancer-related functions of Sec62 in cell migration, drug resistance, and Ca2+-homeostasis

Proteomic insights into non-small cell lung cancer: New ideas for cancer diagnosis and therapy from a functional viewpoint

AbstractWe recently characterized SEC62 as an oncogene in non-small-cell lung cancer (NSCLC). Here we aimed to gain further insight into the molecular mechanisms of the cancer-related functions of this oncogene. We performed 2D-DIGE proteome analysis of tumor material from patients with NSCLC and of HEK293 cells stably overexpressing plasmid-encoded SEC62, combined with investigation of the Sec62 interactome. Furthermore, we analyzed the proteomic effects of siRNA-mediated depletion of the Sec62-interacting protein Sec63. We identified a comprehensive list of differentially regulated proteins, providing new insights into the molecular mechanisms of the cancer-related functions of Sec62 in cell migration, drug resistance, and Ca2+-homeostasis

Proteome data of Amphistegina gibbosa

The proliferation of key marine ecological engineers and carbonate producers often relies on their association with photosymbiotic algae. Evaluating stress responses of these organisms is important to predict their fate under future climate projections. Physiological approaches are limited in their ability to resolve the involved molecular mechanisms and attribute stress effects to the host or symbiont, while probing and partitioning of proteins cannot be applied in organisms where the host and symbiont are small and cannot be physically separated. Here we apply a label-free quantitative proteomics approach to detect changes of proteome composition in the diatom-bearing benthic foraminifera Amphistegina gibbosa experimentally exposed to three thermal-stress scenarios. We developed a workflow for protein extraction from less than ten specimens and simultaneously analysed host and symbiont proteomes. Despite little genomic data for the host, 1,618 proteins could be partially assembled and assigned. The proteomes revealed identical pattern of stress response among stress scenarios as that indicated by physiological measurements, but allowed identification of compartment-specific stress reactions. In the symbiont, stress-response and proteolysis-related proteins were up regulated while photosynthesis-related proteins declined. In contrast, host homeostasis was maintained through chaperone up-regulation associated with elevated proteosynthesis and proteolysis, and the host metabolism shifted to heterotrophy

A pioneer protein is part of a large complex involved in trans-splicing of a group II intron in the chloroplast of Chlamydomonas reinhardtii

Splicing of organellar introns requires the activity of numerous nucleus-encoded factors. In the chloroplast of Chlamydomonas reinhardtii, maturation of psaA mRNA encoding photosystem I subunit A involves two steps of trans-splicing. The exons, located on three separate transcripts, are flanked by sequences that fold to form the conserved structures of two group II introns. A fourth transcript contributes to assembly of the first intron, which is thus tripartite. The raa7 mutant (RNA maturation of psaA 7) is deficient in trans-splicing of the second intron of psaA, and may be rescued by transforming the chloroplast genome with an intron-less version of psaA. Using mapped-based cloning, we identify the RAA7 locus, which encodes a pioneer protein with no previously known protein domain or motif. The Raa7 protein, which is not associated with membranes, localizes to the chloroplast. Raa7 is a component of a large complex and co-sediments in sucrose gradients with the previously described splicing factors Raa1 and Raa2. Based on tandem affinity purification of Raa7 and mass spectrometry, Raa1 and Raa2 were identified as interacting partners of Raa7. Yeast two-hybrid experiments indicate that the interaction of Raa7 with Raa1 and Raa2 may be direct. We conclude that Raa7 is a component of a multimeric complex that is required for trans-splicing of the second intron of psaA. The characterization of this psaA trans-splicing complex is also of interest from an evolutionary perspective because the nuclear spliceosomal introns are thought to derive from group II introns, with which they show mechanistic and structural similarity

Proteome Profiling and Ultrastructural Characterization of the Human RCMH Cell Line: Myoblastic Properties and Suitability for Myopathological Studies

Artículo de publicación ISIStudying (neuro)muscular disorders is a major topic in biomedicine with a demand for suitable model systems. Continuous cell culture (in vitro) systems have several technical advantages over in vivo systems and became widely used tools for discovering physiological/ pathophysiological mechanisms in muscle. In particular, myoblast cell lines are suitable model systems to study complex biochemical adaptations occurring in skeletal muscle and cellular responses to altered genetic/ environmental conditions. Whereas most in vitro studies use extensively characterized murine C2C12 cells, a comprehensive description of an equivalent human cell line, not genetically manipulated for immortalization, is lacking. Therefore, we characterized human immortal myoblastic RCMH cells using scanning (SEM) and transmission electron microscopy (TEM) and proteomics. Among more than 6200 identified proteins we confirm the known expression of proteins important for muscle function. Comparing the RCMH proteome with two well-defined nonskeletal muscle cells lines (HeLa, U2OS) revealed a considerable enrichment of proteins important for muscle function. SEM/TEM confirmed the presence of agglomerates of cytoskeletal components/intermediate filaments and a prominent rough ER. In conclusion, our results indicate RMCH as a suitable in vitro model for investigating muscle function-related processes such as mechanical stress burden and mechanotransduction, EC coupling, cytoskeleton, muscle cell metabolism and development, and (ER-associated) myopathic disorders.Ministerium fur Innovation, Wissenschaft and Forschung des Landes Nordrhein-Westfale