Project description:Ovarian cancer is a major cause of cancer mortality among women largely due to late diagnosis of advanced stage metastatic disease. More extensive molecular analysis of metastatic ovarian cancer is needed to identify post-translational modifications of proteins particularly associated with metastatic disease so that we can better understand the metastatic process and identify potential therapeutic targets. Proteins are the target of many recently developed cancer treatments, but an often neglected aspect of biomarker discovery is protein glycosylation, especially those involving tumor-associated carbohydrate antigens (TACAs) such as sialyl-Lewis(x) (SLe(x) and sialyl-Lewis(a) (SLe(a), which have been identified in various cancers. Although it is already known that considerable changes in protein glycosylation are major contributors to the initiation, progression and metastasis of tumors, specifics about glycosylation changes particularly important to the metastatic process are still lacking. In this report we describe the results of a combined glycomic and proteomic study of metastatic ovarian cancer (OC) ascites fluids. Glycoproteins in ascites fluid were enriched by affinity binding to lectins (ConA or WGA) and other affinity matrices. Separate glycomic and proteomic analyses were performed as well as glycopeptide analyses. Relative abundances of different N-glycan groups and proteins were identified from original ascites fluids and corresponding lectin bound samples. Levels of biomarkers CA125, MUC1 and fibronectin were also monitored in these samples by Western blot analysis. N-glycan analysis of ascites fluids showed the presence of large, highly fucosylated and sialylated, complex and hybrid glycans, some of which were not observed in normal serum. Proteins in OC ascites that were more abundant or not present in the serum control, were haptoglobin, fibronectin, lumican, fibulin, hemopexin, ceruloplasmin, alpha-1-antitrypsin and alpha-1-antichymotrypsin. Glycopeptide analysis identified N- and O-glycans in clusterin, hemopexin, and fibulin that were present in OC ascites.
Project description:Synapses are the brain’s functional units connecting neurons into circuits that underlie memory and behavior. These specialized neuronal junctions are heterogenic in function and molecular composition, reflecting diverse health and disease states. Progress over the past years has shown that trans-synaptic adhesion molecules mediate synapse formation, specification, and differentiation during development. Among the prominently expressed synaptic cleft proteins are SynCAMs, a group of immunoglobulin molecules that engage in homo- and heterophilic interactions and that instruct synapse formation and guide synaptic maturation and that are specific for excitatory synapses. The current study describes the use of peroxidase-mediated proximity labeling to map the proteome of excitatory synapses using SynCAM1 as a reporter protein.
Project description:Poly(ADP-ribosylation)(PARylation)is apost-translational modification mediated by asubset of ADP-ribosyl transferases (ARTs). Although PARylation-inhibition based therapies areconsidered as an avenue to combat debilitating diseases such as cancerand myopathies, the roleof thismodificationinphysiological processes such ascell differentiation remainsunclear. Here we showthat Tankyrase1 (TNKS1), aPARylating ART, plays a major role in myogenesis, a vital process known to drivemuscle fiber formation and regeneration.Althoughallbona fidePARPs are expressed in muscle cells, experiments using siRNA-mediated knockdownor pharmacological inhibitionshow thatTKNS1 is the enzyme responsible ofcatalyzing PARylation during myogenesis. Via this activity,TKNS1controlsthe turnoverof mRNAs encoding myogenic regulatory factors such as nucleophosmin(NPM) and myogenin.TKNS1 mediates these effectsby targeting RNA-binding proteinssuch as Human Antigen R (HuR). HuR harbors a conserved TNKS-binding motif (TBM), the mutation of which not only prevents theassociation of HuR with TKNS1 anditsPARylation, but also precludes HuR from regulating the turnover of NPMand myogeninmRNAsas well as from promoting myogenesis. Therefore, our data uncovera new role forTNKS1as akeymodulator of RBP-mediated post transcriptionaleventsrequired for vitalprocesses suchasmyogenesis.
Project description:Kinetoplastids rely heavily on post-transcriptional mechanisms for control of gene expression, and on RNA-binding proteins that regulate mRNA splicing, translation and decay. Trypanosoma brucei ERBP1 (Tb927.10.14150) and ERBP2 (Tb927.9.9550) were previously identified as mRNA binding proteins that lack canonical RNA-binding domains. We here show that ERBP1 is associated with the endoplasmic reticulum, like ERBP2, and that the two proteins interact in vivo. Loss of ERBP1 from bloodstream-form T. brucei initially resulted in a growth defect but proliferation was restored after more prolonged cultivation. Results from a pull-down of tagged ERBP1 suggest that it preferentially binds to ribosomal protein mRNAs. The ERBP1 sequence resembles that of Saccharomyces cerevisiae Bfr1, which also localises to the endoplasmic reticulum and binds to ribosomal protein mRNAs. However, unlike Bfr1, ERBP1 does not bind to mRNAs encoding secreted proteins, and it is also not recruited to stress granules after starvation.
Project description:The myosin light chain protein family consists of two classes, the regulatory myosin light chains (RLC) and the essential myosin light chains (ELC). Functionally, the MLC proteins are directly involved in sarcomeric activity and force transmission contributing to cardiac contractility. Besides regulating contractility by protein-protein interactions alone, MLCs modulate force transmission through posttranslational phosphorylation. The MLC phosphorylation status in human cardiac disease is under investigation. In contrast to RLC, the phosphorylation pattern of ELC in human heart disease is not well understood. Here, 2-dimensional (2D) gel electrophoresis followed by ELC specific immunoblot (IB) was performed to detect the ELC phosphorylation pattern of human left ventricular tissue. Cardiac proteins were separated by isoelectric point (pI) and molecular weight (MW) (vELC: MW 21,932 kDa, pI 5.03; vRLC: MW 19 kDa, pI 4.89). Next, we performed mass-spectrometry analysis after cutting the spot-regions in question from silver-stained 2D gels of proteins from the human tissue. The aim was 1) to validate the detection of the MLC migration pattern seen in 2D IB by mass spectrometry, 2) to distinguish between atrial and ventricular MLC isoforms as these two forms might converge on 2D gel (vELC: MW 21.932 kDa, pI 5.03; aELC: 21.550 kDa, pI 4.98) and 3) to detect phosphorylated amio acid residues of ELC and RLC in human ventricular tissue.
Project description:Metabolic dysfunction is a primary feature of Werner syndrome (WS), a human premature aging disease caused by mutations in the gene encoding the Werner (WRN) DNA helicase. WS patients exhibit severe metabolic phenotypes, but the underlying mechanisms are not understood, and whether the metabolic deficit can be targeted for therapeutic intervention has not been determined. Here we report impaired mitophagy and depletion of NAD+, a fundamental ubiquitous molecule, in WS patient samples and WS invertebrate models. WRN regulates transcription of a key NAD+ biosynthetic enzyme nicotinamide nucleotide adenylyltransferase 1 (NMNAT1). NAD+ repletion restores NAD+ metabolic profiles and improves mitochondrial quality through DCT-1 and ULK-1-dependent mitophagy. At the organismal level, NAD+ repletion remarkably extends lifespan and delays accelerated aging, including stem cell dysfunction, in C. elegans and Drosophila melanogaster models of WS. Our findings suggest that accelerated aging in WRN syndrome is mediated by impaired mitochondrial function and mitophagy, and that bolstering cellular NAD+ levels counteracts WS phenotypes.
Project description:The essential myosin light chain (ELC) is part of the cardiac myosin motor regulating the contractility of the heart. Recently, we have demonstrated ELC phosphorylation to be responsible not only for basal maintenance of cardiac contractility, but also for the adaptation of sarcomere function to increased demands in adult zebrafish (Scheid et al. Cardiovascular Research, 2016). However, the direct interacting kinases, phosphatases as well as the signaling pathway mediating the ELC phosphorylation are mostly unknown. Here, we aim to identify ELC interacting proteins in zebrafish heart tissue using an ELC-specific precipitation assay followed by mass spectrometry. In brief, ELC specific co-immunoprecipitation was performed by using the Dynabeads Co-immunoprecipitation Kit (Invitrogen, #14321D). Custom-specific polyclonal ELC antibodies (Eurogentec, peptide for immunization: NH2-CAPVPETPKEPEVDLK-CONH2) against zebrafish ELC were covalently coupled to DynabeadsTM M-270 Epoxy (Invitrogen, #14302D) and whole zebrafish heart protein was used as input. All steps were carried out according to the manufacturer's instructions.
Project description:NIMA-related kinase 9 (NEK9) was identified to interact with the essential myosin light chain (ELC). To validate these findings ascorbate peroxidase (APEX) catalyzed proximity labeling (Hung et al. Nat Protoc. 2016) was performed. To do so, human ELC fused to APEX was overexpressed in HEK293 cells. Empty pcDNA3-APEX2-NES vector serves as negative control. Half of each sample was treated with H2O2, which catalyzes protein labeling in close proximity of ELC. After streptavidin pull down of labeled proteins, the samples were analyzed by mass spectrometry.