Project description:Microbacterium sediminis YLB-01, a marine microbacterium tolerant to pressure and low temperature, was subjected to high pressure (accompanied by low temperature) and proteomic analysis was carried out to explore the mechanism of its adaptation to the high-pressure environment of the deep sea.
Project description:Resistance to inhibitors of cholinesterases (Ric-8 proteins) are involved in modulating G-protein function but little is known of their potential physiological importance in the heart. In the present study, we assessed the role of resistance to inhibitors of cholinesterase 8b (Ric-8b) in determining cardiac contractile function using a mouse model. Deletion of Ric-8b in cardiac tissue led to severely reduced contractility as measured using echocardiography days after administration of tamoxifen. Histological analysis of the ventricular tissue showed highly variable myocyte size, prominent fibrosis and an increase in cellular apoptosis. RNA sequencing revealed transcriptional remodelling in response to cardiac Ric-8b deletion involving the extracellular matrix and inflammation. Phosphoproteomic analysis revealed substantial downregulation of phosphopeptides related to myosin light chain 2. At the cellular level, the deletion of Ric-8b led to loss of activation of the L-type calcium channel through the β-adrenergic pathways. Using fluorescence resonance energy transfer-based assays in heterologous expression systems we showed Ric-8b protein selectively interacts with the stimulatory G-protein, Gαs. We explored if deletion of Gnas (the gene encoding Gαs) in cardiac tissue using a similar approach in the mouse led to an equivalent phenotype. The conditional deletion of the Gαs gene in the ventricle led to comparable effects on contractile function and cardiac histology. We conclude that Ric-8b is essential to preserve cardiac contractile function likely through an interaction with the stimulatory G-protein and downstream phosphorylation of myosin light chain 2.
Project description:Genomic response of C. elegans after infection with Microbacterium nematophilum.<br><br>The interaction between the nematode Caenorhabditis elegans and a Gram-positive bacterial pathogen, Microbacterium nematophilum, provides a model for an innate immune response in nematodes. This pathogen adheres to the rectal and post-anal cuticle of the worm, causing slowed growth, constipation, and a defensive swelling response of rectal hypodermal cells. To explore the genomic responses that the worm activates after pathogenic attack we used microarray analysis of transcriptional changes induced after 6 hr infection, comparing virulent with avirulent infection.
Project description:Argonaute proteins are essential players in RNA silencing pathways, and their N-terminal extensions, particularly in the PIWI clade, often harbor conserved sequences like RG motifs. Despite their prevalence in Argonaute proteins, the role of these motifs remains poorly understood. In this study, we focus on the RG motifs within the N-terminal region of Caenorhabditis elegans PRG-1, a PIWI clade Argonaute. Using sequence alignment across Caenorhabditis species, we identify three conserved RG motifs that are methylated, as confirmed by mass spectrometry. The region surrounding these motifs is intrinsically disordered, as predicted by disorder algorithms and structural modeling. While the RG motifs are critical for fertility, germline morphology, and piRNA silencing, they are not required for PRG-1 expression, localization, or piRNA loading. Notably, mutation of the RG motifs results in defects in downstream small RNA production, specifically the depletion of mutator class siRNAs, without affecting piRNA biogenesis. These findings suggest that the RG motifs of PRG-1 play a crucial role in linking piRNA-mediated silencing to siRNA production, and that their function is critical for fertility and germline maintenance in C. elegans. Despite these defects, the phenotypic severity in the RG mutant is milder than in a PRG-1 null mutant, highlighting the complexity of PRG-1 function and its post-translational modifications.
Project description:Analysis of recombinant mouse Ric-8B protein phosphorylation. Ric-8B was purified from insect cells and E.coli and both treated with protein kinase CK2 and re-purified prior to LC-MS/MS. Another Ric-8B sample from insect cells was not treated with CK2 and also subjected to LC-MS/MS.