10 research outputs found
Über eine neue Methode zur Bestimmung der drei Methylamine und des Ammoniaks nebeneinander
Analysis of the interactome of <i>Schistosoma mansoni</i> histone deacetylase 8
<div><p>Background</p><p>Histone deacetylase 8 from <i>Schistosoma mansoni</i> (<i>Sm</i>HDAC8) is essential to parasite growth and development within the mammalian host and is under investigation as a target for the development of selective inhibitors as novel schistosomicidal drugs. Although some protein substrates and protein partners of human HDAC8 have been characterized, notably indicating a role in the function of the cohesin complex, nothing is known of the partners and biological function of <i>Sm</i>HDAC8.</p><p>Methodology/Principal findings</p><p>We therefore employed two strategies to characterize the <i>Sm</i>HDAC8 interactome. We first used <i>Sm</i>HDAC8 as a bait protein in yeast two-hybrid (Y2H) screening of an <i>S</i>. <i>mansoni</i> cDNA library. This allowed the identification of 49 different sequences encoding proteins. We next performed co-immunoprecipitation (Co-IP) experiments on parasite extracts with an anti-<i>Sm</i>HDAC8 antibody. Mass spectrometry (MS) analysis allowed the identification of 160 different proteins.</p><p>Conclusions/Significance</p><p><i>Sm</i>HDAC8 partners are involved in about 40 different processes, included expected functions such as the cohesin complex, cytoskeleton organization, transcriptional and translational regulation, metabolism, DNA repair, the cell cycle, protein dephosphorylation, proteolysis, protein transport, but also some proteasome and ribosome components were detected. Our results show that <i>Sm</i>HDAC8 is a versatile deacetylase, potentially involved in both cytosolic and nuclear processes.</p></div
Raman microspectroscopy reveals unsaturation heterogeneity at the lipid droplet level and validates an in vitro model of bone marrow adipocyte subtypes.
Bone marrow adipocytes (BMAds) constitute the most abundant stromal component of adult human bone marrow. Two subtypes of BMAds have been described, the more labile regulated adipocytes (rBMAds) and the more stable constitutive adipocytes (cBMAds), which develop earlier in life and are more resilient to environmental and metabolic disruptions. In vivo, rBMAds are enriched in saturated fatty acids, contain smaller lipid droplets (LDs) and more readily provide hematopoietic support than their cBMAd counterparts. Mouse models have been used for BMAds research, but isolation of primary BMAds presents many challenges, and thus in vitro models remain the current standard to study nuances of adipocyte differentiation. No in vitro model has yet been described for the study of rBMAds/cBMAds. Here, we present an in vitro model of BM adipogenesis with differential rBMAd and cBMAd-like characteristics. We used OP9 BM stromal cells derived from a (C57BL/6xC3H)F2-op/op mouse, which have been extensively characterized as feeder layer for hematopoiesis research. We observed similar canonical adipogenesis transcriptional signatures for spontaneously-differentiated (sOP9) and induced (iOP9) cultures, while fatty acid composition and desaturase expression of Scd1 and Fads2 differed at the population level. To resolve differences at the single adipocyte level we tested Raman microspectroscopy and show it constitutes a high-resolution method for studying adipogenesis in vitro in a label-free manner, with resolution to individual LDs. We found sOP9 adipocytes have lower unsaturation ratios, smaller LDs and higher hematopoietic support than iOP9 adipocytes, thus functionally resembling rBMAds, while iOP9 more closely resembled cBMAds. Validation in human primary samples confirmed a higher unsaturation ratio for lipids extracted from stable cBMAd-rich sites (femoral head upon hip-replacement surgery) versus labile rBMAds (iliac crest after chemotherapy). As a result, the 16:1/16:0 fatty acid unsaturation ratio, which was already shown to discriminate BMAd subtypes in rabbit and rat marrow, was validated to discriminate cBMAds from rBMAd in both the OP9 model in vitro system and in human samples. We expect our model will be useful for cBMAd and rBMAd studies, particularly where isolation of primary BMAds is a limiting step
Confirmation of interactions by candidate-specific Y2H experiments.
<p>In order to confirm the screening results, the isolated clones CtBP, MBD2, tensin and actin-1 (cloned into pGADT7 vector) transformed in the AH109 strain were mated with the bait construct <i>Sm</i>HDAC8 pGBKT7 transformed in the Y187 strain. After incubation, diploid yeasts were plated on selective medium lacking leucine and tryptophan (left panel) and then on another high stringency selective medium lacking adenine, histidine, leucine and tryptophan (right panel) showing, as expected, that <i>Sm</i>HDAC8 interacts with the clones tested.</p
Venn diagram presenting the proteins identified by Y2H and Co-IP/MS as <i>Sm</i>HDAC8 interactors.
<p>Forty-nine and 160 different proteins were identified by Y2H and Co-IP/MS respectively. Four proteins are common between Y2H and Co-IP/MS: the Proliferation-associated protein 2G4, 38kDa (PA2G4, n°G4LXR6), Cathepsin-B1 (SmCB1, n°Q8MNY2), putative NADH-ubiquinone oxidoreductase (n°G4VK53) and microsomal glutathione <i>S</i>-transferase 3 (GST-3, n°G4VH65).</p
Biological processes involving the proteins identified by Y2H and Co-IP/MS as <i>Sm</i>HDAC8 interactors.
<p>Histogram (left for Y2H and right for Co-IP/MS) showing the biological processes in which the identified protein partners of <i>Sm</i>HDAC8 are involved. The processes were defined using the Blast2GO software. Bars represent the protein percentage belonging to one biological process. Red and black bars highlight common and specific processes between Y2H and Co-IP/MS, respectively.</p