Project description:Protein O-mannosyltransferases (PMTs) are conserved endoplasmic reticulum membrane embedded enzymes responsible for the transfer of mannose from dolichol phosphate-mannose (Dol-P-Man) to serine/threonine-rich protein substrates or unfolded proteins. PMTs from three subfamilies form obligate dimers with different substrate specificities, and require the concerted action of their transmembrane domains (TMDs) and a luminal MIR domain for catalysis. Here, we present structures, native mass spectrometry and structure-based mutagenesis of the Chaetomium thermophilum and Saccharomyces cerevisiae Pmt4 homodimers. The core fold of the TMDs and MIR domain is conserved with the Pmt1-Pmt2 heterodimer, indicating a shared catalytic mechanism. Distinct to Pmt4, the MIR domain interacts in cis with the TMDs of the same subunit and has a beta-hairpin insertion required for O-mannosylation of substrates. We further identify a cytosolic binding site for substrate Dol33 P-Man within the Pmt4 TMDs, which is conserved amongst PMTs and important for in vivo activity. Thus, we provide a framework to understand the substrate specificity and regulation of the Pmt4 homodimer.
Project description:The goal of this study was to compare the transcriptome between wild type strain of Listeria monocytogenes and delete nmlR mutant strain of L. monocytogenes using NGS. Method: Duplicate samples of rRNA depleted RNA from wild type and mutants were used to study transcriptomes by ion torrent platform. Transcriptomes of wild type and nmlR mutant were compared by EDGE-pro program. Result: Differential expression by EDGE-pro showed 74 genes with differential expressions between wild type and nmlR null mutant (46 genes were negatively regluated and 28 genes were positively regulated by NmlR). rRNA-depleted RNA samples from stationary phase wilde type and nmlR null mutant cultures were used to compare transcriptomes. Some affected genes from RNAseq result were selected for confirmation by quantitative reverse transcriptase PCR.
Project description:Human colorectal cancer organoids were injected intrasplenically to form liver metastasis in mice were treated with the TROP2-targeting antibody-drug conjugate Sacituzumab Govitecan (SG) or non-targeting control IgG1-SN-38 (ADC). Tumors were harvested at different time points: 0 h, 6 h, 24 h, 48 h, 72 h and 120 h} and subjected to single-cell RNA-seq using sample hashtag multiplexing.