Project description:Many animal species employ a chromosome-based mechanism of sex determination, which has led to coordinate evolution of dosage compensation systems. Dosage compensation not only corrects the imbalance in the number of X-chromosomes between the sexes, but is also hypothesized to correct dosage imbalance within cells due to mono-allelic X expression and bi-allelic autosomal expression, by upregulating X-linked genes (termed M-CM-"M-BM-^@M-BM-^XOhnoM-CM-"M-BM-^@M-BM-^Ys hypothesisM-CM-"M-BM-^@M-BM-^Y). Although this hypothesis is well supported by expression analyses of individual X-linked genes and by array-based transcriptome analyses, a recent study claimed that no such X upregulation exists in mammals and C. elegans based on RNA-sequencing and proteomics analyses. We provide RNA-seq RNA-seq analysis of mouse female PGK12.1 ES cells with two active X chromosomes and confirmed that the X chromosome is upregulated, consistent with the previous microarray study. Examination of expression of X-linked and autosomal genes in mouse female ES cells with two active X chromosomes.
Project description:Many proteins undergo glycosylation in the endoplasmic reticulum (ER) and the Golgi apparatus. Altered glycosylation can manifest in serious, sometimes fatal malfunctions. We recently showed that mutations in the cytoplasmic protein GDP-mannose pyrophosphorylase A (GMPPA) cause a syndrome characterized by alacrima, achalasia, mental retardation and myopathic alterations. GMPPA acts as feedback inhibitor of GDP-mannose pyrophosphorylase B (GMPPB), which provides GDP-mannose as a substrate for protein glycosylation. Loss of GMPPA enhances incorporation of mannose into glycochains of various proteins, including α-dystroglycan (α-DG), a protein that links the extracellular matrix with the cytoskeleton. Here, we show that loss of GMPPA affects the functionality of the Golgi apparatus using different approaches. First, we show a fragmentation of the Golgi apparatus in skeletal muscle fibers and in neurons of GMPPA KO mice. A major reorganization is also evident by mass spectrometry of KO tissues with a regulation of several ER- and Golgi-resident proteins. We further show that loss of GMPPA increases the retention of α-DG in the ER. Notably, mannose supplementation can mimic changes in ER and Golgi structure and function in WT cells. In summary, our data underline the importance of a balanced mannose homeostasis for structure and function of the secretory pathway.
Project description:Lysine crotonylation has attracted widespread attention in recent years. However, little is known about bacterial crotonylation, particularly crotonyltransferase and decrotonylase, and how it affects antibiotic resistance. Our study demonstrated the ubiquitous presence of crotonylation in E.coli and its connection to bacterial resistance to polymyxin. We first identified the crotonyltransferase YjgM and its regulatory pathways in E. coli with a focus on crotonylation. Further studies suggested that YjgM upregulates the crotonylation of the substrate protein PmrA, which is crucial for controlling E. coli's resistance to polymycin, thereby boosting PmrA's affinity for binding to the promoter of eptA, which in turn promotes EptA expression and confers polymyxin resistance in E. coli. Additionally, we discovered that PmrA's crucial crotonylation site and functional site is Lys 164. These significant discoveries highlight the role of crotonylation in bacterial drug resistance and offer a fresh perspective for creating antibacterial medicines.
Project description:we first expressed AAa and AAg in a eukaryotic system to investigate whether the proteins could be sulfated in vivo. For this purpose we used insect cells as a model of the Anopheles mosquito. Specifically, codon-optimized sequences encoding AAa and AAg were designed as N-terminal fusions with the honeybee mellitin signal sequence in order to direct the recombinant proteins to the secretion pathway and were expressed in Trichoplusia ni insect cells. Following expression, the cell medium containing the secreted proteins was analyzed by nanoliquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS).
Project description:We further expanded on previous optimizations to isolate the plasma peptidome and compared various methods to maximize identifications with speed and ease. Previous studies have reported loss of polypeptides binding to high abundant proteins during depletion strategies. We hypothesized that rapid chaotropic denaturation of plasma with urea under reducing conditions would liberate non-covalently bound peptides to improve recovery during protein depletion. We also compared depletion strategies to isolate the peptidome including protein precipitation and removal with either TCA, acetone or acetonitrile (AcN). Following centrifugation of precipitated proteins, the supernatant containing peptides was collected. For acetone and AcN precipitations, an additional vacuum centrifugation step was required followed by resuspension of peptides in aqueous buffer. Our comparison of peptidome isolation also included removal of proteins with size-exclusion 10 kDa MWCO filters. All peptide isolations were acidified to 0.1% TFA, adjusted to 5% acetonitrile and desalted with HLB-SPE. Peptides were analysed by single-shot nanoUHPLC-MS/MS employing both HCD and EThcD and quantified by LFQ
Project description:We previously reported that gut microbiota induce de novo DNA methylation of the TLR4 gene in intestinal epithelial cells. Since DNA methyltransferase (DNMT) 3 mediates the transfer of methyl groups to any gene undergoing de novo methylation, the question of how gut microbiota induce the recruitment of DNMT3 to specific genes, including TLR4, remains to be addressed. In this study, we tried to identify an adaptor molecule that recruits DNMT3b to the TLR4 gene by comparing expression of DNMT3-interacting proteins between IEC lines with hypermethylated and hypomethylated TLR4 gene.
Project description:Recent technological advances have made it possible to detect circulating breast cancer cells as precursors of distant metastasis and as prognosis marker in nonmetastatic breast cancer patients. Association of circulating tumor cells (CTCs) with molecular alteration in the primary tumor is not widely explored. We reported differential profile of altered genome, copy number alteration and copy-neutral loss of heterogeneity in 14 primary tumors when comparing patients with CTCs+ versus CTCs- using single-nucleotide polymorphism array. The most prevalent copy number alteration in CTCs+ patients was at 8q and particularly at the cytoband 8q24 (MYC loci). As the role of MYC in the process of tumor cell invasion and migration is controversial, we further validated in a larger series of patients whether altered MYC (amplification or gained) in primary tumors was correlated with the presence of CTCs in peripheral blood (as a surrogate of micrometastais). No correlation between MYC alteration and presence of CTCs was observed, providing clinical support to the recent data that MYC suppresses cancer metastasis or at least suggesting that MYC alteration could be contributory but insufficient for the generation of CTCs. This molecular association needs to be further characterized in preclinical model and especially clinically. We analyzed CN and LOH of CTC+ and CTC-
Project description:Here, we used APEX2 proximity labelling technique coupled with mass spectrometry to define Cveolin-1 interactome at steady state and after hypo-osmotic shock. We created a stable cell line expressing Caveolin1-APEX2-EGFP in human retinal pigmented epithelial cells (RPE-1). Hypo osmotic shock was performed accordingly to Sinha et al, 2011. Briefly, cells were incubated in 30mOsm medium for 5 minutes (Hypo condition). To analyse the ability to recover, after the osmotic shock cells were allowed to recover in isotonic medium for 30 minutes (REC condition). Cells grown in iso-tonic medium were used as normal non treated condition (NT). RPE-1 cells expressing APEX2-EGFP in the cytoplasm (NES-APEX2-EGFP) was used as control to exclude highly abundant cytosolic proteins.
Project description:The purpose of this study is to find the differently expressed genes in Aspergillus cristatus at different osmotic pressure. Examination of 3 transcriptomes in different osmotic pressure to find their different gene expression style.