Project description:Mouse was sacrificed at postnatal day 46 by cervical dislocation. Retina were prepared seamlessly with suspension trap (S-TRAP) followed by high-pH reversed-phase peptide fractionation. Peptides fractions were identified with data-dependent acquisition by quadrupole time-of-flight mass spectrometry.
Project description:Aminoacyl-tRNA synthetases (aaRSs) catalyze the ligation of each amino acid to the 3’ hydroxyl group of the cognate tRNA and thereby establish the genetic code for protein synthesis. AaRSs form a complicated network through assembly into a multi-synthetase complex (MSC), which is comprised of nine aaRSs (ArgRS, AspRS, GlnRS, GluProRS, IleRS, LeuRS, LysRS, and MetRS) and three auxiliary proteins (aaRS-interacting multifunctional proteins, p43, p38, and p18 or AIMP1, 2, and 3) in vertebrates. IleRS is one of the least characterized aaRSs in the MSC. It is a class 1a aaRS and has an UNE-I domain at its C-terminal end that is formed by tandem ~90 residue repeats. Systematic depletion and yeast two-hybrid (Y2H) analyses suggest that IleRS binds to the WHEP domain of GluProRS through its UNE-I domain. However, crosslinking and mass spectrometry (XL-MS) analyses revealed that IleRS interacts with ArgRS, LeuRS, and MetRS in the MSC. While such difference may be due to the different dynamic states of the MSC under various cellular environments, it remains to be resolved how IleRS associates with other components of the MSC. Therefore, we performed mass spectrometry analysis to study IleRS interactome using cells expressing wild-type IleRS, C-terminal-deleted IleRS, and the IleRS UNE-I domain alone.
Project description:Non-alcoholic steatohepatitis (NASH) and type 2 diabetes are closely linked, yet the pathophysiological mechanisms underpinning this bidirectional relationship remain unresolved. Using proteomic approaches, we interrogated hepatocyte protein secretion in two models of murine NASH to reveal striking hepatokine remodelling that is associated with insulin resistance and maladaptive lipid metabolism.
Project description:PROteolysis TArgeting Chimeras (PROTACs) are bifunctional small molecules that can simultaneously recruit target protein and E3 ligase to form a ternary complex (target protein / PROTAC / E3 ligase), leading to target protein ubiquitination and degradation via the Ubiquitin-Proteasome System (UPS). PROTACs have gained increasing attention in recent years due to certain advantages over traditional therapeutic modalities enabling targeting of previously "undruggable" proteins. To better understand the mechanism of PROTAC-induced Target Protein Degradation (TPD), several computational approaches have recently been developed to study and predict ternary complex formation. However, mounting evidence suggests that ubiquitination can also be a rate-limiting step in PROTAC induced TPD. Here, we propose a structure-based computational approach to predict target protein ubiquitination induced by CRBN-based PROTACs,
Project description:We examine the changes in HEK293T overexpressing alpha-synucleinA53T-GFP when treated with kinase inhibitors (SB203580, GF109203X,SB202190, Enzastaurin, VX-745). Main findings is altered protein translation, with an increase in lysosomal proteins for all compounds save VX-745
Project description:C-mannosylation stabilizes proteins bearing a thrombospondin repeat (TSR) domain in metazoans. Here we show that Plasmodium falciparum expresses a DPY19 C-mannosyltransferase in the endoplasmic reticulum and that DPY19-deficiency abolishes C-glycosylation, destabilizes members of the TRAP adhesin family and inhibits transmission to mosquitoes. P. falciparum gametogenesis was imaged in its entirety in four dimensions using lattice light-sheet microscopy. This revealed defects in egress and exflagellation for DPY19 microgametes. While exflagellation was diminished, DPY19 microgametes still fertilized macrogametes, forming ookinetes but these were abrogated for mosquito infection. The gametogenesis defects corresponded with destabilization of MTRAP, which we show is C-mannosylated in P. falciparum, and the ookinete defect was concordant with defective CTRP secretion on the DPY19 background. Genetic complementation of DPY19 restored ookinete infectivity, sporozoite production and C-mannosylation activity. Therefore, tryptophan C-mannosylation by DPY19 in the early secretory pathway ensures TSR protein quality control at two lifecycle stages for successful transmission of the human malaria parasite.
Project description:Mass spectrometry-based proteomics has emerged as a powerful technique in the field of clinical diagnostics but requires a multistep process for sample preparation. The purpose of clinical mass spectrometry is to detect and quantify protein targets using well-established methods. Here, we found that the osmotic shock (OS) process allowed us to reduce the salt concentration and simultaneously enrich periplasmic proteins, specifically target proteins associated with antibiotic resistance in Gram-negative bacteria. Furthermore, we demonstrated that an optimized condition of the OS lysis allows the highest sensitivity in the MS analysis, and the OS lysate containing the proteins of interest (e.g., KPC-2) can be directly injected into multiple mass spectrometers without desalting process. Our study provides a detergent-free, straightforward method for periplasmic protein analysis using mass spectrometry.
Project description:Non-alcoholic steatohepatitis (NASH) and type 2 diabetes are closely linked, yet the pathophysiological mechanisms underpinning this bidirectional relationship remain unresolved. Using proteomic approaches, we interrogated hepatocyte protein secretion in two models of murine NASH to reveal striking hepatokine remodelling that is associated with insulin resistance and maladaptive lipid metabolism. We identify arylsulfatase A (ARSA) as a novel hepatokine that is upregulated in NASH and type 2 diabetes. This submission contains proteomic analysis of quadracep lipid rafts with or without overexpression of ARSA.