Project description:Angiotensin converting enzyme (ACE) is known for converting inactive angiotensin I into the potent vasoconstrictor angiotensin II, playing a critical role in blood pressure regulation. However, there is evidence that the dicarboxypeptidase activity of ACE is also essential for other physiological processes likely through processing of various peptide substrates. This study used mass spectrometry for the comprehensive detection and identification of natural substrates and products of ACE within the mouse plasma peptidome. The plasma peptidome of ACE KO mice was obtained through a multi-step purification process which included organic TCA precipitation as well as size exclusion and reversed phase chromatography. The obtained complex mixture of endogenous peptides was then subjected to in vitro cleavage by ACE. ACE-treated and untreated samples were then analyzed by LC/MS on an Orbitrap mass spectrometer, followed by alignment of MS1 data by Progenesis QI software. The MS1 signals that gained or lost intensity after treatment with ACE, were considered as possible products and substrates of ACE, respectively, and were selected for a targeted MS/MS analysis, and subsequently identified with PEAKS 8.5 and Proteome Discoverer software. Results. Close to 250 natural peptides were identified as possible substrates and products of ACE, demonstrating the high promiscuity of the enzyme. The use of internal standards as well as detection of some expected endogenous peptides, such as angiotensin II and bradykinin, supported the validity of the approach. Some of the newly identified substrates of ACE are known for their biological activities. For example, a fragment of complement C3, the 17-amino acid peptide C3f, exhibits spasminogenic activity and was processed by ACE. ACE cleavage of select peptides was further confirmed in vitro. Also, concentrations of ACE substrates in plasma from mice with variant genetic ACE domain backgrounds were determined by LC/MS using multiple reaction monitoring on a triple quadrupole mass spectrometer. The in vivo results were consistent with the in vitro results, in the sense that higher levels of the ACE substrates were observed when the respective processing domain was knocked out. The use of transgenic mice as well as ACE with single active domain allowed clarifying the ACE domain selectivity towards individual peptide substrates. This study resulted in creation of a library of substrates and products of ACE that can be further tested for their biological function and can help to elucidate the link between ACE and the numerous physiological effects attributed to its activity.
Project description:ABC (“ATP-Binding Cassette”) exporters of the type IV subfamily gather transporters involved in the efflux of many compounds and notably those capable to confer multidrug resistance like the mammalian P-glycoprotein or the bacterial transporter BmrA. They function according to an alternating access mechanism between inward-facing (IF) and outward-facing (OF) conformations, but the extent of physical separation between the two nucleotide-binding domains (NBDs) in different states is still unsettled. Small Angle Neutron Scattering (SANS) and hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) were used to highlight different conformational states of BmrA during its catalytic cycle. In particular, mutation of the invariant Lysine residue of the Walker-A motif (K380A) captures BmrA in an ATP-bound IF conformation prior to NBD closure. While in the transition-like state induced by vanadate, wild-type BmrA is mainly in an OF conformation, it and populates only IF conformations only in the presence of ADP/Mg. Importantly, in this post-hydrolytic step, distances between the two NBDs of BmrA appears to be more separated than in the apo state, but they remains shorter than in the widest opening found in the related MsbA transporter. Overall, our results highlight the main steps of the catalytic cycle of a homodimeric bacterial multidrug transporter and underline structural and functional commonalities as well as oddities among the type IV subfamily of ABC exporters.
Project description:The molecular mechanism controlling the zygotic genome activation (ZGA) in mammals remains poorly understood. The 2C-like cells spontaneously emerging from cultures of mouse embryonic stem cells (ESCs) share some key transcriptional and epigenetic programs with 2-cell stage embryos. By studying the transition of ESCs into 2C-like cells, we identified Dppa2/4 as important regulators controlling zygotic transcriptional program through directly upregulating the expression of Dux. In addition, we found that DPPA2 protein is sumoylated and its activity is negatively regulated by Sumo E3 ligase PIAS4. PIAS4 is downregulated during zygotic genome activation process and during transitioning of ESCs into 2C-like cells. Depleting Pias4 or overexpressing Dppa2/4 is sufficient to upregulateactivate 2C-like transcriptional program, while depleting Dppa2/4 or forced expression of PIAS4 or Sumo2-Dppa2 inhibits 2C-like transcriptional program. Furthermore, ectopic expression of Pias4 or Sumo2-Dppa2 impairs early mouse embryo development. In summary, our study identifies key molecular rivals consisting of transcription factors and a Sumo2 E3 ligase that regulate the transition of ESCs into 2C-like cells and zygotic transcriptional program upstream of Dux.
Project description:HeLa cells were synchronized with a double thymidine block procedure. Briefly, the exponentially growing HeLa cells were maintained with 2 mM thymidine for 18 h, followed by a release of 9 h in fresh medium, and then cells were re-cultured in 2 mM thymidine for additional 15 h. After a release of 7.5 h in fresh medium, DMSO or G6PD inhibitor was added to the medium. 1 h later, the cells entered into M phase. Mitotic cells were harvested by mitotic shake-off. Then, the samples were subjected to LC-MS/MS analysis. Finally, a Kinase-Substrate Enrichment was performed, which could infer the changes of upstream kinase activity upon the treatment of G6PD inhibitors.
Project description:Despite the major progress made into spliceosome mechanisms through CryoEM, a major obstacle of this approach is its inability to map the positioning of helicases on the RNA substrate. Here we present a method ‘psiCLIP’ which probes protein-RNA interactions in specific spliceosomal states. The method is based on iCLIP, but is different to previous iCLIP studies in that it is performed on step-specific spliceosomes that are prepared from in vitro splicing extracts, similarly to those prepared for CryoEM. We applied psiCLIP to SmB (C complex), Prp16 (C complex) and Prp22 (C* and P complexes), using pre-mRNA substrates based on UBC4 and ACT1. We also used ATPase deficient dominant negative (dn) mutants of Prp16 and Prp22 to determine the contribution of ATPase activity to binding patterns.
Project description:Phosphorylation, as one of the most important and well-studied post-translational modifications, is tightly associated with protein activity and protein functional regulation. Here in this study, we generated a global protein phosphorylation atlas within the pathological site of human RCT patients by using Tandem Mass Tag (TMT) labeling combining with mass spectrometry. GO enrichment analyses and KEGG pathway analyses were performed. At last, a weighted kinase-site phosphorylation network was built to identify potentially core kinase.
Project description:Mycobacterial Ser/Thr kinases play a critical role in bacterial physiology and pathogenesis. Linking kinases to the substrates they phosphorylate in vivo, thereby elucidating their exact functions, is still a challenge. The aim of this work was to associate protein phosphorylation in mycobacteria with important subsequent macro cellular events by identifying the physiological substrates of PknG in Mycobacterium bovis BCG. The study compared the phosphoproteome dynamics during the batch growth of M. bovis BGC versus the respective PknG knock-out mutant (ΔPknG-BCG) strains.
Project description:Exposure to mild early-life stresses can slow down the aging process, in which protein phosphorylation might be an essential regulator. Currently, the understanding of phosphorylation-based regulatory networks under mild early-life stress remains elusive. Herein, we systematically analyzed the phosphoproteomes of C. elegans, which were treated with three kinds of mild temperature (15°C, 20°C, and 25°C) from two different short-term groups (10 min and 60 min). By utilizing iTRAQ-based quantitative phosphoproteomic approach, a total of 18187 phosphorylation sites from 3330 proteins were identified. Volcano plots illustrated that the phosphorylation abundance of 374 proteins (15°C) and 347 proteins (25°C), were significantly changed, respectively. Gene ontology, KEGG pathway and protein-protein interaction network analysis revealed that these phosphoproteins were mainly involved in metabolism, translation, development, and lifespan determination. Motif analysis of kinase substrates suggested that MAPK, CK and CAMK were most likely involved in the adaption processes. Moreover, 16 and 14 aging-regulated proteins underwent phosphorylation modifications under the mild stress of 15°C and 25°C, respectively, indicating these proteins might be important for maintaining long-term health. Further experiments of lifespan confirmed that the candidate phosphoproteins, e.g.; EGL-27, XNP-1, and GTBP-1 could regulate lifespan at 15°C, 20°C, and 25°C, and showed increased tolerance to heat and oxidative stresses. In summary, our findings provided a wealth of datasets to better understand the phosphorylation mechanism of mild early-life stresses in C. elegans.
Project description:Iso-Seq (PacBio) sequencing was performed to generate a reference library of H. perforatum. We generated genome-wide transcriptome data from in vitro cell suspensions and shoot cultures of H. perforatum.