Project description:Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report the first structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this previously uncharacterized complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains.
Project description:Condensation (C) domains are the key component linking different monomers together, typically forming peptide bonds and occasionally ester bonds, during the nonribosomal peptide synthetase (NRPS). While A domains have been well characterised due to their role in selectivity of the monomers and functioning as a gate keeper in the NRPS biosynthesis, C domains have been a subject of debate as they have not demonstrated signs of “A-domain like” side chain selectivity of its acceptor side. Here, we report our biochemical and structural characterisation of the selectivity of the fuscachelin C3-domain showing that it is not broadly flexible for monomers at the acceptor site, suggesting the need to consider C-domain mutation regarding future NRPS engineering.
Project description:Top-Down proteomics pilot experiment of unfractionated Bovine Heart Mitochondria (BHM) using ultra high resolution Q-ToF tandem mass spectrometry (maXis 4G ETD, Bruker Daltonics).
Project description:In the present study, the whole cell protein extracts of B. abortus and B. melitensis were separated using SDS-PAGE and western blotting was carried out with the antiserum from naturally infected host animals (cow, buffalo, goat and sheep). The proteins bands that matched with western blot signals were excised, trypsin digested and subjected to MALDI identification.
Project description:Glycoproteins play important roles in numerous physiological processes and are often implicated in disease. Analysis of site-specific protein glycobiology through glycoproteomics is evolving rapidly in recent years thanks to hardware and software innovations. Introduction of Parallel Accumulation Serial Fragmentation (PASEF) on hybrid trapped ion mobility time-of-flight instruments combined deep proteome sequencing with separation of (near-)isobaric precursor ions or converging isotope envelopes through ion mobility separation. Despite these advantages, the use of PASEF in integrated glycoproteomics workflows to comprehensively capture the glycoproteome has received little attention. To address this gap, we have developed an integrated methodology using the timsTOF Pro2 to enhance N-glycopeptide identifications in complex mixtures. We explored its potential by systematically evaluating the impact of ion optics tuning, collision energies, mobility isolation width, and the use of dopant-enriched nitrogen gas (DEN) on glycopeptide identification rates. This comprehensive approach showed a marked increase in unique glycopeptide identification rates compared to standard proteomics settings while evaluating key parameters on a large set of glycopeptides. With short liquid chromatography gradients of 30 minutes, we increased the number of unique N-glycopeptide identifications in full human plasma glycopeptide samples from around 100 identifications under standard proteomics condition to over 1500 with our optimized glycoproteomics approach, highlighting the need for tailored solutions.
Project description:RAD51 protein is an evolutionarily conserved recombinase that plays a central role in homologous recombination (HR) and DNA double strand break (DSB) repair. RAD51 inactivation by small molecules has been proposed as a strategy to impair the BRCA2/RAD51 binding and, ultimately, the HR pathway, with the aim to make cancer cells more sensitive to PARP inhibitors (PARPi). This strategy, which mimics a synthetic lethality (SL) approach, has been successfully assayed in vitro by using myr-BRC4, a peptide derived from the fourth BRC repeat of BRCA2, being the strongest reported natural RAD51 binder. The present study applies a method to obtain a proteomic fingerprint for RAD51 inhibition by the myr-BRC4 peptide (designed for a more efficient cell entry) using a mass spectroscopy (MS) proteomic approach. We performed a comparative proteomic profiling of the myr-BRC4 treated vs. untreated BxPC-3 pancreatic cancer cells and evaluated the differential expression of proteins. Among the identified proteomic hits, we focused our attention on proteins shared by both the RAD51 and the BRCA2 interactomes, and on those whose reduction showed high statistical significance. Three downregulated proteins were identified (FANCI, FANCD2, and RPA3) and protein downregulation was confirmed through immunoblotting analysis, validating the MS approach. Our results suggest that, being a direct consequence of RAD51 inhibition, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring HR impairment.
Project description:One of the main Ca2+ decoders in plants are calcium-dependent protein kinases (CDPKs). Among them, AtCPK1 is one of the best studied as a positive regulator in the plant response to biotic and abiotic stress. Inactivation of the autoinhibitory domain of AtCPK1 (the mutated form of AtCPK1-Ca) provides constitutive activity of the kinase via imitation of the stress-induced Ca2+ increase. For the first time in the present study, we performed a proteomic analysis of the overexpressed mutant AtCPK1-Ca form of Arabidopsis thaliana in transformed Vitis amurensis calli. In our previous studies, we have shown that overexpression of this mutated form led to dramatically enhanced specialised metabolism in plant cell cultures, including resveratrol in V. amurensis.
Project description:Summary: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac disorder. It is classified as the second most common cause of unexpected sudden death by cardiac arrest in the young. ARVC has devastating psychosocial consequences, especially as many patients are young adults, and current therapy is limited to extreme exercise restriction and defibrillator implantation. More than 40% of the reported genetic variants linked to ARVC reside in a gene called PKP2 , which encodes for the plakophilin-2 (PKP2) protein. The pathogenic mechanisms linking this protein to ARVC remain to be elucidated, which is the focus of this project. I aim to identify exactly which proteins in the heart are dysregulated and to identify why exercise is detrimental for carriers of PKP2-deficient hearts. Our approach is based on state-of-the-art mass spectrometry (MS) technologies that allow us to measure all proteins in the heart simultaneously. We used both human heart biopsy material as well as a murine disease model I anticipate to elucidate novel roles of PKP2 that are of utmost importance for understanding ARVC pathogenesis.
Project description:Glial cells (microglia and astrocytes) have recently became appreciated as an important target for antidepressant drugs. Here we report on the results of comprehensive proteomic analysis of the alteration in protein profile of rat primary mixed glial culture exposed to imipramine. Two-dimensional differential in gel electrophoresis method allowed to identify 62 proteins regulated by imipramine hydrochloride. Functional analysis revealed the impact of imipramine on the level of proteins involved in oxidative stress. Imipramine upregulated proteins related to glycolysis but downregulated many mitochondrial proteins also enzymes of oxidative phosphorylation. Moreover, imipramine influenced the proteins engaged in phagocytosis and cell migration. Alteration in the level of large number of structural and plasma membrane associated proteins evidenced a widespread cytoskeleton and membrane rearrangement. Imipramine triggered decrease of mitochondrial membrane potential, impairment of protein synthesis and downregulation of chaperon proteins, what could be related to increased apoptosis. Many imipramine regulated proteins, among them chaperons, cathepsins and annexins are evidenced to be engaged in immunity response. Overall these experimental findings suggest that in response to imipramine, glial cells (mainly microglia) undergo a transition toward more quiescent, metabolically less demanding phenotype.