To improve proteome coverage and protein C-terminal identification, we characterized the Methanosarcina acetivorans thermophilic proteinase LysargiNase, which cleaves before lysine and arginine up to 55 °C. Unlike trypsin, LysargiNase-generated peptides had N-terminal lysine or arginine residues and fragmented with b ion-dominated spectra. This improved protein C terminal-peptide identification and several arginine-rich phosphosite assignments. Notably, cleavage also occurred at methylated or dimethylated lysine and arginine, facilitating detection of these epigenetic modifications.
Secreted and membrane tethered matrix metalloproteinases (MMPs) are key homeostatic proteases regulating the extracellular signaling and structural matrix environment of cells and tissues. For drug targeting of proteases, selectivity for individual molecules is highly desired and can be met by high yield active site specificity profiling. Using the high throughput Proteomic Identification of protease Cleavage Sites (PICS) method to simultaneously profile both the prime and non-prime sides of the cleavage sites of nine human MMPs, we identified more than 4300 cleavages from P6 to P6' in biologically diverse human peptide libraries. MMP specificity and kinetic efficiency were mainly guided by aliphatic and aromatic residues in P1' (with a ~32-93% preference for leucine depending on the MMP), and basic and small residues in P2' and P3', respectively. A wide differential preference for the hallmark P3 proline was found between MMPs ranging from 15 to 46%, yet when combined in the same peptide with the universally preferred P1' leucine, an unexpected negative cooperativity emerged. This was not observed in previous studies, probably due to the paucity of approaches that profile both the prime and non-prime sides together, and the masking of subsite cooperativity effects by global heat maps and iceLogos. These caveats make it critical to check for these biologically highly important effects by fixing all 20 amino acids one-by-one in the respective subsites and thorough assessing of the inferred specificity logo changes. Indeed an analysis of bona fide MEROPS physiological substrate cleavage data revealed that of the 37 natural substrates with either a P3-Pro or a P1'-Leu only 5 shared both features, confirming the PICS data. Upon probing with several new quenched-fluorescent peptides, rationally designed on our specificity data, the negative cooperativity was explained by reduced non-prime side flexibility constraining accommodation of the rigidifying P3 proline with leucine locked in S1'. Similar negative cooperativity between P3 proline and the novel preference for asparagine in P1 cements our conclusion that non-prime side flexibility greatly impacts MMP binding affinity and cleavage efficiency. Thus, unexpected sequence cooperativity consequences were revealed by PICS that uniquely encompasses both the non-prime and prime sides flanking the proteomic-pinpointed scissile bond.
The ability of pathogenesis-related proteins of family 10 to bind a broad spectrum of ligands is considered to play a key role for their physiological and pathological functions. In particular, Bet v 1, an archetypical allergen from birch pollen, is described as a highly promiscuous ligand acceptor. However, the detailed recognition mechanisms, including specificity factors discriminating binding properties of naturally occurring Bet v 1 variants, are poorly understood.Here, we report crystal structures of Bet v 1 variants in complex with an array of ligands at a resolution of up to 1.2 Å. Residue 30 within the hydrophobic pocket not only discriminates in high and low IgE binding Bet v 1 isoforms but also induces a drastic change in the binding mode of the model ligand deoxycholate. Ternary crystal structure complexes of Bet v 1 with several ligands together with the fluorogenic reporter 1-anilino-8-naphthalene sulfonate explain anomalous fluorescence binding curves obtained from 1-anilino-8-naphthalene sulfonate displacement assays. The structures reveal key interaction residues such as Tyr83 and rationalize both the binding specificity and promiscuity of the so-called hydrophobic pocket in Bet v 1.The intermolecular interactions of Bet v 1 reveal an unexpected complexity that will be indispensable to fully understand its roles within the physiological and allergenic context.
Collagen constitutes one third of the body protein in humans, reflecting its extraordinary role in health and disease. Of similar importance, therefore, are the idiosyncratic proteases that nature evolved for collagen remodeling. Intriguingly, the most efficient collagenases are those that enable clostridial bacteria to colonize their host tissues, but despite intense studies, the structural and mechanistic basis of these enzymes has remained elusive. Here we present the crystal structure of collagenase G from Clostridium histolyticum at 2.55 Å resolution. By combining the structural data with enzymatic and mutagenesis studies, we derive a conformational two-state model of bacterial collagenolysis, in which the recognition and unraveling of collagen microfibrils into triple helices as well as the unwinding of the latter go hand in hand with collagenase opening and closing.
During inflammation, local tissue responses are augmented by complement and acute phase proteins that exude into the tissue because of increased blood vessel permeability mediated by bradykinin, which is proteolytically released from kininogen. Moreover, proteases govern inflammatory responses by processing extracellular matrix proteins and soluble bioactive mediators; however, the consequences of their complex interplay in inflamed mammalian tissues are largely unknown. We quantified changes in the proteome and the nature of protein N-termini (the N-terminome) and the altered abundance of murine proteases and inhibitors during skin inflammation. Through analysis of the N-terminome by iTRAQ-terminal amine isotopic labeling of substrates (TAILS), we identified cotranslational and posttranslational αN-acetylation motifs, quantitative increases in protein abundance, and qualitative changes in the proteolytic signature during inflammation. Of the proteins identified in normal skin, 50% were cleaved, which increased to 60% during inflammation caused by phorbol esters, including chemokines and complement in which we identified previously uncharacterized cleavage sites. In mice deficient in matrix metalloproteinase 2 (MMP2), exudation of serum proteins was diminished compared to that in wild-type mice, and their proteolytic networks differed. We found that the complement 1 (C1) inhibitor was a key regulator linking these inflammatory responses. Cleavage and inactivation of the C1 inhibitor by MMP2 increased complement activation and bradykinin generation by plasma kallikrein in wild-type mice, leading to increased vessel permeability during inflammation. Thus, our degradomics analysis dissected proteolysis in skin inflammation and demonstrated perturbance of the proteolytic signaling network and its functional consequences arising from lack of a single protease.
Proteases enzymatically hydrolyze peptide bonds in substrate proteins, resulting in a widespread, irreversible posttranslational modification of the protein's structure and biological function. Often regarded as a mere degradative mechanism in destruction of proteins or turnover in maintaining physiological homeostasis, recent research in the field of degradomics has led to the recognition of two main yet unexpected concepts. First, that targeted, limited proteolytic cleavage events by a wide repertoire of proteases are pivotal regulators of most, if not all, physiological and pathological processes. Second, an unexpected in vivo abundance of stable cleaved proteins revealed pervasive, functionally relevant protein processing in normal and diseased tissue-from 40 to 70% of proteins also occur in vivo as distinct stable proteoforms with undocumented N- or C-termini, meaning these proteoforms are stable functional cleavage products, most with unknown functional implications. In this Review, we discuss the structural biology aspects and mechanisms of catalysis by different protease classes. We also provide an overview of biological pathways that utilize specific proteolytic cleavage as a precision control mechanism in protein quality control, stability, localization, and maturation, as well as proteolytic cleavage as a mediator in signaling pathways. Lastly, we provide a comprehensive overview of analytical methods and approaches to study activity and substrates of proteolytic enzymes in relevant biological models, both historical and focusing on state of the art proteomics techniques in the field of degradomics research.
A myriad of co- and post-translational modifications occur at protein N- and C-termini, resulting in an extra layer of proteome complexity and an additional source of protein regulation. Here, we review N- and C-terminal modifications and the contemporary positional proteomics techniques used to isolate protein terminal peptides from complex protein mixtures and characterize their diversity and occurrence in biological systems. Furthermore, these degradomics strategies--often referred to as N- and C-terminomics--represent dedicated high-throughput techniques to study proteolysis in dynamic living systems. Over the past decade, terminomics studies have provided indispensable information on the functional states of individual proteins, cell types, tissues, and biological processes and delivered fundamental new data for the Human Proteome Project, including high confidence identifications of many so-called "missing proteins", which had not been identified by traditional proteomics analyses.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.