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Special Issue "Biocatalysis: Mechanisms of Proteolytic Enzymes"

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biophysics".

Deadline for manuscript submissions: 31 October 2020.

Special Issue Editor

Dr. Peter Goettig
Website
Guest Editor
Structural Biology Group, Department of Biosciences, University of Salzburg, Billrothstrasse 11, 5020 Salzburg, Austria
Interests: protein crystallography, proteases, kallikrein-related peptidases, metalloproteinases, enzyme kinetics, inhibitors, gylcosylation, human physiology, protein folding
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Special Issue Information

Dear Colleagues,

Proteases are traditionally among the most studied enzymes and over the decades a wealth of structural, functional and theoretical information has accumulated. However, the understanding of the molecular mechanisms underlying their activities and regulation remains incomplete. Although the basic principles of peptide bond hydrolysis were delineated a long time ago, there is a lack of experimental evidence for many aspects of substrate recognition, turnover, energetics, time course of the catalytic steps, and of the fine-tuned physiological regulation of activity. Thus, it is worth investigating and comparing the molecular mechanisms for the numerous serine, cysteine, metallo and aspartic proteases, as well as for the much rarer threonine, asparagine and glutamic type. In order to add more biological and medical relevance, we welcome articles that analyze molecular interactions of proteases with natural substrates and inhibitors or their synthetic small molecule counterparts.

The goal of this Special Issue is to present experimental, computational and comparative studies, which promote a deeper understanding of the common mechanistic principles of proteases. Also, structural and analytical data from crystallography, cryo-EM, NMR and MS that support functional and mechanistic research are welcome. Therefore, submissions to this Special Issue on the “Biocatalysis: Mechanisms of Proteolytic Enzymes” should be either original research papers, including short communications, or reviews and perspectives. Eventually, we hope to draw a topical picture of the current status and developments, which could have an impact on other fields of research, such as medicine and pharmacy.

Accepted papers are published in the joint Special Issue in Catalysts (https://www.mdpi.com/journal/catalysts/special_issues/proteolytic_enzymes) or International Journal of Molecular Sciences.

Dr. Peter Goettig
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All papers will be peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. There is an Article Processing Charge (APC) for publication in this open access journal. For details about the APC please see here. Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • aspartic proteases
  • catalytic mechanism
  • conformational selection
  • cysteine proteases
  • enzyme kinetics
  • metalloproteases
  • nucleophilic attack
  • peptide bond hydrolysis
  • serine protease

Published Papers (7 papers)

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Research

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Open AccessArticle
Specificity Studies of the Venezuelan Equine Encephalitis Virus Non-Structural Protein 2 Protease Using Recombinant Fluorescent Substrates
Int. J. Mol. Sci. 2020, 21(20), 7686; https://doi.org/10.3390/ijms21207686 - 16 Oct 2020
Abstract
The non-structural protein 2 (nsP2) of alphavirus Venezuelan equine encephalitis virus (VEEV) is a cysteine protease that is responsible for processing of the viral non-structural polyprotein and is an important drug target owing to the clinical relevance of VEEV. In this study we [...] Read more.
The non-structural protein 2 (nsP2) of alphavirus Venezuelan equine encephalitis virus (VEEV) is a cysteine protease that is responsible for processing of the viral non-structural polyprotein and is an important drug target owing to the clinical relevance of VEEV. In this study we designed two recombinant VEEV nsP2 constructs to study the effects of an N-terminal extension on the protease activity and to investigate the specificity of the elongated enzyme in vitro. The N-terminal extension was found to have no substantial effect on the protease activity. The amino acid preferences of the VEEV nsP2 protease were investigated on substrates representing wild-type and P5, P4, P2, P1, P1′, and P2′ variants of Semliki forest virus nsP1/nsP2 cleavage site, using a His6-MBP-mEYFP recombinant substrate-based protease assay which has been adapted for a 96-well plate-based format. The structural basis of enzyme specificity was also investigated in silico by analyzing a modeled structure of VEEV nsP2 complexed with oligopeptide substrate. To our knowledge, in vitro screening of P1′ amino acid preferences of VEEV nsP2 protease remains undetermined to date, thus, our results may provide valuable information for studies and inhibitor design of different alphaviruses or other Group IV viruses. Full article
(This article belongs to the Special Issue Biocatalysis: Mechanisms of Proteolytic Enzymes)
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Open AccessArticle
Kallikrein-Related Peptidase 14 Activates Zymogens of Membrane Type Matrix Metalloproteinases (MT-MMPs)—A CleavEx Based Analysis
Int. J. Mol. Sci. 2020, 21(12), 4383; https://doi.org/10.3390/ijms21124383 - 19 Jun 2020
Abstract
Kallikrein-related peptidases (KLKs) and matrix metalloproteinases (MMPs) are secretory proteinases known to proteolytically process components of the extracellular matrix, modulating the pericellular environment in physiology and in pathologies. The interconnection between these families remains elusive. To assess the cross-activation of these families, we [...] Read more.
Kallikrein-related peptidases (KLKs) and matrix metalloproteinases (MMPs) are secretory proteinases known to proteolytically process components of the extracellular matrix, modulating the pericellular environment in physiology and in pathologies. The interconnection between these families remains elusive. To assess the cross-activation of these families, we developed a peptide, fusion protein-based exposition system (Cleavage of exposed amino acid sequences, CleavEx) aiming at investigating the potential of KLK14 to recognize and hydrolyze proMMP sequences. Initial assessment identified ten MMP activation domain sequences which were validated by Edman degradation. The analysis revealed that membrane-type MMPs (MT-MMPs) are targeted by KLK14 for activation. Correspondingly, proMMP14-17 were investigated in vitro and found to be effectively processed by KLK14. Again, the expected neo-N-termini of the activated MT-MMPs was confirmed by Edman degradation. The effectiveness of proMMP activation was analyzed by gelatin zymography, confirming the release of fully active, mature MT-MMPs upon KLK14 treatment. Lastly, MMP14 was shown to be processed on the cell surface by KLK14 using murine fibroblasts overexpressing human MMP14. Herein, we propose KLK14-mediated selective activation of cell-membrane located MT-MMPs as an additional layer of their regulation. As both, KLKs and MT-MMPs, are implicated in cancer, their cross-activation may constitute an important factor in tumor progression and metastasis. Full article
(This article belongs to the Special Issue Biocatalysis: Mechanisms of Proteolytic Enzymes)
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Open AccessArticle
A Peptidomimetic Fluorescent Probe to Detect the Trypsin β2 Subunit of the Human 20S Proteasome
Int. J. Mol. Sci. 2020, 21(7), 2396; https://doi.org/10.3390/ijms21072396 - 31 Mar 2020
Abstract
This work describes the chemical synthesis, combinatorial selection, and enzymatic evaluation of peptidomimetic fluorescent substrates specific for the trypsin-like (β2) subunit of the 20S human proteasome. After deconvolution of a library comprising nearly 6000 compounds composed of peg substituted diaminopropionic acid DAPEG building [...] Read more.
This work describes the chemical synthesis, combinatorial selection, and enzymatic evaluation of peptidomimetic fluorescent substrates specific for the trypsin-like (β2) subunit of the 20S human proteasome. After deconvolution of a library comprising nearly 6000 compounds composed of peg substituted diaminopropionic acid DAPEG building blocks, the sequence ABZ–Dap(O2(Cbz))–Dap(GO1)–Dap(O2(Cbz))–Arg–ANB–NH2, where ABZ is 2-aminobenzoic acid, and ANB- 5 amino 2- nitro benzoic acid was selected. Its cleavage followed sigmoidal kinetics, characteristic for allosteric enzymes, with Km = 3.22 ± 0.02 μM, kcat = 245 s−1, and kcat/Km = 7.61 × 107 M−1 s−1. This process was practically halted when a selective inhibitor of the β2 subunit of the 20S human proteasome was supplemented to the reaction system. Titration of the substrate resulting in decreased amounts of proteasome 20S produced a linear signal up to 10−11 M. Using this substrate, we detected human proteasome 20S in human urine samples taken from the bladders of cancer patients. This observation could be useful for the noninvasive diagnosis of this severe disease. Full article
(This article belongs to the Special Issue Biocatalysis: Mechanisms of Proteolytic Enzymes)
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Open AccessArticle
Dimer Interface Organization is a Main Determinant of Intermonomeric Interactions and Correlates with Evolutionary Relationships of Retroviral and Retroviral-Like Ddi1 and Ddi2 Proteases
Int. J. Mol. Sci. 2020, 21(4), 1352; https://doi.org/10.3390/ijms21041352 - 17 Feb 2020
Cited by 3
Abstract
The life cycles of retroviruses rely on the limited proteolysis catalyzed by the viral protease. Numerous eukaryotic organisms also express endogenously such proteases, which originate from retrotransposons or retroviruses, including DNA damage-inducible 1 and 2 (Ddi1 and Ddi2, respectively) proteins. In this study, [...] Read more.
The life cycles of retroviruses rely on the limited proteolysis catalyzed by the viral protease. Numerous eukaryotic organisms also express endogenously such proteases, which originate from retrotransposons or retroviruses, including DNA damage-inducible 1 and 2 (Ddi1 and Ddi2, respectively) proteins. In this study, we performed a comparative analysis based on the structural data currently available in Protein Data Bank (PDB) and Structural summaries of PDB entries (PDBsum) databases, with a special emphasis on the regions involved in dimerization of retroviral and retroviral-like Ddi proteases. In addition to Ddi1 and Ddi2, at least one member of all seven genera of the Retroviridae family was included in this comparison. We found that the studied retroviral and non-viral proteases show differences in the mode of dimerization and density of intermonomeric contacts, and distribution of the structural characteristics is in agreement with their evolutionary relationships. Multiple sequence and structure alignments revealed that the interactions between the subunits depend mainly on the overall organization of the dimer interface. We think that better understanding of the general and specific features of proteases may support the characterization of retroviral-like proteases. Full article
(This article belongs to the Special Issue Biocatalysis: Mechanisms of Proteolytic Enzymes)
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Open AccessArticle
Mdm2 and MdmX RING Domains Play Distinct Roles in the Regulation of p53 Responses: A Comparative Study of Mdm2 and MdmX RING Domains in U2OS Cells
Int. J. Mol. Sci. 2020, 21(4), 1309; https://doi.org/10.3390/ijms21041309 - 15 Feb 2020
Cited by 1
Abstract
Dysfunction of the tumor suppressor p53 occurs in most human cancers. Mdm2 and MdmX are homologous proteins from the Mdm (Murine Double Minute) protein family, which play a critical role in p53 inactivation and degradation. The two proteins interact with one another via [...] Read more.
Dysfunction of the tumor suppressor p53 occurs in most human cancers. Mdm2 and MdmX are homologous proteins from the Mdm (Murine Double Minute) protein family, which play a critical role in p53 inactivation and degradation. The two proteins interact with one another via the intrinsic RING (Really Interesting New Gene) domains to achieve the negative regulation of p53. The downregulation of p53 is accomplished by Mdm2-mediated p53 ubiquitination and proteasomal degradation through the ubiquitin proteolytic system and by Mdm2 and MdmX mediated inhibition of p53 transactivation. To investigate the role of the RING domain of Mdm2 and MdmX, an analysis of the distinct functionalities of individual RING domains of the Mdm proteins on p53 regulation was conducted in human osteosarcoma (U2OS) cell line. Mdm2 RING domain was observed mainly localized in the cell nucleus, contrasting the localization of MdmX RING domain in the cytoplasm. Mdm2 RING was found to possess an endogenous E3 ligase activity, whereas MdmX RING did not. Both Mdm2 and MdmX RING domains were able to dimerize with endogenous full-length Mdm2 and MdmX protein and affect their cellular function. The results showed that overexpression of the Mdm2 or MdmX RING domains interfered with the endogenous full-length Mdm2 and MdmX activity and resulted in p53 stabilization and p53 target gene activation. However, both Mdm RING domains showed oncogenic activity in a colony formation assay, suggesting that the Mdm RING domains possess p53-independent oncogenic properties. This study highlights the distinct structural and functional traits of the RING domain of Mdm2 and MdmX and characterized their role in cellular responses through interfering with p53 dependent signaling pathway. Full article
(This article belongs to the Special Issue Biocatalysis: Mechanisms of Proteolytic Enzymes)
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Open AccessArticle
Development of Chemical Tools to Monitor Human Kallikrein 13 (KLK13) Activity
Int. J. Mol. Sci. 2019, 20(7), 1557; https://doi.org/10.3390/ijms20071557 - 28 Mar 2019
Cited by 2
Abstract
Kallikrein 13 (KLK13) was first identified as an enzyme that is downregulated in a subset of breast tumors. This serine protease has since been implicated in a number of pathological processes including ovarian, lung and gastric cancers. Here we report the design, synthesis [...] Read more.
Kallikrein 13 (KLK13) was first identified as an enzyme that is downregulated in a subset of breast tumors. This serine protease has since been implicated in a number of pathological processes including ovarian, lung and gastric cancers. Here we report the design, synthesis and deconvolution of libraries of internally quenched fluorogenic peptide substrates to determine the specificity of substrate binding subsites of KLK13 in prime and non-prime regions (according to the Schechter and Berger convention). The substrate with the consensus sequential motive ABZ-Val-Arg-Phe-Arg-ANB-NH2 demonstrated selectivity towards KLK13 and was successfully converted into an activity-based probe by the incorporation of a chloromethylketone warhead and biotin bait. The compounds described may serve as suitable tools to detect KLK13 activity in diverse biological samples, as exemplified by overexpression experiments and targeted labeling of KLK13 in cell lysates and saliva. In addition, we describe the development of selective activity-based probes targeting KLK13, to our knowledge the first tool to analyze the presence of the active enzyme in biological samples. Full article
(This article belongs to the Special Issue Biocatalysis: Mechanisms of Proteolytic Enzymes)
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Review

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Open AccessReview
Regulation of the Proteolytic Activity of Cysteine Cathepsins by Oxidants
Int. J. Mol. Sci. 2020, 21(6), 1944; https://doi.org/10.3390/ijms21061944 - 12 Mar 2020
Cited by 3
Abstract
Besides their primary involvement in the recycling and degradation of proteins in endo-lysosomal compartments and also in specialized biological functions, cysteine cathepsins are pivotal proteolytic contributors of various deleterious diseases. While the molecular mechanisms of regulation via their natural inhibitors have been exhaustively [...] Read more.
Besides their primary involvement in the recycling and degradation of proteins in endo-lysosomal compartments and also in specialized biological functions, cysteine cathepsins are pivotal proteolytic contributors of various deleterious diseases. While the molecular mechanisms of regulation via their natural inhibitors have been exhaustively studied, less is currently known about how their enzymatic activity is modulated during the redox imbalance associated with oxidative stress and their exposure resistance to oxidants. More specifically, there is only patchy information on the regulation of lung cysteine cathepsins, while the respiratory system is directly exposed to countless exogenous oxidants contained in dust, tobacco, combustion fumes, and industrial or domestic particles. Papain-like enzymes (clan CA, family C1, subfamily C1A) encompass a conserved catalytic thiolate-imidazolium pair (Cys25-His159) in their active site. Although the sulfhydryl group (with a low acidic pKa) is a potent nucleophile highly susceptible to chemical modifications, some cysteine cathepsins reveal an unanticipated resistance to oxidative stress. Besides an introductory chapter and peculiar attention to lung cysteine cathepsins, the purpose of this review is to afford a concise update of the current knowledge on molecular mechanisms associated with the regulation of cysteine cathepsins by redox balance and by oxidants (e.g., Michael acceptors, reactive oxygen, and nitrogen species). Full article
(This article belongs to the Special Issue Biocatalysis: Mechanisms of Proteolytic Enzymes)
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