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Biocatalysis: Mechanisms of Proteolytic Enzymes 2.0

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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: closed (15 October 2023) | Viewed by 10150

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Special Issue Editor

Dr. Peter Goettig

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Guest Editor

Structural Biology Group, Faculty of Molecular Biology, University of Salzburg, Billrothstr 11, A-5020 Salzburg, Austria
Interests: protein crystallography; proteases; kallikrein-related peptidases; metalloproteinases; enzyme kinetics; inhibitors; glycosylation; human physiology; molecular dynamics; protein folding
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Our previous special issue on “Mechanisms of Proteolytic Enzymes” was quite successful with overall 14 articles and reviews in IJMS (two of them in Catalysts). Beginning of December 2021, these publications were cited 35 times - two of them in the joint issue Catalysts -. 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 aspartic/glutamic, cysteine, metallo, serine, threonine and the rare asparagine type. In order to add more biological and medical relevance, we welcome studies of molecular interactions of proteases with natural substrates and inhibitors.

The goal of this Special Issue is to present experimental, computational and comparative studies, which promote a deeper understanding of the mechanistic principles of proteases. Also, structural and analytical data from crystallography, cryo-EM, NMR and MS that support functional and mechanistic research are welcome. Recent progress in 3D structure prediction by the AlphaFold project may facilitate the exploration of protease mechanisms. Novel promising research areas are real time studies of protease activity and the incorporation of non-natural amino acids into proteases, their substrates and inhibitors or specific probes. Submissions to this Special Issue on “Biocatalysis: Mechanisms of Proteolytic Enzymes 2.0” should be original research papers, including short communications, reviews and perspectives. Eventually, we hope to draw a topical picture of the current status and developments, which could have an impact on pharmaceutical research and medicine.

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 submissions that pass pre-check are 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.

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Keywords

aspartic proteases
catalytic mechanisms
cysteine proteases
enzyme kinetics
metalloproteases
non-natural amino acids
protease engineering
real time activity monitoring
serine proteases
viral proteases

Published Papers (6 papers)

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Research

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17 pages, 9525 KiB

Open AccessArticle

Investigations on Primary Cilia of Nthy-ori 3-1 Cells upon Cysteine Cathepsin Inhibition or Thyrotropin Stimulation

by Alara Gaye Doğru, Maren Rehders and Klaudia Brix

Int. J. Mol. Sci. 2023, 24(11), 9292; https://doi.org/10.3390/ijms24119292 - 26 May 2023

Cited by 1 | Viewed by 1464

Abstract

In the thyroid gland, cysteine cathepsins are secreted upon thyrotropin stimulation for thyroglobulin processing, and they are present at the primary cilia of thyroid epithelial cells. Treatment with protease inhibitors resulted in the loss of cilia from rodent thyrocytes and caused redistribution of the thyroid co-regulating G protein-coupled receptor Taar1 to the endoplasmic reticulum. These findings suggest that ciliary cysteine cathepsins are important to maintain sensory and signaling properties for the proper regulation and homeostasis of thyroid follicles. Therefore, it is important to better understand how cilia structure and frequencies are maintained in human thyroid epithelial cells. Hence, we aimed to investigate the potential role of cysteine cathepsins for the maintenance of primary cilia in the normal human Nthy-ori 3-1 thyroid cell line. This was approached by determining cilia lengths and frequencies in cysteine peptidase inhibition conditions in Nthy-ori 3-1 cell cultures. Cilia lengths were shortened upon 5 h of cysteine peptidase inhibition with cell-impermeable E64. Likewise, cilia lengths and frequencies were decreased upon additional overnight treatment with the cysteine peptidase-targeting, activity-based probe DCG-04. The results suggest that cysteine cathepsin activity is required for the maintenance of the cellular protrusions not only in rodents, but also in human thyrocytes. Hence, thyrotropin stimulation was used to simulate physiological conditions that eventually lead to cathepsin-mediated thyroglobulin proteolysis, which is initiated in the thyroid follicle lumen. Immunoblotting revealed that thyrotropin stimulation conditions result in the secretion of little procathepsin L and some pro- and mature cathepsin S but no cathepsin B from the human Nthy-ori 3-1 cells. Unexpectedly, however, 24 h incubation periods with thyrotropin shortened the cilia although higher amounts of cysteine cathepsins were present in the conditioned media. These data point to the necessity of further studies to delineate which of the cysteine cathepsins plays the most prominent role in cilia shortening and/or elongation. Collectively, the results of our study provide corroboration for the hypothesis of thyroid autoregulation by local mechanisms that our group previously proposed. Full article

(This article belongs to the Special Issue Biocatalysis: Mechanisms of Proteolytic Enzymes 2.0)

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16 pages, 2599 KiB

Open AccessArticle

Legumain Activity Is Controlled by Extended Active Site Residues and Substrate Conformation

by Tasneem Elamin, Hans Brandstetter and Elfriede Dall

Int. J. Mol. Sci. 2022, 23(20), 12548; https://doi.org/10.3390/ijms232012548 - 19 Oct 2022

Cited by 3 | Viewed by 1869

Abstract

Legumain is a lysosomal cysteine protease with strict specificity for cleaving after asparagine residues. By sequence comparison, legumain belongs to MEROPS clan CD of the cysteine proteases, which indicates its structural and mechanistic relation to caspases. Contrasting caspases, legumain harbors a pH-dependent ligase activity in addition to the protease activity. Although we already have a significant body of knowledge on the catalytic activities of legumain, many mechanistic details are still elusive. In this study, we provide evidence that extended active site residues and substrate conformation are steering legumain activities. Biochemical experiments and bioinformatics analysis showed that the catalytic Cys189 and His148 residues are regulated by sterically close Glu190, Ser215 and Asn42 residues. While Glu190 serves as an activity brake, Ser215 and Asn42 have a favorable effect on legumain protease activity. Mutagenesis studies using caspase-9 as model enzyme additionally showed that a similar Glu190 activity brake is also implemented in the caspases. Furthermore, we show that the substrate’s conformational flexibility determines whether it will be hydrolyzed or ligated by legumain. The functional understanding of the extended active site residues and of substrate prerequisites will allow us to engineer proteases with increased enzymatic activity and better ligase substrates, with relevance for biotechnological applications. Full article

(This article belongs to the Special Issue Biocatalysis: Mechanisms of Proteolytic Enzymes 2.0)

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12 pages, 1507 KiB

Open AccessArticle

Protealysin Targets the Bacterial Housekeeping Proteins FtsZ and RecA

by Olga Tsaplina, Sofia Khaitlina, Ksenia Chukhontseva, Maria Karaseva, Ilya Demidyuk, Irina Bakhlanova, Dmitry Baitin, Tatiana Artamonova, Alexey Vedyaykin, Mikhail Khodorkovskii and Innokentii Vishnyakov

Int. J. Mol. Sci. 2022, 23(18), 10787; https://doi.org/10.3390/ijms231810787 - 15 Sep 2022

Cited by 2 | Viewed by 1314

Abstract

Serratia proteamaculans synthesizes the intracellular metalloprotease protealysin. This work was aimed at searching for bacterial substrates of protealysin among the proteins responsible for replication and cell division. We have shown that protealysin unlimitedly cleaves the SOS response protein RecA. Even 20% of the cleaved RecA in solution appears to be incorporated into the polymer of uncleaved monomers, preventing further polymerization and inhibiting RecA ATPase activity. Transformation of Escherichia coli with a plasmid carrying the protealysin gene reduces the bacterial UV survival up to 10 times. In addition, the protealysin substrate is the FtsZ division protein, found in both E. coli and Acholeplasma laidlawii, which is only 51% identical to E. coli FtsZ. Protealysin cleaves FtsZ at the linker between the globular filament-forming domain and the C-terminal peptide that binds proteins on the bacterial membrane. Thus, cleavage of the C-terminal segment by protealysin can lead to the disruption of FtsZ’s attachment to the membrane, and thereby inhibit bacterial division. Since the protealysin operon encodes not only the protease, but also its inhibitor, which is typical for the system of interbacterial competition, we assume that in the case of penetration of protealysin into neighboring bacteria that do not synthesize a protealysin inhibitor, cleavage of FtsZ and RecA by protealysin may give S. proteamaculans an advantage in interbacterial competition. Full article

(This article belongs to the Special Issue Biocatalysis: Mechanisms of Proteolytic Enzymes 2.0)

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18 pages, 1341 KiB

Open AccessArticle

Probing the Conformational States of Thimet Oligopeptidase in Solution

by Marcelo F. M. Marcondes, Gabriel S. Santos, Fellipe Bronze, Mauricio F. M. Machado, Kátia R. Perez, Renske Hesselink, Marcel P. de Vries, Jaap Broos and Vitor Oliveira

Int. J. Mol. Sci. 2022, 23(13), 7297; https://doi.org/10.3390/ijms23137297 - 30 Jun 2022

Viewed by 1791

Abstract

Thimet oligopeptidase (TOP) is a metallopeptidase involved in the metabolism of oligopeptides inside and outside cells of various tissues. It has been proposed that substrate or inhibitor binding in the TOP active site induces a large hinge-bending movement leading to a closed structure, in which the bound ligand is enclosed. The main goal of the present work was to study this conformational change, and fluorescence techniques were used. Four active TOP mutants were created, each equipped with a single-Trp residue (fluorescence donor) and a p-nitro-phenylalanine (pNF) residue as fluorescence acceptor at opposite sides of the active site. pNF was biosynthetically incorporated with high efficiency using the amber codon suppression technology. Inhibitor binding induced shorter Donor-Acceptor (D-A) distances in all mutants, supporting the view that a hinge-like movement is operative in TOP. The activity of TOP is known to be dependent on the ionic strength of the assay buffer and D-A distances were measured at different ionic strengths. Interestingly, a correlation between the D-A distance and the catalytic activity of TOP was observed: the highest activities corresponded to the shortest D-A distances. In this study for the first time the hinge-bending motion of a metallopeptidase in solution could be studied, yielding insight about the position of the equilibrium between the open and closed conformation. This information will contribute to a more detailed understanding of the mode of action of these enzymes, including therapeutic targets like neurolysin and angiotensin-converting enzyme 2 (ACE2). Full article

(This article belongs to the Special Issue Biocatalysis: Mechanisms of Proteolytic Enzymes 2.0)

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Review

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14 pages, 3091 KiB

Open AccessReview

The Inhibition of Serine Proteases by Serpins Is Augmented by Negatively Charged Heparin: A Concise Review of Some Clinically Relevant Interactions

by Edward D. Chan, Paul T. King, Xiyuan Bai, Allen M. Schoffstall, Robert A. Sandhaus and Ashley M. Buckle

Int. J. Mol. Sci. 2024, 25(3), 1804; https://doi.org/10.3390/ijms25031804 - 2 Feb 2024

Cited by 1 | Viewed by 858

Abstract

Serine proteases are members of a large family of hydrolytic enzymes in which a particular serine residue in the active site performs an essential role as a nucleophile, which is required for their proteolytic cleavage function. The array of functions performed by serine proteases is vast and includes, among others, the following: (i) the ability to fight infections; (ii) the activation of blood coagulation or blood clot lysis systems; (iii) the activation of digestive enzymes; and (iv) reproduction. Serine protease activity is highly regulated by multiple families of protease inhibitors, known collectively as the SERine Protease INhibitor (SERPIN). The serpins use a conformational change mechanism to inhibit proteases in an irreversible way. The unusual conformational change required for serpin function provides an elegant opportunity for allosteric regulation by the binding of cofactors, of which the most well-studied is heparin. The goal of this review is to discuss some of the clinically relevant serine protease–serpin interactions that may be enhanced by heparin or other negatively charged polysaccharides. The paired serine protease–serpin in the framework of heparin that we review includes the following: thrombin–antithrombin III, plasmin–anti-plasmin, C1 esterase/kallikrein–C1 esterase inhibitor, and furin/TMPRSS2 (serine protease Transmembrane Protease 2)–alpha-1-antitrypsin, with the latter in the context of COVID-19 and prostate cancer. Full article

(This article belongs to the Special Issue Biocatalysis: Mechanisms of Proteolytic Enzymes 2.0)

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52 pages, 23202 KiB

Open AccessReview

Non-Canonical Amino Acids in Analyses of Protease Structure and Function

by Peter Goettig, Nikolaj G. Koch and Nediljko Budisa

Int. J. Mol. Sci. 2023, 24(18), 14035; https://doi.org/10.3390/ijms241814035 - 13 Sep 2023

Cited by 5 | Viewed by 2074

Abstract

All known organisms encode 20 canonical amino acids by base triplets in the genetic code. The cellular translational machinery produces proteins consisting mainly of these amino acids. Several hundred natural amino acids serve important functions in metabolism, as scaffold molecules, and in signal transduction. New side chains are generated mainly by post-translational modifications, while others have altered backbones, such as the β- or γ-amino acids, or they undergo stereochemical inversion, e.g., in the case of D-amino acids. In addition, the number of non-canonical amino acids has further increased by chemical syntheses. Since many of these non-canonical amino acids confer resistance to proteolytic degradation, they are potential protease inhibitors and tools for specificity profiling studies in substrate optimization and enzyme inhibition. Other applications include in vitro and in vivo studies of enzyme kinetics, molecular interactions and bioimaging, to name a few. Amino acids with bio-orthogonal labels are particularly attractive, enabling various cross-link and click reactions for structure-functional studies. Here, we cover the latest developments in protease research with non-canonical amino acids, which opens up a great potential, e.g., for novel prodrugs activated by proteases or for other pharmaceutical compounds, some of which have already reached the clinical trial stage. Full article

(This article belongs to the Special Issue Biocatalysis: Mechanisms of Proteolytic Enzymes 2.0)

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