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Biocatalysis: Mechanisms of Proteolytic Enzymes

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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 (31 October 2020) | Viewed by 51139

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Dr. Peter Goettig

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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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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 or International Journal of Molecular Sciences.

Dr. Peter Goettig
Guest Editor

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Keywords

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

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Published Papers (12 papers)

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Research

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22 pages, 4732 KiB

Open AccessArticle

Procathepsin V Is Secreted in a TSH Regulated Manner from Human Thyroid Epithelial Cells and Is Accessible to an Activity-Based Probe

by Alaa Al-Hashimi, Vaishnavi Venugopalan, Maren Rehders, Naphannop Sereesongsaeng, Zeynep Hein, Sebastian Springer, Ekkehard Weber, Dagmar Führer, Matthew S. Bogyo, Christopher J. Scott, Roberta E. Burden and Klaudia Brix

Int. J. Mol. Sci. 2020, 21(23), 9140; https://doi.org/10.3390/ijms21239140 - 30 Nov 2020

Cited by 5 | Viewed by 3700

Abstract

The significance of cysteine cathepsins for the liberation of thyroid hormones from the precursor thyroglobulin was previously shown by in vivo and in vitro studies. Cathepsin L is most important for thyroglobulin processing in mice. The present study aims at specifying the possible contribution of its closest relative, cysteine cathepsin L2/V, to thyroid function. Immunofluorescence analysis on normal human thyroid tissue revealed its predominant localization at the apical plasma membrane of thyrocytes and within the follicle lumen, indicating the secretion of cathepsin V and extracellular tasks rather than its acting within endo-lysosomes. To explore the trafficking pathways of cathepsin V in more detail, a chimeric protein consisting of human cathepsin V tagged with green fluorescent protein (GFP) was stably expressed in the Nthy-ori 3-1 thyroid epithelial cell line. Colocalization studies with compartment-specific markers and analyses of post-translational modifications revealed that the chimeric protein was sorted into the lumen of the endoplasmic reticulum and subsequently transported to the Golgi apparatus, while being N-glycosylated. Immunoblotting showed that the chimeric protein reached endo-lysosomes and it became secreted from the transduced cells. Astonishingly, thyroid stimulating hormone (TSH)-induced secretion of GFP-tagged cathepsin V occurred as the proform, suggesting that TSH upregulates its transport to the plasma membrane before it reaches endo-lysosomes for maturation. The proform of cathepsin V was found to be reactive with the activity-based probe DCG-04, suggesting that it possesses catalytic activity. We propose that TSH-stimulated secretion of procathepsin V is the default pathway in the thyroid to enable its contribution to thyroglobulin processing by extracellular means. Full article

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

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24 pages, 3994 KiB

Open AccessArticle

Substrate Specificity and Structural Modeling of Human Carboxypeptidase Z: A Unique Protease with a Frizzled-Like Domain

by Javier Garcia-Pardo, Sebastian Tanco, Maria C. Garcia-Guerrero, Sayani Dasgupta, Francesc Xavier Avilés, Julia Lorenzo and Lloyd D. Fricker

Int. J. Mol. Sci. 2020, 21(22), 8687; https://doi.org/10.3390/ijms21228687 - 18 Nov 2020

Cited by 3 | Viewed by 3769

Abstract

Metallocarboxypeptidase Z (CPZ) is a secreted enzyme that is distinguished from all other members of the M14 metallocarboxypeptidase family by the presence of an N-terminal cysteine-rich Frizzled-like (Fz) domain that binds Wnt proteins. Here, we present a comprehensive analysis of the enzymatic properties and substrate specificity of human CPZ. To investigate the enzymatic properties, we employed dansylated peptide substrates. For substrate specificity profiling, we generated two different large peptide libraries and employed isotopic labeling and quantitative mass spectrometry to study the substrate preference of this enzyme. Our findings revealed that CPZ has a strict requirement for substrates with C-terminal Arg or Lys at the P1′ position. For the P1 position, CPZ was found to display specificity towards substrates with basic, small hydrophobic, or polar uncharged side chains. Deletion of the Fz domain did not affect CPZ activity as a carboxypeptidase. Finally, we modeled the structure of the Fz and catalytic domains of CPZ. Taken together, these studies provide the molecular elucidation of substrate recognition and specificity of the CPZ catalytic domain, as well as important insights into how the Fz domain binds Wnt proteins to modulate their functions. Full article

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

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26 pages, 3827 KiB

Open AccessArticle

Specificity Studies of the Venezuelan Equine Encephalitis Virus Non-Structural Protein 2 Protease Using Recombinant Fluorescent Substrates

by Beáta Bozóki, János András Mótyán, Gyula Hoffka, David S. Waugh and József Tőzsér

Int. J. Mol. Sci. 2020, 21(20), 7686; https://doi.org/10.3390/ijms21207686 - 16 Oct 2020

Cited by 7 | Viewed by 3593

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 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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22 pages, 3211 KiB

Open AccessArticle

by Katherine Falkowski, Ewa Bielecka, Ida B. Thøgersen, Oliwia Bocheńska, Karolina Płaza, Magdalena Kalińska, Laura Sąsiadek, Małgorzata Magoch, Aleksandra Pęcak, Magdalena Wiśniewska, Natalia Gruba, Magdalena Wysocka, Anna Wojtysiak, Magdalena Brzezińska-Bodal, Kamila Sychowska, Anastasija Pejkovska, Maren Rehders, Georgina Butler, Christopher M Overall, Klaudia Brix, Grzegorz Dubin, Adam Lesner, Andrzej Kozik, Jan J. Enghild, Jan Potempa and Tomasz Kantyka Show full author list Hide full author list

Int. J. Mol. Sci. 2020, 21(12), 4383; https://doi.org/10.3390/ijms21124383 - 19 Jun 2020

Cited by 6 | Viewed by 3326

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 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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16 pages, 6439 KiB

Open AccessArticle

A Peptidomimetic Fluorescent Probe to Detect the Trypsin β2 Subunit of the Human 20S Proteasome

by Magdalena Wysocka, Anita Romanowska, Natalia Gruba, Michalina Michalska, Artur Giełdoń and Adam Lesner

Int. J. Mol. Sci. 2020, 21(7), 2396; https://doi.org/10.3390/ijms21072396 - 31 Mar 2020

Cited by 4 | Viewed by 2452

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 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 K_m = 3.22 ± 0.02 μM, k_cat = 245 s⁻¹, and k_cat/K_m = 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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23 pages, 4886 KiB

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

by János András Mótyán, Márió Miczi and József Tőzsér

Int. J. Mol. Sci. 2020, 21(4), 1352; https://doi.org/10.3390/ijms21041352 - 17 Feb 2020

Cited by 10 | Viewed by 4474

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, 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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16 pages, 1360 KiB

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

by Olga Egorova, Heather HC Lau, Kate McGraphery and Yi Sheng

Int. J. Mol. Sci. 2020, 21(4), 1309; https://doi.org/10.3390/ijms21041309 - 15 Feb 2020

Cited by 12 | Viewed by 4207

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 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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18 pages, 1858 KiB

Open AccessArticle

Development of Chemical Tools to Monitor Human Kallikrein 13 (KLK13) Activity

by Natalia Gruba, Ewa Bielecka, Magdalena Wysocka, Anna Wojtysiak, Magdalena Brzezińska-Bodal, Kamila Sychowska, Magdalena Kalińska, Małgorzata Magoch, Aleksandra Pęcak, Katherine Falkowski, Magdalena Wiśniewska, Laura Sąsiadek, Karolina Płaza, Eileen Kroll, Anastasija Pejkovska, Maren Rehders, Klaudia Brix, Grzegorz Dubin, Tomasz Kantyka, Jan Potempa and Adam Lesner Show full author list Hide full author list

Int. J. Mol. Sci. 2019, 20(7), 1557; https://doi.org/10.3390/ijms20071557 - 28 Mar 2019

Cited by 15 | Viewed by 3680

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 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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26 pages, 3528 KiB

Open AccessReview

Mechanisms of Proteolytic Enzymes and Their Inhibition in QM/MM Studies

by Brigitta Elsässer and Peter Goettig

Int. J. Mol. Sci. 2021, 22(6), 3232; https://doi.org/10.3390/ijms22063232 - 22 Mar 2021

Cited by 25 | Viewed by 6066

Abstract

Experimental evidence for enzymatic mechanisms is often scarce, and in many cases inadvertently biased by the employed methods. Thus, apparently contradictory model mechanisms can result in decade long discussions about the correct interpretation of data and the true theory behind it. However, often such opposing views turn out to be special cases of a more comprehensive and superior concept. Molecular dynamics (MD) and the more advanced molecular mechanical and quantum mechanical approach (QM/MM) provide a relatively consistent framework to treat enzymatic mechanisms, in particular, the activity of proteolytic enzymes. In line with this, computational chemistry based on experimental structures came up with studies on all major protease classes in recent years; examples of aspartic, metallo-, cysteine, serine, and threonine protease mechanisms are well founded on corresponding standards. In addition, experimental evidence from enzyme kinetics, structural research, and various other methods supports the described calculated mechanisms. One step beyond is the application of this information to the design of new and powerful inhibitors of disease-related enzymes, such as the HIV protease. In this overview, a few examples demonstrate the high potential of the QM/MM approach for sophisticated pharmaceutical compound design and supporting functions in the analysis of biomolecular structures. Full article

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

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20 pages, 2020 KiB

Open AccessReview

Origin and Expansion of the Serine Protease Repertoire in the Myelomonocyte Lineage

by Stefanie A. I. Weiss, Salome R. T. Rehm, Natascha C. Perera, Martin L. Biniossek, Oliver Schilling and Dieter E. Jenne

Int. J. Mol. Sci. 2021, 22(4), 1658; https://doi.org/10.3390/ijms22041658 - 7 Feb 2021

Cited by 6 | Viewed by 4293

Abstract

The deepest evolutionary branches of the trypsin/chymotrypsin family of serine proteases are represented by the digestive enzymes of the gastrointestinal tract and the multi-domain proteases of the blood coagulation and complement system. Similar to the very old digestive system, highly diverse cleavage specificities emerged in various cell lineages of the immune defense system during vertebrate evolution. The four neutrophil serine proteases (NSPs) expressed in the myelomonocyte lineage, neutrophil elastase, proteinase 3, cathepsin G, and neutrophil serine protease 4, collectively display a broad repertoire of (S1) specificities. The origin of NSPs can be traced back to a circulating liver-derived trypsin-like protease, the complement factor D ancestor, whose activity is tightly controlled by substrate-induced activation and TNFα-induced locally upregulated protein secretion. However, the present-day descendants are produced and converted to mature enzymes in precursor cells of the bone marrow and are safely sequestered in granules of circulating neutrophils. The potential site and duration of action of these cell-associated serine proteases are tightly controlled by the recruitment and activation of neutrophils, by stimulus-dependent regulated secretion of the granules, and by various soluble inhibitors in plasma, interstitial fluids, and in the inflammatory exudate. An extraordinary dynamic range and acceleration of immediate defense responses have been achieved by exploiting the high structural plasticity of the trypsin fold. Full article

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

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33 pages, 2302 KiB

Open AccessReview

Mechanisms Applied by Protein Inhibitors to Inhibit Cysteine Proteases

by Livija Tušar, Aleksandra Usenik, Boris Turk and Dušan Turk

Int. J. Mol. Sci. 2021, 22(3), 997; https://doi.org/10.3390/ijms22030997 - 20 Jan 2021

Cited by 15 | Viewed by 5532

Abstract

Protein inhibitors of proteases are an important tool of nature to regulate and control proteolysis in living organisms under physiological and pathological conditions. In this review, we analyzed the mechanisms of inhibition of cysteine proteases on the basis of structural information and compiled kinetic data. The gathered structural data indicate that the protein fold is not a major obstacle for the evolution of a protease inhibitor. It appears that nature can convert almost any starting fold into an inhibitor of a protease. In addition, there appears to be no general rule governing the inhibitory mechanism. The structural data make it clear that the “lock and key” mechanism is a historical concept with limited validity. However, the analysis suggests that the shape of the active site cleft of proteases imposes some restraints. When the S1 binding site is shaped as a pocket buried in the structure of protease, inhibitors can apply substrate-like binding mechanisms. In contrast, when the S1 binding site is in part exposed to solvent, the substrate-like inhibition cannot be employed. It appears that all proteases, with the exception of papain-like proteases, belong to the first group of proteases. Finally, we show a number of examples and provide hints on how to engineer protein inhibitors. Full article

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

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20 pages, 1434 KiB

Open AccessReview

Regulation of the Proteolytic Activity of Cysteine Cathepsins by Oxidants

by Gilles Lalmanach, Ahlame Saidi, Paul Bigot, Thibault Chazeirat, Fabien Lecaille and Mylène Wartenberg

Int. J. Mol. Sci. 2020, 21(6), 1944; https://doi.org/10.3390/ijms21061944 - 12 Mar 2020

Cited by 23 | Viewed by 4896

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 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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