International Journal of Molecular Sciences

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Open AccessEditorial

Peptidases: Role and Function in Health and Disease

by Janko Kos

Int. J. Mol. Sci. 2023, 24(9), 7823; https://doi.org/10.3390/ijms24097823 - 25 Apr 2023

Cited by 2 | Viewed by 1528

Abstract

Peptidases represent a large family of hydrolases present in all living organisms, which catalyze the degradation of peptide bonds in different biological processes Full article

(This article belongs to the Special Issue Peptidases: Role and Function in Health and Disease)

Research

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7 pages, 849 KiB

Open AccessCommunication

Cell Type-Specific Anti-Viral Effects of Novel SARS-CoV-2 Main Protease Inhibitors

by Nina Geiger, Viktoria Diesendorf, Valeria Roll, Eva-Maria König, Helena Obernolte, Katherina Sewald, Julian Breidenbach, Thanigaimalai Pillaiyar, Michael Gütschow, Christa E. Müller and Jochen Bodem

Int. J. Mol. Sci. 2023, 24(4), 3972; https://doi.org/10.3390/ijms24043972 - 16 Feb 2023

Cited by 4 | Viewed by 1333

Abstract

Recently, we have described novel pyridyl indole esters and peptidomimetics as potent inhibitors of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) main protease. Here, we analysed the impact of these compounds on viral replication. It has been shown that some antivirals [...] Read more.

Recently, we have described novel pyridyl indole esters and peptidomimetics as potent inhibitors of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) main protease. Here, we analysed the impact of these compounds on viral replication. It has been shown that some antivirals against SARS-CoV-2 act in a cell line-specific way. Thus, the compounds were tested in Vero, Huh-7, and Calu-3 cells. We showed that the protease inhibitors at 30 µM suppress viral replication by up to 5 orders of magnitude in Huh-7 cells, while in Calu-3 cells, suppression by 2 orders of magnitude was achieved. Three pyridin-3-yl indole-carboxylates inhibited viral replication in all cell lines, indicating that they might repress viral replication in human tissue as well. Thus, we investigated three compounds in human precision-cut lung slices and observed donor-dependent antiviral activity in this patient-near system. Our results provide evidence that even direct-acting antivirals may act in a cell line-specific manner. Full article

(This article belongs to the Special Issue Peptidases: Role and Function in Health and Disease)

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25 pages, 7100 KiB

Open AccessArticle

Crystal Structure of Inhibitor-Bound Bacterial Oligopeptidase B in the Closed State: Similarity and Difference between Protozoan and Bacterial Enzymes

by Dmitry E. Petrenko, David M. Karlinsky, Veronika D. Gordeeva, Georgij P. Arapidi, Elena V. Britikova, Vladimir V. Britikov, Alena Y. Nikolaeva, Konstantin M. Boyko, Vladimir I. Timofeev, Inna P. Kuranova, Anna G. Mikhailova, Eduard V. Bocharov and Tatiana V. Rakitina

Int. J. Mol. Sci. 2023, 24(3), 2286; https://doi.org/10.3390/ijms24032286 - 24 Jan 2023

Cited by 3 | Viewed by 1424

Abstract

The crystal structure of bacterial oligopeptidase B from Serratia proteamaculans (SpOpB) in complex with a chloromethyl ketone inhibitor was determined at 2.2 Å resolution. SpOpB was crystallized in a closed (catalytically active) conformation. A single inhibitor molecule bound simultaneously to the catalytic residues [...] Read more.

The crystal structure of bacterial oligopeptidase B from Serratia proteamaculans (SpOpB) in complex with a chloromethyl ketone inhibitor was determined at 2.2 Å resolution. SpOpB was crystallized in a closed (catalytically active) conformation. A single inhibitor molecule bound simultaneously to the catalytic residues S532 and H652 mimicked a tetrahedral intermediate of the catalytic reaction. A comparative analysis of the obtained structure and the structure of OpB from Trypanosoma brucei (TbOpB) in a closed conformation showed that in both enzymes, the stabilization of the D-loop (carrying the catalytic D) in a position favorable for the formation of a tetrahedral complex occurs due to interaction with the neighboring loop from the β-propeller. However, the modes of interdomain interactions were significantly different for bacterial and protozoan OpBs. Instead of a salt bridge (as in TbOpB), in SpOpB, a pair of polar residues following the catalytic D617 and a pair of neighboring arginine residues from the β-propeller domain formed complementary oppositely charged surfaces. Bioinformatics analysis and structural modeling show that all bacterial OpBs can be divided into two large groups according to these two modes of D-loop stabilization in closed conformations. Full article

(This article belongs to the Special Issue Peptidases: Role and Function in Health and Disease)

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21 pages, 2284 KiB

Open AccessArticle

Trypanosoma brucei rhodesiense Inhibitor of Cysteine Peptidase (ICP) Is Required for Virulence in Mice and to Attenuate the Inflammatory Response

by Tatiana F. R. Costa, Amy Goundry, Alexandre Morrot, Dennis J. Grab, Jeremy C. Mottram and Ana Paula C. A. Lima

Int. J. Mol. Sci. 2023, 24(1), 656; https://doi.org/10.3390/ijms24010656 - 30 Dec 2022

Cited by 3 | Viewed by 1532

Abstract

The protozoan Trypanosoma brucei rhodesiense causes Human African Trypanosomiasis, also known as sleeping sickness, and penetrates the central nervous system, leading to meningoencephalitis. The Cathepsin L-like cysteine peptidase of T. b. rhodesiense has been implicated in parasite penetration of the blood–brain barrier and [...] Read more.

The protozoan Trypanosoma brucei rhodesiense causes Human African Trypanosomiasis, also known as sleeping sickness, and penetrates the central nervous system, leading to meningoencephalitis. The Cathepsin L-like cysteine peptidase of T. b. rhodesiense has been implicated in parasite penetration of the blood–brain barrier and its activity is modulated by the chagasin-family endogenous inhibitor of cysteine peptidases (ICP). To investigate the role of ICP in T. b. rhodesiense bloodstream form, ICP-null (Δicp) mutants were generated, and lines re-expressing ICP (Δicp:ICP). Lysates of Δicp displayed increased E-64-sensitive cysteine peptidase activity and the mutant parasites traversed human brain microvascular endothelial cell (HBMEC) monolayers in vitro more efficiently. Δicp induced E-selectin in HBMECs, leading to the adherence of higher numbers of human neutrophils. In C57BL/6 mice, no Δicp parasites could be detected in the blood after 6 days, while mice infected with wild-type (WT) or Δicp:ICP displayed high parasitemia, peaking at day 12. In mice infected with Δicp, there was increased recruitment of monocytes to the site of inoculation and higher levels of IFN-γ in the spleen. At day 14, mice infected with Δicp exhibited higher preservation of the CD4⁺, CD8⁺, and CD19⁺ populations in the spleen, accompanied by sustained high IFN-γ, while NK1.1⁺ populations receded nearly to the levels of uninfected controls. We propose that ICP helps to downregulate inflammatory responses that contribute to the control of infection. Full article

(This article belongs to the Special Issue Peptidases: Role and Function in Health and Disease)

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21 pages, 1714 KiB

Open AccessArticle

Complex of Proline-Specific Peptidases in the Genome and Gut Transcriptomes of Tenebrionidae Insects and Their Role in Gliadin Hydrolysis

by Valeriia F. Tereshchenkova, Irina Y. Filippova, Irina A. Goptar, Yakov E. Dunaevsky, Mikhail A. Belozersky and Elena N. Elpidina

Int. J. Mol. Sci. 2023, 24(1), 579; https://doi.org/10.3390/ijms24010579 - 29 Dec 2022

Cited by 1 | Viewed by 1696

Abstract

A detailed analysis of the complexes of proline-specific peptidases (PSPs) in the midgut transcriptomes of the larvae of agricultural pests Tenebrio molitor and Tribolium castaneum and in the genome of T. castaneum is presented. Analysis of the T. castaneum genome revealed 13 PSP [...] Read more.

A detailed analysis of the complexes of proline-specific peptidases (PSPs) in the midgut transcriptomes of the larvae of agricultural pests Tenebrio molitor and Tribolium castaneum and in the genome of T. castaneum is presented. Analysis of the T. castaneum genome revealed 13 PSP sequences from the clans of serine and metal-dependent peptidases, of which 11 sequences were also found in the gut transcriptomes of both tenebrionid species’ larvae. Studies of the localization of PSPs, evaluation of the expression level of their genes in gut transcriptomes, and prediction of the presence of signal peptides determining secretory pathways made it possible to propose a set of peptidases that can directly participate in the hydrolysis of food proteins in the larvae guts. The discovered digestive PSPs of tenebrionids in combination with the post-glutamine cleaving cysteine cathepsins of these insects effectively hydrolyzed gliadins, which are the natural food substrates of the studied pests. Based on the data obtained, a hypothetical scheme for the complete hydrolysis of immunogenic gliadin peptides by T. molitor and T. castaneum digestive peptidases was proposed. These results show promise regarding the development of a drug based on tenebrionid digestive enzymes for the enzymatic therapy of celiac disease and gluten intolerance. Full article

(This article belongs to the Special Issue Peptidases: Role and Function in Health and Disease)

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10 pages, 1489 KiB

Open AccessArticle

FSAP Protects against Histone-Mediated Increase in Endothelial Permeability In Vitro

by Xue Yan Cui, Benedicte Stavik, Bernd Thiede, Per Morten Sandset and Sandip M. Kanse

Int. J. Mol. Sci. 2022, 23(22), 13706; https://doi.org/10.3390/ijms232213706 - 8 Nov 2022

Cited by 3 | Viewed by 1653

Abstract

Factor-VII-activating protease (FSAP) is involved in the regulation of hemostasis and inflammation. Extracellular histones play a role in inflammation and the conversion of latent pro-FSAP into active FSAP. FSAP has been shown to regulate endothelial permeability, but the mechanisms are not clear. Here, [...] Read more.

Factor-VII-activating protease (FSAP) is involved in the regulation of hemostasis and inflammation. Extracellular histones play a role in inflammation and the conversion of latent pro-FSAP into active FSAP. FSAP has been shown to regulate endothelial permeability, but the mechanisms are not clear. Here, we have investigated the effects of FSAP on endothelial permeability in vitro. A mixture of histones from calf thymus stimulated permeability, and the wild-type (WT) serine protease domain (SPD) of FSAP blocked this effect. WT–SPD–FSAP did not influence permeability on its own, nor that stimulated by thrombin or vascular endothelial growth factor (VEGF)-A₁₆₅. Histones induced a large-scale rearrangement of the junction proteins VE-cadherin and zona occludens-1 from a clear junctional distribution to a diffuse pattern. The presence of WT–SPD–FSAP inhibited these changes. Permeability changes by histones were blocked by both TLR-2 and TLR4 blocking antibodies. Histones upregulated the expression of TLR-2, but not TLR-4, in HUVEC cells, and WT–SPD–FSAP abolished the upregulation of TLR-2 expression. An inactive variant, Marburg I (MI)–SPD–FSAP, did not have any of these effects. The inhibition of histone-mediated permeability may be an important function of FSAP with relevance to sepsis, trauma, and stroke and the need to be investigated further in in vivo experiments. Full article

(This article belongs to the Special Issue Peptidases: Role and Function in Health and Disease)

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13 pages, 2996 KiB

Open AccessArticle

Profiling Substrate Specificity of the SUMO Protease Ulp1 by the YESS–PSSC System to Advance the Conserved Mechanism for Substrate Cleavage

by Faying Zhang, Hui Zheng, Yufan Xian, Haoyue Song, Shengchen Wang, Yueli Yun, Li Yi and Guimin Zhang

Int. J. Mol. Sci. 2022, 23(20), 12188; https://doi.org/10.3390/ijms232012188 - 13 Oct 2022

Cited by 2 | Viewed by 2259

Abstract

SUMO modification is a vital post-translational regulation process in eukaryotes, in which the SUMO protease is responsible for the maturation of the SUMO precursor and the deconjugation of the SUMO protein from modified proteins by accurately cleaving behind the C-terminal Gly–Gly motif. To [...] Read more.

SUMO modification is a vital post-translational regulation process in eukaryotes, in which the SUMO protease is responsible for the maturation of the SUMO precursor and the deconjugation of the SUMO protein from modified proteins by accurately cleaving behind the C-terminal Gly–Gly motif. To promote the understanding of the high specificity of the SUMO protease against the SUMO protein as well as to clarify whether the conserved Gly–Gly motif is strictly required for the processing of the SUMO precursor, we systematically profiled the specificity of the S. cerevisiae SUMO protease (Ulp1) on Smt3 at the P2–P1↓P1’ (Gly–Gly↓Ala) position using the YESS–PSSC system. Our results demonstrated that Ulp1 was able to cleave Gly–Gly↓ motif-mutated substrates, indicating that the diglycine motif is not strictly required for Ulp1 cleavage. A structural-modeling analysis indicated that it is the special tapered active pocket of Ulp1 conferred the selectivity of small residues at the P1–P2 position of Smt3, such as Gly, Ala, Ser and Cys, and only which can smoothly deliver the scissile bond into the active site for cleavage. Meanwhile, the P1’ position Ala of Smt3 was found to play a vital role in maintaining Ulp1’s precise cleavage after the Gly–Gly motif and replacing Ala with Gly in this position could expand Ulp1 inclusivity against the P1 and P2 position residues of Smt3. All in all, our studies advanced the traditional knowledge of the SUMO protein, which may provide potential directions for the drug discovery of abnormal SUMOylation-related diseases. Full article

(This article belongs to the Special Issue Peptidases: Role and Function in Health and Disease)

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18 pages, 4117 KiB

Open AccessArticle

Recombinant Cathepsin L of Tribolium castaneum and Its Potential in the Hydrolysis of Immunogenic Gliadin Peptides

by Elena A. Dvoryakova, Maria A. Klimova, Tatiana R. Simonyan, Ivan A. Dombrovsky, Marina V. Serebryakova, Valeriia F. Tereshchenkova, Yakov E. Dunaevsky, Mikhail A. Belozersky, Irina Y. Filippova and Elena N. Elpidina

Int. J. Mol. Sci. 2022, 23(13), 7001; https://doi.org/10.3390/ijms23137001 - 23 Jun 2022

Cited by 4 | Viewed by 1844

Abstract

Wheat gliadins contain a large amount of glutamine- and proline-rich peptides which are not hydrolyzed by human digestive peptidases and can cause autoimmune celiac disease and other forms of gluten intolerance in predisposed people. Peptidases that efficiently cleave such immunogenic peptides can be [...] Read more.

Wheat gliadins contain a large amount of glutamine- and proline-rich peptides which are not hydrolyzed by human digestive peptidases and can cause autoimmune celiac disease and other forms of gluten intolerance in predisposed people. Peptidases that efficiently cleave such immunogenic peptides can be used in enzyme therapy. The stored product insect pest Tribolium castaneum efficiently hydrolyzes gliadins. The main digestive peptidase of T. castaneum is cathepsin L, which is from the papain C1 family with post-glutamine cleavage activity. We describe the isolation and characterization of T. castaneum recombinant procathepsin L (rpTcCathL1, NP_001164001), which was expressed in Pichia pastoris cells. The activation of the proenzyme was conducted by autocatalytic processing. The effects of pH and proenzyme concentration in the reaction mixture on the processing were studied. The mature enzyme retained high activity in the pH range from 5.0 to 9.0 and displayed high pH-stability from 4.0 to 8.0 at 20 °C. The enzyme was characterized according to electrophoretic mobility under native conditions, activity and stability at various pH values, a sensitivity to various inhibitors, and substrate specificity, and its hydrolytic effect on 8-, 10-, 26-, and 33-mer immunogenic gliadins peptides was demonstrated. Our results show that rTcCathL1 is an effective peptidase that can be used to develop a drug for the enzyme therapy of various types of gluten intolerance. Full article

(This article belongs to the Special Issue Peptidases: Role and Function in Health and Disease)

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15 pages, 2207 KiB

Open AccessArticle

A Study of Type II ɛ-PL Degrading Enzyme (pldII) in Streptomyces albulus through the CRISPRi System

by Qinyu Li, Xiaojia Chen, Yuanjie Wu, Zheng Chen, Yang Han, Peng Zhou, Jiping Shi and Zhijun Zhao

Int. J. Mol. Sci. 2022, 23(12), 6691; https://doi.org/10.3390/ijms23126691 - 15 Jun 2022

Cited by 4 | Viewed by 1454

Abstract

ε-Poly-L-lysine (ε-PL) is a widely used antibacterial peptide polymerized of 25–35 L-lysine residues. The antibacterial effect of ε-PL is closely related to the polymerization degree. However, the mechanism of ε-PL degradation in S. albulus remains unclear. This study utilized the integrative plasmid pSET152-based [...] Read more.

ε-Poly-L-lysine (ε-PL) is a widely used antibacterial peptide polymerized of 25–35 L-lysine residues. The antibacterial effect of ε-PL is closely related to the polymerization degree. However, the mechanism of ε-PL degradation in S. albulus remains unclear. This study utilized the integrative plasmid pSET152-based CRISPRi system to transcriptionally repress the ε-PL degrading enzyme (pldII). The expression of pldII is regulated by changing the recognition site of dCas9. Through the ε-PL bacteriostatic experiments of repression strains, it was found that the repression of pldII improves the antibacterial effect of the ε-PL product. The consecutive MALDI-TOF-MS results confirmed that the molecular weight distribution of the ε-PL was changed after repression. The repression strain S1 showed a particular peak with a polymerization degree of 44, and other repression strains also generated ε-PL with a polymerization degree of over 40. Furthermore, the homology modeling and substrate docking of pldII, a typical endo-type metallopeptidase, were performed to resolve the degradation mechanism of ε-PL in S. albulus. The hydrolysis of ε-PL within pldII, initiated from the N-terminus by two amino acid-binding residues, Thr194 and Glu281, led to varying levels of polymerization of ε-PL. Full article

(This article belongs to the Special Issue Peptidases: Role and Function in Health and Disease)

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Review

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15 pages, 1765 KiB

Open AccessReview

The Magic of Proteases: From a Procoagulant and Anticoagulant Factor V to an Equitable Treatment of Its Inherited Deficiency

by Juan A. De Pablo-Moreno, Andrea Miguel-Batuecas, María de Sancha and Antonio Liras

Int. J. Mol. Sci. 2023, 24(7), 6243; https://doi.org/10.3390/ijms24076243 - 26 Mar 2023

Cited by 5 | Viewed by 1745

Abstract

Proteostasis, i.e., the homeostasis of proteins, responsible for ensuring protein turnover, is regulated by proteases, which also participate in the etiopathogenesis of multiple conditions. The magic of proteases is such that, in blood coagulation, one same molecule, such as coagulation factor V, for [...] Read more.

Proteostasis, i.e., the homeostasis of proteins, responsible for ensuring protein turnover, is regulated by proteases, which also participate in the etiopathogenesis of multiple conditions. The magic of proteases is such that, in blood coagulation, one same molecule, such as coagulation factor V, for example, can perform both a procoagulant and an anticoagulant function as a result of the activity of proteases. However, this magic has an insidious side to it, as it may also prevent the completion of the clinical value chain of factor V deficiency. This value chain encompasses the discovery of knowledge, the transfer of this knowledge, and its translation to clinical practice. In the case of rare and ultra-rare diseases like factor V deficiency, this value chain has not been completed as the knowledge acquisition phase has dragged out over time, holding up the transfer of knowledge to clinical practice. The reason for this is related to the small number of patients afflicted with these conditions. As a result, new indications must be found to make the therapies cost-effective. In the case of factor V, significant research efforts have been directed at developing a recombinant factor V capable of resisting the action of the proteases capable of inactivating this factor. This is where bioethics and health equity considerations come into the equation. Full article

(This article belongs to the Special Issue Peptidases: Role and Function in Health and Disease)

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15 pages, 2306 KiB

Open AccessReview

Distinguishing Plasmin-Generating Microvesicles: Tiny Messengers Involved in Fibrinolysis and Proteolysis

by Laurent Plawinski, Audrey Cras, José Rubicel Hernández Lopez, Aurora de la Peña, Angéline Van der Heyden, Catherine Belle, Florence Toti and Eduardo Anglés-Cano

Int. J. Mol. Sci. 2023, 24(2), 1571; https://doi.org/10.3390/ijms24021571 - 13 Jan 2023

Cited by 5 | Viewed by 1943

Abstract

A number of stressors and inflammatory mediators (cytokines, proteases, oxidative stress mediators) released during inflammation or ischemia stimulate and activate cells in blood, the vessel wall or tissues. The most well-known functional and phenotypic responses of activated cells are (1) the immediate expression [...] Read more.

A number of stressors and inflammatory mediators (cytokines, proteases, oxidative stress mediators) released during inflammation or ischemia stimulate and activate cells in blood, the vessel wall or tissues. The most well-known functional and phenotypic responses of activated cells are (1) the immediate expression and/or release of stored or newly synthesized bioactive molecules, and (2) membrane blebbing followed by release of microvesicles. An ultimate response, namely the formation of extracellular traps by neutrophils (NETs), is outside the scope of this work. The main objective of this article is to provide an overview on the mechanism of plasminogen reception and activation at the surface of cell-derived microvesicles, new actors in fibrinolysis and proteolysis. The role of microvesicle-bound plasmin in pathological settings involving inflammation, atherosclerosis, angiogenesis, and tumour growth, remains to be investigated. Further studies are necessary to determine if profibrinolytic microvesicles are involved in a finely regulated equilibrium with pro-coagulant microvesicles, which ensures a balanced haemostasis, leading to the maintenance of vascular patency. Full article

(This article belongs to the Special Issue Peptidases: Role and Function in Health and Disease)

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22 pages, 1149 KiB

Open AccessReview

The Mammalian Cysteine Protease Legumain in Health and Disease

by Rigmor Solberg, Ngoc Nguyen Lunde, Karl Martin Forbord, Meshail Okla, Moustapha Kassem and Abbas Jafari

Int. J. Mol. Sci. 2022, 23(24), 15983; https://doi.org/10.3390/ijms232415983 - 15 Dec 2022

Cited by 15 | Viewed by 2948

Abstract

The cysteine protease legumain (also known as asparaginyl endopeptidase or δ-secretase) is the only known mammalian asparaginyl endopeptidase and is primarily localized to the endolysosomal system, although it is also found extracellularly as a secreted protein. Legumain is involved in the regulation of [...] Read more.

The cysteine protease legumain (also known as asparaginyl endopeptidase or δ-secretase) is the only known mammalian asparaginyl endopeptidase and is primarily localized to the endolysosomal system, although it is also found extracellularly as a secreted protein. Legumain is involved in the regulation of diverse biological processes and tissue homeostasis, and in the pathogenesis of various malignant and nonmalignant diseases. In addition to its proteolytic activity that leads to the degradation or activation of different substrates, legumain has also been shown to have a nonproteolytic ligase function. This review summarizes the current knowledge about legumain functions in health and disease, including kidney homeostasis, hematopoietic homeostasis, bone remodeling, cardiovascular and cerebrovascular diseases, fibrosis, aging and senescence, neurodegenerative diseases and cancer. In addition, this review addresses the effects of some marketed drugs on legumain. Expanding our knowledge on legumain will delineate the importance of this enzyme in regulating physiological processes and disease conditions. Full article

(This article belongs to the Special Issue Peptidases: Role and Function in Health and Disease)

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