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Keywords = serine protease inhibitory activity

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11 pages, 1717 KiB  
Article
Isolation of Allosteric Tryptase Inhibitor from Methanol Extract of Rhubarb and Enhancement of Its Tryptase Inhibitory Activity by Compounds That Were Screened by In Silico Screening
by Hidetoshi Fujii, Moeno Ito, Kentaro Nishioka, Katsutoshi Nishino, Takanao Otsuka, Kazuhiro Irie, Takashi Tanaka and Masaya Nagao
Molecules 2025, 30(6), 1341; https://doi.org/10.3390/molecules30061341 - 17 Mar 2025
Viewed by 618
Abstract
Tryptase, which is abundant in human mast cells and is involved in allergic inflammations such as asthma, is a serine protease. We isolated a tryptase inhibitor, procyanidin B8 3,3′-di-O-gallate (PB8GG’), a tannin, from the methanol extract of rhubarb (RHEI RHIDOMA), which [...] Read more.
Tryptase, which is abundant in human mast cells and is involved in allergic inflammations such as asthma, is a serine protease. We isolated a tryptase inhibitor, procyanidin B8 3,3′-di-O-gallate (PB8GG’), a tannin, from the methanol extract of rhubarb (RHEI RHIDOMA), which is a traditional Chinese medicine (Kampo medicine in Japan). Since it did not inhibit another serine protease trypsin, PB8GG’ specifically inhibited tryptase. A standard kinetic analysis of the inhibitory fashion of PB8GG’ against tryptase suggested that PB8GG’ inhibited tryptase in an allosteric manner. We searched for other tannins like PB8GG’ expected to bind tryptase using AutoDock vina. Two ellagitannins, carpinins B and E, isolated from young leaves of Carpinus japonica were selected as candidates of tryptase inhibitors. Carpinins B and E themselves had almost no inhibitory activity against tryptase but enhanced the inhibitory activity of PB8GG’ against tryptase. This is an example that shows that a combination of an allosteric inhibitor with other compounds that bind but have no inhibitory activity can be used to develop a clinically useful combinatorial enzyme inhibitor. Full article
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13 pages, 9161 KiB  
Article
Improvement in XIa Selectivity of Snake Venom Peptide Analogue BF9-N17K Using P2′ Amino Acid Replacements
by Li Ding, Zhiping Zhai, Tianxiang Qin, Yuexi Lin, Zhicheng Shuang, Fang Sun, Chenhu Qin, Hongyi Luo, Wen Zhu, Xiangdong Ye, Zongyun Chen and Xudong Luo
Toxins 2025, 17(1), 23; https://doi.org/10.3390/toxins17010023 - 5 Jan 2025
Viewed by 993
Abstract
Coagulation factor XIa is a new serine-protease family drug target for next-generation anticoagulants. With the snake venom Kunitz-type peptide BF9 as the scaffold, we obtained a highly active XIa inhibitor BF9-N17K in our previous work, but it also inhibited the hemostatic target plasmin. [...] Read more.
Coagulation factor XIa is a new serine-protease family drug target for next-generation anticoagulants. With the snake venom Kunitz-type peptide BF9 as the scaffold, we obtained a highly active XIa inhibitor BF9-N17K in our previous work, but it also inhibited the hemostatic target plasmin. Here, in order to enhance the selectivity of BF9-N17K toward XIa, four mutants, BF9-N17K-L19A, BF9-N17K-L19S, BF9-N17K-L19D, and BF9-N17K-L19K, were further designed using the P2′ amino acid classification scanning strategy. The anticoagulation assay showed that the four P2′ single-point mutants still had apparent inhibitory anticoagulation activity that selectively inhibited the human intrinsic coagulation pathway and had no influence on the extrinsic coagulation pathway or common coagulation pathway, which indicated that the single-point mutants had minimal effects on the anticoagulation activity of BF9-N17K. Interestingly, the enzyme inhibitor assay experiments showed that the XIa and plasmin inhibitory activities were significantly changed by the P2′ amino acid replacements. The XIa inhibitory activity of BF9-N17K-L19D was apparently enhanced, with an IC50 of 19.28 ± 2.53 nM, and its plasmin inhibitory was significantly weakened, with an IC50 of 459.33 ± 337.40 nM. BF9-N17K-L19K was the opposite to BF9-N17K-L19D, which had enhanced plasmin inhibitory activity and reduced XIa inhibitory activity. For BF9-N17K-L19A and BF9-N17K-L19S, no apparent changes were found in the serine protease inhibitory activity, and they had similar XIa and plasmin inhibitory activities to the template peptide BF9-N17K. These results suggested that the characteristics of the charge of the P2′ site might be associated with the drug selectivity between the anticoagulant target XIa and hemostatic target plasmin. In addition, according to the molecular diversity and sequence conservation, a common motif GR/PCR/KA/SXIP-XYGGC is proposed in the XIa-inhibitory Kunitz-type peptides, which might provide a new clue for further peptide engineering. In conclusion, through P2′ amino acid classification scanning with the snake venom Kunitz-type peptide scaffold, a new potent and selective XIa inhibitor, BF9-N17K-L19D, was discovered, which provides a new XIa-targeting lead drug template for the treatment of thrombotic-related diseases. Full article
(This article belongs to the Special Issue Animals Venom in Drug Discovery: A Valuable Therapeutic Tool)
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15 pages, 4360 KiB  
Article
Characterization of Kunitz-Domain Anticoagulation Peptides Derived from Acinetobacter baumannii Exotoxin Protein F6W77
by Fang Sun, Xiaolin Deng, Huanhuan Gao, Li Ding, Wen Zhu, Hongyi Luo, Xiangdong Ye, Xudong Luo, Zongyun Chen and Chenhu Qin
Toxins 2024, 16(10), 450; https://doi.org/10.3390/toxins16100450 - 21 Oct 2024
Cited by 1 | Viewed by 1639
Abstract
Recent studies have revealed that the coagulation system plays a role in mammalian innate defense by entrapping bacteria in clots and generating antibacterial peptides. So, it is very important for the survival of bacteria to defend against the host coagulation system, which suggests [...] Read more.
Recent studies have revealed that the coagulation system plays a role in mammalian innate defense by entrapping bacteria in clots and generating antibacterial peptides. So, it is very important for the survival of bacteria to defend against the host coagulation system, which suggests that bacterial exotoxins might be a new source of anticoagulants. In this study, we analyzed the genomic sequences of Acinetobacter baumannii and a new bacterial exotoxin protein, F6W77, with five Kunitz-domains, KABP1-5, was identified. Each Kunitz-type domain features a classical six-cysteine framework reticulated by three conserved disulfide bridges, which was obviously similar to animal Kunitz-domain peptides but different from plant Kunitz-domain peptides. Anticoagulation function evaluation showed that towards the intrinsic coagulation pathway, KABP1 and KABP5 had apparently inhibitory activity, KABP4 had weak inhibitory activity, and KBAP2 and KABP3 had no effect even at a high concentration of 20 μg/mL. All five Kunitz-domain peptides, KABP1-5, had no inhibitory activity towards the extrinsic coagulation pathway. Enzyme-inhibitor experiments showed that the high-activity anticoagulant peptide KABP1 had apparently inhibitory activity towards two key coagulation factors, Xa and XIa, which was further confirmed by pull-down experiments that showed that KABP1 can bind to coagulation factors Xa and XIa directly. Structure-function relationship analyses of five Kunitz-type domain peptides showed that the arginine of the P1 site of three new bacterial anticoagulants, KABP1, KABP4 and KABP5, might be the key residue for their anticoagulation activity. In conclusion, with bioinformatics analyses, peptide recombination, and functional evaluation, we firstly found bacterial-exotoxin-derived Kunitz-type serine protease inhibitors with selectively inhibiting activity towards intrinsic coagulation pathways, and highlighted a new interaction between pathogenic bacteria and the human coagulation system. Full article
(This article belongs to the Section Bacterial Toxins)
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17 pages, 3341 KiB  
Article
In Vitro Evaluation of Antipseudomonal Activity and Safety Profile of Peptidomimetic Furin Inhibitors
by Sara Maluck, Rivka Bobrovsky, Miklós Poór, Roman W. Lange, Torsten Steinmetzer, Ákos Jerzsele, András Adorján, Dávid Bajusz, Anita Rácz and Erzsébet Pászti-Gere
Biomedicines 2024, 12(9), 2075; https://doi.org/10.3390/biomedicines12092075 - 11 Sep 2024
Cited by 3 | Viewed by 1585
Abstract
Inhibitors of the serine protease furin have been widely studied as antimicrobial agents due to their ability to block the cleavage and activation of certain viral surface proteins and bacterial toxins. In this study, the antipseudomonal effects and safety profiles of the furin [...] Read more.
Inhibitors of the serine protease furin have been widely studied as antimicrobial agents due to their ability to block the cleavage and activation of certain viral surface proteins and bacterial toxins. In this study, the antipseudomonal effects and safety profiles of the furin inhibitors MI-1851 and MI-2415 were assessed. Fluorescence quenching studies suggested no relevant binding of the compounds to human serum albumin and α1-acid glycoprotein. Both inhibitors demonstrated significant antipseudomonal activity in Madin–Darby canine kidney cells, especially compound MI-1851 at very low concentrations (0.5 µM). Using non-tumorigenic porcine IPEC-J2 cells, neither of the two furin inhibitors induced cytotoxicity (CCK-8 assay) or altered significantly the intracellular (Amplex Red assay) or extracellular (DCFH-DA assay) redox status even at a concentration of 100 µM. The same assays with MI-2415 conducted on primary human hepatocytes also resulted in no changes in cell viability and oxidative stress at up to 100 µM. Microsomal and hepatocyte-based CYP3A4 activity assays showed that both inhibitors exhibited a concentration-dependent inhibition of the isoenzyme at high concentrations. In conclusion, this study indicates a good safety profile of the furin inhibitors MI-1851 and MI-2415, suggesting their applicability as antimicrobials for further in vivo investigations, despite some inhibitory effects on CYP3A4. Full article
(This article belongs to the Special Issue Drug Discovery for Infectious Diseases—Second Edition)
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13 pages, 4921 KiB  
Article
Anti-SARS-CoV-2 Viral Activity of Sweet Potato Trypsin Inhibitor via Downregulation of TMPRSS2 Activity and ACE2 Expression In Vitro and In Vivo
by Wen-Ping Jiang, Jeng-Shyan Deng, Chia-Chen Yu, Jaung-Geng Lin and Guan-Jhong Huang
Int. J. Mol. Sci. 2024, 25(11), 6067; https://doi.org/10.3390/ijms25116067 - 31 May 2024
Cited by 1 | Viewed by 1734
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. Known as COVID-19, it has affected billions of people worldwide, claiming millions of lives and posing a continuing threat to humanity. This is considered one of the most extensive pandemics ever [...] Read more.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. Known as COVID-19, it has affected billions of people worldwide, claiming millions of lives and posing a continuing threat to humanity. This is considered one of the most extensive pandemics ever recorded in human history, causing significant losses to both life and economies globally. However, the available evidence is currently insufficient to establish the effectiveness and safety of antiviral drugs or vaccines. The entry of the virus into host cells involves binding to angiotensin-converting enzyme 2 (ACE2), a cell surface receptor, via its spike protein. Meanwhile, transmembrane protease serine 2 (TMPRSS2), a host surface protease, cleaves and activates the virus’s S protein, thus promoting viral infection. Plant protease inhibitors play a crucial role in protecting plants against insects and/or microorganisms. The major storage proteins in sweet potato roots include sweet potato trypsin inhibitor (SWTI), which accounts for approximately 60% of the total water-soluble protein and has been found to possess a variety of health-promoting properties, including antioxidant, anti-inflammatory, ACE-inhibitory, and anticancer functions. Our study found that SWTI caused a significant reduction in the expression of the ACE2 and TMPRSS2 proteins, without any adverse effects on cells. Therefore, our findings suggest that the ACE2 and TMPRSS2 axis can be targeted via SWTI to potentially inhibit SARS-CoV-2 infection. Full article
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18 pages, 1983 KiB  
Article
4-Chloroisocoumarins as Chlamydial Protease Inhibitors and Anti-Chlamydial Agents
by Matthew J. A. Phillips, Wilhelmina M. Huston, Andrew M. McDonagh and Tristan Rawling
Molecules 2024, 29(7), 1519; https://doi.org/10.3390/molecules29071519 - 28 Mar 2024
Cited by 1 | Viewed by 1410
Abstract
4-Chloroisocoumarin compounds have broad inhibitory properties against serine proteases. Here, we show that selected 3-alkoxy-4-chloroisocoumarins preferentially inhibit the activity of the conserved serine protease High-temperature requirement A of Chlamydia trachomatis. The synthesis of a new series of isocoumarin-based scaffolds has been developed, [...] Read more.
4-Chloroisocoumarin compounds have broad inhibitory properties against serine proteases. Here, we show that selected 3-alkoxy-4-chloroisocoumarins preferentially inhibit the activity of the conserved serine protease High-temperature requirement A of Chlamydia trachomatis. The synthesis of a new series of isocoumarin-based scaffolds has been developed, and their anti-chlamydial properties were investigated. The structure of the alkoxy substituent was found to influence the potency of the compounds against High-temperature requirement A, and modifications to the C-7 position of the 3-alkoxy-4-chloroisocoumarin structure attenuate anti-chlamydial properties. Full article
(This article belongs to the Section Medicinal Chemistry)
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14 pages, 3098 KiB  
Article
Targeted Library of Phosphonic-Type Inhibitors of Human Neutrophil Elastase
by Karolina Torzyk-Jurowska, Jaroslaw Ciekot and Lukasz Winiarski
Molecules 2024, 29(5), 1120; https://doi.org/10.3390/molecules29051120 - 1 Mar 2024
Cited by 3 | Viewed by 1787
Abstract
Despite many years of research, human neutrophil elastase (HNE) still remains an area of interest for many researchers. This multifunctional representative of neutrophil serine proteases is one of the most destructive enzymes found in the human body which can degrade most of the [...] Read more.
Despite many years of research, human neutrophil elastase (HNE) still remains an area of interest for many researchers. This multifunctional representative of neutrophil serine proteases is one of the most destructive enzymes found in the human body which can degrade most of the extracellular matrix. Overexpression or dysregulation of HNE may lead to the development of several inflammatory diseases. Previously, we presented the HNE inhibitor with kinact/KI value over 2,000,000 [M−1s−1]. In order to optimize its structure, over 100 novel tripeptidyl derivatives of α-aminoalkylphosphonate diaryl esters were synthesized, and their activity toward HNE was checked. To confirm the selectivity of the resultant compounds, several of the most active were additionally checked against the two other neutrophil proteases: proteinase 3 and cathepsin G. The developed modifications allowed us to obtain a compound with significantly increased inhibitory activity against human neutrophil elastase with high selectivity toward cathepsin G, but none toward proteinase 3. Full article
(This article belongs to the Special Issue Design, Synthesis, and Evaluation of Protease Inhibitors)
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15 pages, 3242 KiB  
Article
Effect of Conventional and Microwave Heating on Protein and Odor Profile in Soymilk Powder
by Walailak Khotchai, Nantawan Therdthai and Aussama Soontrunnarudrungsri
Sustainability 2023, 15(16), 12395; https://doi.org/10.3390/su151612395 - 15 Aug 2023
Cited by 5 | Viewed by 3135
Abstract
Soymilk contained serine protease enzymes with inhibitory activity against trypsin, causing a negative effect on nutritional absorption. This project aimed to investigate the effects of conventional heating (100 °C/20 min) and microwave heating (360–900 W/1–6 min) on trypsin inhibitor, protein digestibility, and odor [...] Read more.
Soymilk contained serine protease enzymes with inhibitory activity against trypsin, causing a negative effect on nutritional absorption. This project aimed to investigate the effects of conventional heating (100 °C/20 min) and microwave heating (360–900 W/1–6 min) on trypsin inhibitor, protein digestibility, and odor profiles. Soymilk contained 46–47% protein, regardless of heating conditions. Using scanning electron microscopy, it can be seen that the conventionally heated sample had a smooth surface and a porous structure, whereas microwave-heated samples contained a protein matrix in clusters with rough surfaces. The molecular weight of proteins in SDS-PAGE was reduced to 19 kDa and <16 kDa after conventional and microwave heating, respectively, resulting in the reduction of trypsin inhibitor from 36.66 to 0.91 and 0.56–0.66 mg/g, respectively. Protein digestibility was significantly improved when either the conventional heating at 100 °C for 20 min or the microwave heating at 900 W for 6 min was applied. From e-nose analysis, the beany flavor was reduced after the microwave heating for 3–6 min, and the highest intensity of the sweet odor compounds were observed after the microwave heating at 600 W for 1 min. Due to the short processing time, microwave heating was then recommended for inhibition of trypsin inhibitors and improving protein digestibility. Full article
(This article belongs to the Special Issue Sustainable Food Production and Processing Development)
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14 pages, 2758 KiB  
Article
Trapa bispinosa Roxb. Pericarp Extract Exerts 5α-Reductase Inhibitory Activity in Castrated Benign Prostatic Hyperplasia Model Mice
by Takashi Fujita, Tomoko Aoyama, Tomohiro Uemura, Shouko Takeshita, Takuto Yamasaki, Hiroko Heijou and Koji Morimoto
Int. J. Mol. Sci. 2023, 24(14), 11765; https://doi.org/10.3390/ijms241411765 - 21 Jul 2023
Cited by 1 | Viewed by 1992
Abstract
Trapa bispinosa Roxb. pericarp extract (TBE) has a polyphenol-rich composition and exhibits potent antioxidant and anti-glycation activities in vitro. In the present study, we investigated the inhibitory effects of TBE on 5α-reductase in vitro using LNCaP cells and in vivo using a mouse [...] Read more.
Trapa bispinosa Roxb. pericarp extract (TBE) has a polyphenol-rich composition and exhibits potent antioxidant and anti-glycation activities in vitro. In the present study, we investigated the inhibitory effects of TBE on 5α-reductase in vitro using LNCaP cells and in vivo using a mouse model of castrated benign prostatic hyperplasia. TBE showed concentration-dependent inhibitory effects in the 5α-reductase (5αR) activity assay. In a reporter assay using AR-Luc/LNCaP cells, TBE inhibited the activity induced by testosterone, but not that induced by dihydrotestosterone. TBE also suppressed prostate cell proliferation, prostate-specific antigens, and transmembrane protease serine 2 expression in a castrated benign prostatic hyperplasia mouse model. In addition, ellagic acid, but not gallic acid, decreased 5αR and AR-Luc activities. Together, these results suggest a potential role for TBE in benign prostatic hyperplasia through inhibition of 5αR. Full article
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14 pages, 4059 KiB  
Article
Epitope-Specific Anti-SerpinB3 Antibodies for SerpinB3 Recognition and Biological Activity Inhibition
by Alessandra Biasiolo, Michele Sandre, Stefania Ferro, Santina Quarta, Mariagrazia Ruvoletto, Gianmarco Villano, Cristian Turato, Maria Guido, Oriano Marin and Patrizia Pontisso
Biomolecules 2023, 13(5), 739; https://doi.org/10.3390/biom13050739 - 25 Apr 2023
Cited by 4 | Viewed by 2103
Abstract
SerpinB3 is a serine protease inhibitor that plays a relevant role in disease progression and cancer by increasing fibrosis, cell proliferation, and invasion, besides conferring resistance to apoptosis. The mechanisms underlying these biological activities are not yet fully understood. The aim of this [...] Read more.
SerpinB3 is a serine protease inhibitor that plays a relevant role in disease progression and cancer by increasing fibrosis, cell proliferation, and invasion, besides conferring resistance to apoptosis. The mechanisms underlying these biological activities are not yet fully understood. The aim of this study was to generate antibodies directed against different SerpinB3 epitopes to better investigate their biological role. Five exposed epitopes were identified using the software DNASTAR Lasergene and the corresponding synthetic peptides were used for NZW rabbit immunization. Anti-P#2 and anti-P#4 antibodies were able to recognize both SerpinB3 and SerpinB4 by ELISA. Anti-P#5 antibody, produced against the reactive site loop of SerpinB3, showed the greatest specific reactivity for human SerpinB3. This antibody was able to recognize SerpinB3 at nuclear level, while anti-P#3 antibody recognized SerpinB3 only at cytoplasmic level, both by immunofluorescence and by immunohistochemistry. The biological activity of each antibody preparation was assessed in HepG2 cells overexpressing SerpinB3 and anti-P#5 antibody reduced proliferation by 12% cell and cell invasion by 75%, while trivial results were obtained with the other antibody preparations. These findings indicate that the reactive site loop of SerpinB3 is essential for the invasiveness features induced by this serpin and it could become a novel druggable target. Full article
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22 pages, 5841 KiB  
Review
Diversity, Biosynthesis and Bioactivity of Aeruginosins, a Family of Cyanobacteria-Derived Nonribosomal Linear Tetrapeptides
by Jiameng Liu, Mengli Zhang, Zhenkuai Huang, Jiaqi Fang, Zhongyuan Wang, Chengxu Zhou and Xiaoting Qiu
Mar. Drugs 2023, 21(4), 217; https://doi.org/10.3390/md21040217 - 29 Mar 2023
Cited by 13 | Viewed by 3266
Abstract
Aeruginosins, a family of nonribosomal linear tetrapeptides discovered from cyanobacteria and sponges, exhibit in vitro inhibitory activity on various types of serine proteases. This family is characterized by the existence of the 2-carboxy-6-hydroxy-octahydroindole (Choi) moiety occupied at the central position of the tetrapeptide. [...] Read more.
Aeruginosins, a family of nonribosomal linear tetrapeptides discovered from cyanobacteria and sponges, exhibit in vitro inhibitory activity on various types of serine proteases. This family is characterized by the existence of the 2-carboxy-6-hydroxy-octahydroindole (Choi) moiety occupied at the central position of the tetrapeptide. Aeruginosins have attracted much attention due to their special structures and unique bioactivities. Although many studies on aeruginosins have been published, there has not yet been a comprehensive review that summarizes the diverse research ranging from biogenesis, structural characterization and biosynthesis to bioactivity. In this review, we provide an overview of the source, chemical structure as well as spectrum of bioactivities of aeruginosins. Furthermore, possible opportunities for future research and development of aeruginosins were discussed. Full article
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21 pages, 15070 KiB  
Article
VPg Impact on Ryegrass Mottle Virus Serine-like 3C Protease Proteolysis and Structure
by Gints Kalnins, Rebeka Ludviga, Ieva Kalnciema, Gunta Resevica, Vilija Zeltina, Janis Bogans, Kaspars Tars, Andris Zeltins and Ina Balke
Int. J. Mol. Sci. 2023, 24(6), 5347; https://doi.org/10.3390/ijms24065347 - 10 Mar 2023
Cited by 1 | Viewed by 1876
Abstract
Sobemoviruses encode serine-like 3C proteases (Pro) that participate in the processing and maturation of other virus-encoded proteins. Its cis and trans activity is mediated by the naturally unfolded virus-genome-linked protein (VPg). Nuclear magnetic resonance studies show a Pro–VPg complex interaction and VPg tertiary [...] Read more.
Sobemoviruses encode serine-like 3C proteases (Pro) that participate in the processing and maturation of other virus-encoded proteins. Its cis and trans activity is mediated by the naturally unfolded virus-genome-linked protein (VPg). Nuclear magnetic resonance studies show a Pro–VPg complex interaction and VPg tertiary structure; however, information regarding structural changes of the Pro–VPg complex during interaction is lacking. Here, we solved a full Pro–VPg 3D structure of ryegrass mottle virus (RGMoV) that demonstrates the structural changes in three different conformations due to VPg interaction with Pro. We identified a unique site of VPg interaction with Pro that was not observed in other sobemoviruses, and observed different conformations of the Pro β2 barrel. This is the first report of a full plant Pro crystal structure with its VPg cofactor. We also confirmed the existence of an unusual previously unmapped cleavage site for sobemovirus Pro in the transmembrane domain: E/A. We demonstrated that RGMoV Pro in cis activity is not regulated by VPg and that in trans, VPg can also mediate Pro in free form. Additionally, we observed Ca2+ and Zn2+ inhibitory effects on the Pro cleavage activity. Full article
(This article belongs to the Collection Feature Papers in “Molecular Biology”)
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20 pages, 7184 KiB  
Article
Amino Acid Substitutions at P1 Position Change the Inhibitory Activity and Specificity of Protease Inhibitors BmSPI38 and BmSPI39 from Bombyx mori
by Youshan Li, Meng Wei, Jie Zhang, Rui Zhu, Yuan Wang, Zhaofeng Zhang, Changqing Chen and Ping Zhao
Molecules 2023, 28(5), 2073; https://doi.org/10.3390/molecules28052073 - 22 Feb 2023
Cited by 4 | Viewed by 2317
Abstract
It was found that silkworm serine protease inhibitors BmSPI38 and BmSPI39 were very different from typical TIL-type protease inhibitors in sequence, structure, and activity. BmSPI38 and BmSPI39 with unique structure and activity may be good models for studying the relationship between the structure [...] Read more.
It was found that silkworm serine protease inhibitors BmSPI38 and BmSPI39 were very different from typical TIL-type protease inhibitors in sequence, structure, and activity. BmSPI38 and BmSPI39 with unique structure and activity may be good models for studying the relationship between the structure and function of small-molecule TIL-type protease inhibitors. In this study, site-directed saturation mutagenesis at the P1 position was conducted to investigate the effect of P1 sites on the inhibitory activity and specificity of BmSPI38 and BmSPI39. In-gel activity staining and protease inhibition experiments confirmed that BmSPI38 and BmSPI39 could strongly inhibit elastase activity. Almost all mutant proteins of BmSPI38 and BmSPI39 retained the inhibitory activities against subtilisin and elastase, but the replacement of P1 residues greatly affected their intrinsic inhibitory activities. Overall, the substitution of Gly54 in BmSPI38 and Ala56 in BmSPI39 with Gln, Ser, or Thr was able to significantly enhance their inhibitory activities against subtilisin and elastase. However, replacing P1 residues in BmSPI38 and BmSPI39 with Ile, Trp, Pro, or Val could seriously weaken their inhibitory activity against subtilisin and elastase. The replacement of P1 residues with Arg or Lys not only reduced the intrinsic activities of BmSPI38 and BmSPI39, but also resulted in the acquisition of stronger trypsin inhibitory activities and weaker chymotrypsin inhibitory activities. The activity staining results showed that BmSPI38(G54K), BmSPI39(A56R), and BmSPI39(A56K) had extremely high acid–base and thermal stability. In conclusion, this study not only confirmed that BmSPI38 and BmSPI39 had strong elastase inhibitory activity, but also confirmed that P1 residue replacement could change their activity and inhibitory specificity. This not only provides a new perspective and idea for the exploitation and utilization of BmSPI38 and BmSPI39 in biomedicine and pest control, but also provides a basis or reference for the activity and specificity modification of TIL-type protease inhibitors. Full article
(This article belongs to the Special Issue Design, Synthesis, and Evaluation of Protease Inhibitors)
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17 pages, 8446 KiB  
Article
Tandem Multimerization Can Enhance the Structural Homogeneity and Antifungal Activity of the Silkworm Protease Inhibitor BmSPI39
by Youshan Li, Yuan Wang, Rui Zhu, Xi Yang, Meng Wei, Zhaofeng Zhang, Changqing Chen and Ping Zhao
Cells 2023, 12(5), 693; https://doi.org/10.3390/cells12050693 - 22 Feb 2023
Cited by 1 | Viewed by 1807
Abstract
Previous studies have shown that BmSPI39, a serine protease inhibitor of silkworm, can inhibit virulence-related proteases and the conidial germination of insect pathogenic fungi, thereby enhancing the antifungal capacity of Bombyx mori. The recombinant BmSPI39 expressed in Escherichia coli has poor structural [...] Read more.
Previous studies have shown that BmSPI39, a serine protease inhibitor of silkworm, can inhibit virulence-related proteases and the conidial germination of insect pathogenic fungi, thereby enhancing the antifungal capacity of Bombyx mori. The recombinant BmSPI39 expressed in Escherichia coli has poor structural homogeneity and is prone to spontaneous multimerization, which greatly limits its development and application. To date, the effect of multimerization on the inhibitory activity and antifungal ability of BmSPI39 remains unknown. It is urgent to explore whether a BmSPI39 tandem multimer with better structural homogeneity, higher activity and a stronger antifungal ability can be obtained by protein engineering. In this study, the expression vectors of BmSPI39 homotype tandem multimers were constructed using the isocaudomer method, and the recombinant proteins of tandem multimers were obtained by prokaryotic expression. The effects of BmSPI39 multimerization on its inhibitory activity and antifungal ability were investigated by protease inhibition and fungal growth inhibition experiments. In-gel activity staining and protease inhibition assays showed that tandem multimerization could not only greatly improve the structural homogeneity of the BmSPI39 protein, but also significantly increase its inhibitory activity against subtilisin and proteinase K. The results of conidial germination assays showed that tandem multimerization could effectively enhance the inhibitory ability of BmSPI39 on the conidial germination of Beauveria bassiana. A fungal growth inhibition assay showed that BmSPI39 tandem multimers had certain inhibitory effects on both Saccharomyces cerevisiae and Candida albicans. The inhibitory ability of BmSPI39 against these the above two fungi could be enhanced by tandem multimerization. In conclusion, this study successfully achieved the soluble expression of tandem multimers of the silkworm protease inhibitor BmSPI39 in E. coli and confirmed that tandem multimerization can improve the structural homogeneity and antifungal ability of BmSPI39. This study will not only help to deepen our understanding of the action mechanism of BmSPI39, but also provide an important theoretical basis and new strategy for cultivating antifungal transgenic silkworms. It will also promote its exogenous production and development and application in the medical field. Full article
(This article belongs to the Special Issue Cellular Events in Insect Development, Immunity, and Reproduction)
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15 pages, 5875 KiB  
Article
Alpha-1 Antitrypsin Inhibits Tumorigenesis and Progression of Colitis-Associated Colon Cancer through Suppression of Inflammatory Neutrophil-Activated Serine Proteases and IGFBP-3 Proteolysis
by Qing Cai, Minsun Kim, Aki Harada, Michael O. Idowu, Gobalakrishnan Sundaresan, Jamal Zweit and Youngman Oh
Int. J. Mol. Sci. 2022, 23(22), 13737; https://doi.org/10.3390/ijms232213737 - 8 Nov 2022
Cited by 12 | Viewed by 2821
Abstract
Colitis-associated colon cancer (CAC) accompanies the massive infiltration of neutrophils during tumorigenesis and progression of CAC. Depletion of neutrophils in circulation results in significant inhibition of tumor incidence in CAC. However, the underlying mechanisms are largely unclear. In this study, we provide evidence [...] Read more.
Colitis-associated colon cancer (CAC) accompanies the massive infiltration of neutrophils during tumorigenesis and progression of CAC. Depletion of neutrophils in circulation results in significant inhibition of tumor incidence in CAC. However, the underlying mechanisms are largely unclear. In this study, we provide evidence for the crucial involvement of inflammatory neutrophil-activated serine proteases (NSPs) on the dysregulation of the anti-inflammatory and antitumor IGFBP-3/IGFBP-3R signaling axis in CAC using a chronic AOM/DSS mouse model. We also provide preclinical evidence for α1-antitrypsin (AAT) as a preventive and as a therapeutic for CAC. AAT administration not only prevented colitis-associated tumorigenesis but also inhibited established CAC. AOM/DSS treatment results in the significant activation of NSPs, leading to CAC through increased pro-inflammatory cytokines and decreased anti-inflammatory and antitumor IGFBP-3. Collectively, these data suggest that the NSPs proteolyze IGFBP-3, whereas AAT inhibits chronic colonic inflammation-induced NSP activity and subsequently suppresses IGFBP-3 proteolysis. Therefore, the anti-inflammatory and antitumor functions of the IGFBP-3/IGFBP-3R axis are restored. AAT mimicking small peptides also showed their inhibitory effects on NSP-induced IGFBP-3 proteolysis. These results suggest that targeting the NSP-IGFBP-3/IGFBP-3R axis using NSP inhibitors such as AAT and the AAT mimics and IGFBP-3R agonists could lead to novel approaches for the prevention and treatment of CAC. Full article
(This article belongs to the Special Issue Focus on Gastrointestinal Diseases 2.0: Inflammation)
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