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Characterization of a Bowman-Birk type trypsin inhibitor purified from seeds of Solanum surattense.

ABSTRACT: A Bowman-Birk type trypsin inhibitor protein (SSTI) from seeds of the medicinal plant Solanum surattense was isolated, purified and characterized. SSTI showed a single band on SDS-PAGE corresponding to 11.4 kDa molecular weight. It is a glycoprotein (2.8% glycosylation) that differentially interacted with trypsin and chymotrypsin in a concentration-dependent manner. Its peptide sequence is similar to other Bowman-Birk type protease inhibitors found in Glycine max and Phaseolus acutifolius. The inhibitory activity was stable over a wide range of pH (1-10) and temperatures (10-100° C). Far-UV Circular Dichroism (CD) studies showed that SSTI contains β sheets (~ 23%) and α helix (~ 6%) and demonstrated structural stability at wide pH and high temperature. The kinetic analysis revealed a noncompetitive (mixed) type nature of SSTI and low inhibitor constant (Ki) values (16.6 × 10^-8 M) suggested strong inhibitory activity. Isothermal titration calorimetric analysis revealed its high affinity towards trypsin with dissociation constant (K_d) 2.28 µM.

SUBMITTER: Herwade AP

PROVIDER: S-EPMC8060351 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Identification and Target-Modification of SL-BBI: A Novel Bowman-Birk Type Trypsin Inhibitor from Sylvirana latouchii.

Project description:The peptides from the ranacyclin family share similar active disulphide loop with plant-derived Bowman-Birk type inhibitors, some of which have the dual activities of trypsin inhibition and antimicrobial. Herein, a novel Bowman-Birk type trypsin inhibitor of the ranacyclin family was identified from the skin secretion of broad-folded frog (Sylvirana latouchii) by molecular cloning method and named as SL-BBI. After chemical synthesis, it was proved to be a potent inhibitor of trypsin with a Ki value of 230.5 nM and showed weak antimicrobial activity against tested microorganisms. Modified analogue K-SL maintains the original inhibitory activity with a Ki value of 77.27 nM while enhancing the antimicrobial activity. After the substitution of active P1 site to phenylalanine and P2' site to isoleucine, F-SL regenerated its inhibitory activity on chymotrypsin with a Ki value of 309.3 nM and exhibited antiproliferative effects on PC-3, MCF-7 and a series of non-small cell lung cancer cell lines without cell membrane damage. The affinity of F-SL for the β subunits in the yeast 20S proteasome showed by molecular docking simulations enriched the understanding of the possible action mode of Bowman-Birk type inhibitors. Further mechanistic studies have shown that F-SL can activate caspase 3/7 in H157 cells and induce apoptosis, which means it has the potential to become an anticancer agent.

| S-EPMC7565067 | biostudies-literature

A fungal effector and a rice NLR protein have antagonistic effects on a Bowman-Birk trypsin inhibitor.

Project description:Bowman-Birk trypsin inhibitors (BBIs) play important roles in animal and plant immunity, but how these protease inhibitors are involved in the immune system remains unclear. Here, we show that the rice (Oryza sativa) BBI protein APIP4 is a common target of a fungal effector and an NLR receptor for innate immunity. APIP4 exhibited trypsin inhibitor activity in vitro and in vivo. Knockout of APIP4 in rice enhanced susceptibility, and overexpression of APIP4 increased resistance to the fungal pathogen Magnaporthe oryzae. The M. oryzae effector AvrPiz-t interacted with APIP4 and suppressed APIP4 trypsin inhibitor activity. By contrast, the rice NLR protein Piz-t interacted with APIP4, enhancing APIP4 transcript and protein levels, and protease inhibitor activity. Our findings reveal a novel host defence mechanism in which a host protease inhibitor targeted by a fungal pathogen is protected by an NLR receptor.

| S-EPMC7589341 | biostudies-literature

A Bowman-Birk type chymotrypsin inhibitor peptide from the amphibian, Hylarana erythraea.

Project description:The first amphibian skin secretion-derived Bowman-Birk type chymotrypsin inhibitor is described here from the Asian green frog, Hylarana erythraea, and was identified by use of molecular cloning and tandem mass spectrometric amino acid sequencing. It was named Hylarana erythraea chymotrypsin inhibitor (HECI) and in addition to inhibition of chymotrypsin (Ki = 3.92 ± 0.35 μM), the peptide also inhibited the 20 S proteasome (Ki = 8.55 ± 1.84 μM). Additionally, an analogue of HECI, named K9-HECI, in which Phe9 was substituted by Lys9 at the P1 position, was functional as a trypsin inhibitor. Both peptides exhibited anti-proliferation activity against the human cancer cell lines, H157, PC-3 and MCF-7, up to a concentration of 1 mM and possessed a low degree of cytotoxicity on normal cells, HMEC-1. However, HECI exhibited higher anti-proliferative potency against H157. The results indicate that HECI, inhibiting chymotryptic-like activity of proteasome, could provide new insights in treatment of lung cancer.

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Multimeric Association of Purified Novel Bowman-Birk Inhibitor From the Medicinal Forage Legume Mucuna pruriens (L.) DC.

Project description:A Bowman-Birk protease, i.e., Mucuna pruriens trypsin inhibitor (MPTI), was purified from the seeds by 55.702-fold and revealed a single trypsin inhibitor on a zymogram with a specific activity of 202.31 TIU/mg of protein. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions, the protease trypsin inhibitor fraction [i.e., trypsin inhibitor non-reducing (TINR)] exhibited molecular weights of 74 and 37 kDa, and under reducing conditions [i.e., trypsin inhibitor reducing (TIR)], 37 and 18 kDa. TINR-37 revealed protease inhibitor activity on native PAGE and 37 and 18 kDa protein bands on SDS-PAGE. TINR-74 showed peaks corresponding to 18.695, 37.39, 56.085, and 74.78 kDa on ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization/quadrupole time-of-flight-mass spectrometry (ESI/QTOF-MS). Similarly, TINR-37 displayed 18.695 and 37.39 kDa peaks. Furthermore, TIR-37 and TIR-18 exhibited peaks corresponding to 37.39 and 18.695 kDa. Multiple peaks observed by the UPLC-ESI/QTOF analysis revealed the multimeric association, confirming the characteristic and functional features of Bowman-Birk inhibitors (BBIs). The multimeric association helps to achieve more stability, thus enhancing their functional efficiency. MPTI was found to be a competitive inhibitor which again suggested that it belongs to the BBI family of inhibitors, displayed an inhibitor constant of 1.3 × 10-6 M, and further demonstrates potent anti-inflammatory activity. The study provided a comprehensive basis for the identification of multimeric associates and their therapeutic potential, which could elaborate the stability and functional efficiency of the MPTI in the native state from M. pruriens.

| S-EPMC8655843 | biostudies-literature

Crystal structure of the Bowman-Birk Inhibitor from Vigna unguiculata seeds in complex with beta-trypsin at 1.55 A resolution and its structural properties in association with proteinases.

Project description:The structure of the Bowman-Birk inhibitor from Vigna unguiculata seeds (BTCI) in complex with beta-trypsin was solved and refined at 1.55 A to a crystallographic R(factor) of 0.154 and R(free) of 0.169, and represents the highest resolution for a Bowman-Birk inhibitor structure to date. The BTCI-trypsin interface is stabilized by hydrophobic contacts and hydrogen bonds, involving two waters and a polyethylene glycol molecule. The conformational rigidity of the reactive loop is characteristic of the specificity against trypsin, while hydrophobicity and conformational mobility of the antichymotryptic subdomain confer the self-association tendency, indicated by atomic force microscopy, of BTCI in complex and free form. When BTCI is in binary complexes, no significant differences in inhibition constants for producing a ternary complex with trypsin and chymotrypsin were detected. These results indicate that binary complexes present no conformational change in their reactive site for both enzymes confirming that these sites are structurally independent. The free chymotrypsin observed in the atomic force microscopy assays, when the ternary complex is obtained from BTCI-trypsin binary complex and chymotrypsin, could be related more to the self-association tendency between chymotrypsin molecules and the flexibility of the reactive site for this enzyme than to binding-related conformational changes.

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A structural and functional analogue of a Bowman-Birk-type protease inhibitor from Odorrana schmackeri.

Project description:Frog skin secretions contain complex peptidomes and peptidic protease inhibitors that are one of the biologically and structurally described groups of components. In the present study, by use of molecular 'shotgun' cloning and LC MS/MS fractionation sequencing, a novel Bowman-Birk-type heptadecapeptide (AALKGCWTKSIPPKPCF-amide), named Odorrana schmackeriTrypsin Inhibitor (OSTI), with a canonical Cys6-Cys16 disulfide bridge, was isolated and identified in piebald odorous frog (O. schmackeri) skin secretion. A synthetic replicate of OSTI-exhibited trypsin inhibitory activity with a Ki value of 0.3 ± 0.04 nM and also a tryptase inhibitory effect with a Ki of 2.5 ± 0.6 μM. This is the first time that this property has been reported for a peptide originating from amphibian sources. In addition, substituting lysine (K) with phenylalanine (F) at the presumed P1 position, completely abrogated the trypsin and tryptase inhibition, but produced a strong chymotrypsin inhibition with a Ki of 1.0 ± 0.1 μM. Thus, the specificity of this peptidic protease inhibitor could be optimized through modifying the amino acid residue at the presumed P1 position and this novel native OSTI, along with its analogue, [Phe9]-OSTI, have expanded the potential drug discovery and development pipeline directed towards alleviation of serine protease-mediated pathologies.

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Bowman‒Birk Inhibitor Suppresses Herpes Simplex Virus Type 2 Infection of Human Cervical Epithelial Cells.

Project description:The Bowman‒Birk inhibitor (BBI), a protease inhibitor derived from soybeans, has been extensively studied in anti-tumor and anti-inflammation research. We recently reported that BBI has an anti-HIV-1 property in primary human macrophages. Because HSV-2 infection plays a role in facilitating HIV-1 sexual transmission, we thus examined whether BBI has the ability to inhibit HSV-2 infection. We demonstrated that BBI could potently inhibit HSV-2 replication in human cervical epithelial cells (End1/E6E7). This BBI-mediated HSV-2 inhibition was partially through blocking HSV-2-mediated activation of NF-κB and p38 MAPK pathways. In addition, BBI could activate the JAK/STAT pathway and enhance the expression of several antiviral interferon-stimulated genes (ISGs). Furthermore, BBI treatment of End1/E6E7 cells upregulated the expression of tight junction proteins and reduced HSV-2-mediated cellular ubiquitinated proteins' degradation through suppressing the ubiquitin‒proteasome system. These observations indicate that BBI may have therapeutic potential for the prevention and treatment of HSV-2 infections.

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Project description:Bowman-Birk inhibitor (BBI) is a soybean-derived protease inhibitor that has anti-inflammation and anti-HIV effect. Here, we further investigated the anti-HIV action of BBI in macrophages, focusing on its effect on viral entry. We found that BBI could significantly block HIV entry into macrophages. Investigation of the mechanism(s) of the BBI action on HIV inhibition showed that BBI down-regulated the expression of CD4 receptor (as much as 80%) and induced the production of the CC chemokines (up to 60 folds at protein level) in macrophages. This inhibitory effect of BBI on HIV entry could be blocked by the neutralization antibodies to CC chemokines. These findings indicate that BBI may have therapeutic potential as a viral entry inhibitor for the prevention and treatment of HIV infection.

| S-EPMC5890813 | biostudies-literature

Bowman birk inhibitor concentrate and oral leukoplakia: a randomized phase IIb trial.

Project description:Oral premalignancy serves as an ideal model for study of chemopreventive agents. Although 13-cis-retinoic acid showed reversal of oral premalignancy, toxicity, and reversal of clinical response after cessation of therapy obviated its widespread use. A search for nontoxic agents with cancer preventive activity led us to evaluate Bowman Birk Inhibitor (BBI) formulated as BBI Concentrate (BBIC). We previously reported encouraging results in a phase IIa trial of BBIC in patients with oral leukoplakia with measurable clinical responses and favorable biomarker changes. On the basis of these results, we undertook a randomized, placebo controlled phase IIb trial with patients receiving BBIC or placebo for 6 months, with assessment of clinical response and change in lesion area as primary end point and an intent-to-treat analysis. One hundred and thirty two subjects were randomized; and 89 subjects completed six months on study drug or placebo. Both placebo and BBIC showed a statistically significant decrease in mean lesion area of 17.1% and 20.6%, respectively, and partial or greater clinical responses of 30% and 28% respectively. No significant difference between placebo and study drug arms was observed. Histologic review, review of photographs of lesions, and comparison of serum neu protein and oral mucosal cell protease activity also did not show significant differences between study arms. Probable reasons for these negative results were considered, are discussed, and include a placebo with non-BBIC clinical activity and reduced pharmacokinetic availability of the second batch of BBIC. This experience should be a strong cautionary note to those considering "Green" chemoprevention.

| S-EPMC3730525 | biostudies-literature

Discovery and Rational Design of a Novel Bowman-Birk Related Protease Inhibitor.

Project description:Anuran amphibian skin secretions are a rich source of peptides, many of which represent novel protease inhibitors and can potentially act as a source for protease inhibitor drug discovery. In this study, a novel bioactive Bowman-Birk type inhibitory hexadecapeptide of the Ranacyclin family from the defensive skin secretion of the Fukien gold-striped pond frog, Pelophlax plancyi fukienesis, was successfully isolated and identified, named PPF-BBI. The primary structure of the biosynthetic precursor was deduced from a cDNA sequence cloned from a skin-derived cDNA library, which contains a consensus motif representative of the Bowman-Birk type inhibitor. The peptide was chemically synthesized and displayed a potent inhibitory activity against trypsin (Ki of 0.17 µM), as well as an inhibitory activity against tryptase (Ki of 30.73 µM). A number of analogues of this peptide were produced by rational design. An analogue, which substituted the lysine (K) at the predicted P1 position with phenylalanine (F), exhibited a potent chymotrypsin inhibitory activity (Ki of 0.851 µM). Alternatively, a more potent protease inhibitory activity, as well as antimicrobial activity, was observed when P16 was replaced by lysine, forming K16-PPF-BBI. The addition of the cell-penetrating peptide Tat with a trypsin inhibitory loop resulted in a peptide with a selective inhibitory activity toward trypsin, as well as a strong antifungal activity. This peptide also inhibited the growth of two lung cancer cells, H460 and H157, demonstrating that the targeted modifications of this peptide could effectively and efficiently alter its bioactivity.

| S-EPMC6681222 | biostudies-literature

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