Search Results (14)

Impaired endothelial function, excessive oxidative stress, and persistent bacterial infection collectively contribute to delayed healing of diabetic chronic wounds. Methylglyoxal (MGO)-induced metabolic stress is a critical driver of endothelial injury; however, effective multifunctional strategies capable of restoring vascular function and maintaining cellular homeostasis remain limited. In this study, the endothelial protective potential of an amphibian-derived Bowman–Birk inhibitor (BBI)-type peptide, OSTI-1872, and its rationally designed structural analogues were investigated using an MGO-induced injury model in human umbilical vein endothelial cells (HUVECs). Among the tested peptides, the optimised analogue OSTI-2337 exhibited superior protective activity. OSTI-2337 markedly attenuated MGO-induced oxidative stress, restored nitric oxide bioavailability, enhanced VEGF expression, promoted endothelial migration and tube formation, and reduced oxidative DNA damage. Mechanistically, these effects were associated with coordinated regulation of MGO detoxification and redox homeostasis, as evidenced by enhanced GLO1 expression and modulation of the PI3K/AKT/GSK3β/Nrf2 axis, accompanied by increased expression of downstream antioxidant proteins HO-1 and NQO1. In addition, OSTI-1872 and OSTI-2337 displayed antibacterial activity against representative bacterial strains, suggesting their potential advantages for complex diabetic wound environments. Collectively, these findings demonstrate that structural optimisation significantly enhances the biological activity of amphibian BBI-type peptides and identify OSTI-2337 as a multifunctional peptide candidate capable of integrating endothelial protection, MGO detoxification, redox regulation, and antibacterial activity for the management of diabetes-associated vascular injury and chronic wound complications. Full article

Cell-penetrating peptide (CPP) conjugation represents a promising strategy for enhancing the biological activity of therapeutic peptides. In this study, three analogues were designed by conjugating the trypsin inhibitory loop (TIL) derived from a Bowman–Birk-type inhibitor with the transactivator of transcription (TAT) peptide to improve their bioactivity. All TAT-TIL conjugates exhibited significantly enhanced antimicrobial activity compared with the parent peptide. Notably, the analogue containing a glycine linker (-GG-) showed further improvement in antiproliferative activity against cancer cells, indicating the potential role of linker design in optimizing peptide function. All analogues exhibited low hemolytic activity at the highest tested concentrations, although increased cytotoxicity toward normal HaCaT cells was observed, suggesting the need for further optimization of selectivity. Interestingly, comparable antimicrobial activities were observed regardless of protease inhibitory capacity, indicating that protease inhibition is not essential for the enhanced biological effects. Overall, TAT conjugation significantly improves the biological activity of Bowman–Birk-type inhibitor-derived peptides, and the incorporation of a glycine linker further enhances their functional properties. These findings support CPP-mediated peptide modification as an effective strategy for developing potential antimicrobial and anticancer peptide candidates. Full article

Chickpea (Cicer arietinum L.) generally contains lower levels of these compounds than many other legumes, yet information on Hungarian chickpea cultivars is scarce. This study aimed to characterize protein-based antinutritional factors in twenty chickpeas grown under different agroclimatic conditions over three consecutive years (15 samples from seven Hungarian cultivars from three cultivation areas, and five commercially available foreign genotypes). Protein profiles were examined by SDS-PAGE and native PAGE, while trypsin inhibitor activity (TIA) was quantified spectrophotometrically according to ISO 14902, and lectin activity was determined using a hemagglutination assay. SDS-PAGE revealed highly similar protein patterns among samples, indicating comparable overall protein composition. Native PAGE combined with activity staining confirmed the presence of Kunitz-type trypsin inhibitors, with multiple isoforms detected, but no Bowman–Birk-type inhibitor activity was observed. TIA values were low (0.49–4.07 mg inhibited trypsin/g), and lectin activities were generally low (1–2.5 HU/mg flour; only one sample reached 5 HU/mg) or undetectable. Neither cultivation area nor growing year had a significant effect on TIA or lectin activity, confirmed by statistical analyses. Overall, Hungarian chickpea varieties exhibited low and stable levels of antinutritional proteins, supporting their favorable nutritional quality and suitability for human consumption and expanded cultivation under Hungarian agroclimatic conditions. Full article

Drug-resistant bacteria cause millions of global infections each year, and the development of alternative antimicrobial drugs has become a serious undertaking. Currently, peptides with antimicrobial activity represent potential candidates for new antibiotic discovery as they are less likely to cause drug resistance in bacteria. In this study, bifunctional peptides with potent trypsin-inhibitory activity and antimicrobial activity were obtained by rational computation-based structural modifications to a novel Bowman–Birk-type inhibitor (BBI) peptide. The analogues not only displayed potent bacterial killing ability against two drug-resistant bacteria strains of E. coli but also an excellent safety profile, as assessed by low haemolytic activity and low anti-proliferation activity on HaCaT cells. Throughout the molecular dynamics simulations, the peptides exhibited stable adsorption onto the mixed POPE/POPG membrane; most amino acid residues of the AMPs remained bound to the membrane surface, with a few amino acid residues partially penetrating the membrane interior. This showed that the electrostatic interactions were the dominant driving force mediating the peptide–membrane associations. In addition, the tested peptides displayed a degree of stability in the presence of salt ions, serum, and trypsin. These modified peptides thus possess potential as clinical antibacterial agents, and the strategies used in structural modification may also provide a different path to developing new antimicrobial peptides. Full article

Soybean meal (SBM) is extensively used as a predominant protein source in animal feed. However, raw soybean cannot be directly utilized in animal feed, due to the presence of the Kunitz trypsin inhibitor (KTi) and the Bowman–Birk protease inhibitor (BBi). These antinutritional factors inhibit the digestive enzymes in animals, trypsin and chymotrypsin, resulting in poor animal performance. To inactivate the activity of protease inhibitors, SBM is subjected to heat processing, a procedure that can negatively impact the soybean protein quality. Thus, it would be beneficial to develop soybean varieties with little or no trypsin inhibitors. In this study, we report on the creation of experimental soybean lines with significantly reduced levels of Bowman–Birk protease inhibitors. RNA interference (RNAi) technology was employed to generate several transgenic soybean lines. Some of these BBi knockdown soybean lines showed significantly lower amounts of both trypsin and chymotrypsin inhibitor activities. Western blot analysis revealed the complete absence of BBi in selected RNAi-derived lines. RNA sequencing (RNAseq) analysis demonstrated a drastic reduction in the seed-specific expression of BBi genes in the transgenic soybean lines during seed development. Confocal fluorescence immunolabeling studies showed that the accumulation of BBi was drastically diminished in BBi knockdown lines compared to wild-type soybeans. The absence of BBi in the transgenic soybean did not alter the overall protein, oil, and sulfur amino acid content of the seeds compared to wild-type soybeans. The seed protein from the BBi knockdown lines were more rapidly hydrolyzed by trypsin and chymotrypsin compared to the wild type, indicating that the absence of BBi enhances protein digestibility. Our study suggests that these BBi knockdown lines could be a valuable resource in order for plant breeders to incorporate this trait into commercial soybean cultivars, potentially enabling the use of raw soybeans in animal feed. Full article

Despite the high quality of soybean protein, raw soybeans and soybean meal cannot be directly included in animal feed mixtures due to the presence of Kunitz (KTi) and Bowman–Birk protease inhibitors (BBis), which reduces animal productivity. Heat treatment can substantially inactivate trypsin and chymotrypsin inhibitors (BBis), but such treatment is energy-intensive, adds expense, and negatively impacts the quality of seed proteins. As an alternative approach, we have employed CRISPR/Cas9 gene editing to create mutations in BBi genes to drastically lower the protease inhibitor content in soybean seed. Agrobacterium-mediated transformation was used to generate several stable transgenic soybean events. These independent CRISPR/Cas9 events were examined in comparison to wild-type plants using Sanger sequencing, proteomic analysis, trypsin/chymotrypsin inhibitor activity assays, and qRT-PCR. Collectively, our results demonstrate the creation of an allelic series of loss-of-function mutations affecting the major BBi gene in soybean. Mutations in two of the highly expressed seed-specific BBi genes lead to substantial reductions in both trypsin and chymotrypsin inhibitor activities. Full article

This study explores the impact of RNAi in terms of selectively inhibiting the expression of the OsBBTI5 gene, with the primary objective of uncovering its involvement in the molecular mechanisms associated with salt tolerance in rice. OsBBTI5, belonging to the Bowman–Birk inhibitor (BBI) family gene, is known for its involvement in plant stress responses. The gene was successfully cloned from rice, exhibiting transcriptional self-activation in yeast. A yeast two-hybrid assay confirmed its specific binding to OsAPX2 (an ascorbate peroxidase gene). Transgenic OsBBTI5-RNAi plants displayed insensitivity to varying concentrations of 24-epibrassinolide in the brassinosteroid sensitivity assay. However, they showed reduced root and plant height at high concentrations (10 and 100 µM) of GA₃ immersion. Enzyme activity assays revealed increased peroxidase (POD) and superoxide dismutase (SOD) activities and decreased malondialdehyde (MDA) content under 40-60 mM NaCl. Transcriptomic analysis indicated a significant upregulation of photosynthesis-related genes in transgenic plants under salt stress compared to the wild type. Notably, this study provides novel insights, suggesting that the BBI gene is part of the BR signaling pathway, and that OsBBTI5 potentially enhances stress tolerance in transgenic plants through interaction with the salt stress-related gene OsAPX2. Full article

We previously reported that Lys175 in the region of the active site of chymotrypsin (Csin) could be site-selectively modified by using an N-hydroxy succinimide (NHS) ester of the peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester [NHS-Suc-Ala-Ala-Phe^P(OPh)₂]. In this study, the Lys175-selective modification method was expanded to incorporate functional groups into Lys 175 in Csin. Two types of peptidyl phosphonate derivatives with the dansyl group (Dan) as a functional molecule, Dan-β-Ala-[Asp(NHS) or Glu(NHS)]-Ala-Ala-(R)-Phe^P(OPh)₂ (DanD and DanE, respectively), were synthesized, and their action was evaluated when modifying Lys175 in Csin. Ion-exchange chromatography (IEC), fluorescence spectroscopy, and LC-MS/MS were used to analyze the products from the reaction of Csin with DanD or DanE. By IEC and LC-MS/MS, the results showed that DanE reacted with Csin more effectively than DanD to produce the modified Csin (DanMCsin) bearing Dan at Lys175. DanMCsin exhibited an enzymatic activity corresponding to 1/120 of Csin against Suc-Ala-Ala-Phe-pNA. In addition, an effect of Lys175 modification on the access of the proteinaceous Bowman–Birk inhibitor to the active site of DanMCsin was investigated. In conclusion, by using a peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester, we demonstrated that a functional group could be incorporated into Lys175 in Csin. Full article

Plant-derived protease inhibitors (PI), such as Bowman-Birk inhibitors and Kunitz-type inhibitors, have been suggested to negatively affect dietary protein digestion by blocking the activity of trypsin and chymotrypsin in the human gastrointestinal system. In addition, some PIs may possess proinflammatory activities. However, there is also scientific evidence on some beneficial effects of PIs, for example, gut-related anti-inflammatory and chemopreventive activities in vitro and in vivo. Some PIs are sensitive to processing and digestion; thus, their survival is an important aspect when considering their positive and negative bioactivities. The aim of this review was to evaluate the relevance of PIs in protein digestion in humans and to discuss the potential of PIs from whole foods and as purified compounds in decreasing symptoms of bowel-related conditions. Based on the reviewed literature, we concluded that while the complex interactions affecting plant protein digestibility and bioavailability remain unclear, PI supplements could be considered for targeted purposes to mitigate inflammation and gastric pain. Full article

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. Full article

Serine protease inhibitors are found in plants, animals and microorganisms, where they play important roles in many physiological and pathological processes. Inhibitor scaffolds based on natural proteins and peptides have gradually become the focus of current research as they tend to bind to their targets with greater specificity than small molecules. In this report, a novel Bowman–Birk type inhibitor, named ranacyclin-NF (RNF), is described and was identified in the skin secretion of the East Asian frog, Pelophylax nigromaculatus. A synthetic replicate of the peptide was subjected to a series of functional assays. It displayed trypsin inhibitory activity with an inhibitory constant, Ki, of 447 nM and had negligible direct cytotoxicity. No observable direct antimicrobial activity was found but RNF improved the therapeutic potency of Gentamicin against Methicillin-resistant Staphylococcus aureus (MRSA). RNF shared significant sequence similarity to previously reported and related inhibitors from Odorrana grahami (ORB) and Rana esculenta (ranacyclin-T), both of which were found to be multi-functional. Two analogues of RNF, named ranacyclin-NF1 (RNF1) and ranacyclin-NF3L (RNF3L), were designed based on some features of ORB and ranacyclin-T to study structure–activity relationships. Structure–activity studies demonstrated that residues outside of the trypsin inhibitory loop (TIL) may be related to the efficacy of trypsin inhibitory activity. Full article

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. Full article

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. Full article

The Maize Wip1 gene encodes a wound-induced Bowman-Birk inhibitor (BBI) protein which is a type of serine protease inhibitor, and its expression is induced by wounding or infection, conferring resistance against pathogens and pests. In this study, the maize Wip1 promoter was isolated and its function was analyzed. Different truncated Wip1 promoters were fused upstream of the GUS reporter gene and transformed into Arabidopsis, tobacco and rice plants. We found that (1) several truncated maize Wip1 promoters led to strong GUS activities in both transgenic Arabidopsis and tobacco leaves, whereas low GUS activity was detected in transgenic rice leaves; (2) the Wip1 promoter was not wound-induced in transgenic tobacco leaves, but was induced by wounding in transgenic rice leaves; (3) the truncated Wip1 promoter had different activity in different organs of transgenic tobacco plants; (4) the transgenic plant leaves containing different truncated Wip1 promoters had low GUS transcripts, even though high GUS protein level and GUS activities were observed; (5) there was one transcription start site of Wip1 gene in maize and two transcription start sites of GUS in Wip1::GUS transgenic lines; (6) the adjacent 35S promoter which is present in the transformation vectors enhanced the activity of the truncated Wip1 promoters in transgenic tobacco leaves, but did not influence the disability of truncated Wip₁₂₃₁ promoter to respond to wounding signals. We speculate that an ACAAAA hexamer, several CAA trimers and several elements similar to ACAATTAC octamer in the 5'-untranslated region might contribute to the strong GUS activity in Wip₁₂₃₁ transgenic lines, meanwhile, compared to the 5'-untranslated region from Wip₁₂₃₁ transgenic lines, the additional upstream open reading frames (uORFs) in the 5'-untranslated region from Wip₁₇₃₇ transgenic lines might contribute to the lower level of GUS transcript and GUS activity. Full article