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BACKGROUND: The green peach aphid (Myzus persicae) is a severe agricultural crop pest that has developed resistance to most current control methods, requiring the urgent development of novel strategies. Plant proteinase inhibitors (PINs) are small proteins that protect plants against pathogens and/or herbivores, likely by preventing efficient protein digestion. RESULTS: We identified 67 protease genes in the transcriptomes of three M. persicae lineages (USDA‐Red, G002 and G006). Comparison of gene expression levels in aphid guts and whole aphids showed that several proteases, including a highly expressed serine protease, are significantly overexpressed in the guts. Furthermore, we identified three genes encoding serine protease inhibitors (SerPIN‐II1, 2 and 3) in Nicotiana benthamiana, which is a nonpreferred host for M. persicae. Using virus‐induced gene silencing (VIGS) with a tobacco rattle virus (TRV) vector and overexpression with a turnip mosaic virus (TuMV) vector, we demonstrated that N. benthamiana SerPIN‐II1 and SerPIN‐II2 cause reduced survival and growth, but do not affect aphid protein content. Likewise, SerPIN‐II3 overexpression reduced survival and growth, and serpin‐II3 knockout mutations, which we generated using CRISPR/Cas9, increased survival and growth. Protein content was significantly increased in aphids fed on SerPIN‐II3 overexpressing plants, yet it was decreased in aphids fed on serpin‐II3 mutants. CONCLUSION: Our results show that three PIN‐IIs from N. benthamiana, a nonpreferred host plant, effectively inhibit M. persicae survival and growth, thereby representing a new resource for the development of aphid‐resistant crop plants. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

• Growth of cotton (Gossypium arboreum) plant and application of salt-stress. • Identification of salt stress responsive proteinase inhibitor (PI) gene at transcript level using differential display technique. • Characterizations and expression profiling under various abiotic stresses of PI gene. Cotton production in the world is constrained due to various abiotic stresses. Among these, salt-stress plays a key role in declining the cotton yield. The study of genetic makeup in cotton plants for salt-stress responsive factors may open a way to understand the molecular mechanism of the plant against salt-stress. The differentially expressed gene, proteinase inhibitor (gpi) was isolated at the transcript level from salt-tolerant Gossypium arboreum (local cultivar FDH-171) by differential display technique, which was made full-length using gene RACER kit and was characterized through expression profiling under salt, cold and drought stresses, and predicted 3-D structure of gpi protein. The size of full-length gpi was found 496 bps with one intronic region from 74 to 156 bases in sequence. The gpi showed maximum homology with pi of Macleaya cordata 73%, Lactuca sativa 72%, Daucus carota 71%, Corchorus olitorius 70%, Corchorus capsularis 70%, Artemisia annua 70%, Trema orientale 67%, and Ricinus communis 65%. The protein homology search in protein database revealed that gpi has 63% homology with Linum usitatissimum trypsin inhibitor (LUTI). When gpi was subjected to predictive secondary structure, sheets, beta turns, coils and helixes were evident but with the most solvent buried area rather than exposed area according to solvent accessibility standards. The expression analysis of gpi in different tissues of salt-stressed G. arboreum plant indicated that gpi has 31-fold higher expression in salt-stressed root tissue, 13-fold in leaf tissue and no expression in stem tissue compared to control; while gpi indicated 13-fold expression under salt, 7-fold in cold and 4-fold in drought stress compared to control. Subsequent analysis showed that gpi gene expression was up-regulated and its transcription might enhance salt tolerance in plants. Therefore, gpi gene would be helpful to develop salt-stress tolerant cotton plants. [ABSTRACT FROM AUTHOR]

Context α2-Macroglobulin (α2-M) is believed to be a potential anti-irradiation agent, but related mechanisms remains unclear.Objective We investigated the irradiation protective effect of α2-M.Materials and methods A total of 10 Gy dose of irradiation was used to damage human skin fibroblasts. The influence of α2-M (100 µg/mL) on the proliferation, migration, invasion and apoptosis of fibroblasts was observed using Cell Counting Kit-8 (CCK8), wound healing, transwell, and flow cytometry. Malondialdehyde, superoxide dismutase and catalase was measured using related ELISA kits. The levels of mitochondrial membrane potential and calcium were detected using flow cytometry. The expression of transient receptor potential melastatin 2 (TRPM2) was investigated through western blotting and immunofluorescence staining.Results High purity of α2-M was isolated from Cohn fraction IV. α2-M significantly increased cell proliferation, migration, invasion, but suppressed cell apoptosis after irradiation. The promotion of cell proliferation, migration and invasion by α2-M exceeded over 50% compared group irradiation. The increased cell ratio in the S phase and decreased cell ratio in the G2 phase induced by irradiation were remarkably reversed by α2-M. α2-M markedly suppressed the increased oxidative stress level caused by irradiation. The mitochondrial damage induced by irradiation was improved by α2-M through inhibiting mitochondrial membrane potential loss, calcium and TRPM2 expression.Discussion and conclusions α2-M significantly promoted the decreased fibroblast viability and improved the mitochondria dysfunction caused by irradiation. α2-M might present anti-radiation effect through alleviating mitochondrial dysfunction caused by irradiation. This study could provide a novel understanding about the improvement of α2-M on irradiation-induced injury.

Urogenital malignancy accounts for one of the major causes of cancer-related deaths globally. Numerous studies have investigated novel molecular markers in the blood circulation, tumor tissue, or urine in order to assist in the clinical identification of tumors at early stages, predict the response of therapeutic strategies, and give accurate prognosis assessment. As an endogenous inhibitor of lysosomal cysteine proteinases, cystatin C plays an integral role in diverse processes. A substantial number of studies have indicated that it may be such a potential promising biomarker. Therefore, this review was intended to provide a detailed overview of the role of cystatin C in urogenital malignancy. [ABSTRACT FROM AUTHOR]

Context: a2-Macroglobulin (a2-M) is believed to be a potential anti-irradiation agent, but related mechanisms remains unclear. Objective: We investigated the irradiation protective effect of a2-M. Objective: We investigated the irradiation protective effect of a2-M. Materials and methods: A total of 10 Gy dose of irradiation was used to damage human skin fibroblasts. The influence of a2-M (100 mg/mL) on the proliferation, migration, invasion and apoptosis of fibroblasts was observed using Cell Counting Kit-8 (CCK8), wound healing, transwell, and flow cytometry. Malondialdehyde, superoxide dismutase and catalase was measured using related ELISA kits. The levels of mitochondrial membrane potential and calcium were detected using flow cytometry. The expression of transient receptor potential melastatin 2 (TRPM2) was investigated through western blotting and immunofluorescence staining. Results: High purity of a2-M was isolated from Cohn fraction IV. a2-M significantly increased cell proliferation, migration, invasion, but suppressed cell apoptosis after irradiation. The promotion of cell proliferation, migration and invasion by a2-M exceeded over 50% compared group irradiation. The increased cell ratio in the S phase and decreased cell ratio in the G2 phase induced by irradiation were remarkably reversed by a2-M. a2-M markedly suppressed the increased oxidative stress level caused by irradiation. The mitochondrial damage induced by irradiation was improved by a2-M through inhibiting mitochondrial membrane potential loss, calcium and TRPM2 expression. Discussion and conclusions: a2-M significantly promoted the decreased fibroblast viability and improved the mitochondria dysfunction caused by irradiation. a2-M might present anti-radiation effect through alleviating mitochondrial dysfunction caused by irradiation. This study could provide a novel understanding about the improvement of a2-M on irradiation-induced injury. [ABSTRACT FROM AUTHOR]

Background: In establishing the diagnosis — type 2 diabetes, there is a trend of multiple increases every year, which is a health issue. According to the state register, the statistics of patients with diabetes in Russia is about 17% of the total population of the country. The prevalence of diabetes is steadily increasing every year. Today, diabetes mellitus is the leader among the prevalence of non-communicable diseases after cancer, cardiovascular diseases, often leading to disability and death.Aim: To study the relationship between oxidative stress and the «proteinase-inhibitor» system in diabetes mellitus 2.Materials and methods: An observational single-center single-stage sample study was conducted with the participation of patients with type 2 diabetes mellitus and a control group (practically healthy individuals). the activity of elastase-, trypsin-like proteinases, α1-proteinase inhibitor, the content of bityrosine as an indicator of oxidative modification of proteins and the state of lipid peroxidation were determined by spectrophotometric method in all subjects in blood plasma: the concentrations of TBK-active products and antioxidants-the activity of superoxide dismutase, catalase.Results: It was shown that in type 2 diabetes mellitus, the content of TBA of active products (an indicator of lipid peroxidation) increases by 2.34 times and the concentration of bityrosine — an indicator of oxidative modification of proteins, which was negatively dependent on the activity of a proteinase inhibitor (-0.79, p=0.03) increases by 7 times. A decrease in the activity of the proteinase inhibitor (by 23%) was accompanied by an increase in the activity of elastase — and trypsin-like proteinases, respectively, by 1.2 and 2.3 times, compared with practically healthy individuals. The activity of antioxidant enzymes, superoxide dismutase and catalase increased by 8.2 and 6.4 times, respectively.Conclusion: An increase in oxidative stress and oxidative modification of proteins is accompanied by a decrease in the activity of the proteinase inhibitor and an increase in the activity of elastase-, trypsin-like proteinases, as well as antioxidant enzymes.

To elucidate the application value of insect endogenous protease and its inhibitor genes in pest control, we analyzed in detail the transcriptome sequence of the Asian corn borer, Ostrinia furnacalis. We obtained 12 protease genes and 11 protease inhibitor genes, and comprehensively analyzed of their spatiotemporal expression by qRT-PCR. In which, a previous unstudied serine protease inhibitor gene attracted our attention. It belongs to the canonical serine proteinase inhibitor family, a trypsin inhibitor-like cysteine-rich domain (TIL)-type protease inhibitor, but its TIL domain lacks two cysteine residues, and it was named as ACB-TIL. Its expression level is relatively very low in the absence of pathogen stimulation, and can be up-regulated expression induced by Gram-negative bacteria (Escherichia coli), virus (BmNPV), and dsRNA (dsEGFP), but cannot be induced by fungus spores (Metarrhizium anisopliae). Prokaryotic expressed ACB-TIL protein can significantly inhibit the melanization in vitro. Injecting this protein into insect body can inhibit the production of antimicrobial peptides of attacin, lebocin and gloverin. Inhibition of ACB-TIL by RNAi can cause the responses of other immune-, protease- and inhibitor-related genes. ACB-TIL is primarily involved in Asian corn borer humoral immunity in responses to Gram-negative bacteria and viruses. This gene can be a potential target for pest control since this will mainly affect insect immune response. [ABSTRACT FROM AUTHOR]

To elucidate the application value of insect endogenous protease and its inhibitor genes in pest control, we analyzed in detail the transcriptome sequence of the Asian corn borer, Ostrinia furnacalis. We obtained 12 protease genes and 11 protease inhibitor genes, and comprehensively analyzed of their spatiotemporal expression by qRT-PCR. In which, a previous unstudied serine protease inhibitor gene attracted our attention. It belongs to the canonical serine proteinase inhibitor family, a trypsin inhibitor-like cysteine-rich domain (TIL)-type protease inhibitor, but its TIL domain lacks two cysteine residues, and it was named as ACB-TIL. Its expression level is relatively very low in the absence of pathogen stimulation, and can be up-regulated expression induced by Gram-negative bacteria (Escherichia coli), virus (BmNPV), and dsRNA (dsEGFP), but cannot be induced by fungus spores (Metarrhizium anisopliae). Prokaryotic expressed ACB-TIL protein can significantly inhibit the melanization in vitro. Injecting this protein into insect body can inhibit the production of antimicrobial peptides of attacin, lebocin and gloverin. Inhibition of ACB-TIL by RNAi can cause the responses of other immune-, protease- and inhibitor-related genes. ACB-TIL is primarily involved in Asian corn borer humoral immunity in responses to Gram-negative bacteria and viruses. This gene can be a potential target for pest control since this will mainly affect insect immune response.

The pandemic of coronavirus disease 2019 (COVID-19) is changing the world like never before. This crisis is unlikely contained in the absence of effective therapeutics or vaccine. The papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays essential roles in virus replication and immune evasion, presenting a charming drug target. Given the PLpro proteases of SARS-CoV-2 and SARS-CoV share significant homology, inhibitor developed for SARS-CoV PLpro is a promising starting point of therapeutic development. In this study, we sought to provide structural frameworks for PLpro inhibitor design. We determined the unliganded structure of SARS-CoV-2 PLpro mutant C111S, which shares many structural features of SARS-CoV PLpro. This crystal form has unique packing, high solvent content and reasonable resolution 2.5 Å, hence provides a good possibility for fragment-based screening using crystallographic approach. We characterized the protease activity of PLpro in cleaving synthetic peptide harboring nsp2/nsp3 juncture. We demonstrate that a potent SARS-CoV PLpro inhibitor GRL0617 is highly effective in inhibiting protease activity of SARS-CoV-2 with the IC50 of 2.2 ± 0.3 μmol/L. We then determined the structure of SARS-CoV-2 PLpro complexed by GRL0617 to 2.6 Å, showing the inhibitor accommodates the S3–S4 pockets of the substrate binding cleft. The binding of GRL0617 induces closure of the BL2 loop and narrows the substrate binding cleft, whereas the binding of a tetrapeptide substrate enlarges the cleft. Hence, our results suggest a mechanism of GRL0617 inhibition, that GRL0617 not only occupies the substrate pockets, but also seals the entrance to the substrate binding cleft hence prevents the binding of the LXGG motif of the substrate.

Abstract Diseases caused by Phytophthora species seriously affect global crop production and food security. Identification of key factors involved in plant resistance is valuable for disease management. Previously, we characterized the transcriptome of Nicotiana benthamiana which was infected with Phytophthora parasitica. Here, we selected NbERF173, one of the most strongly up-regulated genes of N. benthamiana in response to P. parasitica infection, for further investigation. First, NbERF173 encodes a conserved transcription factor in our tested plant species. Second, overexpression of NbERF173 in N. benthamiana enhanced its resistance to P. parasitica, and silencing of NbERF173 significantly promoted the infection of both P. parasitica and Botrytis cinerea. Additionally, we demonstrated that NbERF173 can participate in reprograming of gene expression during P. parasitica infection and manipulate expression patterns of many defense-related genes, including two proteinase inhibitors encoding genes (PI1-B and KTI1). Furthermore, overexpression of PI1-B and KTI1 strengthened plant resistance to P. parasitica and partially restored the deficiency in resistance of NbERF173-silenced N. benthamiana plants. Finally, we found that NbERF173 could not bind to the promoters of PI1-B and KTI1 using yeast one-hybrid assay. Together, our results suggest that NbERF173 positively regulate the disease resistance, probably by reprograming of defense-related genes.

In natural and agricultural ecosystems, plants are often simultaneously or sequentially exposed to combinations of stressors. Here we tested whether limited water availability (LWA) affects plant response to insect herbivory using two populations of Eruca sativa from desert and Mediterranean habitats that differ in their induced defenses. Considering that such differences evolved as responses to biotic and possibly abiotic stress factors, the two populations offered an opportunity to study ecological aspects in plant response to combined stresses. Analysis of chemical defense mechanisms showed that LWA significantly induced total glucosinolate concentrations in the Mediterranean plants, but their concentrations were reduced in the desert plants. However, LWA, with and without subsequent jasmonate elicitation, significantly induced the expression of proteinase inhibitor in the desert plants. Results of a no-choice feeding experiment showed that LWA significantly increased desert plant resistance to Spodoptera littoralis larvae, whereas it did not affect the relatively strong basal resistance of the Mediterranean plants. LWA and subsequent jasmonate elicitation increased resistance against the generalist insect in Mediterranean plants, possibly due to both increased proteinase inhibitor expression and glucosinolate accumulation. The effect of LWA on the expression of genes involved in phytohormone signaling, abscisic acid (ABA-1) and jasmonic acid (AOC1), and the jasmonate responsive PDF1.2, suggested the involvement of abscisic acid in the regulation of defense mechanisms in the two populations. Our results indicate that specific genotypic responses should be considered when estimating general patterns in plant response to herbivory under water deficiency conditions. [ABSTRACT FROM AUTHOR]

Kunitz trypsin inhibitor (TfgKTI) isolated from fenugreek (Trigonella foenum-graecum) seeds inhibited the enzyme trypsin. The inhibitor is resistant to wide range of temperatures (37–90 °C) and pHs (3–10) but reduction of the inhibitor with dithiothreitol abolished trypsin inhibitory activity. Reduction of the disulfide linkages with dithiothreitol affected the tertiary structure significantly, whereas the secondary structure was not affected considerably. The kinetic plots of the reduction as followed by protein activity and loss in tertiary structure could be comparable. Chemical denaturation of TfgKTI with guanidine hydrochloride and fluorescence quenching studies by two neutral quenchers (acrylamide and succinimide), an anionic quencher Iodide ion (I−) and a cationic quencher Cesium ion (Cs+) suggested higher unfolding of protein under reduced conditions compared with native protein. Chemically denatured TfgKTI showed refolding, while it is irreversible with reduced protein. Native TfgKTI showed high binding affinity with porphyrin, but lost its binding capacity under reduced condition. Thus, disulfide linkages play a predominant role in maintaining the three-dimensional structure of the TfgKTI. The loss of inhibitory activity is correlated with loss in tertiary structure. [ABSTRACT FROM AUTHOR]

It is essential to identify intermediate marker endpoints of carcinogenesis for the evaluation of the effectiveness of cancer-chemopreventive agents. We have observed that levels of proteolytic activities (as detected by 4 different substrates) are increased 2-3-fold (P < 0.003) in oral buccal mucosa cells of smokers and patients with oral leukoplakia or erythroplakia as compared to a nonsmoking comparison group. In addition, proteolytic activity levels in the buccal cells were increased nearly 3-fold in patients with oral trauma (P < 0.01) or diabetes (P < 0.02), as well as pregnant women (P < 0.04). Excluding these subgroups of patients in epidemiological studies increase the differences in levels of proteolytic activities between both the nonsmoking comparison group and smokers and between the comparison group and patients with oral leukoplakia or erythroplakia. Evaluation of prerandomization levels of proteolytic activities of patients in cancer chemoprevention trials will increase the statistical power by allowing stratified randomization based on levels of proteolytic activities. The observed increases in levels of proteolytic activities in tissues at higher than normal risk of cancer development suggest that levels of proteolytic activities should be used as immediate marker endpoints in human cancer prevention trials using protease inhibitors as potential anticarcinogenic agents.

Background: Alpha-1 antitrypsin deficiency (AATD) is a hereditary disorder involving lungs, characterized by low serum concentration of the protein alpha-1 antitrypsin (AAT) also called proteinase inhibitor (PI). Asthma is common in AATD patients, but there are only few data on respiratory function in asthmatic patients with AATD. Objectives: The aim of the study was to evaluate lung function in asthmatic outpatients with mutation in the SERPINA1 gene coding for AAT versus asthmatic subjects without mutation. Methods: We performed the quantitative analysis of the serum concentration of AAT in 600 outpatients affected by mild to moderate asthma from the University Hospital of Parma, Italy. Fifty-seven of them underwent the genetic analysis subsequently; they were subdivided into mutated and non-mutated subjects. All the mutated patients had a heterozygous genotype, except 1 (PI*SS). We assessed the lung function through a flow-sensing spirometer and the small airway parameters through an impulse oscillometry system. Results: The values of forced vital capacity (% predicted) and those of the residual volume to total lung capacity ratio (%) were, respectively, lower and higher in patients mutated versus patients without mutation, showing a significantly greater air trapping (p = 0.014 and p = 0.017, respectively). Moreover, patients with mutation in comparison to patients without mutation showed lower forced expiratory volume in 3 s (% predicted) and forced expiratory volume in 6 s (L) spirometric values, reflecting a smaller airways contribution. Conclusions: In asthmatic patients, heterozygosity for AAT with PI*MZ and PI*MS genotypes was associated with small airway dysfunction and with lung air trapping. [ABSTRACT FROM AUTHOR]

Organophosphate class of pesticides causes neurotoxicity and carcinogenicity in humans. Once inside the human body, these pesticides often interact with plasma proteins, such as alpha-2-macroglobulin (α2M) which is the key anti-proteinase. Our work focuses on the structural and functional alteration of α2M by chlorpyrifos (CPF), a member of organophosphates. We explored the binding interaction between alpha-2-macroglobulin and CPF by using UV absorption and fluorescence spectroscopy (steady state and synchronous), circular dichroism and molecular docking approach. The functional activity of α2M was analyzed by anti-proteinase trypsin inhibitory assay which showed dose-dependent decrease in alpha-2-macroglobulin antiproteolytic potential. UV absorption studies and fluorescence quenching experiments suggested the formation of a complex between α2M and CPF. The CD spectra suggested a reduction in the beta helical (β helix) content of α2M. Analysis of thermodynamic parameters suggested the process is spontaneous and endothermic with the ΔG and ΔH values being −5.501 kJ/mol, 11.49 kJ/mol, respectively. CPF binds with Ile-1390, Pro-1391, Leu-1392, Lys-1393, Val-1396, Lys-1397, Arg-1407, Thr-1408, Glu-1409, Val-1410, Asp-282, Glu-281 of α2M as suggested by molecular docking. [ABSTRACT FROM AUTHOR]

The pandemic of coronavirus disease 2019 (COVID-19) is changing the world like never before. This crisis is unlikely contained in the absence of effective therapeutics or vaccine. The papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays essential roles in virus replication and immune evasion, presenting a charming drug target. Given the PLpro proteases of SARS-CoV-2 and SARS-CoV share significant homology, inhibitor developed for SARS-CoV PLpro is a promising starting point of therapeutic development. In this study, we sought to provide structural frameworks for PLpro inhibitor design. We determined the unliganded structure of SARS-CoV-2 PLpro mutant C111S, which shares many structural features of SARS-CoV PLpro. This crystal form has unique packing, high solvent content and reasonable resolution 2.5 Å, hence provides a good possibility for fragment-based screening using crystallographic approach. We characterized the protease activity of PLpro in cleaving synthetic peptide harboring nsp2/nsp3 juncture. We demonstrate that a potent SARS-CoV PLpro inhibitor GRL0617 is highly effective in inhibiting protease activity of SARS-CoV-2 with the IC 50 of 2.2 ± 0.3 μmol/L. We then determined the structure of SARS-CoV-2 PLpro complexed by GRL0617 to 2.6 Å, showing the inhibitor accommodates the S3–S4 pockets of the substrate binding cleft. The binding of GRL0617 induces closure of the BL2 loop and narrows the substrate binding cleft, whereas the binding of a tetrapeptide substrate enlarges the cleft. Hence, our results suggest a mechanism of GRL0617 inhibition, that GRL0617 not only occupies the substrate pockets, but also seals the entrance to the substrate binding cleft hence prevents the binding of the LXGG motif of the substrate. This study indicates a mechanism of GRL0617 inhibition and provides a framework for further inhibitor development targeting SARS-CoV-2 PLpro. Image 1 [ABSTRACT FROM AUTHOR]

The Caribbean king crab Maguimithrax spinosissimus is an omnivorous crustacean with high feeding plasticity. We characterized the main digestive enzymes of this crab species. Alpha-amylase and esterase activities were higher in the hepatopancreas, while those of trypsin and chymotrypsin were higher in the gastric fluid. Only one α-amylase isoform of 40 kDa generates a high α-amylase activity in this crab with a maximum at pH 5.5, 40–60°C, and 2.5 mM CaCl2. A high proteolytic activity was observed in the gastric chamber, while the activity of these enzymes was low in hepatopancreas extracts due to the presence of an inhibitor that was further demonstrated by enzyme assays and reverse zymography. Several proteases from 10 to 45 kDa are synthesized in the hepatopancreas and selectively secreted into the gut. Trypsin-like and chymotrypsin-like activities were highest at pH 7, 60°C, 0.02–1.5 M NaCl, 25 mM CaCl2. These activities were poorly influenced by Ca2+. Both enzymes were stable at a neutral to alkaline pH and a temperature up to 40°C. Esterase activity peaked at pH 9.0, 37°C, 2 M NaCl, being also poorly affected by Ca2+ but temperature sensitive. In summary, high α-amylase and protease activities, a high protease abundance, as well as a tight control over their activity by an unknown inhibitor-based mechanism appear suited for M. spinsissimus to feed opportunistically on a diet of benthic algae and epifauna with eventual carnivorous preferences. [ABSTRACT FROM AUTHOR]

α1‐Antitrypsin (α1‐AT) serves as an archetypal example for the serine proteinase inhibitor (serpin) protein family and has been used as a scaffold for protein engineering for >35 years. Techniques used to engineer α1‐AT include targeted mutagenesis, protein fusions, phage display, glycoengineering, and consensus protein design. The goals of engineering have also been diverse, ranging from understanding serpin structure–function relationships, to the design of more potent or more specific proteinase inhibitors with potential therapeutic relevance. Here we summarize the history of these protein engineering efforts, describing the techniques applied to engineer α1‐AT, specific mutants of interest, and providing an appended catalog of the >200 α1‐AT mutants published to date. [ABSTRACT FROM AUTHOR]

Cancer chemoprevention is a rapidly evolving approach to reverse or inhibit carcinogenesis, and there is active interest in development of effective chemopreventive agents against head and neck cancers. The retinoids are archetypal chemopreventive agents for oral premalignant lesions. They have significant clinical effect, but widespread use is limited by significant clinical toxicity. The Bowman-Birk Inhibitor is one of several nontoxic compounds exhibiting both potent anticarcinogenic activity and minimal toxicity. The purposes of the study were to summarize the preclinical and clinical development of Bowman-Birk Inhibitor and a Bowman-Birk Inhibitor concentrate against oral premalignant lesions and to evaluate Neu immunohistochemical staining intensity for lesions and simultaneously obtained biopsy specimens of normal-appearing mucosa from the Phase IIa Bowman-Birk Inhibitor concentrate oral leukoplakia chemoprevention trial.Part I is a selected literature review. Part II is a retrospective analysis of pathological specimens prospectively obtained from the Phase IIa clinical trial of Bowman-Birk Inhibitor concentrate.Thirty-two sets of biopsy specimens from lesions and uninvolved oral mucosa before and after treatment with Bowman-Birk Inhibitor concentrate in doses ranging from 200 to 1066 chymotrypsin inhibitory units were examined in blinded fashion for Neu immunohistochemical staining intensity using the 3B-5 monoclonal antibody. Staining intensity scores among the lesion and control biopsy specimens before and after Bowman-Birk Inhibitor concentrate treatment were analyzed and compared with previously obtained values for serum Neu, oral mucosal cell Neu, protease activity, and clinical response to treatment.Mean Neu staining score was significantly higher in lesions compared with uninvolved mucosa (P

Microarray was used to investigate the gene expression profile of potato tubers during its complete life cycle. The potato life cycle was divided into 8 different stages and total RNA was isolated and used as probe after labeling of cDNA generated from RNA samples. cDNAs were used to hybridize with potato microarray. Results of this study revealed that there is upregulation of many genes during developing stages (3b, 3c, 3d, 4a, 4b) compared to mature stages (6, 7). Many of those genes regulate growth, storage of protein and sugar, defense, inhibition of various proteinases, and oxidation. Some genes showed upregulation during certain growth stage (4a) compared to other growth stages, whereas, other genes showed upregulation in mature stages (stage 8). Profiling of gene expression during potato life cycle is essential step for enhancing potato for some highly economic traits like tuber nutrition quality, resistance to diseases, or using tubers as source for proteinase inhibitors. Also, this allows for isolation of some genes for biotechnology applications like proteinase inhibitors and improving nutrition and industrial value of tubers. [ABSTRACT FROM AUTHOR]

Three out of 17 Streptomycetes strains – Streptomyces sp. 35 LBG09, Streptomyces sp. 36 LBG09, and Streptomyces sp. 39 LBG09, were selected based on the high production of proteinase inhibitors with trypsin serine proteinase activity. The strains were isolated from soil samples taken from the area around the Bulgarian station on Livingston Island, Antarctica. Biosynthesis of proteinase inhibitors by the promising strains started at different stages of their development but was generally not associated with the growth of the producers. Peak levels were reached in the stationary phase (96–120 h) of their cultivation. Inducing effects on strain development, biomass accumulation, and proteinase inhibitor biosynthesis were based on the composition of the nutrient medium: the polypeptones contained in Taguchi medium and glucose as a carbon source. The most productive out of the three strains was Streptomyces sp. 36 LBG09. Its maximum inhibitory activity was reached at 96 h in culturing media modified by three different carbon sources. The active proteinase inhibitor biosynthesis proceeded at pH values between 6.8 and 8.6 and the dynamics of production depended on the type of carbon source. Peak levels of extracellular protein and dry biomass were reached at 120 h in the stationary growth phase. The residual sugars were minimal at the end of the process when using soluble starch as a carbon source, and maximal when glucose was used. [ABSTRACT FROM AUTHOR]

Abstract Acute liver injury (ALI) is highly lethal acute liver failure caused by different etiologies. Transforming growth factor β (TGF-β) is a multifunctional cytokine and a well-recognized inducer of apoptotic and necrotic cell death in hepatocytes. Latent TGF-β is activated partly through proteolytic cleavage by a serine protease plasma kallikrein (PLK) between the R58 and L59 residues of its propeptide region. Recently, we developed a specific monoclonal antibody to detect the N-terminal side LAP degradation products ending at residue R58 (R58 LAP-DPs) that reflect PLK-dependent TGF-β activation. This study aimed to explore the potential roles of PLK-dependent TGF-β activation in the pathogenesis of ALI. We established a mouse ALI model via the injection of anti-Fas antibodies (Jo2) and observed increases in the TGF-β1 mRNA level, Smad3 phosphorylation, TUNEL-positive apoptotic hepatocytes and R58-positive cells in the liver tissues of Jo2-treated mice. The R58 LAP-DPs were observed in/around F4/80-positive macrophages, while macrophage depletion with clodronate liposomes partly alleviated the Jo2-induced liver injury. Blocking PLK-dependent TGF-β activation using either the serine proteinase inhibitor FOY305 or the selective PLK inhibitor PKSI-527 or blocking the TGF-β receptor-mediated signaling pathway using SB431542 significantly prevented Jo2-induced hepatic apoptosis and mortality. Furthermore, similar phenomena were observed in the mouse model of ALI with the administration of acetaminophen (APAP). In summary, R58 LAP-DPs reflecting PLK-dependent TGF-β activation may serve as a biomarker for ALI, and targeting PLK-dependent TGF-β activation has potential as a therapeutic strategy for ALI. Highlights • TGF-β contributes to the pathogenesis of acute liver injury (ALI). • PLK-dependent TGF-β activation occurs in/around macrophages in ALI mice. • R58 latency associated protein-degradation products serve as an indicator for ALI. • Inhibiting PLK suppresses apoptosis and improves survival rate of ALI mice. [ABSTRACT FROM AUTHOR]

Sea anemone mucus, due to its multiple and vital functions, is a valuable substance for investigation of new biologically active peptides. In this work, compounds of Heteractis magnifica mucus were separated by multistage liquid chromatography and resulting fractions were analyzed by MALDI-TOF MS. Peptide maps constructed according to the molecular masses and hydrophobicity showed presence of 326 both new and known peptides. Several major peptides from mucus were identified, including the sodium channel toxin RpII isolated earlier from H . magnifica , and four Kunitz-type proteinase inhibitors identical to H . crispa ones. Kunitz-type transcript diversity was studied and sequences of mature peptides were deduced. New β-defensin α-amylase inhibitor, a homolog of helianthamide from Stichodactyla helianthus , was isolated and structurally characterized. Overall, H . magnifica is a source of biologically active peptides with great pharmacological potential. Biological significance Proteinase and α-amylase inhibitors along with toxins are major components of H . magnifica mucus which play an important role in the successful existence of sea anemones. Obtained peptide maps create a basis for more accurate identification of peptides during future transcriptomic/genomic studies of sea anemone H . magnifica . [ABSTRACT FROM AUTHOR]

The water-soluble protein fraction obtained from Plumeria pudica (LPPp) latex has previously been demonstrated to have anti-inflammatory and antinociceptive effects. In the present study, LPPp was tested for activity against diarrhea induced by castor oil, prostaglandin E 2 (PGE 2 ) or cholera toxin. Different doses of LPPp (10, 20 or 40 mg/kg) significantly inhibited the percentage of diarrheal stools (31.18%, 42.97% and 59.70%, respectively) induced by castor oil. This event was followed by significant reduction of both intestinal fluid accumulation (31.42%; LPPp 40 mg/kg) and intestinal transit (68.4%; LPPp 40 mg/kg). The pretreatment of animals with LPPp (40 mg/kg) prevented glutathione and malondialdehyde alterations induced by castor oil. The effects of LPPp against diarrhea induced by castor oil were lost when the fraction was submitted to protein denaturing treatment with heat. LPPp (40 mg/kg) also inhibited the average volume of intestinal fluid induced by PGE 2 (inhibition of 46.0%). Furthermore, LPPp (40 mg/kg) prevented intestinal fluid secretion accumulation (37.7%) and chloride ion concentration (50.2%) induced by cholera toxin. In parallel, colorimetric assays demonstrated that proteinases, chitinases and proteinase inhibitors were found in LPPp. Our data suggest that the antidiarrheal effect of LPPp is due to its protein content and is probably associated with its anti-inflammatory properties. [ABSTRACT FROM AUTHOR]

A regulatory sequence from a serine proteinase inhibitor gene (BvSTIpro) that is induced by the sugar beet root maggot was fused to the ß-glucuronidase (GUS) reporter gene to characterize its expression patterns in transgenic Nicotiana benthamiana plants. In 2- and 6-week old BvSTIpro-GUS transformed plants, GUS expression was observed throughout leaves and roots that was comparable to GUS staining detected in plants transformed with the positive control 35S-GUS gene construct. At 10-14 weeks, GUS expression in the leaves and roots was reduced as compared to the younger plants and the 35S-GUS control but was up-regulated by abiotic and biotic stress. At 6 to 24 hours after mechanical wounding, GUS expression increased throughout the wounded leaves and roots of 14-week old plants to constitutive levels observed with the 35S-GUS gene. Fall armyworm (Spodoptera frugiperda) feeding on BvSTIpro-GUS leaves and roots similarly induced GUS gene activity like those observed with the 35S-GUS control. We demonstrate that the BvSTI promoter induced gene expression in response to abiotic and biotic stresses. Fusion of the BvSTI promoter to resistance genes should provide expression at sites of pest and pathogen invasion to augment the capacity of the plant to defend itself. [ABSTRACT FROM AUTHOR]

Helicoverpa armigera is an orthodox rival of many crop plants affecting agricultural economy. Plant leaves found to accumulate proteinase inhibitors, although this insect pest chooses leaves for laying eggs. Plant defense response at this juncture is not fully explored. In this context, here we are reporting proteinase inhibitor ( ppLPI-1) having significant homology with the I13 family from leaves of pigeonpea (cv. BSMR 736). The isolation of ppLPI-1 was carried out from leaves of field-grown pigeonpea under an outbreak of H. armigera. The acetone precipitated ppLPI-1 (125 µg) displayed substantial inhibition potential towards bovine trypsin (56.5 ± 1.8%) and HaGPs (52.6 ± 1.7%) on solution assay. These results were corroborated with dot-blot analysis. The molecular form of ppLPI-1 was characterized by reverse zymography and GXCP. The optimum condition was found to be pH 8 and temperature in the range of 30-40 °C. The protein identification via MASCOT-PMF and NCBI-BLAST search showed substantial homology with an inducible subtilisin inhibitor of Fabaceae comprising Vigna angularis (96%), Canavalia lineata (78%), Cicer arietinum (76%), Glycine max (75%), Medicago truncatula (73%) and Vicia faba (73%) consists of conserved domain of potato inhibitor I family. [ABSTRACT FROM AUTHOR]

Alpha-2-macroglobulin (α 2 M), a pan-proteinase inhibitor, inhibits a variety of endogenous and exogenous proteinases and constitutes an important part of body’s innate defense system. In the present study, we explored how trifluoroethanol (TFE) may modulate the structure, antiproteinase activity and aggregation of α 2 M. TFE was sequentially added over a range of 0–20% (v/v) and the effects induced were studied by activity assay, intrinsic fluorescence, ANS fluorescence, circular dichroism, turbidity assay, Rayleigh scattering measurement and ThT fluorescence measurement. Decrease in activity and increase in fluorescence intensity of α 2 M upon addition of TFE shows structural deviation from the native structure and suggests aggregation of protein upon solvent addition. Increase in turbidity and Rayleigh scattering of modified α 2 M confirms the formation of aggregates. Insignificant ThT fluorescence intensity of TFE treated α 2 M is indicative of amorphous or non-amyloid aggregation. Further, circular dichroism results indicate the changes in secondary structure of native α 2 M as negative ellipticity decreased on addition of the polar solvent to the inhibitor. The turbidometric analysis, Rayleigh scattering, ThT fluorescence intensity of modified α 2 M suggests that the protein might be driven towards non-amyloid or amorphous aggregation. Our studies provide important mechanistic insight how α 2 M undergoes conformational and functional changes when exposed to TFE. [ABSTRACT FROM AUTHOR]

Ferulic acid is a major phenolic acid found in numerous plant species in conjugated form. It binds to enzymes and oligomeric proteins and modifies their structure and function. This study was designed to examine the interaction of ferulic acid, an active ingredient of some important medicines, with α 2 M, a key serum proteinase, under physiological conditions. The mechanism of interaction was studied by spectroscopic techniques such as, UV–visible absorption, fluorescence spectroscopy, circular dichroism along with isothermal titration calorimetry. Fluorescence quenching of α 2 M by ferulic acid demonstrated the formation of α 2 M-ferulic acid complex by static quenching mechanism. Binding parameters calculated by Stern-Volmer method showed that ferulic acid binds to α 2 M with moderate affinity of the order of ∼10 4 M −1 . The thermodynamic signatures reveal that binding was enthalpy driven and hydrogen bonding played a major role in ferulic acid-α 2 M binding. CD spectra analysis suggests very little conformational changes in α 2 M on ferulic acid binding. [ABSTRACT FROM AUTHOR]

Plant proteinase inhibitors (PIs) are plant defense proteins and considered as potential candidates for engineering plant resistances against herbivores. Capsicum annuum proteinase inhibitor (CanPI7) is a multi-domain potato type II inhibitor (Pin-II) containing four inhibitory repeat domains (IRD), which target major classes of digestive enzymes in the gut of Helicoverpa armigera larvae. Stable integration and expression of the transgene in T1 transgenic generation, were confirmed by established molecular techniques. Protein extract of transgenic tomato lines showed increased inhibitory activity against H. armigera gut proteinases, supporting those domains of CanPI7 protein to be effective and active. When T1 generation plants were analyzed, they exhibited antibiosis effect against first instar larvae of H. armigera. Further, larvae fed on transgenic tomato leaves showed delayed growth relative to larvae fed on control plants, but did not change mortality rates significantly. Thus, better crop protection can be achieved in transgenic tomato by overexpression of multi-domain proteinase inhibitor CanPI7 gene against H. armigera larvae. [ABSTRACT FROM AUTHOR]

The shrimp multifunctional protein alpha-2-macroglobulin (A2M) is abundantly expressed in plasma, highly up-regulated upon microbial infection and involved in several immune pathways such as blood clotting system, phagocytosis and melanization. Herein, the function of Lv A2M from Litopenaeus vannamei on the prophenoloxidase (proPO) system is reported. The recombinant (r) Lv A2M produced strongly and specifically inhibited trypsin and the PO activity in shrimp plasma in a dose-dependent manner. Silencing of Lv A2M led to an increase in the PO activity in shrimp plasma although the expression of proPO-associated genes, proPO-activating enzyme ( PPAE ) and prophenoloxidase ( proPO ) but not the proPO-activating factor ( PPAF ) was down-regulated. In Vibrio parahaemolyticus AHPND-infected shrimp, the Lv A2M activity was suppressed in an early phase of infection while the PO activity was increased. Thus, the proPO-activating system was regulated by the Lv A2M. [ABSTRACT FROM AUTHOR]

Proteinase inhibitors play an important role in plant resistance of insects and pathogens. In this study, we characterized the serine proteinase inhibitor SaPIN2b, which is constitutively expressed in Solanum americanum trichomes and contains two conserved motifs of the proteinase inhibitor II (PIN2) family. The recombinant SaPIN2b (rSaPIN2b), which was expressed in Escherichia coli, was demonstrated to be a potent proteinase inhibitor against a panel of serine proteinases, including subtilisin A, chymotrypsin and trypsin. Moreover, rSaPIN2b also effectively inhibited the proteinase activities of midgut trypsin-like proteinases that were extracted from the devastating pest Helicoverpa armigera. Furthermore, the overexpression of SaPIN2b in transgenic tobacco plants resulted in enhanced resistance against H. armigera. Taken together, our results demonstrated that SaPIN2b is a potent serine proteinase inhibitor that may act as a protective protein in plant defense against insect attacks.

Root-knot nematodes (RKN) cause substantial yield decline in eggplant and sustainable management options to minimize crop damage due to nematodes are still limited. A number of genetic engineering strategies have been developed to disrupt the successful plant-nematode interactions. Among them, delivery of proteinase inhibitors from the plant to perturb nematode development and reproduction is arguably the most effective strategy. In the present study, transgenic eggplant expressing a modified rice cystatin (OC-IΔD86) gene under the control of the root-specific promoter, TUB-1, was generated to evaluate the genetically modified nematode resistance. Five putative transformants were selected through PCR and genomic Southern blot analysis. Expression of the cystatin transgene was confirmed in all the events using western blotting, ELISA and qPCR assay. Upon challenge inoculation, all the transgenic events exhibited a detrimental effect on RKN development and reproduction. The best transgenic line (a single copy event) showed 78.3% inhibition in reproductive success of RKN. Our results suggest that cystatins can play an important role for improving nematode resistance in eggplant and their deployment in gene pyramiding strategies with other proteinase inhibitors could ultimately enhance crop yield.