Types Of Proteases Research Articles

Naphthalen-1-ylethanamine–containing small molecule inhibitors of the papain-like protease of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has not yet been eradicated. SARS-CoV-2 has two types of proteases, a main protease (Mpro) and a papain-like protease (PLpro), which together process two translated non-structural polyproteins, pp1a and pp1ab, to produce functional viral proteins. In this study, effective inhibitors against PLpro of SARS-CoV-2 were designed and synthesized using GRL-0048 as a lead. A docking simulation of GRL-0048 and SARS-CoV-2 PLpro showed that GRL-0048 noncovalently interacts with PLpro, and there is a newly identified binding pocket in PLpro. Structure-activity relationship studies were next performed on GRL-0048, resulting in the development of several inhibitors, specifically compounds 1, 2b, and 3h, that have more potent inhibitory activity than GRL-0048.

Isolation of Bacillus altitudinis 5-DSW with Protease Activity from Deep-Sea Mineral Water and Preparation of Functional Active Peptide Fractions from Chia Seeds.

In this study, we successfully isolated Bacillus strains with high protease activity from deep-sea mineral water in Korea and used them to obtain functional peptide fractions from chia seeds. The obtained Bacillus strains showed a high similarity of 99% with B. altitude with a long rod type (named B. altitudinis 5-DSW) and high protease activity at 40 °C, and 70% of the activity remained even at 70 °C. The defatted chia seed protein (15-50 kDa) was treated with crude protease from B. altitudinis 5-DSW and digested into small peptides below 20 kDa. The obtained chia seed peptides showed 3 times and 1.5 times higher antioxidant activity in DPPH and ABT radical scavenging assays, respectively. Moreover, chia seed peptides showed enhanced AChE inhibitory activity with an IC50 value of 14.48 ± 0.88 μg/mL and BChE inhibition activity with an IC50 value of 10.90 ± 0.80 μg/mL. Our results indicate that the newly isolated B. altitudinis 5-DSW and chia seed protein hydrolysates have potential applications in biotechnology and functional food development, enhancing the nutritional quality and value-added utilization of chia byproducts.

Engineering inhibitory repeat domains of Pin-II type proteinase inhibitors indicate their high structural-functional tolerance to mutagenesis

Plant proteinase inhibitors (PIs) are critical in defending against biotic stress. Most PIs contain an inhibitory repeat domain (IRD), which serves as the functional component, displaying a high degree of sequence and structural conservation. In this study, we examined the structural and functional resilience of IRDs using a combination of computational modeling and experimental validation. We have taken an evolution-based approach to enhance the PIs effectiveness of two previously identified Capsicum annuum IRDs, IRD4 and IRD10. Through in silico site-saturation mutagenesis of IRD4 and IRD10, we identified key sites associated with enhanced PI activity for targeted mutagenesis. Binding energy predictions for a mutant IRD library, tested against target proteases, suggested that positions R11 and N32 in IRD4 and N32 and H33 in IRD10 were promising candidates for further modification to improve inhibitory potential. Subsequent experimental validation revealed that the mutant proteins IRD4_R11K and IRD4_N32S exhibited stronger chymotrypsin inhibition than the wild-type (WT) IRD4. Similarly, the mutants IRD10_N32S and IRD10_H33 N demonstrated improved trypsin inhibition relative to the WT IRD10. These findings indicate that engineered IRD variants can tolerate structural changes while maintaining or enhancing their inhibitory activity against target proteases. Overall, this study demonstrates the potential of engineering PIs to increase their structural and functional resilience, offering new opportunities for biotechnological applications.

A new Bowman-Birk type protease inhibitor regulated by MeJA pathway in maize exhibits anti-feedant activity against the Ostrinia furnacalis.

Jasmonic acid (JA), an important plant hormone, plays a crucial role in defending against herbivorous insects. In this study, we have identified a new Bowman-Birk type protease inhibitor (BBTI) protein in maize that is regulated by the JA pathway and exhibits significant antifeedant activity, which is notably induced by exogenous Methyl Jasmonate and Ostrinia furnacalis feeding treatments. Bioinformatics analysis revealed significant differences in the BBTI protein among different maize inbred lines, except for the conserved domain. Prokaryotic and eukaryotic expression systems were constructed and expressed, and combined with bioassays, it was demonstrated that the antifeedant activity of BBTI is determined by protein modifications and conserved domains. Through RT-qPCR detection of BBTI and JA regulatory pathway-related genes' temporal expression in different maize inbred lines, we identified the regulatory mechanism of BBTI synthesis under the JA pathway. This study successfully cloned and identified the MeJA-induced anti-feedant activity gene BBTI and conducted functional validation in different maize inbred lines, providing valuable insights into the response mechanism of insect resistance induced by the plant JA pathway. The increased expression of the anti-feedant activity gene BBTI through exogenous MeJA induction may offer a potential new strategy for mediating plant defense against Lepidoptan insects.

Association between TMPRSS2 rs2070788 polymorphism and COVID-19 severity: a case-control study in multiple cities of Iran.

Host genetic variations have been identified as potential influencers of COVID-19 infection. This study aimed to examine the association between transmembrane serine protease type 2 (TMPRSS2) rs2070788 single nucleotide polymorphism (SNP) and the prognosis of COVID-19 in Iranian populations. This case-control study was performed on 756 COVID-19 patients and 59 healthy individuals across Iran. Clinical data, blood samples, and the presence of the TMPRSS2 rs2070788: G>A SNP were determined using T-ARMS-PCR. Additionally, serum levels of tumor necrosis factor α (TNF-α), C-reactive protein (CRP), interleukin-6 (IL-6), and IL-1β were evaluated in the collected blood samples. No significant association was found between the genotypes and allele frequencies of TMPRSS2 rs2070788 SNP and susceptibility to or mortality from COVID-19 infection. However, we observed a substantial increase in IL-6 and CRP levels associated with the severity of COVID-19, while no such trend was observed for IL-1β and TNF-α. This study showed a considerable rise in TNF-α and IL-1β serum levels exclusively in COVID-19 patients with TT rs2070788 TMPRSS2 SNP genotype compared to healthy controls. In this study conducted across multiple cities in Iran, no significant association was found between the TMPRSS2 rs2070788 SNP genotypes and COVID-19 severity or mortality.

Aprotinin (I): Understanding the Role of Host Proteases in COVID-19 and the Importance of Pharmacologically Regulating Their Function.

Proteases are produced and released in the mucosal cells of the respiratory tract and have important physiological functions, for example, maintaining airway humidification to allow proper gas exchange. The infectious mechanism of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), takes advantage of host proteases in two ways: to change the spatial conformation of the spike (S) protein via endoproteolysis (e.g., transmembrane serine protease type 2 (TMPRSS2)) and as a target to anchor to epithelial cells (e.g., angiotensin-converting enzyme 2 (ACE2)). This infectious process leads to an imbalance in the mucosa between the release and action of proteases versus regulation by anti-proteases, which contributes to the exacerbation of the inflammatory and prothrombotic response in COVID-19. In this article, we describe the most important proteases that are affected in COVID-19, and how their overactivation affects the three main physiological systems in which they participate: the complement system and the kinin-kallikrein system (KKS), which both form part of the contact system of innate immunity, and the renin-angiotensin-aldosterone system (RAAS). We aim to elucidate the pathophysiological bases of COVID-19 in the context of the imbalance between the action of proteases and anti-proteases to understand the mechanism of aprotinin action (a panprotease inhibitor). In a second-part review, titled "Aprotinin (II): Inhalational Administration for the Treatment of COVID-19 and Other Viral Conditions", we explain in depth the pharmacodynamics, pharmacokinetics, toxicity, and use of aprotinin as an antiviral drug.

Effect of ‘Procumbenase’ a serine protease from Tridax procumbens aqueous extract on wound healing: A scar free healing of full thickness wounds

Wound healing involves several cellular and molecular pathways. Tridax procumbens activates genetic pathways with antibacterial, antioxidant, anticancer, and anti-inflammatory properties, aiding wound healing. This study purified Procumbenase, a serine protease from T. procumbens extract, using gel filtration (Sephadex G-75) and ion exchange (CM-Sephadex C-50) chromatography. Characterization involved analyses of protease activity, RP-HPLC, SDS-PAGE, gelatin zymogram, PAS staining, mass spectrometry, and circular dichroism. Optimal pH and temperature were determined. Protease type was identified using inhibitors. Wound-healing potential was evaluated through tensile strength, wound models, hydroxyproline estimation, and NIH 3T3 cell scratch analysis. In incision wound rat models, Procumbenase increased tensile strength on day 14 more than saline and Povidone‑iodine. It increased wound contraction by 89 % after 10 days in excision wound models, attaining full contraction by day 15 and closure by day 21. Scarless wound healing was enhanced by 18 days of epithelialization against 22 and 21 days for saline and povidone‑iodine. Procumbenase increased hydroxyproline concentration 2.53-fold (59.93 ± 2.89 mg/g) compared to saline (23.67 ± 1.86 mg/g). In NIH 3 T3 cell scratch assay, Procumbenase increased migration by 60.93 % (50 μg) and 60.57 % (150 μg) after 48 h. Thus, Procumbenase is the primary bioactive molecule in T. procumbens, demonstrates scar-free wound healing properties.

New Method of Obtaining Proteolytic Enzymatic Preparation from Fungal Strain Fusarium gibbosum CNMN FD 12

Proteases, that are able to hydrolyze the peptide bonds in proteins and polypeptides, occupy a key position in diverse fields, such as food, agriculture, animal feed. Considering the increasing demand for microbial proteases, identification of new way for improving the productivity of producers is of great importance. The aim of the present study was to evaluate the effect of a new coordination compound [Fe(H2L)(H2O)2](NO3)3∙2.5H2O (where H2L = 2,6-diacetylpyridine bis(picolinoylhydrazone)) on the activity of proteolytic complex synthesized by the micromycete Fusarium gibbosum CNMN FD 12 under the submerged fermentations in shake flask, as well as in lab-scale fermenter. It was found that [Fe(H2L)(H2O)2](NO3)3∙2.5H2O exerts a significant positive effect on proteolytic activity in F. gibbosum CNMN FD 12 both in flasks and bioreactor and can be used as stimulator and regulator of proteases activity. By varying the concentration of the coordination compound and the duration of cultivation, it is possible to obtain proteolytic complexes enriched in different types of proteases (acid, neutral or alkaline), depending on the practical needs.

Characterization and evaluation of the in vitro and in vivo anti-diabetic activities of camel milk protein hydrolysates derived with different protease digestions

The study aimed to determine the potential anti-diabetic activities of the hydrolysates of camel milk proteins (CMPH). Camel milk proteins were hydrolyzed independently by using 11 proteases, and their anti-diabetic activities were analyzed by in vitro, and in vivo. The results showed that CMPH-Flavourzyme exhibited the best α-glucosidase and dipeptidyl peptidase-IV inhibitory activity, while CMPH-Papain exhibited the highest α-amylase inhibitory activity, and best proliferative effect on streptozotocin-induced NIT-1 cells in vitro. Furthermore, CMPH-Papain exhibited the most effect in decreasing the serum levels of fasting blood glucose, oral glucose tolerance test, pro-inflammatory factors (IL-1β, IL-6) and triglyceride, as well as increasing insulin levels on in vivo streptozotocin-induced diabetic mice. Peptides identification followed with molecular docking and network pharmacology analysis also found the potential anti-diabetic peptides from CMPH-Flavourzyme and CMPH-Papain. Our results indicated the type of protease used for the treatment significantly influences the anti-diabetic activities of the resulting CMPH.

PSII-17 Effect of protease supplementation on amino acids digestibility of soybean meal fed to growing pigs of different ages

Abstract Protease supplementation can increase amino acids (AA) availability; however, the effectiveness of protease in swine diets varies and has been associated with the type and dose of protease, dietary ingredients, and potentially animal age. This study investigated the interactive effect of protease inclusion level and pig age on standardized ileal digestibility (SID) of protein (CP) and AA of soybean meal (SBM) fed to growing pigs. A corn starch–based diet was formulated with SBM as the sole source of AA and containing titanium as an indigestible marker. Protease was supplemented at 0, 15,000; 30,000 and 45,000 NFP/kg of feed (ProAct 360). An N-free diet was used to estimate basal ileal endogenous AA losses. Ten cannulated pigs (body weight = 21 kg ± 2 kg) were assigned to experimental diets in a duplicate 5 × 5 Latin square design. In Phase I (23 to 30 kg-pigs, 90 ± 17 d of age), ileal digesta was collected in 5 periods of 7 d (5 d adaptation and 2 d ileal digesta collection). In Phase II (50 to 65 kg-pigs. 140 ± 17 d of age), using the same diets, ileal digesta was collected in 5 more periods of 7 d. Pigs were fed at 4% of body weight (BW) adjusted at the end of each period. Diets and ileal digesta were analyzed for CP, AA, and titanium and SID was calculated. Orthogonal polynomial contrasts were used to determine the linear and quadratic effects of protease supplementation. The combined data were analyzed as 4 x 2 factorial, including the fixed effects of protease inclusion (excluding N-free diet), age, and their interactions. In phase I, increasing levels of protease resulted in linear improved (P < 0.1) of SID for the majority of indispensable AA (Except Arg, His, Met + Cys, and Trp) and the average of dispensable AA. In phase II, the SID of Ile, Leu, Met, Met + Cys, Val, the average of indispensable AA, and the majority of dispensable AA was quadratically improved. Overall (Phase I and II), the SID of Ile, Leu, Met + Cys, Val, Cys, Glu, Tyr, and the average of dispensable AA was quadratically improved (P < 0.1). In most cases, the supplementation of 30,000 NFP/kg of feed resulted in the greatest AA digestibility improvements. However, the linear response in the first phase for some AA suggests that diets for younger pigs could be supplemented with a greater level (45,000 NFP/kg of feed). There were no interactions between protease levels and age. Younger pigs had improved (P < 0.1) SID of CP and most AA (Except Cys, Gly, and Ser). In conclusion, dietary protease supplementation can improve SID of AA in soybean mean independent of pig age. Pigs age can influence AA digestibility where younger pigs have greater AA digestibility.

Effect of thermal pretreatment and gastrointestinal digestion on the bioactivity of dry-cured ham bone enzymatic hydrolyzates

This study reports the effect of thermal pretreatment and the use of different commercial proteolytic enzymes (Protamex, Flavourzyme, Protana prime, and Alcalase) on the free amino acid content (FAA), peptide profile, and antioxidant, antidiabetic, antihypertensive, and anti-inflammatory potential (DPPH, FRAP, and ABTS assay, DPP-IV, ACE-I, and NEP inhibitory activities) of dry-cured ham bone hydrolyzates. The effect of in vitro digestion was also determined. Thermal pretreatment significantly increased the degree of hydrolysis, the FAA, and the DPP-IV and ACE-I inhibitory activities. The type of peptidase used was the most significant factor influencing antioxidant activity and neprilysin inhibitory activity. Protana prime hydrolyzates failed to inhibit DPP-IV and neprilysin enzymes and had low values of ACE-I inhibitory activity. After in vitro digestion, bioactivities kept constant in most cases or even increased in ACE-I inhibitory activity. Therefore, hydrolyzates from dry-cured ham bones could serve as a potential source of functional food ingredients for health benefits.

Integrated machine learning-based virtual screening and biological evaluation for identification of potential inhibitors against cathepsin K.

Cathepsin K is a type of cysteine proteinase that is primarily expressed in osteoclasts and has a key role in the breakdown of bone matrix protein during bone resorption. Many studies suggest that the deficiency of cathepsin K is concomitant with a suppression of osteoclast functioning, therefore rendering the resorptive properties of cathepsin K the most prominent target for osteoporosis. This innovative work has identified a novel anti-osteoporotic agent against Cathepsin K by using a comparison of machine learning and deep learning-based virtual screening followed by their biological evaluation. Out of ten shortlisted compounds, five of the compounds (JFD02945, JFD02944, RJC01981, KM08968 and SB01934) exhibit more than 50% inhibition of the Cathepsin K activity at 0.1μM concentration and are considered to have a promising inhibitory effect against Cathepsin K. The comprehensive docking, MD simulation, and MM/PBSA investigations affirm the stable and effective interaction of these compounds with Cathepsin K to inhibit its function. Furthermore, the compounds RJC01981, KM08968 and SB01934 are represented to have promising anti-osteoporotic properties for the management of osteoporosis owing to their significantly well predicted ADMET properties.

Airborne indoor allergen serine proteases and their contribution to sensitisation and activation of innate immunity in allergic airway disease.

Common airborne allergens (pollen, animal dander and those from fungi and insects) are the main triggers of type I allergic disorder in the respiratory system and are associated with allergic rhinitis, allergic asthma, as well as immunoglobulin E (IgE)-mediated allergic bronchopulmonary aspergillosis. These allergens promote IgE crosslinking, vasodilation, infiltration of inflammatory cells, mucosal barrier dysfunction, extracellular matrix deposition and smooth muscle spasm, which collectively cause remodelling of the airways. Fungus and insect (house dust mite and cockroaches) indoor allergens are particularly rich in proteases. Indeed, more than 40 different types of aeroallergen proteases, which have both IgE-neutralising and tissue-destructive activities, have been documented in the Allergen Nomenclature database. Of all the inhaled protease allergens, 85% are classed as serine protease activities and include trypsin-like, chymotrypsin-like and collagenolytic serine proteases. In this article, we review and compare the allergenicity and proteolytic effect of allergen serine proteases as listed in the Allergen Nomenclature and MEROPS databases and highlight their contribution to allergic sensitisation, disruption of the epithelial barrier and activation of innate immunity in allergic airways disease. The utility of small-molecule inhibitors of allergen serine proteases as a potential treatment strategy for allergic airways disease will also be discussed.

Precise Determination of Selenium Forms and Contents in Selenium-Enriched Rapeseed Seedlings and Flowering Stalks by HPLC-ICP-MS.

Rapeseed (Brassica napus L.) has the ability of selenium (Se) enrichment. Identification of selenides in Se-rich rapeseed products will promote the development and utilization of high value. By optimizing the Se species extraction process (protease type, extraction reagent, enzyme sample ratio, extraction time, etc.) and chromatographic column, an efficient, stable, and accurate method was established for the identification of Se species and content in rapeseed seedlings and flowering stalks, which were cultured by inorganic Se hydroponics. Five Se compounds, including selenocystine (SeCys2), methylselenocysteine (MeSeCys), selenomethionine (SeMet), selenite (SeIV), and selenate (SeVI) were qualitatively and quantitatively identified. Organoselenium was absolutely dominant in both seedlings and flowering stalks among the detected rapeseed varieties, with 64.18-90.20% and 94.38-98.47%, respectively. Further, MeSeCys, a highly active selenide, predominated in rapeseed flowering stalks with a proportion of 56.36-72.93% and a content of 1707.3-5030.3 μg/kg. This study provides a new source of MeSeCys supplementation for human Se fortification.

Evaluating a Retro-Inverso Type Inhibitor of HTLV-1 Protease as a Competitive Inhibitor.

The inhibition mode of a retro-inverso (RI) inhibitor containing a hydroxyethylamine dipeptide isostere against the human T-cell leukemia virus type-1 (HTLV-1) protease was examined. Enzymatic evaluation of the RI-modified inhibitor containing a D-allo-Ile residue revealed that HTLV-1 was competitively inhibited. IC50 values of the RI-modified inhibitor and pepstatin A, a standard inhibitor of aspartic proteases, were nearly equivalent.

Effect of different pretreatments on the hydrolysis efficiency and flavor of squid viscera (Dosidicus gigas)

In order to better utilize the proteins in squid viscera (Dosidicus gigas), the effects of different pretreatments on the degree of hydrolysis (DH) and volatile compounds of hydrolysates were studied. The DH was used as an indicator to select alkaline protease as the optimal protease from 8 types of proteases. The hydrolysis conditions of alkaline protease and the flavor of the hydrolysates were investigated through single-factor, response surface methodology, and pretreatment-assisted hydrolysis experiments. The results showed that alkaline protease had the highest DH (42.08 ± 0.58%), with enzyme dosage of 1.5%, hydrolysis temperature of 45 °C, hydrolysis time of 5 h, and liquid-solid ratio of 4:1. The DH of the ultrasound group was significantly higher than other groups, with the highest DH (45.67 ± 0.34%) after ultrasound for 40 min (U40). The proportion of molecular weight < 1 kDa and the content of free amino acids (1613.64 mg/100 g) were the highest at U40. The electronic nose results indicated that the composition of organic sulfur and aliphatic compounds in U40 showed significant differences compared to other products. The U40 sample had the highest type and content of volatile compounds (especially benzaldehyde), which was more conducive to the volatilization of flavor substances. In conclusion, U40 pretreatment can effectively improve the DH and flavor of hydrolysates.

Isolation and identification of milk-clotting proteases from Prinsepia utilis Royle and its application in cheese processing

The aim of this study was to isolate and identify the main milk-clotting proteases from Prinsepia utilis Royle. Protein isolates obtained using precipitation with 20 %–50 % ammonium sulfate (AS) showed higher milk-clotting activity (MCA) at 154.34 + 0.35 SU. Two milk-clotting proteases, namely P191 and P1831, with molecular weight of 49.665 kDa and 68.737 kDa, respectively, were isolated and identified using liquid chromatography-mass spectrometry (LC-MS/MS). Bioinformatic analysis showed that the two identified milk-clotting proteases were primarily involved in hydrolase activity and catabolic processes. Moreover, secondary structure analysis showed that P191 structurally consisted of 40.85 % of alpha-helices, 15.96 % of beta-strands, and 43.19 % of coiled coil motifs, whereas P1831 consisted of 70 % of alpha-helices, 7.5 % of beta-strands, and 22.5 % of coiled coil motifs. P191 and P1831 were shown to belong to the aspartic protease and metalloproteinase types, and exhibited stability within the pH range of 4–6 and good thermal stability at 30–80 °C. The addition of CaCl2 (<200 mg/L) increased the MCA of P191 and P1831, while the addition of NaCl (>3 mg/mL) inhibited their MCA. Moreover, P191 and P1831 preferably hydrolyzed kappa-casein, followed by alpha-casein, and to a lesser extent beta-casein. Additionally, cheese processed with the simultaneous use of the two proteases isolated in the present study exhibited good sensory properties, higher protein content, and denser microstructure compared with cheese processed using papaya rennet or calf rennet. These findings unveil the characteristics of two proteases isolated from P. utilis, their milk-clotting properties, and potential application in the cheese-making industry.

Production and characterization of an organic solvent activated protease from haloalkaliphilic bacterium Halobiforma sp. strain BNMIITR

An unusual haloalkaliphilic bacterium known as Halobiforma sp. strain BNMIITR, which was noticed to produce an extracellular alkaline protease, was found in a soil sample from Northern India's Sambhar Lake. On the generation of protease, the effects of dietary elements including nitrogen and carbon sources, amino acids, and growth conditions like temperature and pH were investigated. When low-cost agricultural by-products were employed as nitrogen sources, the manufacturing of enzymes was significantly boosted. In the present study, protease production was enhanced by 2.94 fold and 2.17 fold. By solvent precipitation and Hydrophobic interaction chromatography (HIC) on Phenyl Sepharose 6 Fast Flow matrix, the enzyme was purified 31.67 fold. It was determined that the apparent molecular mass was 21 kDa. The pH range where the enzyme was most stable was 6.0–12.0, with a temperature of 50 °C as optimum. When there was alkaline earth metals and heavy metals, protease was discovered to be active. It was evident that the enzyme was a serine type of protease because it was active in the presence of a variety of surfactants, oxidizing and reducing chemicals, and phenylmethylsulfonyl fluoride (PMSF) completely inhibited activity. Enzyme exhibited a wide range of substrate specificity. Amazingly, enzyme remained stable both in polar and nonpolar solvents. The most interesting aspect of this enzyme is enhanced activity in polar solvents like dimethylformamide (DMF) and dimethyl sulfoxide (DMSO). It was discovered that the protease was stable and compatible with a number of widely available detergents.

Harmaline to Human Mitochondrial Caseinolytic Serine Protease Activation for Pediatric Diffuse Intrinsic Pontine Glioma Treatment.

Diffuse intrinsic pontine glioma (DIPG), affecting children aged 4-7 years, is a rare, aggressive tumor that originates in the pons and then spreads to nearby tissue. DIPG is the leading cause of death for pediatric brain tumors due to its infiltrative nature and inoperability. Radiotherapy has only a palliative effect on stabilizing symptoms. In silico and preclinical studies identified ONC201 as a cytotoxic agent against some human cancer cell lines, including DIPG ones. A single-crystal X-ray analysis of the complex of the human mitochondrial caseinolytic serine protease type C (hClpP) and ONC201 (PDB ID: 6DL7) allowed hClpP to be identified as its main target. The hyperactivation of hClpP causes damage to mitochondrial oxidative phosphorylation and cell death. In some DIPG patients receiving ONC201, an acquired resistance was observed. In this context, a wide program was initiated to discover original scaffolds for new hClpP activators to treat ONC201-non-responding patients. Harmaline, a small molecule belonging to the chemical class of β-carboline, was identified through Fingerprints for Ligands and Proteins (FLAP), a structure-based virtual screening approach. Molecular dynamics simulations and a deep in vitro investigation showed interesting information on the interaction and activation of hClpP by harmaline.

Enzymatic Preparation and Processing Properties of DPP-IV Inhibitory Peptides Derived from Wheat Gluten: Effects of Pretreatment Methods and Protease Types.

The choice of appropriate proteases and pretreatment methods significantly influences the preparation of bioactive peptides. This study aimed to investigate the effects of different pretreatment methods on the hydrolytic performance of diverse proteases during the production of dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides derived from wheat and their foaming and emulsion properties. Dry heating, aqueous heating, and ultrasound treatment were employed as pretreatments for the protein prior to the enzymatic hydrolysis of wheat gluten. FTIR analysis results indicated that all pretreatment methods altered the secondary structure of the protein; however, the effects of dry heating treatment on the secondary structure content were opposite to those of aqueous heating and ultrasound treatment. Nevertheless, all three methods enhanced the protein solubility and surface hydrophobicity. By using pretreated proteins as substrates, five different types of proteases were employed for DPP-IV inhibitory peptide production. The analysis of the DPP-IV inhibitory activity, degree of hydrolysis, and TCA-soluble peptide content revealed that the specific pretreatments had a promoting or inhibiting effect on DPP-IV inhibitory peptide production depending on the protease used. Furthermore, the pretreatment method and the selected type of protease collectively influenced the foaming and emulsifying properties of the prepared peptides.