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The reversible attachment of ubiquitin (Ub) and ubiquitin like modifiers (Ubls) to proteins are crucial post-translational modifications (PTMs) for many cellular processes. Not only do cells possess hundreds of ligases to mediate substrate specific modification with Ub and Ubls, but they also have a repertoire of more than 100 dedicated enzymes for the specific removal of ubiquitin (Deubiquitinases or DUBs) and Ubl modifications (Ubl-specific proteases or ULPs). Over the past two decades, there has been significant progress in our understanding of how DUBs and ULPs function at a molecular level and many novel DUBs and ULPs, including several new DUB classes, have been identified. Here, the development of chemical tools that can bind and trap active DUBs has played a key role. Since the introduction of the first activity-based probe for DUBs in 1986, several innovations have led to the development of more sophisticated tools to study DUBs and ULPs. In this review we discuss how chemical biology has led to the development of activity-based probes and substrates that have been invaluable to the study of DUBs and ULPs. We summarise our currently available toolbox, highlight the main achievements and give an outlook of how these tools may be applied to gain a better understanding of the regulatory mechanisms of DUBs and ULPs.

Apart from its obvious agronomic interest in feeding billions of people worldwide, the porcine species represents an irreplaceable experimental model for intestinal physiologists and nutritionists. In this review, we give an overview on the fate of proteins that are not fully digested in the pig small intestine, and thus are transferred into the large intestine. In the large intestine, dietary and endogenous proteins are converted to peptides and amino acids (AA) by the action of bacterial proteases. AA, which cannot, except in the neonatal period, be absorbed to any significant level by the colonocytes, are used by the intestinal microbes for protein synthesis and for the production of numerous metabolites. Of note, the production of the AA-derived metabolites greatly depends on the amount of undigested polysaccharides in the pig’s diet. The effects of these AA-derived bacterial metabolites on the pig colonic epithelium have not yet been largely studied. However, the available data, performed on colonic mucosa, isolated colonic crypts and colonocytes, indicate that some of them, like ammonia, butyrate, acetate, hydrogen sulfide (H2S), and p-cresol are active either directly or indirectly in energy metabolism in colonic epithelial cells. Further studies in that area will certainly gain from the utilization of the pig colonic organoid model, which allows for disposal of functional epithelial unities. Such studies will contribute to a better understanding of the potential causal links between diet-induced changes in the luminal concentrations of these AA-derived bacterial metabolites and effects on the colon epithelial barrier function and water/electrolyte absorption.

The SARS-CoV-2 coronavirus has become one of the most immediate and widely-studied systems since its identification and subsequent global outbreak from 2019-2020. In an effort to understand the biophysical changes as a result of mutations, the mechanics of multiple different proteins within the SARS-CoV-2 virus have been studied and compared with SARS-CoV-1. Focusing on the main protease (mPro), we first explored the long range dynamic-relationship, particularly in cross-chain dynamics, using the Dynamic Coupling Index (DCI) to investigate the dynamic coupling between the catalytic site residues and the rest of the protein, both inter and intra chain for the CoV-1 and CoV-2 mPro. We found that there is significant cross-chain coupling between these active sites and distal residues in the CoV-2 mPro but it was missing in CoV-1. The enhanced long distance interactions, particularly between the two chains, suggest subsequently enhanced cooperativity for CoV-2. A further comparative analysis of the dynamic flexibility using the Dynamic Flexibility Index (DFI) between the CoV-1 and CoV-2 mPros shows that the inhibitor binding near active sites induces change in flexibility to a distal region of the protein, opposite in behavior between the two systems; this region becomes more flexible upon inhibitor binding in CoV-1 while it becomes less flexible in the CoV-2 mPro. Upon inspection, we show that, on average, the dynamic flexibility of the sites substituted from CoV-1 to CoV-2 changes significantly less than the average calculated across all residues within the structure, indicating that the differences in behaviors between the two systems is likely the result of allosteric influence, where the new substitutions in COV-2 induce flexibility and dynamical changes elsewhere in the structure.SIGNIFICANCEHere we have conducted a comparative analysis between the SARS-CoV-1 and SARS-CoV-2 mPro systems to shed mechanistic insight on the biophysical changes associated with the mutations between these two enzymes. Our work shows that the CoV-2 mPro system exhibits enhanced cross-chain communication between catalytic site residues and the rest of the structure. Further, both dynamic coupling and dynamic flexibility analyses indicates that, largely, the dynamic changes as evaluated by DCI and DFI occur at sites other than the mutation sites themselves, indicating that the functional differences between these two proteins are a result of dynamic allostery

Cell-penetrating peptides (CPPs) hold great promise for intracellular delivery of therapeutic proteins. However, endosomal entrapment of transduced cargo is a major bottleneck hampering their successful application. While developing a transducible zinc finger protein-based artificial transcription factor targeting the expression of endothelin receptor A, we identified interaction between the CPP and the endosomal membrane or endosomal entanglement as a main culprit for endosomal entrapment. To achieve endosomal disentanglement, we utilized endosome-resident proteases to sever the artificial transcription factor from its CPP upon arrival inside the endosome. Using this approach, we greatly enhanced the correct subcellular localization of the disentangled artificial transcription factor, significantly increasing its biological activity and distribution in vivo. With rational engineering of proteolytic sensitivity, we propose a new design principle for transducible therapeutic proteins, helping CPPs attain their full potential as delivery vectors for therapeutic proteins.

The extensive use of proteases in many areas, especially in the textile, food, and detergent industries, has led increases in the discovery and characterization of novel proteolytic enzymes. Microorganism from the Antarctic may contain novel proteases because of the extreme environment; however, to our knowledge, few relevant studies have been reported to date. Here, an Antarctic soil metagenomic fosmid library was successfully constructed, and a 36 kb positive fosmid clone was screened using lactose-free skim milk agar. Sequence alignment and phylogenetic analysis indicated that the positive fosmid clone encoded a new protein belonging to the metalloprotease subfamily M43B (36.6 % identity), which was named as Pr18H-2. Enzymatic analysis revealed that Pr18H-2 possess several features: (1). it demonstrated high thermal stability with the optimal temperature was 60℃; (2). it was active in the broad pH range of 5.0–10.0, with the optimal pH of 8.0 which allowed it to be an alkaline protease; (3). it was able to degrade a wide range of protein substrates and exhibit the substrate preferences for small substrates, such as casein hydrolysate and fibrin; (4). it showed that the enzymatic activity was increased by approximately four times in the presence of 5 mM Co2+.

In recent years, many attempts have been made to find new plant proteases to make artisan cheeses. The global increase in cheese consumption, together with a lower supply and increasing cost of calf rennet, religious factors (Islam and Judaism) and food choices (vegetarianism) have led to the search for suitable rennet substitutes for milk clotting. This study describes the milk-clotting and hydrolytic activities of an aspartic protease from Salpichroa origanifolia fruits (SoAP) on individual caseins to explore its potential use as an alternative to animal rennet. The milk-clotting index obtained for SoAP was 8.4 times lower than that obtained for chymosin. SoAP showed a higher degree of hydrolysis on α-casein than on the other fractions under the proposed conditions. RP-HPLC, mass spectrometry analyses and sequencing of the hydrolysates allowed identifying five peptides from α-casein, one peptide from β-casein, and three peptides from k-casein. In silico analysis showed that the peptides identified may display a wide variety of potential biological activities. These results demonstrate the possibility of using SoAP for the manufacture of new types or artisan cheeses, with the simultaneous added value of the potential health-promoting benefits of the bioactive peptides generated during the hydrolysis.

In insects, proteolytic cascades medicated by serine proteases (SPs), serine protease homologs (SPHs) and prophenoloxidases (PPOs) control several physiological processes, notably the innate immunity. However, no attempts have been made to identify and characterize these genes in Spodoptera frugiperda, one of the most destructive agricultural pests. In this study, 83 SPs, 26 SPHs and four PPOs were respectively identified in S. frugiperda genome based on homology blast against those of other insects. We then analyzed the domain organization of these proteins and assigned them into different groups by phylogenetic reconstruction. Furthermore, the mRNA levels of clip-domain SPs/SPHs (cSPs/cSPHs) and PPOs were quantified in response to a mixed infection of Micrococcus luteus and Escherichia coli, and obvious accumulations were recorded in immune tissues, including hemocytes and fat body. In the latter study, we profiled the expression patterns of highly expressed cSPs and PPOs in different developmental stages, including egg, larva, pupa, female and male adults. It was shown that most cSPs were abundantly expressed in adults, while PPOs were detected at high levels in both egg and larval stages. These current findings substantially add to our understanding of the roles of S. frugiperda SPs, SPHs and PPOs in immune regulation and further lay a solid foundation for uncovering the interaction mechanisms between insects and pathogens.

Structure-based antiviral developments in the past two years have been dominated by the structure determination and inhibition of SARS-CoV-2 proteins and new lead molecules for picornaviruses. The SARS-CoV-2 spike protein has been targeted successfully with antibodies, nanobodies, and receptor protein mimics effectively blocking receptor binding or fusion. The two most promising non-structural proteins sharing strong structural and functional conservation across virus families are the main protease and the RNA-dependent RNA polymerase, for which design and reuse of broad range inhibitors already approved for use has been an attractive avenue. For picornaviruses, the increasing recognition of the transient expansion of the capsid as a critical transition towards RNA release has been targeted through a newly identified, apparently widely conserved, druggable, interprotomer pocket preventing viral entry. We summarize some of the key papers in these areas and ponder the practical uses and contributions of molecular modeling alongside empirical structure determination.

Apoptosis plays essential roles in the immune defense mechanism against pathogen infection. Caspase 3 is a family of cysteine proteases involved in apoptosis and the immune response. In this study, the full-length of mud crab (Scylla paramamosain) caspase 3 (designated as Sp-caspase 3) was cloned and characterized. The open reading frame of Sp-caspase 3 was comprised a 1035 bp, which encoded a putative protein of 344 amino acids. Sp-caspase 3 was ubiquitously expressed in various tissues with a high-level expression in hemocytes. Cellular localization analysis revealed that Sp-caspase 3 was located in the cytoplasm and nucleus. Over-expression of Sp-caspase 3 could induce cell apoptosis. In addition, V. Parahaemolyticus infection induced the relative expression of caspase-3 mRNA and increased caspase-3 activity. Knocking down Sp-caspase 3 in vivo significantly reduced cell apoptosis and increased mortality of mud crab after V. parahaemolyticus infection. These results indicated that Sp-caspase 3 played important roles in the immune response and apoptosis against bacterial infection.

Proteases are the most abundant enzyme gene family in vertebrates and they execute essential functions in all living organisms. Their main role is to hydrolase the peptide bond within proteins, a process also called proteolysis. Contrary to the conventional paradigm, proteases are not only random catalytic devices, but can perform highly selective and targeted cleavage of specific substrates, finely modulating multiple essential cellular processes. Lysosomal protease cathepsins comprise 3 families of proteases that preferentially act within acidic cellular compartments, but they can also be found in other cellular locations. They can operate alone or as part of signalling cascades and regulatory circuits, playing important roles in apoptosis, extracellular matrix remodelling, hepatic stellate cell activation, autophagy and metastasis, contributing to the initiation, development and progression of liver disease. In this review, we comprehensively summarise current knowledge on the role of lysosomal cathepsins in liver disease, with a particular emphasis on liver fibrosis, non-alcoholic fatty liver disease and hepatocellular carcinoma., AM’s research was supported by a “Research Challenges" R+D+i Project (RTI2018-097475-A-100) and a Ramon y Cajal contract (RYC-2016-19731) awarded by FEDER/Ministerio de Ciencia e Innovación - Agencia Estatal de Investigación. PRB is supported by a FPU PhD studentship (FPU19/05357) funded by Ministerio de Ciencia, Innovación y Universidades. VP held a PhD studentship funded by University of Naples Federico II. MFF salary was supported by JAE Intro fellowship (JAEINT_20_02409) from the Spanish National Research Council (CSIC) and FI AGAUR (2021 FI_B 00249).

Lumbrokinases (LKs) belong to the group serine proteases capable to prevent thrombosis through the proteolysis of both plasminogen-bound and plasminogen-free fibrin molecules. The article presents improved activity of Lumbrokinase-6 (Lk-6) by suggesting the substitution of a Serine found at position 214 (Lk-6) with three other amino acids namely Glutamic acid, Proline and Valine. To characterize the stability, enzyme-substrate interaction and improved activity of three mutant Lk-6 proteins (Lk-Glu214, Lk-Pro214, Lk-Val214) In Silico tools were utilized. Subsequently, Lk-6 wild type and three mutant proteins were subjected to structure prediction, molecular modeling, phylogeny, molecular docking and Protein-Protein Interaction (PPI) using the In Silico tools. Collection and analysis of results revealed that substituted mutation at Ser214 with Valine214 can appreciably stabilize the overall structure of Lk-6 protein and makes its interaction with plasminogen activator physically powerful for higher plasmin activation. Similarly, Serine214 to Valine214 substitution resulted the direct activation of plasmin breakage at the Arg561-Val562 bond. The Arg-Val at position 561–562 in plasminogen and its connection at catalytic site have significantly shown that the predicted residue Valine214 could be further examined through genetic engineering of Lk-6 protein. Therefore, such results are potential steps towards the engineering of smart and active Lks.

Background Bovine colostrum (BC) and chicken egg contain proteins possessing growth factor activity. Epidermal growth factor (EGF) provides much of the pro-reparative activity within BC. Clinical use of orally administered peptide growth factors is hampered by digestion from pancreatic proteases. Objectives We examined whether adding a protease inhibitor [soybean trypsin inhibitor (SBTI) or ovomucoid] protected bioactivity of BC ± egg or EGF alone against pancreatic digestion using in vitro and in vivo models. Methods BC, egg, or EGF alone or in combination with trypsin inhibitors were tested for proliferative (Alamar blue) activity using human gastric adenocarcinoma (AGS) cells, prior to and after incubation with HCl/pepsin and trypsin/chymotrypsin. Data were analyzed using 2-factor ANOVA. Eight groups (n = 10) of adult female Sprague-Dawley rats (mean: 188.3 ± 0.8 g) received 20 mg/kg/d of BC + egg, 100 μg/d of EGF, 5 mg/d ovomucoid, or 10.8 mg/d SBTI, alone or in combination (in 1 mL 3% NaHCO3) by gavage for 9 d and dextran sodium sulfate (DSS; 5% in drinking water) for the final 7 d. Histology, microscopic damage score, and myeloperoxidase (MPO) were assessed and analyzed using 1-factor ANOVA. Results Proliferative activities of BC, egg, or EGF were reduced 40–57% by HCl/pepsin exposure and further reduced 14–24% by chymotrypsin/trypsin. Co-addition of SBTI or ovomucoid truncated the decrease in proliferative bioactivity caused by chymotrypsin/trypsin by 54–100% (P

Flaxseed (Linum usitatissimum), commonly known as linseed is an oilseed crop, emerging as an important and functional ingredient of food and has been paid more attention due to its nutritional value as well as beneficial effects. It is mainly rich in is α-linolenic acid (ALA, omega-3 fatty acid), fibres and lignans that have potential health benefits in reducing cardiovascular diseases, diabetes, osteoporosis, atherosclerosis, cancer, arthritis, neurological and autoimmune disorders. Due to its richness in omega-3 fatty acid, a group of enzymes known as fatty acid desaturases (FADs) mainly introduce double bonds into fatty acids’ (FAs) hydrocarbon chains that produce unsaturated fatty acids. Fatty acid desaturase 3 (FAD3), the commonest microsomal enzyme of omega-3 fatty acid, synthesizes linolenic acid (C18:3) from linoleic acid located in endoplasmic reticulum (ER) facing towards the cytosol. The emerging field of bioinformatics and large number of databases of bioactive peptides, helps in providing time-saving and efficient method for identification of potential bioactivities of any protein. In this study, 10 unique sequences of FAD3 from flaxseed protein have been used for in silico proteolysis and releasing of various bioactive peptides using three plant proteases, namely ficin, papain and stem bromelain, that are evaluated with the help of BIOPEP database. Overall, 20 biological activities were identified from these proteins. The results showed that FAD3 protein is a potential source of peptides with angiotensin-I-converting enzyme (ACE) inhibitory and dipeptidyl peptidase-IV (DPP-IV) activities, and also various parameters such as ∑A, ∑B, AE, W, BE, V and DHt were also calculated. Furthermore, PeptideRanker have been used for screening of novel promising bioactive peptides. Various bioinformatics tools also used to study protein’s physicochemical properties, peptide’s score, toxicity, allergenicity aggregation, water solubility, and drug likeliness. The present work suggests that flaxseed protein can be a good source of bioactive peptides for the synthesis of good quality and quantity of oil, and in silico method helps in investigating and production of functional peptides.

Using molecular dynamics simulations, we describe how crowded environments affect the internal dynamics and diffusion of the hepatitis C virus proteases NS3/4A. This protease plays a key role in viral replication and is successfully used as a target for antiviral treatment. The NS3 enzyme requires a peptide cofactor, called NS4A, with its central part interacting with the NS3 β-sheet, and flexible, protruding terminal tails that are unstructured in water solution. The simulations describe the enzyme and water molecules at atomistic resolution, whereas crowders are modeled via either all-atom or coarse-grained models to emphasize different aspects of crowding. Crowders reflect the polyethylene glycol (PEG) molecules used in the experiments to mimic the crowded surrounding. A bead-shell model of folded coarse-grained PEG molecules considers mainly the excluded volume effect, whereas all-atom PEG models afford more protein-like crowder interactions. Circular dichroism spectroscopy experiments of the NS4A N-terminal tail show that a helical structure is formed in the presence of PEG crowders. The simulations suggest that crowding may assist in the formation of an NS4A helical fragment, positioned exactly where a transmembrane helix would fold upon the NS4A contact with the membrane. In addition, partially interactive PEGs help the NS4A N-tail to detach from the protease surface, thus enabling the process of helix insertion and potentially helping the virus establish a replication machinery needed to produce new viruses. Results point to an active role of crowding in assisting structural changes in disordered protein fragments that are necessary for their biological function.

Aedes aegypti can transmit dengue, chikungunya, zika and yellow fever. Its control represents the main strategy for reducing the spreading of these diseases. In the present work, Plectranthus barbatus leaf saline extract (SE), decoction and infusion were studied for their effects on the survival of A. aegypti third instar larvae (L3), as well as for the presence of secondary metabolites, lectins, and trypsin inhibitors. The effects of SE (non-heated or heated) on digestive proteases, permeability of peritrophic membrane, morphophysiology, microbiota growth and melanogenesis of L3 are reported. SE killed L3 (LC50 of 0.48%, w/v), while decoction and infusion were not larvicides. HPLC analysis identified caffeic acid and flavonoids in SE, decoction, and infusion. SE also contained a ribose/galactose-binding lectin, trypsin inhibitor activities and polypeptides (34, 63 and 66 kDa) resistant to hydrolysis by L3 gut proteases. The incubation of SE with larval proteases did not alter lectin and trypsin inhibitor activities. Treatment of L3 with SE (at LC50) increased the permeability of peritrophic membrane and prevented the larvae from passing into the fourth instar. The morphology of larval midgut was not affected by SE, but the content of neutral polysaccharides in epithelial cells was reduced. SE stimulated in vitro the proliferation of L3 midgut microbiota and increased in vivo the production of melanin by larvae. The heating (100°C, 5 h) of SE increased lectin, trypsin inhibitor and larvicidal activities. In conclusion, SE larvicidal activity may be linked to the presence of lectin and trypsin inhibitor and involves the inhibition of digestive proteases, damage to larval midgut morphophysiology and induction of dysbiosis.

Papaya is reported to trigger food and respiratory allergy. Here, we identified chymopapain Cari p 2 as an allergen that can sensitize atopic individuals through fruit consumption followed by respiratory hazards through pollen exposure. Recombinant Cari p 2 displayed IgE-reactivity with 78% of papaya allergic sera. rCari p 2 also displayed allergenic activity through basophil degranulation. rCari p 2 is correctly folded and showed irreversible denaturation in the melting curve. rCari p 2 displayed IgE-cross-reactivity with homologous cysteine proteases from kiwi and pineapple. Cari p 2 transcript was also detected in papaya pulps. rCari p 2 was resistant to pepsin digestion and retained IgE-reactivity after 60 minutes of pepsin digestion. In mouse model, rCari p 2 was found to elicit inflammatory responses in the lung and gastrointestinal epithelium. Hence, Cari p 2 is a newly characterized allergen with diagnostic and immunotherapeutic potential for managing allergic disorders in papaya sensitized individuals.

Background Consistent with fulminant endothelial cell activation, elevated plasma von Willebrand factor (VWF) antigen levels have been reported in patients with COVID‐19. The multimeric size and function of VWF are normally regulated through A Disintegrin And Metalloprotease with ThrombSpondin Motif type 1 motif, member 13 (ADAMTS‐13)‐‐mediated proteolysis. Objectives This study investigated the hypothesis that ADAMTS‐13 regulation of VWF multimer distribution may be impaired in severe acute respiratory syndrome‐coronavirus‐2 (SARS‐CoV‐2) infection contributing to the observed microvascular thrombosis. Patients and Methods Patients with COVID‐19 (n = 23) were recruited from the Beaumont Hospital Intensive Care Unit (ICU) in Dublin. Plasma VWF antigen, multimer distribution, ADAMTS‐13 activity, and known inhibitors thereof were assessed. Results We observed markedly increased VWF collagen‐binding activity in patients with severe COVID‐19 compared to controls (median 509.1 versus 94.3 IU/dl). Conversely, plasma ADAMTS‐13 activity was significantly reduced (median 68.2 IU/dl). In keeping with an increase in VWF:ADAMTS‐13 ratio, abnormalities in VWF multimer distribution were common in patients with COVID‐19, with reductions in high molecular weight VWF multimers. Terminal sialylation regulates VWF susceptibility to proteolysis by ADAMTS‐13 and other proteases. We observed that both N‐ and O‐linked sialylation were altered in severe COVID‐19. Furthermore, plasma levels of the ADAMTS‐13 inhibitors interleukin‐6, thrombospondin‐1, and platelet factor 4 were significantly elevated. Conclusions These findings support the hypothesis that SARS‐CoV‐2 is associated with profound quantitative and qualitative increases in plasma VWF levels, and a multifactorial down‐regulation in ADAMTS‐13 function. Further studies will be required to determine whether therapeutic interventions to correct ADAMTS‐13‐VWF multimer dysfunction may be useful in COVID‐microvascular thrombosis and angiopathy.

Furins are a family of proteases involved in the processing of many proproteins including growth regulators, immune proteins, and toxins. However, very few studies have been conducted on furin-like PCs in insects. Herein, we isolated and characterized full-length furin-like cDNAs (Psfurin-1 and Psfurin-2) from larvae of Protaetia brevitarsis seulensis (Kolbe). To search for furin-like PC cDNAs, we used high-throughput RNA sequencing (RNA-seq) expression profiling of data. The Psfurin-1 and Psfurin-2 transcripts, 2,370 and 3,702 nucleotides in length, respectively, encode peptides of 790 and 1,234 amino acids with calculated molecular masses of 88.3 and 135.6 kDa. We identified a consensus site for proprotein convertase that includes Asp, His, Asn, and Ser catalytic residues, and revealed the presence of a subtilisin-like serine protease/putative cell envelope proteinase domain related to the immune system. The predicted structures of the two proteins are very similar, but the cell envelope protease/immune-related domain differs somewhat. To examine the possible immune-related expression of Psfurin-1 and 2 transcripts, late larvae were injected with the Gram-negative bacterium Escherichia coli or the yeast Saccharomyces cerevisiae. Expression of Psfurin-1 mRNA in E. coli K12-challenged larvae was ~15-fold higher at 8 h and ~20-fold higher at 12 h than in LB medium-challenged control larvae. In the case of yeast-challenged larvae, expression of Psfurin-1 mRNA was not as elevated as in E. coli K12-challenged larvae, but expression was 4–5-fold higher than in LB medium-challenged larvae. Expression of Psfurin-2 mRNA in E. coli K12-challenged and yeast-challenged larvae was also significantly higher than in LB medium-challenged controls from 4 to 48 h, but expression levels were lower than for Psfurin-1 mRNA in both E. coli K12- and yeast-challenged larvae.

The emergence of multidrug resistant (MDR) HIV strains severely reduces the effectiveness of antiretroviral therapy. Clinical inhibitor darunavir (1) has picomolar binding affinity for HIV-1 protease (PR), however, drug resistant variants like PR(S17) show poor inhibition by 1, despite the presence of only two mutated residues in the inhibitor-binding site. Antiviral inhibitors that target MDR proteases like PR(S17) would be valuable as therapeutic agents. Inhibitors 2 and 3 derived from 1 through substitutions at P1, P2 and P2ʹ positions exhibit 3.4- to 500-fold better inhibition than clinical inhibitors for PR(S17) with the exception of amprenavir. Crystal structures of PR(S17)/2 and PR(S17)/3 reveal how these inhibitors target the two active site mutations of PR(S17). The substituted methoxy P2 group of 2 forms new interactions with G48V mutation, while the modified bis-fluoro-benzyl P1 group of 3 forms a halogen interaction with V82S mutation, contributing to improved inhibition of PR(S17).

Anticarsia gemmatalis is one of the main defoliators of soybean in Brazil. Bacillus thuringiensis (Bt) transgenic crops are used for their management. In this paper we used RNA-seq to explore the response of A. gemmatalis to Bt HD73, as well as to detect transcriptional differences after Bt infection between resistant and susceptible strains. A total of 3853 and 6224 differentially expressed genes (DGEs) were identified in susceptible and resistant larvae after Bt exposure, respectively. We identified 2143 DEGs between susceptible and resistant larvae and 1991 between susceptible and resistant larvae Bt exposed. Immunity-related genes, Bt toxins receptors, proteases, genes involved in metabolic processes, transporters, cuticle proteins and mobile elements have been identified. qRT-PCR data demonstrated upregulation of five genes in susceptible strain after Bt exposure. These results provide insights to understand the molecular and cellular mechanisms of response to Bt that could be used in strategies to control agricultural pests.

The majority (90%) of the snakebite envenomation in Brazil accounts for Bothrops from the Viperidae family. Some snake venom serine proteases provoke blood coagulation in ophidian accident victims because of their fibrinolytic activity, one of those proteases from Bothrops jararaca (B. jararaca) has been chosen for this study. Our objectives were to isolate and characterize the target serine protease; isolate, purify, and characterize the orange bagasse flavone (hesperetin, Hst), and investigate the interactions between the targets, enzyme, and hesperetin. The purified serine protease was named BjSP24 because of its molecular mass and proteolytic activity. BjSP24 was folded and characterized using circular dichroism and showed low alpha-helix contents (7.7%). BjSP24 exhibited sequence similarity to other known snake venom serine proteases as measured in the enzyme tryptic peptides' LC-MS/MS run. Hesperetin was obtained within the expected yield and with the predominance of 2S isomer (82%). It acted as a mixed inhibitor for the serine protease (SVSP) from Bothrops jararaca snake venom observed in three different in vitro experiments, fluorescence, kinetics, and SSTD-NMR. It is still to determine if hesperetin might aid-in reverting the on site blood clotting problems just after snakebite accidents.

The immune system is continuously challenged with large quantities of exogenous antigens at the barriers between the external environment and internal human tissues. Antimicrobial activity is essential at these sites, though the immune responses must be tightly regulated to prevent tissue destruction by inflammation. Secretory Leukocyte Protease Inhibitor (SLPI) is an evolutionarily conserved, pleiotropic protein expressed at mucosal surfaces, mainly by epithelial cells. SLPI inhibits proteases, exerts antimicrobial activity and inhibits nuclear factor-kappa B (NF-κB)-mediated inflammatory gene transcription. SLPI maintains homeostasis at barrier tissues by preventing tissue destruction and regulating the threshold of inflammatory immune responses, while protecting the host from infection. However, excessive expression of SLPI in cancer cells may have detrimental consequences, as recent studies demonstrate that overexpression of SLPI increases the metastatic potential of epithelial tumors. Here, we review the varied functions of SLPI in the respiratory tract, skin, gastrointestinal tract and genitourinary tract, and then discuss the mechanisms by which SLPI may contribute to cancer.

Food-derived bioactive peptides are gaining popularity for their vast amount of potential health benefits against diseases such as obesity, hypertension, type 2 diabetes, and many more. However, many of these bioactivities are demonstrated using in vitro models, which do not completely reflect the true conditions that peptides experience upon oral ingestion. Furthermore, results obtained from in vivo studies are not entirely reproducible in other in vivo models due to systemic physiological differences. This review discusses the different bioaccessibility challenges, such as gastrointestinal tract hydrolysis by proteases, food matrix interactions, and intestinal barrier interaction that must be considered when developing peptide-based nutraceuticals or functional foods. The paper also discusses the potential avenues for improving bioaccessibility of bioactive peptides, amongst other factors that should be considered, during the design and production phases.

Cell invasion through extracellular matrix (ECM) plays pivotal roles in cell dispersal during development, immune cell trafficking, and cancer metastasis. Many elegant studies have revealed the specialized cellular protrusions, proteases, and distinct modes of migration invasive cells use to overcome ECM barriers. Less clear, however, is how invasive cells provide energy, and specifically ATP, to power the energetically demanding membrane trafficking, F-actin polymerization, and actomyosin machinery that mediate break down, remodeling, and movement through ECMs. Here, we provide an overview of the challenges of examining ATP generation and delivery within invading cells and how recent studies using diverse invasion models, experimental approaches, and energy biosensors are revealing that energy metabolism is an integral component of cell invasive behavior that is dynamically tuned to overcome the ECM environment.

Autophagy is one of the major cellular degradation pathways, which prevents accumulation of cellular wastes including "hazardous" material such as oxidized proteins and lipids and allows removal of aggregates and dysfunctional organelles. Hence, autophagy is meant to preserve cell survival, and is mostly protective. However, autophagy may trigger a feedforward, exaggerated cycle in which cells continue to degrade proteins and organelles, finally leading to autophagy-dependent cell death (ADCD), a process that can be initiated with lysosomotropic detergents, which are protonated within the lysosome and cause a permeabilization of the membrane. Such drugs may be useful to combat cancer. In some paradigms of ADCD, there is evidence that the cellular fate is determined by the integrity of lysosomal membranes, transporters, enzymes and ion gradients. Detergent-like effects of lysosomotropic drugs can over-activate autophagy. A disruption of the lysosomal membrane barrier with leakage of lysosomal enzymes or lipids may trigger a vicious cycle via proteases and accumulation of lipids, which impair the functions of the plasma - and organelle membranes. This review summarizes the current evidence for a crosstalk between lysosomal dysfunction and autophagy and the lysosomal events, which progress toward ADCD with a focus on the role of sphingolipids and cholesterol as cargo and as regulators of ADCD.

Protein homeostasis mechanisms are fundamentally important to match cellular needs and to counteract stress conditions. A fundamental challenge is to understand how defective proteins are recognized and extracted from cellular organelles to be degraded in the cytoplasm. The endoplasmic reticulum (ER)-associated degradation (ERAD) pathway is the best-understood organellar protein quality control system. Here, we review new insights into the mechanism of recognition and retrotranslocation of client proteins in ERAD. In addition to the membrane-integral ERAD E3 ubiquitin ligases, we highlight one protein family that is remarkably often involved in various aspects of membrane protein quality control and protein dislocation: the rhomboid superfamily, which includes derlins and intramembrane serine proteases. Rhomboid-like proteins have been found to control protein homeostasis in the ER, but also in other eukaryotic organelles and in bacteria, pointing toward conserved principles of membrane protein quality control across organelles and evolution.

Serine proteases are fundamental components of biology, including innate immunity, which is systematically orchestrated in an orderly, balanced fashion in the healthy host. Such serine proteases are found in two well-recognized pathways of an innate immune network, coagulation and complement. Both pathways, if uncontrolled due to a variety of causes, are pathogenic in numerous diseases, including coagulation disorders and infectious diseases. Previous studies have reported sequence homologies, functional similarities and interplay between these two pathways with some implications in health and disease. The current study newly reveals that complement component factor B (Bf), the second component of the alternative complement pathway, has thrombin-like activity, which is supported by a characteristic homology of the trypsin-like domain of Bf to that of thrombin. Moreover, we newly report that the trypsin-like domain of Bf is closely related to Limulus clotting factor C, the LPS sensitive clotting factor of the innate immune system. We will also discuss potential implications of our findings in diseases.

Mis-functional βAPP processing is deemed to be the major phenomenon resulting in increased neuronal cell death, impaired neurogenesis and the loss of synapses, which eventually manifest as the complex symptoms of Alzheimer’s disease. Despite of several milestones having been achieved in the field of drug development, the stigma of the disorder as an incurable disease still remains. Some ADAM proteases mediate the physiological non-amyloidogenic α-secretase processing of βAPP that generates neuroprotective sAPPα production. Earlier studies have also pointed out the role of p53 in Alzheimer’s disease neuropathology, although a direct link with metalloprotease activities remains to be established. In this study, we explored the consequences of α-secretase inhibition on p53 status in cultured human neuroblastoma SH-SY5Y cells by means of specific inhibitors of ADAM10 and ADAM17 and the metal chelator and general metalloprotease inhibitor phenanthroline. We establish that, beyond the ability of all inhibitors to affect sAPPα production to varying degrees, phenanthroline specifically and dose-dependently lessened βAPP expression, a phenomenon that correlated with a strong increase in p53 protein levels and a concomitant decrease of the p53-degrading calpain protease. Furthermore, treatment of cells at concentrations of phenanthroline similar to those inducing increased levels of p53 induced cell cycle arrest leading to apoptosis. Altogether, our results identify new roles of phenanthroline in perturbing βAPP, p53 and calpain biology, and suggest that the use of this compound and its derivatives as antimicrobial and anti-cancer therapies might trigger Alzheimer’s disease pathogenesis.

Fungal proteases are well-known allergens. In this issue of Immunity, Wu et al. (2021) observe that allergic airway responses to Candida albicans are mediated by the peptide toxin candidalysin rather than proteases. Candidalysin promotes these responses by stimulating platelets to release the Wnt antagonist Dickkopf-1.

Purpose Dentin endogenous proteases are involved in the progression of dental caries. This study investigated the proteolytic activity of endogenous enzymes of carious and sound dentin of primary teeth and the potential selective inhibitory effect of plant-derived proanthocyanidins (PACs). Methods Carious and sound dentin from forty extracted primary teeth were collected and cryo-pulverized into a fine powder. Two PAC mixtures were prepared from grape seeds (Vitis vinifera, Vv), an oligomer enriched and polymer depleted extract (Vve) as well as a further refined mixture containing primarily trimeric and tetrameric PACs (Vv3+4). Fluorescein conjugate-gelatin was used as cleavage substrate. The inhibition of endogenous protease activity was determined by the fluorescence emission intensity of cleaved gelatin at 535 nm over a 24-hour period. Results Deciduous carious dentin exhibited higher gelatinolytic activity (~7 fold higher) than sound dentin (p Conclusions Endogenous enzymes are highly active in carious dentin, contributing for host-driven degradation along with the breakdown by cariogenic bacteria. The protease inhibitory effect of both the refined Vv3+4 preparation and its Vve precursor appears to be promising as an effective strategy to impair caries progression in primary teeth.

Background The ever-expanding human population demands some novel ideas and ingenious ways to produce foods of sufficient quality and quantity that would meet the protein requirements of humans. Muscle proteins, both lab- and farm-grown, are going to contribute their part and play an important role in near and distant future. However, finite animal and land resources and increasing environmental concerns will limit our capacity to produce adequate muscle proteins for all. A viable option to sustain protein resources is to reduce the food wastage and to fully utilize existing muscle proteins by improving the digestibility of muscle foods. The use of novel food processing techniques may support such objectives. However, this is relatively nascent area and there is an urgent need to expand our understanding by examining the basic knowledge of how muscle proteins processed under different conditions behave in the gastrointestinal environment, both in vivo and in vitro. Scope and approach The present review highlights some of the novel processing techniques of improving muscle protein digestibility and discusses how different processing conditions affect the digestibility of muscle proteins under gastrointestinal digestion environments. The underlying mechanisms have been discussed in detail and supported by the current literature. Key findings and conclusions Protein digestibility of muscle foods can be improved by employing several thermal and non-thermal emerging technologies, which have potential to improve the susceptibility of muscle proteins to gastrointestinal proteases. By affecting the structural (quaternary, tertiary or even secondary) and functional properties of the proteins and muscle microstructure, emerging technologies such as pulsed electric field, high-pressure, ultrasound and hydrodynamic shockwave, can induce denaturation and affect the unfolding and refolding of the proteins, thereby affecting both the diffusion of gastrointestinal proteases deep into the protein matrix as well as their accessibility to cleavage sites. The denaturation patterns of the processed proteins are not always predictable and depends on the processing conditions and the origin of the protein, thereby affecting protein digestibility positively or negatively. Extensive research is required to understand optimum processing conditions of these emerging technologies towards the favourable effects on the protein digestibility as affected by protein structure and muscle configuration to enable processing conditions suitable for different meat cuts and from different species. There is a need to improve and standardise the methods used to study meat digestion particularly ‘in vitro’.

Plant-derived proteases, bromelain, papain, and ficin, are broad-acting enzymes with generally recognized as safe status for foods and have current application in several food industries. These proteases have also been reported to have antimicrobial properties. This study investigated the efficacy of commercially prepared bromelain, papain, and ficin, individually and combined (2,500 ppm of crude extract), for inactivation of hepatitis A virus (HAV) and human norovirus surrogates, Tulane virus (TV), and murine norovirus (MNV). Various treatment temperatures (45, 50, or 55°C), times (10 or 60 min), and pH values (5.5 or 7.0) in the presence of cysteine (2 mM) were evaluated. Inactivation was assessed by infectivity in plaque assay for TV and MNV and by median tissue culture infective dose for HAV. No reduction in infectious TV or HAV was attributed to the plant-derived proteases at any of the conditions tested. Infectious MNV was reduced by 1 to 3 log PFU/mL; the most effective treatment was bromelain at pH 7 and 50°C for 10 min. A time course study with MNV in bromelain at 50°C indicated that a 2-log PFU/mL reduction could be achieved within 6 min, but extended treatment of 15 min was still insufficient to eliminate infectious MNV. The lack of or limited efficacy of bromelain, papain, and ficin on HAV, TV, and MNV, even at elevated temperatures and exposure times, suggests the plant-derived proteases are not commercially applicable for inactivation of virus on commodities or materials that could not also withstand mild heat treatment. The variable susceptibilities observed between TV and MNV illustrate limitations in utilization of surrogates for predicting pathogen behavior for a structure-specific treatment. HIGHLIGHTS

Though numerous proteases have been isolated and screened for the dehairing purpose, their use in the leather industry is limited mainly due to high cost, the need for expertise, and control during unit operation and alterations in the quality of leather due to lack of the right kind of substrate specificity of the enzymes used. This paper deals with the comparative specificity and dehairing efficiency of proteases isolated from Bacillus cereus VITSP01 (PE2) and Brevibacterium luteolum VITSP02 (PE). PE2 and PE were found to be trypsin-like and elastase-like serine proteases respectively. The protease of VITSP02 degraded the proteoglycans efficiently in comparison to that of VITSP01. The results suggest that the possible targets of the studied proteases might be skin proteoglycans, including those cementing the hair root bulb. Hence, an in-depth study on the substrate specificity of the dehairing proteases would help in designing an improved screening method for isolating potent dehairing enzymes.

Graphical abstract, Highlights • The SARS-CoV-2 main protease is a prime drug target. • Coronavirus main proteases share a structurally conserved substrate-binding region. • The structure and sequence similarity of SARS-CoV-2 to SARS-CoV readily allows testing of inhibitors designed for the latter. • Both covalent and non-covalent inhibitors of the SARS-CoV-2 main protease have been designed., The coronavirus disease 2019 (COVID-19) pandemic has prompted an urgent need for new treatment strategies. No target-specific drugs are currently available for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but new drug candidates targeting the viral replication cycle are being explored. A prime target of drug-discovery efforts is the SARS-CoV-2 main protease (Mpro). The main proteases of different coronaviruses, including SARS-CoV-2, SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), share a structurally conserved substrate-binding region that can be exploited to design new protease inhibitors. With the recent reporting of the X-ray crystal structure of the SARS-CoV-2 Mpro, studies to discover Mpro inhibitors using both virtual and in vitro screening are progressing rapidly. This review focusses on the recent developments in the search for small-molecule inhibitors targeting the SARS-CoV-2 Mpro.

In this paper, the effect of glycerol at different concentrations and pH values on the stabilities of 13 immobilized enzymes has been analyzed. Among the enzymes, we have selected 5 lipases (immobilized on octyl or amino-glutaraldehyde supports), 3 proteases (immobilized on glyoxyl or amino-glutaraldehyde supports), 2 glycosidases, 1 laccase, 1 catalase (immobilized on glutaraldehyde matrix) and one penicillin acylase (immobilized on glyoxyl supports). The effect was completely enzyme-dependent. The results indicate that the stabilizing effect of glycerol is not universal, as in certain cases it produced even some destabilization of the immobilized enzyme (e.g., in the case of ficin extract, a protease). The glycerol effect was strongly concentration dependent (but not always the most significant results were observed at the highest glycerol concentrations), and it also strongly depends on the inactivation pH (at pH 9.0 this agent showed the strongest destabilization effects). Very interestingly, the effect of glycerol on enzyme stability was much dependent on the used immobilization protocol, which determines not only the intensity but even the sense of this effect. Thus, before using glycerol as enzyme stabilizer agent, it is necessary to confirm that the effect is positive for a specific preparation under the specific used conditions.

Objective: To study mRNA and protein expression of SARS-CoV-2 entry receptors (ACE2, CD147) and proteases (TMPRSS2, CTSL) in human oocytes, embryos, cumulus and granulosa cells. Design: Research Study. Setting: Clinical IVF treatment center. Patients: IVF patients treated at the Colorado Center for Reproductive Medicine. Interventions: Oocytes (GV, MII) and embryos (1 cell (1C); blastocyst (BL)) were donated to research at disposition by IVF patients. Follicular cells (cumulus (CC); granulosa (GC)) were collected from women undergoing egg retrieval after ovarian stimulation without an ovulatory trigger for IVM/IVF treatment cycles. Main Outcome Measures: The presence or absence of ACE2, CD147, TMPRSS2, and CTSL mRNA using RT-qPCR and protein using capillary Western blotting in human oocytes, embryos and ovarian follicular cells. Result s: RT- q-PCR analysis revealed high abundance of ACE2 gene transcripts in GV and MII oocytes compared to CC, GC, and BL. ACE2 protein was only present in MII oocytes, 1C, and BL embryos, but other ACE2 protein variants were observed in all the samples. TMPRSS2 protein was present in all samples while mRNA was observed only at the blastocyst stage. All the samples were positive for CD147 and CTSL mRNA expression. However, cumulus and granulosa cells were the only samples that showed co-expression of both CD147 and CTSL proteins in low abundance. Conclusions: Cumulus and granulosa cells are least susceptible to SARS-CoV-2 infection due to the lack of required receptors and proteases combination (ACE2/TMPRSS2 or CD147/CTSL) in high abundance. Co-expression of the ACE2 and TMPRSS2 proteins in MII oocytes, zygotes, and blastocysts demonstrate that these gametes and embryos have the cellular machinery required and thus are potentially susceptible to SARS-CoV-2 infection if exposed to the virus. However, we do not yet know if infection occurs in vivo, or in vitro in an ART setting.

Contact activation is triggered when blood is exposed to compounds or "surfaces" that promote conversion of the plasma zymogens factor XII (FXII) and prekallikrein to the active proteases FXIIa and kallikrein. FXIIa promotes blood coagulation by converting zymogen factor XI (FXI) to the protease FXIa. Contact activation appears to represent an enhancement of the propensity for FXII and prekallikrein to reciprocally activate each other by surface-independent limited proteolysis. The nature of the activities that perpetuate this process, and that trigger contact activation, are debated. FXII and prekallikrein, like most members of the chymotrypsin/trypsin protease family, are synthesized as single polypeptides that are presumed to be in an inactive state. Internal cleavage leads to conformational changes in the protease domain that convert the enzyme active site from a closed conformation to an open conformation accessible to substrates. We observed that FXII expresses a low level of activity as a single-chain zymogen that catalyzes prekallikrein activation in solution, as well as surface-dependent activation of prekallikrein, FXI, and FXII (autoactivation). Prekallikrein also expresses activity that promotes cleavage of kininogen to release bradykinin, and surface-dependent FXII activation. Modeling suggests that a glutamine residue at position 156 in the FXII and prekallikrein protease domains stabilizes an open active site conformation by forming hydrogen bonds with Asp194. The activity inherent in FXII and prekallikrein suggests a mechanism for sustaining reciprocal activation of the proteins and for initiating contact activation, and supports the premise that zymogens of some trypsin-like enzymes are active proteases.

Atherosclerosis is a degenerative disease characterized by lesions that develop in the wall of large- and medium-sized arteries due to the accumulation of low-density lipoproteins (LDLs) in the intima. A growing bulk of evidence suggests that cholesterol oxidation products, known as oxysterols, and the aldehyde 4-hydroxy-2-nonenal (HNE), the major pro-atherogenic components of oxidized LDLs, significantly contribute to atherosclerotic plaque progression and destabilization, with eventual plaque rupture. The involvement of certain members of the protein convertase subtilisin/kexin proteases (PCSKs) in atherosclerosis has been recently hypothesized. Among them, PCSK6 has been associated with plaque instability, mainly thanks to its ability to stimulate the activity of matrix metalloproteinases (MMPs) involved in extracellular matrix remodeling and to enhance inflammation. In U937 promonocytic cells and in human umbilical vein endothelial cells, an oxysterol mixture and HNE were able to up-regulate the level and activity of PCSK6, resulting in MMP-9 activation as demonstrated by PCSK6 silencing. Inflammation, enhanced by these lipid oxidation products, plays a key role in the up-regulation of PCSK6 activity as demonstrated by cell pretreatment with NS-398, with epigallocatechin gallate or with acetylsalicylic acid, all with anti-inflammatory effects. For the first time, we demonstrated that both oxysterols and HNE, which substantially accumulate in the atherosclerotic plaque, up-regulate the activity of PCSK6. Of note, we also suggest a potential association between PCSK6 activity and MMP-9 activation, pointing out that PCSK6 could contribute to atherosclerotic plaque development.

The complex COVID-19-associated coagulopathy appears to impair prognosis. Recently, we presented the hypothesis that children are to some extent protected by higher α2-macroglobulin (α2-M) levels from severe COVID-19. In addition to endothelial cells, thrombin, and platelets, neutrophil granulocytes also appear to play an important role. Neutrophils extrude extracellular nets, which are histone- and protease-coated web-like DNA structures; activate coagulation and platelets; and release radicals and proteases such as elastase. The unique phylogenetically ancient and “versatile” inhibitor α2-M contributes particularly during childhood to the antithrombin activity of plasma, binds a broad spectrum of proteases, and interacts with other mediators of inflammation such as cytokines. It is suggested that the scope of basic research and clinical studies would include the potential role of α2-M in COVID-19.

Marine organisms are rich sources of enzymes and their inhibitors having enormous therapeutic potential. Among different proteolytic enzymes, serine proteases, which can be obtained from various marine organisms show a potential to biomedical application as thrombolytic agents. Although this type of proteases plays a crucial role in almost all biological processes, their uncontrolled activity often leads to several diseases. Accordingly, the actions of these types of proteases are regulated by serine protease inhibitors (SPIs). Marine SPIs control complement activation and various other physiological functions, such as inflammation, immune function, fibrinolysis, blood clotting, and cancer metastasis. This review highlights the potential use of serine proteases and their inhibitors as the new wave of promising drugs.

Neutrophil extravasation is a critical event during immune responses to infection or injury that ensures survival. However, given the destructive potential of neutrophil effector molecules such as proteases and reactive oxygen species, strict control of neutrophil recruitment is required to avoid tissue damage. Neutrophil extravasation is a multistep process of adhesive interactions between neutrophils and components of the venular wall, that is, endothelium, basement membrane and pericytes. A plethora of proteins have been identified as critical regulators of each step that provide for compensatory mechanisms thus ensuring that extravasation can happen even if one mechanism is defective. Here, we discuss recent discoveries of how extravasation is regulated with emphasis on neutrophil-specific mechanisms; and discuss potential implications for future research.

Traditional therapy for malignant neoplasms involving surgical procedures, radiotherapy and chemotherapy aims to kill neoplastic cells, but also affects normal cells. Therefore, exogenous proteases are the target of studies in cancer therapy, as they have been shown to be effective in suppressing tumors and reducing metastases. Pluronic F127 (F127) is a copolymer of amphiphilic blocks that has shown significant potential for drug administration, as it is capable of incorporating hydrophobic drugs and self-assembling in micrometers of nanometric size. This study investigated the effects of immobilization of the alkaline protease PR4A3 with pluronic F127 micelles on the enzyme-induced cytotoxicity. Protease immobilization was demonstrated through UV-visible and circular dichroism (CD) spectroscopies, as the enzyme interacts with the polymeric micelle of Pluronic F127 without changing its secondary structure. In addition, the immobilized form of the enzyme showed greater bioavailability after passing through the simulated gastrointestinal transit. Cell viability was assessed using the tetrazoic methylthiazole (MTT) assay. The results open perspectives for new research and development for PR4A3 in the treatment of colorectal carcinoma.

Proteases are critical virulence determinants of Porphyromonas gingivalis, an emerging Alzheimer's disease, cancer, and arthritis pathogen and established agent of periodontitis. Transposon sequencing has been employed to define the core essential genome of this bacterium and genes conditionally essential in multiple environments - abscess formation; epithelial colonization; and cigarette smoke toxin exposure; as well as to elucidate genes required for iron acquisition and a functional type 9 secretion system. Validated and predicted protein catabolism genes identified include a combination of established virulence factors and a larger set of seemingly more mundane proteolytic genes. The functions and relevance of genes that share essentiality in multiple disease-relevant conditions are examined. These common stress-related genes may represent particularly attractive therapeutic targets for the control of P. gingivalis infections.

Proteases are highlighted group of enzymes which corresponds to approximately 60% of enzymes used globally in commercial applications. Present study describes isolation of a new protease from TSA5 strain. Sequencing of 16SrRNA revealed identity to Bacillus sps with common five restriction sites (Acc651, Btscl, Fok1, Plel, Kpnl). The enzyme from TSA5 strain partial purified by, salting and dialysis which resulted a homogeneity of 6-fold with the yield of 34%. Enzyme activity was characterised for a broad range of pH (4–10.5) and temperature (0–120 °C). Optimum activity was observed at pH 8.5 and 80 °C. The enzyme showed substrate specificity to casein. The effect of different solvents and chemicals were assayed. It is found to be more active in presence of polar solvents. EDTA (Ethylenediaminetetraacetic acid) and β-mercaptoethanol shows potent inhibition. The purified fraction showed silver recovery from X-ray films. The protease also exhibited detergent activity and antibacterial property suggesting the use of enzymes for enzyme hydrolysis in industries for commercial applications.

Brain-derived neurotrophic factor (BDNF) is involved in pathophysiological mechanisms in neuropsychiatric diseases, including depression, anxiety, and schizophrenia (SZ), as well as neurodegenerative diseases like Parkinson's disease (PD) and Alzheimer's disease (AD). An imbalance or insufficient pro-brain-derived neurotrophic factor (proBDNF) transformation into mature BDNF (mBDNF) is potentially critical to the disease pathogenesis by impairing neuronal plasticity as suggested by results from many studies. Thus, promoting proBDNF transformation into mBDNF is therefore hypothesized as beneficial for the treatment of neuropsychiatric and neurodegenerative diseases. ProBDNF is proteolytically cleaved into the mBDNF by intracellular furin/proprotein convertases and extracellular proteases (plasmin/matrix metallopeptidases). This article reviews the mechanisms of the conversion of proBDNF to mBDNF and the research status of intracellular/extracellular proteolytic proteases for neuropsychiatric and neurodegenerative disorders.

Caspases are a family of proteases that play critical roles in controlling inflammation and cell death. Apoptosis is a caspase-3 mainly controlled behavior to avoid inflammation and damage to surrounding cells, whereas anomalistic cell apoptosis may be associated with many diseases. The detection and imaging of caspase-3 will be of great significance in evaluating the early therapeutic effect of tumors. Developing smart fluorescent probes may be helpful for the visualization of therapeutic effect compared with “always on” probes. Thus, more and more works toward activatable fluorescent probes for caspase-3 imaging have been reported. In addition, multifunctional probes have also been designed to further improve the imaging of caspase-3. Herein, this review systematically summarized the representative work of caspase-3 from the perspective of molecular design that it will play a guiding role in the design of probes that respond to caspase-3. Also, challenges and perspectives toward the field for imaging of cell apoptosis (caspase-3) are also discussed.

The coronavirus disease 2019 (COVID-19) pandemic caused by 2019 novel coronavirus (2019-nCoV) has been a crisis of global health, whereas the effective vaccines against 2019-nCoV are still under development. Alternatively, utilization of old drugs or available medicine that can suppress the viral activity or replication may provide an urgent solution to suppress the rapid spread of 2019-nCoV. Andrographolide is a highly abundant natural product of the medicinal plant, Andrographis paniculata, which has been clinically used for inflammatory diseases and anti-viral therapy. We herein demonstrate that both andrographolide and its fluorescent derivative, the nitrobenzoxadiazole-conjugated andrographolide (Andro- NBD), suppressed the main protease (Mpro) activities of 2019-nCoV and severe acute respiratory syndrome coronavirus (SARS-CoV). Moreover, Andro-NBD was shown to covalently link its fluorescence to these proteases. Further mass spectrometry (MS) analysis suggests that andrographolide formed a covalent bond with the active site Cys145 of either 2019-nCoV Mpro or SARS-CoV Mpro. Consistently, molecular modeling analysis supported the docking of andrographolide within the catalytic pockets of both viral Mpros. Considering that andrographolide is used in clinical practice with acceptable safety and its diverse pharmacological activities that could be beneficial for attenuating COVID-19 symptoms, extensive investigation of andrographolide on the suppression of 2019-nCoV as well as its application in COVID-19 therapy is suggested., Highlights • 2019-nCoV and SARS-CoV main proteases exhibit similar enzyme kinetics. • Andrographolide inhibits enzyme activity of 2019-nCoV and SARS-CoV main proteases. • Andrographolide is covalently linked to active cysteine of 2019-nCoV main protease.

Lactoferrin is a glycoprotein with a molecular weight of 80 kDa, which produced in many mammalian excretions. LF is involved in various physiological processes and known to possess prominent biocidal activities, serving as an effective agent against a wide range of pathogens. This effective biocidal activity of LF in association with immune system response has made this protein an attractive therapeutic candidate. Interaction of proteins with nanoparticles (NPs) gives rise to the formation of a dynamic NP-protein complex and can induce conformational changes in the adsorbed proteins which may lead to the change in their function. With the recent advances in nanotechnology, NPs may provide the protection and stabilization of LF from hydrolysis by some proteases and increase their uptake by targeted cells. These nanoformulations of LF can be used as diagnosis, disease targeting and drug delivery tools. Owing to its multiple functionalities, LF is a promising active ingredient to be loaded or adsorbed to NPs for preparing a stable, controlled surface NPs. Thus, LF NPs can potentially empower the resulting nanocomplex with attracting functionalities and might be useful in many applications, e.g., to modify the optical or rheological properties of products, or to encapsulate and deliver bioactive ingredients.

Huanglongbing (HLB) is the most devastating and widespread citrus disease. All commercial citrus varieties are susceptible to the HLB-associated bacterium, Candidatus Liberibacter asiaticus (CLas), which resides in the phloem. The phloem is part of the plant vascular system and is involved in sugar transport. To investigate the plant response to CLas, we enriched for proteins surrounding the phloem in an HLB susceptible sweet orange variety, Washington navel (Citrus sinensis (L) Osbeck). Quantitative proteomics revealed global changes in the citrus proteome after CLas inoculation. Plant metabolism and translation were suppressed, while defense-related proteins such as peroxidases, proteases and protease inhibitors were induced in the vasculature. Transcript accumulation and enzymatic activity of plant peroxidases in CLas infected sweet orange varieties under greenhouse and field conditions were assessed. While peroxidase transcript accumulation was induced in CLas infected sweet orange varieties, peroxidase enzymatic activity varied. Specific serine proteases were upregulated in Washington navel in the presence of CLas based on quantitative proteomics. Subsequent activity-based protein profiling revealed increased activity of two serine proteases, and reduced activity of one protease in two C. sinensis sweet orange varieties under greenhouse and field conditions. The observations in the current study highlight global reprogramming of the citrus vascular proteome and differential regulation of enzyme classes in response to CLas infection. These results open an avenue for further investigation of diverse responses to HLB across different environmental conditions and citrus genotypes.