Elucidation of potential relationship between endogenous proteases and key flavor substances in dry-cured pork coppa

Dry-cured meat products are considerably popular around the world due to unique flavor.Proteolysis is one of the enzymatic reactions from which flavor substances are derived,which is affected by endogenous proteases.The purpose aimed to reveal the potential relationship between endogenous proteases and key flavor substances in dry-cured pork coppa in this paper.The dynamic changes of endogenous proteases activity,free amino acids,and volatiles during dry-cured pork coppa processing were characterized.The results showed that 5 kinds of free amino acids,Glu,Lys,Val,Ala,and Leu,were identified as significant contributors to taste.Meanwhile,key volatiles,such as hexanal,nonanal,octanal,benzaldehyde,3-methyl butanoic acid,2-methyl propanoic acid,and ethyl octanoate,greatly contributed to the flavor characteristics of dry-cured pork coppa.Further partial correlation analysis was performed to better elucidate the relationship among parameters.The results revealed that close relationship between endogenous proteases and key substances.RAP not only significantly affected the accumulation of key active-amino acids,but also affected the accumulation of ethyl octanoate,2,3-pentanedione,and 2,3-octanedione by regulating the accumulation of octanoic acid and Leu.In addition,cathepsin B and D,DPP II,DPP IV and RAP notably affected accumulation of hexanal.

Roles of host proteases in the entry of SARS-CoV-2

The spike protein(S)of SARS-CoV-2 is responsible for viral attachment and entry,thus a major factor for host suscep-tibility,tissue tropism,virulence and pathogenicity.The S is divided with S1 and S2 region,and the S1 contains the receptor-binding domain(RBD),while the S2 contains the hydrophobic fusion domain for the entry into the host cell.Numerous host proteases have been implicated in the activation of SARS-CoV-2 S through various c leavage sites.In this article,we review host proteases including furin,trypsin,transmembrane protease serine 2(TMPRSS2)and cathepsins in the activation of SARS-CoV-2 S.Many betacoronaviruses including SARS-CoV-2 have polybasic residues at the S1/S2 site which is subjected to the cleavage by furin.The S1/S2 cleavage facilitates more assessable RBD to the receptor ACE2,and the binding triggers further conformational changes and exposure of the S2'site to proteases such as type Il transmembrane serine proteases(TTPRs)including TMPRSS2.In the presence of TMPRSS2 on the target cells,SARS-CoV-2 can utilize a direct entry route by fusion of the viral envelope to the cellular membrane.In the absence of TMPRSS2,SARS-CoV-2 enter target cells via endosomes where multiple cathepsins cleave the S for the successful entry.Additional host proteases involved in the cleavage of the S were discussed.This article also includes roles of 3C-like protease inhibitors which have inhibitory activity against cathepsin L in the entry of SARS-CoV-2,and discussed the dual roles of such inhibitors in virus replication.

STRUCTURE-FUNCTION FEATURES AND EFFECTS ON BLOOD COAGULATION OF SNAKE VENOM SERINE PROTEASES*

Snake venoms,especially those from the two subfamilies,Crotalinae and Viperinae,contained a lot of serine proteases. They were responsible for the hemorrhage,shock,or disorder of blood coagulation after envenomation. They acted,by activating,inactivating,or other converting effects,on almost all the components of hemostatic and fibrinolytic systems. Their sequences were homologous to trypsin-kallikrein serine proteases. Variation of primary sequences out of active center results in the difference of substrate specificities and the further difference of biological and pharmacological activities. Because of their common and unique properties compared to their physiological corresponding factors,snake venom proteases are proved to be an excellent model for the study of protease substrate discriminating mechanism. Furthermore,they have found an important position both in basic research and application of hemostasis and thrombosis in clinic.

Effects of Different Light Qualities on Activity and Gene Expression of Caspase-like Proteases in Tobacco Leaves

[Objective] The study aimed to investigate the activity and gene expression of caspase-like proteases in tobacco leaves growing under different light qualities. [Method] By covering tobacco plants with white, red, yellow, blue and purple films to obtain different light quality, the changes of chlorophyll content, activity and gene expression of caspase-like proteases in the tobacco leaves were studied. [Results] Compared with treatments of white, red and yellow film, blue and purple films delayed the decrease of chlorophyll content and senescence of tobacco leaves at the late growth stage, and relatively lowered the activity and gene expression of caspase-like proteases during growth, development and senescence periods. [Conclusion] Different light qualities exhibited various effects on the growth, development and senescence of tobacco leaves, possibly by affecting the activity and gene expression of caspase-like proteases to some extent.

Regulation of intestinal permeability: The role of proteases

The gastrointestinal barrier is-with approximately 400 m^2-the human body's largest surface separating the external environment from the internal milieu. This barrier serves a dual function: permitting the absorption of nutrients, water and electrolytes on the one hand, while limiting host contact with noxious luminal antigens on the other hand. To maintain this selective barrier, junction protein complexes seal the intercellular space between adjacent epithelial cells and regulate the paracellular transport. Increased intestinal permeability is associated with and suggested as a player in the pathophysiology of various gastrointestinal and extraintestinal diseases such as inflammatory bowel disease, celiac disease and type 1 diabetes. The gastrointestinal tract is exposed to high levels of endogenous and exogenous proteases, both in the lumen and in the mucosa. There is increasing evidence to suggest that a dysregulation of the protease/antiprotease balance in the gut contributes to epithelial damage and increased permeability. Excessive proteolysis leads to direct cleavage of intercellular junction proteins, or to opening of the junction proteins via activation of protease activated receptors. In addition, proteases regulate the activity and availability of cytokines and growth factors, which are also known modulators of intestinal permeability. This review aims at outlining the mechanisms by which proteases alter the intestinal permeability. More knowledge on the role of proteases in mucosal homeostasis and gastrointestinal barrier function will definitely contribute to the identification of new therapeutic targets for permeability-related diseases.

Visceral hypersensitivity in inflammatory bowel diseases and irritable bowel syndrome: The role of proteases

Proteases, enzymes catalyzing the hydrolysis of peptide bonds, are present at high concentrations in the gastrointestinal tract. Besides their well-known role in the digestive process, they also function as signaling molecules through the activation of protease-activated receptors(PARs). Based on their chemical mechanism for catalysis, proteases can be classified into several classes: serine, cysteine, aspartic, metallo- and threonine proteases represent the mammalian protease families. In particular, the class of serine proteases will play a significant role in this review. In the last decades, proteases have been suggested to play a key role in the pathogenesis of visceral hypersensitivity, which is a major factor contributing to abdominal pain in patients with inflammatory bowel diseases and/or irritable bowel syndrome. So far, only a few preclinical animal studies have investigated the effect of protease inhibitors specifically on visceral sensitivity while their effect on inflammation is described in more detail. In our accompanying review we describe their effect on gastrointestinal permeability. On account of their promising results in the field of visceral hypersensitivity, further research is warranted. The aim of this review is to give an overview on the concept of visceral hypersensitivity as well as on the physiological and pathophysiological functions of proteases herein.

Family-level diversity of extracellular proteases of sedimentary bacteria from the South China Sea

Protease-producing bacteria and their extracellular proteases are key players in degrading organic nitrogen to drive marine nitrogen cycling and yet knowledge on both of them is still very limited. This study screened protease-producing bacteria from the South China Sea sediments and analyzed the diversity of their extracellular proteases at the family level through N-terminal amino acid sequencing. Results of the 16 S rRNA gene sequence analysis showed that all screened protease-producing bacteria belonged to the class Gammaproteobacteria and most of them were affiliated with different genera within the orders Alteromonadales and Vibrionales. The Nterminal amino acid sequence analysis for fourteen extracellular proteases from fourteen screened bacterial strains revealed that all these proteases belonged to the M4 family of metalloproteases or the S8 family of serine proteases. This study presents new details on taxa of marine sedimentary protease-producing bacteria and types of their extracellular proteases, which will help to comprehensively understand the process and mechanism of the microbial enzymatic degradation of marine sedimentary organic nitrogen.

Effects of Dietary Soybean Stachyose and Phytic Acid on Gene Expressions of Serine Proteases in Japanese Flounder(Paralichthys olivaceus)

Soybean stachyose (SBS) and phytic acid (PA) are anti-nutritional factors (ANF) which have deleterious effects on the growth and digestibility in fish. The present research studied the effects of dietary SBS and PA on the expression of three serine protease genes in the liver of Japanese flounder (Paralichthys olivaceus). These genes are trypsinogen 1 (poTRY), elastase 1 (poEL) and chymotrypsinogen 1 (poCTRY). Eight artificial diets with graded levels of supplemented ANFs were formulated to 4 levels of SBS (0.00, 0.40, 0.80 and 1.50%), 4 levels of PA (0.00, 0.20, 0.40 and 0.80), respectively.Japanese flounder (initial weight 2.45 g ± 0.01 g) were fed with these diets for 10 weeks with three replications per treatment. At the end of 10 weeks, supplementation of 0.40% of dietary SBS or PA significantly increased the gene expression of poTRY and poCTRY (P<0.05). The same level of dietary SBS significantly decreased the gene expression of poEL. In comparison with the control group (ANF-free),dietary PA (0.2% and 0.8%) significantly decreased the gene expression of poTRY, poCTRY and poEL (P<0.05). However, excessive supplement of dietary SBS (1.5%) has no significant effects on these gene expressions (P>0.05). These results suggested that dietary SBS and dietary PA could directly affect the serine protease genes at the transcriptional level in Japanese flounder, and these genes'expression was more sensitive to dietary PA than to SBS under the current experimental conditions.

Emerging potential of ubiquitin-specific proteases and ubiquitinspecific proteases inhibitors in breast cancer treatment

Breast cancer is the most frequently diagnosed cancer in women,accounting for 30%of new diagnosing female cancers.Emerging evidence suggests that ubiquitin and ubiquitination played a role in a number of breast cancer etiology and progression processes.As the primary deubiquitinases in the family,ubiquitin-specific peptidases(USPs)are thought to represent potential therapeutic targets.The role of ubiquitin and ubiquitination in breast cancer,as well as the classification and involvement of USPs are discussed in this review,such as USP1,USP4,USP7,USP9X,USP14,USP18,USP20,USP22,USP25,USP37,and USP39.The reported USPs inhibitors investigated in breast cancer were also summarized,along with the signaling pathways involved in the investigation and its study phase.Despite no USP inhibitor has yet been approved for clinical use,the biological efficacy indicated their potential in breast cancer treatment.With the improvements in phenotypic discovery,we will know more about USPs and USPs inhibitors,developing more potent and selective clinical candidates for breast cancer.

Versatility of microbial proteases

Proteases or peptidases constitute the largest group of enzymes in bio-industry with a long array of uses. They play an invincible role in industrial biotechnology, especially in detergent, food and pharmaceutical arena. This focused review encompasses an overview on alkaline proteases, mainly of microbial sources in a handy module. Following an introduction and general classification with evolutionary insight, major sources of proteases (animal, plant and microbial including fungal, bacterial), their general properties with mechanism of action and molecular masses are discussed. Proteases fromBacillusspp. have been given special attention. In addition to this, an overview on the applications of proteases in detergent, tannery, food, metal recovery and waste treatment industries is also addressed briefly.

Contribution of lysosomal cysteine proteases in cardiac and renal diseases

Cardiac and renal diseases(CRDs) are characterized by extensive remodeling of the extracellular matrix(ECM)architecture of the cardiorenal system. Among the many extracellular proteolytic enzymes present in cardiorenal cells and involved in ECM remodeling, members of the matrix metalloproteinase family and serine protease family have received the most attention. However, recent findings from laboratory and clinical studies have indicated that cysteine protease cathepsins also participate in pathogenesis of the heart and kidney.Deficiency and pharmacological inhibition of cathepsins have allowed their in vivo evaluation in the setting of pathological conditions. Furthermore, recent studiesevaluating the feasibility of cathepsins as a diagnostic tool have suggested that the serum levels of cathepsins L, S and K and their endogenous inhibitor cystatin C have predictive value as biomarkers in patients with coronary artery disease and heart and renal failure. The goal of this review is to highlight recent discoveries regarding the contributions of cathepsins in CRDs, particularly hypertensive heart failure and proteinuric kidney disease.

Degradation of naturally occurring and engineered antimicrobial peptides by proteases

We hypothesized that current antimicrobial peptides should be susceptible to proteolytic digestion. The antimicrobial peptides: Griffithinsin, RC-101, LL-37, LSA-5, PSC-RANTES and DJ007 were degraded by commercially available proteases. Two different species of anaerobic vaginal flora, Prevotella bivia and Porphyromonas asaccharolytica also degraded the materials. Griffithsin was resistant to digestion by 8 of the 9 proteases and the bacteria while LL-37 was the most sensitive to protease digestion. These data suggests most of the molecules may not survive for very long in the proteolytic rich environments in which they are intended to function.

May bacterial or pancreatic proteases play a critical role in inflammatory bowel disease?

In a recent review paper,Carroll and Maharshak discussed a critical role of enteric bacterial proteases in the pathogenesis of inflammatory bowel disease(IBD).I take a great interest in this paper as I also suspected proteases,not from the bacteria,but those originated from the pancreas that failed to be inactivated in the lower gut due to a reduction in gut bacteria,may have played a critical role in the pathogenesis of IBD,which was first published more than a decade ago and discussed again in more detail in a recent paper published in this journal.Antibiotics may result in a big reduction in gut bacteria and bacterial proteases,but multiple studies demonstrated dramatic increased of pancreatic proteases like trypsin and chymotrypsin in the feces of animals or patients treated with antibiotics.Multiple large-scale studies also demonstrated use of antibiotics caused an increase but not decrease in the risk of developing IBD,suggesting impaired inactivation and degradation of pancreatic proteases may have played a more critical role in the pathogenesis of IBD.

Impaired inactivation of digestive proteases:The possible key factor for the high susceptibility of germ-free and antibiotic-treated animals to gut epithelial injury

Recent study shows that germ-free and antibiotic-treated animals are highly susceptible to gut epithelial injury. This paper addresses that impaired inactivation of digestive proteases may be the key factor for the increased susceptibility.

Enhanced Free Radical Scavenging and Inhibition of DPP-4 and ACE Activities by Compounds from Salmon Tissues Digested in Vitro with Gastrointestinal Proteases

Research on marine bioactive peptides has mainly focused on characterization of peptides in hydrolysates prepared with commercial industrial enzymes and the usefulness of such hydrolysates in health and functional foods. However, a relevant question is whether digestion of fish proteins with gastrointestinal proteases per se generates peptides that also can have health promoting properties and can reduce, e.g., diabetes 2, inflammation and hypertension either in relation to gastrointestinal digestion or as alternative to industrial proteases. The aim of the study was to investigate hydrolysates obtained from in vitro sequential digestion of salmon muscle and skin with gastrointestinal proteases including pepsin, pancreatic and pancreatic ＋ mucosal proteases for their ability to scavenge ABTS^＋ radicals and inhibit activity of angiotensin I-converting enzyme （ACE） and dipeptidyl peptidase 4 （DPP-4）. Furthermore, it was the aim to study the inhibitory mechanism and stability towards ACE and DPP-4 activity. Analysis of〈 10 kDa hydrolysates showed that gastrointestinal proteases generated peptides with clear radical scavenging activity and DPP-4 and ACE inhibiting activity as well. Hydrolysates from pepsin digestion exhibited the lowest ECso values for radical scavenging activity and ACE inhibition, whereas ECso increased in hydrolysates after subsequent digestion with pancreatic and mucosal proteases. Interestingly, ECso values for the DPP-4 inhibition were hardly affected by sequential digestion. Inhibition modes for the muscle hydrolysates were both competitive and non-competitive, but prolonged incubation showed that the inhibitory properties were unstable and therefore they were probably digested as competitive substrates by gastrointestinal proteases.

Enteric bacterial proteases in inflammatory bowel diseasepathophysiology and clinical implications

Numerous reports have identified a dysbiosis in the intestinal microbiota in patients suffering from inflammatory bowel diseases(IBD),yet the mechanism(s)in which this complex microbial community initiates or perpetuates inflammation remains unclear.The purpose of this review is to present evidence for one such mechanism that implicates enteric microbial derived proteases in the pathogenesis of IBD.We highlight and discuss studies demonstrating that proteases and protease receptors are abundant in the digestive system.Additionally,we investigate studies demonstrating an association between increased luminal protease activity and activation of protease receptors,ultimately resulting in increased intestinal permeability and exacerbation of colitis in animal models as well as in human IBD.Proteases are essential for the normal functioning of bacteria and in some cases can serve as virulence factors for pathogenic bacteria.Although not classified as traditional virulence factors,proteases originating from commensal enteric bacteria also have a potential association with intestinal inflammation via increased enteric permeability.Reports of increased protease activity in stools from IBD patients support a possible mechanism for a dysbiotic enteric microbiota in IBD.A better understanding of these pathways and characterization of the enteric bacteria involved,their proteases,and protease receptors may pave the way for new therapeutic approaches for these diseases.

Enhanced Production of Salinity-Induced Proteases from <i>Aspergillus flavus</i>and <i>Aspergillus niger</i>

Proteases are important industrial enzymes that account for about 60% of the total enzyme market globally due to their large application in food, feed, textile and pharmaceutical industries. The effect of salt stress on protease production was evaluated on Aspergillus flavus and Aspergillus niger. The enzyme production was enhanced by stepwise optimization of the culture parameters, notably, carbon source, nitrogen source, pH, and temperature of the submerged fermentation process while using a minimal salt media and casein as substrate for the protease activity. The fungi species were found to be good producers of both acid and alkaline proteases under 4% salt stress condition. The optimum culture conditions for alkaline protease production by Aspergillus flavus were sucrose 4%, peptone 1%, pH 8 at 40&deg;C with maximum enzymatic activities of 8.85 mM/min/mg protein, 5.22 mM/min/mg protein, 3.75 mM/min/mg protein, and 1.64 mM/min/mg protein, respectively. Lactose 4%, peptone 1%, pH 6 at 50&deg;C were the optimum culture conditions for acid protease production by Aspergillus flavus with maximum enzymatic activities of 4.59 mM/min/mg protein, 2.06 mM/min/mg protein, 1.24 mM/min/mg protein, and 1.23 mM/min/mg protein, respectively. For Aspergillus niger, the optimum culture conditions for alkaline protease production were corn starch 4%, yeast extract 1%, pH 6 at 40&deg;C with maximum enzymatic activities of 5.99 mM/min/mg protein, 3.85 mM/min/mg protein, 6.18 mM/min/mg protein, and 3.72 mM/min/mg protein, respectively. While lactose 4%, yeast extract 1%, pH 6 at 50&deg;C were the best culture conditions for acid protease production by Aspergillus niger with maximum enzymatic activities of 4.81 mM/min/mg protein, 0.93 mM/min/mg protein, 5.71 mM/min/mg protein, and 3.34 mM/min/mg protein, respectively.

Screening and characterization of proteases produced by deep-sea cold seep bacteria

Fifty protease-producing strains were screened from sediment of deep-sea cold seep,and divided into four diff erent categories:Bacillus,Pseudoalteromonas,Vibrio,and Alteromonas according to the sequences of 16s rRNA.Their abilities to produce protease,amylase,and lipase were determined,and a Bacillus strain gcc-1 displayed very strong alkaline protease activity and stability under different thermal and acidic conditions.The purifi cation of the protease produced by strain gcc-1 was carried out by precipitation with ammonium sulfate,and sequentially chromatographed by anion exchange column and gel filtration.The purifi ed protease showed a single band at the molecular weight of 28 kDa by SDS-PAGE.The characterization results show that the purified protease exhibited a considerable activity and stability in a wide thermal range of 10-80℃and a wide acidic range of pH 6.5-11.5,and displayed highest activity at 40℃ and pH 8.5.Notably,the protease still maintained high activity even at low to 10℃.Furthermore,the protease exhibited good stability in presence of diff erent surfactants,organic solvents,oxidizing agent H_(2)O_(2),and commercial detergents.Therefore,the protease produced by gcc-1 is a cold active and high stable enzyme,and has a promising potential in laundry detergent as an additive.

Characterization of proteases from Planomicrobium sp. L-2 isolated from the gastrointestinal tract of Octopus variabilis (Sasaki)

A crude protease produced from Planomicrobium sp. L-2 is described, and its effectiveness as an additive in liquid detergent evaluated. We isolate the protease-producing Planomicrobium sp. L-2 from the gastrointestinal tract of Octopus variabilis. At least three caseinolytic protease clear bands were observed in zymogram analysis. The crude alkaline protease was highly tolerant of a pH range from 7.0 to 9.0, and temperatures to 50℃ after incubation for 1 h. Proteolytic enzymes were stable towards three surfactants （5% Tween 80, 1% Triton X-100 and 0.05% SDS） and an oxidizing agent （1% hydrogen peroxide）, in addition to being highly stable and compatible with popular commercial laundry powered detergent brands available in China. Our study demonstrates the potential these proteases have for development into novel classes of detergent additive. This study also suggests that the gastrointestinal tract of Octopus variabilis may be a rich source of commercially valuable strains of enzvme.

Heterogenous Expression of Synechocystis sp. PCC 6803 Deg Proteases and Their Possible Roles on Phycobiliprotein Degradation in vitro

The Synechocystis sp. PCC 6803 genome harbours a Deg gene family consisting of three members, htrA （degP, slr1204）, hhoA （degQ, sll1679） and hhoB （degS, sll1427）. This work provided biochemical characterization of HhoA, HtrA and HhoB from Synechocystis sp. PCC 6803. Firstly mature HhoA, HhoB and HtrA from Synechocystis sp. PCC 6803 were cloned and expressed as soluble recombinant his-tagged fusion protein in Escherichia coli. Then the proteolytic activity of HhoA, HhoB and HtrA was tested using casein, bovine serum albumin, five recombinant chromoproteins and cyanobacterial phycocyanin as substrates in vitro. The experimental results showed that HhoA and HtrA had proteolytic activity on casein, five recombinant chromoproteins and cyanobacterial phycocyanin. No proteolytic activity of HhoB was found using all substrates in vitro, indicating functional difference among Deg proteases from Synechocystis sp. PCC 6803. Therefore, the results indicated the biochemical properties of HhoA and HtrA on hydrolysis of proteins and phycobiliproteins in vitro, which implicated that they were proteases possibly involved in phycobiliprotein degradation in vivo.