Ubiquitin-specific protease 21 promotes tumorigenicity and stemness of colorectal cancer by deubiquitinating and stabilizing ZEB1

BACKGROUND Colorectal cancer(CRC)is one very usual tumor together with higher death rate.Ubiquitin-specific protease 21(USP21)has been confirmed to take part into the regulation of CRC progression through serving as a facilitator.Interestingly,the promotive function of USP21 has also discovered in the progression of CRC.ZEB1 has illustrated to be modulated by USP7,USP22 and USP51 in cancers.However,the regulatory functions of USP21 on ZEB1 in CRC progression need more invest-igations.AIM To investigate the relationship between USP21 and ZEB1 in CRC progression.METHODS The mRNA and protein expressions were assessed through RT-qPCR,western blot and IHC assay.The interaction between USP21 and ZEB1 was evaluated through Co-IP and GST pull down assays.The cell proliferation was detected through colony formation assay.The cell migration and invasion abilities were determined through Transwell assay.The stemness was tested through sphere formation assay.The tumor growth was evaluated through in vivo mice assay.RESULTS In this work,USP21 and ZEB1 exhibited higher expression in CRC,and resulted into poor prognosis.Moreover,the interaction between USP21 and ZEB1 was further investigated.It was demonstrated that USP21 contributed to the stability of ZEB1 through modulating ubiquitination level.In addition,USP21 streng-thened cell proliferation,migration and stemness through regulating ZEB1.At last,through in vivo assays,it was illustrated that USP21/ZEB1 axis aggravated tumor growth.CONCLUSION For the first time,these above findings manifested that USP21 promoted tumorigenicity and stemness of CRC by deubiquitinating and stabilizing ZEB1.This discovery suggested that USP21/ZEB1 axis may provide novel sights for the treatment of CRC.

Novel mutations in ubiquitin-specific protease 26 gene might cause spermatogenesis impairment and male infertility

Aim： To study the incidence of single nucleotide polymorphisms in ubiquitin-specific protease 26 （USP26） gene and its involvement in idiopathic male infertility in China. Methods： Routine semen analysis was performed. Infertility factors such as immunological, infectious and biochemical disorders were examined to select patients with idiopathic infertility. DNA was isolated from peripheral blood of the selected patients and control population, which were examined for mutations using polymerase chain reaction-single strand conformation polymorphism analysis. Furthermore, nucleotide sequences were sequenced in some patients and controls. Results： Of 41 infertile men, 9 （22.0%, P = 0.01） had changes in USP26 gene on the X chromosome. A compound mutation （364insACA; 460G→A） was detected in 8 patients （19.5%, P = 0.01） and a 1044T→A substitution was found in 1 patient （2.4%, P 〉 0.05）. All three variations led to changes in the coding amino acids. Two substitutions predict some changes： 460G→ A changes a valine into an isoleucine, and 1044T → A substitutes a leucine for a phenylalanine. Another insertion of three nucleotides ACA causes an insertion of threonine. No other changes were found in the remaining patients and fertile controls. Conclusion： The USP26 gene might be of importance in male reproduction. Mutations in this gene might be associated with male infertility, and might negatively affect testicular function. Further research on this issue is in progress.

Ubiquitin-specific protease 15 contributes to gastric cancer progression by regulating the Wnt/β-catenin signaling pathway

BACKGROUND Ubiquitin-specific protease 15(USP15)is an important member of the ubiquitinspecific protease family,the largest deubiquitinase subfamily,whose expression is dysregulated in many types of cancer.However,the biological function and the underlying mechanisms of USP15 in gastric cancer(GC)progression have not been elucidated.AIM To explore the biological role and underlying mechanisms of USP15 in GC progression.METHODS Bioinformatics databases and western blot analysis were utilized to determine the expression of USP15 in GC.Immunohistochemistry was performed to evaluate the correlation between USP15 expression and clinicopathological characteristics of patients with GC.A loss-and gain-of-function experiment was used to investigate the biological effects of USP15 on GC carcinogenesis.RNA sequencing,immunofluorescence,and western blotting were performed to explore the potential mechanism by which USP15 exerts its oncogenic functions.RESULTS USP15 was up-regulated in GC tissue and cell lines.The expression level of USP15 was positively correlated with clinical characteristics(tumor size,depth of invasion,lymph node involvement,tumor-node-metastasis stage,perineural invasion,and vascular invasion),and was related to poor prognosis.USP15 knockdown significantly inhibited cell proliferation,invasion and epithelialmesenchymal transition(EMT)of GC in vitro,while overexpression of USP15 promoted these processes.Knockdown of USP15 inhibited tumor growth in vivo.Mechanistically,RNA sequencing analysis showed that USP15 regulated the Wnt signaling pathway in GC.Western blotting confirmed that USP15 silencing led to significant down-regulation ofβ-catenin and Wnt/β-catenin downstream genes(c-myc and cyclin D1),while overexpression of USP15 yielded an opposite result and USP15 mutation had no change.Immunofluorescence indicated that USP15 promoted nuclear translocation ofβ-catenin,suggesting activation of the Wnt/β-catenin signaling pathway,which may be the critical mechanism promoting GC progression.Finally,rescue experiments showed that the effect of USP15 on gastric cancer progression was dependent on Wnt/β-catenin pathway.CONCLUSION USP15 promotes cell proliferation,invasion and EMT progression of GC via regulating the Wnt/β-catenin pathway,which suggests that USP15 is a novel potential therapeutic target for GC.

Association of 370-371insACA, 494T〉C, and 1423C〉T haplotype in ubiquitin-specific protease 26 gene and male infertility： a meta-analysis

Whether the 370-371insACA, 494T〉C, and 1423C〉T haplotype in ubiquitin-specific protease 26 （USP26） gene is associated with male infertility is controversial. To clarify this issue, we conducted a meta-analysis based on the most recent studies. Eligible studies were screened by using PubMed and Embase. Pooled odd ratio （OR） with 95% confidence interval （CI） was calculated with fixed effect models. Ten studies with 1603 patients and 2505 controls were included, Overall, the results indicated that there was an association between the haplotype and male infertile risk （OR = 1.74, 95% CI： 1.09-2.77）. The OR calculated based on the five studies in Asia and three in Europe was 1.96 （95% CI： 1,05-3.67） and 1.54 （95% Ch 0.75-3.16） respectively, however, the OR was 0.86 （95% Ch 0.05-15,29） based on the two investigations in America. In addition, the data from the patients with azoospermia （AZO） showed an increased pooled OR of 2.35 （95% Cl： 1.22-4.50）. In contrast, the studies with oligoasthenoteratozoospermia （OAT） exhibited that the pooled OR was 0,97 （95% Ch 0.43-2.16）. Our analyses indicate that there is an association of alteration in USP26 with male infertility, especially in AZO and Asian population.

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.

Inhibition of Ubiquitin-specific Protease 4 Attenuates Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells via Transforming Growth Factor Beta Receptor Type Ⅰ

Objective Ubiquitin-specific protease 4(USP4)facilitates the development of transforming growth factor-beta 1(TGF-β1)-induced epithelial-mesenchymal transition(EMT)in various cancer cells.Moreover,EMT of renal tubular epithelial cells(RTECs)is required for the progression of renal interstitial fibrosis.However,the role of USP4 in EMT of RTECs remains unknown.The present study aimed to explore the effect of USP4 on the EMT of RTECs as well as the involved mechanism.Methods In established unilateral ureteral obstruction(UUO)rats and NRK-52E cells,immunohistochemistry and Western blot assays were performed.Results USP4 expression was increased significantly with obstruction time.In NRK-52E cells stimulated by TGF-β1,USP4 expression was increased in a time-dependent manner.In addition,USP4 silencing with specific siRNA indicated that USP4 protein was suppressed effectively.Meanwhile,USP4 siRNA treatment restored E-cadherin and weakened alpha smooth muscle actin(α-SMA)expression,indicating that USP4 may promote EMT.After treatment with USP4 siRNA and TGF-β1 for 24 h,the expression of TGF-β1 receptor type I(TβRI)was decreased.Conclusion USP4 promotes the EMT of RTECs through upregulating TβRI,thereby facilitating renal interstitial fibrosis.These findings may provide a potential target of USP4 in the treatment of renal fibrosis.

Ubiquitin-specific protease 22 enhances intestinal cell proliferation and tissue regeneration after intestinal ischemia reperfusion injury

BACKGROUND Intestinal ischemia reperfusion(I/R) injury is a serious but common pathophysiological process of many diseases, resulting in a high mortality rate in clinical practice. Ubiquitin-specific protease 22(USP22) acts as regulator of cell cycle progression, proliferation, and tumor invasion. Depleted USP22 expression has been reported to contribute to arrested cell cycle and disrupted generation of differentiated cell types in crypts and villi. However, the role of USP22 in intestinal damage recovery has not been investigated. Therefore, elucidation of the underlying mechanism of USP22 in intestinal I/R injury may help to improve the tissue repair and patient prognosis in clinical practice.AIM To investigate the role of USP22 in intestinal cell proliferation and regeneration after intestinal I/R injury.METHODS An animal model of intestinal I/R injury was generated in male Sprague-Dawley rats by occlusion of the superior mesenteric artery followed by reperfusion.Chiu's scoring system was used to grade the damage to the intestinal mucosa. An in vitro model was developed by incubating rat intestinal epithelial IEC-6 cells in hypoxia/reoxygenation conditions in order to simulate I/R in vivo. siRNA and overexpression plasmid were used to regulate the expression of USP22. USP22,Cyclin D1, and proliferating cell nuclear antigen(PCNA) expression levels were measured by Western blot analysis and immunohistochemistry staining. Cell survival(viability) and cell cycle were evaluated using the Cell Counting Kit-8and flow cytometry, respectively.RESULTS USP22 expression was positively correlated with the expression levels of PCNA and Cyclin D1 both in vivo and in vitro, which confirmed that USP22 was involved in cell proliferation and intestinal regeneration after intestinal I/R injury. Decreased levels of Cyclin D1 and cell cycle arrest were observed in the USP22 knockdown group(P < 0.05), while opposite results were observed in the USP22 overexpression group(P < 0.05). In addition, increased expression of USP22 was related to improved intestinal pathology or IEC-6 cell viability after I/R or hypoxia/reoxygenation. These results suggested that USP22 may exert a protective effect on intestinal I/R injury by regulating cell proliferation and facilitating tissue regeneration.CONCLUSION USP22 is correlated with promoting intestinal cell proliferation and accelerating intestinal tissue regeneration after intestinal I/R injury and may serve as a potential target for therapeutic development for tissue repair during intestinal I/R injury.

Ubiquitin-specific protease 24 promotes EV71 infection by restricting K63-linked polyubiquitination of TBK1

TANK-binding kinase 1(TBK1)is an essential protein kinase for activation of interferon regulatory factor 3(IRF3)and induction of the type I interferons(IFN-I).Although the biochemical regulation of TBK1 activation has been studied,little is known about how enterovirus 71(EV71)employs the deubiquitinases(DUBs)to regulate TBK1 activation for viral immune evasion.Here,we found that EV71 infection upregulated the expression of ubiquitinspecific protease 24(USP24).Further studies revealed that USP24 physically interacted with TBK1,and can reduce K63-linked polyubiquitination of TBK1.Knockdown of USP24 upregulated TBK1 K63-linked polyubiquitination,promoted the phosphorylation and nuclear translocation of IRF3,and in turn improved IFN-I production during EV71 infection.As a consequence,USP24 knockdown dramatically inhibited EV71 infection.This study revealed USP24 as a novel regulator of TBK1 activation,which promotes the understanding of immune evasion mechanisms of EV71 and could provide a potential strategy for treatment of EV71 infection.

Ubiquitin-Specific Protease 14 （UBP14） Is Involved in Root Responses to Phosphate Deficiency in Arabidopsis

A mutant isolated from a screen of EMS-mutagenized Arabidopsis lines, per1, showed normal root hair development under control conditions but displayed an inhibited root hair elongation phenotype upon Pi deficiency. Additionally, the per1 mutant exhibited a pleiotropic phenotype under control conditions, resembling Pi-deficient plants in several aspects. Inhibition of root hair elongation upon growth on low Pi media was reverted by treatment with the Pi analog phosphite, suggesting that the mutant phenotype is not caused by a lack of Pi. Reciprocal grafting experiments revealed that the mutant rootstock is sufficient to cause the phenotype. Complementation analyses showed that the PER1 gene encodes an ubiquitin-specific protease, UBP14. The mutation caused a synonymous substitution in the 12th exon of this gene, resulting in a lower abundance of the UBP14 protein, probably as a consequence of reduced translation efficiency. Transcriptional profiling of per1 and wild-type plants subjected to short-term Pi starvation revealed genes that may be important for the signaling of Pi deficiency. We conclude that UBP14 function is crucial for adapting root development to the prevailing local availability of phosphate.

Ubiquitin-specific protease 47 regulates intestinal inflammation through deubiquitination of TRAF6 in epithelial cells

Deubiquitinates(DUBs) alter the stabilities, localizations or activities of substrates by removing their ubiquitin conjugates,which are closely related to the development of inflammatory response. Here, we show that ubiquitin-specific protease 47(USP47) prevents inflammation development in inflammatory bowel disease(IBD). Compared with wild-type mice, Usp47 knockout mice are more susceptible to dextran sodium sulfate(DSS)-induced acute and chronic colitis with higher inflammatory cytokines expression and severe intestinal tissue damage. Chimeric mouse experiments suggest that non-hematopoietic cells mainly contribute to the phenotype. And, DSS-induced colitis of the Usp47 knockout mice depends on commensal bacteria.Mechanistically, down-regulation of USP47 aggravates the activation of NF-κB signaling pathway by increasing the K63-linked poly-ubiquitination of tumor necrosis factor receptor-associated factor 6(TRAF6) in intestinal epithelial cells. Furthermore, the expression of USP47, negatively correlated with the degree of inflammation, is lower at colonic inflammatory lesions than that non-inflammatory sites from the intestine from ulcerative colitis(UC) and Crohn's disease(CD) patients. These data, taken together, indicate that USP47 regulates intestinal inflammation through de-ubiquitination of K63-linked poly-ubiquitination TRAF6 in intestinal epithelial cells.

Deubiquitinase ubiquitin-specific protease 3 (USP3) inhibits HIV-1 replication via promoting APOBEC3G (A3G) expression in both enzyme activity-dependent and -independent manners

Background: Ubiquitination plays an essential role in many biological processes, including viral infection, and can be reversed by deubiquitinating enzymes (DUBs). Although some studies discovered that DUBs inhibit or enhance viral infection by various mechanisms, there is lack of information on the role of DUBs in virus regulation, which needs to be further investigated.Methods: Immunoblotting, real-time polymerase chain reaction,in vivo/in vitro deubiquitination, protein immunoprecipitation, immunofluorescence, and co-localization biological techniques were employed to examine the effect of ubiquitin-specific protease 3 (USP3) on APOBEC3G (A3G) stability and human immunodeficiency virus (HIV) replication. To analyse the relationship between USP3 and HIV disease progression, we recruited 20 HIV-infected patients to detect the levels of USP3 and A3G in peripheral blood and analysed their correlation with CD4^(+) T-cell counts. Correlation was estimated by Pearson correlation coefficients (for parametric data).Results: The results demonstrated that USP3 specifically inhibits HIV-1 replication in an A3G-dependent manner. Further investigation found that USP3 stabilized 90% to 95% of A3G expression by deubiquitinating Vif-mediated polyubiquitination and blocking its degradation in an enzyme-dependent manner. It also enhances the A3G messenger RNA (mRNA) level by binding to A3G mRNA and stabilizing it in an enzyme-independent manner. Moreover, USP3 expression was positively correlated with A3G expression (r= 0.5110) and CD4^(+) T-cell counts (r= 0.5083) in HIV-1-infected patients.Conclusions: USP3 restricts HIV-1 viral infections by increasing the expression of the antiviral factor A3G. Therefore, USP3 may be an important target for drug development and serve as a novel therapeutic strategy against viral infections.

The association between mutations in ubiquitin-specific protease 26(USP26)and male infertility:a systematic review and meta-analysis

During recent decades,the association between mutations in ubiquitin-specific protease 26(USP26)and male infertility remains doubtful.We conducted this meta-analysis to evaluate the association between mutations in USP26 and male infertility according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses(PRISMA)2020 guidelines.It was registered in the International Prospective Register of Systematic Reviews(PROSPERO;CRD42021225251).PubMed,Web of Science,and Scopus were systematically searched for comparative clinical studies,which were written in English and provided eligible data.Studies were included when they compared USP26 mutations in azoospermic,oligozoospermic,and asthenozoospermic patients with controls with normal sperm parameter values or whose partners had experienced spontaneous pregnancy.Pooled odds ratio(OR)with 95%confidence interval(CI)was calculated with random effect models.Overall,twelve studies with 3927 infertility patients and 4648 healthy controls were included.The association between overall USP26 mutations and infertility was not significant(OR=1.60,95%CI:0.51-5.01).For specific mutations,the pooled ORs were 1.65(95%CI:1.02-2.69)for cluster mutation(including 370-371insACA,494T>C,and 1423C>T),1.80(95%CI:0.35-9.15)for c.576G>A,1.43(95%CI:0.79-2.56)for c.1090C>T,and 3.59(95%CI:2.30-5.59)for c.1737G>A.Our results suggest that several mutations(cluster mutation,c.1737G>A)may play roles in male infertility,while others(c.576G>A and c.1090C>T)do not show notable associations with male infertility.More high-quality clinical researches are needed for validation.

A dicistrovirus increases pupal mortality in Spodoptera frugiperda by suppressing protease activity and inhibiting larval diet consumption

Understanding interactions between viruses and their hosts is conducive to enabling better application of viruses as biocontrol agents.Certain viruses carried by parasitic wasps enhance the parasitic efficiency of wasp-larvae by protecting them against the immune system of their Lepidopteran host.However,the relationship between prey pests and viruses found in predatory natural enemies remains unclear.Herein,we report the interaction between Arma chinensis virus-1(AcV-1),originally isolated from a predatory natural enemy,Arma chinensis(Hemiptera:Pentatomidae),and one of its prey species,Spodoptera frugiperda(Lepidoptera:Noctuidae).The results showed that the AcV-1 virus appeared harmful to the novel host S.frugiperda by inhibiting larval diet consumption and increasing pupal mortality.Meanwhile,sequencing data indicated that the virus altered the gene expression profiles of S.frugiperda.KEGG analysis showed that the proteasome and phagosome pathways related to protein degradation and immune response were significantly enriched.Although the expression levels of digestive enzyme genes did not change significantly,the total protease activity of AcV-1 virus-positive individuals was significantly decreased,suggesting that the virus inhibited diet consumption of S.frugiperda via the down-regulation of digestive enzyme activities.These results indicate that a virus initially isolated in a predatory natural enemy can decrease the fitness of its prey species.The virus was found to impact the host proteasome and phagosome pathways related to protein degradation and immunity,providing a potential mechanism to enhance controlling efficiency.

Comprehensive analysis of the role of ubiquitin-specific peptidases in colorectal cancer:A systematic review

BACKGROUND Colorectal cancer(CRC)is the third most frequent and the second most fatal cancer.The search for more effective drugs to treat this disease is ongoing.A better understanding of the mechanisms of CRC development and progression may reveal new therapeutic strategies.Ubiquitin-specific peptidases(USPs),the largest group of the deubiquitinase protein family,have long been implicated in various cancers.There have been numerous studies on the role of USPs in CRC;however,a comprehensive view of this role is lacking.AIM To provide a systematic review of the studies investigating the roles and functions of USPs in CRC.METHODS We systematically queried the MEDLINE(via PubMed),Scopus,and Web of Science databases.RESULTS Our study highlights the pivotal role of various USPs in several processes implicated in CRC:Regulation of the cell cycle,apoptosis,cancer stemness,epithelial–mesenchymal transition,metastasis,DNA repair,and drug resistance.The findings of this study suggest that USPs have great potential as drug targets and noninvasive biomarkers in CRC.The dysregulation of USPs in CRC contributes to drug resistance through multiple mechanisms.CONCLUSION Targeting specific USPs involved in drug resistance pathways could provide a novel therapeutic strategy for overcoming resistance to current treatment regimens in CRC.

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.

Transmembrane serine protease 4 expression in the prognosis of radical resection for biliary tract cancer

BACKGROUND Recent advancements in biliary tract cancer(BTC)treatment have expanded beyond surgery to include adjuvant therapy,yet the prognosis remains poor.Identifying prognostic biomarkers could enhance the assessment of patients who have undergone radical resection for BTC.AIM To determine transmembrane serine protease 4(TMPRSS4)utility as a prognostic biomarker of radical resection for BTC.METHODS Medical records of patients who underwent radical resection for BTC,excluding intrahepatic cholangiocarcinoma,were retrospectively reviewed.The associations between TMPRSS4 expression and clinicopathological factors,overall survival,and recurrence-free survival were analyzed.RESULTS Among the 85 patients undergoing radical resection for BTC,46(54%)were TMPRSS4-positive.The TMPRSS4-positive group exhibited significantly higher preoperative carbohydrate antigen 19-9(CA19-9)values and greater lymphatic invasion than the TMPRSS4-negative group(P=0.019 and 0.039,respectively).Postoperative overall survival and recurrence-free survival were significantly worse in the TMPRSS4-positive group(median survival time:25.3 months vs not reached,P<0.001;median survival time:28.7 months vs not reached,P=0.043,respectively).Multivariate overall survival analysis indicated TMPRSS4 positivity,pT3/T4,and resection status R1 were independently associated with poor prognosis(P=0.032,0.035 and 0.030,respectively).TMPRSS4 positivity correlated with preoperative CA19-9 values≥37 U/mL and pathological tumor size≥30 mm(P=0.016 and 0.038,respectively).CONCLUSION TMPRSS4 is a potential prognostic biomarker of radical resection for BTC.

A constitutive serine protease inhibitor suppresses herbivore performance in tea(Camellia sinensis)

Protease inhibitors promote herbivore resistance in diverse plant species.Although many inducible protease inhibitors have been identified,there are limited reports available on the biological relevance and molecular basis of constitutive protease inhibitors in herbivore resistance.Here,we identified a serine protease inhibitor,CsSERPIN1,from the tea plant(Camellia sinensis).Expression of CsSERPIN1 was not strongly affected by the assessed biotic and abiotic stresses.In vitro and in vivo experiments showed that CsSERPIN1 strongly inhibited the activities of digestive protease activities of trypsin and chymotrypsin.Transient or heterologous expression of CsSERPIN1 significantly reduced herbivory by two destructive herbivores,the tea geometrid and fall armyworm,in tea and Arabidopsis plants,respectively.The expression of CsSERPIN1 in Arabidopsis did not negatively influence the growth of the plants under the measured parameters.Our findings suggest that CsSERPIN1 can inactivate gut digestive proteases and suppress the growth and development of herbivores,making it a promising candidate for pest prevention in agriculture.

Purification of Moringa oleifera Leaves Protease by Three-Phase Partitioning and Investigation of Its Potential Antibacterial Activity

One of plant-based products for dental care is plant-based proteolytic enzymes which are principally proteases. In order not to damage the protein and bioactive content, an efficient method should be employed for their purifications. As such, three-phase partitioning (TPP) was used to purify protease from moringa (Moringa oleifera). TPP is an emerging, promising, non-chromatographic and economical technology which is simple, quick, efficient and often one-step process for the separation and purification of bioactive molecules from natural sources. It involves the addition of salt (ammonium sulphate) to the crude extract followed by the addition of an organic solvent (butanol). The protein appears as an interfacial precipitate between upper organic solvent and lower aqueous phases. The various conditions such as ammonium sulphate, ratio of crude extract to t-butanol and pH which are required for attaining efficient purification of the protease fractions were optimized. Under optimized conditions, it was seen that, 35% of ammonium sulphate saturation with 1:0.75 ratio of crude extract to t-butanol at pH 7 gave 4.94-fold purification with 96.20% activity yield of protease in the middle phase of the TPP system. The purified enzyme from Moringa oleifera has no antimicrobial effect on the pathogenic bacteria tested. However, this purified enzyme, can be considered as a promising agent, cheap, and safe source which is suitable for using in various industries.

Systematic functional analysis and potential application of a serine protease from cold-adapted Planococcus bacterium

In this study, a gene encoding serine protease(PmSpr288)from cold-adapted bacterium, namely Planococcus maritimus XJ11, was cloned and overexpressed in Escherichia coli BL21(DE3). Bioinformatics analysis revealed that PmSpr288 belongs to serine protease S8 superfamily with a classical catalytic triad comprised by the Asp49, His86 and Ser251. Moreover, PmSpr288 was found to be active over broad alkaline pH and low-moderate temperature, and exhibited wide range of protein substrate specificity. In addition, PmSpr288 was able to hydrolyze the meat proteins actin and myosin, and molecular docking results suggested that the crucial interaction between PmSpr288 and actin/myosin complexes was mainly occupied by hydrogen bonds. The muscle protein hydrolysates of silver carp prepared by PmSpr288 was shown to have antioxidant activity via DPPH radical scavenging assay, which presented an IC_(50) valve of 1.309 mg/mL. In conclusion, these characteristics imply that PmSpr288 has potential biotechnological application prospect for the production of bioactive peptides.