Arabidopsis SBT5.2 and SBT1.7 subtilases mediate C-terminal cleavage of flg22 epitope from bacterial flagellin.

Plants initiate specific defense responses by recognizing conserved epitope peptides within the flagellin proteins derived from bacteria. Proteolytic cleavage of epitope peptides from flagellin by plant apoplastic proteases is thought to be crucial for the perception of the epitope by the plant receptor. However, the identity of the plant proteases involved in this process remains unknown. Here, we establish an efficient identification system for the target proteases in Arabidopsis apoplastic fluid; the method employs native two-dimensional electrophoresis followed by an in-gel proteolytic assay using a fluorescence-quenching peptide substrate. We designed a substrate to specifically detect proteolytic activity at the C-terminus of the flg22 epitope in flagellin and identified two plant subtilases, SBT5.2 and SBT1.7, as specific proteases responsible for the C-terminal cleavage of flg22. In the apoplastic fluid of Arabidopsis mutant plants deficient in these two proteases, we observe a decrease in the C-terminal cleavage of the flg22 domain from flagellin, leading to a decrease in the efficiency of flg22 epitope liberation. Consequently, defensive reactive oxygen species (ROS) production is delayed in sbt5.2 sbt1.7 double-mutant leaf disks compared to wild type following flagellin exposure.

Structural, Functional, and Mutational Studies of a Potent Subtilisin Inhibitor from Budgett's Frog, Lepidobatrachus laevis.

Subtilases play a significant role in microbial pathogen infections by degrading the host proteins. Subtilisin inhibitors are crucial in fighting against these harmful microorganisms. LL-TIL, from skin secretions of Lepidobatrachus laevis, is a cysteine-rich peptide belonging to the I8 family of inhibitors. Protease inhibitory assays demonstrated that LL-TIL acts as a slow-tight binding inhibitor of subtilisin Carlsberg and proteinase K with inhibition constants of 91 pM and 2.4 nM, respectively. The solution structures of LL-TIL and a mutant peptide reveal that they adopt a typical TIL-type fold with a canonical conformation of a reactive site loop (RSL). The structure of the LL-TIL-subtilisin complex and molecular dynamics (MD) simulations provided an in-depth view of the structural basis of inhibition. NMR relaxation data and molecular dynamics simulations indicated a rigid conformation of RSL, which does not alter significantly upon subtilisin binding. The energy calculation for subtilisin inhibition predicted Ile31 as the highest contributor to the binding energy, which was confirmed experimentally by site-directed mutagenesis. A chimeric mutant of LL-TIL broadened the inhibitory profile and attenuated subtilisin inhibition by 2 orders of magnitude. These results provide a template to engineer more specific and potent TIL-type subtilisin inhibitors.

Activation of the Plasmodium Egress Effector Subtilisin-Like Protease 1 Is Mediated by Plasmepsin X Destruction of the Prodomain.

Following each round of replication, daughter merozoites of the malaria parasite Plasmodium falciparum escape (egress) from the infected host red blood cell (RBC) by rupturing the parasitophorous vacuole membrane (PVM) and the RBC membrane (RBCM). A proteolytic cascade orchestrated by a parasite serine protease, subtilisin-like protease 1 (SUB1), regulates the membrane breakdown. SUB1 activation involves primary autoprocessing of the 82-kDa zymogen to a 54-kDa (p54) intermediate that remains bound to its inhibitory propiece (p31) postcleavage. A second processing step converts p54 to the terminal 47-kDa (p47) form of SUB1. Although the aspartic protease plasmepsin X (PM X) has been implicated in the activation of SUB1, the mechanism remains unknown. Here, we show that upon knockdown of PM X, the inhibitory p31-p54 complex of SUB1 accumulates in the parasites. Using recombinant PM X and SUB1, we show that PM X can directly cleave both p31 and p54. We have mapped the cleavage sites on recombinant p31. Furthermore, we demonstrate that the conversion of p54 to p47 can be effected by cleavage at either SUB1 or PM X cleavage sites that are adjacent to one another. Importantly, once the p31 is removed, p54 is fully functional inside the parasites, suggesting that the conversion to p47 is dispensable for SUB1 activity. Relief of propiece inhibition via a heterologous protease is a novel mechanism for subtilisin activation. IMPORTANCE Malaria parasites replicate inside a parasitophorous vacuole within the host red blood cells. The exit of mature progeny from the infected host cells is essential for further dissemination. Parasite exit is a highly regulated, explosive process that involves membrane breakdown. To do this, the parasite utilizes a serine protease called SUB1 that proteolytically activates various effector proteins. SUB1 activity is dependent on an upstream protease called PM X, although the mechanism was unknown. Here, we describe the molecular basis for PM X-mediated SUB1 activation. PM X proteolytically degrades the inhibitory segment of SUB1, thereby activating it. The involvement of a heterologous protease is a novel mechanism for subtilisin activation.

Heterologous expression of recombinant nattokinase in Escherichia coli BL21(DE3) and media optimization for overproduction of nattokinase using RSM.

Nattokinase, a serine protease, was discovered in Bacillus subtilis during the fermentation of a soybean byproduct. Nattokinase is essential for the lysis of blood clots and the treatment of cardiac diseases including atherosclerosis, thrombosis, high blood pressure, and stroke. The demand for thrombolytic drugs rises as the prevalence of cardiovascular disease rises, and nattokinase is particularly effective for the treatment of cardiovascular diseases due to its long duration of action. In this study, we cloned the nattokinase gene from the Bacillus subtilis strain into the pET32a vector and expressed the protein in the E. coli BL21(DE3) strain. The active recombinant nattokinase was purified using Ni-NTA affinity chromatography and then evaluated for fibrinolytic and blood clot lysis activity. Physiological parameters for optimizing protein production at optimal pH, temperature, IPTG concentration, and incubation time were investigated. A statistical technique was used to optimize media components for nattokinase overproduction, and Central Composite Design-Response Surface Methodology-based optimization was used to select significant components for protein production. The optimized media produced 1805.50 mg/L of expressed nattokinase and 42.80 gm/L of bacterial mass. The fibrinolytic activity obtained from refolded native protein was 58FU/mg, which was five times higher than the available orokinase drug (11FU/mg). The efficiency with which a statistical technique for media optimization was implemented improved recombinant nattokinase production and provides new information for scale - up nattokinase toward industrial applications.

Biodegradation of Pine Processionary Caterpillar Silk Is Mediated by Elastase- and Subtilisin-like Proteases.

Pine processionary caterpillar nests are made from raw silk. Fibroin protein is the main component of silk which, in the case of pine processionary caterpillar, has some unusual properties such as a higher resistance to chemical hydrolysis. Isolation of microorganisms naturally present in silk nests led to identification of Bacillus licheniformis and Pseudomonas aeruginosa strains that in a defined minimal medium were able to carry out extensive silk biodegradation. A LasB elastase-like protein from P. aeruginosa was shown to be involved in silk biodegradation. A recombinant form of this protein expressed in Escherichia coli and purified by affinity chromatography was able to efficiently degrade silk in an in vitro assay. However, silk biodegradation by B. licheniformis strain was mediated by a SubC subtilisin-like protease. Homologous expression of a subtilisin Carlsberg encoding gene (subC) allowed faster degradation compared to the biodegradation kinetics of a wildtype B. licheniformis strain. This work led to the identification of new enzymes involved in biodegradation of silk materials, a finding which could lead to possible applications for controlling this pest and perhaps have importance from sanitary and biotechnological points of view.

Loss of hypothalamic Furin affects POMC to proACTH cleavage and feeding behavior in high-fat diet-fed mice.

OBJECTIVE: The hypothalamus regulates feeding and glucose homeostasis through the balanced action of different neuropeptides, which are cleaved and activated by the proprotein convertases PC1/3 and PC2. However, the recent association of polymorphisms in the proprotein convertase FURIN with type 2 diabetes, metabolic syndrome, and obesity, prompted us to investigate the role of FURIN in hypothalamic neurons controlling glucose and feeding. METHODS: POMC-Cre+/- mice were bred with Furinfl/fl mice to generate conditional knockout mice with Furin-deletion in neurons expressing proopiomelanocortin (POMCFurKO), and Furinfl/fl mice were used as controls. POMCFurKO and controls were periodically monitored on both normal chow diet and high fat diet (HFD) for body weight and glucose tolerance by established in-vivo procedures. Food intake was measured in HFD-fed FurKO and controls. Hypothalamic Pomc mRNA was measured by RT-qPCR. ELISAs quantified POMC protein and resulting peptides in the hypothalamic extracts of POMCFurKO mice and controls. The in-vitro processing of POMC was studied by biochemical techniques in HEK293T and CHO cell lines lacking FURIN. RESULTS: In control mice, Furin mRNA levels were significantly upregulated on HFD feeding, suggesting an increased demand for FURIN activity in obesogenic conditions. Under these conditions, the POMCFurKO mice were hyperphagic and had increased body weight compared to Furinfl/fl mice. Moreover, protein levels of POMC were elevated and ACTH concentrations markedly reduced. Also, the ratio of α-MSH/POMC was decreased in POMCFurKO mice compared to controls. This indicates that POMC processing was significantly reduced in the hypothalami of POMCFurKO mice, highlighting for the first time the involvement of FURIN in the cleavage of POMC. Importantly, we found that in vitro, the first stage in processing where POMC is cleaved into proACTH was achieved by FURIN but not by PC1/3 or the other proprotein convertases in cell lines lacking a regulated secretory pathway. CONCLUSIONS: These results suggest that FURIN processes POMC into proACTH before sorting into the regulated secretory pathway, challenging the dogma that PC1/3 and PC2 are the only convertases responsible for POMC cleavage. Furthermore, its deletion affects feeding behaviors under obesogenic conditions.

Proprotein convertase subtilisin/kexin type 9 is a psoriasis-susceptibility locus that is negatively related to IL36G.

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a posttranslational regulator of the LDL receptor (LDLR). Recent studies have proposed a role for PCSK9 in regulating immune responses. Using RNA-Seq-based variant discovery, we identified a possible psoriasis-susceptibility locus at 1p32.3, located within PCSK9 (rs662145 C > T). This finding was verified in independently acquired genomic and RNA-Seq data sets. Single-cell RNA-Seq (scRNA-Seq) identified keratinocytes as the primary source of PCSK9 in human skin. PCSK9 expression, however, was not uniform across keratinocyte subpopulations. scRNA-Seq and IHC demonstrated an epidermal gradient of PCSK9, with expression being highest in basal and early spinous layer keratinocytes and lowest in granular layer keratinocytes. IL36G expression followed the opposite pattern, with expression highest in granular layer keratinocytes. PCSK9 siRNA knockdown experiments confirmed this inverse relationship between PCSK9 and IL36G expression. Other immune genes were also linked to PCSK9 expression, including IL27RA, IL1RL1, ISG20, and STX3. In both cultured keratinocytes and nonlesional human skin, homozygosity for PCSK9 SNP rs662145 C > T was associated with lower PCSK9 expression and higher IL36G expression, when compared with heterozygous skin or cell lines. Together, these results support PCSK9 as a psoriasis-susceptibility locus and establish a putative link between PCSK9 and inflammatory cytokine expression.

Identification of novel non-HFE mutations in Chinese patients with hereditary hemochromatosis.

BACKGROUNDS: Hereditary hemochromatosis (HH) is mainly caused by homozygous p.C282Y mutations in HFE in the Caucasians. We recently reported non-HFE mutations constitute the major cause of HH in Chinese. However, there is still a relatively high proportion of cases with primary iron overload from unexplained causes. We aimed to explore novel non-HFE mutations in Chinese patients with primary iron overload. METHODS: Whole exome sequence was conducted to screen mutations in novel HH-related genes in the 9 cases with unexplained primary iron overload. Then the representative candidate genes were screened for mutations in another cohort of 18 HH cases. The biological function of the selected genes and variants were analyzed in vitro. RESULTS: Whole exome sequencing of 9 cases with unexplained primary iron overload identified 42 missense variants in 40 genes associated with iron metabolism pathway genes such as UBE2O p.K689R and PCSK7 p.R711W. Subsequent Sanger sequencing of the UBE2O and PCSK7 genes in the 27 cases with primary iron overload identified p.K689R in UBE2O, p.R711W and p.V143F in PCSK7 at frequency of 2/27,1/27 and 2/27 respectively. In vitro siRNA interference of UBE2O and PCSK7 resulted in down-regulated HAMP mRNA expression. Adenovirus generation of UBE2O p.K689R in cell lines resulted in increased expression of SMAD6 and SMAD7 and downregulation of p-SMAD1/5 and HAMP expression, and the reduction of hepcidin level. CONCLUSIONS: Our study identified a series of novel candidate non-HFE mutations in Chinese patients with HH. These may provide insights into the genetic basis of unexplained primary iron overload.

Bioinformatics analysis of extracellular subtilisin E from Bacillus subtilis.

Bacillus spp. are the main sources of subtilisin E, which has several applications in biotechnology. The 3D structure of subtilisin E has a significant impact on its efficacy. In this study, we evaluated subtilisin E from Bacillus subtilis subsp. subtilis str. 168 by bioinformatic methods. The results revealed that the subtilisin E sequence from B. subtilis contains highly conserved amino acids, including histidine (H), aspartic acid (D) and serine (S). Subtilisin E cleaves the bonds between hydrophobic and polar amino acids in keratin-associated proteins. The effects of point mutations on the crystal structure of subtilisin E (PDB ID: 1SCJ) showed that changes of asparagine 123 (N123) to valine (V) and serine 331 (S331) to leucine (L) respectively, were the most stabilizing. Genomic analysis of the subtilisin E-coding gene (aprE) indicated that this gene and the yhfN gene are expressed through a σA promoter. The analysis of TBFs revealed AbrB, ScoC, DegU, Hpr, σA, SinR, TenA, and DegU as relevant regulators of aprE expression. Phylogenetic analysis showed that subtilisin Es have highly conserved structures among Bacillus spp., sharing a common ancestor, where their coding genes were duplicated and evolved within the Bacillus spp. As the conclusion, our in silico study demonstrated that the overexpression of the aprE gene and stability of the produced subtilisin E can be improved though system biology methods such as point mutations and identifying the involved transcription factors (TFs) or/and TBFs.Communicated by Ramaswamy H. Sarma.

Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity.

BACKGROUND: Nattokinase is a fibrinolytic enzyme that has huge market value as a nutritional supplement for health promotion. In order to increase nattokinase yields, fermentation conditions, strains, cultivation media, and feeding strategies have been optimized. Nattokinase has been expressed using several heterologous expression systems. Pichia pastoris heterologous expression system was the alternative. RESULTS: This report aimed to express high levels of nattokinase from B. subtilis natto (NK-Bs) using a Pichia pastoris heterologous expression system and assess its fibrinolytic activity in vivo. Multicopy expression strains bearing 1-7 copies of the aprN gene were constructed. The expression level of the target protein reached a maximum at five copies of the target gene. However, multicopy expression strains were not stable in shake-flask or high-density fermentation, causing significant differences in the yield of the target protein among batches. Therefore, P. pastoris bearing a single copy of aprN was used in shake-flask and high-density fermentation. Target protein yield was 320 mg/L in shake-flask fermentation and approximately 9.5 g/L in high-density fermentation. The recombinant nattokinase showed high thermo- and pH-stability. The present study also demonstrated that recombinant NK-Bs had obvious thrombolytic activity. CONCLUSIONS: This study suggests that the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of nattokinase.

The rs508487, rs236911, and rs236918 Genetic Variants of the Proprotein Convertase Subtilisin-Kexin Type 7 (PCSK7) Gene Are Associated with Acute Coronary Syndrome and with Plasma Concentrations of HDL-Cholesterol and Triglycerides.

Dyslipidemia has a substantial role in the development of acute coronary syndrome (ACS). Previous reports, including genome-wide associations studies (GWAS), have shown that some genetic variants of the proprotein convertase subtilisin-kexin type 7 (PCSK7) gene are associated with plasma lipid levels. In the present study, we evaluated whether PCSK7 gene polymorphisms are significantly associated with the plasma lipid profile and ACS. Three PCSK7 gene polymorphisms (rs508487 T/C, rs236911 C/A, and rs236918 C/G) were determined using TaqMan genotyping assays in a group of 603 ACS patients and 622 healthy controls. The plasma lipid profile was determined in the study groups by enzymatic/colorimetric assays. Under the recessive model, the rs236918 C allele was associated with a high risk of ACS (OR = 2.11, pC = 0.039). In the same way, under the recessive and additive models, the rs236911 C allele was associated with a high risk of ACS (OR = 1.95, pC = 0.037, and OR = 1.28, pC = 0.037, respectively). In addition, under the co-dominant model, the rs508487 T allele was associated with a higher risk of ACS (OR = 1.78, pC = 0.010). The CCC and TCC haplotypes were associated with a high risk of ACS (OR = 1.21, pC = 0.047, and OR = 1.80, pC = 0.001, respectively). The rs236911 CC and rs236918 CC genotypes were associated with lower high-density lipoproteins-cholesterol (HDL-C) plasma concentrations, whereas the rs236911 CC genotype was associated with a higher concentration of triglycerides, as demonstrated in the control individuals who were not receiving antidyslipidemic drugs. Our data suggest that the PCSK7 rs508487 T/C, rs236911 C/A, and rs236918 C/G polymorphisms are associated with the risk of developing ACS, and with plasma concentrations of HDL-C and triglycerides.

Novel recombinant keratin degrading subtilisin like serine alkaline protease from Bacillus cereus isolated from marine hydrothermal vent crabs.

Microbial secondary metabolites from extreme environments like hydrothermal vents are a promising source for industrial applications. In our study the protease gene from Bacillus cereus obtained from shallow marine hydrothermal vents in the East China Sea was cloned, expressed and purified. The protein sequence of 38 kDa protease SLSP-k was retrieved from mass spectrometry and identified as a subtilisin serine proteinase. The novel SLSP-k is a monomeric protein with 38 amino acid signal peptides being active over wide pH (7-11) and temperature (40-80 °C) ranges, with maximal hydrolytic activities at pH 10 and at 50 °C temperature. The hydrolytic activity is stimulated by Ca2+, Co2+, Mn2+, and DTT. It is inhibited by Fe2+, Cd2+, Cu2+, EDTA, and PMSF. The SLSP-k is stable in anionic, non-anionic detergents, and solvents. The ability to degrade keratin in chicken feather and hair indicates that this enzyme is suitable for the degradation of poultry waste without the loss of nutritionally essential amino acids which otherwise are lost in hydrothermal processing. Therefore, the proteinase is efficient in environmental friendly bioconversion of animal waste into fertilizers or value added products such as secondary animal feedstuffs.

Peptidic boronic acids are potent cell-permeable inhibitors of the malaria parasite egress serine protease SUB1.

Malaria is a devastating infectious disease, which causes over 400,000 deaths per annum and impacts the lives of nearly half the world's population. The causative agent, a protozoan parasite, replicates within red blood cells (RBCs), eventually destroying the cells in a lytic process called egress to release a new generation of parasites. These invade fresh RBCs to repeat the cycle. Egress is regulated by an essential parasite subtilisin-like serine protease called SUB1. Here, we describe the development and optimization of substrate-based peptidic boronic acids that inhibit Plasmodium falciparum SUB1 with low nanomolar potency. Structural optimization generated membrane-permeable, slow off-rate inhibitors that prevent Pfalciparum egress through direct inhibition of SUB1 activity and block parasite replication in vitro at submicromolar concentrations. Our results validate SUB1 as a potential target for a new class of antimalarial drugs designed to prevent parasite replication and disease progression.

Recombinant Production and Characterization of an Extracellular Subtilisin-Like Serine Protease from Acinetobacter baumannii of Fermented Food Origin.

Acinetobacter baumannii is a ubiquitous bacteria that is increasingly becoming a formidable nosocomial pathogen. Due to its clinical relevance, studies on the bacteria's secretory molecules especially extracellular proteases are of interest primarily in relation to the enzyme's role in virulence. Besides, favorable properties that extracellular proteases possess may be exploited for commercial use thus there is a need to investigate extracellular proteases from Acinetobacter baumannii to gain insights into their catalytic properties. In this study, an extracellular subtilisin-like serine protease from Acinetobacter baumannii designated as SPSFQ that was isolated from fermented food was recombinantly expressed and characterized. The mature catalytically active form of SPSFQ shared a high percentage sequence identity of 99% to extracellular proteases from clinical isolates of Acinetobacter baumannii and Klebsiella pneumoniae as well as a moderately high percentage identity to other bacterial proteases with known keratinolytic and collagenolytic activity. The homology model of mature SPSFQ revealed its structure is composed of 10 ß-strands, 8 α-helices, and connecting loops resembling a typical architecture of subtilisin-like α/ß motif. SPSFQ is catalytically active at an optimum temperature of 40 °C and pH 9. Its activity is stimulated in the presence of Ca2+ and severely inhibited in the presence of PMSF. SPSFQ also displayed the ability to degrade several tissue-associated protein substrates such as keratin, collagen, and fibrin. Accordingly, our study shed light on the catalytic properties of a previously uncharacterized extracellular serine protease from Acinetobacter baumannii that warrants further investigations into its potential role as a virulence factor in pathogenicity and commercial applications.

Subtilase activity in intrusive cells mediates haustorium maturation in parasitic plants.

Parasitic plants that infect crops are devastating to agriculture throughout the world. These parasites develop a unique inducible organ called the haustorium that connects the vascular systems of the parasite and host to establish a flow of water and nutrients. Upon contact with the host, the haustorial epidermal cells at the interface with the host differentiate into specific cells called intrusive cells that grow endophytically toward the host vasculature. Following this, some of the intrusive cells re-differentiate to form a xylem bridge (XB) that connects the vasculatures of the parasite and host. Despite the prominent role of intrusive cells in host infection, the molecular mechanisms mediating parasitism in the intrusive cells remain poorly understood. In this study, we investigated differential gene expression in the intrusive cells of the facultative parasite Phtheirospermum japonicum in the family Orobanchaceae by RNA-sequencing of laser-microdissected haustoria. We then used promoter analyses to identify genes that are specifically induced in intrusive cells, and promoter fusions with genes encoding fluorescent proteins to develop intrusive cell-specific markers. Four of the identified intrusive cell-specific genes encode subtilisin-like serine proteases (SBTs), whose biological functions in parasitic plants are unknown. Expression of SBT inhibitors in intrusive cells inhibited both intrusive cell and XB development and reduced auxin response levels adjacent to the area of XB development. Therefore, we propose that subtilase activity plays an important role in haustorium development in P. japonicum.

Mapping proteolytic neo-N termini at the surface of living cells.

N terminomics is a powerful strategy for profiling proteolytic neo-N termini, but its application to cell surface proteolysis has been limited by the low relative abundance of plasma membrane proteins. Here we apply plasma membrane-targeted subtiligase variants (subtiligase-TM) to efficiently and specifically capture cell surface N termini in live cells. Using this approach, we sequenced 807 cell surface N termini and quantified changes in their abundance in response to stimuli that induce proteolytic remodeling of the cell surface proteome. To facilitate exploration of our datasets, we developed a web-accessible Atlas of Subtiligase-Captured Extracellular N Termini (ASCENT; http://wellslab.org/ascent). This technology will facilitate greater understanding of extracellular protease biology and reveal neo-N termini biomarkers and targets in disease.

Insight into the collagen-degrading activity of a serine protease in the latex of Ficus carica cultivar Masui Dauphine.

Ficus carica produces, in addition to the cysteine protease ficin, a serine protease. Earlier study on a serine protease from F. carica cultivar Brown Turkey showed that it specifically degraded collagen. In this study, we characterized the collagenolytic activity of a serine protease in the latex of F. carica cultivar Masui Dauphine. The serine protease degraded denatured, but not undenatured, acid-solubilized type I collagen. It also degraded bovine serum albumin, while the collagenase from Clostridium histolyticum did not. These results indicated that the serine protease in Masui Dauphine is not collagen-specific. The protease was purified to homogeneity by two-dimensional gel electrophoresis, and its partial amino acid sequence was determined by liquid chromatography-tandem mass spectrometry. BLAST searches against the Viridiplantae (green plants) genome database revealed that the serine protease was a subtilisin-like protease. Our results contrast with the results of the earlier study stating that the serine protease from F. carica is collagen-specific.

Spatially Resolved Tagging of Proteolytic Neo-N termini with Subtiligase-TM.

Mass spectrometry-based proteomics has been used successfully to identify substrates for proteases. Identification of protease substrates at the cell surface, however, can be challenging since cleavages are less abundant compared to other cellular events. Precise methods are required to delineate cleavage events that take place in these compartmentalized areas. This article by up-and-coming scientist Dr. Amy Weeks, an Assistant Professor at the University of Wisconsin-Madison, provides an overview of methods developed to identify protease substrates and their cleavage sites at the membrane. An overview is presented with the pros and cons for each method and in particular the N-terminomics subtiligase-TM method, developed by Dr. Weeks as a postdoctoral fellow in the lab of Dr. Jim Wells at University of California, San Francisco, is described in detail. Subtiligase-TM is a genetically engineered subtilisin protease variant that acts to biotinylate newly generated N termini, hence revealing new cleavage events in the presence of a specific enzyme, and furthermore can precisely identify the cleavage P1 site. Importantly, this proteomics method is compatible with living cells. This method will open the door to understanding protein shedding events at the biological membrane controlled by proteases to regulate biological processes.

Identification of two subtilisin-like serine proteases engaged in the degradation of recombinant proteins in Nicotiana benthamiana.

The tobacco variant Nicotiana benthamiana has recently emerged as a versatile host for the manufacturing of protein therapeutics, but the fidelity of many recombinant proteins generated in this system is compromised by inadvertent proteolysis. Previous studies have revealed that the anti-HIV-1 antibodies 2F5 and PG9 as well as the protease inhibitor α1 -antitrypsin (A1AT) are particularly susceptible to N. benthamiana proteases. Here, we identify two subtilisin-like serine proteases (NbSBT1 and NbSBT2) whose combined action is sufficient to account for all major cleavage events observed upon expression of 2F5, PG9 and A1AT in N. benthamiana. We propose that downregulation of NbSBT1 and NbSBT2 activities could constitute a powerful means to optimize the performance of this promising platform for the production of biopharmaceuticals. DATABASES: NbSBT sequence data are available in the DDBJ/EMBL/GenBank databases under the accession numbers MN534996 to MN535005.

Molecular analysis of a fibrin-degrading enzyme from Bacillus subtilis K2 isolated from the Indonesian soybean-based fermented food moromi.

The screening of proteolytic and fibrinolytic bacteria from moromi (an Indonesian soybean-based fermented food) yielded a number of isolates. Based on morphological and biochemical analyses and sequencing of the 16S rRNA gene, the isolate that exhibited the highest proteolytic and fibrinolytic activity was identified as Bacillus subtilis K2. The study was performed to analyze molecular characteristic of a fibrin-degrading enzyme from B. subtilis K2. BLASTn analysis of the nucleotide sequence encoding this fibrinolytic protein demonstrated 73.6% homology with the gene encoding the fibrin-degrading enzyme nattokinase of the B. subtilis subsp. natto, which was isolated from fermented soybean in Japan. An analysis of the putative amino-acid sequence of this protein indicated that it is a serine protease enzyme with aspartate, histidine, and serine in the catalytic triad. This enzyme was determined to be a 26-kDa molecule, as confirmed with a zymogram assay. Further bioinformatic analysis using Protparam demonstrated that the enzyme has a pI of 6.02, low instability index, high aliphatic index, and low GRAVY value. Molecular docking analysis using HADDOCK indicated that there are favorable interactions between subtilisin K2 and the fibrin substrate, as demonstrated by a high binding affinity (ΔG: - 19.4 kcal/mol) and low Kd value (6.3E-15 M). Overall, the study concluded that subtilisin K2 belong to serine protease enzyme has strong interactions with its fibrin substrate and fibrin can be rapidly degraded by this enzyme, suggesting its application as a treatment for thrombus diseases.