HtrAs are essential for the survival of the haloarchaeon Natrinema gari J7-2 in response to heat, high salinity, and toxic substances.

Bacterial and eukaryotic HtrAs can act as an extracytoplasmic protein quality control (PQC) system to help cells survive in stress conditions, but the functions of archaeal HtrAs remain unknown. Particularly, haloarchaea route most secretory proteins to the Tat pathway, enabling them to fold properly in well-controlled cytoplasm with cytosolic PQC systems before secretion. It is unclear whether HtrAs are required for haloarchaeal survival and stress response. The haloarchaeon Natrinema gari J7-2 encodes three Tat signal peptide-bearing HtrAs (NgHtrA, NgHtrB, and NgHtrC), and the signal peptides of NgHtrA and NgHtrC contain a lipobox. Here, the in vitro analysis reveals that the three HtrAs show different profiles of temperature-, salinity-, and metal ion-dependent proteolytic activities and could exhibit chaperone-like activities to prevent the aggregation of reduced lysozyme when their proteolytic activities are inhibited at low temperatures or the active site is disrupted. The gene deletion and complementation assays reveal that NgHtrA and NgHtrC are essential for the survival of strain J7-2 at elevated temperature and/or high salinity and contribute to the resistance of this haloarchaeon to zinc and inhibitory substances generated from tryptone. Mutational analysis shows that the lipobox mediates membrane anchoring of NgHtrA or NgHtrC, and both the membrane-anchored and free extracellular forms of the two enzymes are involved in the stress resistance of strain J7-2, depending on the stress conditions. Deletion of the gene encoding NgHtrB in strain J7-2 causes no obvious growth defect, but NgHtrB can functionally substitute for NgHtrA or NgHtrC under some conditions.IMPORTANCEHtrA-mediated protein quality control plays an important role in the removal of aberrant proteins in the extracytoplasmic space of living cells, and the action mechanisms of HtrAs have been extensively studied in bacteria and eukaryotes; however, information about the function of archaeal HtrAs is scarce. Our results demonstrate that three HtrAs of the haloarchaeon Natrinema gari J7-2 possess both proteolytic and chaperone-like activities, confirming that the bifunctional nature of HtrAs is conserved across all three domains of life. Moreover, we found that NgHtrA and NgHtrC are essential for the survival of strain J7-2 under stress conditions, while NgHtrB can serve as a substitute for the other two HtrAs under certain circumstances. This study provides the first biochemical and genetic evidence of the importance of HtrAs for the survival of haloarchaea in response to stresses.

A TrmBL2-like transcription factor mediates the growth phase-dependent expression of halolysin SptA in a concentration-dependent manner in Natrinema gari J7-2.

To cope with a high-salinity environment, haloarchaea generally employ the twin-arginine translocation (Tat) pathway to transport secretory proteins across the cytoplasm membrane in a folded state, including Tat-dependent extracellular subtilases (halolysins) capable of autocatalytic activation. Some halolysins, such as SptA of Natrinema gari J7-2, are produced at late-log phase to prevent premature enzyme activation and proteolytic damage of cellular proteins in haloarchaea; however, the regulation mechanism for growth phase-dependent expression of halolysins remains largely unknown. In this study, a DNA-protein pull-down assay was performed to identify the proteins binding to the 5'-flanking sequence of sptA encoding halolysin SptA in strain J7-2, revealing a TrmBL2-like transcription factor (NgTrmBL2). The ΔtrmBL2 mutant of strain J7-2 showed a sharp decrease in the production of SptA, suggesting that NgTrmBL2 positively regulates sptA expression. The purified recombinant NgTrmBL2 mainly existed as a dimer although monomeric and higher-order oligomeric forms were detected by native-PAGE analysis. The results of electrophoretic mobility shift assays (EMSAs) showed that NgTrmBL2 binds to the 5'-flanking sequence of sptA in a non-specific and concentration-dependent manner and exhibits an increased DNA-binding affinity with the increase in KCl concentration. Moreover, we found that a distal cis-regulatory element embedded in the neighboring upstream gene negatively regulates trmBL2 expression and thus participates in the growth phase-dependent biosynthesis of halolysin SptA. IMPORTANCE: Extracellular proteases play important roles in nutrient metabolism, processing of functional proteins, and antagonism of haloarchaea, but no transcription factor involved in regulating the expression of haloaechaeal extracellular protease has been reported yet. Here we report that a TrmBL2-like transcription factor (NgTrmBL2) mediates the growth phase-dependent expression of an extracellular protease, halolysin SptA, of haloarchaeon Natrinema gari J7-2. In contrast to its hyperthermophilic archaeal homologs, which are generally considered to be global transcription repressors, NgTrmBL2 functions as a positive regulator for sptA expression. This study provides new clues about the transcriptional regulation mechanism of extracellular protease in haloarchaea and the functional diversity of archaeal TrmBL2.

Nanoclay Drug-Delivery System Loading Potassium Iodide Promotes Endocytosis and Targeted Therapy in Anaplastic Thyroid Cancer.

The rapid proliferative biological behavior of primary foci of anaplastic thyroid cancer (ATC) makes it a lethal tumor. According to the specific iodine uptake capacity of thyroid cells and enhanced endocytosis of ATC cells, we designed a kind of nanoclay drug-loading system and showed a promising treatment strategy for ATC. Introducing potassium iodide (KI) improves the homoaggregation of clay nanoparticles and then affects the distribution of nanoparticles in vivo, which makes KI@DOX-KaolinMeOH enriched almost exclusively in thyroid tissue. Simultaneously, the improvement of dispersibility of KI@DOX-KaolinMeOH changes the target uptake of ATC cells by improving the endocytosis and nanoparticle-induced autophagy, which regulate the production of autolysosomes and autophagy-enhanced chemotherapy, eventually contributing to a tumor inhibition rate of more than 90% in the primary foci of ATC. Therefore, this facile strategy to improve the homoaggregation of nanoclay by introducing KI has the potential to become an advanced drug delivery vehicle in ATC treatment.

Diagnosis and treatment of biliary mucinous cystic neoplasms: A single-center experience.

BACKGROUND: Biliary mucinous cystic neoplasms (BMCNs) are rare hepatobiliary cystic tumors, which can be divided into noninvasive and invasive types. This study aimed to investigate the diagnosis, treatment, and prognosis of BMCNs in a large single center. METHODS: We analyzed 49 patients with BMCNs confirmed by postoperative pathology at the First Affiliated Hospital, Zhejiang University School of Medicine between January 2007 and December 2021. RESULTS: Among the 49 patients, 37 were female (75.5%), and the average age was 57.04 years. Common symptoms included abdominal discomfort, jaundice and fever, while 22 patients (44.9%) had no symptoms. Serum carbohydrate antigen (CA) 19-9 and CA125 concentrations were elevated in 34.8% and 19.6% of patients, respectively. Forty-eight patients had tumors in the intrahepatic bile ducts and only one had a tumor in the extrahepatic bile duct. Forty-eight patients with noninvasive intrahepatic BMCNs were further analyzed in terms of pathological features: 34 (70.8%) had low-grade intraepithelial neoplasms (LGINs), and 14 (29.2%) had high-grade intraepithelial neoplasms (HGINs). The potential immunohistochemical markers of BMCNs were cytokeratin (CK) 19, CK7, estrogen receptor and progesterone receptor. Follow-up data for 37 patients with intrahepatic BMCNs were obtained. The median overall survival (OS) of BMCNs was not reached. The longest survival time was 137 months.The 5- and 10-year OS rates were 100% and 85.4%, respectively. The 5- and 10-year recurrence-free survival (RFS) rates were 93.9% and 80.2%, respectively. CONCLUSIONS: BMCNs are rare cystic neoplasms that commonly occur in middle-aged females. BMCNs can only be diagnosed and classified by postoperative pathology, as there are no specific clinical presentations, serological indicators or imaging modalities for preoperative diagnosis. Complete surgical resection is necessary for BMCNs, and the postoperative prognosis is favorable.

Maturation Process and Characterization of a Novel Thermostable and Halotolerant Subtilisin-Like Protease with High Collagenolytic Activity but Low Gelatinolytic Activity.

Enzymatic degradation of collagen is of great industrial and environmental significance; however, little is known about thermophile-derived collagenolytic proteases. Here, we report a novel collagenolytic protease (TSS) from thermophilic Brevibacillus sp. WF146. The TSS precursor comprises a signal peptide, an N-terminal propeptide, a subtilisin-like catalytic domain, a ß-jelly roll (ßJR) domain, and a prepeptidase C-terminal (PPC) domain. The maturation of TSS involves a stepwise autoprocessing of the N-terminal propeptide and the PPC domain, and the ßJR rather than the PPC domain is necessary for correct folding of the enzyme. Purified mature TSS displayed optimal activity at 70°C and pH 9.0, a half-life of 1.5 h at 75°C, and an increased thermostability as the NaCl concentration increased up to 4 M. TSS possesses an increased number of surface acidic residues and ion pairs, as well as four Ca2+-binding sites, which contribute to its high thermostability and halotolerance. At high temperatures, TSS exhibited high activity toward insoluble type I collagen and azocoll but showed a low gelatinolytic activity, with a strong preference for Arg and Gly at the P1 and P1' positions, respectively. Both the ßJR and PPC domains could bind but not swell collagen, and thus facilitate TSS-mediated collagenolysis via improving the accessibility of the enzyme to the substrate. Additionally, TSS has the ability to efficiently degrade fish scale collagen at high temperatures. IMPORTANCE Proteolytic degradation of collagen at high temperatures has the advantages of increasing degradation efficiency and minimizing the risk of microbial contamination. Reports on thermostable collagenolytic proteases are limited, and their maturation and catalytic mechanisms remain to be elucidated. Our results demonstrate that the thermophile-derived TSS matures in an autocatalytic manner and represents one of the most thermostable collagenolytic proteases reported so far. At elevated temperatures, TSS prefers hydrolyzing insoluble heat-denatured collagen rather than gelatin, providing new insight into the mechanism of collagen degradation by thermostable collagenolytic proteases. Moreover, TSS has the potential to be used in recycling collagen-rich wastes such as fish scales.

Auto- and Hetero-Catalytic Processing of the N-Terminal Propeptide Promotes the C-Terminal Fibronectin Type III Domain-Mediated Dimerization of a Thermostable Vpr-like Protease.

Microbial Vpr-like proteases are extracellular multidomain subtilases with diverse functions and can form oligomers, but their maturation and oligomerization mechanisms remain to be elucidated. Here, we report a novel Vpr-like protease (BTV) from thermophilic bacterium Brevibacillus sp. WF146. The BTV precursor comprises a signal peptide, an N-terminal propeptide, a subtilisin-like catalytic domain with an inserted protease-associated (PA) domain, two tandem fibronectin type III domains (Fn1 and Fn2), and a C-terminal propeptide. The BTV proform (pro-BTV) could be autoprocessed into the mature form (mBTV) via two intermediates lacking the N- or C-terminal propeptide, respectively, and the C-terminal propeptide delays the autocatalytic maturation of the enzyme. By comparison, pro-BTV is more efficiently processed into mBTV by protease TSS from strain WF146. Purified mBTV is a Ca2+-dependent thermostable protease, showing optimal activity at 60°C and retaining more than 60% of activity after incubation at 60°C for 8 h. The PA domain is important for enzyme stability and contributes to the substrate specificity of BTV by restricting the access of protein substrates to the active site. The proform and mature form of BTV exist as a monomer and a homodimer, respectively, and the dimerization is mediated by the Fn1 and Fn2 domains. The N-terminal propeptide of BTV not only acts as intramolecular chaperone and enzymatic inhibitor but also inhibits the homodimerization of the enzyme. The removal of the N-terminal propeptide leads to a structural adjustment of the enzyme and thus promotes enzyme dimerization. IMPORTANCE Vpr-like proteases are widely distributed in bacteria and fungi and are involved in processing lantibiotics, degrading collagen, keratin, and fibrin, and pathogenesis of microbes. The dissection of the roles of individual domains in enzyme maturation and oligomerization is crucial for understanding the action mechanisms of these multidomain proteases. Our results demonstrate that hetero-catalytic maturation of the extracellular Vpr-like protease BTV of Brevibacillus sp. WF146 is more efficient than autocatalytic maturation of the enzyme. Moreover, we found that the C-terminal tandem fibronectin type III domains rather than the PA domain mediate the dimerization of mature BTV, while the N-terminal propeptide inhibits the dimerization of the BTV proform. This study provides new insight into the activation and oligomerization mechanisms of Vpr-like proteases.

Sec-Dependent Secretion of Subtilase SptE in Haloarchaea Facilitates Its Proper Folding and Heterocatalytic Processing by Halolysin SptA Extracellularly.

In response to high-salt conditions, haloarchaea export most secretory proteins through the Tat pathway in folded states; however, it is unclear why some haloarchaeal proteins are still routed to the Sec pathway. SptE is an extracellular subtilase of Natrinema sp. strain J7-2. Here, we found that SptE precursor comprises a Sec signal peptide, an N-terminal propeptide, a catalytic domain, and a long C-terminal extension (CTE) containing seven domains (C1 to C7). SptE is produced extracellularly as a mature form (M180) in strain J7-2 and a proform (ΔS) in the ΔsptA mutant strain, indicating that halolysin SptA mediates the conversion of the secreted proform into M180. The proper folding of ΔS is more efficient in the presence of NaCl than KCl. ΔS requires SptA for cleavage of the N-terminal propeptide and C-terminal C6 and C7 domains to generate M180, accompanied by the appearance of autoprocessing product M120 lacking C5. At lower salinities or elevated temperatures, M180 and M120 could be autoprocessed into M90, which comprises the catalytic and C1 domains and has a higher activity than M180. When produced in Haloferax volcanii, SptE could be secreted as a properly folded proform, but its variant (TSptE) with a Tat signal peptide does not fold properly and suffers from severe proteolysis extracellularly; meanwhile, TSptE is more inclined to aggregate intracellularly than SptE. Systematic domain deletion analysis reveals that the long CTE is an important determinant for secretion of SptE via the Sec rather than Tat pathway to prevent enzyme aggregation before secretion. IMPORTANCE While Tat-dependent haloarchaeal subtilases (halolysins) have been extensively studied, the information about Sec-dependent subtilases of haloarchaea is limited. Our results demonstrate that proper maturation of Sec-dependent subtilase SptE of Natrinema sp. strain J7-2 depends on the action of halolysin SptA from the same strain, yielding multiple hetero- and autocatalytic mature forms. Moreover, we found that the different extra- and intracellular salt types (NaCl versus KCl) of haloarchaea and the long CTE are extrinsic and intrinsic factors crucial for routing SptE to the Sec rather than Tat pathway. This study provides new clues about the secretion and adaptation mechanisms of Sec substrates in haloarchaea.

Enhancing extracellular production of recombinant proteins in Escherichia coli by co-expressing with a haloarchaeal protein containing a putative LolA-like domain.

Escherichia coli represents one of the most widely used hosts for recombinant protein production, but its limited capacity for producing extracellular proteins is often cited as a drawback. NJ7G_0991 is an extracellular protein of the haloarchaeon Natrinema sp. J7-2 and comprises a signal peptide, a putative LolA-like domain, and a C-terminal domain of unknown function. Here, we found that the full-length (0991) and the C-terminal domain-deletion variant (0991ΔC) of NJ7G_0991, but not its signal peptide-deletion variant (0991ΔS), were efficiently released into the culture supernatant of E. coli without extensive cell lysis as determined by ß-galactosidase activity assay. After lysozyme treatment, E. coli cells producing 0991 or 0991ΔC, but not 0991ΔS, were converted from rod-shaped forms to spheres, suggesting that the secretion of 0991 or 0991ΔC into the periplasm leads to an increase of outer membrane permeability of E. coli. A pelB signal peptide was fused to the N-terminus of the LolA-like domain, and the resulting variant PelB-0991ΔC could be released into the culture supernatant of E. coli more efficiently than 0991ΔC. By using PelB-0991ΔC as a co-expression partner, the extracellular production level of a recombinant thermostable subtilase WF146 could be enhanced by up to 14-fold, and the extracellular concentration of an active site variant of WF146 (WF146-SA) reached up to 129 mg/l. To the best of our knowledge, this is the first report on archaeal protein-based co-expression system for extracellular production of recombinant proteins in E. coli. KEY POINTS: • The haloarchaeal protein NJ7G_0991 can be efficiently released into the culture supernatant of E. coli. • The recombinant NJ7G_0991 increases the outer membrane permeability of E. coli. • The LolA-like domain of NJ7G_0991 can be used as a co-expression partner to improve extracellular production of recombinant proteins in E. coli.

Comparison and development of advanced machine learning tools to predict nonalcoholic fatty liver disease: An extended study.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a public health challenge and significant cause of morbidity and mortality worldwide. Early identification is crucial for disease intervention. We recently proposed a nomogram-based NAFLD prediction model from a large population cohort. We aimed to explore machine learning tools in predicting NAFLD. METHODS: A retrospective cross-sectional study was performed on 15 315 Chinese subjects (10 373 training and 4942 testing sets). Selected clinical and biochemical factors were evaluated by different types of machine learning algorithms to develop and validate seven predictive models. Nine evaluation indicators including area under the receiver operating characteristic curve (AUROC), area under the precision-recall curve (AUPRC), accuracy, positive predictive value, sensitivity, F1 score, Matthews correlation coefficient (MCC), specificity and negative prognostic value were applied to compare the performance among the models. The selected clinical and biochemical factors were ranked according to the importance in prediction ability. RESULTS: Totally 4018/10 373 (38.74%) and 1860/4942 (37.64%) subjects had ultrasound-proven NAFLD in the training and testing sets, respectively. Seven machine learning based models were developed and demonstrated good performance in predicting NAFLD. Among these models, the XGBoost model revealed the highest AUROC (0.873), AUPRC (0.810), accuracy (0.795), positive predictive value (0.806), F1 score (0.695), MCC (0.557), specificity (0.909), demonstrating the best prediction ability among the built models. Body mass index was the most valuable indicator to predict NAFLD according to the feature ranking scores. CONCLUSIONS: The XGBoost model has the best overall prediction ability for diagnosing NAFLD. The novel machine learning tools provide considerable beneficial potential in NAFLD screening.

Membrane raft redox signalling contributes to endothelial dysfunction and vascular remodelling of thoracic aorta in angiotensin II-infused rats.

NEW FINDINGS: What is the central question of this study? Is the membrane raft redox signalling pathway involved in blood pressure increase, endothelial dysfunction and vascular remodelling in an angiotensin II-induced hypertensive animal model? What is the main finding and its importance? The membrane raft redox signalling pathway was involved in endothelial dysfunction and medial remodelling in angiotensin II-induced hypertension. ABSTRACT: The membrane raft (MR) redox pathway is characterized by NADPH oxidase activation via the clustering of its subunits through lysosome fusion and the activation of acid sphingomyelinase (ASMase). Our previous study shows that the MR redox signalling pathway is associated with angiontensin II (AngII)-induced production of reactive oxygen species (ROS) and endothelial dysfunction in rat mesenteric arteries. In the present study, we hypothesized that this signalling pathway is involved in blood pressure increase, endothelial dysfunction and vascular remodelling in an AngII-induced hypertensive animal model. Sixteen-week-old male Sprague-Dawley rats were subjected to AngII infusion for 2 weeks with or without treatment with the lysosome fusion inhibitor bafilomycin A1 and ASMase inhibitor amitriptyline. After treatments, aortas were harvested for further study. The results showed that the MR redox signalling pathway was activated as indicated by the increase of MR formation, ASMase activity and ROS production in aorta from AngII-infused rats compared with that from control rats. MR formation and ROS production were significantly inhibited in thoracic aorta from AngII-induced rats treated with bafilomycin A1 and amitriptyline. Both treatments significantly attenuated blood pressure increase, endothelial dysfunction and vascular remodelling including medial hypertrophy, and increased collagen and fibronectin deposition in thoracic aortas from AngII-infused rats. Finally, both treatments significantly prevented the increase of inflammatory factors including monocyte chemotactic protein 1, intercellular adhesion molecule 1 and tumour necrosis factor α in thoracic aorta from AngII-infused rats. In conclusion, the present study demonstrates that the MR redox signalling pathway was involved in endothelial dysfunction and medial remodelling in AngII-induced hypertension.

Membrane rafts-redox signalling pathway contributes to renal fibrosis via modulation of the renal tubular epithelial-mesenchymal transition.

KEY POINTS: Membrane rafts (MRs)-redox signalling pathway is activated in response to transforming growth factor-ß1 (TGF-ß1) stimulation in renal tubular cells. This pathway contributes to TGF-1ß-induced epithelial-mesenchymal transition (EMT) in renal tubular cells. The the MRs-redox signalling pathway is activated in renal tubular cells isolated from angiotensin II (AngII)-induced hypertensive rats. Inhibition of this pathway attenuated renal inflammation and fibrosis in AngII-induced hypertension. ABSTRACT: The membrane rafts (MRs)-redox pathway is characterized by NADPH oxidase subunit clustering and activation through lysosome fusion, V-type proton ATPase subunit E2 (encoded by the Atp6v1e2 gene) translocation and sphingomyelin phosphodiesterase 1 (SMPD1, encoded by the SMPD1 gene) activation. In the present study, we hypothesized that the MRs-redox-derived reactive oxygen species (ROS) are involved in renal inflammation and fibrosis by promoting renal tubular epithelial-mesenchymal transition (EMT). Results show that transforming growth factor-ß1 (TGF-ß1) acutely induced MR formation and ROS production in NRK-52E cells, a rat renal tubular cell line. In addition, transfection of Atp6v1e2 small hairpin RNAs (shRNA) and SMPD1 shRNA attenuated TGF-ß1-induced changes in EMT markers, including E-cadherin, α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1) in NRK-52E cells. Moreover, Erk1/2 activation may be a downstream regulator of the MRs-redox-derived ROS, because both shRNAs significantly inhibited TGF-ß1-induced Erk1/2 phosphorylation. Further in vivo study shows that the renal tubular the MRs-redox signalling pathway was activated in angiotensin II (AngII)-induced hypertension, as indicated by the increased NADPH oxidase subunit Nox4 fraction in the MR domain, SMPD1 activation and increased ROS content in isolated renal tubular cells. Finally, renal transfection of Atp6v1e2 shRNA and SMPD1 shRNA significantly prevented renal fibrosis and inflammation, as indicated by the decrease of α-SMA, fibronectin, collagen I, monocyte chemoattractant protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1) and tumour necrosis factor-α (TNF-α) in kidneys from AngII-infused rats. It was concluded that the the MRs-redox signalling pathway is involved in TGF-ß1-induced renal tubular EMT and renal inflammation/fibrosis in AngII-induced hypertension.

Four Inserts within the Catalytic Domain Confer Extra Stability and Activity to Hyperthermostable Pyrolysin from Pyrococcus furiosus.

Pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus is the prototype of the pyrolysin family of the subtilisin-like serine protease superfamily (subtilases). It contains four inserts (IS147, IS29, IS27, and IS8) of unknown function in the catalytic domain. We performed domain deletions and showed that three inserts are either essential (IS147 and IS27) or important (IS8) for efficient maturation of pyrolysin at high temperatures, whereas IS29 is dispensable. The large insert IS147 contains Ca3 and Ca4, two calcium-binding Dx[DN]xDG motifs that are conserved in many pyrolysin-like proteases. Mutagenesis revealed that the Ca3 site contributes to enzyme thermostability and the Ca4 site is necessary for pyrolysin to fold into a maturation-competent conformation. Mature insert-deletion variants were characterized and showed that IS29 and IS8 contribute to enzyme activity and stability, respectively. In the presence of NaCl, pyrolysin undergoes autocleavage at two sites: one within IS29 and the other in IS27 Disrupting the ion pairs in IS27 and IS8 induces autocleavage in the absence of salts. Interestingly, autocleavage products combine noncovalently to form an active, nicked enzyme that is resistant to SDS and urea denaturation. Additionally, a single mutation in IS29 increases resistance to salt-induced autocleavage and further increases enzyme thermostability. Our results suggest that these extra structural elements play a crucial role in adapting pyrolysin to hyperthermal environments.IMPORTANCE Pyrolysin-like proteases belong to the subtilase superfamily and are characterized by large inserts and long C-terminal extensions; however, the role of the inserts in enzyme function is unclear. Our results demonstrate that four inserts in the catalytic domain of hyperthermostable pyrolysin contribute to the folding, maturation, stability, and activity of the enzyme at high temperatures. The modification of extra structural elements in pyrolysin-like proteases is a promising strategy for modulating global structure stability and enzymatic activity of this class of protease.

Alternative Translation Initiation of a Haloarchaeal Serine Protease Transcript Containing Two In-Frame Start Codons.

UNLABELLED: Recent studies have shown that haloarchaea employ leaderless and Shine-Dalgarno (SD)-less mechanisms for translation initiation of leaderless transcripts with a 5' untranslated region (5' UTR) of <10 nucleotides (nt) and leadered transcripts with a 5' UTR of ≥10 nt, respectively. However, whether the two mechanisms can operate on the same naturally occurring haloarchaeal transcript carrying multiple potential start codons is unknown. In this study, the transcript of the sptA gene (encoding an extracellular serine protease of Natrinema sp. strain J7-2) was experimentally determined and found to contain two potential in-frame AUG codons (AUG(1) and AUG(2)) located 5 and 29 nt, respectively, downstream of the transcription start site. Mutational analysis revealed that both AUGs can function as the translation start codon for production of active SptA, although AUG(1) is more efficient than AUG(2) for translation initiation. Insertion of a stable stem-loop structure between the two AUGs completely abolished initiation at AUG(1) but did not affect initiation at AUG(2), indicating that AUG(2)-initiated translation does not involve ribosome scanning from the 5' end of the transcript. Furthermore, the efficiency of AUG(2)-initiated translation was not influenced by an upstream SD-like sequence. In addition, both AUG(1) and AUG(2) contribute to transcript stability, probably by recruiting ribosomes to protect the transcript against degradation. These data suggest that depending on which of two in-frame start codons is used, the sptA transcript can act as either a leaderless or a leadered transcript for SptA production in haloarchaea. IMPORTANCE: In eukaryotes and bacteria, alternative translation start sites contribute to proteome complexity and can be used as a functional mechanism to increase translation efficiency. However, little is known about alternative translation initiation in archaea. Our results demonstrate that leaderless and SD-less mechanisms can be used for translation initiation of the sptA transcript from two in-frame start codons, raising the possibility that in haloarchaea, alternative translation initiation on one transcript functions to increase translation efficiency and/or contribute to proteome complexity.

Secretion of Tat-dependent halolysin SptA capable of autocatalytic activation and its relation to haloarchaeal growth.

Halolysins are Tat-dependent extracellular subtilases of haloarchaea. Whether halolysins can be activated before transport across the cytoplasmic membrane in a folded state and how haloarchaea minimize the risk of intracellular activation of halolysins and proteolysis of cellular proteins are unknown. Here, we report that both the precursor and proform of halolysin SptA from Natrinema sp. J7-2 mature autocatalytically, and the SptA maturation proceeds less efficiently in the presence of KCl than NaCl. When produced in Haloferax volcanii, most SptA molecules are secreted into the culture medium, but a small number of molecules can be activated intracellularly, affecting the cell's growth. Furthermore, retardation of SptA secretion in Hfx. volcanii via mutation of the Tat signal peptide leads to intracellular accumulation of the active enzyme and subsequent cell death. Although the Sec signal peptide can mediate SptA secretion in Hfx. volcanii, the secreted protein undergoes proteolysis. In Natrinema sp. J7-2, SptA is secreted primarily during stationary phase, and the intracellular accumulation of mature enzyme occurs during the stationary and death phases. The growth phase-dependent synthesis of SptA, highly efficient secretion system, and high intracellular KCl concentration, contribute to the suppression of premature activation of this enzyme in Natrinema sp. J7-2.

Protease-activated receptor 1 and 2 contribute to angiotensin II-induced activation of adventitial fibroblasts from rat aorta.

Adventitial fibroblasts (AFs) can be activated by angiotensin II (Ang II) and exert pro-fibrotic and pro-inflammatory effects in vascular remodeling. Protease-activated receptor (PAR) 1 and 2 play a significant role in fibrogenic and inflammatory diseases. The present study hypothesized that PAR1 and PAR2 are involved in Ang II-induced AF activation and contribute to adventitial remodeling. We found that direct activation of PAR1 and PAR2 with PAR1-AP and PAR2-AP led to AF activation, including proliferation and differentiation of AFs, extracellular matrix synthesis, as well as production of pro-fibrotic cytokine TGF-ß and pro-inflammatory cytokines IL-6 and MCP-1. Furthermore, PAR1 and PAR2 mediated Ang II-induced AF activation, since both PAR1 and PAR2 antagonists inhibited Ang II-induced proliferation, migration, differentiation, extracellular matrix synthesis and production of pro-fibrotic and pro-inflammatory cytokines in AFs. Finally, mechanistic study showed that Ang II, via Ang II type I receptor (AT1R), upregulated both PAR1 and PAR2 expression, and transactivated PAR1 and PAR2, as denoted by internalization of both proteins. In conclusion, our results suggest that PAR1 and PAR2 play a critical role in Ang II-induced AF activation, and this may contribute to adventitia-related pathological changes.

Maturation of Fibrinolytic Bacillopeptidase F Involves both Hetero- and Autocatalytic Processes.

Bacillopeptidase F (Bpr) is a fibrinolytic serine protease produced by Bacillus subtilis. Its precursor is composed of a signal peptide, an N-terminal propeptide, a catalytic domain, and a long C-terminal extension (CTE). Several active forms of Bpr have been previously reported, but little is known about the maturation of this enzyme. Here, a gene encoding a Bpr (BprL) was cloned from B. subtilis LZW and expressed in B. subtilis WB700, and three fibrinolytic mature forms with apparent molecular masses of 45, 75, and 85 kDa were identified in the culture supernatant. After treatment with urea, the 75-kDa mature form had the same molecular mass as the 85-kDa mature form, from which we infer that they adopt different conformations. Mutational analysis revealed that while the 85-kDa mature form is generated via heterocatalytic processing of a BprL proform by an unidentified protease of B. subtilis, the production of the 75- and 45-kDa mature forms involves both hetero- and autocatalytic events. From in vitro analysis of BprL and its sequential C-terminal truncation variants, it appears that partial removal of the CTE is required for the initiation of autoprocessing of the N-terminal propeptide, which is composed of a core domain (N*) and a 15-residue linker peptide, thereby yielding the 45-kDa mature form. These data suggest that the differential processing of BprL, either heterocatalytically or autocatalytically, leads to the formation of multiple mature forms with different molecular masses or conformations.

Autocatalytic activation of a thermostable glutamyl endopeptidase capable of hydrolyzing proteins at high temperatures.

Glutamyl endopeptidases (GSEs) specifically hydrolyze peptide bonds formed by α-carboxyl groups of Glu and Asp residues. We cloned the gene for a thermophilic GSE (designated TS-GSE) from Thermoactinomyces sp. CDF. A proform of TS-GSE that contained a 61-amino acid N-terminal propeptide and a 218-amino acid mature domain was produced in Escherichia coli. We found that the proform possessed two processing sites and was capable of autocatalytic activation via multiple pathways. The N-terminal propeptide could be autoprocessed at the Glu-1-Ser1 bond to directly generate the mature enzyme. It could also be autoprocessed at the Glu-12-Lys-11 bond to yield an intermediate, which was then converted into the mature form after removal of the remaining part of the propeptide. The segment surrounding the two processing sites was flexible, which allowed the proform and the intermediate form to be trans-processed into the mature form by either active TS-GSE or heterogeneous proteases. Deletion analysis revealed that the N-terminal propeptide is important for the correct folding and maturation of TS-GSE. The propeptide, even its last 11-amino acid peptide segment, could inhibit the activity of its cognate mature domain. The mature TS-GSE displayed a temperature optimum of 85 °C and retained approximately 90 % of its original activity after incubation at 70 °C for 6 h, representing the most thermostable GSE reported to date. Mutational analysis suggested that the disulfide bonds Cys32-Cys48 and Cys180-Cys183 cumulatively contributed to the thermostability of TS-GSE.

Improving the Thermostability and Activity of a Thermophilic Subtilase by Incorporating Structural Elements of Its Psychrophilic Counterpart.

The incorporation of the structural elements of thermostable enzymes into their less stable counterparts is generally used to improve enzyme thermostability. However, the process of engineering enzymes with both high thermostability and high activity remains an important challenge. Here, we report that the thermostability and activity of a thermophilic subtilase were simultaneously improved by incorporating structural elements of a psychrophilic subtilase. There were 64 variable regions/residues (VRs) in the alignment of the thermophilic WF146 protease, mesophilic sphericase, and psychrophilic S41. The WF146 protease was subjected to systematic mutagenesis, in which each of its VRs was replaced with those from S41 and sphericase. After successive rounds of combination and screening, we constructed the variant PBL5X with eight amino acid residues from S41. The half-life of PBL5X at 85°C (57.1 min) was approximately 9-fold longer than that of the wild-type (WT) WF146 protease (6.3 min). The substitutions also led to an increase in the apparent thermal denaturation midpoint temperature (Tm) of the enzyme by 5.5°C, as determined by differential scanning calorimetry. Compared to the WT, PBL5X exhibited high caseinolytic activity (25 to 95°C) and high values of Km and kcat (25 to 80°C). Our study may provide a rational basis for developing highly stable and active enzymes, which are highly desired in industrial applications.

Degradation of intact chicken feathers by Thermoactinomyces sp. CDF and characterization of its keratinolytic protease.

Thermoactinomyces is known for its resistance to extreme environmental conditions and its ability to digest a wide range of hard-to-degrade compounds. Here, Thermoactinomyces sp. strain CDF isolated from soil was found to completely degrade intact chicken feathers at 55 °C, with the resulting degradation products sufficient to support growth as the primary source of both carbon and nitrogen. Although feathers were not essential for the expression of keratinase, the use of this substrate led to a further 50-300 % increase in enzyme production level under different nutrition conditions, with extracellular keratinolytic activity reaching its highest level (∼400 U/mL) during the late-log phase. Full degradation of feathers required the presence of living cells, which are thought to supply reducing agents necessary for the cleavage of keratin disulfide bonds. Direct contact between the hyphae and substrate may enhance the reducing power and protease concentrations present in the local microenvironment, thereby facilitating keratin degradation. The gene encoding the major keratinolytic protease (protease C2) of strain CDF was cloned, revealing an amino acid sequence identical to that of subtilisin-like E79 protease from Thermoactinomyces sp. E79, albeit with significant differences in the upstream flanking region. Exogenous expression of protease C2 in Escherichia coli resulted in the production of inclusion bodies with proteolytic activity, which could be solubilized to an alkaline solution to produce mature protease C2. Purified protease C2 was able to efficiently hydrolyze α- and ß-keratins at 60-80 °C and pH 11.0, representing a promising candidate for enzymatic processing of hard-to-degrade proteins such as keratinous wastes.

Proteomic analysis of the secretome of haloarchaeon Natrinema sp. J7-2.

Although in silico predictions have revealed that haloarchaea can be distinguished from other organisms in that the Tat pathway is used more extensively than the Sec pathway for haloarchaeal protein secretion, only a few haloarchaeal-secreted proteins have been experimentally confirmed. Here, the culture supernatant and membrane fraction of the haloarchaeon Natrinema sp. J7-2 grown at 23% salt concentration were subjected to RPLC-ESI-MS/MS analysis. In total, 46 predicted Tat substrates, 14 predicted Sec substrates, and 3 class III signal peptide-bearing proteins were detected. Approximately 65% of the detected Tat substrates contain lipoboxes, emphasizing the role of the Tat pathway in haloarchaeal lipoprotein secretion. Most of the detected Tat substrates are extracellular substrate (solute)-binding proteins and redox proteins. Despite the small number of Sec substrates, two of them, a cell surface glycoprotein and a putative lipoprotein carrier protein, were identified to be high-abundance secreted proteins. While limited proteins were detected in the culture supernatant, most of the secreted proteins were found in the membrane fraction. The anchoring of secreted proteins to the cell surface via a lipobox or a PGF-CTERM seems to be an adaptation strategy of haloarchaea to handle the harsh extracellular environment. Additionally, ∼15% of the integral membrane proteins (IMPs) detected in the membrane fraction possess putative Sec signal peptides or signal anchors, implying that the Sec pathway is important for membrane insertion of IMPs. This is the first report to describe the experimental secretome of haloarchaea and provide new information for better understanding of haloarchaeal protein secretion patterns.