HtrA Family Research Articles

High resolution analysis of proteolytic substrate processing

Members of the widely conserved high temperature requirement A (HtrA) family of serine proteases are involved in multiple aspects of protein quality control. In this context, they have been shown to efficiently degrade misfolded proteins or protein fragments. However, recent reports suggest that folded proteins can also be native substrates. To gain a deeper understanding of how folded proteins are initially processed and subsequently degraded into short peptides by human HTRA1, we established an integrated and quantitative approach using time-resolved mass spectrometry, CD spectroscopy, and bioinformatics. The resulting data provide high-resolution information on up to 178 individual proteolytic sites within folded ANXA1 (consisting of 346 amino acids), the relative frequency of cuts at each proteolytic site, the preferences of the protease for the amino acid sequence surrounding the scissile bond, as well as the degrees of sequential structural relaxation and unfolding of the substrate that occur during progressive degradation. Our workflow provides precise molecular insights into protease-substrate interactions, which could be readily adapted to address other posttranslational modifications such as phosphorylation in dynamic protein complexes.

The CssRS two-component system of Bacillus subtilis contributes to teicoplanin and polymyxin B response.

CssRS is a two-component system that plays a pivotal role in mediating the secretion stress response in Bacillus subtilis. This system upregulates the synthesis of membrane-bound HtrA family proteases that cope with misfolded proteins that accumulate within the cell envelope as a result of overexpression or heat shock. Recent studies have shown the induction of CssRS-regulated genes in response to cell envelope stress. We investigated the induction of the CssRS-regulated htrA promoter in the presence of different cell wall- and membrane-active substances and observed induction of the CssRS-controlled genes by glycopeptides (vancomycin and teicoplanin), polymyxins B and E, certain β-lactams, and detergents. Teicoplanin was shown to elicit remarkably stronger induction than vancomycin and polymyxin B. Teicoplanin and polymyxin B induced the spxO gene expression in a CssRS-dependent fashion, resulting in increased activity of Spx, a master regulator of disulfide stress in Bacillus subtilis. The CssRS signaling pathway and Spx activity were demonstrated to be involved in Bacillus subtilis resistance to teicoplanin and polymyxin B.

Exploring the Role of HtrA Family Genes in Cancer: A Systematic Review.

HtrA1, HtrA2, HtrA3 and HtrA4 appear to be involved in the development of pathologies such as cancer. This systematic review reports the results of a literature search performed to compare the expression of HtrA family genes and proteins in cancer versus non-cancer tissues and cell lines, assess relationships between HtrA expression and cancer clinical features in cancer, and analyse the molecular mechanism, by which HtrA family affects cancer. The literature search was conducted according to the PRISMA statement among four databases (PubMed, Web of Science, Embase and Scopus). A total of 38 articles met the inclusion criteria and involved the expression of HtrA family members and concerned the effect of HtrA expression on cancer and metastasis development or on the factor that influences it. Additionally, 31 reports were retrieved manually. Most articles highlighted that HtrA1 and HtrA3 exhibited tumour suppressor activity, while HtrA2 was associated with tumour growth and metastasis. There were too few studies to clearly define the role of the HtrA4 protease in tumours. Although the expression of serine proteases of the HtrA family was dependent on tumour type, stage and the presence of metastases, most articles indicated that HtrA1 and HtrA3 expression in tumours was downregulated compared with healthy tissue or cell lines. The expression of HtrA2 was completely study dependent. The limited number of studies on HtrA4 expression made it impossible to draw conclusions about differences in expression between healthy and tumour tissue. The conclusions drawn from the study suggest that HtrA1 and HtrA3 act as tumour suppressors.

Functional characterization and transcriptional analysis of degQ of Xanthomonas campestris pathovar campestris.

High-temperature-requirement protein A (HtrA) family proteins play important roles in controlling protein quality and are recognized as virulence factors in numerous animal and human bacterial pathogens. The role of HtrA family proteins in plant pathogens remains largely unexplored. Here, we investigated the HtrA family protein, DegQ, in the crucifer black rot pathogen Xanthomonas campestris pathovar campestris (Xcc). DegQ is essential for bacterial attachment and full virulence of Xcc. Moreover, the degQ mutant strain showed increased sensitivity to heat treatment and sodium dodecyl sulfate. Expressing the intact degQ gene in trans in the degQ mutant could reverse the observed phenotypic changes. In addition, we demonstrated that the DegQ protein exhibited chaperone-like activity. Transcriptional analysis displayed that degQ expression was induced under heat treatment. Our results contribute to understanding the function and expression of DegQ of Xcc for the first time and provide a novel perspective about HtrA family proteins in plant pathogen.

HTRA1 expression is associated with immune-cell infiltration and survival in breast cancer.

High temperature requirement A1 (HTRA1), a member of the HTRA family, is a serine peptidase involved in many crucial bioprocesses such as proliferation, mitochondrial homeostasis, apoptosis, and protein quality control. It also plays an important role in the development of various tumors. However, the potential role and mechanisms of action of HTRA1 in breast cancer (BRCA) remain unclear. We conducted a bioinformatics-based study to investigate HTRA1 expression in BRCA alongside its associations with immune-cell infiltrates and survival outcomes. The expression of HTRA1 in BRCA samples was analyzed using RNAseq datasets from The Cancer Genome Atlas and Gene Expression Omnibus. R software was employed to assess the relationship between HTRA1 expression and clinicopathological characteristics, tumor-infiltrating immune cells, and immunity-associated biomarkers in BRCA. MethSurv and cBioPortal database were utilized to evaluate DNA methylation and genovariation within the HTRA1 DNA. Receiver operating characteristic curves, Kaplan-Meier analysis, and Cox regression were performed to estimate the impact of HTRA1 on diagnosis, prognosis, and response to chemotherapy in BRCA. HTRA1 expression was significantly downregulated in BRCA tissues compared to adjacent normal breast tissue controls. Differentially expressed genes associated with HTRA1 expression primarily enriched in cell proliferation pathways. Furthermore, altered HTRA1 expression significantly correlated with patient age, tumor histological type, T stage, progesterone receptor/estrogen receptor status, and PAM50 subtype of BRCA. Both positive and negative associations were observed between HTRA1 levels and the abundance of different types of immune cells, as well as immune biomarkers, including resting mast cells, follicular helper T cells, PD-L1, p53, and Ki67. Low HTRA1 expression was related with pathological complete response in luminal B BRCA patients undergoing chemotherapy. Additionally, lower HTRA1 expression in BRCA was associated with inferior overall survival and relapse-free survival. HTRA1 expression is associated with immune-cell infiltration, response to chemotherapy, and survival outcomes in BRCA. HTRA1 has the potential to serve as a promising biomarker and therapeutic target moving forward.

The structure of MucD from Pseudomonas syringae revealed N-terminal loop-mediated trimerization of HtrA-like serine protease

Protein quality control mechanisms are essential for maintaining cellular integrity, and the HtrA family of serine proteases plays a crucial role in handling folding stress in prokaryotic periplasm. Escherichia coli harbors three HtrA members, namely, DegS, DegP, and DegQ, which share a common domain structure. MucD, a putative HtrA family member that resembles DegP, is involved in alginate biosynthesis regulation and the stress response. Pseudomonas syringae causes plant diseases and opportunistic infections in humans. This study presents the high-resolution structure of MucD from Pseudomonas syringae (psMucD), revealing its composition as a typical HtrA family serine protease with protease and PDZ domains. Its findings suggest that psMucD containing one PDZ domain is a trimer in solution, and psMucD trimerization is mediated by its N-terminal loop. Sequence and structural analyses revealed similarities and differences with other HtrA family members. Additionally, this study provides a model of psMucD's catalytic process, comparing it with other members of the HtrA family of serine proteases.

HtrA3: a promising prognostic biomarker and therapeutic target for head and neck squamous cell carcinoma.

The dysregulation of the human high-temperature requirement A (HtrA) family of serine proteases is associated with many malignancies. However, there are few reports on HtrAs in head and neck squamous cell carcinoma (HNSCC). The aim of this study was to investigate the expression, prognostic value, and biological functions of HtrAs in HNSCC. The RNA-sequencing data and clinical data of HNSCC were downloaded from The Cancer Genome Atlas (TCGA) database. The GSE30784 and GSE31056 datasets from the Gene Expression Omnibus (GEO) database were used for further verification. This study explored the differential expression of HtrAs and assessed their potential impact on the prognosis of HNSCC patients using a survival module. Correlations between clinical characteristics and HtrA expression levels were then explored using a Wilcoxon rank sum test. A Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were performed using "clusterProfile" in the R software. A Pearson/Spearman correlation test was applied to analyze the relationship between HtrAs and immune infiltration level/checkpoint genes. Validation of HtrA expression levels were carried out by RT-PCR and western blot in human squamous carcinoma cell lines (Fadu and Cal-27) and human non-tumorigenic bronchial epithelium cells (BEAS-2B). Finally, through cell transfection, CCK-8, Ki-67 immunofluorescence, and flow cytometry assays, the effect of HtrA3 knockdown on the malignant biological behavior of HNSCC cells was explored. The gene expression levels of HtrAs were significantly upregulated and associated with patient age, TNM stage, clinical stage, and TP53 mutation status in the TCGA-HNSCC cohort. High expressions of HtrA1/3 were associated with shorter overall survival, shorter progress-free interval, and lower disease-specific survival in HNSCC. A nomogram for HtrAs was constructed and validated. HtrA-related genes were significantly enriched in the immune response and cell apoptosis pathway. In addition, the expression of HtrAs showed significant correlations with B cells, M cells, DC cell infiltration, and immune infiltration checkpoint (CD276, TNFRSF14). Validation of HtrA expression was carried out by RT-PCR and western blot. Results of in vitro experiments indicated that HtrA3 gene knockdown inhibits the proliferation of FaDu and Cal-27 cells while concurrently promoting apoptosis. HtrA3 shows significant potential as both a prognostic marker and a promising therapeutic target for HNSCC, highlighting its relevance and importance in future research and potential clinical applications.

Identification and Characterization of an HtrA Sheddase Produced by Coxiella burnetii.

Having previously shown that soluble E-cadherin (sE-cad) is found in sera of Q fever patients and that infection of BeWo cells by C. burnetii leads to modulation of the E-cad/β-cat pathway, our purpose was to identify which sheddase(s) might catalyze the cleavage of E-cad. Here, we searched for a direct mechanism of cleavage initiated by the bacterium itself, assuming the possible synthesis of a sheddase encoded in the genome of C. burnetii or an indirect mechanism based on the activation of a human sheddase. Using a straightforward bioinformatics approach to scan the complete genomes of four laboratory strains of C. burnetii, we demonstrate that C. burnetii encodes a 451 amino acid sheddase (CbHtrA) belonging to the HtrA family that is differently expressed according to the bacterial virulence. An artificial CbHtrA gene (CoxbHtrA) was expressed, and the CoxbHtrA recombinant protein was found to have sheddase activity. We also found evidence that the C. burnetii infection triggers an over-induction of the human HuHtrA gene expression. Finally, we demonstrate that cleavage of E-cad by CoxbHtrA on macrophages-THP-1 cells leads to an M2 polarization of the target cells and the induction of their secretion of IL-10, which "disarms" the target cells and improves C. burnetii replication. Taken together, these results demonstrate that the genome of C. burnetii encodes a functional HtrA sheddase and establishes a link between the HtrA sheddase-induced cleavage of E-cad, the M2 polarization of the target cells and their secretion of IL-10, and the intracellular replication of C. burnetii.

Crystal structures and solution conformations of HtrA from Helicobacter pylori reveal pH-dependent oligomeric conversion and conformational rearrangements

Helicobacter pylori is a Gram-negative microaerophilic bacterium that infects over 50 % of the world's population, making it a major risk factor for chronic gastritis, ulcer diseases of the stomach and duodenum, MALT lymphoma, and gastric cancer. The clinical consequences of H. pylori infection are closely linked with the expression of virulence factors secreted by the bacterium. One such virulence factor is high temperature requirement A (HtrA), which possesses chaperone and serine protease activity. In the host stomach, HtrA secreted from H. pylori (HpHtrA) disrupts intercellular adhesions by cleaving epithelial adhesion proteins including E-cadherin and desmoglein-2. This disruption causes intercellular junctions to open, allowing the bacterium to pass through the epithelial barrier, access the intercellular space, and colonize the gastric mucosa. HtrA proteases are well known for their structural complexity, reflected in their diverse oligomer forms and multi-tasking activities in both prokaryotes and eukaryotes. In this study, we determined crystal structures and solution conformations of HpHtrA monomer and trimer, which revealed large domain rearrangements between them. Notably, this is the first report of a monomeric structure in the HtrA family. We further found a pH-dependent dynamic trimer-to-monomer conversion and concurrent conformational changes that seem closely linked with a pH-sensing ability through the protonation of certain Asp residues. These results advance our understanding of the functional roles and the related mechanisms of this protease in bacterial infection, which may shed light on the development of HtrA-targeted therapies for H. pylori-associated diseases.

Dual role of an essential HtrA2/Omi protease in the human malaria parasite: Maintenance of mitochondrial homeostasis and induction of apoptosis-like cell death under cellular stress

Members of the HtrA family of serine proteases are known to play roles in mitochondrial homeostasis as well as in programmed cell death. Mitochondrial homeostasis and metabolism are crucial for the survival and propagation of the malaria parasite within the host. Here we have functionally characterized a Plasmodium falciparum HtrA2 (PfHtrA2) protein, which harbours trypsin-like protease activity that can be inhibited by its specific inhibitor, ucf-101. A transgenic parasite line was generated, using the HA-glmS C-terminal tagging approach, for localization as well as for inducible knock-down of PfHtrA2. The PfHtrA2 was localized in the parasite mitochondrion during the asexual life cycle. Genetic ablation of PfHtrA2 caused significant parasite growth inhibition, decreased replication of mtDNA, increased mitochondrial ROS production, caused mitochondrial fission/fragmentation, and hindered parasite development. However, the ucf-101 treatment did not affect the parasite growth, suggesting the non-protease/chaperone role of PfHtrA2 in the parasite. Under cellular stress conditions, inhibition of PfHtrA2 by ucf-101 reduced activation of the caspase-like protease as well as parasite cell death, suggesting the involvement of protease activity of PfHtrA2 in apoptosis-like cell death in the parasite. Under these cellular stress conditions, the PfHtrA2 gets processed but remains localized in the mitochondrion, suggesting that it acts within the mitochondrion by cleaving intra-mitochondrial substrate(s). This was further supported by trans-expression of PfHtrA2 protease domain in the parasite cytosol, which was unable to induce any cell death in the parasite. Overall, we show the specific roles of PfHtrA2 in maintaining mitochondrial homeostasis as well as in regulating stress-induced cell death.

Allosteric inhibition of HTRA1 activity by a conformational lock mechanism to treat age-related macular degeneration

The trimeric serine protease HTRA1 is a genetic risk factor associated with geographic atrophy (GA), a currently untreatable form of age-related macular degeneration. Here, we describe the allosteric inhibition mechanism of HTRA1 by a clinical Fab fragment, currently being evaluated for GA treatment. Using cryo-EM, X-ray crystallography and biochemical assays we identify the exposed LoopA of HTRA1 as the sole Fab epitope, which is approximately 30 Å away from the active site. The cryo-EM structure of the HTRA1:Fab complex in combination with molecular dynamics simulations revealed that Fab binding to LoopA locks HTRA1 in a non-competent conformational state, incapable of supporting catalysis. Moreover, grafting the HTRA1-LoopA epitope onto HTRA2 and HTRA3 transferred the allosteric inhibition mechanism. This suggests a conserved conformational lock mechanism across the HTRA family and a critical role of LoopA for catalysis, which was supported by the reduced activity of HTRA1-3 upon LoopA deletion or perturbation. This study reveals the long-range inhibition mechanism of the clinical Fab and identifies an essential function of the exposed LoopA for activity of HTRA family proteases.

High temperature requirement A3 attenuates hypoxia/reoxygenation induced injury in H9C2 cells via suppressing inflammatory responses

High temperature requirement A3 (HtrA3) belongs to the HtrA family, and its role in inflammation and myocardial ischemia-reperfusion injury remains unknown. Herein, the study aimed to explore the role of HtrA3 in inflammatory cytokine secretion and the nuclear factor kappa B (NF-κB) signaling pathway in hypoxia-reoxygenation (H/R)-induced H9C2 cardiomyoblasts. H9C2 cells were treated with H/R to mimic myocardial ischemia-reperfusion in vitro. Results showed that HtrA3 expression was significantly downregulated and the expression of inflammatory cytokines was regulated in response to H/R. HtrA3 overexpression decreased the secretion of inflammatory cytokines, whereas HtrA3 knockdown led to increase levels of inflammatory cytokines. And H/R-induced inflammation in H9C2 cells was inhibited by the regulation of the NF-κB signaling pathway. Our findings demonstrate that HtrA3 alleviates H/R-induced inflammatory responses in H9C2 cardiomyoblasts, possibly by suppressing the pro-inflammatory NF-κB signaling pathway.

Unraveling the Dichotomy of Enigmatic Serine Protease HtrA2.

Mitochondrial high-temperature requirement protease A2 (HtrA2) is an integral member of the HtrA family of serine proteases that are evolutionarily conserved from prokaryotes to humans. Involvement in manifold intricate cellular networks and diverse pathophysiological functions make HtrA2 the most enigmatic moonlighting protease amongst the human HtrAs. Despite perpetuating the oligomeric architecture and overall structural fold of its homologs that comprises serine protease and regulatory PDZ domains, subtle conformational alterations and dynamic enzymatic regulation through the distinct allosteric mode of action lead to its functional diversity. This mitochondrial protease upon maturation, exposes its one-of-a-kind N-terminal tetrapeptide (AVPS) motif that binds and subsequently cleaves Inhibitor of Apoptosis Proteins (IAPs) thus promoting cell death, and posing as an important molecule for therapeutic intervention. Interestingly, unlike its other human counterparts, HtrA2 has also been implicated in maintaining the mitochondrial integrity through a bi-functional chaperone-protease activity, the on-off switch of which is yet to be identified. Furthermore, its ability to activate a wide repertoire of substrates through both its N- and C-terminal regions presumably has calibrated its association with several cellular pathways and hence diseases including neurodegenerative disorders and cancer. Therefore, the exclusive structural attributes of HtrA2 that involve multimodal activation, intermolecular PDZ-protease crosstalk, and an allosterically-modulated trimeric active-site ensemble have enabled the protease to evolve across species and partake functions that are fine-tuned for maintaining cellular homeostasis and mitochondrial proteome quality control in humans. These unique features along with its multitasking potential make HtrA2 a promising therapeutic target both in cancer and neurodegeneration.

Overview of Human HtrA Family Proteases and Their Distinctive Physiological Roles and Unique Involvement in Diseases, Especially Cancer and Pregnancy Complications.

The mammalian high temperature requirement A (HtrA) proteins are a family of evolutionarily conserved serine proteases, consisting of four homologs (HtrA1-4) that are involved in many cellular processes such as growth, unfolded protein stress response and programmed cell death. In humans, while HtrA1, 2 and 3 are widely expressed in multiple tissues with variable levels, HtrA4 expression is largely restricted to the placenta with the protein released into maternal circulation during pregnancy. This limited expression sets HtrA4 apart from the rest of the family. All four HtrAs are active proteases, and their specific cellular and physiological roles depend on tissue type. The dysregulation of HtrAs has been implicated in many human diseases such as cancer, arthritis, neurogenerative ailments and reproductive disorders. This review first discusses HtrAs broadly and then focuses on the current knowledge of key molecular characteristics of individual human HtrAs, their similarities and differences and their reported physiological functions. HtrAs in other species are also briefly mentioned in the context of understanding the human HtrAs. It then reviews the distinctive involvement of each HtrA in various human diseases, especially cancer and pregnancy complications. It is noteworthy that HtrA4 expression has not yet been reported in any primary tumour samples, suggesting an unlikely involvement of this HtrA in cancer. Collectively, we accentuate that a better understanding of tissue-specific regulation and distinctive physiological and pathological roles of each HtrA will improve our knowledge of many processes that are critical for human health.

Over-activation of a nonessential bacterial protease DegP as an antibiotic strategy

Rising antibiotic resistance urgently begs for novel targets and strategies for antibiotic discovery. Here, we report that over-activation of the periplasmic DegP protease, a member of the highly conserved HtrA family, can be a viable strategy for antibiotic development. We demonstrate that tripodal peptidyl compounds that mimic DegP-activating lipoprotein variants allosterically activate DegP and inhibit the growth of an Escherichia coli strain with a permeable outer membrane in a DegP-dependent fashion. Interestingly, these compounds inhibit bacterial growth at a temperature at which DegP is not essential for cell viability, mainly by over-proteolysis of newly synthesized proteins. Co-crystal structures show that the peptidyl arms of the compounds bind to the substrate-binding sites of DegP. Overall, our results represent an intriguing example of killing bacteria by activating a non-essential enzyme, and thus expand the scope of antibiotic targets beyond the traditional essential proteins or pathways.

Cage-like polyhedrons of DegQ from Cytophaga hutchinsonii show stable proteolytic activity and strong chaperone activity

DegQ is a member of highly conserved htrA family proteins, acting as a chaperone-protease facilitating refolding or degradation of periplasmic misfolded proteins. In the current regulatory model of DegQ, the resting state was inactive hexamers and they would reassemble into cage-like polyhedrons to refold or degrade substrates. Here, the characteristics of DegQ from Cytophaga hutchinsonii were reported. The resting state of ChuDegQ was trimers and no hexamers were detected, suggesting a different regulatory mechanism. Cage-like polyhedrons were formed depending on the concentration of substrates, which were able to degrade unfolded BSA and prevent the aggregation of unfolded lysozyme, suggesting that ChuDegQ harbored both proteolytic and chaperone-like activity. Further study showed that trimers possessed robust proteolytic activity as cage-like polyhedrons. However, when degrading unfolded substrates, trimers suffered serious self-degradation while cage-like polyhedrons were stable. Moreover, trimers were able to prevent the aggregation of unfolded lysozyme and refold chemically denatured MalS, indicating that trimers had a certain chaperone activity. While the chaperone activity of cage-like polyhedrons was much higher than that of trimers. These results suggested that the formation of cage-like polyhedrons was important for both stable protease activity and robust chaperone activity. This study enriched our knowledge of both the proteolytic activity and chaperone activity of the trimeric DegQ and shed light on the reason for the cage formation of DegQ.

Mitochondrial serine protease Omi/HtrA2 accentuates brain ischemia/reperfusion injury in rats and oxidative stress injury in vitro by modulating mitochondrial stress proteins CHOP and ClpP and physically interacting with mitochondrial fusion protein OPA1

ABSTRACT Serine protease Omi/HtrA2, a member of the HtrA family, is closely related to the maintenance of mitochondrial integrity and participates in apoptosis but its role in cerebral ischemia/reperfusion (I/R) injury and cellular oxidative stress response remains unclear. In this study, we found that I/R injury resulted in a time-dependent increase in Omi/HtrA2 expression in rat brain tissue. Inhibition of Omi/HtrA2 significantly inhibited XIAP cleavage in H2O2-induced PC12 cells. In addition, inhibition of Omi/HtrA2 significantly inhibited the up-regulation of mitochondrial stress proteins CHOP and ClpP, significantly reduced mitochondrial aggregation, and attenuated the decline of mitochondrial ΔΨm in PC12 cells. Studies show that there is a physical interaction between Omi/HtrA2 and OPA1. We found that Omi/HtrA2 and OPA1 are closely related to the oxidative stress mitochondrial response in PC12 cells. The current study has demonstrated that Omi/HtrA2 is upregulated in brain I/R injury in vivo and is implicated in mitochondrial response to oxidative stress in vitro by regulating mitochondrial stress proteins CHOP and CLpP and by interacting with mitochondrial cristae remodeling protein OPA1. These findings suggest that Omi/HtrA2 could be a candidate molecular target in diseases that involve oxidative stress such as in I/R injury. Abbreviation: ATP: Adenosine tripHospHate; Bax: BCL2-Associated X; Bcl-2: B-cell lympHoma-2; BSA: Albumin from bovine serum; DMEM: Dulbecco’s Minimum Essential Medium; DMSO: Dimethyl sulfoxide; HSP60: Heat shock protein60, 70; L-OPA1: Long forms of OPA1; Omi/HtrA2: high-temperature-regulated A2; MCAO: Middle cerebral artery occlusion; OPA1: Optic AtropHy; PBS: PHospHate buffered saline; PMSF: pHenylmethyl sulfonylfluoride; ROS: reactive oxygen species; SDS: Sodium dodecyl sulfate; S-OPA1: Short forms of OPA1; TTC: TripHenyltetrazalium chloride; XIAP: X-linked inhibitor apoptosis protein

Cellular substrates and pro-apoptotic function of the human HtrA4 protease

The human HtrA4 protein, belonging to the HtrA family of proteases/chaperones, participates in oncogenesis and placentation, and plays a role in preeclampsia. As the knowledge concerning the biochemical features of this protein and its role at the molecular level is limited, in this work we characterized the HtrA4 molecule and searched for its cellular function. We found that recombinant HtrA4 composed of the protease and PDZ domains is a trimeric protein of intermediate thermal stability whose activity is considerably lower compared to other human HtrA proteases. By pull-down combined with mass spectrometry we identified a large array of potential HtrA4 partners. Using other experimental approaches, including immunoprecipitation, enzyme-linked immunosorbent assay and fluorescence microscopy we confirmed that HtrA4 formed complexes in vitro and in cellulo with proteins such as XIAP (inhibitor of apoptosis protein), caspases 7 and 9, β-tubulin, actin, TCP1α and S100A6. The recombinant HtrA4 degraded XIAP, the caspases, β-tubulin and actin but not TCP1α or S100A6. Together, these results suggest that HtrA4 may influence various cellular functions, including apoptosis. Furthermore, the panel of potential HtrA4 partners may serve as a basis for future studies of HtrA4 function.

Assessing the Contribution of an HtrA Family Serine Protease During Borrelia turicatae Mammalian Infection.

Tick-borne relapsing fever (TBRF), characterized by recurring febrile episodes, is globally distributed and among the most common bacterial infections in some African countries. Despite the public health concern that this disease represents, little is known regarding the virulence determinants required by TBRF Borrelia during infection. Because the chromosomes of TBRF Borrelia show extensive colinearity with those of Lyme disease (LD) Borrelia, the exceptions represent unique genes encoding proteins that are potentially essential to the disparate enzootic cycles of these two groups of spirochetes. One such exception is a gene encoding an HtrA family protease, BtpA, that is present in TBRF Borrelia, but not in LD spirochetes. Previous work suggested that btpA orthologs may be important for resistance to stresses faced during mammalian infection. Herein, proteomic analyses of the TBRF spirochete, Borrelia turicatae, demonstrated that BtpA, as well as proteins encoded by adjacent genes in the B. turicatae genome, were produced in response to culture at mammalian body temperature, suggesting a role in mammalian infection. Further, transcriptional analyses revealed that btpA was expressed with the genes immediately upstream and downstream as part of an operon. To directly assess if btpA is involved in resistance to environmental stresses, btpA deletion mutants were generated. btpA mutants demonstrated no growth defect in response to heat shock, but were more sensitive to oxidative stress produced by t-butyl peroxide compared to wild-type B. turicatae. Finally, btpA mutants were fully infectious in a murine relapsing fever (RF) infection model. These results indicate that BtpA is either not required for mammalian infection, or that compensatory mechanisms exist in TBRF spirochetes to combat environmental stresses encountered during mammalian infection in the absence of BtpA.

Antagonism between HTRA3 and TGFβ1 Contributes to Metastasis in Non-Small Cell Lung Cancer.

High temperature requirement A3 (HTRA3, long and short isoforms) is a member of the HtrA family and has been implicated as a tumor suppressor in cancer progression in multiple cancer types, yet its molecular functions in non-small cell lung cancer (NSCLC) are not well understood. Here, we report that decreased levels of HTRA3 negatively correlate with elevated TGFβ1 in lung tumor tissue with metastasis. Furthermore, high expression of HTRA3 indicated better prognosis independent of TGFβ1 expression. In NSCLC cell lines, exogenous TGFβ1 significantly downregulated the level of HTRA3, especially the long isoform, during induction of epithelial-mesenchymal transition (EMT). Mechanistically, c-Jun, which is elevated by TGFβ1, directly bound the promoter of HTRA3-L and inhibited its transcription. As a negative feedback loop, overexpression of HTRA3-L attenuated TGFβ1-mediated invasion-metastasis cascades via activation of SMAD2/3 and sensitized cells to anti-PD-L1 treatment. Taken together, our findings suggest that in the early stages of cancer, overexpressed HTRA3 acts as a brake on the oncogenic effects of TGFβ1 and inhibits tumor metastasis. In later stages, the role of HTRA3 is weakened and TGFβ1 efficiently promotes EMT in the absence of the HTRA3 brake. SIGNIFICANCE: This study provides new mechanistic insight of the interaction between HTRA3 and TGFβ in lung cancer by illustrating that HTRA3 is a novel mediator acting as a suppressor of TGFβ1-related oncogenic effects.