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1
NIE, Gui-Ying, Anne HAMPTON, Ying LI, Jock K. FINDLAY, and Lois A. SALAMONSEN. "Identification and cloning of two isoforms of human high-temperature requirement factor A3 (HtrA3), characterization of its genomic structure and comparison of its tissue distribution with HtrA1 and HtrA2." Biochemical Journal 371, no. 1 (April 1, 2003): 39–48. http://dx.doi.org/10.1042/bj20021569.
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In the present study, we identified an additional member of the human high-temperature requirement factor A (HtrA) protein family, called pregnancy-related serine protease or HtrA3, which was most highly expressed in the heart and placenta. We cloned the full-length sequences of two forms (long and short) of human HtrA3 mRNA, located the gene on chromosome 4p16.1, determined its genomic structure and revealed how the two mRNA variants are produced through alternative splicing. The alternative splicing was also verified by Northern blotting. Four distinct domains were found for the long form HtrA3 protein: (i) an insulin/insulin-like growth factor binding domain, (ii) a Kazal-type S protease-inhibitor domain, (iii) a trypsin protease domain and (iv) a PDZ domain. The short form is identical to the long form except it lacks the PDZ domain. Comparison of all members of human HtrA proteins, including their isoforms, suggests that both isoforms of HtrA3 represent active serine proteases, that they may have different substrate specificities and that HtrA3 may have similar functions to HtrA1. All three HtrA family members showed very different mRNA-expression patterns in 76 human tissues, indicating a specific function for each. Interestingly, both HtrA1 and HtrA3 are highly expressed in the placenta. Identification of the tissue-specific function of each HtrA family member is clearly of importance.
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Bowden, M. A., L. A. Di Nezza, T. Jobling, L. A. Salamonsen, and G. Nie. "284.Expression of HtrA1, 2 and 3 in human endometrial cancer." Reproduction, Fertility and Development 16, no. 9 (2004): 284. http://dx.doi.org/10.1071/srb04abs284.
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The mammalian HtrA family consists of serine proteases with distinct domains homologous to the bacterial high temperature requirement factor (HtrA). Three human HtrA members have been reported: HtrA1 (PRSS11 or L56), HtrA2 (OMI) and HtrA3 (PRSP). The function of HtrA1 is not well characterised, but it has been shown to be downregulated in malignant tissues (1–3) indicating that the downregulation of HtrA1 is associated with cancer progression. HtrA2 regulates apoptosis by interacting with X-linked inhibitors of apoptosis (XIAP) thus preventing the caspase-inhibitory function of XIAP (4). The function of newly identified HtrA3 is not known, however it shares a high degree of sequence and domain homologies with HtrA1 and may therefore share a functional similarity with HtrA1 (5). Endometrial cancer (EC) is a prevalent gynaecological cancer, commonly affecting women after menopause. In this study we examined the expression of HtrA1, 2 and 3 in EC. Reverse transcriptase-PCR (semi-quantitative) analysis showed decreased mRNA expression of both HtrA1 and HtrA3, but no significant change for HtrA2, in EC tissue samples compared to normal endometrium. We then determined the protein level of expression and the cellular localisation of all three HtrA members in EC progression using immunohistochemistry. HtrA1 and HtrA3 showed a similar pattern of expression and both decreased dramatically with the progression of cancer from grade 1 through to 3. Surprisingly, HtrA2 protein expression was also decreased with cancer progression, but the decline was not as dramatic as that for HtrA1 and HtrA3. Interestingly, considerably less staining was observed for all three HtrA proteins in grade 3 cancer tissues. These data suggest that decreased expression of HtrA proteins, particularly HtrA1 and HtrA3, is associated with the progression of endometrial cancer. (1) Nie, G., Hampton, A., Li, Y., Findlay, J., Salamonsen, L.A. (2003) Identification and cloning of two isoforms of human high-temperature requirement factor A3 (HtrA3), characterization of its genomic structure and comparison of its tissue distribution with HtrA1 and HtrA2. Biochem. J. 371, 39–48. (2) van Loo, G., van Gurp, M., Depuydt, B., Srinivasula, S.M., Rodriguez, I., Alnemri, E.S., Gevaert, K., Vandekerckhove, J., Declercq, W., Vandenabeele, P. (2002) The serine protease OMI/HtrA2 is released from mitochondria during apoptosis. OMI interacts with caspase-inhibitor XIAP and induces enhanced caspase activity. Cell Death Diff. 9, 20–26. (3) Chien, J., Staub, J., Hu, S., Erickson-Johnson, M.R., Couch, F.J., Smith, D.I., Crowl, R.M., Kaufmann, S., Shridhar, V. (2004) A candidate tumour supressor HtrA1 is down-regulated in ovarian cancer. Oncogene 23, 1636–1644. (4) Shridhar, V., Sen, A., Chien, J., Staub, J., Avula, R., Kovats, S., Lee, J., Lillie, J., Smith, D.I. (2002) Identification of underexpressed genes in early- and late-stage primary ovarian tumours by suppression subtraction hybridization. Cancer Res. 62, 262–270. (5) Baldi, A., De Luca, A., Morini, M., Battista, T., Felsani, A., Baldi, F., Catricala, C., Amantea, A., Noonan, D. M., Albini, A., Ciorgio, P., Lombardi, D., Paggi, M. G. (2002) The HtrA1 serine protease is down-regulated during human melanoma progression and represses growth of metastatic melanoma cells. Oncogene 21, 6684–6688.
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Singh, Khundrakpam Herojit, Savita Yadav, Deepak Kumar, and Bichitra Kumar Biswal. "The crystal structure of an essential high-temperature requirement protein HtrA1 (Rv1223) from Mycobacterium tuberculosis reveals its unique features." Acta Crystallographica Section D Structural Biology 74, no. 9 (September 1, 2018): 906–21. http://dx.doi.org/10.1107/s205979831800952x.
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High-temperature requirement A (HtrA) proteins, which are members of the heat-shock-induced serine protease family, are involved in extracytoplasmic protein quality control and bacterial survival strategies under stress conditions, and are associated with the virulence of several pathogens; they are therefore major drug targets. Mycobacterium tuberculosis possesses three putative HtrAs: HtrA1 (Rv1223), HtrA2 (Rv0983) and HtrA3 (Rv0125). Each has a cytoplasmic region, a transmembrane helix and a periplasmic region. Here, the crystal structure of the periplasmic region consisting of a protease domain (PD) and a PDZ domain from an M. tuberculosis HtrA1 mutant (mHtrA1S387A) is reported at 2.7 Å resolution. Although the mHtrA1S387A PD shows structural features similar to those of other HtrAs, its loops, particularly L3 and LA, display different conformations. Loop L3 communicates between the PDs of the trimer and the PDZ domains and undergoes a transition from an active to an inactive conformation, as reported for an equivalent HtrA (DegS). Loop LA, which is responsible for higher oligomer formation owing to its length (50 amino acids) in DegP, is very short in mHtrA1S387A (five amino acids), as in mHtrA2 (also five amino acids), and therefore lacks essential interactions for the formation of higher oligomers. Notably, a well ordered loop known as the insertion clamp in the PDZ domain interacts with the protease domain of the adjacent molecule, which possibly aids in the stabilization of a trimeric functional unit of this enzyme. The three-dimensional structure of mHtrA1S387A presented here will be useful in the design of enzyme-specific antituberculosis inhibitors.
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Bæk, Kristoffer T., Christina S. Vegge, Joanna Skórko-Glonek, and Lone Brøndsted. "Different Contributions of HtrA Protease and Chaperone Activities toCampylobacter jejuniStress Tolerance and Physiology." Applied and Environmental Microbiology 77, no. 1 (November 12, 2010): 57–66. http://dx.doi.org/10.1128/aem.01603-10.
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ABSTRACTThe microaerophilic bacteriumCampylobacter jejuniis the most common cause of bacterial food-borne infections in the developed world. Tolerance to environmental stress relies on proteases and chaperones in the cell envelope, such as HtrA and SurA. HtrA displays both chaperone and protease activities, but little is known about how each of these activities contributes to stress tolerance in bacteria.In vitroexperiments showed temperature-dependent protease and chaperone activities ofC. jejuniHtrA. AC. jejunimutant lacking only the protease activity of HtrA was used to show that the HtrA chaperone activity is sufficient for growth at high temperature or under oxidative stress, whereas the HtrA protease activity is essential only under conditions close to the growth limit forC. jejuni. However, the protease activity was required to prevent induction of the cytoplasmic heat shock response even under optimal growth conditions. Interestingly, the requirement of HtrA at high temperatures was found to depend on the oxygen level, and our data suggest that HtrA may protect oxidatively damaged proteins. Finally, protease activity stimulates HtrA production and oligomer formation, suggesting that a regulatory role depends on the protease activity of HtrA. Studying a microaerophilic organism encoding only two known periplasmic chaperones (HtrA and SurA) revealed an efficient HtrA chaperone activity and proposed multiple roles of the protease activity, increasing our understanding of HtrA in bacterial physiology.
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Bernegger, Sabine, Evelyn Hutterer, Urszula Zarzecka, Thomas P. Schmidt, Markus Huemer, Isabella Widlroither, Gernot Posselt, Joanna Skorko-Glonek, and Silja Wessler. "E-Cadherin Orthologues as Substrates for the Serine Protease High Temperature Requirement A (HtrA)." Biomolecules 12, no. 3 (February 24, 2022): 356. http://dx.doi.org/10.3390/biom12030356.
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Helicobacter pylori (H. pylori) expresses the serine protease and chaperone High temperature requirement A (HtrA) that is involved in periplasmic unfolded protein stress response. Additionally, H. pylori-secreted HtrA directly cleaves the human cell adhesion molecule E-cadherin leading to a local disruption of intercellular adhesions during pathogenesis. HtrA-mediated E-cadherin cleavage has been observed in response to a broad range of pathogens, implying that it is a prevalent mechanism in humans. However, less is known whether E-cadherin orthologues serve as substrates for bacterial HtrA. Here, we compared HtrA-mediated cleavage of human E-cadherin with murine, canine, and simian E-cadherin in vitro and during bacterial infection. We found that HtrA targeted mouse and dog E-cadherin equally well, whereas macaque E-cadherin was less fragmented in vitro. We stably re-expressed orthologous E-cadherin (Cdh1) in a CRISPR/Cas9-mediated cdh1 knockout cell line to investigate E-cadherin shedding upon infection using H. pylori wildtype, an isogenic htrA deletion mutant, or complemented mutants as bacterial paradigms. In Western blot analyses and super-resolution microscopy, we demonstrated that H. pylori efficiently cleaved E-cadherin orthologues in an HtrA-dependent manner. These data extend previous knowledge to HtrA-mediated E-cadherin release in mammals, which may shed new light on bacterial infections in non-human organisms.
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Padmanabhan, Nirmala, Lars Fichtner, Achim Dickmanns, Ralf Ficner, Jörg B. Schulz, and Gerhard H. Braus. "The Yeast HtrA Orthologue Ynm3 Is a Protease with Chaperone Activity that Aids Survival Under Heat Stress." Molecular Biology of the Cell 20, no. 1 (January 2009): 68–77. http://dx.doi.org/10.1091/mbc.e08-02-0178.
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Ynm3 is the only budding yeast protein possessing a combination of serine protease and postsynaptic density 95/disc-large/zona occludens domains, a defining feature of the high temperature requirement A (HtrA) protein family. The bacterial HtrA/DegP is involved in protective stress response to aid survival at higher temperatures. The role of mammalian mitochondrial HtrA2/Omi in protein quality control is unclear, although loss of its protease activity results in susceptibility toward Parkinson's disease, in which mitochondrial dysfunction and impairment of protein folding and degradation are key pathogenetic features. We studied the role of the budding yeast HtrA, Ynm3, with respect to unfolding stresses. Similar to Escherichia coli DegP, we find that Ynm3 is a dual chaperone-protease. Its proteolytic activity is crucial for cell survival at higher temperature. Ynm3 also exhibits strong general chaperone activity, a novel finding for a eukaryotic HtrA member. We propose that the chaperone activity of Ynm3 may be important to improve the efficiency of proteolysis of aberrant proteins by averting the formation of nonproductive toxic aggregates and presenting them in a soluble state to its protease domain. Suppression studies with Δynm3 led to the discovery of chaperone activity in a nucleolar peptidyl-prolyl cis-trans isomerase, Fpr3, which could partly relieve the heat sensitivity of Δynm3.
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Gupta, Arvind Kumar, Debashree Behera, and Balasubramanian Gopal. "The crystal structure of Mycobacterium tuberculosis high-temperature requirement A protein reveals an autoregulatory mechanism." Acta Crystallographica Section F Structural Biology Communications 74, no. 12 (November 29, 2018): 803–9. http://dx.doi.org/10.1107/s2053230x18016217.
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The crystal structure of Mycobacterium tuberculosis high-temperature requirement A (HtrA) protein was determined at 1.83 Å resolution. This membrane-associated protease is essential for the survival of M. tuberculosis. The crystal structure reveals that interactions between the PDZ domain and the catalytic domain in HtrA lead to an inactive conformation. This finding is consistent with its proposed role as a regulatory protease that is conditionally activated upon appropriate environmental triggers. The structure provides a basis for directed studies to evaluate the role of this essential protein and the regulatory pathways that are influenced by this protease.
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Ye, Meiping, Kavita Sharma, Meghna Thakur, Alexis A. Smith, Ozlem Buyuktanir, Xuwu Xiang, Xiuli Yang, et al. "HtrA, a Temperature- and Stationary Phase-Activated Protease Involved in Maturation of a Key Microbial Virulence Determinant, Facilitates Borrelia burgdorferi Infection in Mammalian Hosts." Infection and Immunity 84, no. 8 (June 6, 2016): 2372–81. http://dx.doi.org/10.1128/iai.00360-16.
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High-temperature requirement protease A (HtrA) represents a family of serine proteases that play important roles in microbial biology. Unlike the genomes of most organisms, that ofBorrelia burgdorferinotably encodes a single HtrA gene product, termed BbHtrA. Previous studies identified a few substrates of BbHtrA; however, their physiological relevance could not be ascertained, as targeted deletion of the gene has not been successful. Here we show that BbhtrAtranscripts are induced during spirochete growth either in the stationary phase or at elevated temperature. Successful generation of a BbhtrAdeletion mutant and restoration by genetic complementation suggest a nonessential role for this protease in microbial viability; however, its remarkable growth, morphological, and structural defects during cultivation at 37°C confirm a high-temperature requirement for protease activation and function. The BbhtrA-deficient spirochetes were unable to establish infection of mice, as evidenced by assessment of culture, PCR, and serology. We show that transcript abundance as well as proteolytic processing of a borrelial protein required for cell fission and infectivity, BB0323, is impaired in BbhtrAmutants grown at 37°C, which likely contributed to their inability to survive in a mammalian host. Together, these results demonstrate the physiological relevance of a unique temperature-regulated borrelial protease, BbHtrA, which further enlightens our knowledge of intriguing aspects of spirochete biology and infectivity.
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Kummari, Raghupathi, Shubhankar Dutta, Lalith K. Chaganti, and Kakoli Bose. "Discerning the mechanism of action of HtrA4: a serine protease implicated in the cell death pathway." Biochemical Journal 476, no. 10 (May 21, 2019): 1445–63. http://dx.doi.org/10.1042/bcj20190224.
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Abstract High-temperature requirement protease A4 (HtrA4) is a secretary serine protease whose expression is up-regulated in pre-eclampsia (PE) and hence is a possible biomarker of PE. It has also been altered in cancers such as glioblastoma, breast carcinoma, and prostate cancer making it an emerging therapeutic target. Among the human HtrAs, HtrA4 is the least characterized protease pertaining to both structure and its functions. Although the members of human HtrA family share a significant structural and functional conservation, subtle structural changes have been associated with certain distinct functional requirements. Therefore, intricate dissection of HtrA4 structural and functional properties becomes imperative to understand its role in various biological and pathophysiological processes. Here, using inter-disciplinary approaches including in silico, biochemical and biophysical studies, we have done a domain-wise dissection of HtrA4 to delineate the roles of the domains in regulating oligomerization, stability, protease activity, and specificity. Our findings distinctly demonstrate the importance of the N-terminal region in oligomerization, stability and hence the formation of a functional enzyme. In silico structural comparison of HtrA4 with other human HtrAs, enzymology studies and cleavage assays with X-linked inhibitor of apoptosis protein (XIAP) show overall structural conservation and allosteric mode of protease activation, which suggest functional redundancy within this protease family. However, significantly lower protease activity as compared with HtrA2 indicates an additional mode of regulation of the protease activity in the cellular milieu. Overall, these studies provide first-hand information on HtrA4 and its interaction with antiapoptotic XIAP thus implicating its involvement in the apoptotic pathway.
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Wang, Yao, and Guiying Nie. "Overview of Human HtrA Family Proteases and Their Distinctive Physiological Roles and Unique Involvement in Diseases, Especially Cancer and Pregnancy Complications." International Journal of Molecular Sciences 22, no. 19 (October 6, 2021): 10756. http://dx.doi.org/10.3390/ijms221910756.
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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.
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Zhang, Luhua, Ying Li, Yiping Wen, Gee W. Lau, Xiaobo Huang, Rui Wu, Qigui Yan, et al. "HtrA Is Important for Stress Resistance and Virulence in Haemophilus parasuis." Infection and Immunity 84, no. 8 (May 23, 2016): 2209–19. http://dx.doi.org/10.1128/iai.00147-16.
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Haemophilus parasuisis an opportunistic pathogen that causes Glässer's disease in swine, with polyserositis, meningitis, and arthritis. The high-temperature requirement A (HtrA)-like protease, which is involved in protein quality control, has been reported to be a virulence factor in many pathogens. In this study, we showed that HtrA ofH. parasuis(HpHtrA) exhibited both chaperone and protease activities. Finally, nickel import ATP-binding protein (NikE), periplasmic dipeptide transport protein (DppA), and outer membrane protein A (OmpA) were identified as proteolytic substrates for HpHtrA. The protease activity reached its maximum at 40°C in a time-dependent manner. Disruption of thehtrAgene from strain SC1401 affected tolerance to temperature stress and resistance to complement-mediated killing. Furthermore, increased autoagglutination and biofilm formation were detected in thehtrAmutant. In addition, thehtrAmutant was significantly attenuated in virulence in the murine model of infection. Together, these data demonstrate that HpHtrA plays an important role in the virulence ofH. parasuis.
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Bakker, Dennis, Anthony M. Buckley, Anne de Jong, Vincent J. C. van Winden, Joost P. A. Verhoeks, Oscar P. Kuipers, Gillian R. Douce, Ed J. Kuijper, Wiep Klaas Smits, and Jeroen Corver. "The HtrA-Like Protease CD3284 Modulates Virulence of Clostridium difficile." Infection and Immunity 82, no. 10 (July 21, 2014): 4222–32. http://dx.doi.org/10.1128/iai.02336-14.
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ABSTRACTIn the past decade,Clostridium difficilehas emerged as an important gut pathogen. Symptoms ofC. difficileinfection range from mild diarrhea to pseudomembranous colitis. Besides the two main virulence factors toxin A and toxin B, other virulence factors are likely to play a role in the pathogenesis of the disease. In other Gram-positive and Gram-negative pathogenic bacteria, conserved high-temperature requirement A (HtrA)-like proteases have been shown to have a role in protein homeostasis and quality control. This affects the functionality of virulence factors and the resistance of bacteria to (host-induced) environmental stresses. We found that theC. difficile630 genome encodes a single HtrA-like protease (CD3284; HtrA) and have analyzed its rolein vivoandin vitrothrough the creation of an isogenic ClosTron-basedhtrAmutant ofC. difficilestrain 630Δerm(wild type). In contrast to the attenuated phenotype seen withhtrAdeletion in other pathogens, this mutant showed enhanced virulence in the Golden Syrian hamster model of acuteC. difficileinfection. Microarray data analysis showed a pleiotropic effect ofhtrAon the transcriptome ofC. difficile, including upregulation of the toxin A gene. In addition,the htrAmutant showed reduced spore formation and adherence to colonic cells. Together, our data show thathtrAcan modulate virulence inC. difficile.
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Zhang, Zhemin, Qi Huang, Xuan Tao, Guobing Song, Peng Zheng, Hongyan Li, Hongzhe Sun, and Wei Xia. "The unique trimeric assembly of the virulence factor HtrA from Helicobacter pylori occurs via N-terminal domain swapping." Journal of Biological Chemistry 294, no. 20 (April 1, 2019): 7990–8000. http://dx.doi.org/10.1074/jbc.ra119.007387.
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Knowledge of the molecular mechanisms of specific bacterial virulence factors can significantly contribute to antibacterial drug discovery. Helicobacter pylori is a Gram-negative microaerophilic bacterium that infects almost half of the world's population, leading to gastric disorders and even gastric cancer. H. pylori expresses a series of virulence factors in the host, among which high-temperature requirement A (HpHtrA) is a newly identified serine protease secreted by H. pylori. HpHtrA cleaves the extracellular domain of the epithelial cell surface adhesion protein E-cadherin and disrupts gastric epithelial cell junctions, allowing H. pylori to access the intercellular space. Here we report the first crystal structure of HpHtrA at 3.0 Å resolution. The structure revealed a new type of HtrA protease trimer stabilized by unique N-terminal domain swapping distinct from other known HtrA homologs. We further observed that truncation of the N terminus completely abrogates HpHtrA trimer formation as well as protease activity. In the presence of unfolded substrate, HpHtrA assembled into cage-like 12-mers or 24-mers. Combining crystallographic, biochemical, and mutagenic data, we propose a mechanistic model of how HpHtrA recognizes and cleaves the well-folded E-cadherin substrate. Our study provides a fundamental basis for the development of anti-H. pylori agents by using a previously uncharacterized HtrA protease as a target.
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Fahrenkrog, Birthe. "Nma111p, the pro-apoptotic HtrA-like nuclear serine protease in Saccharomyces cerevisiae: a short survey." Biochemical Society Transactions 39, no. 5 (September 21, 2011): 1499–501. http://dx.doi.org/10.1042/bst0391499.
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The baker's yeast, Saccharomyces cerevisiae, is also capable of undergoing programmed cell death or apoptosis, for example in response to viral infection as well as during chronological and replicative aging. Intrinsically, programmed cell death in yeast can be induced by, for example, H2O2, acetic acid or the mating-type pheromone. A number of evolutionarily conserved apoptosis-regulatory proteins have been identified in yeast, one of which is the HtrA (high-temperature requirement A)-like serine protease Nma111p (Nma is nuclear mediator of apoptosis). Nma111p is a nuclear serine protease of the HtrA family, which targets Bir1p, the only known inhibitor-of-apoptosis protein in yeast. Nma111p mediates apoptosis in a serine-protease-dependent manner and exhibits its activity exclusively in the nucleus. How the activity of Nma111p is regulated has remained largely elusive, but some evidence points to a control by phosphorylation. Current knowledge of Nma111p's function in apoptosis will be discussed in the present review.
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Huesgen, Pitter F., Helder Miranda, XuanTam Lam, Manuela Perthold, Holger Schuhmann, Iwona Adamska, and Christiane Funk. "Recombinant Deg/HtrA proteases from Synechocystis sp. PCC 6803 differ in substrate specificity, biochemical characteristics and mechanism." Biochemical Journal 435, no. 3 (April 13, 2011): 733–42. http://dx.doi.org/10.1042/bj20102131.
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Cyanobacteria require efficient protein-quality-control mechanisms to survive under dynamic, often stressful, environmental conditions. It was reported that three serine proteases, HtrA (high temperature requirement A), HhoA (HtrA homologue A) and HhoB (HtrA homologue B), are important for survival of Synechocystis sp. PCC 6803 under high light and temperature stresses and might have redundant physiological functions. In the present paper, we show that all three proteases can degrade unfolded model substrates, but differ with respect to cleavage sites, temperature and pH optima. For recombinant HhoA, and to a lesser extent for HtrA, we observed an interesting shift in the pH optimum from slightly acidic to alkaline in the presence of Mg2+ and Ca2+ ions. All three proteases formed different homo-oligomeric complexes with and without substrate, implying mechanistic differences in comparison with each other and with the well-studied Escherichia coli orthologues DegP (degradation of periplasmic proteins P) and DegS. Deletion of the PDZ domain decreased, but did not abolish, the proteolytic activity of all three proteases, and prevented substrate-induced formation of complexes higher than trimers by HtrA and HhoA. In summary, biochemical characterization of HtrA, HhoA and HhoB lays the foundation for a better understanding of their overlapping, but not completely redundant, stress-resistance functions in Synechocystis sp. PCC 6803.
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Zaide, Galia, Uri Elia, Inbar Cohen-Gihon, Ma’ayan Israeli, Shahar Rotem, Ofir Israeli, Sharon Ehrlich, et al. "Comparative Analysis of the Global Transcriptomic Response to Oxidative Stress of Bacillus anthracis htrA-Disrupted and Parental Wild Type Strains." Microorganisms 8, no. 12 (November 30, 2020): 1896. http://dx.doi.org/10.3390/microorganisms8121896.
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We previously demonstrated that the HtrA (High Temperature Requirement A) protease/chaperone active in the quality control of protein synthesis, represents an important virulence determinant of Bacillus anthracis. Virulence attenuation of htrA-disrupted Bacillus anthracis strains was attributed to susceptibility of ΔhtrA strains to stress insults, as evidenced by affected growth under various stress conditions. Here, we report a comparative RNA-seq transcriptomic study generating a database of differentially expressed genes in the B. anthracis htrA-disrupted and wild type parental strains under oxidative stress. The study demonstrates that, apart from protease and chaperone activities, HtrA exerts a regulatory role influencing expression of more than 1000 genes under stress. Functional analysis of groups or individual genes exhibiting strain-specific modulation, evidenced (i) massive downregulation in the ΔhtrA and upregulation in the WT strains of various transcriptional regulators, (ii) downregulation of translation processes in the WT strain, and (iii) downregulation of metal ion binding functions and upregulation of sporulation-associated functions in the ΔhtrA strain. These modulated functions are extensively discussed. Fifteen genes uniquely upregulated in the wild type strain were further interrogated for their modulation in response to other stress regimens. Overexpression of one of these genes, encoding for MazG (a nucleoside triphosphate pyrophosphohydrolase involved in various stress responses in other bacteria), in the ΔhtrA strain resulted in partial alleviation of the H2O2-sensitive phenotype.
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Sharafutdinov, Irshad, Nicole Tegtmeyer, Mathias Müsken, and Steffen Backert. "Campylobacter jejuni Serine Protease HtrA Induces Paracellular Transmigration of Microbiota across Polarized Intestinal Epithelial Cells." Biomolecules 12, no. 4 (March 30, 2022): 521. http://dx.doi.org/10.3390/biom12040521.
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Campylobacter jejuni represents an eminent zoonotic germ responsible for foodborne infections causing campylobacteriosis. In addition, infections with C. jejuni constitute a risk factor for the occurrence of inflammatory bowel disease (IBD). In the latter case, patients show inflammatory reactions not only against C. jejuni, but also against the non-infectious microbiota. However, the involved mechanisms and molecular basis are still largely unclear. We recently reported that C. jejuni breaches the intestinal epithelial barrier by secretion of serine protease HtrA (high temperature requirement A), which cleaves several major tight and adherens junction proteins. In the present study, we aimed to study if HtrA-expressing C. jejuni may also trigger the transepithelial migration of non-pathogenic gastrointestinal microbiota. Using confocal immunofluorescence and scanning electron microscopy, we demonstrate that C. jejuni wild-type (wt) as well as the isogenic ∆htrA mutant bind to the surface of polarized intestinal Caco-2 epithelial cells, but do not invade them at the apical side. Instead, C. jejuni wt, but not ∆htrA mutant, disrupt the cellular junctions and transmigrate using the paracellular route between neighboring cells. Using transwell filter systems, we then co-incubated the cells with C. jejuni and non-invasive microbiota strains, either Escherichia coli or Lactococcus lactis. Interestingly, C. jejuni wt, but not ∆htrA mutant, induced the efficient transmigration of these microbiota bacteria into the basal compartment. Thus, infection of the intestinal epithelium with C. jejuni causes local opening of cellular junctions and paracellular translocation in an HtrA-dependent manner, which paves the way for transmigration of microbiota that is otherwise non-invasive. Taken together, these findings may have impacts on various Campylobacter-associated diseases such as IBD, which are discussed here.
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Renke, Joanna, Eliza Wasilewska, Sabina Kędzierska-Mieszkowska, Katarzyna Zorena, Sylwia Barańska, Tomasz Wenta, Anna Liberek, et al. "Tumor Suppressors—HTRA Proteases and Interleukin-12—in Pediatric Asthma and Allergic Rhinitis Patients." Medicina 56, no. 6 (June 17, 2020): 298. http://dx.doi.org/10.3390/medicina56060298.
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Background and objective: Allergy belongs to a group of mast cell-related disorders and is one of the most common diseases of childhood. It was shown that asthma and allergic rhinitis diminish the risk of various cancers, including colon cancer and acute lymphoblastic leukemia. On the other hand, asthma augments the risk of lung cancer and an increased risk of breast cancer in patients with allergy has been observed. Thus, the relation between allergy and cancer is not straightforward and furthermore, its biological mechanism is unknown. The HTRA (high temperature requirement A) proteases promote apoptosis, may function as tumor suppressors and HTRA1 is known to be released by mast cells. Interleukin-12 (Il-12) is an important cytokine that induces antitumor immune responses and is produced mainly by dendritic cells that co-localize with mast cells in superficial organs. Material and methods: In the present study we have assessed with ELISA plasma levels of the HTRA proteins, Il-12, and of the anti-HTRA autoantibodies in children with allergy (40) and in age matched controls (39). Children are a special population, since they usually do not have comorbidities and take not many drugs the processes we want to observe are not influenced by many other factors. Results: We have found a significant increase of HTRA1, 2 and 3, and of the Il-12 levels in the children with atopy (asthma and allergic rhinitis) compared to controls. Conclusion: Our results suggest that the HTRA1–3 and Il-12 levels might be useful in analyzing the pro- and antioncogenic potential in young atopic patients.
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Wahlberg, Patrik, Åsa Nylander, Nina Ahlskog, Kui Liu, and Tor Ny. "Expression and Localization of the Serine Proteases High-Temperature Requirement Factor A1, Serine Protease 23, and Serine Protease 35 in the Mouse Ovary." Endocrinology 149, no. 10 (June 19, 2008): 5070–77. http://dx.doi.org/10.1210/en.2007-1736.
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Proteolytic degradation of extracellular matrix components has been suggested to play an essential role in the occurrence of ovulation. Recent studies in our laboratory have indicated that the plasminogen activator and matrix metalloproteinase systems, which were previously believed to be crucial for ovulation, are not required in this process. In this study we have used a microarray approach to identify new proteases that are involved in ovulation. We found three serine proteases that were relatively highly expressed during ovulation: high-temperature requirement factor A1 (HtrA1), which was not regulated much during ovulation; serine protease 23 (PRSS23), which was down-regulated by gonadotropins; and serine protease 35 (PRSS35), which was up-regulated by gonadotropins. We have further investigated the expression patterns of these proteases during gonadotropin-induced ovulation in immature mice and in the corpus luteum (CL) of pseudopregnant mice. We found that HtrA1 was highly expressed in granulosa cells throughout follicular development and ovulation, as well as in the forming and regressing CL. PRSS23 was highly expressed in atretic follicles, and it was expressed in the ovarian stroma and theca tissues just before ovulation. PRSS35 was expressed in the theca layers of developing follicles. It was also highly induced in granulosa cells of preovulatory follicles. PRSS35 was also expressed in the forming and regressing CL. These data suggest that HtrA1 and PRSS35 may be involved in ovulation and CL formation and regression, and that PRSS23 may play a role in follicular atresia.
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Sun, Hongyu, Luyan Shen, Ping Zhang, Fu Lin, Jiaoyan Ma, Ying Wu, Huimei Yu, and Liankun Sun. "Inhibition of High-Temperature Requirement Protein A2 Protease Activity Represses Myogenic Differentiation via UPRmt." International Journal of Molecular Sciences 23, no. 19 (October 4, 2022): 11761. http://dx.doi.org/10.3390/ijms231911761.
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Skeletal muscles require muscle satellite cell (MuSC) differentiation to facilitate the replenishment and repair of muscle fibers. A key step in this process is called myogenic differentiation. The differentiation ability of MuSCs decreases with age and can result in sarcopenia. Although mitochondria have been reported to be involved in myogenic differentiation by promoting a bioenergetic remodeling, little is known about the interplay of mitochondrial proteostasis and myogenic differentiation. High-temperature-requirement protein A2 (HtrA2/Omi) is a protease that regulates proteostasis in the mitochondrial intermembrane space (IMS). Mice deficient in HtrA2 protease activity show a distinct phenotype of sarcopenia. To investigate the role of IMS proteostasis during myogenic differentiation, we treated C2C12 myoblasts with UCF101, a specific inhibitor of HtrA2 during differentiation process. A key step in this process is called myogenic differentiation. The differentiation ability of MuSCs decreases with age and can result in sarcopenia. Further, CHOP, p-eIF2α, and other mitochondrial unfolded protein response (UPRmt)-related proteins are upregulated. Therefore, we suggest that imbalance of mitochondrial IMS proteostasis acts via a retrograde signaling pathway to inhibit myogenic differentiation via the UPRmt pathway. These novel mechanistic insights may have implications for the development of new strategies for the treatment of sarcopenia.
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Tennstaedt, Annette, Simon Pöpsel, Linda Truebestein, Patrick Hauske, Anke Brockmann, Nina Schmidt, Inga Irle, et al. "Human High Temperature Requirement Serine Protease A1 (HTRA1) Degrades Tau Protein Aggregates." Journal of Biological Chemistry 287, no. 25 (April 25, 2012): 20931–41. http://dx.doi.org/10.1074/jbc.m111.316232.
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Campbell, Robert A., Mark Cody, Yasuhiro Kosaka, Heather D. Campbell, and Christian Yost. "Placental HTRA1 Protease Cleaves Alpha-1-Antitrypsin and Generates Neonatal NET-Inhibitory Factor." Blood 132, Supplement 1 (November 29, 2018): 273. http://dx.doi.org/10.1182/blood-2018-99-111195.
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Abstract BACKGROUND: Neutrophil extracellular traps (NET) are extracellular lattices of decondensed chromatin associated with anti-microbial proteins and degradative enzymes released by polymorphonuclear leukocytes (PMN) to trap and kill invading microbes. Dysregulated NET formation, however, contributes to inflammatory tissue damage. We have identified a novel NET-inhibitory peptide, neonatal NET-Inhibitory Factor (nNIF), present in the fetal circulation. nNIF is formed as a carboxy-terminus cleavage fragment of alpha-1 antitrypsin (AAT), an abundant, circulating protease inhibitor with homologs in human and mouse blood. However, the exact mechanisms by which nNIF is generated in fetal and neonatal blood remains unknown. OBJECTIVE: High temperature requirement A 1 (HTRA1) is expressed in the placenta during fetal development and inhibits AAT. We hypothesized that placentally expressed HTRA1, a serine protease, regulates the formation of NET-inhibitory peptides, such as nNIF, through cleavage of AAT. DESIGN/METHODS: Term and preterm placenta were lysed and probed for HTRA1 expression. HTRA1 and AAT plasma expression from term and preterm infants and adults were determined by ELISA. Recombinant, bioactive HTRA1 or placenta-eluted HTRA1 were incubated with AAT and the generation of carboxy-terminus fragments of AAT was assessed using western blotting and mass spectrometry. Fragments of AAT generated by HTRA1 were incubated with LPS-stimulated PMNs and NET formation was examined qualitatively using live cell imaging and quantitatively using a high throughput fluorescence assay. The effect of the HTRA-AAT cleavage fragment on reactive oxygen species generation, neutrophil chemotaxis, phagocytosis, and bacterial killing was measured using flow cytometry, a modified Boyden chamber asssay, neutrophil labeled Escherichia coli uptake assay, and a bacterial killing assay with a pathogenic strain of Escherichia coli, respectively. Finally, NET formation was evaluated qualitatively and quantitatively in murine PMNs isolated from neonatal WT and HTRA1-/- pups between 1-3, 4-6 and 7-10 days after birth to determine when PMNs become NET-competent. RESULTS: Term and preterm infant placentas express HTRA1, and we detected significantly (p<0.05) higher levels of HTRA1 in plasma from term (465.1±71.8 µg/mL) and preterm (385.9±71.3 µg/mL) infant cord blood compared to adults (58.6±11.6 µg/mL). Recombinant, bioactive HTRA1 and placenta-derived HTRA1 incubated with AAT generate a 4kD AAT fragment based on western blot and mass spectrometry similar to the nNIF fragment found in cord blood from term and preterm infants. Pre-incubation of this fragment with LPS-stimulated PMNs significantly inhibits NET formation (p<0.05). The cleavage fragment from HTRA1-AAT, however, has no effect on reactive oxygen species generation, chemotaxis, or phagocytosis. However, incubation of this fragment with LPS-stimulated PMNs significantly (p<0.05) reduces NET-associated bacterial killing by 62% compared to a scrambled HTRA-AAT control peptide. In addition, the HTRA1-AAT fragment significantly (p<0.05) reduces nuclear decondensation by 93% compared to LPS-stimulated PMN, suggesting this fragment inhibits PAD4 activation similar to other NIFs previously examined. Neonatal murine plasma contains a 4kD AAT fragment which inhibits NET formation by adult mouse PMNs, indicating that nNIF generation is conserved in mice. Neonatal PMNs stimulated with LPS exhibit delayed NET formation following birth with PMNs becoming NET-competent by day 8 of life. However, neonatal PMNs from pups born from HTRA1-/- deficient mice generate significantly (p<0.05) more NETs between day 4-6 of life compared to WT controls, suggesting that HTRA1 regulates NET formation through nNIF production. CONCLUSIONS: Placental HTRA1 interacts with AAT to generate a carboxy-terminus cleavage fragment of AAT with identical NET-inhibitory properties to nNIF. Our data strongly indicate that placental HTRA1 generates nNIF in the fetal circulation. We speculate that nNIF participates in the required tolerance to new microbial antigens encountered during the transition to extrauterine life. Disclosures No relevant conflicts of interest to declare.
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Ciferri, Claudio, Michael T. Lipari, Wei-Ching Liang, Alberto Estevez, Julie Hang, Scott Stawicki, Yan Wu, et al. "The trimeric serine protease HtrA1 forms a cage-like inhibition complex with an anti-HtrA1 antibody." Biochemical Journal 472, no. 2 (November 13, 2015): 169–81. http://dx.doi.org/10.1042/bj20150601.
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The high temperature requirement A1 (HtrA1) protease is implicated in many pathological processes, including the age-related macular degeneration (AMD). We identified a blocking antibody binding to the HTRA1 complex trimer and were able to elucidate an unusual inhibitory mechanism by structural and biochemical experiments.
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Zhang, Dingwa, Deyong He, Xiaoliang Pan, Yaping Xu, and Lijun Liu. "Molecular design of orthogonal stacking system at the complex interface of HtrA PDZ domain with its peptide ligands." Journal of the Serbian Chemical Society 84, no. 12 (2019): 1367–79. http://dx.doi.org/10.2298/jsc181221029z.
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The high temperature requirement A (HtrA) protease plays a crucial role in protein quality control and cell fate. The enzyme contains a catalytic protease domain and a regulatory PDZ domain; the latter determines the substrate specificity of the former by specifically binding to the C-terminal hydrophobic stretch of its partner proteins. Previously, a pentapeptide ligand H3C1 was identified as the potential binder of HtrA PDZ domain using phage display technique. Here, an orthogonal ??cation?? stacking system at the crystal domain?peptide complex interface was analysed by integrating theoretical calculations and experimental assays. It was demonstrated that there is a strong (positive) synergistic effect between the two wings of the stacking system; breaking of cation?? interaction in one wing can largely impair the interaction strength of another wing. The ?-electron contributes primarily to the synergistic effect, although geometric property is also (marginally) responsible for it. Next, the systematic combinations between the four aromatic amino acids (Phe, Tyr, Trp and His) plus one non-aromatic amino acid (Ala) at the two wings of ??cation?? stacking were investigated. It was found that two aromatic substitutions (Phe-4Tyr and Phe-4Trp) at a wing can considerably and moderately improve peptide affinity by 3.2- and 1.5-fold, respectively, whereas the non-aromatic mutations at each wing and at both of them can significantly reduce the affinity with Kd increase from 1.8 (wild type) to 34 ?M and 160 ?M (single-point mutations), as well as 210 ?M (double-point mutation), suggesting that just breaking of one wing can substantially undermine the synergism of orthogonal ??cation?? stacking.
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Dynon, K., and G. Nie. "309. DEVELOPMENT AND VALIDATION OF AN ELISA FOR HIGH TEMPERATURE REQUIREMENT FACTOR A3 (HtrA3): EARLY DETECTION OF PREECLAMPSIA." Reproduction, Fertility and Development 22, no. 9 (2010): 109. http://dx.doi.org/10.1071/srb10abs309.
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Preeclampsia (PE) is a multisystemic condition in pregnant women that can be life threatening for both mother and baby. PE is a hypertensive disorder that develops concurrently with proteinuria after 20 weeks of gestation. Abnormal placental development during early pregnancy precedes the onset of PE later in gestation. Early diagnosis of PE is essential to reduce PE-related mortality and morbidity. To date there is no clinically useful biochemical diagnostic method that can detect PE during early pregnancy. Our laboratory discovered and cloned the serine protease HtrA3 and has shown that HtrA3 protein levels are intimately involved in placentation (1). Persistently high serum levels of HtrA3 are detected at the end of the first trimester in pregnant women who subsequently develop PE, suggesting that monitoring HtrA3 in maternal blood during early pregnancy may identify women at risk for PE. To develop monoclonal antibodies specific for HtrA3; and to develop an enzyme-linked immunosorbent assay (ELISA) to detect HtrA3 in human sera. Monoclonal antibodies were generated against full length human HtrA3 and small HtrA3 peptides and tested on recombinant HtrA3, human sera and first trimester decidual and villous tissues using western blot, immunoprecipitation and Amplified luminescent proximity homogeneous assay (Alpha)LISA technology. Three antibody pairs were identified that detected either short and/or long isoforms of HtrA3 in sera and placental tissues. Recombinant HtrA3 was detected by AlphaLISA and higher levels of HtrA3 were detected in serum of PE women compared to gestation-matched controls in preliminary testing. These antibody pairs can now be used for the development of specific and high throughput assays. The AlphaLISA will then be used to validate that abnormal serum HtrA3 levels during early pregnancy can predict preeclampsia. (1) Nie et al, (2006) Biol Reprod 74, 366–374.
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Spiers, Alison, Heather K. Lamb, Simon Cocklin, Kerry A. Wheeler, Jo Budworth, Anna L. Dodds, Mark J. Pallen, Duncan J. Maskell, Ian G. Charles, and Alastair R. Hawkins. "PDZ Domains Facilitate Binding of High Temperature Requirement Protease A (HtrA) and Tail-specific Protease (Tsp) to Heterologous Substrates through Recognition of the Small Stable RNA A (ssrA)-encoded Peptide." Journal of Biological Chemistry 277, no. 42 (August 12, 2002): 39443–49. http://dx.doi.org/10.1074/jbc.m202790200.
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Acharya, Saujanya, Shubhankar Dutta, and Kakoli Bose. "A distinct concerted mechanism of structural dynamism defines activity of human serine protease HtrA3." Biochemical Journal 477, no. 2 (January 30, 2020): 407–29. http://dx.doi.org/10.1042/bcj20190706.
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Human HtrA3 (high-temperature requirement protease A3) is a trimeric multitasking propapoptotic serine protease associated with critical cellular functions and pathogenicity. Implicated in diseases including cancer and pre-eclampsia, its role as a tumor suppressor and potential therapeutic target cannot be ignored. Therefore, elucidating its mode of activation and regulatory switch becomes indispensable towards modulating its functions with desired effects for disease intervention. Using computational, biochemical and biophysical tools, we delineated the role of all domains, their combinations and the critical phenylalanine residues in regulating HtrA3 activity, oligomerization and specificity. Our findings underline the crucial roles of the N-terminus as well as the PDZ domain in oligomerization and formation of a catalytically competent enzyme, thus providing new insights into its structure–function coordination. Our study also reports an intricate ligand-induced allosteric switch, which redefines the existing hypothesis of HtrA3 activation besides opening up avenues for modulating protease activity favorably through suitable effector molecules.
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Hwang, Jimin, Sonya Mros, Allan B. Gamble, Joel D. A. Tyndall, and Arlene McDowell. "Improving Antibacterial Activity of a HtrA Protease Inhibitor JO146 against Helicobacter pylori: A Novel Approach Using Microfluidics-Engineered PLGA Nanoparticles." Pharmaceutics 14, no. 2 (February 1, 2022): 348. http://dx.doi.org/10.3390/pharmaceutics14020348.
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Nanoparticle drug delivery systems have emerged as a promising strategy for overcoming limitations of antimicrobial drugs such as stability, bioavailability, and insufficient exposure to the hard-to-reach bacterial drug targets. Although size is a vital colloidal feature of nanoparticles that governs biological interactions, the absence of well-defined size control technology has hampered the investigation of optimal nanoparticle size for targeting bacterial cells. Previously, we identified a lead antichlamydial compound JO146 against the high temperature requirement A (HtrA) protease, a promising antibacterial target involved in protein quality control and virulence. Here, we reveal that JO146 was active against Helicobacter pylori with a minimum bactericidal concentration of 18.8–75.2 µg/mL. Microfluidic technology using a design of experiments approach was utilized to formulate JO146-loaded poly(lactic-co-glycolic) acid nanoparticles and explore the effect of the nanoparticle size on drug delivery. JO146-loaded nanoparticles of three different sizes (90, 150, and 220 nm) were formulated with uniform particle size distribution and drug encapsulation efficiency of up to 25%. In in vitro microdilution inhibition assays, 90 nm nanoparticles improved the minimum bactericidal concentration of JO146 two-fold against H. pylori compared to the free drug alone, highlighting that controlled engineering of nanoparticle size is important in drug delivery optimization.
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Frochaux, Violette, Diana Hildebrand, Anja Talke, Michael W. Linscheid, and Hartmut Schlüter. "Alpha-1-Antitrypsin: A Novel Human High Temperature Requirement Protease A1 (HTRA1) Substrate in Human Placental Tissue." PLoS ONE 9, no. 10 (October 20, 2014): e109483. http://dx.doi.org/10.1371/journal.pone.0109483.
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Yamauchi, Shota, Yan Yan Hou, Alvin Kunyao Guo, Hiroaki Hirata, Wataru Nakajima, Ai Kia Yip, Cheng-han Yu, et al. "p53-mediated activation of the mitochondrial protease HtrA2/Omi prevents cell invasion." Journal of Cell Biology 204, no. 7 (March 24, 2014): 1191–207. http://dx.doi.org/10.1083/jcb.201309107.
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Oncogenic Ras induces cell transformation and promotes an invasive phenotype. The tumor suppressor p53 has a suppressive role in Ras-driven invasion. However, its mechanism remains poorly understood. Here we show that p53 induces activation of the mitochondrial protease high-temperature requirement A2 (HtrA2; also known as Omi) and prevents Ras-driven invasion by modulating the actin cytoskeleton. Oncogenic Ras increases accumulation of p53 in the cytoplasm, which promotes the translocation of p38 mitogen-activated protein kinase (MAPK) into mitochondria and induces phosphorylation of HtrA2/Omi. Concurrently, oncogenic Ras also induces mitochondrial fragmentation, irrespective of p53 expression, causing the release of HtrA2/Omi from mitochondria into the cytosol. Phosphorylated HtrA2/Omi therefore cleaves β-actin and decreases the amount of filamentous actin (F-actin) in the cytosol. This ultimately down-regulates p130 Crk-associated substrate (p130Cas)-mediated lamellipodia formation, countering the invasive phenotype initiated by oncogenic Ras. Our novel findings provide insights into the mechanism by which p53 prevents the malignant progression of transformed cells.
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M’Angale, P. Githure, and Brian E. Staveley. "The HtrA2 Drosophila model of Parkinson’s disease is suppressed by the pro-survival Bcl-2 Buffy." Genome 60, no. 1 (January 2017): 1–7. http://dx.doi.org/10.1139/gen-2016-0069.
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Mutations in High temperature requirement A2 (HtrA2), also designated PARK13, which lead to the loss of its protease activity, have been associated with Parkinson’s disease (PD). HtrA2 is a mitochondrial protease that translocates to the cytosol upon the initiation of apoptosis where it participates in the abrogation of inhibitors of apoptosis (IAP) inhibition of caspases. Here, we demonstrate that the loss of the HtrA2 function in the dopaminergic neurons of Drosophila melanogaster results in PD-like phenotypes, and we attempt to restore the age-dependent loss in locomotor ability by co-expressing the sole pro-survival Bcl-2 homologue Buffy. The inhibition of HtrA2 in the dopaminergic neurons of Drosophila resulted in shortened lifespan and impaired climbing ability, and the overexpression of Buffy rescued the reduction in lifespan and the age-dependent loss of locomotor ability. In supportive experiments, the inhibition of HtrA2 in the Drosophila eye results in eye defects, marked by reduction in ommatidia number and increased disruption of the ommatidial array; phenotypes that are suppressed by the overexpression of Buffy.
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Vierkotten, Sascha, and Victoria Korinek. "HTRA1 enhance signaling pathway of uveitis via modulation of the TGF-β signaling cascade." American Journal of BioMedicine 4, no. 3 (August 30, 2016): 276–88. http://dx.doi.org/10.18081/2333-5106/016-276-288.
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High temperature requirement protein A1 (HtrA1) is a serine protease that is mostly secreted to degrade numerous extracellular matrix proteins, They are involved in the development and progression of several pathological processes such as cancer, neurodegenerative disorders and arthritic diseases, but it also exists within cells for some partially understood. The purpose of this study was to investigate the role of HTRA1 in the uveitis, and the possible mechanisms involved. Interphotoreceptor retinoid-binding protein peptide R14 to induced uveitis in rat model. A recombinant lentiviral vector carrying HTRA1-shRNA to knockdown HTRA1 expression. ELISA to detected proinflammatory cytokines and standard molecular biological techniques were used for subcloning of HtrA1. The knockdown of HTRA1 was associated with decreased the cellularlevel expression of proinflammatory cytokine resulted in reduced cellular damage, and increased mRNA levels of TGF-β1.
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Canfield, A. E., K. D. Hadfield, C. F. Rock, E. C. Wylie, and F. L. Wilkinson. "HtrA1: a novel regulator of physiological and pathological matrix mineralization?" Biochemical Society Transactions 35, no. 4 (July 20, 2007): 669–71. http://dx.doi.org/10.1042/bst0350669.
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HtrA1 (high-temperature requirement protein A1) is a secreted multidomain protein with proven serine protease activity and the ability to regulate TGF-β (transforming growth factor-β)/BMP (bone morphogenetic protein) signalling. There is increasing evidence that HtrA1 regulates several pathological processes, including tumour development, Alzheimer's disease, age-related macular degeneration and osteoarthritis, although the mechanism(s) by which it regulates these processes have not been fully elucidated. Using overexpression and knock-down strategies, we have evidence demonstrating that HtrA1 is also a key regulator of physiological and pathological matrix mineralization in vitro. We propose that HtrA1 regulates mineralization by inhibiting TGF-β/BMP signalling and/or by cleaving specific matrix proteins, including decorin and MGP (matrix Gla protein). Taken together, these studies suggest that HtrA1 may be a novel therapeutic target for several diseases.
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Tiaden, André N., Marina Klawitter, Vanda Lux, Ali Mirsaidi, Gregor Bahrenberg, Stephan Glanz, Lilian Quero, et al. "Detrimental Role for Human High Temperature Requirement Serine Protease A1 (HTRA1) in the Pathogenesis of Intervertebral Disc (IVD) Degeneration." Journal of Biological Chemistry 287, no. 25 (May 3, 2012): 21335–45. http://dx.doi.org/10.1074/jbc.m112.341032.
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Catalano, V., A. Baldi, V. Shridhar, M. P. Staccioli, J. Chien, P. Giordani, D. Rossi, et al. "HtrA1 expression as a predictive factor of response to cisplatin-based regimen in patients with advanced gastric cancer." Journal of Clinical Oncology 24, no. 18_suppl (June 20, 2006): 4077. http://dx.doi.org/10.1200/jco.2006.24.18_suppl.4077.
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4077 Background: Human HtrA1 is a member of the HtrA (High temperature requirement) family of serine proteases. Recent reports suggest that htrA1 plays a protective role in varous malignancies due to its tumour suppressive properties. This study was performed to estimate HtrA1 expression as a predictor of the response to chemotherapy of patients with gastric cancer. Methods: HtrA1 was measured immunohistochemically on archival specimens of primary gastric cancer from 51 patients treated consecutively at our institution with a weekly chemotherapy including cisplatin 40 mg/m2, epirubicin 35 mg/m2, 6S-leucovorin 100 mg/m2, 5-fluorouracil 500 mg/m2, with the support of filgrastim 5 μg/Kg from the day 2 to 7 (PELF regimen), or cisplatin 40 mg/m2, epirubicin 35 mg/m2, 6S-leucovorin 100 mg/m2, 5-fluorouracil 500 mg/m2 (PLF regimen). Response to chemotherapy was assessed after 8 weekly treatments according to the WHO criteria. Results: our population consisted of M/F 32/19; median age 64 years (range, 46–79). The prevalent metastatic sites were liver (17 pts), peritoneum (13 pts), lymph nodes (21 pts), locoregional disease (16 pts); 31/16/4 pts had 1/2/3 or more sites of disease. 23 pts had a low expression of HtrA1 (0/1+) versus 28 patients with higher expression (2+). Of the total 51 patients, there were 28 responders: 8 showing complete response (CR) and 20, partial response (PR). Of the 28 responders, 20 were in the higher HtrA1 staining group (2+), while of the 23 non-responders, 15 were in the higher HtrA1 staining group (0/1+). A statistically significant correlation between HtrA1 expression (HtrA1 2+ versus HtrA1 0/1+) and the clinical response was observed (response rate in patients with 2+ and 0/1+: 71.4% versus 34.8%, P < 0.01, respectively). Interestingly, among 16 pts with locoregional disease (stomach, gastric bed, anastomosis), 1/6 pts had HtrA1 1+ expression compared to 8/10 pts with HtrA1 2+ (17% versus 80%, respectively; p = 0.025). Conclusions: The immunohistochemical identification of HtrA1 on the primary gastric cancer prior to chemotherapy may be a useful predictor for choice of potentially responders to a cisplatin-based chemotherapy. No significant financial relationships to disclose.
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Kummari, Raghupathi, Shubhankar Dutta, Shubhangi Patil, Snehal Pandav Mudrale, and Kakoli Bose. "Elucidating the role of GRIM-19 as a substrate and allosteric activator of pro-apoptotic serine protease HtrA2." Biochemical Journal 478, no. 6 (March 19, 2021): 1241–59. http://dx.doi.org/10.1042/bcj20200923.
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HtrA2 (high-temperature requirement A2) and GRIM-19 (gene associated with retinoic and interferon-induced mortality 19 protein) are involved in various biological functions with their deregulation leading to multiple diseases. Although it is known that the interaction between GRIM-19 with HtrA2 promotes the pro-apoptotic activity of the latter, the mechanistic details remained elusive till date. Moreover, designing allosteric modulators of HtrA2 remains obscure due to lack of adequate information on the mode of interaction with its natural substrates cum binding partners. Therefore, in this study, we have unfolded the interaction between HtrA2 and GRIM-19 so as to understand its subsequent functional repercussions. Using in silico analyses and biochemical assays, we identified the region in GRIM-19 that is involved in protein–protein interaction with HtrA2. Furthermore, we have presented a comprehensive illustration of HtrA2's cleavage site specificity. Quantitative analysis using enzyme kinetics underscored the role of GRIM-19 in significant allosteric activation of HtrA2. Overall, this is an extensive study that not only defines HtrA2–GRIM-19 interaction, but also creates a framework for developing strategies toward allosteric regulation of HtrA2 for future therapeutic interventions.
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Tom, Irene, Victoria C. Pham, Kenneth J. Katschke, Wei Li, Wei-Ching Liang, Johnny Gutierrez, Andrew Ah Young, et al. "Development of a therapeutic anti-HtrA1 antibody and the identification of DKK3 as a pharmacodynamic biomarker in geographic atrophy." Proceedings of the National Academy of Sciences 117, no. 18 (April 28, 2020): 9952–63. http://dx.doi.org/10.1073/pnas.1917608117.
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Genetic polymorphisms in the region of the trimeric serine hydrolase high-temperature requirement 1 (HTRA1) are associated with increased risk of age-related macular degeneration (AMD) and disease progression, but the precise biological function of HtrA1 in the eye and its contribution to disease etiologies remain undefined. In this study, we have developed an HtrA1-blocking Fab fragment to test the therapeutic hypothesis that HtrA1 protease activity is involved in the progression of AMD. Next, we generated an activity-based small-molecule probe (ABP) to track target engagement in vivo. In addition, we used N-terminomic proteomic profiling in preclinical models to elucidate the in vivo repertoire of HtrA1-specific substrates, and identified substrates that can serve as robust pharmacodynamic biomarkers of HtrA1 activity. One of these HtrA1 substrates, Dickkopf-related protein 3 (DKK3), was successfully used as a biomarker to demonstrate the inhibition of HtrA1 activity in patients with AMD who were treated with the HtrA1-blocking Fab fragment. This pharmacodynamic biomarker provides important information on HtrA1 activity and pharmacological inhibition within the ocular compartment.
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Bowden, Marissa A., Ann E. Drummond, Peter J. Fuller, Lois A. Salamonsen, Jock K. Findlay, and Guiying Nie. "High-temperature requirement factor A3 (Htra3): A novel serine protease and its potential role in ovarian function and ovarian cancers." Molecular and Cellular Endocrinology 327, no. 1-2 (October 7, 2010): 13–18. http://dx.doi.org/10.1016/j.mce.2010.06.001.
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Toyama, Yuki, Robert W. Harkness, Tim Y. T. Lee, Jason T. Maynes, and Lewis E. Kay. "Oligomeric assembly regulating mitochondrial HtrA2 function as examined by methyl-TROSY NMR." Proceedings of the National Academy of Sciences 118, no. 11 (March 10, 2021): e2025022118. http://dx.doi.org/10.1073/pnas.2025022118.
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Human High temperature requirement A2 (HtrA2) is a mitochondrial protease chaperone that plays an important role in cellular proteostasis and in regulating cell-signaling events, with aberrant HtrA2 function leading to neurodegeneration and parkinsonian phenotypes. Structural studies of the enzyme have established a trimeric architecture, comprising three identical protomers in which the active sites of each protease domain are sequestered to form a catalytically inactive complex. The mechanism by which enzyme function is regulated is not well understood. Using methyl transverse relaxation optimized spectroscopy (TROSY)-based solution NMR in concert with biochemical assays, a functional HtrA2 oligomerization/binding cycle has been established. In the absence of substrates, HtrA2 exchanges between a heretofore unobserved hexameric conformation and the canonical trimeric structure, with the hexamer showing much weaker affinity toward substrates. Both structures are substrate inaccessible, explaining their low basal activity in the absence of the binding of activator peptide. The binding of the activator peptide to each of the protomers of the trimer occurs with positive cooperativity and induces intrasubunit domain reorientations to expose the catalytic center, leading to increased proteolytic activity. Our data paint a picture of HtrA2 as a finely tuned, stress-protective enzyme whose activity can be modulated both by oligomerization and domain reorientation, with basal levels of catalysis kept low to avoid proteolysis of nontarget proteins.
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Campbell, Robert A., Heather D. Campbell, J. Samuel Bircher, Claudia Valeria de Araujo, Frederik Denorme, Jacob L. Crandell, John L. Rustad, et al. "Placental HTRA1 cleaves α1-antitrypsin to generate a NET-inhibitory peptide." Blood 138, no. 11 (June 30, 2021): 977–88. http://dx.doi.org/10.1182/blood.2020009021.
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Abstract Neutrophil extracellular traps (NETs) are important components of innate immunity. Neonatal neutrophils (polymorphonuclear leukocytes [PMNs]) fail to form NETs due to circulating NET-inhibitory peptides (NIPs), cleavage fragments of α1-antitrypsin (A1AT). How fetal and neonatal blood NIPs are generated remains unknown, however. The placenta expresses high-temperature requirement serine protease A1 (HTRA1) during fetal development, which can cleave A1AT. We hypothesized that placentally expressed HTRA1 regulates the formation of NIPs and that NET competency changed in PMNs isolated from neonatal HTRA1 knockout mice (HTRA1−/−). We found that umbilical cord blood plasma has elevated HTRA1 levels compared with adult plasma and that recombinant and placenta-eluted HTRA1 cleaves A1AT to generate an A1AT cleavage fragment (A1ATM383S-CF) of molecular weight similar to previously identified NIPs that block NET formation by adult neutrophils. We showed that neonatal mouse pup plasma contains A1AT fragments that inhibit NET formation by PMNs isolated from adult mice, indicating that NIP generation during gestation is conserved across species. Lipopolysaccharide-stimulated PMNs isolated from HTRA1+/+ littermate control pups exhibit delayed NET formation after birth. However, plasma from HTRA1−/− pups had no detectable NIPs, and PMNs from HTRA1−/− pups became NET competent earlier after birth compared with HTRA1+/+ littermate controls. Finally, in the cecal slurry model of neonatal sepsis, A1ATM383S-CF improved survival in C57BL/6 pups by preventing pathogenic NET formation. Our data indicate that placentally expressed HTRA1 is a serine protease that cleaves A1AT in utero to generate NIPs that regulate NET formation by human and mouse PMNs.
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Li, Dapeng, Jiawei Yue, Lu Jiang, Yonghui Huang, Jifu Sun, and Yan Wu. "Correlation Between Expression of High Temperature Requirement Serine Protease A1 (HtrA1) in Nucleus Pulposus and T2 Value of Magnetic Resonance Imaging." Medical Science Monitor 23 (April 22, 2017): 1940–46. http://dx.doi.org/10.12659/msm.904018.
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Hur, Kang, Kim, Lee, Kim, Nam, Rhim, and Yoon. "Serine Protease HtrA2/Omi Deficiency Impairs Mitochondrial Homeostasis and Promotes Hepatic Fibrogenesis via Activation of Hepatic Stellate Cells." Cells 8, no. 10 (September 20, 2019): 1119. http://dx.doi.org/10.3390/cells8101119.
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The loss of mitochondrial function impairs intracellular energy production and potentially results in chronic liver disease. Increasing evidence suggests that mitochondrial dysfunction in hepatocytes contributes to the activation of hepatic stellate cells (HSCs), thereby resulting in hepatic fibrogenesis. High-temperature requirement protein A2 (HtrA2/Omi), a mitochondrial serine protease with various functions, is responsible for quality control in mitochondrial homeostasis. However, little information is available regarding its role in mitochondrial damage during the development of liver fibrosis. This study examined whether HtrA2/Omi regulates mitochondrial homeostasis in hepatocyte during the development of hepatic fibrogenesis. In this study, we demonstrated that HtrA2/Omi expression considerably decreased in liver tissues from the CCl4-induced liver fibrotic mice model and from patients with liver cirrhosis. Knockdown of HtrA2/Omi in hepatocytes induced the accumulation of damaged mitochondria and provoked mitochondrial reactive oxygen species (mtROS) stress. We further show that the damaged mtDNA isolated from HtrA2/Omi-deficient hepatocytes as a form of damage-associated molecular patterns can induce HSCs activation. Moreover, we found that motor neuron degeneration 2-mutant mice harboring the missense mutation Ser276Cys in the protease domain of HtrA2/Omi displayed altered mitochondrial morphology and function, which increased oxidative stress and promoted liver fibrosis. Conversely, the overexpression of HtrA2/Omi via hydrodynamics-based gene transfer led to the antifibrotic effects in CCl4-induced liver fibrosis mice model through decreasing collagen accumulation and enhancing anti-oxidative activity by modulating mitochondrial homeostasis in the liver. These results suggest that suppressing HtrA2/Omi expression promotes hepatic fibrogenesis via modulating mtROS generation, and these novel mechanistic insights involving the regulation of mitochondrial homeostasis by HtrA2/Omi may be of importance for developing new therapeutic strategies for hepatic fibrosis.
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Tossetta, Giovanni, Sonia Fantone, Rosaria Gesuita, Gian Carlo Di Renzo, Arun Meyyazhagan, Chiara Tersigni, Giovanni Scambia, Nicoletta Di Simone, and Daniela Marzioni. "HtrA1 in Gestational Diabetes Mellitus: A Possible Biomarker?" Diagnostics 12, no. 11 (November 5, 2022): 2705. http://dx.doi.org/10.3390/diagnostics12112705.
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Background: The high-temperature requirement A 1 (HtrA1) is a multidomain secretory protein with serine-protease activity, expressed in many tissues, including placenta, where its expression is higher in the first trimester, suggesting an association of this serine protease in early phases of human placenta development. In this study, we evaluated maternal serum HtrA1 levels in the first and third trimester of gestation. In particular, we evaluated a possible role of HtrA1 as an early marker of gestational diabetes mellitus (GDM) in the first trimester of gestation. Methods: We evaluated HtrA1 serum levels in the third trimester (36–40 weeks) in normal pregnancies (n = 20) and GDM pregnancies (n = 20) by using ELISA analysis. Secondly, we performed the same analysis by using the first trimester sera (10–12 weeks) of healthy pregnant women that will develop a normal pregnancy (n = 210) or GDM (n = 28) during pregnancy. Results: We found that HtrA1 serum levels in the third trimester were higher in pregnancies complicated by GDM. Interestingly, higher HtrA1 serum levels were also found in the first trimester in women developing GDM later during the second–third trimester. No significant differences in terms of maternal age and gestational age were found between cases and controls. Women with GDM shown significantly higher pre-pregnancy BMI values compared to controls. Moreover, the probability of GDM occurrence significantly increased with increasing HtrA1 levels and BMI values. The ROC curve showed a good accuracy in predicting GDM, with an AUC of 0.74 (95%CI: 0.64–0.92). Conclusions: These results suggest an important role of HtrA1 as an early predictive marker of GDM in the first trimester of gestation, showing a significative clinical relevance for prevention of this disease.
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Williams, Brandi L., Nathan A. Seager, Jamie D. Gardiner, Chris M. Pappas, Monica C. Cronin, Cristina Amat di San Filippo, Robert A. Anstadt, et al. "Chromosome 10q26–driven age-related macular degeneration is associated with reduced levels of HTRA1 in human retinal pigment epithelium." Proceedings of the National Academy of Sciences 118, no. 30 (July 22, 2021): e2103617118. http://dx.doi.org/10.1073/pnas.2103617118.
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Genome-wide association studies have identified the chromosome 10q26 (Chr10) locus, which contains the age-related maculopathy susceptibility 2 (ARMS2) and high temperature requirement A serine peptidase 1 (HTRA1) genes, as the strongest genetic risk factor for age-related macular degeneration (AMD) [L.G. Fritsche et al., Annu. Rev. Genomics Hum. Genet. 15, 151–171, (2014)]. To date, it has been difficult to assign causality to any specific single nucleotide polymorphism (SNP), haplotype, or gene within this region because of high linkage disequilibrium among the disease-associated variants [J. Jakobsdottir et al. Am. J. Hum. Genet. 77, 389–407 (2005); A. Rivera et al. Hum. Mol. Genet. 14, 3227–3236 (2005)]. Here, we show that HTRA1 messenger RNA (mRNA) is reduced in retinal pigment epithelium (RPE) but not in neural retina or choroid tissues derived from human donors with homozygous risk at the 10q26 locus. This tissue-specific decrease is mediated by the presence of a noncoding, cis-regulatory element overlapping the ARMS2 intron, which contains a potential Lhx2 transcription factor binding site that is disrupted by risk variant rs36212733. HtrA1 protein increases with age in the RPE–Bruch’s membrane (BM) interface in Chr10 nonrisk donors but fails to increase in donors with homozygous risk at the 10q26 locus. We propose that HtrA1, an extracellular chaperone and serine protease, functions to maintain the optimal integrity of the RPE–BM interface during the aging process and that reduced expression of HTRA1 mRNA and protein in Chr10 risk donors impairs this protective function, leading to increased risk of AMD pathogenesis. HtrA1 augmentation, not inhibition, in high-risk patients should be considered as a potential therapy for AMD.
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Morin, Gilles, Maria Nouri, Oliver Brandau, Nebal Saadi, Mohammed Nouri, Florence van den Broek, Holger Prokisch, Johannes Mayr, Saskia Wortmann, and Reka Kovacs-Nagy. "HTRA2 Defect: A Recognizable Inborn Error of Metabolism with 3-Methylglutaconic Aciduria as Discriminating Feature Characterized by Neonatal Movement Disorder and Epilepsy—Report of 11 Patients." Neuropediatrics 49, no. 06 (August 16, 2018): 373–78. http://dx.doi.org/10.1055/s-0038-1667345.
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AbstractNeonatal-onset movement disorders, especially in combination with seizures, are rare and often related to mitochondrial disorders. 3-methylglutaconic aciduria (3-MGA-uria) is a marker for mitochondrial dysfunction. In particular, consistently elevated urinary excretion of 3-methylglutaconic acid is the hallmark of a small but growing group of inborn errors of metabolism (IEM) due to defective phospholipid remodeling or mitochondrial membrane-associated disorders (mutations in TAZ, SERAC1, OPA3, CLPB, DNAJC19, TMEM70, TIMM50). Exome/genome sequencing is a powerful tool for the diagnosis of the clinically and genetically heterogeneous mitochondrial disorders. Here, we report 11 individuals, of whom 2 are previously unpublished, with biallelic variants in high temperature requirement protein A2 (HTRA2) encoding a mitochondria-localized serine protease. All individuals presented a recognizable phenotype with neonatal- or infantile-onset neurodegeneration and death within the first month of life. Hallmark features were central hypopnea/apnea leading to respiratory insufficiency, seizures, neutropenia, 3-MGA-uria, tonus dysregulation, and dysphagia. Tremor, jitteriness, dystonia, and/or clonus were also common. HTRA2 defect should be grouped under the IEM with 3-MGA-uria as discriminating feature. Clinical characteristics overlap with other disorders of this group suggesting a common underlying pathomechanism. Urinary organic acid analysis is a noninvasive and inexpensive test that can guide further genetic testing in children with suggestive clinical findings.
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Hortmann, Marcus, Samuel Robinson, Moritz Mohr, Maximillian Mauler, Daniela Stallmann, Jochen Reinöhl, Daniel Duerschmied, et al. "The mitochondria-targeting peptide elamipretide diminishes circulating HtrA2 in ST-segment elevation myocardial infarction." European Heart Journal: Acute Cardiovascular Care 8, no. 8 (May 23, 2017): 695–702. http://dx.doi.org/10.1177/2048872617710789.
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Background: The extent of myocardial damage in patients with ST-segment elevation myocardial infarction (STEMI) depends on both the time to reperfusion as well as injury induced by ischaemia–reperfusion resulting in a cascade of cellular and humoral reactions. As a consequence of ischaemia–reperfusion in the heart, the high-temperature requirement serine peptidase 2 (HtrA2) is translocated from the mitochondria to the cytosol, whereupon it induces protease activity-dependent apoptosis mediated via caspases. Myocardial damage induced by reperfusion cannot be monitored due to a current lack in specific biomarkers. We examined the serum level of HtrA2 as a potentially novel biomarker for mitochondrial-induced cardiomyocyte apoptosis. Methods: After informed consent, peripheral blood was obtained from patients ( n=19) with first-time acute anterior STEMI after percutaneous coronary intervention. Within this group, 10 of the patients received the mitochondria-targeting peptide elamipretide (phase 2a clinical study EMBRACE (NCT01572909)). Blood was also obtained from a control group of healthy donors ( n=16). The serum level of HtrA2 was measured by an enzyme-linked immunosorbent assay (ELISA). In a murine model of myocardial ischaemia–reperfusion injury, HtrA2 was determined in plasma by ELISA after left anterior descending artery occlusion. Results: HtrA2 median was significantly increased in patients with STEMI compared to healthy controls 392.4 (240.7–502.8) pg/mL vs. 1805.5 (981.3–2220.1) pg/mL ( P⩽0.05). Elamipretide significantly reduced the HtrA2 median serum level after myocardial infarction 1805.5 (981.3–2220.1) pg/mL vs. 496.5 (379.4–703.8) pg/mL ( P⩽0.05). Left anterior descending artery occlusion in mice significantly increased HtrA2 mean in plasma (117.4 fg/ml±SEM 28.1 vs. 525.2 fg/ml±SEM 96; P⩽0.05). Conclusion: Compared to healthy controls, we found significantly increased serum levels of HtrA2 in patients with STEMI. The result was validated in a murine model of myocardial ischaemia–reperfusion injury. In humans the increased serum level was significantly reduced by the mitochondria-targeting peptide elamipretide. In conclusion, HtrA2 is detectable in serum of patients with STEMI and might present a novel biomarker for mitochondrial-induced cardiomyocyte apoptosis. Consequently, HtrA2 may also show promise as a biomarker for the identification of ischaemia–reperfusion injury. However, this must be validated in a lager clinical trial.
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Yao, Tingyan, Junge Zhu, Xiao Wu, Xuying Li, Yongjuan Fu, Yuan Wang, Zhanjun Wang, et al. "HeterozygousHTRA1Mutations Cause Cerebral Small Vessel Diseases." Neurology Genetics 8, no. 6 (December 2022): e200044. http://dx.doi.org/10.1212/nxg.0000000000200044.
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Background and ObjectivesCerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is a rare hereditary cerebrovascular disease caused by homozygous or compound heterozygous variations in the high-temperature requirement A serine peptidase 1 (HTRA1) gene. However, several studies in recent years have found that some heterozygousHTRA1mutations also cause cerebral small vessel disease (CSVD). The current study aims to report the novel genotypes, phenotypes, and histopathologic results of 3 pedigrees of CSVD with heterozygousHTRA1mutation.MethodsThree pedigrees of familiar CSVD, including 11 symptomatic patients and 3 asymptomatic carriers, were enrolled. Whole-exome sequencing was conducted in the probands for identifying rare variants, which were then evaluated for pathogenicity according to the American College of Medical Genetics and Genomics guidelines. Sanger sequencing was performed for validation of mutations in the probands and other family members. The protease activity was assayed for the novel mutations. All the participants received detailed clinical and imaging examinations and the corresponding results were concluded. Hematoma evacuation was performed for an intracerebral hemorrhage patient with the p.Q318H mutation, and the postoperative pathology including hematoma and cerebral small vessels were examined.ResultsThree novel heterozygousHTRA1mutations (p.Q318H, p.V279M, and p.R274W) were detected in the 3 pedigrees. The protease activity was largely lost for all the mutations, confirming that they were loss-of-function mutations. The patients in each pedigree presented with typical clinical and imaging features of CVSD, and some of them displayed several new phenotypes including color blindness, hydrocephalus, and multiple arachnoid cysts. In addition, family 1 is the largest pedigree with heterozygousHTRA1mutation so far and includes homozygous twins, displaying some variation in clinical phenotypes. More importantly, pathologic study of a patient with p.Q318H mutation showed hyalinization, luminal stenosis, loss of smooth muscle cells, splitting of the internal elastic lamina, and intramural hemorrhage/dissection-like structures.DiscussionThese findings broaden the mutational and clinical spectrum of heterozygousHTRA1-related CSVD. Pathologic features were similar with the previous heterozygous and homozygous cases. Moreover, clinical heterogeneity was revealed within the largest single family, and the mechanisms of the phenotypic heterogenetic remain unclear. Overall, heterozygous HTRA1-related CSVD should not be simply taken as a mild type of CARASIL as previously considered.
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Wang, Chang-Chun, Hsiao-Fan Lo, Shu-Yu Lin, and Hungwen Chen. "RACK1 (receptor for activated C-kinase 1) interacts with FBW2 (F-box and WD-repeat domain-containing 2) to up-regulate GCM1 (glial cell missing 1) stability and placental cell migration and invasion." Biochemical Journal 453, no. 2 (June 28, 2013): 201–8. http://dx.doi.org/10.1042/bj20130175.
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GCM1 (glial cell missing 1) is a short-lived transcription factor essential for placental development. The F-box protein, FBW2 (F-box and WD-repeat domain-containing 2), which contains five WD (tryptophan–aspartate) repeats, recognizes GCM1 and mediates its ubiquitination via the SCFFBW2 E3 ligase complex. Although the interaction between GCM1 and FBW2 is facilitated by GCM1 phosphorylation, it is possible that this interaction might be regulated by additional cellular factors. In the present study, we perform tandem-affinity purification coupled with MS analysis identifying RACK1 (receptor for activated C-kinase 1) as an FBW2-interacting protein. RACK1 is a multifaceted scaffold protein containing seven WD repeats. We demonstrate that the WD repeats in both RACK1 and FBW2 are required for the interaction of RACK1 and FBW2. Furthermore, RACK1 competes with GCM1 for FBW2 and thereby prevents GCM1 ubiquitination, which is also supported by the observation that GCM1 is destabilized in RACK1-knockdown BeWo placental cells. Importantly, RACK1 knockdown leads to decreased expression of the GCM1 target gene HTRA4 (high-temperature requirement protein A4), which encodes a serine protease crucial for cell migration and invasion. As a result, migration and invasion activities are down-regulated in RACK1-knockdown BeWo cells. The present study reveals a novel function for RACK1 to regulate GCM1 activity and placental cell migration and invasion.
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Lewis, Claire, Henrieta Skovierova, Gary Rowley, Bronislava Rezuchova, Dagmar Homerova, Andrew Stevenson, Janice Spencer, Jacinta Farn, Jan Kormanec, and Mark Roberts. "Salmonella enterica Serovar Typhimurium HtrA: regulation of expression and role of the chaperone and protease activities during infection." Microbiology 155, no. 3 (March 1, 2009): 873–81. http://dx.doi.org/10.1099/mic.0.023754-0.
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HtrA is a bifunctional stress protein required by many bacterial pathogens to successfully cause infection. Salmonella enterica serovar Typhimurium (S. Typhimurium) htrA mutants are defective in intramacrophage survival and are highly attenuated in mice. Transcription of htrA in Escherichia coli is governed by a single promoter that is dependent on σ E (RpoE). S. Typhimurium htrA also possesses a σ E-dependent promoter; however, we found that the absence of σ E had little effect on production of HtrA by S. Typhimurium. This suggests that additional promoters control expression of htrA in S. Typhimurium. We identified three S. Typhimurium htrA promoters. Only the most proximal promoter, htrAp3, was σ E dependent. The other promoters, htrAp1 and htrAp2, are probably recognized by the principal sigma factor σ 70. These two promoters were constitutively expressed but were also slightly induced by heat shock. Thus expression of htrA is different in S. Typhimurium and E. coli. The role of HtrA is to deal with misfolded/damaged proteins in the periplasm. It can do this either by degrading (protease activity) or folding/capturing (chaperone/sequestering, C/S, activity) the aberrant protein. We investigated which of these functions are important to S. Typhimurium in vitro and in vivo. Point or deletion mutants of htrA that encode variant HtrA molecules have been used in previous studies to investigate the role of different regions of HtrA in C/S and protease activity. These htrA variants were placed under the control of the S. Typhimurium htrAP123 promoters and expressed in a S. Typhimurium htrA mutant, GVB1343. Both wild-type HtrA and HtrA (HtrA S210A) lacking protease activity enabled GVB1343 to grow at high temperature (46 °C). Both molecules also significantly enhanced the growth/survival of GVB1343 in the liver and spleen of mice during infection. However, expression of wild-type HtrA enabled GVB1343 to grow to much higher levels than expression of HtrA S210A. Thus both the protease and C/S functions of HtrA operate in vivo during infection but the protease function is probably more important. Absence of either PDZ domain completely abolished the ability of HtrA to complement the growth defects of GVB1343 in vitro or in vivo.
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Loosmore, Sheena M., Yan-ping Yang, Ray Oomen, Jean M. Shortreed, Debbie C. Coleman, and Michel H. Klein. "The Haemophilus influenzae HtrA Protein Is a Protective Antigen." Infection and Immunity 66, no. 3 (March 1, 1998): 899–906. http://dx.doi.org/10.1128/iai.66.3.899-906.1998.
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ABSTRACT The htrA gene from two strains of nontypeableHaemophilus influenzae has been cloned and sequenced, and the encoded approximately 46-kDa HtrA proteins were found to be highly conserved. H. influenzae HtrA has approximately 55% identity with the Escherichia coli and Salmonella typhimurium HtrA stress response proteins, and expression of theH. influenzae htrA gene was inducible by high temperature. Recombinant HtrA (rHtrA) was expressed from E. coli, and the purified protein was found to have serine protease activity. rHtrA was found to be very immunogenic and partially protective in both the passive infant rat model of bacteremia and the active chinchilla model of otitis media. Immunoblot analysis indicated that HtrA is antigenically conserved in encapsulated and nontypeable H. influenzae species. Site-directed mutagenesis was performed on the htrA gene to ablate the endogenous serine protease activity of wild-type HtrA, and it was found that eight of nine recombinant mutant proteins had no measurable residual proteolytic activity. Two mutant proteins were tested in the animal protection models, and one, H91A, was found to be partially protective in both models. H91A HtrA may be a good candidate antigen for a vaccine against invasive H. influenzae type b disease and otitis media and is currently in phase I clinical trials.