HTRA2 Research Articles

Abstract 28: Proteomic Signatures of Adiposity in Middle-Aged Adults: The Atherosclerosis Risk in Communities (ARIC) Study

Background: Excess adiposity is linked to adverse cardiovascular outcomes via direct and indirect effects on metabolic pathways. There is limited large-scale data assessing proteomic signatures of adiposity. Methods: We quantified 4,955 plasma proteins using SomaLogic v4.0 in stored plasma samples of 9,940 ARIC Visit 2 participants (1990-92, mean age: 57±6 years, 55% women, 22% Black adults, 28% with obesity). We used linear regression and Bonferroni-corrected p-values to identify proteins associated with body mass index (BMI) in 2/3 discovery and 1/3 validation samples. We performed additional analyses to assess key pathways associated with obesity. Results: In the discovery sample (28% with obesity), after adjusting for age, sex, race-center, eGFR, 1,312 proteins were significantly associated with BMI. After further adjustments for clinical and lifestyle risk factors (Figure), 975 proteins were significantly associated with BMI (p<10 -5 ). There were 713 proteins confirmed in the validation sample (p<0.05/975). Among the confirmed proteins, there were >500 proteins that did not have previously described BMI associations in population studies (including serine protease HTRA1, seizure 6-like protein) as well as proteins with known BMI associations (including leptin, fatty acid binding proteins, insulin-like growth factor-binding proteins). Acute phase response, atherosclerosis signaling, hepatic fibrosis, pancreatic cancer and lipid metabolism were among the top biological pathways overrepresented by the proteins associated with BMI (Figure). The upstream regulators of these proteins included interleukins (IL-17A, IL-13, IL-4), as well as tumor necrosis factor (TNF), which was activated. Conclusion: We discovered several novel proteins associated with adiposity and confirmed previously identified proteins. Alteration in the plasma proteome related to adiposity is linked to differential regulation of inflammatory and metabolic pathways independent of cardiovascular risk mediators.

The involvement of CiaR and the CiaR-regulated serine protease HtrA in thermal adaptation of Streptococcus pneumoniae.

The in vivo temperature can vary according to the host tissue and the response to infection. Streptococcus pneumoniae has evolved mechanisms to survive these temperature differences, but neither the consequences of different temperatures for pneumococcal phenotype nor the genetic basis of thermal adaptation are known in detail. In our previous study [16], we found that CiaR, which is a part of two-component regulatory system CiaRH, as well as 17 genes known to be controlled by CiaRH, were identified to be differentially expressed with temperature. One of the CiaRH-regulated genes shown to be differentially regulated by temperature is for the high-temperature requirement protein (HtrA), coded by SPD_2068 (htrA). In this study, we hypothesized that the CiaRH system plays an important role in pneumococcal thermal adaptation through its control over htrA. This hypothesis was evaluated by testing strains mutated or overexpressing ciaR and/or htrA, in in vitro and in vivo assays. The results showed that in the absence of ciaR, the growth, haemolytic activity, amount of capsule and biofilm formation were considerably diminished at 40 °C only, while the cell size and virulence were affected at both 34 and 40 °C. The overexpression of htrA in the ∆ciaR background reconstituted the growth at all temperatures, and the haemolytic activity, biofilm formation and virulence of ∆ciaR partially at 40 °C. We also showed that overexpression of htrA in the wild-type promoted pneumococcal virulence at 40 °C, while the increase of capsule was observed at 34 °C, suggesting that the role of htrA changes at different temperatures. Our data suggest that CiaR and HtrA play an important role in pneumococcal thermal adaptation.

Circ_0001326 suppresses trophoblast cell proliferation, invasion, migration and epithelial-mesenchymal transition progression in preeclampsia by miR-188-3p/HtrA serine peptidase 1 axis.

It has been reported that the alteration of circular RNAs (circRNAs) during preeclampsia (PE) can be associated with the pathogenesis of this disease. Herein, this work investigated the potential functions and mechanism of circ_0001326 in PE process. The levels of genes and proteins were evaluated by quantitative real-time PCR (qRT-PCR) and western blotting. The functional experiments were conducted using cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell, and wound healing assays, respectively. The binding between miR-188-3p and circ_0001326 or HtrA serine peptidase 1 (HTRA1) was verified by bioinformatics analysis and dual-luciferase reporter assays. Circ_0001326 and HTRA1 expression was increased, while miR-188-3p expression was decreased in the placental tissues of preeclamptic singleton pregnant women compared with the normal pregnant women. Functionally, up-regulation of circ_0001326 or HTRA1, or down-regulation of miR-188-3p led to the arrest of cell growth, invasion, migration and epithelial-mesenchymal transition (EMT) process in trophoblast cells. Mechanistically, circ_0001326 acted as a sponge for miR-188-3p, which directly targeted HTRA1. Moreover, circ_0001326 could regulate HTRA1 through sequestering miR-188-3p. A series of rescue experiments showed that miR-188-3p reversed the inhibitory effects of circ_0001326 knockdown on above behaviors of trophoblast cells. Besides that, HTRA1 silencing attenuated the action of miR-188-3p inhibitor on trophoblast cell phenotype alteration. Our study demonstrated that circ_0001326 could promote trophoblast cell proliferation, invasion, migration and EMT in PE by miR-188-3p/HTRA1 axis, indicating a novel insight into the pathogenesis of PE.

Rol polimorfizmiv heniv HTRA serynovoi peptydazy 1, faktora rostu endoteliiu sudyn, faktora nekrozu pukhlyn u likuvanni «volohoi» formy vikovoi makuliarnoi deheneratsii

Introduction. Age-related macular degeneration (AMD) of the retina is still considered the leading cause of vision loss in the elderly. The multifactoriality of the disease impairs the clinical effectiveness of modern AMD treatment methods. however, the study of single-nucleotide polymorphisms, in particular, of the HtrA serine peptidase 1 (HTRA1), vascular endothelial growth factor A (VEGF A) and tumor necrosis factor (TNF) genesis a promising link on the way to improve and develope more effective treatment strategies of the disease. The Aim of the Study. To investigate role of HTRA 1, VEGF A and TNF gene polymorphisms in the treatment of wet age-related macular degeneration. Materials and Methods. 162 people with diagnosed wet AMD took part in the investigation. They received anti-VEGF A therapy in the form of injections of aflibercept monthly for half a year. Structural changes of the eyes were studied using optical coherence tomography (OCT); polymerase chain reaction (PCR) studies were performed using a Bio-Rad CFX 96 apparatus (BioRad, USA) using a reagent package (Lytech, Russia). Statistical analysis of the obtained results was performed using a set of software packages Statistica 10 (StatSoft, Inc., USA) and SPSS 23.0. Results. It was revealed best prognostic significance in patients with the TC rs2010963 genotype of the VEGFA gene was registered during the analysis of OCT 2 (RR=2.7; 95% CI 1.556 – 4.8), OCT 4 (RR=2.9 ; 95% CI 1.7 – 5.03) and OCT 8 (RR=2.6; 95% CI 1.6 – 4.12) sections, while in patients with the CC genotype these indicators in the OCT 2 section were: RR= 6.1; 95% CI 3.66 – 10.27; in OCT zone 4 RR=4.9; 95% CI 2.9 – 8.29, and in the OCT section 8: RR=4.23; 95% CI 2.7 – 6.556, which indicates a more pronounced influence of the CC genotype. When analyzing rs1800629 of the TNF gene, the best prognostic significance of the GA genotype was established in the OCT 4 (RR=1.77; 95% CI 1.218 – 2.56) and OCT 8 (RR=1.9; 95% CI 1.17 – 3.175) areas (p-value less than 0.05), with the AA genotype in OCT 4 (RR=3.77; 95% CI 2.17 – 6.58), OCT 8 (RR=3.1; 95% CI 1 .7 – 5.59) zones and when evaluating changes in visual acuity of patients with wet AMD (RR=4.2; 95% CI 2 – 8.98). No statistically significant results were found in the evaluation of the HTRA1 gene rs11200638 (p-value more than 0.05). Conclusions. The data obtained in our study indicate a direct influence of the vascular endothelial growth factor A (rs2010963) and tumor necrosis factor (rs1800629) polymorphisms on the emergence of resistance to aflibercept. However, the study of this influence in the presence of the HtrA serine peptidase 1 gene rs11200638 requires further research.

Clinical Pathogenesis, Molecular Mechanisms of Gastric Cancer Development.

The human pathogen Helicobacter pylori is the strongest known risk factor for gastric disease and cancer, and gastric cancer remains a leading cause of cancer-related death across the globe. Carcinogenic mechanisms associated with H. pylori are multifactorial and are driven by bacterial virulence constituents, host immune responses, environmental factors such as iron and salt, and the microbiota. Infection with strains that harbor the cytotoxin-associated genes (cag) pathogenicity island, which encodes a type IV secretion system (T4SS) confer increased risk for developing more severe gastric diseases. Other important H. pylori virulence factors that augment disease progression include vacuolating cytotoxin A (VacA), specifically type s1m1 vacA alleles, serine protease HtrA, and the outer-membrane adhesins HopQ, BabA, SabA and OipA. Additional risk factors for gastric cancer include dietary factors such as diets that are high in salt or low in iron, H. pylori-induced perturbations of the gastric microbiome, host genetic polymorphisms, and infection with Epstein-Barr virus. This chapter discusses in detail host factors and how H. pylori virulence factors augment the risk of developing gastric cancer in human patients as well as how the Mongolian gerbil model has been used to define mechanisms of H. pylori-induced inflammation and cancer.

HTRA1 methylation in peripheral blood as a potential marker for the preclinical detection of stroke: a case–control study and a prospective nested case–control study

BackgroundStroke is the leading cause of mortality in China. DNA methylation has essential roles in multiple diseases, but its association with stroke was barely studied. We hereby explored the association between blood-based HTRA serine protease 1 (HTRA1) methylation and the risk of stroke.ResultsThe association was discovered in a hospital-based case–control study (cases/controls = 190:190) and further validated in a prospective nested case–control study including 139 cases who developed stroke within 2 years after recruitment and 144 matched stroke-free controls. We observed stroke-related altered HTRA1 methylation and expression in both case–control study and prospective study. This blood-based HTRA1 methylation was associated with stroke independently from the known risk factors and mostly affected the older population. The prospective results further showed that the altered HTRA1 methylation was detectable 2 years before the clinical determination of stroke and became more robust with increased discriminatory power for stroke along with time when combined with other known stroke-related variables [onset time ≤ 1 year: area under the curve (AUC) = 0.76].ConclusionsIn our study, altered HTRA1 methylation was associated with stroke at clinical and preclinical stages and thus may provide a potential biomarker in the blood for the risk evaluation and preclinical detection of stroke.

Antibody Characterization Report for Serine protease HTRA1

Antibody Characterization Report for Serine protease HTRA1

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

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

Early and late genome-wide gastric epithelial transcriptome response during infection with the human carcinogen Helicobacterpylori.

Infection of the stomach by Helicobacter pylori is a major risk factor for the development of gastric cancer. Colonization of the gastric epithelium leads to the activation of multiple disease-related signaling pathways. Serine protease HtrA represents an important secreted virulence factor that mediates cleavage of cellular junctions. However, its potential role in nuclear responses is unknown. Here, we performed a genome-wide RNA-seq analysis of polarized gastric epithelial cells infected by wild-type (wt) and ΔhtrA mutant bacteria. Fluorescence microscopy showed that H.pylori wt, but not ΔhtrA bacteria, preferably localized at cellular junctions. Our results pinpointed early (2h) and late (6h) transcriptional responses, with most differentially expressed genes at 6h post infection. The transcriptomes revealed HtrA-dependent targeting of genes associated with inflammation and apoptosis (e.g. IL8, ZFP36, TNF). Accordingly, infection with the ΔhtrA mutant induced increased apoptosis rates in host cells, which was associated with reduced H.pylori CagA expression. In contrast, transcription of various carcinogenesis-associated genes (e.g. DKK1, DOCK8) was affected by H.pylori independent of HtrA. These findings suggest that H.pylori disturbs previously unknown molecular pathways in an HtrA-dependent and HtrA-independent manner, and provide valuable new insights of this significant pathogen in humans and thus potential targets for better controlling the risk of malignant transformation.

PD36-09 DIFFERENTIAL EXPRESSION OF SEMINAL PLASMA PROTEINS IN RESPONDERS AND NON-RESPONDERS TO VARICOCELE TREATMENT

You have accessJournal of UrologyCME1 May 2022PD36-09 DIFFERENTIAL EXPRESSION OF SEMINAL PLASMA PROTEINS IN RESPONDERS AND NON-RESPONDERS TO VARICOCELE TREATMENT Timur Bitsoev, Vitaliy Chagovets, Taras Shatylko, Alisa Tokareva, Safar Gamidov, Natalia Starodubtseva, Alina Popova, and Vladimir Frankevich Timur BitsoevTimur Bitsoev More articles by this author , Vitaliy ChagovetsVitaliy Chagovets More articles by this author , Taras ShatylkoTaras Shatylko More articles by this author , Alisa TokarevaAlisa Tokareva More articles by this author , Safar GamidovSafar Gamidov More articles by this author , Natalia StarodubtsevaNatalia Starodubtseva More articles by this author , Alina PopovaAlina Popova More articles by this author , and Vladimir FrankevichVladimir Frankevich More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002594.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Despite large amounts of evidence linking varicocele to male infertility, the role of varicocele repair in infertile men is controversial. Even after technically perfect operation not all couples achieve pregnancy or at least have a significant improvement in semen parameters. Probably, there are cases of infertility which share a causal relationship with varicocele, and there are cases when idiopathic infertility co-exists with harmless varicocele. Unfortunately, there is no way to predict response to varicocele treatment among infertile males. Since sperm are transcriptionally inert, the proteome of seminal plasma is relatively stable and may reflect the true extent of deleterious effects of varicocele, such as oxidative stress and hyperthermia. Our goal was to assess whether proteomic signature of seminal plasma may help to distinguish responders and non-responders to varicocele repair. METHODS: We used mass-spectrometry to perform a proteomic analysis of seminal plasma specimens obtained from 10 infertile patients (age: 25–44 years) with bilateral varicocele. All patients underwent routine semen analysis and had at least two altered core parameters out of three (total sperm count, progressive motility, percentage of sperm with normal morphology). Microsurgical bilateral subinguinal varicocelectomy was performed in all patients. Routine semen analysis and proteomic analysis were repeated at follow-up, which was scheduled at 6 months after the surgery. Responders were defined as patients whose spouse became pregnant during follow-up period or whose total motile sperm count increased by at least 50%. Proteomic profiles of responders and non-responders were compared. RESULTS: Five patients were responders to varicocele repair (partners of two of them became pregnant). Proteomic analysis identified 18 proteins with differential expression in responders and non-responders: melanophilin, kallikrein-2, alpha-actinin-4, Kunitz-type protease inhibitor 3, alpha-1-acid glycoprotein 1, CD177 antigen, alpha-1-acid glycoprotein 2, protein-glutamine gamma-glutamyltransferase 4, phosphatidylethanol amine-binding protein 4, acyl-CoA-binding protein, hemoglobin subunit alpha, antileukoproteinase, collagen alpha-1(XVIII) chain, vitronectin, serine protease HTRA1, V-type proton ATPase catalytic subunit A, endoplasmic reticulum resident protein 44, NADP-dependent malic enzyme. CONCLUSIONS: If validated, semen proteomics may become a useful tool to predict response to varicocele repair and avoid unnecessary surgery. Source of Funding: N/A © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e636 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Timur Bitsoev More articles by this author Vitaliy Chagovets More articles by this author Taras Shatylko More articles by this author Alisa Tokareva More articles by this author Safar Gamidov More articles by this author Natalia Starodubtseva More articles by this author Alina Popova More articles by this author Vladimir Frankevich More articles by this author Expand All Advertisement PDF DownloadLoading ...

Campylobacter jejuni Serine Protease HtrA Induces Paracellular Transmigration of Microbiota across Polarized Intestinal Epithelial Cells.

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.

An allosteric HTRA1-calpain 2 complex with restricted activation profile.

SignificanceClassic serine proteases are synthesized as inactive precursors that are proteolytically processed, resulting in irreversible activation. We report an alternative and reversible mechanism of activation that is executed by an inactive protease. This mechanism involves a protein complex between the serine protease HTRA1 and the cysteine protease calpain 2. Surprisingly, activation is restricted as it improves the proteolysis of soluble tau protein but not the dissociation and degradation of its amyloid fibrils, a task that free HTRA1 is efficiently performing. These data exemplify a challenge for protein quality control proteases in the clearing of pathogenic fibrils and suggest a potential for unexpected side effects of chemical modulators targeting PDZ or other domains located at a distance to the active site.

Forward and reverse degradomics defines the proteolytic landscape of human knee osteoarthritic cartilage and the role of the serine protease HtrA1

Proteolytic destruction of articular cartilage, a major pathogenic mechanism in osteoarthritis (OA), was not previously investigated by terminomics strategies. We defined the degradome of human knee OA cartilage and the contribution therein of the protease HtrA1 using Terminal Amine Isotopic Labeling of Substrates (TAILS). Proteins from OA cartilage taken at knee arthroplasty (n=6) or separately, from healthy cartilage incubated in triplicate with/without active HtrA1, were labeled at natural and proteolytically cleaved N-termini by reductive dimethylation, followed by trypsin digestion, enrichment of N-terminally labeled/blocked peptides, tandem mass spectrometry and positional peptide annotation to identify cleavage sites. Biglycan proteolysis by HtrA1 was validated biochemically and Amino-Terminal Oriented Mass Spectrometry of Substrates (ATOMS) was used to define the HtrA1 cleavage sites. We identified 10,155 unique internal peptides from 2,162 proteins, suggesting at least 10,797 cleavage sites in OA cartilage. 7,635 internal peptides originated in 371 extracellular matrix/secreted components, many undergoing extensive proteolysis. Rampant ragging of protein termini suggested pervasive exopeptidase activity. HtrA1, the most abundant protease in OA cartilage, experimentally generated 323 cleavages in 109 cartilage proteins, accounting for 171 observed cleavages in the OA degradome. ATOMS identified HtrA1 cleavage sites in a selected substrate, biglycan, whose direct cleavage by HtrA1 was thus orthogonally validated. OA cartilage demonstrates widespread proteolysis by endo- and exopeptidases. HtrA1 contributes broadly to cartilage proteolysis. Forward degradomics of OA cartilage together with reverse degradomics of proteases active in OA, e.g., HtrA1, can potentially fully annotate OA proteolytic pathways and provide new biomarkers.

Evaluation of Utilizing the Distinct Genes as Predictive Biomarkers in Late-Onset Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disease that is characterized by a devastating decline in cognitive activities among all types of dementia, and it severely affects the quality of life. Late-onset AD (LOAD) occurs after the age of 65 years and develops sporadically. Although aging comes first along the main risk factors underlying LOAD, disease-causing susceptibility genes have been associated with disease pathogenesis. In our study, we included the genes PARP1 , POLB , HTRA2 , SLC1A2 , HS1BP3 , and DRD3 to be investigated in LOAD patients based on their expression levels. Within this framework, we aimed to determine the possible functions of these genes in the pathophysiology of the disease. We investigated whether the utilization of these genes as biomarkers in the early diagnosis of LOAD may help the treatment scheme to be applied in the clinic. We involved 50 individuals in the study and collected peripheral blood samples from the patients and control groups for molecular genetic analysis. Subsequently, RNA was extracted from the peripheral blood samples, and expression analyzes were performed using qualitative reverse transcription polymerase chain reaction. The results obtained were evaluated by using proper statistical methods. Our results demonstrated that there was no difference between patient and control groups in terms of HTRA2 , DRD3 , HS1BP3 , and POLB genes. The expression levels of the SLC1A2 and PARP1 genes were significantly lower in the patient group compared with the control group. In conclusion, we presume that the PARP1 and SLC1A2 genes can be utilized as molecular biomarkers for LOAD.

A new function for the serine protease HtrA2 in controlling radiation-induced senescence in cancer cells.

Radiation therapy can induce cellular senescence in cancer cells, leading to short‐term tumor growth arrest but increased long‐term recurrence. To better understand the molecular mechanisms involved, we developed a model of radiation‐induced senescence in cultured cancer cells. The irradiated cells exhibited a typical senescent phenotype, including upregulation of p53 and its main target, p21, followed by a sustained reduction in cellular proliferation, changes in cell size and cytoskeleton organization, and senescence‐associated beta‐galactosidase activity. Mass spectrometry‐based proteomic profiling of the senescent cells indicated downregulation of proteins involved in cell cycle progression and DNA repair, and upregulation of proteins associated with malignancy. A functional siRNA screen using a cell death‐related library identified mitochondrial serine protease HtrA2 as being necessary for sustained growth arrest of the senescent cells. In search of direct HtrA2 substrates following radiation, we determined that HtrA2 cleaves the intermediate filament protein vimentin, affecting its cytoplasmic organization. Ectopic expression of active cytosolic HtrA2 resulted in similar changes to vimentin filament assembly. Thus, HtrA2 is involved in the cytoskeletal reorganization that accompanies radiation‐induced senescence and the continuous maintenance of proliferation arrest.

Role of conserved regulatory loop residues in allosteric propagation of serine protease HtrA2

High temperature requirement protease A2 (HtrA2) is a mitochondrial serine protease that demonstrates multifaceted roles including protein quality control and proapoptotic properties in humans, making it a potential therapeutic target. Current literature suggests involvement of flexible regulatory loops in governing the allosteric propagation within the trimeric HtrA2 ensemble. Here, we have identified three important residues – R147, P148 (L3 loop) and F131 (LD loop) surrounding the catalytic-site that play crucial roles in stabilizing HtrA2 active conformation during its multimodal activation. Although mutagenesis of these residues does not affect the structural integrity, it renders the protease inactive by affecting the regulatory inter-subunit PDZ-protease crosstalk. This is further emphasized by the inactivity observed during N-terminal mediated activation of the HtrA2 loop mutants via BIR2 domain of the antiapoptotic protein XIAP. Overall, our results demonstrate the importance of L3 loop dynamics in mediating the inter-molecular allostery via R147-P148 residues. Understanding the on-off switch that regulates HtrA2 activation might help in designing HtrA2 modulators for therapeutic applications.

Design, synthesis and biological evaluation of P2-modified proline analogues targeting the HtrA serine protease in Chlamydia

High temperature requirement A (HtrA) serine proteases have emerged as a novel class of antibacterial target, which are crucial in protein quality control and are involved in the pathogenesis of a wide array of bacterial infections. Previously, we demonstrated that HtrA in Chlamydia is essential for bacterial survival, replication and virulence. Here, we report a new series of proline (P2)-modified inhibitors of Chlamydia trachomatis HtrA (CtHtrA) developed by proline ring expansion and Cγ-substitutions. The structure-based drug optimization process was guided by molecular modelling and in vitro pharmacological evaluation of inhibitory potency, selectivity and cytotoxicity. Compound 25 from the first-generation 4-substituted proline analogues increased antiCtHtrA potency and selectivity over human neutrophil elastase (HNE) by approximately 6- and 12-fold, respectively, relative to the peptidic lead compound 1. Based on this compound, second-generation substituted proline residues containing 1,2,3-triazole moieties were synthesized by regioselective azide-alkyne click chemistry. Compound 49 demonstrated significantly improved antichlamydial activity in whole cell assays, diminishing the bacterial infectious progeny below the detection limit at the lowest dose tested. Compound 49 resulted in approximately 9- and 22-fold improvement in the inhibitory potency and selectivity relative to 1, respectively. To date, compound 49 is the most potent HtrA inhibitor developed against Chlamydia spp.

APP deficiency and HTRA2 modulates PrPc proteostasis in human cancer cells

Cellular protein homeostasis (proteostasis) requires an accurate balance between protein biosynthesis, folding, and degradation, and its instability is causally related to human diseases and cancers. Here, we created numerous engineered cancer cell lines targeting APP (amyloid ß precursor protein) and/or PRNP (cellular prion) genes and we showed that APP knocking-down impaired PRNP mRNA level and vice versa, suggesting a link between their gene regulation. PRNPKD, APPKD and PRNPKD/APPKD HeLa cells encountered major difficulties to grow in a 3D tissue-like environment. Unexpectedly, we found a cytoplasmic accumulation of the PrPc protein without PRNP gene up regulation, in both APPKD and APPKO HeLa cells. Interestingly, APP and/or PRNP gene ablation enhanced the chaperone/serine protease HTRA2 gene expression, which is a protein processing quality factor involved in Alzheimer's disease. Importantly, HTRA2 gene silencing decreased PRNP mRNA level and lowered PrPc protein amounts, and conversely, HTRA2 overexpression increased PRNP gene regulation and enhanced membrane-anchored and cytoplasmic PrPc fractions. PrPc, APP and HTRA2 destabilized membrane-associated CD24 protein, suggesting changes in the lipid raft structure. Our data show for the first time that APP and the dual chaperone/serine protease HTRA2 protein could modulate PrPc proteostasis hampering cancer cell behavior.

Optimization of peptide-based inhibitors targeting the HtrA serine protease in Chlamydia: Design, synthesis and biological evaluation of pyridone-based and N-Capping group-modified analogues

The obligate intracellular bacterium Chlamydia trachomatis (C.trachomatis) is responsible for the most common bacterial sexually transmitted infection and is the leading cause of preventable blindness, representing a major global health burden. While C.trachomatis infection is currently treatable with broad-spectrum antibiotics, there would be many benefits of a chlamydia-specific therapy. Previously, we have identified a small-molecule lead compound JO146 [Boc-Val-Pro-ValP(OPh)2] targeting the bacterial serine protease HtrA, which is essential in bacterial replication, virulence and survival, particularly under stress conditions. JO146 is highly efficacious in attenuating infectivity of both human (C.trachomatis) as well as koala (C.pecorum) species invitro and invivo, without host cell toxicity. Herein, we present our continuing efforts on optimizing JO146 by modifying the N-capping group as well as replacing the parent peptide structure with the 2-pyridone scaffold at P3/P2. The drug optimization process was guided by molecular modelling, enzyme and cell-based assays. Compound 18b from the pyridone series showed improved inhibitory activity against CtHtrA by 5-fold and selectivity over human neutrophil elastase (HNE) by 109-fold compared to JO146, indicating that 2-pyridone is a suitable bioisostere of the P3/P2 amide/proline for developing CtHtrA inhibitors. Most pyridone-based inhibitors showed superior anti-chlamydial potency to JO146 especially at lower doses (25 and 50μM) in C.trachomatis and C.pecorum cell culture assays. Modifications of the N-capping group of the peptidyl inhibitors did not have much influence on the anti-chlamydial activities, providing opportunities for more versatile alterations and future optimization. In summary, we present 2-pyridone based analogues as a new generation of non-peptidic CtHtrA inhibitors, which hold better promise as anti-chlamydial drug candidates.

Carasil and htra1: an early adulthood syndrome

Cerebral Autosomal Recessive Arteriopathy Subcortical Infracts Leukoencephalopathy (CARASIL), is an autosomal recessive disorder inherited by two copies of an abnormal gene from some trait, one from each parent. Person with this disorder may have a change in deep white matter in brain; and this disorder can be characterized by damage to small blood vessels in brain. Increasing muscle tone, slurred speech, stiff movement of legs, gait disturbance, spondylosis, sparse hair are the major symptoms. But the ultimate cause of this disorder can lead to loss of memory, cognitive impairment. This disorder can be caused in early adulthood i.e., between 20-40 years of age. CARASIL has first reported in Japan, later occurred in China and Caucasian population. A neurological disorder named Binswanger’s disease (BD) and chronic immunological disorder multiple sclerosis are main different alternation of this disease. Mutation requires the high temperature resultant, A serine peptidase 1 causes CARASIL, so it is a serine protease.
 Serine protease HTRA1 is an enzyme that in humans is encoded by HTRA1 gene. HTRA1 protein is composed of four distinct protein domains. This is located on the long (q) arm of chromosome 10 in a region known as 10q26. This protein is a secreted enzyme that is proposed to regulate the availability of insulin like growth factors (IGF) by cleaving IGF binding proteins. HTRA1 enzyme helps breakdown many other kinds of proteins in this space surrounding cells (extracellular matrix). This is attached to protein in Transforming Growth Factor Beta (TGF-beta) and slows down their ability to send chemical signals. Variant known as rs11200638 is found in an area of gene called promoter region which starts the production HTRA1 enzyme. It is unclear how of polymorphism in the HTRA1 gene might be an age-related macular degeneration.
 So at last, we can conclude that CARASIL is related to brain inherited from the abnormal genes caused to small arteries and to the specific area of deep brain in higher function; tissue loss due to lack of oxygen where small arteries get blocked destruction to myelin an oily substance covers and protects the nerve fibers in CNS.