Tumor Suppressors-HTRA Proteases and Interleukin-12-in Pediatric Asthma and Allergic Rhinitis Patients.

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.

Expression of HTRA Genes and Its Association with Microsatellite Instability and Survival of Patients with Colorectal Cancer.

HtrA proteases regulate cellular homeostasis and cell death. Their dysfunctions have been correlated with oncogenesis and response to therapeutic treatment. We investigated the relation between HtrA1-3 expression and clinicopathological, and survival data, as well as the microsatellite status of tumors. Sixty-five colorectal cancer patients were included in the study. The expression of HTRA1-3 was estimated at the mRNA and protein levels by quantitative PCR and immunoblotting. Microsatellite status was determined by high-resolution-melting PCR. We found that the HTRA1 mRNA level was higher in colorectal cancer tissue as compared to the unchanged mucosa, specifically in primary lesions of metastasizing cancer. The levels of HtrA1 and HtrA2 proteins were reduced in tumor tissue when compared to unchanged mucosa, specifically in primary lesions of metastasizing disease. Moreover, a decrease in HTRA1 and HTRA2 transcripts' levels in cancers with a high level of microsatellite instability compared to microsatellite stable ones has been observed. A low level of HtrA1 or/and HtrA2 in cancer tissue correlated with poorer patient survival. The expression of HTRA1 and HTRA2 changes during colorectal carcinogenesis and microsatellite instability may be, at least partially, associated with these changes. The alterations in the HTRA1/2 genes' expression are connected with metastatic potential of colorectal cancer and may affect patient survival.

HtrA4 Protease Promotes Chemotherapeutic-Dependent Cancer Cell Death.

The HtrA4 human protease is crucial in placentation and embryo implantation, and its altered level is connected with pre-eclampsia. The meta-analyses of microarray assays revealed that the HtrA4 level is changed in brain tumors and breast and prostate cancers, which suggests its involvement in oncogenesis. In spite of the HtrA4 involvement in important physiological and pathological processes, its function in the cell is poorly understood. In this work, using lung and breast cancer cell lines, we showed for the first time that the full-length HtrA4 and its N-terminally deleted variant promote cancer cell death induced by chemotherapeutic drugs by enhancing apoptosis. The effect is dependent on the HtrA4 proteolytic activity, and the N-terminally deleted HtrA4 is more efficient in the cell death stimulation. Furthermore, HtrA4 increases the effect of chemotherapeutics on the clonogenic potential and motility of cancer cells, and it increases cell cycle arrest at the G2/M phase. HtrA4 may modulate cell death by degrading the anti-apoptotic XIAP protein and also by proteolysis of the executioner pro-caspase 7 and cytoskeletal proteins, actin and ß-tubulin. These findings provide new insight into the mechanism of the HtrA4 protease function in cell death and oncogenesis, and they may help to develop new anti-cancer therapeutic strategies.

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

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

The HtrA3 protease promotes drug-induced death of lung cancer cells by cleavage of the X-linked inhibitor of apoptosis protein (XIAP).

HtrA3 is a proapoptotic protease shown to promote drug-induced cytotoxicity in lung cancer cells and proposed to have an antitumor effect. However, at the molecular level, the role of HtrA3 in cell death induction is poorly understood. There are two HtrA3 isoforms, a long and a short one, termed HtrA3L and HtrA3S. By performing pull down assays, co-immunoprecipitation and ELISA, we showed that HtrA3 formed complexes with the X-linked inhibitor of apoptosis protein (XIAP). The recombinant HtrA3 variants ΔN-HtrA3L and -S, lacking the N-terminal regions that are not essential for protease activity, cleaved XIAP with a comparable efficiency, though ΔN-HtrA3S was more active in the presence of cellular extract, suggesting the existence of an activating factor. Immunofluorescence and proximity ligation assays indicated that HtrA3 partially co-localized with XIAP. Exogenous ΔN-HtrA3L/S promoted apoptotic death of lung cancer cells treated with etoposide and caused a significant decrease of cellular XIAP levels, in a way dependent on HtrA3 proteolytic activity. These results collectively indicate that both HtrA3 isoforms stimulate drug-induced apoptotic death of lung cancer cells via XIAP cleavage and thus help to understand the molecular mechanism of HtrA3 function in apoptosis and in cancer cell death caused by chemotherapy.

Properties of the HtrA Protease From Bacterium Helicobacter pylori Whose Activity Is Indispensable for Growth Under Stress Conditions.

The protease high temperature requirement A from the gastric pathogen Helicobacter pylori (HtrA Hp ) belongs to the well conserved family of serine proteases. HtrA Hp is an important secreted virulence factor involved in the disruption of tight and adherens junctions during infection. Very little is known about the function of HtrA Hp in the H. pylori cell physiology due to the lack of htrA knockout strains. Here, using a newly constructed ΔhtrA mutant strain, we found that bacteria deprived of HtrA Hp showed increased sensitivity to certain types of stress, including elevated temperature, pH and osmotic shock, as well as treatment with puromycin. These data indicate that HtrA Hp plays a protective role in the H. pylori cell, presumably associated with maintenance of important periplasmic and outer membrane proteins. Purified HtrA Hp was shown to be very tolerant to a wide range of temperature and pH values. Remarkably, the protein exhibited a very high thermal stability with the melting point (Tm) values of above 85°C. Moreover, HtrA Hp showed the capability to regain its active structure following treatment under denaturing conditions. Taken together, our work demonstrates that HtrA Hp is well adapted to operate under harsh conditions as an exported virulence factor, but also inside the bacterial cell as an important component of the protein quality control system in the stressed cellular envelope.

Mast cells in mastocytosis and allergy - Important player in metabolic and immunological homeostasis.

The role of mast cell (MC) activity in pathophysiology is complex and challenging and its clinical effects are difficult to predict. Apart from the known role of MCs in basic immunological processes and allergy, underlined is their importance in bone mineralization and in regulation of autoimmune reactions. Mast cell mediators, especially those released from mast cells in degranulation, but also those released constitutively, are important both in metabolic and immunological processes. Mastocytosis is a heterogeneous group of disorders characterized by accumulation of MC in one or more organs. There are scientific data indicating that mastocytosis patients are at increased risk of osteoporosis in the systemic form of the disease and children with cutaneous mastocytosis have a higher rate of hypogammaglobulinemia. Moreover, the origin of osteoporosis in patients with allergy is no longer considered as linked to steroid therapy only, but to the mast cell mediators' activity as well. There are indications that osteoporosis symptoms in this group of patients may develop independently of the cumulative steroids' dose. Thus, the influence of mast cells on metabolic and immunologic processes in allergic patients should be investigated. The assessment of mast cell activity and burden in mastocytosis may be used to guide clinical management of patients with allergy.

Proteomic analysis of protein homeostasis and aggregation.

Protein homeostasis (proteostasis) refers to the ability of cells to preserve the correct balance between protein synthesis, folding and degradation. Proteostasis is essential for optimal cell growth and survival under stressful conditions. Various extracellular and intracellular stresses including heat shock, oxidative stress, proteasome malfunction, mutations and aging-related modifications can result in disturbed proteostasis manifested by enhanced misfolding and aggregation of proteins. To limit protein misfolding and aggregation cells have evolved various strategies including molecular chaperones, proteasome system and autophagy. Molecular chaperones assist folding of proteins, protect them from denaturation and facilitate renaturation of the misfolded polypeptides, whereas proteasomes and autophagosomes remove the irreversibly damaged proteins. The impairment of proteostasis results in protein aggregation that is a major pathological hallmark of numerous age-related disorders, such as cataract, Alzheimer's, Parkinson's, Huntington's, and prion diseases. To discover protein markers and speed up diagnosis of neurodegenerative diseases accompanied by protein aggregation, proteomic tools have increasingly been used in recent years. Systematic and exhaustive analysis of the changes that occur in the proteomes of affected tissues and biofluids in humans or in model organisms is one of the most promising approaches to reveal mechanisms underlying protein aggregation diseases, improve their diagnosis and develop therapeutic strategies. Significance: In this review we outline the elements responsible for maintaining cellular proteostasis and present the overview of proteomic studies focused on protein-aggregation diseases. These studies provide insights into the mechanisms responsible for age-related disorders and reveal new potential biomarkers for Alzheimer's, Parkinson's, Huntigton's and prion diseases.

Immune response against HtrA proteases in children with cutaneous mastocytosis.

Mast cells play an important role in both, the innate and adaptive immunity, however, clonal proliferation of abnormal mast cells in various organs leads to mastocytosis. A skin variant of the disease, cutaneous mastocytosis (CM) is the most frequent form of mastocytosis in children. HtrA proteases are modulators of important cellular processes, including cell signaling and apoptosis, and are related to development of several pathologies. The above and the observation that mast cells constitutively release the HtrA1 protein, prompted us to investigate a possible involvement of the HtrA proteins in pediatric CM. Levels of the serum autoantibodies (IgG) against the recombinant HtrA proteins (HtrA1-4) in children with CM (n=36) and in healthy controls (n=62) were assayed. Anti-HtrA IgGs were detected using enzyme linked immunosorbent assay (ELISA) and Western-blotting. In the CM sera, levels of the anti-HtrA1 and anti-HtrA3 autoantibodies were significantly increased when compared to the control group, while the HtrA protein levels were comparable. No significant differences in the anti-HtrA2 IgG level were found; for the anti-HtrA4 IgGs lower levels in CM group were revealed. In healthy children, the IgG levels against the HtrA1, -3 and -4 increased significantly with the age of children; no significant changes were observed for the anti-HtrA2 IgG. Our results suggest involvement of the HtrA1 and HtrA3 proteins in pediatric CM; involvement of the HtrA4 protein is possible but needs to be investigated further. In healthy children, the autoantibody levels against HtrA1, -3 and -4, but not against HtrA2, increase with age.

HtrA3 is a cellular partner of cytoskeleton proteins and TCP1α chaperonin.

The human HtrA3 protease is involved in placentation, mitochondrial homeostasis, stimulation of apoptosis and proposed to be a tumor suppressor. Molecular mechanisms of the HtrA3 functions are poorly understood and knowledge concerning its cellular targets is very limited. There are two HtrA3 isoforms, the long (HtrA3L) and short (HtrA3S). Upon stress, their N-terminal domains are removed, resulting in the more active ΔN-HtrA3. By pull down and mass spectrometry techniques, we identified a panel of putative ΔN-HtrA3L/S substrates. We confirmed that ΔN-HtrA3L/S formed complexes with actin, ß-tubulin, vimentin and TCP1α in vitro and in a cell and partially co-localized with the actin and vimentin filaments, microtubules and TCP1α in a cell. In vitro, both isoforms cleaved the cytoskeleton proteins, promoted tubulin polymerization and displayed chaperone-like activity, with ΔN-HtrA3S being more efficient in proteolysis and ΔN-HtrA3L - in polymerization. TCP1α, essential for the actin and tubulin folding, was directly bound by the ΔN-HtrA3L/S but not cleaved. These results indicate that actin, ß-tubulin, vimentin, and TCP1α are HtrA3 cellular partners and suggest that HtrA3 may influence cytoskeleton dynamics. They also suggest different roles of the HtrA3 isoforms and a possibility that HtrA3 protease may also function as a co-chaperone. SIGNIFICANCE: The HtrA3 protease stimulates apoptosis and is proposed to be a tumor suppressor and a therapeutic target, however little is known about its function at the molecular level and very few HtrA3 physiological substrates have been identified so far. Furthermore, HtrA3 is the only member of the HtrA family of proteins which, apart from the long isoform possessing the PD and PDZ domains (HtrA3L), has a short isoform (HtrA3S) lacking the PDZ domain. In this work we identified a large panel (about 150) of the tentative HtrA3L/S cellular partners which provides a good basis for further research concerning the HtrA3 function. We have shown that the cytoskeleton proteins actin, ß-tubulin and vimentin, and the TCP1α chaperonin are cellular partners of both HtrA3 isoforms. Our findings indicate that HtrA3 may promote destabilization of the actin and vimentin cytoskeleton and suggest that it may influence the dynamics of the microtubule network, with the HtrA3S being more efficient in cytoskeleton protein cleavage and HtrA3L - in tubulin polymerization. Also, we have shown for the first time that HtrA3 has a chaperone-like, holdase activity in vitro - activity typical for co-chaperone proteins. The proposed HtrA3 influence on the cytoskeleton dynamics may be one of the ways in which HtrA3 promotes cell death and affects cancerogenesis. We believe that the results of this study provide a new insight into the role of HtrA3 in a cell and further confirm the notion that HtrA3 should be considered as a target of new anti-cancer therapies.

Biochemical properties of the HtrA homolog from bacterium Stenotrophomonas maltophilia.

The HtrA proteins due to their proteolytic, and in many cases chaperone activity, efficiently counteract consequences of stressful conditions. In the environmental bacterium and nosocomial pathogen Stenotrophomonas maltophilia HtrA (HtrASm) is induced as a part of adaptive response to host temperature (37°C). We examined the biochemical properties of HtrASm and compared them with those of model HtrAEc from Escherichia coli. We found that HtrASm is a protease and chaperone that operates over a wide range of pH and is highly active at temperatures between 35 and 37°C. The temperature-sensitive activity corresponded well with the lower thermal stability of the protein and weaker stability of the oligomer. Interestingly, the enzyme shows slightly different substrate cleavage specificity when compared to other bacterial HtrAs. A computational model of the three-dimensional structure of HtrASm indicates differences in the S1 substrate specificity pocket and suggests weaker inter-trimer interactions when compared to HtrAEc. The observed features of HtrASm suggest that this protein may play a protective role under stressful conditions acting both as a protease and a chaperone. The optimal temperatures for the protein activity may reflect the evolutionary adaptation of S. maltophilia to life in soil or aqueous environments, where the temperatures are usually much below 37°C.

Vitamin D status in patients with rheumatoid arthritis: a correlation analysis with disease activity and progression, as well as serum IL-6 levels.

OBJECTIVES: Recent epidemiological studies suggested an association between a poor vitamin D [25(OH)D] status, inflammatory mediators, and rheumatoid arthritis (RA). We have recently proposed that pro-inflammatory interleukin 6 (IL-6) may represent a good marker for disease activity of RA. The aim of this study was to investigate the relationship between serum 25(OH)D levels and disease activity, joint damage, as well as serum IL-6 levels in a Polish RA population. MATERIALS AND METHODS: Serum 25(OH)D levels were measured in 35 female RA patients and 38 age- and gender-matched healthy controls. Statistical correlations between 25(OH)D levels and the disease activity score 28 (DAS 28), joint damage based on the Steinbrocker criteria, as well as serum IL-6 levels were performed. RESULTS: There was no statistically significant difference between levels of 25(OH)D in RA (16.89±8.57 ng/ml) and healthy controls (14.12±7.51 ng/ml), and the vitamin D deficiency (<20 ng/ml) was found in 71.43% of RA patients and 73.68 % of healthy controls. While vitamin D status did not correlate with DAS 28 (r=0.265, p=0.149) and joint damage based on the Steinbrocker criteria (r=0.367, p=0.065), a positive correlation between 25(OH)D and IL-6 (r=0.537, p=0.002) was observed in RA. CONCLUSION: Although further studies on a larger group of patients will be needed to confirm the data presented here, it seems that hypovitaminosis D is common in the RA patients and middle-aged non-RA healthy women in the Polish population. 25(OH)D levels were similar in the RA patients and age- and gender-matched healthy controls, and were not associated with joint damage and disease activity in patients.

Selection of Effective HTRA3 Activators Using Combinatorial Chemistry.

Herein, we report selection, synthesis, and enzymatic evaluation of a peptidomimetic library able to increase proteolytic activity of HtrA3 (high temperature requirement A) protease. Iterative deconvolution in solution of synthesized modified pentapeptides yielded two potent HtrA3 activators acting in the micromolar range (HCOO-CH2O-C6H4-OCH2-CO-Tyr-Asn-Phe-His-Asn-OH and HCOO-CH2O-C6H4-OCH2-CO-Tyr-Asn-Phe-His-Glu-OH). Both compounds increased proteolysis of an artificial HtrA3 substrate over 40-fold in a selective manner. On the basis of molecular modeling, the selected compounds bind strongly to the PDZ domain.

The role of the LB structural loop and its interactions with the PDZ domain of the human HtrA3 protease.

Human HtrA3 protease is a proapoptotic protein, involved in embryo implantation and oncogenesis. In stress conditions the protease is activated by removal of its N-terminal domain. The activated form, ΔN-HtrA3L is a homotrimer composed of the protease (PD) and PDZ domains. The LB structural loop of the PD is longer by six amino acid residues than its counterparts of other human HtrA proteins and interacts with the PDZ in a way not observed in other known HtrA structures. By size exclusion chromatography of the ΔN-HtrA3L mutated variants we found that removal of the additional LB loop residues caused a complete loss of the proper trimeric structure while impairing their interactions with the PDZ domain decreased the amount of the trimers. This indicates that the LB loop participates in stabilization of the ΔN-HtrA3L oligomer structure and suggests involvement of the LB-PDZ interactions in the stabilization. Removal of the additional LB loop residues impaired the ΔN-HtrA3L activity against the peptide and protein substrates, including the antiapoptotic XIAP protein, while a decrease in the LB-PDZ interaction caused a diminished efficiency of the peptide cleavage. These results indicate that the additional LB residues are important for the ΔN-HtrA3L proteolytic activity. Furthermore, a monomeric form of the ΔN-HtrA3L is proteolytically inactive. In conclusion, our results suggest that the expanded LB loop promotes the ΔN-HtrA3L activity by stabilizing the protease native trimeric structure.

Structural insights into the activation mechanisms of human HtrA serine proteases.

Human HtrA1-4 proteins belong to the HtrA family of evolutionarily conserved serine proteases and function as important modulators of many physiological processes, including maintenance of mitochondrial homeostasis, cell signaling and apoptosis. Disturbances in their action are linked to severe diseases, including oncogenesis and neurodegeneration. The HtrA1-4 proteins share structural and functional features of other members of the HtrA protein family, however there are several significant differences in structural architecture and mechanisms of action which makes each of them unique. Our goal is to present recent studies regarding human HtrAs. We focus on their physiological functions, structure and regulation, and describe current models of activation mechanisms. Knowledge of molecular basis of the human HtrAs' action is a subject of great interest; it is crucial for understanding their relevance in cellular physiology and pathogenesis as well as for using them as targets in future therapies of diseases such as neurodegenerative disorders and cancer.

The Extracellular Bacterial HtrA Proteins as Potential Therapeutic Targets and Vaccine Candidates.

BACKGROUND: An increasing resistance of bacteria to the commonly used antimicrobials forces to search for alternative or supportive ways to cure infections. Targeting virulence factors is one of such approaches. The bacterial HtrA proteins are strongly involved in virulence and the lack of functional HtrA in many cases impairs invasiveness of pathogens. HtrAs act by protecting the cells under stressful conditions as well as they take direct part in invasion of the host. The latter function is played predominantly by the recently identified extracellular fraction of HtrA. This review aims to evaluate HtrAs as therapeutic targets, including design of chemical inhibitors and vaccines. METHODS: We undertook a thorough search of bibliographic databases for peer-reviewed scientific literature. RESULTS: One hundred and sixty-four papers were included in the review. First, we briefly summarized key structural and functional properties of known HtrA proteins with the special focus on the extracellular HtrA fraction. Then we provided an overview of efforts and advancements to target HtrAs of pathogenic bacteria as a promising antimicrobial therapy. In some cases, encouraging results were obtained and application of HtrAspecific inhibitors protected tissues from damage and killed bacteria. Also promising reports concerning the use of HtrA as a protective antigen in several disease models have recently been published. CONCLUSION: The findings of this review suggest that the exported HtrA proteins are very attractive therapeutic targets due to their accessibility, significance in virulence and immunogenicity. However, further extensive studies are still needed to develop a safe antimicrobial treatment.

Distinct 3D Architecture and Dynamics of the Human HtrA2(Omi) Protease and Its Mutated Variants.

HtrA2(Omi) protease controls protein quality in mitochondria and plays a major role in apoptosis. Its HtrA2S306A mutant (with the catalytic serine routinely disabled for an X-ray study to avoid self-degradation) is a homotrimer whose subunits contain the serine protease domain (PD) and the regulatory PDZ domain. In the inactive state, a tight interdomain interface limits penetration of both PDZ-activating ligands and PD substrates into their respective target sites. We successfully crystalized HtrA2V226K/S306A, whose active counterpart HtrA2V226K has had higher proteolytic activity, suggesting higher propensity to opening the PD-PDZ interface than that of the wild type HtrA2. Yet, the crystal structure revealed the HtrA2V226K/S306A architecture typical of the inactive protein. To get a consistent interpretation of crystallographic data in the light of kinetic results, we employed molecular dynamics (MD). V325D inactivating mutant was used as a reference. Our simulations demonstrated that upon binding of a specific peptide ligand NH2-GWTMFWV-COOH, the PDZ domains open more dynamically in the wild type protease compared to the V226K mutant, whereas the movement is not observed in the V325D mutant. The movement relies on a PDZ vs. PD rotation which opens the PD-PDZ interface in a lid-like (budding flower-like in trimer) fashion. The noncovalent hinges A and B are provided by two clusters of interfacing residues, harboring V325D and V226K in the C- and N-terminal PD barrels, respectively. The opening of the subunit interfaces progresses in a sequential manner during the 50 ns MD simulation. In the systems without the ligand only minor PDZ shifts relative to PD are observed, but the interface does not open. Further activation-associated events, e.g. PDZ-L3 positional swap seen in any active HtrA protein (vs. HtrA2), were not observed. In summary, this study provides hints on the mechanism of activation of wtHtrA2, the dynamics of the inactive HtrA2V325D, but does not allow to explain an increased activity of HtrA2V226K.

The LD loop as an important structural element required for transmission of the allosteric signal in the HtrA (DegP) protease from Escherichia coli.

High-temperature requirement A (HtrA; DegP) from Escherichia coli, an important element of the extracytoplasmic protein quality-control system, is a member of the evolutionarily conserved family of serine proteases. The characteristic feature of this protein is its allosteric mode of activation. The regulatory loops, L3, L2, L1 and LD, play a crucial role in the transmission of the allosteric signal. Yet, the role of LD has not been fully elucidated. Therefore, we undertook a study to explain the role of the individual LD residues in inducing and maintaining the proteolytic activity of HtrA. We investigated the influence of amino acid substitutions located within the LD loop on the kinetics of a model substrate cleavage as well as on the dynamics of the oligomeric structure of HtrA. We found that the mutations that were expected to disturb the loop's structure and/or interactions with the remaining regulatory loops severely diminished the proteolytic activity of HtrA. The opposite effect, that is, increased activity, was observed for G174S substitution, which was predicted to strengthen the interactions mediated by LD. HtrAG174S protein had an equilibrium shifted toward the active enzyme and formed preferentially high-order oligomeric forms.

Intra- and intersubunit changes accompanying thermal activation of the HtrA2(Omi) protease homotrimer.

HtrA2(Omi) protease is involved in the maintenance of mitochondrial homeostasis and stimulation of apoptosis as well as in development of cancer and neurodegenerative disorders. The protein is a homotrimer whose subunits comprise serine protease domain (PD) and PDZ regulatory domain. In the basal, inactive state, a tight interdomain interface limits access both to the PDZ peptide (carboxylate) binding site and to the PD catalytic center. The molecular mechanism of activation is not well understood. To further the knowledge of HtrA2 thermal activation we monitored the dynamics of the PDZ-PD interactions during temperature increase using tryptophan-induced quenching (TrIQ) method. The TrIQ results suggested that during activation the PDZ domain changed its position versus PD inside a subunit, including a prominent change affecting the L3 regulatory loop of PD, and also changed its interactions with the PD of the adjacent subunit (PD*), specifically with its L1* regulatory loop containing the active site serine. The α5 helix of PDZ was involved in both, the intra- and intersubunit changes of interactions and thus seems to play an important role in HtrA2 activation. The amino acid substitutions designed to decrease the PDZ interactions with the PD or PD* promoted protease activity at a wide range of temperatures, which supports the conclusions based on the TrIQ analysis. The model presented in this work describes PDZ movement in relation to PD and PD*, resulting in an increased access to the peptide binding and active sites, and conformational changes of the L3 and L1* loops.

Structural and Functional Analysis of Human HtrA3 Protease and Its Subdomains.

Human HtrA3 protease, which induces mitochondria-mediated apoptosis, can be a tumor suppressor and a potential therapeutic target in the treatment of cancer. However, there is little information about its structure and biochemical properties. HtrA3 is composed of an N-terminal domain not required for proteolytic activity, a central serine protease domain and a C-terminal PDZ domain. HtrA3S, its short natural isoform, lacks the PDZ domain which is substituted by a stretch of 7 C-terminal amino acid residues, unique for this isoform. This paper presents the crystal structure of the HtrA3 protease domain together with the PDZ domain (ΔN-HtrA3), showing that the protein forms a trimer whose protease domains are similar to those of human HtrA1 and HtrA2. The ΔN-HtrA3 PDZ domains are placed in a position intermediate between that in the flat saucer-like HtrA1 SAXS structure and the compact pyramidal HtrA2 X-ray structure. The PDZ domain interacts closely with the LB loop of the protease domain in a way not found in other human HtrAs. ΔN-HtrA3 with the PDZ removed (ΔN-HtrA3-ΔPDZ) and an N-terminally truncated HtrA3S (ΔN-HtrA3S) were fully active at a wide range of temperatures and their substrate affinity was not impaired. This indicates that the PDZ domain is dispensable for HtrA3 activity. As determined by size exclusion chromatography, ΔN-HtrA3 formed stable trimers while both ΔN-HtrA3-ΔPDZ and ΔN-HtrA3S were monomeric. This suggests that the presence of the PDZ domain, unlike in HtrA1 and HtrA2, influences HtrA3 trimer formation. The unique C-terminal sequence of ΔN-HtrA3S appeared to have little effect on activity and oligomerization. Additionally, we examined the cleavage specificity of ΔN-HtrA3. Results reported in this paper provide new insights into the structure and function of ΔN-HtrA3, which seems to have a unique combination of features among human HtrA proteases.