Not an immune cell, but they may act like one-cells with immune properties outside the immune system.

The cells presented in this work are not classified as cells that make up the immune system. They, however, present functions and molecules, which are characteristic of immune cells. These characteristic functions are, for example, sensing threat, performing phagocytosis, presentation of foreign antigens, cytokine release or enhancing immune memory. The enlisted immune response mechanisms are carried out by the possession of molecules such as Toll-like receptors, receptors for the Fc fragment of IgG, major histocompatibility complex class II molecules, costimulatory CD80/CD86 proteins and molecules needed for NLRP3 (NOD-like family pyrin domain containing 3) inflammasome activation. Thanks to these properties, the described nonimmune cells play an important role in the local immune response and support of the entire body in the fight against pathogens. They constitute the first line of defense of tissues and organs against pathogens and molecules recognized as harmful. The cells described in this article are particularly important in immunologically privileged places (e.g. the Bowman's capsule in the kidney), where "typical" immune cells normally do not have access. In this paper, we present immune-like functions and molecule suites of resident kidney cells (podocytes and mesangial cells), cochlear resident cells, fibrocytes and fibroblasts, as well as some stem cells (mesenchymal stem cells and umbilical cord Wharton's jelly-derived cells).

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.

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.

Distinct Expression Patterns of Two Tumor Necrosis Factor Superfamily Member 15 Gene Isoforms in Human Colon Cancer.

BACKGROUND: Tumor necrosis factor superfamily member 15 (TNFSF15) gene is involved in development of several cancers. It encodes two proteins: tumor necrosis factor ligand-related molecule 1A (TL1A) and vascular endothelial growth inhibitor 192 (VEGI-192). The main receptor for TL1A is death receptor 3 (DR3). AIMS: We investigated expression of TL1A, VEGI-192, and DR3 transcripts in different stages of colon cancer and compared them with survival of patients. We also aimed to reveal possible effects of microsatellite instability (MSI) and selected TNFSF15 single-nucleotide polymorphisms (SNPs) on expression of this gene. METHODS: Forty-five healthy individuals and 95 colon cancer patients were included in the study. Expression of VEGI-192, TL1A, and DR3 was measured by quantitative PCR. SNP and MSI analyses were performed on DNA isolated from normal or cancer tissue. RESULTS: Expression of VEGI-192 and TL1A was elevated in colon cancer, although the level of VEGI-192 decreased, while the level of TL1A increased with the progression of cancer. Patients with low expression of TL1A and/or high expression of VEGI-192 in tumor-transformed tissue showed longer survival. DR3 expression was decreased in the cancer, but it did not change with the tumor progression. Alleles T of rs6478108 and G of rs6478109 SNPs were associated with elevated expression of the TNFSF15 gene. There was no relation between the MSI status and TNFSF15 expression levels. CONCLUSIONS: Expression of the TNFSF15 gene isoforms was associated with the progression of colon cancer. Levels of TL1A and VEGI-192 transcripts can be considered as independent prognostic factors for colon cancer.

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.

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.

Temperature-induced conformational changes within the regulatory loops L1-L2-LA of the HtrA heat-shock protease from Escherichia coli.

The present investigation was undertaken to characterize mechanism of thermal activation of serine protease HtrA (DegP) from Escherichia coli. We monitored the temperature-induced structural changes within the regulatory loops L1, L2 and LA using a set of single-Trp HtrA mutants. The accessibility of each Trp residue to aqueous medium at temperature range 25-45 degrees C was assessed by steady-state fluorescence quenching using acrylamide and these results in combination with mean fluorescence lifetimes (tau) and wavelength emission maxima (lambda(em)max) were correlated with the induction of the HtrA proteolytic activity. Generally the temperature shift caused better exposure of Trps to the quencher; although, each of the loops was affected differently. The LA loop seemed to be the most prone to temperature-induced conformational changes and a significant opening of its structure was observed even at the lowest temperatures tested (25-30 degrees C). To the contrary, the L1 loop, containing the active site serine, remained relatively unchanged up to 40 degrees C. The L2 loop was the most exposed element and showed the most pronounced changes at temperatures exceeding 35 degrees C. Summing up, the HtrA structure appears to open gradually, parallel to the gradual increase of its proteolytic activity.

Expression of human HtrA1, HtrA2, HtrA3 and TGF-beta1 genes in primary endometrial cancer.

The HtrA family of serine proteases takes part in cellular stress response including heat shock, inflammation and cancer. Downregulation of human HtrA1 and HtrA3 genes has been reported in some cancers, including endometrial cancer (EC), suggesting a tumor-suppressor role for both genes. The mechanism of the HtrA function is not known, however, evidence exists showing that both HtrA1 and HtrA3 regulate biological processes by modulating TGF-beta signaling. In the presented study the expression of human HtrA1, HtrA2, HtrA3 and TGF-beta1 genes was examined in 124 endometrial tissue specimens including 88 cancers and 36 normal endometria. The expression of the tested genes was evaluated at mRNA and protein levels by semi-quantitative RT-PCR and western blotting methods, respectively. Our results showed significant decrease of HtrA1 and HtrA3 mRNA and protein levels in EC compared to normal tissues. The most dramatic decrease was found for HtrA3 at both mRNA and protein levels (3.2- and 5.6-fold, respectively). Moreover, the HtrA3 protein (short isoform) was not detected in 19% of the cancers, and its level decreased from the premenopausal to the postmenopausal group. The HtrA2 protein levels were significantly lower in EC tissues compared to normal tissues. We also found a significant increase of the TGF-beta1 protein level in EC as well as a significant negative correlation between HtrA1/2/3 and TGF-beta1 relative protein levels. Our results showing downregulation of HtrA1 and HtrA3 gene expression support previous studies suggesting a tumor suppressor role for these genes. Furthermore, our data suggest that HtrA2 may be involved in EC development as well as suggest the involvement of HtrA1, HtrA2 and HtrA3 in the inhibition of TGF-beta signaling in endometrial tissues.

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.

Changes in expression of serine proteases HtrA1 and HtrA2 during estrogen-induced oxidative stress and nephrocarcinogenesis in male Syrian hamster.

Serine proteases HtrA1 and HtrA2 are involved in cellular stress response and development of several diseases, including cancer. Our aim was to examine the involvement of the HtrA proteins in acute oxidative stress response induced in hamster kidney by estrogen treatment, and in nephrocarcinogenesis caused by prolonged estrogenization of male Syrian hamster. We used semi-quantitative RT-PCR to estimate the HtrA1 and HtrA2 mRNA levels in kidney tissues, and Western blotting to monitor the amount of the HtrA proteins. Within the first five hours following estrogen administration both HtrA1 mRNA and the protein levels were increased significantly. No changes in the expression of HtrA2 were observed. This indicates that HtrA1 may be involved in the response against oxidative stress induced by estrogen treatment in hamster kidney. During prolonged estrogenization, a significant reduction of the HtrA1 mRNA and protein levels was observed after 6 months of estradiol treatment, while the expression of HtrA2 was significantly elevated starting from the third month. This suggests an involvement of the HtrA proteins in estrogen-induced nephrocarcinogenesis in hamster. Using fluorescence in situ hybridization we localized the HtrA1 gene at the qb3-4 region of Syrian hamster chromosome 2, the region known to undergo a nonrandom deletion upon prolonged estrogenization. It is possible that the reduced level of HtrA1 expression is due to this chromosomal aberration. A full-length cDNA sequence of the hamster HtrA1 gene was obtained. It codes for a 50 kDa protein which has 98 and 96% identity with mouse and human counterparts, respectively.

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.

Dynamics of estrogen-induced oxidative stress.

The objective of this study was to assess the dynamics of oxidative damage to cellular macromolecules such as proteins, lipids and DNA under conditions of oxidative stress triggering early stages of estrogen-dependent carcinogenesis. A rodent model of carcinogenesis was used. Syrian hamsters were sacrificed after 1, 3, 5 h and one month from the initial implantation of estradiol. Matching control groups were used. Kidneys as target organs for estradiol-mediated oxidative stress were excised and homogenized for biochemical assays. Subcellular fractions were isolated. Carbonyl groups (as a marker of protein oxidation) and lipid hydroxyperoxides were assessed. DNA was isolated and 8-oxodGuo was assessed. Electron paramagnetic resonance spectroscopy was used to confirm the results for lipid peroxidation. Exposition to estradiol in the rodent model leads to damage of macromolecules of the cell, including proteins and DNA, but not lipids. Proteins appear to be the primary target of the damage but are closely followed by DNA. It has previously been speculated that protein peroxides can increase DNA modifications. This time sequence was observed in our study. Nevertheless, the direct relation between protein and DNA damage still remains unsolved.

[Characterization of the HtrA family of proteins]. / Charakterystyka bialek z rodziny HtrA.

The HtrA family of proteins consists of evolutionary conserved serine proteases, which are homologues of the HtrA protein from a model bacterium Escherichia coli. They are widely distributed among organisms, prokaryotic as well as eukaryotic including humans. HtrA proteins participate in defense against stresses causing aberrations in protein structure, for example heat or oxidative stress. At least four human homologues have been identified. They are involved in cell growth and differentiation, apoptosis, and disturbances in their function may induce carcinogenesis, arthritic and neurodegenerative disorders. This article summarizes recent studies regarding the HtrA family of proteins, their structure, regulation and function. It also presents practical applications of this knowledge and perspective of its use in the future.

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.

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.

Analysis of the link between the redox state and enzymatic activity of the HtrA (DegP) protein from Escherichia coli.

Bacterial HtrAs are proteases engaged in extracytoplasmic activities during stressful conditions and pathogenesis. A model prokaryotic HtrA (HtrA/DegP from Escherichia coli) requires activation to cleave its substrates efficiently. In the inactive state of the enzyme, one of the regulatory loops, termed LA, forms inhibitory contacts in the area of the active center. Reduction of the disulfide bond located in the middle of LA stimulates HtrA activity in vivo suggesting that this S-S bond may play a regulatory role, although the mechanism of this stimulation is not known. Here, we show that HtrA lacking an S-S bridge cleaved a model peptide substrate more efficiently and exhibited a higher affinity for a protein substrate. An LA loop lacking the disulfide was more exposed to the solvent; hence, at least some of the interactions involving this loop must have been disturbed. The protein without S-S bonds demonstrated lower thermal stability and was more easily converted to a dodecameric active oligomeric form. Thus, the lack of the disulfide within LA affected the stability and the overall structure of the HtrA molecule. In this study, we have also demonstrated that in vitro human thioredoxin 1 is able to reduce HtrA; thus, reduction of HtrA can be performed enzymatically.

Escherichia coli small heat shock proteins IbpA/B enhance activity of enzymes sequestered in inclusion bodies.

Escherichia coli small heat shock proteins, IbpA/B, function as molecular chaperones and protect misfolded proteins against irreversible aggregation. IbpA/B are induced during overproduction of recombinant proteins and bind to inclusion bodies in E. coli cells. We investigated the effect of DeltaibpA/B mutation on formation of inclusion bodies and biological activity of enzymes sequestered in the aggregates in E. coli cells. Using three different recombinant proteins: Cro-beta-galactosidase, beta-lactamase and rat rHtrA1 we demonstrated that deletion of the ibpA/B operon did not affect the level of produced inclusion bodies. However, in aggregates containing IbpA/B a higher enzymatic activity was detected than in the IbpA/B-deficient inclusion bodies. These results confirm that IbpA/B protect misfolded proteins from inactivation in vivo.