There Are No Insurmountable Barriers: Passage of the Helicobacter pylori VacA Toxin from Bacterial Cytoplasm to Eukaryotic Cell Organelle.

The Gram-negative bacterium Helicobacter pylori is a very successful pathogen, one of the most commonly identified causes of bacterial infections in humans worldwide. H. pylori produces several virulence factors that contribute to its persistence in the hostile host habitat and to its pathogenicity. The most extensively studied are cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA). VacA is present in almost all H. pylori strains. As a secreted multifunctional toxin, it assists bacterial colonization, survival, and proliferation during long-lasting infections. To exert its effect on gastric epithelium and other cell types, VacA undergoes several modifications and crosses multiple membrane barriers. Once inside the gastric epithelial cell, VacA disrupts many cellular-signaling pathways and processes, leading mainly to changes in the efflux of various ions, the depolarization of membrane potential, and perturbations in endocytic trafficking and mitochondrial function. The most notable effect of VacA is the formation of vacuole-like structures, which may lead to apoptosis. This review focuses on the processes involved in VacA secretion, processing, and entry into host cells, with a particular emphasis on the interaction of the mature toxin with host membranes and the formation of transmembrane pores.

E-Cadherin Orthologues as Substrates for the Serine Protease High Temperature Requirement A (HtrA).

Helicobacter pylori (H. pylori) expresses the serine protease and chaperone High temperature requirement A (HtrA) that is involved in periplasmic unfolded protein stress response. Additionally, H. pylori-secreted HtrA directly cleaves the human cell adhesion molecule E-cadherin leading to a local disruption of intercellular adhesions during pathogenesis. HtrA-mediated E-cadherin cleavage has been observed in response to a broad range of pathogens, implying that it is a prevalent mechanism in humans. However, less is known whether E-cadherin orthologues serve as substrates for bacterial HtrA. Here, we compared HtrA-mediated cleavage of human E-cadherin with murine, canine, and simian E-cadherin in vitro and during bacterial infection. We found that HtrA targeted mouse and dog E-cadherin equally well, whereas macaque E-cadherin was less fragmented in vitro. We stably re-expressed orthologous E-cadherin (Cdh1) in a CRISPR/Cas9-mediated cdh1 knockout cell line to investigate E-cadherin shedding upon infection using H. pylori wildtype, an isogenic htrA deletion mutant, or complemented mutants as bacterial paradigms. In Western blot analyses and super-resolution microscopy, we demonstrated that H. pylori efficiently cleaved E-cadherin orthologues in an HtrA-dependent manner. These data extend previous knowledge to HtrA-mediated E-cadherin release in mammals, which may shed new light on bacterial infections in non-human organisms.

The Periplasmic Oxidoreductase DsbA Is Required for Virulence of the Phytopathogen Dickeya solani.

In bacteria, the DsbA oxidoreductase is a crucial factor responsible for the introduction of disulfide bonds to extracytoplasmic proteins, which include important virulence factors. A lack of proper disulfide bonds frequently leads to instability and/or loss of protein function; therefore, improper disulfide bonding may lead to avirulent phenotypes. The importance of the DsbA function in phytopathogens has not been extensively studied yet. Dickeya solani is a bacterium from the Soft Rot Pectobacteriaceae family which is responsible for very high economic losses mainly in potato. In this work, we constructed a D. solani dsbA mutant and demonstrated that a lack of DsbA caused a loss of virulence. The mutant bacteria showed lower activities of secreted virulence determinants and were unable to develop disease symptoms in a potato plant. The SWATH-MS-based proteomic analysis revealed that the dsbA mutation led to multifaceted effects in the D. solani cells, including not only lower levels of secreted virulence factors, but also the induction of stress responses. Finally, the outer membrane barrier seemed to be disturbed by the mutation. Our results clearly demonstrate that the function played by the DsbA oxidoreductase is crucial for D. solani virulence, and a lack of DsbA significantly disturbs cellular physiology.

SurA-like and Skp-like Proteins as Important Virulence Determinants of the Gram Negative Bacterial Pathogens.

In the Gram-negative bacteria, many important virulence factors reach their destination via two-step export systems, and they must traverse the periplasmic space before reaching the outer membrane. Since these proteins must be maintained in a structure competent for transport into or across the membrane, they frequently require the assistance of chaperones. Based on the results obtained for the model bacterium Escherichia coli and related species, it is assumed that in the biogenesis of the outer membrane proteins and the periplasmic transit of secretory proteins, the SurA peptidyl-prolyl isomerase/chaperone plays a leading role, while the Skp chaperone is rather of secondary importance. However, detailed studies carried out on several other Gram-negative pathogens indicate that the importance of individual chaperones in the folding and transport processes depends on the properties of client proteins and is species-specific. Taking into account the importance of SurA functions in bacterial virulence and severity of phenotypes due to surA mutations, this folding factor is considered as a putative therapeutic target to combat microbial infections. In this review, we present recent findings regarding SurA and Skp proteins: their mechanisms of action, involvement in processes related to virulence, and perspectives to use them as therapeutic targets.

SecA - a multidomain and multitask bacterial export protein.

Most bacterial secretory proteins destined to the extracytoplasmic space are secreted posttranslationally by the Sec translocase. SecA, a key component of the Sec system, is the ATPase motor protein, directly responsible for transferring the preprotein across the cytoplasmic membrane. SecA is a large protein, composed of several domains, capable of binding client preproteins and a variety of partners, including the SecYEG inner membrane channel complex, membrane phospholipids and ribosomes. SecA-mediated translocation can be divided into two major steps: (1) targeting of the preproteins to the membrane translocation apparatus and (2) transport across the membrane through the SecYEG channel. In this review we present current knowledge regarding SecA structure and function of this protein in both translocation steps. The most recent model of the SecA-dependent preprotein mechanical translocation across the bacterial cytoplasmic membrane is described. A possibility of targeting SecA with inhibitory compounds as a strategy to combat pathogenic bacteria will be discussed as well.

Importance of two PDZ domains for the proteolytic and chaperone activities of Helicobacter pylori serine protease HtrA.

The Helicobacter pylori HtrA protein (HtrAHp ) is an important virulence factor involved in the infection process by proteolysis of components of the tight (claudin-8 and occludin) and adherens junctions (E-cadherin) between epithelial cells. As a protease and chaperone, HtrAHp is involved in protein quality control, which is particularly important under stress conditions. HtrAHp contains a protease domain and two C-terminal PDZ domains (PDZ1 and PDZ2). In the HtrA protein family, the PDZ domains are proposed to play important roles, including regulation of proteolytic activity. We therefore mutated the PDZ1 and PDZ2 domains in HtrAHp and studied the maintenance of proteolytic activity, assembly and rearrangement of the corresponding oligomeric forms. Our in vitro experiments demonstrated that at least PDZ1 is important for efficient substrate cleavage, while both PDZ domains are dispensable for the chaperone-like activity. However, in living H. pylori cells, only the mutant containing at least PDZ1, but not PDZ2, ensured bacterial growth under stressful conditions. Moreover, we can demonstrate that PDZ1 is crucial for HtrAHp oligomerization. Interestingly, all truncated proteolytically active HtrAHp variants were functional in the in vitro infection assay and caused damage to the E-cadherin-based adherens junctions. These findings provide valuable new insights into the function of HtrAHp in an important pathogen of humans.

Functional analysis and cryo-electron microscopy of Campylobacterjejuni serine protease HtrA.

Campylobacter jejuni is a predominant zoonotic pathogen causing gastroenteritis and other diseases in humans. An important bacterial virulence factor is the secreted serine protease HtrA (HtrA Cj ), which targets tight and adherens junctional proteins in the gut epithelium. Here we have investigated the function and structure of HtrA Cj using biochemical assays and cryo-electron microscopy. Mass spectrometry analysis identified differences and similarities in the cleavage site specificity for HtrA Cj by comparison to the HtrA counterparts from Helicobacter pylori and Escherichia coli. We defined the architecture of HtrA Cj at 5.8 Å resolution as a dodecamer, built of four trimers. The contacts between the trimers are quite loose, a fact that explains the flexibility and mobility of the dodecameric assembly. This flexibility has also been studied through molecular dynamics simulation, which revealed opening of the dodecamer to expose the proteolytically active site of the protease. Moreover, we examined the rearrangements at the level of oligomerization in the presence or absence of substrate using size exclusion chromatography, which revealed hexamers, dodecamers and larger oligomeric forms, as well as remarkable stability of higher oligomeric forms (> 12-mers) compared to previously tested homologs from other bacteria. Extremely dynamic decay of the higher oligomeric forms into lower forms was observed after full cleavage of the substrate by the proteolytically active variant of HtrA Cj . Together, this is the first report on the in-depth functional and structural analysis of HtrA Cj , which may allow the construction of therapeutically relevant HtrA Cj inhibitors in the near future.

Lon Protease Is Important for Growth Under Stressful Conditions and Pathogenicity of the Phytopathogen, Bacterium Dickeya solani.

The Lon protein is a protease implicated in the virulence of many pathogenic bacteria, including some plant pathogens. However, little is known about the role of Lon in bacteria from genus Dickeya. This group of bacteria includes important potato pathogens, with the most aggressive species, D. solani. To determine the importance of Lon for pathogenicity and response to stress conditions of bacteria, we constructed a D. solani Δlon strain. The mutant bacteria showed increased sensitivity to certain stress conditions, in particular osmotic and high-temperature stresses. Furthermore, qPCR analysis showed an increased expression of the lon gene in D. solani under these conditions. The deletion of the lon gene resulted in decreased motility, lower activity of secreted pectinolytic enzymes and finally delayed onset of blackleg symptoms in the potato plants. In the Δlon cells, the altered levels of several proteins, including virulence factors and proteins associated with virulence, were detected by means of Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) analysis. These included components of the type III secretion system and proteins involved in bacterial motility. Our results indicate that Lon protease is important for D. solani to withstand stressful conditions and effectively invade the potato plant.

Chaperone activity of serine protease HtrA of Helicobacter pylori as a crucial survival factor under stress conditions.

BACKGROUND: Serine protease HtrA exhibits both proteolytic and chaperone activities, which are involved in cellular protein quality control. Moreover, HtrA is an important virulence factor in many pathogens including Helicobacter pylori, for which the crucial stage of infection is the cleavage of E-cadherin and other cell-to-cell junction proteins. METHODS: The in vitro study of H. pylori HtrA (HtrAHp) chaperone activity was carried out using light scattering assays and investigation of lysozyme protein aggregates. We produced H. pylori ∆htrA deletion and HtrAHp point mutants without proteolytic activity in strain N6 and investigated the survival of the bacteria under thermal, osmotic, acidic and general stress conditions as well as the presence of puromycin or metronidazole using serial dilution tests and disk diffusion method. The levels of cellular and secreted proteins were examined using biochemical fraction and Western blotting. We also studied the proteolytic activity of secreted HtrAHp using zymography and the enzymatic digestion of ß-casein. Finally, the consequences of E-cadherin cleavage were determined by immunofluorescence microscopy. RESULTS: We demonstrate that HtrAHp displays chaperone activity that inhibits the aggregation of lysozyme and is stable under various pH and temperature conditions. Next, we could show that N6 expressing only HtrA chaperone activity grow well under thermal, pH and osmotic stress conditions, and in the presence of puromycin or metronidazole. In contrast, in the absence of the entire htrA gene the bacterium was more sensitive to a number of stresses. Analysing the level of cellular and secreted proteins, we noted that H. pylori lacking the proteolytic activity of HtrA display reduced levels of secreted HtrA. Moreover, we compared the amounts of secreted HtrA from several clinical H. pylori strains and digestion of ß-casein. We also demonstrated a significant effect of the HtrAHp variants during infection of human epithelial cells and for E-cadherin cleavage. CONCLUSION: Here we identified the chaperone activity of the HtrAHp protein and have proven that this activity is important and sufficient for the survival of H. pylori under multiple stress conditions. We also pinpointed the importance of HtrAHp chaperone activity for E- cadherin degradation and therefore for the virulence of this eminent pathogen.

Establishment of serine protease htrA mutants in Helicobacter pylori is associated with secA mutations.

Helicobacter pylori plays an essential role in the pathogenesis of gastritis, peptic ulcer disease, and gastric cancer. The serine protease HtrA, an important secreted virulence factor, disrupts the gastric epithelium, which enables H. pylori to transmigrate across the epithelium and inject the oncogenic CagA protein into host cells. The function of periplasmic HtrA for the H. pylori cell is unknown, mainly due to unavailability of the htrA mutants. In fact, htrA has been described as an essential gene in this bacterium. We have screened 100 worldwide H. pylori isolates and show that only in the N6 strain it was possible to delete htrA or mutate the htrA gene to produce proteolytically inactive HtrA. We have sequenced the wild-type and mutant chromosomes and we found that inactivation of htrA is associated with mutations in SecA - a component of the Sec translocon apparatus used to translocate proteins from the cytoplasm into the periplasm. The cooperation of SecA and HtrA has been already suggested in Streptococcus pneumonia, in which these two proteins co-localize. Hence, our results pinpointing a potential functional relationship between HtrA and the Sec translocon in H. pylori possibly indicate for the more general mechanism responsible to maintain bacterial periplasmic homeostasis.

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.

The Role of Proteases in the Virulence of Plant Pathogenic Bacteria.

A pathogenic lifestyle is inextricably linked with the constant necessity of facing various challenges exerted by the external environment (both within and outside the host). To successfully colonize the host and establish infection, pathogens have evolved sophisticated systems to combat the host defense mechanisms and also to be able to withstand adverse environmental conditions. Proteases, as crucial components of these systems, are involved in a variety of processes associated with infection. In phytopathogenic bacteria, they play important regulatory roles and modulate the expression and functioning of various virulence factors. Secretory proteases directly help avoid recognition by the plant immune systems, and contribute to the deactivation of the defense response pathways. Finally, proteases are important components of protein quality control systems, and thus enable maintaining homeostasis in stressed bacterial cells. In this review, we discuss the known protease functions and protease-regulated signaling processes associated with virulence of plant pathogenic bacteria.

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.

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.

Amino-Terminal Processing of Helicobacter pylori Serine Protease HtrA: Role in Oligomerization and Activity Regulation.

The HtrA family of serine proteases is found in most bacteria, and plays an essential role in the virulence of the gastric pathogen Helicobacter pylori. Secreted H. pylori HtrA (HtrA Hp ) cleaves various junctional proteins such as E-cadherin disrupting the epithelial barrier, which is crucial for bacterial transmigration across the polarized epithelium. Recent studies indicated the presence of two characteristic HtrA Hp forms of 55 and 52 kDa (termed p55 and p52, respectively), in worldwide strains. In addition, p55 and p52 were produced by recombinant HtrA Hp , indicating auto-cleavage. However, the cleavage sites and their functional importance are yet unclear. Here, we determined the amino-terminal ends of p55 and p52 by Edman sequencing. Two proteolytic cleavage sites were identified (H46/D47 and K50/D51). Remarkably, the cleavage site sequences are conserved in HtrA Hp from worldwide isolates, but not in other Gram-negative pathogens, suggesting a highly specific assignment in H. pylori. We analyzed the role of the amino-terminal cleavage sites on activity, secretion and function of HtrA Hp . Three-dimensional modeling suggested a trimeric structure and a role of amino-terminal processing in oligomerization and regulation of proteolytic activity of HtrA Hp . Furthermore, point and deletion mutants of these processing sites were generated in the recently reported Campylobacter jejuni ΔhtrA/htrAHp genetic complementation system and the minimal sequence requirements for processing were determined. Polarized Caco-2 epithelial cells were infected with these strains and analyzed by immunofluorescence microscopy. The results indicated that HtrA Hp processing strongly affected the ability of the protease to disrupt the E-cadherin-based cell-to-cell junctions. Casein zymography confirmed that the amino-terminal region is required for maintaining the proteolytic activity of HtrA Hp . Furthermore, we demonstrated that this cleavage influences the secretion of HtrA Hp in the extracellular space as an important prerequisite for its virulence activity. Taken together, our data demonstrate that amino-terminal cleavage of HtrA Hp is conserved in this pathogen and affects oligomerization and thus, secretion and regulatory activities, suggesting an important role in the pathogenesis of H. pylori.

Extracellular HtrA serine proteases: An emerging new strategy in bacterial pathogenesis.

The HtrA family of chaperones and serine proteases is important for regulating stress responses and controlling protein quality in the periplasm of bacteria. HtrA is also associated with infectious diseases since inactivation of htrA genes results in significantly reduced virulence properties by various bacterial pathogens. These virulence features of HtrA can be attributed to reduced fitness of the bacteria, higher susceptibility to environmental stress and/or diminished secretion of virulence factors. In some Gram-negative and Gram-positive pathogens, HtrA itself can be exposed to the extracellular environment promoting bacterial colonisation and invasion of host tissues. Most of our knowledge on the function of exported HtrAs stems from research on Helicobacter pylori, Campylobacter jejuni, Borrelia burgdorferi, Bacillus anthracis, and Chlamydia species. Here, we discuss recent progress showing that extracellular HtrAs are able to cleave cell-to-cell junction factors including E-cadherin, occludin, and claudin-8, as well as extracellular matrix proteins such as fibronectin, aggrecan, and proteoglycans, disrupting the epithelial barrier and producing substantial host cell damage. We propose that the export of HtrAs is a newly discovered strategy, also applied by additional bacterial pathogens. Consequently, exported HtrA proteases represent highly attractive targets for antibacterial treatment by inhibiting their proteolytic activity or application in vaccine development.

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.

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.