Persistent infections with the human pathogen Helicobacter pylori (H. pylori) have been closely associated with the induction and progression of a wide range of gastric disorders, including acute and chronic gastritis, ulceration in the stomach and duodenum, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric adenocarcinoma. The pathogenesis of H. pylori is determined by a complicated network of manifold mechanisms of pathogen–host interactions, which involves a coordinated interplay of H. pylori pathogenicity and virulence factors with host cells. While these molecular and cellular mechanisms have been intensively investigated to date, the knowledge about outer membrane vesicles (OMVs) derived from H. pylori and their implication in bacterial pathogenesis is not well developed. In this review, we summarize the current knowledge on H. pylori-derived OMVs.

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.

HtrA2(Omi), belonging to the high-temperature requirement A (HtrA) family of stress proteins, is involved in the maintenance of mitochondrial homeostasis and in the stimulation of apoptosis, as well as in cancer and neurodegenerative disorders. The protein comprises a serine protease domain and a postsynaptic density of 95 kDa, disk large, and zonula occludens 1 (PDZ) regulatory domain and functions both as a protease and a chaperone. Based on the crystal structure of the HtrA2 inactive trimer, it has been proposed that PDZ domains restrict substrate access to the protease domain and that during protease activation there is a significant conformational change at the PDZ–protease interface, which removes the inhibitory effect of PDZ from the active site. The crystal structure of the HtrA2 active form is not available yet. HtrA2 activity markedly increases with temperature. To understand the molecular basis of this increase in activity, we monitored the temperature-induced structural changes using a set of single-Trp HtrA2 mutants with Trps located at the PDZ–protease interface. The accessibility of each Trp to aqueous medium was assessed by fluorescence quenching, and these results, in combination with mean fluorescence lifetimes and wavelength emission maxima, indicate that upon an increase in temperature the HtrA2 structure relaxes, the PDZ–protease interface becomes more exposed to the solvent, and significant conformational changes involving both domains occur at and above 30 °C. This conclusion correlates well with temperature-dependent changes of HtrA2 proteolytic activity and the effect of amino acid substitutions (V226K and R432L) located at the domain interface, on HtrA2 activity. Our results experimentally support the model of HtrA2 activation and provide an insight into the mechanism of temperature-induced changes in HtrA2 structure.

HtrA3 is a proapoptotic protease shown to promote drug‐induced cytotoxicity in lung cancer cells and proposed to have an anti‐tumor 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.