Przemyslaw Glaza

HtrA Protease Family as Therapeutic Targets

The HtrA proteases degrade damaged proteins and thus control the quality of proteins and protect cells against the consequences of various stresses; they also recognize specific protein substrates and in this way participate in regulation of many pathways. In many pathogenic bacteria strains lacking the HtrA function lose virulence or their virulence is decreased. This is due to an increased vulnerability of bacteria to stresses or to a decrease in secretion of virulence factors. In some cases HtrA is secreted outside the cell, where it promotes the pathogens invasiveness. Thus, the HtrA proteases of bacterial pathogens are attractive targets for new therapeutic approaches aimed at inhibiting their proteolytic activity. The exported HtrAs are considered as especially promising targets for chemical inhibitors. In this review, we characterize the model prokaryotic HtrAs and HtrAs of pathogenic bacteria, focusing on their role in virulence. In humans HtrA1, HtrA2(Omi) and HtrA3 are best characterized. We describe their role in promoting cell death in stress conditions and present evidence indicating that HtrA1 and HtrA2 function as tumor suppressors, while HtrA2 stimulates cancer cell death induced by chemotherapeutic agents. We characterize the HtrA2 involvement in pathogenesis of Parkinsons and Alzheimers diseases, and briefly describe the involvement of human HtrAs in other diseases. We hypothesize that stimulation of the HtrAs proteolytic activity might be beneficial in therapies of cancer and neurodegenerative disorders, and discuss the possibilities of modulating HtrA proteolytic activity considering the present knowledge about their structure and regulation.

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.

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.

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, β-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, β-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, β-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.

Interaction of substrate-mimicking peptides with the AAA+ ATPase ClpB from Escherichia coli

A molecular chaperone ClpB disaggregates and reactivates aggregated proteins in cooperation with DnaK, DnaJ, and GrpE. Within a cellular environment, ClpB must distinguish between properly folded and aggregated proteins by recognizing specific physical and/or chemical surface properties of the aggregates. However, the molecular mechanism of substrate binding to ClpB is poorly understood. We hypothesized that ClpB recognizes those polypeptide segments that promote protein aggregation because they are likely present at the surface of growing aggregates. We used an algorithm TANGO (Fernandez-Escamilla et al., Nat. Biotech. 2004, 22, 1302) to predict the aggregation-prone segments within the model ClpB-binding peptides and investigated interactions of the FITC-labeled peptides with ClpB using fluorescence anisotropy. We found that ClpB binds the substrate-mimicking peptides with positive cooperativity, which is consistent with an allosteric linkage between substrate binding and ClpB oligomerization. The apparent affinity towards ClpB for peptides displaying different predicted aggregation propensities correlates with the peptide length. However, discrete aggregation-prone segments within the peptides are neither sufficient nor necessary for efficient interaction with ClpB. Our results suggest that the substrate recognition mechanism of ClpB may rely on global surface properties of aggregated proteins rather than on local sequence motifs.

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.

HTRA3 (HtrA serine peptidase 3)

Review on HTRA3 (HtrA serine peptidase 3), with data on DNA, on the protein encoded, and where the gene is implicated.