Structural and functional analysis of the ligand specificity of the HtrA2/Omi PDZ domain

The mitochondrial serine protease HtrA2/Omi helps to maintain mitochondrial function by handling misfolded proteins in the intermembrane space. In addition, HtrA2/Omi has been implicated as a proapoptotic factor upon release into the cytoplasm during the cell death cascade. The protein contains a C-terminal PDZ domain that packs against the protease active site and inhibits proteolytic activity. Engagement of the PDZ domain by peptide ligands has been shown to activate the protease and also has been proposed to mediate substrate recognition. We report a detailed structural and functional analysis of the human HtrA2/Omi PDZ domain using peptide libraries and affinity assays to define specificity, X-ray crystallography to view molecular details of PDZ-ligand interactions, and alanine-scanning mutagenesis to probe the peptide-binding groove. We show that the HtrA2/Omi PDZ domain recognizes both C-terminal and internal stretches of extended, hydrophobic polypeptides. High-affinity ligand recognition requires contacts with up to five hydrophobic side chains by distinct sites on the PDZ domain. However, no particular residue type is absolutely required at any position, and thus, the HtrA2/Omi PDZ domain appears to be a promiscuous module adapted to recognize unstructured, hydrophobic polypeptides. This type of specificity is consistent with the biological role of HtrA2/Omi in mitochondria, which requires the recognition of diverse, exposed stretches of hydrophobic sequences in misfolded proteins. The findings are less consistent with, but do not exclude, a role for the PDZ domain in targeting the protease to specific substrates during apoptosis.     

Structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3

High-temperature requirement A (HtrA) and its homologs contain a serine protease domain followed by one or two PDZ domains. Bacterial HtrA proteins and the mitochondrial protein HtrA2/Omi maintain cell function by acting as both molecular chaperones and proteases to manage misfolded proteins. The biological roles of the mammalian family members HtrA1 and HtrA3 are less clear. We report a detailed structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3 using peptide libraries and affinity assays to define specificity, structural studies to view the molecular details of ligand recognition, and alanine scanning mutagenesis to investigate the energetic contributions of individual residues to ligand binding. In common with HtrA2/Omi, we show that the PDZ domains of HtrA1 and HtrA3 recognize hydrophobic polypeptides, and while C-terminal sequences are preferred, internal sequences are also recognized. However, the details of the interactions differ, as different domains rely on interactions with different residues within the ligand to achieve high affinity binding. The results suggest that mammalian HtrA PDZ domains interact with a broad range of hydrophobic binding partners. This promiscuous specificity resembles that of bacterial HtrA family members and suggests a similar function for recognizing misfolded polypeptides with exposed hydrophobic sequences. Our results support a common activation mechanism for the HtrA family, whereby hydrophobic peptides bind to the PDZ domain and induce conformational changes that activate the protease. Such a mechanism is well suited to proteases evolved for the recognition and degradation of misfolded proteins.     

The C-terminal tail of presenilin regulates Omi/HtrA2 protease activity

Presenilin mutations are responsible for most cases of autosomal dominant inherited forms of early onset Alzheimer disease. Presenilins play an important role in amyloid beta-precursor processing, NOTCH receptor signaling, and apoptosis. However, the molecular mechanisms by which presenilins regulate apoptosis are not fully understood. Here, we report that presenilin-1 (PS1) regulates the proteolytic activity of the serine protease Omi/HtrA2 through direct interaction with its regulatory PDZ domain. We show that a peptide corresponding to the cytoplasmic C-terminal tail of PS1 dramatically increases the proteolytic activity of Omi/HtrA2 toward the inhibitor of apoptosis proteins and beta-casein and induces cell death in an Omi/HtrA2-dependent manner. Consistent with these results, ectopic expression of full-length PS1, but not PS1 lacking the C-terminal PDZ binding motif, potentiated Omi/HtrA2-induced cell death. Our results suggest that the C terminus of PS1 is an activation peptide ligand for the PDZ domain of Omi/HtrA2 and may regulate the protease activity of Omi/HtrA2 after its release from the mitochondria during apoptosis. This mechanism of Omi/HtrA2 activation is similar to the mechanism of activation of the related bacterial DegS protease by the outer-membrane porins.     

Molecular adaptation of the DegQ protease to exert protein quality control in the bacterial cell envelope

To react to distinct stress situations and to prevent the accumulation of misfolded proteins, all cells employ a number of proteases and chaperones, which together set up an efficient protein quality control system. The functionality of proteins in the cell envelope of Escherichia coli is monitored by the HtrA proteases DegS, DegP, and DegQ. In contrast with DegP and DegS, the structure and function of DegQ has not been addressed in detail. Here, we show that substrate binding triggers the conversion of the resting DegQ hexamer into catalytically active 12- and 24-mers. Interestingly, substrate-induced oligomer reassembly and protease activation depends on the first PDZ domain but not on the second. Therefore, the regulatory mechanism originally identified in DegP should be a common feature of HtrA proteases, most of which encompass only a single PDZ domain. Using a DegQ mutant lacking the second PDZ domain, we determined the high resolution crystal structure of a dodecameric HtrA complex. The nearly identical domain orientation of protease and PDZ domains within 12- and 24-meric HtrA complexes reveals a conserved PDZ1 → L3 → LD/L1/L2 signaling cascade, in which loop L3 senses the repositioned PDZ1 domain of higher order, substrate-engaged particles and activates protease function. Furthermore, our in vitro and in vivo data imply a pH-related function of DegQ in the bacterial cell envelope.     

Structural insights into the pro-apoptotic function of mitochondrial serine protease HtrA2/Omi

HtrA2/Omi, a mitochondrial serine protease in mammals, is important in programmed cell death. However, the underlining mechanism of HtrA2/Omi-mediated apoptosis remains unclear. Analogous to the bacterial homolog HtrA (DegP), the mature HtrA2 protein contains a central serine protease domain and a C-terminal PDZ domain. The 2.0 A crystal structure of HtrA2/Omi reveals the formation of a pyramid-shaped homotrimer mediated exclusively by the serine protease domains. The peptide-binding pocket of the PDZ domain is buried in the intimate interface between the PDZ and the protease domains. Mutational analysis reveals that the monomeric HtrA2/Omi mutants are unable to induce cell death and are deficient in protease activity. The PDZ domain modulates HtrA2/Omi-mediated cell death activity by regulating its serine protease activity. These structural and biochemical observations provide an important framework for deciphering the mechanisms of HtrA2/Omi-mediated apoptosis.     

HtrA1 serine protease inhibits signaling mediated by Tgfbeta family proteins

HtrA1, a member of the mammalian HtrA serine protease family, has a highly conserved protease domain followed by a PDZ domain. Because HtrA1 is a secretory protein and has another functional domain with homology to follistatin, we examined whether HtrA1 functions as an antagonist of Tgfbeta family proteins. During embryo development, mouse HtrA1 was expressed in specific areas where signaling by Tgfbeta family proteins plays important regulatory roles. The GST-pulldown assay showed that HtrA1 binds to a broad range of Tgfbeta family proteins, including Bmp4, Gdf5, Tgfbetas and activin. HtrA1 inhibited signaling by Bmp4, Bmp2, and Tgfbeta1 in C2C12 cells, presumably by preventing receptor activation. Experiments using a series of deletion mutants indicated that the binding activity of HtrA1 required the protease domain and a small linker region preceding it, and that inhibition of Tgfbeta signaling is dependent on the proteolytic activity of HtrA1. Misexpression of HtrA1 near the developing chick eye led to suppression of eye development that was indistinguishable from the effects of noggin. Taken together, these data indicate that HtrA1 protease is a novel inhibitor of Tgfbeta family members.     

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.     

Identification of a Novel HtrA1-susceptible Cleavage Site in Human Aggrecan: EVIDENCE FOR THE INVOLVEMENT OF HtrA1 IN AGGRECAN PROTEOLYSIS IN VIVO

Mass spectrometry-based proteomic analyses performed on cartilage tissue extracts identified the serine protease HtrA1/PRSS11 as a major protein component of human articular cartilage, with elevated levels occurring in association with osteoarthritis. Overexpression of a catalytically active form of HtrA1, but not an active site mutant (S328A), caused a marked reduction in proteoglycan content in chondrocyte-seeded alginate cultures. Aggrecan degradation fragments were detected in conditioned media from the alginate cultures overexpressing active HtrA1. Incubation of native or recombinant aggrecan with wild type HtrA1 resulted in distinct cleavage of these substrates. Cleavage of aggrecan by HtrA1 was strongly enhanced by HtrA1 agonists such as CPII, a C-terminal hexapeptide derived from the C-propeptide of procollagen IIα1 (i.e. chondrocalcin). A novel HtrA1-susceptible cleavage site within the interglobular domain (IGD) of aggrecan was identified, and an antibody that specifically recognizes the neoepitope sequence (VQTV³⁵⁶) generated at the HtrA1 cleavage site was developed. Western blot analysis demonstrated that HtrA1-generated aggrecan fragments containing the VQTV³⁵⁶ neoepitope were significantly more abundant in osteoarthritic cartilage compared with cartilage from healthy joints, implicating HtrA1 as a critical protease involved in proteoglycan turnover and cartilage degradation during degenerative joint disease.The mammalian high-temperature requirement A (HtrA) family of serine proteases is defined by a characteristic trypsin-like serine protease domain and one or two C-terminal PDZ domains. Four mammalian HtrA proteins have been identified to date, HtrA1–4. HtrA1 (also called PRSS11) is a ubiquitously expressed extracellular serine protease which contains a signal sequence for secretion, an insulin-like growth factor (IGF)²-binding protein domain, and a Kazal-type serine protease inhibitor domain in addition to the serine protease domain and one C-terminal PDZ domain (1). HtrA1 has been implicated in the progression of several pathologies including age-related macular degeneration, cancer, Alzheimer disease, rheumatoid arthritis, and osteoarthritis (OA) (2–10). HtrA1 has also been shown to inhibit osteoblast mineralization (11).Expression of HtrA1 has been found to be elevated in articular cartilage in association with OA (5). In addition, HtrA1 levels are up-regulated in murine cartilage after experimentally induced joint damage (6). The physiological role of HtrA1 in OA disease progression as well as in other pathologies is unclear. Preliminary studies using in vitro digestion assays suggest that HtrA1 might be capable of digesting cartilage extracellular matrix (ECM) proteins such as fibromodulin, cartilage oligomeric matrix protein (COMP), fibronectin, decorin, and aggrecan (6, 12, 13). Furthermore, it was recently reported that elevated levels of HtrA1 protein (∼7-fold above normal) are present in synovial fluids obtained from OA patients and that fibronectin fragments generated by HtrA1 cleavage induced the expression of catabolic enzymes such as matrix metalloproteinases-1 (MMP-1) and MMP-3 in synovial fibroblasts (4). HtrA1 has also been shown to modulate multiple signaling pathways in vitro. It binds to transforming growth factor-β family proteins including transforming growth factor-β1 and bone morphogenetic proteins 2 and 4 and inhibits signaling mediated by these factors (14, 15). In addition, HtrA1 has been shown to cleave IGF-binding protein-5 and possibly regulate signaling mediated by IGF (16). These findings suggest that the protease HtrA1 may play a physiological role in cartilage during OA.Articular cartilage is made up of chondrocytes surrounded by the ECM comprised mainly of the proteoglycan, aggrecan, and type II collagen. During normal homeostasis there is a dynamic balance between anabolic activities such as proteoglycan synthesis as well as catabolic activities in which the ECM is destroyed. When the catabolic activities of proteases, such as MMPs and aggrecanases, offset new matrix synthesis, focal degradation and loss of articular cartilage occurs, resulting in the development of OA. In some in vitro digestion studies, we and others have shown degradation of aggrecan by recombinant HtrA1 (6, 12, 13). In the present study we set out to examine the physiological relevance of aggrecan cleavage by HtrA1 in OA disease progression.     

A new principle of oligomerization of plant DEG7 protease based on interactions of degenerated protease domains

Deg/HtrA proteases are a large group of ATP-independent serine endoproteases found in almost every organism. Their usual domain arrangement comprises a trypsin-type protease domain and one or more PDZ domains. All Deg/HtrA proteases form homo-oligomers with trimers as the basic unit, where the active protease domain mediates the interaction between individual monomers. Among the members of the Deg/HtrA protease family, the plant protease DEG7 is unique since it contains two protease domains (one active and one degenerated) and four PDZ domains. In the present study, we investigated the oligomerization behaviour of this unusual protease using yeast two-hybrid analysis in vivo and with recombinant protein in vitro. We show that DEG7 forms trimeric complexes, but in contrast with other known Deg/HtrA proteases, it shows a new principle of oligomerization, where trimerization is based on the interactions between degenerated protease domains. We propose that, during evolution, a duplicated active protease domain degenerated and specialized in protein-protein interaction and complex formation.     

Regulation of bone matrix protein expression and induction of differentiation of human osteoblasts and human bone marrow stromal cells by bone morphogenetic protein-2

We have examined the effects of BMP-2 on the expression of bone matrix proteins in both human bone marrow stromal cells (HBMSC) and human osteoblasts (HOB) and their proliferation and mineralization. Both HBMSC and HOB express BMP-2/-4 type I and type II receptors. Treatment of these two cell types with BMP-2 for 4 weeks in the presence of β-glycerophosphate and ascorbic acid results in mineralization of their matrix. BMP-2 increases the mRNA level and activities of alkaline phosphatase and elevates the mRNA levels and protein synthesis of osteopontin, bone sialoprotein, osteocalcin, and α1(I) collagen in both cell types. Whereas the mRNA level of decorin is increased, the mRNA concentration of biglycan is not altered by BMP-2. No effect on osteonectin is observed. The effect of BMP-2 on bone matrix protein expression is dose dependent from 25 to 100 ng/ml and is evident after 1–7 days treatment. In the presence of BMP-2, proliferation of HBMSC and HOB is decreased under either serum-free condition or in the presence of serum. Thus, BMP-2 has profound effects on the proliferation, expression of most of the bone matrix proteins and the mineralization of both relatively immature human bone marrow stromal preosteoblasts and mature human osteoblasts. J. Cell. Biochem. 67:386–398, 1997. © 1997 Wiley-Liss, Inc.     

Temperature-induced changes of HtrA2(Omi) protease activity and structure

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.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-012-0355-1) contains supplementary material, which is available to authorized users.     

Rapid characterization of the binding property of HtrA2/Omi PDZ domain by validation screening of PDZ ligand library

HtrA2/Omi is a mammalian mitochondrial serine protease, and was found to have dual roles in mammalian cells, not only acting as an apoptosis-inducing protein but also maintaining mitochondrial homeostasis. PDZ domain is one of the most important protein-protein interaction modules and is involved in a variety of important cellular functions, such as signal transduction, degradation of proteins, and formation of cytoskeleton. Recently, it was reported that the PDZ domain of HtrA2/Omi might regulate proteolytic activity through its interactions with ligand proteins. In this study, we rapidly characterized the binding properties of HtrA2/Omi PDZ domain by validation screening of the PDZ ligand library with yeast two-hybrid approach. Then, we predicted its novel ligand proteins in human proteome and reconfirmed them in the yeast two-hybrid system. Finally, we analyzed the smallest networks bordered by the shortest path length between the protein pairs of novel interactions to evaluate the confidence of the identified interactions. The results revealed some novel binding properties of HtrA2/Omi PDZ domain. Besides the reported Class II PDZ motif, it also binds to Class I and Class III motifs, and exhibits restricted variability at P⁻³, which means that the P⁻³ residue is selected according to the composition of the last three residues. Seven novel ligand proteins of HtrA2/Omi PDZ domain were discovered, providing significant clues for further clarifying the roles of HtrA2/Omi. Moreover, this study proves the high efficiency and practicability of the newly developed validation screening of candidate ligand library method for binding property characterization of peptide-binding domains.     

Osteoactivin acts as downstream mediator of BMP-2 effects on osteoblast function

Our laboratory previously showed that osteoactivin (OA) is a novel, osteoblast-related glycoprotein that plays a role in osteoblast differentiation and function. The purpose of this study was to examine the regulation of OA expression by BMP-2 and the role OA plays as a downstream mediator of BMP-2 effects in osteoblast function. Using primary osteoblast cultures, we tested different doses of BMP-2 on the regulation of OA expression during osteoblast development. To test whether Smad-1 signaling is responsible for BMP-2 regulation of OA expression, osteoblast cultures were transfected with Smad1 siRNA, treated with 50 ng/ml of BMP-2 and analyzed by Western blot. BMP-2 treatment increased OA mRNA and protein expression in a dose-dependent manner and this upregulation was blocked in Smad1 siRNA transfected cultures. We next examined whether the role of OA as a downstream mediator of BMP-2 effects on osteoblast differentiation and matrix mineralization. Osteoblast cultures were transfected with OA antisense oligonucleotides and treated with 50 ng/ml of BMP-2. Cultures transfected with OA antisense oligonucleotides and treated with BMP-2 showed a reduction of OA expression associated with a significant reduction in early and late differentiation markers induced by BMP-2. Therefore, OA acts, at least in part, as a downstream mediator of BMP-2 effects on osteoblast differentiation and matrix mineralization. Our findings suggest that BMP-2 regulates OA expression through the Smad1 signaling pathway. Our data also emphasize that OA protein acts as a downstream mediator of BMP-2 effects on osteoblast differentiation and function.     

Bone morphogenetic protein receptor IB signaling mediates apoptosis independently of differentiation in osteoblastic cells

Bone morphogenetic protein-2 (BMP-2) is an important regulator of osteoblast differentiation. However, the regulation of osteoblast apoptosis by BMP signaling remains poorly understood. Here we examined the role of type I BMP receptor (BMP-RI) in osteoblast apoptosis promoted by BMP-2. Despite undetectable BMP-RIB expression in OHS4 cells, BMP-2 or BMP-2 overexpression increased osteoblast differentiation similarly as in SaOS2 cells which express BMP-RIB, as shown by alkaline phosphatase and CBFA1/RUNX2 expression. In contrast to SaOS2 cells, however, BMP-2 or BMP-2 overexpression did not increase caspase-9 and caspases-3, -6, and -7 activity and DNA fragmentation in OHS4 cells. Consistently, BMP-2 increased protein kinase C (PKC) activity, and PKC inhibition suppressed BMP-2-induced caspase activity in SaOS2 but not in OHS4 cells that lack BMP-RIB. A dominant negative BMP-RIB inhibited BMP-2-induced caspase activity, whereas wild-type BMP-RIB promoted caspase activity induced by BMP-2 in SaOS2 and MC3T3-E1 cells. Wild-type BMP-RIB rescued the apoptotic response to BMP-2, and a constitutively active BMP-RIB restored the apoptotic signal in OHS4 cells, supporting an essential role for BMP-RIB in osteoblast apoptosis. We also assessed whether BMP-2-induced apoptosis occurred independently of osteoblast differentiation. General inhibition of caspases did not abolish BMP-2-induced alkaline phosphatase and CBFA1/RUNX2 expression in SaOS2 cells. Furthermore, broad caspases inhibition increased matrix mineralization but did not reverse the BMP-2 effect on mineralization in MC3T3-E1 cells. These results indicate that BMP-2-induced apoptosis was mediated by BMP-RIB in osteoblasts and occurred independently of BMP-2-induced osteoblast differentiation, which provides additional insights into the dual mechanism of BMP-2 action on osteoblast fate.     

PDZ domains facilitate binding of high temperature requirement protease A (HtrA) and tail-specific protease (Tsp) to heterologous substrates through recognition of the small stable RNA A (ssrA)-encoded peptide

The Escherichia coli protease HtrA has two PDZ domains, and sequence alignments predict that the E. coli protease Tsp has a single PDZ domain. PDZ domains are composed of short sequences (80-100 amino acids) that have been implicated in a range of protein:protein interactions. The PDZ-like domain of Tsp may be involved in binding to the extreme COOH-terminal sequence of its substrate, whereas the HtrA PDZ domains are involved in subunit assembly and are predicted to be responsible for substrate binding and subsequent translocation into the active site. E. coli has a system of protein quality control surveillance mediated by the ssrA-encoded peptide tagging system. This system tags misfolded proteins or protein fragments with an 11-amino acid peptide that is recognized by a battery of cytoplasmic and periplasmic proteases as a degradation signal. Here we show that both HtrA and Tsp are able to recognize the ssrA-encoded peptide tag with apparent K(D) values of approximately 5 and 390 nm, respectively, and that their PDZ-like domains mediate this recognition. Fusion of the ssrA-encoded peptide tag to the COOH terminus of a heterologous protein (glutathione S-transferase) renders it sensitive to digestion by Tsp but not HtrA. These observations support the prediction that the HtrA PDZ domains facilitate substrate binding and the differential proteolytic responses of HtrA and Tsp to SsrA-tagged glutathione S-transferase are interpreted in terms of the structure of HtrA.     

Binding specificity and regulation of the serine protease and PDZ domains of HtrA2/Omi

Inhibitor of apoptosis proteins (IAPs) prevent apoptosis through direct inhibition of caspases. The serine protease HtrA2/Omi has an amino-terminal IAP interaction motif like that found in Reaper, which displaces IAPs from caspases, leading to enhanced caspase activity. The cell death-promoting properties of HtrA2/Omi are not only exerted through its capacity to oppose IAP inhibition of caspases but also through its integral serine protease activity. We have used peptide libraries to determine the optimal substrate sequence for cleavage by HtrA2 and also the preferred binding sequence for its PDZ domain. Using these peptides, we show that the PDZ domain of HtrA2/Omi suppresses the proteolytic activity unless it is engaged by a binding partner. Subjecting HtrA2/Omi to heat shock treatment also increases its protease activity. Unexpectedly, binding of X-linked inhibitor of apoptosis protein (XIAP) to the Reaper motif of HtrA2/Omi results in a marked increase in proteolytic activity, suggesting a new role for IAPs. When HtrA2/Omi is released from mitochondria following an apoptotic stimulus, binding to IAPs may switch their function from caspase inhibition to serine protease activation. Thus although IAP overexpression can suppress caspase activation, it could have the opposite effect on HtrA2/Omi-dependent cell death. This, together with the ability of HtrA2/Omi to degrade IAPs, may limit the overall cellular protection that can be provided by these proteins.     

Solution structure of HtrA PDZ domain from Streptococcus pneumoniae and its interaction with YYF-COOH containing peptides

High-temperature requirement A (HtrA), a highly conserved family of serine protease, plays crucial roles in protein quality control in prokaryotes and eukaryotes. The HtrA protein contains a C-terminal PDZ domain that mediates the proteolytic activity. Here we reported the solution structure of the HtrA PDZ domain from Streptococcus pneumoniae by NMR spectroscopy. Our results showed that the structure of HtrA PDZ domain, which contains three α-helices and five β-strands, illustrates conservation within the canonical PDZ domains. In addition, we demonstrated the interactions between S. pneumoniae HtrA PDZ domain and peptides with the motif XXX–YYF–COOH by surface plasmon resonance. Besides, we identified the ligand binding surface and the critical residues responsible for ligand binding of HtrA PDZ domain by chemical shift perturbation and site-directed mutagenesis.     

Rapid characterization of the binding property of HtrA2/Omi PDZ domain by validation screening of PDZ ligand library

HtrA2/Omi is a mammalian mitochondrial serine protease, and was found to have dual roles in mammalian cells, not only acting as an apoptosis-inducing protein but also maintaining mitochondrial homeostasis. PDZ domain is one of the most important protein-protein interaction modules and is involved in a variety of important cellular functions, such as signal transduction, degradation of proteins,and formation of cytoskeleton. Recently, it was reported that the PDZ domain of HtrA2/Omi might regulate proteolytic activity through its interactions with ligand proteins. In this study, we rapidly characterized the binding properties of HtrA2/Omi PDZ domain by validation screening of the PDZ ligand library with yeast two-hybrid approach. Then, we predicted its novel ligand proteins in human proteome and reconfirmed them in the yeast two-hybrid system. Finally, we analyzed the smallest networks bordered by the shortest path length between the protein pairs of novel interactions to evaluate the confidence of the identified interactions. The results revealed some novel binding properties of HtrA2/Omi PDZ domain. Besides the reported Class Ⅱ PDZ motif, it also binds to Class Ⅰ and Class Ⅲ motifs, and exhibits restricted variability at P-3, which means that the P-3 residue is selected according to the composition of the last three residues. Seven novel ligand proteins of HtrA2/Omi PDZ domain were discovered, providing significant clues for further clarifying the roles of HtrA2/Omi.Moreover, this study proves the high efficiency and practicability of the newly developed validation screening of candidate ligand library method for binding property characterization of peptide-binding domains.     

The HtrA family of proteases: implications for protein composition and cell fate

Cells precisely monitor the concentration and functionality of each protein for optimal performance. Protein quality control involves molecular chaperones, folding catalysts, and proteases that are often heat shock proteins. One quality control factor is HtrA, one of a new class of oligomeric serine proteases. The defining feature of the HtrA family is the combination of a catalytic domain with at least one C-terminal PDZ domain. Here, we discuss the properties and roles of this ATP-independent protease chaperone system in protein metabolism and cell fate.     

The role of human HtrA1 in arthritic disease

Human HtrA1 belongs to a widely conserved family of serine proteases involved in various aspects of protein quality control and cell fate. Although HtrA1 has been implicated in the pathology of several diseases, its precise biological functions remain to be established. Through identification of potential HtrA1 targets, studies presented herein propose that within the context of arthritis pathology HtrA1 contributes to cartilage degradation. Elevated synovial HtrA1 levels were detected in fluids obtained from rheumatoid and osteoarthritis patients, with synovial fibroblasts identified as a major source of secreted HtrA1. Mass spectrometry analysis of potential HtrA1 substrates within synovial fluids identified fibronectin as a candidate target, and treatment of fibronectin with recombinant HtrA1 led to the generation of fibronectin-degradation products that may be involved in cartilage catabolism. Consistently, treatment of synovial fibroblasts with HtrA1 or HtrA1-generated fibronectin fragments resulted in the specific induction of matrix metalloprotease 1 and matrix metalloprotease 3 expression, suggesting that HtrA1 contributes to the destruction of extracellular matrix through both direct and indirect mechanisms.