Dual role of an essential HtrA2/Omi protease in the human malaria parasite: Maintenance of mitochondrial homeostasis and induction of apoptosis-like cell death under cellular stress.

Members of the HtrA family of serine proteases are known to play roles in mitochondrial homeostasis as well as in programmed cell death. Mitochondrial homeostasis and metabolism are crucial for the survival and propagation of the malaria parasite within the host. Here we have functionally characterized a Plasmodium falciparum HtrA2 (PfHtrA2) protein, which harbours trypsin-like protease activity that can be inhibited by its specific inhibitor, ucf-101. A transgenic parasite line was generated, using the HA-glmS C-terminal tagging approach, for localization as well as for inducible knock-down of PfHtrA2. The PfHtrA2 was localized in the parasite mitochondrion during the asexual life cycle. Genetic ablation of PfHtrA2 caused significant parasite growth inhibition, decreased replication of mtDNA, increased mitochondrial ROS production, caused mitochondrial fission/fragmentation, and hindered parasite development. However, the ucf-101 treatment did not affect the parasite growth, suggesting the non-protease/chaperone role of PfHtrA2 in the parasite. Under cellular stress conditions, inhibition of PfHtrA2 by ucf-101 reduced activation of the caspase-like protease as well as parasite cell death, suggesting the involvement of protease activity of PfHtrA2 in apoptosis-like cell death in the parasite. Under these cellular stress conditions, the PfHtrA2 gets processed but remains localized in the mitochondrion, suggesting that it acts within the mitochondrion by cleaving intra-mitochondrial substrate(s). This was further supported by trans-expression of PfHtrA2 protease domain in the parasite cytosol, which was unable to induce any cell death in the parasite. Overall, we show the specific roles of PfHtrA2 in maintaining mitochondrial homeostasis as well as in regulating stress-induced cell death.

Oligomeric assembly regulating mitochondrial HtrA2 function as examined by methyl-TROSY NMR.

Human High temperature requirement A2 (HtrA2) is a mitochondrial protease chaperone that plays an important role in cellular proteostasis and in regulating cell-signaling events, with aberrant HtrA2 function leading to neurodegeneration and parkinsonian phenotypes. Structural studies of the enzyme have established a trimeric architecture, comprising three identical protomers in which the active sites of each protease domain are sequestered to form a catalytically inactive complex. The mechanism by which enzyme function is regulated is not well understood. Using methyl transverse relaxation optimized spectroscopy (TROSY)-based solution NMR in concert with biochemical assays, a functional HtrA2 oligomerization/binding cycle has been established. In the absence of substrates, HtrA2 exchanges between a heretofore unobserved hexameric conformation and the canonical trimeric structure, with the hexamer showing much weaker affinity toward substrates. Both structures are substrate inaccessible, explaining their low basal activity in the absence of the binding of activator peptide. The binding of the activator peptide to each of the protomers of the trimer occurs with positive cooperativity and induces intrasubunit domain reorientations to expose the catalytic center, leading to increased proteolytic activity. Our data paint a picture of HtrA2 as a finely tuned, stress-protective enzyme whose activity can be modulated both by oligomerization and domain reorientation, with basal levels of catalysis kept low to avoid proteolysis of nontarget proteins.

Dissecting the role of interprotomer cooperativity in the activation of oligomeric high-temperature requirement A2 protein.

The human high-temperature requirement A2 (HtrA2) mitochondrial protease is critical for cellular proteostasis, with mutations in this enzyme closely associated with the onset of neurodegenerative disorders. HtrA2 forms a homotrimeric structure, with each subunit composed of protease and PDZ (PSD-95, DLG, ZO-1) domains. Although we had previously shown that successive ligand binding occurs with increasing affinity, and it has been suggested that allostery plays a role in regulating catalysis, the molecular details of how this occurs have not been established. Here, we use cysteine-based chemistry to generate subunits in different conformational states along with a protomer mixing strategy, biochemical assays, and methyl-transverse relaxation optimized spectroscopy-based NMR studies to understand the role of interprotomer allostery in regulating HtrA2 function. We show that substrate binding to a PDZ domain of one protomer increases millisecond-to-microsecond timescale dynamics in neighboring subunits that prime them for binding substrate molecules. Only when all three PDZ-binding sites are substrate bound can the enzyme transition into an active conformation that involves significant structural rearrangements of the protease domains. Our results thus explain why when one (or more) of the protomers is fixed in a ligand-binding-incompetent conformation or contains the inactivating S276C mutation that is causative for a neurodegenerative phenotype in mouse models of Parkinson's disease, transition to an active state cannot be formed. In this manner, wild-type HtrA2 is only active when substrate concentrations are high and therefore toxic and unregulated proteolysis of nonsubstrate proteins can be suppressed.

HTRA2/OMI-Mediated Mitochondrial Quality Control Alters Macrophage Polarization Affecting Systemic Chronic Inflammation.

Systemic chronic inflammation (SCI) due to intrinsic immune over-activation is an important factor in the development of many noninfectious chronic diseases, such as neurodegenerative diseases and diabetes mellitus. Among these immune responses, macrophages are extensively involved in the regulation of inflammatory responses by virtue of their polarization plasticity; thus, dysregulation of macrophage polarization direction is one of the potential causes of the generation and maintenance of SCI. High-temperature demand protein A2 (HtrA2/Omi) is an important regulator of mitochondrial quality control, not only participating in the degradation of mis-accumulated proteins in the mitochondrial unfolded protein response (UPRmt) to maintain normal mitochondrial function through its enzymatic activity, but also participating in the regulation of mitochondrial dynamics-related protein interactions to maintain mitochondrial morphology. Recent studies have also reported the involvement of HtrA2/Omi as a novel inflammatory mediator in the regulation of the inflammatory response. HtrA2/Omi regulates the inflammatory response in BMDM by controlling TRAF2 stabilization in a collagen-induced arthritis mouse model; the lack of HtrA2 ameliorates pro-inflammatory cytokine expression in macrophages. In this review, we summarize the mechanisms by which HtrA2/Omi proteins are involved in macrophage polarization remodeling by influencing macrophage energy metabolism reprogramming through the regulation of inflammatory signaling pathways and mitochondrial quality control, elucidating the roles played by HtrA2/Omi proteins in inflammatory responses. In conclusion, interfering with HtrA2/Omi may become an important entry point for regulating macrophage polarization, providing new research space for developing HtrA2/Omi-based therapies for SCI.

Elucidating the role of GRIM-19 as a substrate and allosteric activator of pro-apoptotic serine protease HtrA2.

HtrA2 (high-temperature requirement A2) and GRIM-19 (gene associated with retinoic and interferon-induced mortality 19 protein) are involved in various biological functions with their deregulation leading to multiple diseases. Although it is known that the interaction between GRIM-19 with HtrA2 promotes the pro-apoptotic activity of the latter, the mechanistic details remained elusive till date. Moreover, designing allosteric modulators of HtrA2 remains obscure due to lack of adequate information on the mode of interaction with its natural substrates cum binding partners. Therefore, in this study, we have unfolded the interaction between HtrA2 and GRIM-19 so as to understand its subsequent functional repercussions. Using in silico analyses and biochemical assays, we identified the region in GRIM-19 that is involved in protein-protein interaction with HtrA2. Furthermore, we have presented a comprehensive illustration of HtrA2's cleavage site specificity. Quantitative analysis using enzyme kinetics underscored the role of GRIM-19 in significant allosteric activation of HtrA2. Overall, this is an extensive study that not only defines HtrA2-GRIM-19 interaction, but also creates a framework for developing strategies toward allosteric regulation of HtrA2 for future therapeutic interventions.

Intramitochondrial proteostasis is directly coupled to α-synuclein and amyloid ß1-42 pathologies.

Mitochondrial dysfunction has long been implicated in the neurodegenerative disorder Parkinson's disease (PD); however, it is unclear how mitochondrial impairment and α-synuclein pathology are coupled. Using specific mitochondrial inhibitors, EM analysis, and biochemical assays, we report here that intramitochondrial protein homeostasis plays a major role in α-synuclein aggregation. We found that interference with intramitochondrial proteases, such as HtrA2 and Lon protease, and mitochondrial protein import significantly aggravates α-synuclein seeding. In contrast, direct inhibition of mitochondrial complex I, an increase in intracellular calcium concentration, or formation of reactive oxygen species, all of which have been associated with mitochondrial stress, did not affect α-synuclein pathology. We further demonstrate that similar mechanisms are involved in amyloid-ß 1-42 (Aß42) aggregation. Our results suggest that, in addition to other protein quality control pathways, such as the ubiquitin-proteasome system, mitochondria per se can influence protein homeostasis of cytosolic aggregation-prone proteins. We propose that approaches that seek to maintain mitochondrial fitness, rather than target downstream mitochondrial dysfunction, may aid in the search for therapeutic strategies to manage PD and related neuropathologies.

Dual specificity phosphatase 9: A novel binding partner cum substrate of proapoptotic serine protease HtrA2.

Human high temperature requirement protease A2 (HtrA2) is a trimeric PDZ bearing proapoptotic serine protease, which is involved in various cellular processes and pathologies. Research in the last decade strongly advocates its role as a potential therapeutic target and therefore warrants the need to minutely investigate its mechanism of action, regulation, interactions with other proteins and its binding specificities. In this particular study, we adopted an in silico approach to predict novel interacting partners and/or substrates of HtrA2 by building a peptide library using a binding pattern search. This library was used to look for novel ligand proteins in the human proteome. Thereafter, the putative interaction was validated using biochemical and cell-based studies. In a first, here we report that HtrA2 shows robust interactions with DUSP9 (Dual specificity phosphatase 9) in GST-pulldown and Co-Immunoprecipitation (Co-IP) experiments and cleaves it in vitro. Besides, we also provided a detailed characterization of the interaction interface. Moreover, this study in general provides an efficient, fast and practical method of candidate ligand library screening for exploring the binding properties of HtrA2.

Acetaldehyde dehydrogenase 2 deficiency increases mitochondrial reactive oxygen species emission and induces mitochondrial protease Omi/HtrA2 in skeletal muscle.

Acetaldehyde dehydrogenase 2 (ALDH2) is an enzyme involved in redox homeostasis as well as the detoxification process in alcohol metabolism. Nearly 8% of the world's population have an inactivating mutation in the ALDH2 gene. However, the expression patterns and specific functions of ALDH2 in skeletal muscles are still unclear. Herein, we report that ALDH2 is expressed in skeletal muscle and is localized to the mitochondrial fraction. Oxidative muscles had a higher amount of ALDH2 protein than glycolytic muscles. We next comprehensively investigated whether ALDH2 knockout in mice induces mitochondrial adaptations in gastrocnemius muscle (for example, content, enzymatic activity, respiratory function, supercomplex formation, and functional networking). We found that ALDH2 deficiency resulted in partial mitochondrial dysfunction in gastrocnemius muscle because it increased mitochondrial reactive oxygen species (ROS) emission (2',7'-dichlorofluorescein and MitoSOX oxidation rate during respiration) and the frequency of regional mitochondrial depolarization. Moreover, we determined whether ALDH2 deficiency and the related mitochondrial dysfunction trigger mitochondrial stress and quality control responses in gastrocnemius muscle (for example, mitophagy markers, dynamics, and the unfolded protein response). We found that ALDH2 deficiency upregulated the mitochondrial serine protease Omi/HtrA2 (a marker of the activation of a branch of the mitochondrial unfolded protein response). In summary, ALDH2 deficiency leads to greater mitochondrial ROS production, but homeostasis can be maintained via an appropriate stress response.

Structural modeling and role of HAX-1 as a positive allosteric modulator of human serine protease HtrA2.

HAX-1, a multifunctional protein involved in cell proliferation, calcium homeostasis, and regulation of apoptosis, is a promising therapeutic target. It regulates apoptosis through multiple pathways, understanding of which is limited by the obscurity of its structural details and its intricate interaction with its cellular partners. Therefore, using computational modeling, biochemical, functional enzymology and spectroscopic tools, we predicted the structure of HAX-1 as well as delineated its interaction with one of it pro-apoptotic partner, HtrA2. In this study, three-dimensional structure of HAX-1 was predicted by threading and ab initio tools that were validated using limited proteolysis and fluorescence quenching studies. Our pull-down studies distinctly demonstrate that the interaction of HtrA2 with HAX-1 is directly through its protease domain and not via the conventional PDZ domain. Enzymology studies further depicted that HAX-1 acts as an allosteric activator of HtrA2. This 'allosteric regulation' offers promising opportunities for the specific control and functional modulation of a wide range of biological processes associated with HtrA2. Hence, this study for the first time dissects the structural architecture of HAX-1 and elucidates its role in PDZ-independent activation of HtrA2.

Global Interactome Mapping of Mitochondrial Intermembrane Space Proteases Identifies a Novel Function for HTRA2.

A number of unique proteases localize to specific sub-compartments of the mitochondria, but the functions of these enzymes are poorly defined. Here, in vivo proximity-dependent biotinylation (BioID) is used to map the interactomes of seven proteases localized to the mitochondrial intermembrane space (IMS). In total, 802 high confidence proximity interactions with 342 unique proteins are identified. While all seven proteases co-localized with the IMS markers OPA1 and CLPB, 230 of the interacting partners are unique to just one or two protease bait proteins, highlighting the ability of BioID to differentiate unique interactomes within the confined space of the IMS. Notably, high-temperature requirement peptidase 2 (HTRA2) interacts with eight of 13 components of the mitochondrial intermembrane space bridging (MIB) complex, a multiprotein assembly essential for the maintenance of mitochondrial cristae structure. Knockdown of HTRA2 disrupts cristae in HEK 293 and OCI-AML2 cells, and leads to increased intracellular levels of the MIB subunit IMMT. Using a cell-free assay it is demonstrated that HTRA2 can degrade recombinant IMMT but not two other core MIB complex subunits, SAMM50 and CHCHD3. The IMS protease interactome thus represents a rich dataset that can be mined to uncover novel IMS protease biology.

p53 mediated transcription of Omi/HtrA2 in aging myocardium.

AIMS: Omi/HtrA2 is a pro-apoptotic protein, increased mRNA and protein levels of Omi/HtrA2 in aging myocardium facilitates apoptosis and affects mitochondrial homeostasis. Our previous study found that p53 can bind to the Omi/HtrA2 promoter. The purpose of this study was to determine whether p53 participates in regulating the expression of Omi/HtrA2 in aging myocardium. METHODS AND RESULTS: we used Western blot to detect the expression of Omi/HtrA2 and p53 nucleoprotein, and then found that both of them were elevated in aging heart. Furthermore, we also observed the increased binding of p53 to Omi/HtrA2 promoter by chromatin immunoprecipitation. To initially explore the regulation mechanism of Omi/HtrA2, plasmid transfection and RNA interference in NIH3T3 cells were used to upregulate or knock down p53, respectively. The mRNA and protein levels of Omi/HtrA2 were increased with the overexpression of p53 by real-time PCR and Western blot, and Omi/HtrA2 promoter activity enhanced after transfected with pcDNA3.1-p53. The result from RNA interference was quite the contrary.Our study demonstrated that the binding ability of p53 to Omi/HtrA2 promoter was increased in aging myocardium, and increased p53 promoted the mRNA and protein levels of Omi/HtrA2 by enhancing the promoter activity of Omi/HtrA2. CONCLUSIONS: p53 acts as a transcriptional factor that induces Omi/HtrA2 expression in aged cardiomyocytes.These results provide a new way to explore the mechanism of increased Omi/HtrA2 in the aging process of heart.

Extracellular HtrA2 Induces Apoptosis in Human Umbilical Vein Endothelial Cells.

The serine protease high-temperature-required protein A2 (HtrA2) has been identified as a key intracellular molecule promoting apoptosis in cells during ischemia reperfusion (IR) injury. IR injury in ST-segment elevation myocardial infarction (STEMI) contributes to overall myocardial damage. HtrA2 has further been shown to be significantly increased in the serum of patients with STEMI. In the present pilot study, we use human umbilical vein endothelial cells (HUVECs) to investigate whether extracellular HtrA2 induces apoptosis using Annexin V staining. Furthermore, we examine whether HtrA2 is released extracellularly after staurosporine-induced apoptosis using ELISA. We find that HtrA2 is released upon induction of apoptosis by staurosporine into the cell culture medium. Furthermore, treatment of HUVECs with extracellular HtrA2-induces apoptosis, while the addition of anti-HtrA2 antibodies reduces both HtrA2- and staurosporine-induced endothelial cell apoptosis. In conclusion, we show here that extracellular HtrA2 induces apoptosis in human endothelial cells, although the exact molecular mechanisms have to be investigated in future.

Effect of Mst1 on Endometriosis Apoptosis and Migration: Role of Drp1-Related Mitochondrial Fission and Parkin-Required Mitophagy.

BACKGROUND/AIMS: Mitochondrial homeostasis is implicated in the development and progression of endometriosis through poorly defined mechanisms. Mst1 is the major growth suppressor related to cancer migration, apoptosis and proliferation. However, whether Mst1 is involved in endometriosis apoptosis and migration via regulating the mitochondrial function remains to be elucidated. METHODS: Expression of Mst1 in endometriosis was examined via western blots. Cellular apoptosis was detected via MTT and TUNEL assay. Gain of function assay about Mst1 was conducted via adenovirus over-expression. Mitochondrial functions were evaluated via mitochondrial membrane potential JC-1 staining, ROS flow cytometry analysis, mPTP opening assessment and immunofluorescence of HtrA2/Omi. The mitophagy activity were examined via western blots and immunofluorescence. RESULTS: First, we found that Mst1 was significantly downregulated in the ectopic endometrium of endometriosis compared to the normal endometrium. However, the recovery of Mst1 function was closely associated with the inability of endometrial stromal cells (ESCs) to migrate and survive. A functional study indicated that regaining Mst1 enhanced Drp1 post-transcriptional phosphorylation at Ser616 and repressed Parkin transcription activity via p53, leading to mitochondrial fission activation and mitophagy inhibition. Excessive Drp1-related fission forced the mitochondria to liberate HtrA2/Omi into the cytoplasm. Moreover, Mst1-induced defective mitophagy evoked cellular oxidative stress, energy metabolism and calcium overload. Through excessive mitochondrial fission and aberrant mitophagy, Mst1 launched caspase 9-related mitochondrial apoptosis and abrogated F-actin/lamellipodium-dependent cellular migration. Notably, we also defined NR4A/miR181c as the upstream signal for Mst1 dysfunction in endometriosis. CONCLUSION: Collectively, our results comprehensively described the important role of the NR4A-miR181c-Mst1 pathway in endometriosis, which handled mitochondrial apoptosis and F-actin/ lamellipodium-based migration via the regulation of Drp1-related mitochondrial fission and Parkin-required mitophagy, with a potential application in endometriosis therapy by limiting ESCs migration and promoting apoptosis.

Omi/HtrA2 Regulates a Mitochondria-Dependent Apoptotic Pathway in a Murine Model of Septic Encephalopathy.

BACKGROUND/AIMS: the pathogenesis of sepsis-associated encephalopathy (SAE) is multifactorial, involving neurotransmitter alterations, inflammatory cytokines, oxidative damage, mitochondrial dysfunction, apoptosis, and other factors. Mitochondria are major producers of reactive oxygen species, resulting in cellular injury. Omi/HtrA2 is a proapoptotic mitochondrial serine protease involved in caspase-dependent cell death; it is translocated from mitochondria to the cytosol after an apoptotic insult. We previously found that UCF-101, a specific inhibitor of Omi/HtrA2, has neuroprotective effects on cerebral oxidative injury and cognitive impairment in septic rats. In this study, the mechanisms and molecular pathways underlying these effects were investigated. METHODS: Male Sprague-Dawley rats were subjected to cecal ligation and puncture (CLP) or sham-operated laparotomy and were administered vehicle or UCF-101 (10 µmol/kg). The hippocampus was isolated for subsequent analysis. Omi/HtrA2 expression in the mitochondria or cytosol was evaluated by immunofluorescence or western blotting. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining was utilized to evaluate levels of apoptosis, and western blotting was used to evaluate apoptosis-related proteins, such as cleaved caspase-3, caspase-9, and poly (ADP-ribose) polymerase (PARP). Tight junction expression was assessed by immunofluorescence and western blotting. Mitochondrial function, inflammatory cytokines, and oxidative stress were also assayed. In addition, a wet/dry method was used to evaluate brain edema and Evans blue extravasation was used to evaluate blood-brain barrier (BBB) integrity. RESULTS: After CLP treatment, the hippocampus exhibited a mild increase in Omi/HtrA2 expression; cytosolic Omi/HtrA2 expression increased significantly, whereas mitochondrial Omi/HtrA2 expression was reduced, indicating that CLP-induced oxidative stress resulted in the translocation of Omi/HtrA2 from mitochondria to the cytosol. Hippocampal cleaved caspase-3, caspase-9, and PARP levels were significantly higher in animals treated with CLP than in sham-operated animals, while XIAP expression was lower. Treatment with UCF-101 prevented the mobilization of Omi/HtrA2 from mitochondria to the cytosol, attenuated XIAP degradation, and decreased cleaved caspase-3, caspase-9, and PARP expression as well as apoptosis. UCF-101 also reversed the decreased mitochondrial complex I, II, and III respiration and the reduced ATP caused by CLP. In addition, UCF-101 treatment resulted in a significant improvement in BBB integrity, as demonstrated by increased occludin, claudin-5, and zonula occludens 1 levels and reduced Evans blue extravasation. No significant effects of UCF-101 on brain edema were found. Inflammatory cytokines and oxidative stress were significantly higher in the CLP-treated group than in the sham-operated group. However, the inhibition of Omi/HtrA2 by UCF-101 significantly alleviated these responses. CONCLUSION: Our data indicated that Omi/ HtrA2 regulates a mitochondria-dependent apoptotic pathway in a murine model of septic encephalopathy. Inhibition of Omi/HtrA2 by UCF-101 leads to neuroprotection by inhibiting the cytosolic translocation of Omi/HtrA2 and antagonizing the caspase-dependent apoptosis pathway. Therapeutic interventions that inhibit Omi/HtrA2 translocation or protease activity may provide a novel method to treat SAE.

Quantitative biochemical characterization and biotechnological production of caspase modulator, XIAP: Therapeutic implications for apoptosis-associated diseases.

BACKGROUND: Regulating apoptosis is a common and essential therapeutic strategy for cancer and neurodegenerative disorders. Based on basic studies of apoptotic mechanisms, various researches have attempted to overcome the pathogenesis of such diseases by activating or inhibiting apoptosis. Generally, the biochemical characteristics of the target molecules should be evaluated along with understanding of their mechanisms of action during drug development. Among apoptotic regulators, XIAP serves as a potent negative regulator to block apoptosis through the inhibition of caspase (CASP)-9 and -3/7. Although XIAP is an attractive target with such apoptotic-modulating property, biochemical and biophysical studies of XIAP are still challenging. METHODS: In this study, the CASP-9 and -3/7 inhibitors XIAP, 242Δ and Δ230 were prepared using the pGEX expression system and biochemically characterized. RESULTS: These inhibitors were expressed in Escherichia coli at a concentration of ≥20 mg/L culture under a native condition with 0.01 mM IPTG induction. Notably, using a simple and rapid affinity purification technique, these CASP-9 and -3/7 inhibitors have been purified, yielding ≥5 mg/L culture at approximately 90% purity. CONCLUSIONS: We have determined that HtrA2 specifically binds to the BIR2 and BIR3 of XIAP at a 1:1 molecular ratio. Moreover, in vitro cell-free CASP-9 and -3/7 activation-apoptosis assays have demonstrated that these purified XIAP proteins dramatically inhibit CASP-9 and -3/7 action. GENERAL SIGNIFICANCE: Our system is suitable for biochemical studies, such as quantitation of the number of molecules acting on the apoptosis regulation, and provides a basis and insights that can be applied to the development of therapeutic agents for neurodegenerative disorders and cancer.

YAP Inhibits the Apoptosis and Migration of Human Rectal Cancer Cells via Suppression of JNK-Drp1-Mitochondrial Fission-HtrA2/Omi Pathways.

BACKGROUND/AIMS: The Hippo-Yap pathway is associated with tumor development and progression. However, little evidence is available concerning its role in cancer cell apoptosis and migration via mitochondrial homeostasis. Here, we identify mitochondrial fission as a regulator of the Hippo-Yap pathway in human rectal cancer tumorigenesis and metastasis. METHODS: In this study, we performed loss-of function assays concerning Yap in RCC via shRNA. Cellular viability and apoptosis were measured via MTT, the TUNEL assay and trypan blue staining. Mitochondrial function was assessed via JC1 staining, the mPTP opening assay, mitochondrial respiratory function analysis, electron microscopy and immunofluorescence analysis of HtrA2/Omi. Mitophagy and mitochondrial fission were assessed via western blots and immunofluorescence. Cell migration was evaluated via the Transwell assay, wound-healing assay and immunofluorescence analysis of F-actin. The interaction between JNK and Yap was detected via co-immunoprecipitation and Yap recombinant mutagenic plasmid transfection. Western blots were used to analyze signaling pathways in conjunction with JNK inhibitors or HtrA2/Omi siRNA. RESULTS: Yap is upregulated in human rectal cancer cells, where its expression correlates positively with cell survival and migration. Functional studies established that silencing of Yap drove JNK phosphorylation, which induced Drp1 activation and translocation to the surface of mitochondria, initiating mitochondrial fission. Excessive mitochondrial fission mediated HtrA2/Omi leakage from the mitochondria into the cytoplasm, where HtrA2/Omi triggered cellular apoptosis via the mitochondrial apoptosis pathway. Moreover, released HtrA2/Omi also phosphorylated cofilin and inhibited cofilin-mediated F-actin polymerization. F-actin collapse perturbed lamellipodia formation and therefore impaired cellular migration and invasion. CONCLUSION: Collectively, our results demonstrate that Hippo-Yap can serve as a tumor promoter in human rectal cancer and acts by restricting JNK/Drp1/mitochondrial fission/ HtrA2/Omi, with potential implications for new approaches to human rectal cancer therapy.

Structural basis of substrate recognition and allosteric activation of the proapoptotic mitochondrial HtrA2 protease.

The mitochondrial serine protease HtrA2 is a human homolog of the Escherichia coli Deg-proteins exhibiting chaperone and proteolytic roles. HtrA2 is involved in both apoptotic regulation via its ability to degrade inhibitor-of-apoptosis proteins (IAPs), as well as in cellular maintenance as part of the cellular protein quality control machinery, by preventing the possible toxic accumulation of aggregated proteins. In this study, we use advanced solution NMR spectroscopy methods combined with biophysical characterization and biochemical assays to elucidate the crucial role of the substrate recognizing PDZ domain. This domain regulates the protease activity of HtrA2 by triggering an intricate allosteric network involving the regulatory loops of the protease domain. We further show that divalent metal ions can both positively and negatively modulate the activity of HtrA2, leading to a refined model of HtrA2 regulation within the apoptotic pathway.

Exploring the Role of HtrA Family Genes in Cancer: A Systematic Review.

PURPOSE: HtrA1, HtrA2, HtrA3 and HtrA4 appear to be involved in the development of pathologies such as cancer. This systematic review reports the results of a literature search performed to compare the expression of HtrA family genes and proteins in cancer versus non-cancer tissues and cell lines, assess relationships between HtrA expression and cancer clinical features in cancer, and analyse the molecular mechanism, by which HtrA family affects cancer. METHODS: The literature search was conducted according to the PRISMA statement among four databases (PubMed, Web of Science, Embase and Scopus). RESULTS: A total of 38 articles met the inclusion criteria and involved the expression of HtrA family members and concerned the effect of HtrA expression on cancer and metastasis development or on the factor that influences it. Additionally, 31 reports were retrieved manually. Most articles highlighted that HtrA1 and HtrA3 exhibited tumour suppressor activity, while HtrA2 was associated with tumour growth and metastasis. There were too few studies to clearly define the role of the HtrA4 protease in tumours. CONCLUSION: Although the expression of serine proteases of the HtrA family was dependent on tumour type, stage and the presence of metastases, most articles indicated that HtrA1 and HtrA3 expression in tumours was downregulated compared with healthy tissue or cell lines. The expression of HtrA2 was completely study dependent. The limited number of studies on HtrA4 expression made it impossible to draw conclusions about differences in expression between healthy and tumour tissue. The conclusions drawn from the study suggest that HtrA1 and HtrA3 act as tumour suppressors.

Structures of BIRC6-client complexes provide a mechanism of SMAC-mediated release of caspases.

Tight regulation of apoptosis is essential for metazoan development and prevents diseases such as cancer and neurodegeneration. Caspase activation is central to apoptosis, and inhibitor of apoptosis proteins (IAPs) are the principal actors that restrain caspase activity and are therefore attractive therapeutic targets. IAPs, in turn, are regulated by mitochondria-derived proapoptotic factors such as SMAC and HTRA2. Through a series of cryo-electron microscopy structures of full-length human baculoviral IAP repeat-containing protein 6 (BIRC6) bound to SMAC, caspases, and HTRA2, we provide a molecular understanding for BIRC6-mediated caspase inhibition and its release by SMAC. The architecture of BIRC6, together with near-irreversible binding of SMAC, elucidates how the IAP inhibitor SMAC can effectively control a processive ubiquitin ligase to respond to apoptotic stimuli.

Structural basis for regulation of apoptosis and autophagy by the BIRC6/SMAC complex.

Inhibitor of apoptosis proteins (IAPs) bind to pro-apoptotic proteases, keeping them inactive and preventing cell death. The atypical ubiquitin ligase BIRC6 is the only essential IAP, additionally functioning as a suppressor of autophagy. We performed a structure-function analysis of BIRC6 in complex with caspase-9, HTRA2, SMAC, and LC3B, which are critical apoptosis and autophagy proteins. Cryo-electron microscopy structures showed that BIRC6 forms a megadalton crescent shape that arcs around a spacious cavity containing receptor sites for client proteins. Multivalent binding of SMAC obstructs client binding, impeding ubiquitination of both autophagy and apoptotic substrates. On the basis of these data, we discuss how the BIRC6/SMAC complex can act as a stress-induced hub to regulate apoptosis and autophagy drivers.