Metalloproteases of the Inner Mitochondrial Membrane.

The inner mitochondrial membrane (IM) is among the most protein-rich cellular compartments. The metastable IM subproteome where the concentration of proteins is approaching oversaturation creates a challenging protein folding environment with a high probability of protein malfunction or aggregation. Failure to maintain protein homeostasis in such a setting can impair the functional integrity of the mitochondria and drive clinical manifestations. The IM is equipped with a series of highly conserved, proteolytic complexes dedicated to the maintenance of normal protein homeostasis within this mitochondrial subcompartment. Particularly important is a group of membrane-anchored metallopeptidases commonly known as m-AAA and i-AAA proteases, and the ATP-independent Oma1 protease. Herein, we will summarize the current biochemical knowledge of these proteolytic machines and discuss recent advances in our understanding of mechanistic aspects of their functioning.

Protease OMA1 modulates mitochondrial bioenergetics and ultrastructure through dynamic association with MICOS complex.

Remodeling of mitochondrial ultrastructure is a process that is critical for organelle physiology and apoptosis. Although the key players in this process-mitochondrial contact site and cristae junction organizing system (MICOS) and Optic Atrophy 1 (OPA1)-have been characterized, the mechanisms behind its regulation remain incompletely defined. Here, we found that in addition to its role in mitochondrial division, metallopeptidase OMA1 is required for the maintenance of intermembrane connectivity through dynamic association with MICOS. This association is independent of OPA1, mediated via the MICOS subunit MIC60, and is important for stability of MICOS and the intermembrane contacts. The OMA1-MICOS relay is required for optimal bioenergetic output and apoptosis. Loss of OMA1 affects these activities; remarkably it can be alleviated by MICOS-emulating intermembrane bridge. Thus, OMA1-dependent ultrastructure support is required for mitochondrial architecture and bioenergetics under basal and stress conditions, suggesting a previously unrecognized role for OMA1 in mitochondrial physiology.

Depletion of mitochondrial protease OMA1 alters proliferative properties and promotes metastatic growth of breast cancer cells.

Metastatic competence of cancer cells is influenced by many factors including metabolic alterations and changes in mitochondrial biogenesis and protein homeostasis. While it is generally accepted that mitochondria play important roles in tumorigenesis, the respective molecular events that regulate aberrant cancer cell proliferation remain to be clarified. Therefore, understanding the mechanisms underlying the role of mitochondria in cancer progression has potential implications in the development of new therapeutic strategies. We show that low expression of mitochondrial quality control protease OMA1 correlates with poor overall survival in breast cancer patients. Silencing OMA1 in vitro in patient-derived metastatic breast cancer cells isolated from the metastatic pleural effusion and atypical ductal hyperplasia mammary tumor specimens (21MT-1 and 21PT) enhances the formation of filopodia, increases cell proliferation (Ki67 expression), and induces epithelial-mesenchymal transition (EMT). Mechanistically, loss of OMA1 results in alterations in the mitochondrial protein homeostasis, as reflected by enhanced expression of canonic mitochondrial unfolded protein response genes. These changes significantly increase migratory properties in metastatic breast cancer cells, indicating that OMA1 plays a critical role in suppressing metastatic competence of breast tumors. Interestingly, these results were not observed in OMA1-depleted non-tumorigenic MCF10A mammary epithelial cells. This newly identified reduced activity/levels of OMA1 provides insights into the mechanisms leading to breast cancer development, promoting malignant progression of cancer cells and unfavorable clinical outcomes, which may represent possible prognostic markers and therapeutic targets for breast cancer treatment.

Mitochondrial Quality Control Proteases in Neuronal Welfare.

The functional integrity of mitochondria is a critical determinant of neuronal health and compromised mitochondrial function is a commonly recognized factor that underlies a plethora of neurological and neurodegenerative diseases. Metabolic demands of neural cells require high bioenergetic outputs that are often associated with enhanced production of reactive oxygen species. Unopposed accumulation of these respiratory byproducts over time leads to oxidative damage and imbalanced protein homeostasis within mitochondrial subcompartments, which in turn may result in cellular demise. The post-mitotic nature of neurons and their vulnerability to these stress factors necessitate strict protein homeostatic control to prevent such scenarios. A series of evolutionarily conserved proteases is one of the central elements of mitochondrial quality control. These versatile proteolytic enzymes conduct a multitude of activities to preserve normal mitochondrial function during organelle biogenesis, metabolic remodeling and stress. In this review we discuss neuroprotective aspects of mitochondrial quality control proteases and neuropathological manifestations arising from defective proteolysis within the mitochondrion.

A conserved motif in the ITK PH-domain is required for phosphoinositide binding and TCR signaling but dispensable for adaptor protein interactions.

Binding of the membrane phospholipid phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) to the Pleckstrin Homology (PH) domain of the Tec family protein tyrosine kinase, Inducible T cell Kinase (ITK), is critical for the recruitment of the kinase to the plasma membrane and its co-localization with the TCR-CD3 molecular complex. Three aromatic residues, termed the FYF motif, located in the inner walls of the phospholipid-binding pocket of the ITK PH domain, are conserved in the PH domains of all Tec kinases, but not in other PH-domain containing proteins, suggesting an important function of the FYF motif in the Tec kinase family. However, the biological significance of the FYF amino acid motif in the ITK-PH domain is unknown. To elucidate it, we have tested the effects of a FYF triple mutant (F26S, Y90F, F92S), henceforth termed FYF-ITK mutant, on ITK function. We found that FYF triple mutation inhibits the TCR-induced production of IL-4 by impairing ITK binding to PIP(3), reducing ITK membrane recruitment, inducing conformational changes at the T cell-APC contact site, and compromising phosphorylation of ITK and subsequent phosphorylation of PLCγ(1). Interestingly, however, the FYF motif is dispensable for the interaction of ITK with two of its signaling partners, SLP-76 and LAT. Thus, the FYF mutation uncouples PIP(3)-mediated ITK membrane recruitment from the interactions of the kinase with key components of the TCR signalosome and abrogates ITK function in T cells.

In Vivo Consequences of Disrupting SH3-Mediated Interactions of the Inducible T-Cell Kinase.

ITK-SH3-mediated interactions, both with exogenous ligands and via intermolecular self-association with ITK-SH2, have been shown to be important for regulation of ITK activity. The biological significance of these competing SH3 interactions is not completely understood. A mutant of ITK where substitution of the SH3 domain with that of the related kinase BTK (ITK-BTK((SH3))) was used to disrupt intermolecular self-association of ITK while maintaining canonical binding to exogenous ligands such as SLP-76. ITK-BTK((SH3)) displays reduced association with SLP-76 leading to inefficient transphosphorylation, reduced phosphorylation of PLCγ1, and diminished Th(2) cytokine production. In contrast, ITK-BTK((SH3)) displays no defect in its localization to the T-cell-APC contact site. Another mutation, Y511F, in the activation loop of ITK, impairs ITK activation. T cells expressing ITK-Y511F display defective phosphorylation of ITK and its downstream target PLCγ1, as well as significant inhibition of Th(2) cytokines. In contrast, the inducible localization of ITK-Y511F to the T cell-APC contact site and its association with SLP-76 are not affected. The presented data lend further support to the hypothesis that precise interactions between ITK and its signaling partners are required to support ITK signaling downstream of the TCR.

Correlation between generation of nitric oxide and cell viability in human peripheral blood mononuclear cells and leukemic Jurkat T-cell line.

AIM: To measure nitric oxide (NO) production in the form of nitrite derivative in relation to cell viability and apoptosis development in human peripheral blood mononuclear cells compared to that processes in human leukemic Jurkat T-cell line. METHODS: Apoptosis was induced by dexamethasone (1 microg/ml) or NaNO(2) (7 microg/ml) added in the presence or absence of NO-synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) (27 microg/ml) during cell culturing. Cell viability was determined by trypan blue assay. Apoptosis was measured using DNA "ladder" assay. RESULTS: Dexamethazone and NaNO(2) were shown to cause DNA "laddering" in both cell types. L-NAME prevented the appearance of apoptosis in both normal mononuclear cells of peripheral blood and leukemic Jurkat T-cell in the case of dexamethasone action, but it could not prevent it in the case of NaNO(2) action. The results of cell viability showed that both the dexamethasone and NaNO(2) significantly increased the percentage of dead cells. Their effect was better expressed in Jurkat T-cell line. The levels of nitrite production were higher in the leukemic T-cells comparing to such levels in the normal mononuclear cells. CONCLUSION: Strong positive correlation was demonstrated between NO production and apoptosis development in both studied cell types, however leukemic Jurkat T-cell line responses were better expressed than such responses in normal mononuclear cells of peripheral blood. Potential significance of that correlation as well as possible mechanisms of appearing differences are discussed.