The serine protease HtrA2/Omi cleaves Parkin and irreversibly inactivates its E3 ubiquitin ligase activity

The serine protease HtrA2 is important in regulating not only apoptosis but also cellular homeostasis. Recently, several lines of evidence suggest that HtrA2 may be intimately associated with Parkin; however, little is known about the functional relationships between HtrA2 and Parkin. Here we have shown that HtrA2 is co-localized with Parkin in the cytosol through the release of HtrA2 from the mitochondria upon cellular stresses. Moreover, endogenous levels of Parkin were significantly decreased in wild-type (HtrA2^+/+) mouse embryonic fibroblasts (MEF) compared with those in HtrA2-knockout (HtrA2^−/−) MEF under the same stress conditions. Using cleavage and binding assays, we have demonstrated that HtrA2 specifically binds to and directly cleaves the E3 ubiquitin (Ub) ligase Parkin. Interestingly, the HtrA2-mediated Parkin cleavage irreversibly disrupts Parkin-mediated synphilin-1 ubiquitination and autoubiquitination, indicating that HtrA2 may play a critical role in the Parkin-related pathway involved in the ubiquitin proteasome system.     

Modulation of mitochondrial function and morphology by interaction of Omi/HtrA2 with the mitochondrial fusion factor OPA1

Loss of Omi/HtrA2 function leads to nerve cell loss in mouse models and has been linked to neurodegeneration in Parkinson's and Huntington's disease. Omi/HtrA2 is a serine protease released as a pro-apoptotic factor from the mitochondrial intermembrane space into the cytosol. Under physiological conditions, Omi/HtrA2 is thought to be involved in protection against cellular stress, but the cytological and molecular mechanisms are not clear. Omi/HtrA2 deficiency caused an accumulation of reactive oxygen species and reduced mitochondrial membrane potential. In Omi/HtrA2 knockout mouse embryonic fibroblasts, as well as in Omi/HtrA2 silenced human HeLa cells and Drosophila S2R+ cells, we found elongated mitochondria by live cell imaging. Electron microscopy confirmed the mitochondrial morphology alterations and showed abnormal cristae structure. Examining the levels of proteins involved in mitochondrial fusion, we found a selective up-regulation of more soluble OPA1 protein. Complementation of knockout cells with wild-type Omi/HtrA2 but not with the protease mutant [S306A]Omi/HtrA2 reversed the mitochondrial elongation phenotype and OPA1 alterations. Finally, co-immunoprecipitation showed direct interaction of Omi/HtrA2 with endogenous OPA1. Thus, we show for the first time a direct effect of loss of Omi/HtrA2 on mitochondrial morphology and demonstrate a novel role of this mitochondrial serine protease in the modulation of OPA1. Our results underscore a critical role of impaired mitochondrial dynamics in neurodegenerative disorders.     

HtrA2/Omi influences the stability of LON protease 1 and prohibitin,proteins involved in mitochondrial homeostasis

High temperature requirement A2 (HtrA2)/Omi is a serine protease localized in mitochondria. In response to apoptotic stimuli, HtrA2 is released to the cytoplasm and cleaves many proteins, including XIAP, Apollon/BRUCE, WT1, and Ped/Pea-15, to promote apoptosis. However, the function of HtrA2 in mitochondria under normal conditions remains unclear. Here, we show that the mitochondrial proteins, LON protease 1 (LONP1) and prohibitin (PHB), are overexpressed in HtrA2^−/− mouse embryonic fibroblast (MEF) cells and HtrA2 knock-down HEK293T cells. We also confirm the effect of the HtrA2 protease on the stability of the above mitochondrial quality control proteins in motor neuron degeneration 2 (mnd2) mice, which have a greatly reduced protease activity as a result of a Ser276Cys missense mutation of the HtrA2 gene. In addition, PHB interacts with and is directly cleaved by HtrA2. Luminescence assays demonstrate that the intracellular ATP level is decreased in HtrA2^−/− cells compared to HtrA2^+/+ cells. HtrA2 deficiency causes a decrease in the mitochondrial membrane potential, and reactive oxygen species (ROS) generation is greater in HtrA2^−/− cells than in HtrA2^+/+ cells. Our results implicate that HtrA2 might be an upstream regulator of mitochondrial homeostasis.     

Mitochondrial import of Omi: the definitive role of the putative transmembrane region and multiple processing sites in the amino-terminal segment

The mitochondrial serine protease Omi/HtrA2 has a proapoptotic role in mammalian cells. However, neither the topology nor the processing of Omi in mitochondria is clearly understood. To determine the topology of Omi in the mitochondrial IMS, EGFP fusions were expressed with the entire N-terminal segment of full-length Omi (FL-Omi) (133-EGFP), and that without the transmembrane region (DeltaTM-EGFP) in the cells. Immunocytochemical staining and alkaline extraction experiments revealed that the TM determines the topology of Omi in the IMS and anchors the pro form into the inner membrane. As a result, the protease and the PDZ domains are exposed to the IMS. Mature Omi largely exists in the IMS as a soluble form. The processing sites of the precursor protein were examined by in vitro import experiments. The import of the processing mutants revealed importance of Arg80, Arg91, and Arg93 residues for the processing of the N-terminal segment of FL-Omi. These results suggest that the N-terminal segment of FL-Omi contains multiple processing sites processed by matrix processing proteases.     

TMEM126A is a mitochondrial located mRNA (MLR) protein of the mitochondrial inner membrane

Background

Hereditary optic neuropathies (HONs) are a heterogeneous group of disorders that affect retinal ganglion cells (RGCs) and axons that form the optic nerve. Leber's Hereditary Optic Neuropathy and the autosomal dominant optic atrophy related to OPA1 mutations are the most common forms. Nonsyndromic autosomal recessive optic neuropathies are rare and their existence has been long debated. We recently identified the first gene responsible for these conditions, TMEM126A. This gene is highly expressed in retinal cellular compartments enriched in mitochondria and supposed to encode a mitochondrial transmembrane protein of unknown function.

Methods

A specific polyclonal antibody targeting the TMEM126A protein has been generated. Quantitative fluorescent in situ hybridization, cellular fractionation, mitochondrial membrane association study, mitochondrial sub compartmentalization analysis by both proteolysis assays and transmission electron microscopy, and expression analysis of truncated TMEM126A constructs by immunofluorescence confocal microscopy were carried out.

Results

TMEM126A mRNAs are strongly enriched in the vicinity of mitochondria and encode an inner mitochondrial membrane associated cristae protein. Moreover, the second transmembrane domain of TMEM126A is required for its mitochondrial localization.

Conclusions

TMEM126A is a mitochondrial located mRNA (MLR) that may be translated in the mitochondrial surface and the protein is subsequently imported to the inner membrane. These data constitute the first step toward a better understanding of the mechanism of action of TMEM126A in RGCs and support the importance of mitochondrial dysfunction in the pathogenesis of HON.

General significance

Local translation of nuclearly encoded mitochondrial mRNAs might be a mechanism for rapid onsite supply of mitochondrial membrane proteins.     

Mutation and expression analysis of the IDH1, IDH2, DNMT3A, and MYD88 genes in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of death around the world. Its genetic mechanism was intensively investigated in the past decades with findings of a number of canonical oncogenes and tumor-suppressor genes such as APC, KRAS, and TP53. Recent genome-wide association and sequencing studies have identified a series of promising oncogenes including IDH1, IDH2, DNMT3A, and MYD88 in hematologic malignancies. However, whether these genes are involved in CRC remains unknown. In this study, we screened the hotspot mutations of these four genes in 305 CRC samples from Han Chinese by direct sequencing. mRNA expression levels of these genes were quantified by quantitative real-time PCR (RT-qPCR) in paired cancerous and paracancerous tissues. Association analyses between mRNA expression levels and different cancerous stages were performed. Except for one patient harboring IDH1 mutation p.I99M, we identified no previously reported hotspot mutations in colorectal cancer tissues. mRNA expression levels of IDH1, DNMT3A, and MYD88, but not IDH2, were significantly decreased in the cancerous tissues comparing with the paired paracancerous normal tissues. Taken together, the hotspot mutations of IDH1, IDH2, DNMT3A, and MYD88 gene were absent in CRC. Aberrant mRNA expression of IDH1, DNMT3A, and MYD88 gene might be actively involved in the development of CRC.