Mitochondrial Targeting Signal Peptide Research Articles

Mitochondrial Translocation of DJ-1 Is Mediated by Grp75: Implication in Cardioprotection of Resveratrol Against Hypoxia/Reoxygenation-Induced Oxidative Stress.

Resveratrol (Res) was recently reported to ameliorate hypoxia/reoxygenation (H/R)-caused oxidative stress in H9c2 cardiomyocytes through promoting the mitochondrial translocation of DJ-1 protein and subsequently preserving the activity of mitochondrial complex I. However, it is noteworthy that DJ-1 possesses no mitochondria-targeting sequence. Therefore, how Res induces DJ-1 mitochondrial translocation is an important and interesting question for further exploration. Glucose-regulated protein 75 (Grp75), whose N-terminus contains a 51-amino acid long mitochondrial-targeting signal peptide, is a cytoprotective chaperone that partakes in mitochondrial import of several proteins. Here, the contribution of Grp75 to mitochondrial import of DJ-1 by Res was investigated in a cellular model of H/R. Our results showed that Res upregulated the expression of DJ-1 protein, enhanced the interaction of DJ-1 and Grp75, and promoted DJ-1 translocation to mitochondria from cytosol in H9c2 cardiomyocytes undergoing H/R. Importantly, knockdown of Grp75 markedly reduced the interaction of DJ-1 with Grp75 and subsequent DJ-1 mitochondrial translocation induced by Res. Furthermore, Res pretreatment promoted the association of DJ-1 with ND1 and NDUFA4 subunits of complex I, preserved the activity of complex I, decreased mitochondria-derived reactive oxygen species production, and eventually ameliorated H/R-caused oxidative stress damage. Intriguingly, these effects were largely prevented also by small interfering RNA targeting Grp75. Overall, these results suggested that Grp75 interacts with DJ-1 to facilitate its translocation from cytosol to mitochondria, which is required for Res-mediated preservation of mitochondria complex I and cardioprotection from H/R-caused oxidative stress injury.

A Unique SUMO-Interacting Motif of Trx2 Is Critical for Its Mitochondrial Presequence Processing and Anti-oxidant Activity.

ObjectiveMitochondrial thioredoxin 2 (Trx2) is a vital mitochondrial redox protein that mediates normal protein thiol reduction and provides electrons to peroxiredoxin 3 (Prx3) to scavenge H2O2 in mitochondria. It has been widely reported that Trx2 deletion in cells or mice generates massive reactive oxygen species (ROS) which have been implicated in many pathological processes. On the contrary, how ROS regulate Trx2 processing and activity remains to be elucidated.Approach and ResultsHere we show that excess ROS induce endothelial cell senescence concomitant with an attenuation of Trx2 processing in which Trx2 presequence [i.e., mitochondrial targeting signal peptide (MTS)] is cleaved to generate a mature form. Mutation analyses indicate that Trx2 processing is mediated by mitochondrial processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP)-recognition sites within the MTS. Interestingly, a mutation at a SUMO- interacting motif (SIM), but not the catalytic sites within the mature Trx2 protein, completely blocks Trx2 processing with no effect on Trx2 mitochondrial targeting. Consistently, chemical inhibition of protein SUMOylation attenuates, while SUMOylation agonist promotes, Trx2 processing. Moreover, we identify the α–MPP subunit is a SUMOylated protein that potentially mediates Trx2-binding and cleavage. Furthermore, the unprocessed form of Trx2-SIM is unable to protect cells from both ROS generation and oxidative stress-induced cellular senescence.ConclusionOur study reveals that a unique SUMO-interacting motif of Trx2 is critical for its mitochondrial processing and subsequent anti-oxidant/antisenescence activities.

Identification and functional characterization of nuclear mortalin in human carcinogenesis.

The Hsp70 family protein mortalin is an essential chaperone that is frequently enriched in cancer cells and exists in various subcellular sites, including the mitochondrion, plasma membrane, endoplasmic reticulum, and cytosol. Although the molecular mechanisms underlying its multiple subcellular localizations are not yet clear, their functional significance has been revealed by several studies. In this study, we examined the nuclear fractions of human cells and found that the malignantly transformed cells have more mortalin than the normal cells. We then generated a mortalin mutant that lacked a mitochondrial targeting signal peptide. It was largely localized in the nucleus, and, hence, is called nuclear mortalin (mot-N). Functional characterization of mot-N revealed that it efficiently protects cancer cells against endogenous and exogenous oxidative stress. Furthermore, compared with the full-length mortalin overexpressing cancer cells, mot-N derivatives showed increased malignant properties, including higher proliferation rate, colony forming efficacy, motility, and tumor forming capacity both in in vitro and in vivo assays. We demonstrate that mot-N promotes carcinogenesis and cancer cell metastasis by inactivation of tumor suppressor protein p53 functions and by interaction and functional activation of telomerase and heterogeneous ribonucleoprotein K (hnRNP-K) proteins.

Intracellular observation of nanocarriers modified with a mitochondrial targeting signal peptide

This study focused on the intracellular observation of nanocarriers modified with a mitochondrial targeting signal peptide (MTS). The nanocarriers showed an efficient cellular uptake, and the MTS had a positive effect on their mitochondrial targeting. This is the first report of an intracellular observation of nanocarriers modified with MTS.

Enhancement in selective mitochondrial association by direct modification of a mitochondrial targeting signal peptide on a liposomal based nanocarrier

The focus of this study was on the development of a nano carrier targeted to mitochondria, a promising therapeutic drug target. We synthesized a lipid derivative that is conjugated with a mitochondrial targeting signal peptide (MTS), which permits the selective delivery of certain types of proteins to mitochondria. We then explored the use of an innovative technology in which MTS and the MITO-Porter were integrated. The latter is a liposome that delivers cargos to mitochondria via membrane fusion. The results indicate that the combination of MTS and the MITO-Porter would be useful for selective mitochondrial delivery via membrane fusion.

Betanodavirus B2 Causes ATP Depletion-induced Cell Death via Mitochondrial Targeting and Complex II Inhibition in Vitro and in Vivo

The betanodavirus non-structural protein B2 is a newly discovered necrotic death factor with a still unknown role in regulation of mitochondrial function. In the present study, we examined protein B2-mediated inhibition of mitochondrial complex II activity, which results in ATP depletion and thereby in a bioenergetic crisis in vitro and in vivo. Expression of protein B2 was detected early at 24 h postinfection with red-spotted grouper nervous necrosis virus in the cytoplasm. Later B2 was found in mitochondria using enhanced yellow fluorescent protein (EYFP) and immuno-EM analysis. Furthermore, the B2 mitochondrial targeting signal peptide was analyzed by serial deletion and specific point mutation. The sequence of the B2 targeting signal peptide ((41)RTFVISAHAA(50)) was identified and its presence correlated with loss of mitochondrial membrane potential in fish cells. Protein B2 also was found to dramatically inhibit complex II (succinate dehydrogenase) activity, which impairs ATP synthesis in fish GF-1 cells as well as human embryonic kidney 293T cells. Furthermore, when B2 was injected into zebrafish embryos at the one-cell stage to determine its cytotoxicity and ability to inhibit ATP synthesis, we found that B2 caused massive embryonic cell death and depleted ATP resulting in further embryonic death at 10 and 24 h post-fertilization. Taken together, our results indicate that betanodavirus protein B2-induced cell death is due to direct targeting of the mitochondrial matrix by a specific signal peptide that targets mitochondria and inhibits mitochondrial complex II activity thereby reducing ATP synthesis.

Smac/DIABLO and acute renal ischemia-reperfusion injury

Ischemia-reperfusion after neonatal asphyxia is a key factor in renal injury,which often leads to apoptosis of tubular epithelial cells.Apoptosis is an important form of injury for renal tubular epithelial cells after asphyxia.Smac/DIABLO is released to the cytosol in response to diverse apoptotic stimuli while mitochondrial targeting signal peptide is removed.In the cytosol,Smac/DIABLO interacts and antagonizes inhibitors of apoptosis proteins,thus allowing the activation of caspases and apoptosis.And thus it increases the ischemia-reperfusion renal injury,leading to acute renal failure. Key words: Smac/DIABLO; Inhibitors of apoptosis protein; Apoptosis; Kidney; Ischemia-reperfusion injury

The distinct in vivo roles of the long‐form and short‐form of glutathione peroxidase 4: the short‐form is essential for somatic cell functions and the long‐form is important for male fertility

Glutathione Peroxidase 4 (Gpx4) is an essential antioxidant enzyme that protects cells against membrane lipid peroxidation. The Gpx4 gene encodes for three isoforms: a long‐form (lGpx4), a short form (sGpx4) and a nuclear form. Genetic studies indicate that the nuclear form of Gpx4 plays a role in sperm chromatin condensation. However, the in vivo roles of lGpx4 and sGpx4, which are distinguishable by the presence or lack of a mitochondrial targeting signal peptide at the N‐terminal of the Gpx4 protein, remain unclear. In this study, we generated transgenic mice using mutated human GPX4 genes encoding either lGPX4 [Tg(lGPX4) mice] or sGPX4 [Tg(sGPX4) mice]. Tg(sGPX4) mice had increased total Gpx4 proteins in all tissues whereas Tg(lGPX4) mice had increased total Gpx4 proteins only in the testes. Tg(sGPX4) mice, but not the Tg(lGPX4) mice, were protected against diquat‐induced liver apoptosis. By cross‐breeding the Tg(sGPX4) mice with the Gpx4 knockout mice, we showed that the sGPX4 transgene was able to rescue the lethal phenotype of mouse Gpx4 null mutation. In contrast, the lGPX4 transgene failed to rescue Gpx4 null mice. Gpx4 protein expression data from the sGPX4‐rescued Gpx4 null mice indicated that sGPX4 was the major isoform in the somatic tissue subcellular fractions including mitochondria whereas lGPX4 was predominant in testes. The male sGPX4‐rescued mice were infertile and exhibited low sperm count and reduced sperm mobility, suggesting lGPX4 plays a role in male fertility. Together, our results indicate that the sGpx4 is the major isoform in somatic tissues and it is essential for survival and protection against apoptosis; the lGpx4 is predominant in the testes and it is important in regulating male fertility.

Preliminary molecular characterization and crystallization of mitochondrial respiratory complex II from porcine heart

The mitochondrial respiratory complex II, or succinate:ubiquinone oxidoreductase, is an integral membrane protein complex in both the tricarboxylic acid cycle (Krebs cycle) and aerobic respiration. The gene sequences of each complex II subunit were measured by RT-PCR. N-terminal sequencing work was performed to identify the mitochondrial targeting signal peptide of each subunit. Complex II was extracted from porcine heart and purified by the ammonium sulfate precipitation method. The sample was solubilized by 0.5% (w/v) sugar detergent n-decyl-beta-D-maltoside, stabilized by 200 mM sucrose, and crystallized with 5% (w/v) poly(ethylene glycol) 4000. Important factors for the extraction, purification and crystallization of mitochondrial respiratory complex II are discussed.

The processing of human mitochondrial leucyl-tRNA synthetase in the insect cells

A His-tagged full-length cDNA of human mitochondrial leucyl-tRNA synthetase was expressed in a baculovirus system. The N-terminal sequence of the enzyme isolated from the mitochondria of insect cells was found to be IYSATGKWTKEYTL, indicating that the mitochondrial targeting signal peptide was cleaved between Ser39 and Ile40 after the enzyme precursor was translocated into mitochondria. The enzyme purified from mitochondria catalyzed the leucylation of Escherichia coli tRNA 1 Leu(CAG) and Aquifex aeolicus tRNA Leu(GAG) with higher catalytic activity in the leucylation of E. coli tRNA Leu than that previously expressed in E. coli without the N-terminal 21 residues.

Interorganellar gene transfer in bryophytes: the functional nad7 gene is nuclear encoded in Marchantia polymorpha.

The nad7 gene, encoding subunit 7 of NADH dehydrogenase, is mitochondrially encoded in seed plants. In the liverwort, Marchantia polymorpha, only a pseudogene is located in the mitochondrial genome. We have now identified the functional nad7 gene copy in the nuclear genome of Marchantia, coding for a polypeptide of 468 amino acids. The nuclear-encoded nad7 has lost the two group II introns present in the mitochondrial pseudogene copy. Instead, a typical nuclear intron is found to split an exon encoding the presumptive mitochondrial targeting signal peptide and the mature subunit 7 of NADH dehydrogenase. These results suggest that RNA-mediated gene transfer from the mitochondrial into the nuclear genome occurs not only in seed plants but also in bryophytes.