Mitochondrial Proteins HSP60 Research Articles

Novel Functions of CD147 in the Mitochondria Exacerbates Melanoma Metastasis.

Melanoma is an aggressive form of skin cancer characterized by rapid invasion and metastasis. CD147 is known to be functioning in cell invasion. In this study, we showed that CD147 was translocated from the cell membrane to the mitochondria in advanced melanoma. Melanoma patients with CD147 localized to the mitochondria confer a worse prognosis. The mitochondrial CD147 levels are correlated with the invasion potential of various melanoma cell lines as well as mitochondrial energy metabolism. Depletion of CD147 decreased the activity of mitochondrial complex V. STRING analysis for protein-protein interaction networks (PPIN) in CD147-depleted melanoma cells showed that mitochondrial proteins HSP60 and ATP5B, a subunit of mitochondrial complex V, were node proteins. HSP60 upregulation was correlated with a worse prognosis of melanoma patients. Co-immunoprecipitation (Co-IP) assay indicates that CD147 interacts with HSP60. These data suggested that mitochondrial CD147 may prompt HSP60 to activate ATP5B, thereby promoting the mitochondrial aerobic oxidation and the invasive abilities of melanoma cells. Correlation analysis of the data acquired from patients was helpful to draw a 5-year survival curve for patients who screened positive and negative for mitochondrial CD147. This study unravels the function of CD147 in tumor invasion and highlights it as a potential tumor therapeutic target.

Environmental cadmium exposure induces fetal growth restriction via triggering PERK-regulated mitophagy in placental trophoblasts

Cadmium (Cd), an environmental toxicant, is positively associated with fetal growth restriction (FGR). However, the mechanism by which gestational exposure to Cd induces FGR remains unclear. This study designed in vitro and in vivo experiments to explore the role of placental mitophagy in Cd-impaired fetal growth. Based on our case-control study, we also investigated the association of placental mitophagy with reduced progesterone (P4) level and all-cause FGR. We firstly found environmental Cd exposure lowered the P4 content in maternal sera, placentae and amnioticfluids of mice. The level of three mitochondrial P4 synthases, including StAR, CYP11A1 and 3β-HSD, was also reduced in Cd-treated placentae. Furthermore, Cd triggered mitophagy, as determined by the degradation of two mitochondrial proteins HSP60 and COX IV, and the accumulation of co-localizations of TOM20 with LC3B or Parkin in placental trophoblasts. Correspondingly, Cd elevated mitochondrial Parkin level in placental trophoblasts. Mdivi-1, a mitophagy inhibitor, obviously attenuated Cd-induced reduction of placental P4 and FGR in mice. Moreover, mdivi-1 and Parkin siRNA (siR) markedly reversed Cd-caused P4 synthesis inhibition in human placental trophoblasts. Interestedly, the PERK/ATF4 signaling was activated in Cd-stimulated placental trophoblasts. PERK siR inhibited mitochondrial proteins degradation in Cd-stimulated placental trophoblasts. In particularly, mitophagy activation and P4 synthesis suppression occurred in small-for-gestational-age placentae based on our case-control study. Environmental Cd exposure induced FGR via activating PERK-regulated mitophagy and inhibiting P4 synthesis in placentaltrophoblasts. Furthermore, placental mitophagy was related to the reduced progesterone level and all-cause fetal growth restriction based on our case-control study. As above, placental mitophagy maybe the common mechanism of environmental toxicants-impaired fetal growth.

Antisera against Neisseria gonorrhoeae cross-react with specific brain proteins of the common marmoset monkey and other nonhuman primate species

Prenatal maternal infections with Neisseria gonorrhoeae (NG) correlate with an increased lifetime probability for the offspring to develop psychosis. We could previously demonstrate that in human choroid plexus papilloma cells, anti-NG antibodies (α-NG) bind to mitochondrial proteins HSP60 and ATPB, and interfere with cellular energy metabolism. To assess the in vivo relevance for this, especially during prenatal neural development, we investigated here interactions of NG-specific antisera (α-NG1, α-NG2) with brain, choroid plexus and other non-neural tissues in pre- and perinatal samples of the nonhuman primate (NHP) Callithrix jacchus (CJ), a NHP model for preclinical research. In histological sections at embryonic day E75, immunohistochemistry revealed α-NG1 and -2-staining in choroid plexus, ganglionic hill, optic cup, heart, and liver. Within the cells, organelle-like structures were labeled, which could be identified by immunohistochemical double-labeling as mitochondria. Both one- and two-dimensional Western blot analysis revealed tissue specific patterns of α-NG1 immunoreactive bands and spots, respectively, which were subsequently characterized by mass spectrometry. Thereby we could confirm the interactions of α-NG1 with human HSP60 and ATPB also in CJ choroid plexus and liver. Even more important, in the CJ brain, several new targets, including NCAM1, CRMP2, and SYT1, were identified, which by unrelated studies have been previously suggested to correlate with an increased schizophrenia risk. These findings support the idea that the marmoset monkey is a useful NHP model to investigate the role of maternal bacterial infections during prenatal brain development, and thereby might improve the understanding of this important aspect of schizophrenia pathology.

Systems biology analysis of the proteomic alterations induced by MPP+, a Parkinson's disease-related mitochondrial toxin

Parkinson's disease (PD) is a complex neurodegenerative disease whose etiology has not been completely characterized. Many cellular processes have been proposed to play a role in the neuronal damage and loss: defects in the proteosomal activity, altered protein processing, increased reactive oxygen species burden. Among them, the involvement of a decreased activity and an altered disposal of mitochondria is becoming more and more evident. The mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+), an inhibitor of complex I, has been widely used to reproduce biochemical alterations linked to PD in vitro and its precursor, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), to induce a Parkinson-like syndrome in vivo. Therefore, we performed a meta-analysis of the literature of all the proteomic investigations of neuronal alterations due to MPP+ treatment and compared it with our results obtained with a mitochondrial proteomic analysis of SH-SY5Y cells treated with MPP+. By using open-source bioinformatics tools, we identified the biochemical pathways and the molecular functions mostly affected by MPP+, i.e., ATP production, the mitochondrial unfolded stress response, apoptosis, autophagy, and, most importantly, the synapse funcionality. Eventually, we generated protein networks, based on physical or functional interactions, to highlight the relationships among the molecular actors involved. In particular, we identified the mitochondrial protein HSP60 as the central hub in the protein-protein interaction network. As a whole, this analysis clarified the cellular responses to MPP+, the specific mitochondrial proteome alterations induced and how this toxic model can recapitulate some pathogenetic events of PD.

Sex differences in mitochondrial biogenesis determine neuronal death and survival in response to oxygen glucose deprivation and reoxygenation

BackgroundMitochondrial dysfunction has been linked to neuronal death and a wide array of neurodegenerative diseases. Previously, we have shown sex differences in mitochondria-mediated cell death pathways following hypoxia-ischemia. However, the role of mitochondrial biogenesis in hypoxic-ischemic brain injury between male vs. female has not been studied yet.ResultsPrimary cerebellar granule neurons (CGNs), isolated from P7 male and female mice (CD-1) segregated based on visual inspection of sex, were exposed to 2 h of oxygen glucose deprivation (OGD) followed by 6–24 h of reoxygenation (Reox). Mitochondrial membrane potential (ΔΨm) and cellular ATP levels were reduced significantly in XX CGNs as compared to XY CGNs. Mitochondrial DNA (mtDNA) content was increased (>2-fold) at 2 h OGD in XY CGNs and remained increased up to 24 h of Reox compared to XX neurons and normoxia controls. The expression of mitochondrial transcription factor A (Tfam), the nuclear respiratory factor-1 (NRF-1) and the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis, were up-regulated (2-fold, ***p < 0.001) in XY CGNs but slightly reduced or remained unchanged in XX neurons. Similarly, the TFAM and PGC-1α protein levels and the mitochondrial proteins HSP60 and COXIV were increased in XY neurons only. Supportively, a balanced stimulation of fusion (Mfn 1and Mfn 2) and fission (Fis 1 and Drp 1) genes and enhanced formation of donut-shaped mitochondria were observed in XY CGNs vs. XX neurons (**p < 0.01).ConclusionsOur results demonstrate that OGD/Reox alters mitochondrial biogenesis and morphological changes in a sex-specific way, influencing neuronal injury/survival differently in both sexes.

“Stainomics”: Identification of mitotracker labeled proteins in mammalian cells

Organelle-specific cell-permeable fluorescent dyes are invaluable tools in cell biology as they reveal intracellular dynamics in living cells. Mitrotracker is a family of dyes that strongly label the mitochondrion, a key organelle associated with many crucial cellular functions. Despite the popularity of these dyes, little is known about the molecular mechanism behind their staining specificity. Here, we aimed to identify the protein targets of one member of this dye family, mitotracker red (MTR), by 2DE and MS. MTR bound to cellular proteins covalently, and its fluorescence persisted even after cell lysis, protein solubilization, denaturation, and electrophoresis. This enabled us to display MTR-labeled proteins by 2DE. The MTR-specific fluorescent signals on the gel revealed the spots that contained MTR-conjugated proteins. These spots were analyzed by MS, resulting into the identification of ten proteins. We discovered that one major target is the mitochondrial protein HSP60 and that MTR staining could induce production of HSP60, predisposing cells to heat shock-like responses. The identification of the molecular targets of biological dyes, or "stainomics," can help correlate their intracellular staining properties with biochemical affinities. We believe this approach can be applied to a wide range of fluorescent probes.

Purification of mitochondrial proteins HSP60 and ATP synthase from ascidian eggs: implications for antibody specificity.

Use of antibodies is a cornerstone of biological studies and it is important to identify the recognized protein with certainty. Generally an antibody is considered specific if it labels a single band of the expected size in the tissue of interest, or has a strong affinity for the antigen produced in a heterologous system. The identity of the antibody target protein is rarely confirmed by purification and sequencing, however in many cases this may be necessary. In this study we sought to characterize the myoplasm, a mitochondria-rich domain present in eggs and segregated into tadpole muscle cells of ascidians (urochordates). The targeted proteins of two antibodies that label the myoplasm were purified using both classic immunoaffinity methods and a novel protein purification scheme based on sequential ion exchange chromatography followed by two-dimensional gel electrophoresis. Surprisingly, mass spectrometry sequencing revealed that in both cases the proteins recognized are unrelated to the original antigens. NN18, a monoclonal antibody which was raised against porcine spinal cord and recognizes the NF-M neurofilament subunit in vertebrates, in fact labels mitochondrial ATP synthase in the ascidian embryo. PMF-C13, an antibody we raised to and purified against PmMRF, which is the MyoD homolog of the ascidian Phallusia mammillata, in fact recognizes mitochondrial HSP60. High resolution immunolabeling on whole embryos and isolated cortices demonstrates localization to the inner mitochondrial membrane for both ATP synthase and HSP60. We discuss the general implications of our results for antibody specificity and the verification methods which can be used to determine unequivocally an antibody's target.

Two-Dimensional Electrophoretic Analysis of Compartment- Specific Hepatic Protein Charge Modification Induced by Thioacetamide Exposure in Rats

Thioacetamide (TA) is a well-known hepatotoxicant. It has been reported that an obligate intermediate of TA binds to proteins with the formation of acetylimidolysine derivatives that are responsible for TA-induced hepatotoxic effects. TA has also been reported to cause chemically induced cell death via both apoptosis and necrosis. The objective of this study was 2-fold: first, to investigate the effect of TA exposure on protein charge modifications in the rat liver and second, to study the role of these molecular correlates in the regulation of cell death. Male Sprague–Dawley rats (200–225 g, 7–8 weeks old) were divided into four major groups and treated intraperitoneally with a 12-fold dose range of TA (50, 150, 300, and 600 mg TA/kg) dissolved in water. Using whole liver extracts, alterations in the hepatic protein pattern following treatment with the 12-fold dose range of TA were studied using high-resolution, two-dimensional polyacrylamide gel electrophoresis and computerized image analysis. The results indicate that charge modification was clearly evident as early as 2 hr with the lowest dose of 50 mg TA/kg. At this dose and time endoplasmic reticulum proteins, calreticulin, grp78, and ER60 exhibited acidic charge variants. The effect of TA became more prominent with dose and time. Generally the elevation of charge modification indices (CMI) by TA appeared to reach a peak between 4 and 6 hr and then while CMI either leveled off or declined in the lower two doses of 50 and 150 mg TA/kg, it continued to remain elevated with the higher doses of 300 and 600 mg TA/kg. This dichotomy in the elevation of CMI is in close correspondence to the pattern of cell death observed with a similar dose range of TA, where lower doses (50 and 150 mg TA/kg) predominantly cause cell death via apoptosis while higher doses cause cell death via necrosis. Delayed charge modification was observed with the cytosolic hsc70s with the 300 and 600 mg TA/kg treatments, indicating that the reactive metabolite(s) slowly leak out into the cytosol from the endoplasmic reticulum. There were no alterations in the mitochondrial proteins hsp60 and grp75, suggesting that TA has no effect on the mitochondrion, its effects primarily being confined to the endoplasmic reticulum. The concept of looking at these proteins as biomarkers of tissue injury has validity. These changes may be indicators of bioactivation and adduct formation and also may be signaling events in the regulation of the mode of cell death.

Two-Dimensional Electrophoretic Analysis of Compartment-Specific Hepatic Protein Charge Modification Induced by Thioacetamide Exposure in Rats

Thioacetamide (TA) is a well-known hepatotoxicant. It has been reported that an obligate intermediate of TA binds to proteins with the formation of acetylimidolysine derivatives that are responsible for TA-induced hepatotoxic effects. TA has also been reported to cause chemically induced cell death via both apoptosis and necrosis. The objective of this study was 2-fold: first, to investigate the effect of TA exposure on protein charge modifications in the rat liver and second, to study the role of these molecular correlates in the regulation of cell death. Male Sprague-Dawley rats (200–225 g, 7–8 weeks old) were divided into four major groups and treated intraperitoneally with a 12-fold dose range of TA (50, 150, 300, and 600 mg TA/kg) dissolved in water. Using whole liver extracts, alterations in the hepatic protein pattern following treatment with the 12-fold dose range of TA were studied using high-resolution, two-dimensional polyacrylamide gel electrophoresis and computerized image analysis. The results indicate that charge modification was clearly evident as early as 2 hr with the lowest dose of 50 mg TA/kg. At this dose and time endoplasmic reticulum proteins, calreticulin, grp78, and ER60 exhibited acidic charge variants. The effect of TA became more prominent with dose and time. Generally the elevation of charge modification indices (CMI) by TA appeared to reach a peak between 4 and 6 hr and then while CMI either leveled off or declined in the lower two doses of 50 and 150 mg TA/kg, it continued to remain elevated with the higher doses of 300 and 600 mg TA/kg. This dichotomy in the elevation of CMI is in close correspondence to the pattern of cell death observed with a similar dose range of TA, where lower doses (50 and 150 mg TA/kg) predominantly cause cell death via apoptosis while higher doses cause cell death via necrosis. Delayed charge modification was observed with the cytosolic hsc70s with the 300 and 600 mg TA/kg treatments, indicating that the reactive metabolite(s) slowly leak out into the cytosol from the endoplasmic reticulum. There were no alterations in the mitochondrial proteins hsp60 and grp75, suggesting that TA has no effect on the mitochondrion, its effects primarily being confined to the endoplasmic reticulum. The concept of looking at these proteins as biomarkers of tissue injury has validity. These changes may be indicators of bioactivation and adduct formation and also may be signaling events in the regulation of the mode of cell death.