Mitochondrial Precursor Protein Import Complex Proteins

线粒体前体蛋白导入复合蛋白
  • 文章类型: Journal Article
    SAM50, a 7-8 nm diameter β-barrel channel of the mitochondrial outer membrane, is the central channel of the sorting and assembly machinery (SAM) complex involved in the biogenesis of β-barrel proteins. Interestingly, SAM50 is not known to have channel translocase activity; however, we have recently found that this channel is necessary and sufficient for mitochondrial entry of cytotoxic proteases. Cytotoxic lymphocytes eliminate cells that pose potential hazards, such as virus- and bacteria-infected cells as well as cancer cells. They induce cell death following the delivery of granzyme cytotoxic proteases into the cytosol of the target cell. Although granzyme A and granzyme B (GA and GB), the best characterized of the five human granzymes, trigger very distinct apoptotic cascades, they share the ability to directly target the mitochondria. GA and GB do not have a mitochondrial targeting signal, yet they enter the target cell mitochondria to disrupt respiratory chain complex I and induce mitochondrial reactive oxygen species (ROS)-dependent cell death. We found that granzyme mitochondrial entry requires SAM50 and the translocase of the inner membrane 22 (TIM22). Preventing granzymes\' mitochondrial entry compromises their cytotoxicity, indicating that this event is unexpectedly an important step for cell death. Although mitochondria are best known for their roles in cell metabolism and energy conversion, these double-membrane organelles are also involved in Ca2+ homeostasis, metabolite transport, cell cycle regulation, cell signaling, differentiation, stress response, redox homeostasis, aging, and cell death. This multiplicity of functions is matched with the complexity and plasticity of the mitochondrial proteome as well as the organelle\'s morphological and structural versatility. Indeed, mitochondria are extremely dynamic and undergo fusion and fission events in response to diverse cellular cues. In humans, there are 1500 different mitochondrial proteins, the vast majority of which are encoded in the nuclear genome and translated by cytosolic ribosomes, after which they must be imported and properly addressed to the right mitochondrial compartment. To this end, mitochondria are equipped with a very sophisticated and highly specific protein import machinery. The latter is centered on translocase complexes embedded in the outer and inner mitochondrial membranes working along five different import pathways. We will briefly describe these import pathways to put into perspective our finding regarding the ability of granzymes to enter the mitochondria.
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  • 文章类型: Journal Article
    在蛋白质晶体中,柔性环经常因晶体接触而变形,而在解决方案中,大的运动导致在NMR结构确定中这种柔性回路的收敛性差。我们需要一种实验技术来表征蛋白质分子固有柔性环的结构和动态特性。
    我们在蛋白质晶体中设计了一个预期的晶体接触自由空间(CCFS),并在CCFS中安排了灵活的兴趣循环。选择酵母Tim21蛋白作为模型蛋白,因为其中一个环(环2)被常规晶体中的晶体接触扭曲。
    酵母Tim21通过刚性α-螺旋接头与MBP蛋白融合。在两种蛋白质之间产生的空间用作CCFS。链接器长度为在CCFS中布置环2提供了可调节的自由度。我们重新确定了酵母Tim21的NMR结构,并进行了MD模拟进行比较。使用多维缩放来可视化环2的构象相似性。我们发现,在NMR和MD结构中,环2的晶体无接触构象位于环2构象集合的中心附近。
    CCFS中的酵母Tim21的Loop2采用代表,溶液中的显性构象。
    没有一种强大的技术可用于表征蛋白质分子中的柔性结构。NMR分析和MD模拟提供了有用的,但信息不完整。CCFS晶体学为实现这一目标提供了第三条途径。
    In protein crystals, flexible loops are frequently deformed by crystal contacts, whereas in solution, the large motions result in the poor convergence of such flexible loops in NMR structure determinations. We need an experimental technique to characterize the structural and dynamic properties of intrinsically flexible loops of protein molecules.
    We designed an intended crystal contact-free space (CCFS) in protein crystals, and arranged the flexible loop of interest in the CCFS. The yeast Tim 21 protein was chosen as the model protein, because one of the loops (loop 2) is distorted by crystal contacts in the conventional crystal.
    Yeast Tim21 was fused to the MBP protein by a rigid α-helical linker. The space created between the two proteins was used as the CCFS. The linker length provides adjustable freedom to arrange loop 2 in the CCFS. We re-determined the NMR structure of yeast Tim21, and conducted MD simulations for comparison. Multidimensional scaling was used to visualize the conformational similarity of loop 2. We found that the crystal contact-free conformation of loop 2 is located close to the center of the ensembles of the loop 2 conformations in the NMR and MD structures.
    Loop 2 of yeast Tim21 in the CCFS adopts a representative, dominant conformation in solution.
    No single powerful technique is available for the characterization of flexible structures in protein molecules. NMR analyses and MD simulations provide useful, but incomplete information. CCFS crystallography offers a third route to this goal.
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  • 文章类型: Journal Article
    OBJECTIVE: To use case-parent triad data to investigate if cerebral palsy (CP) is associated with variants of the APOE gene, the rs59007384 SNP of the TOMM40 gene or combined haplotypes of the two genes.
    METHODS: DNA was analyzed in buccal swabs from 235 children with CP, their parents and a sibling. The relative risks (RR) with 95% confidence intervals (CI) that the children would have a distribution of APOE genotypes, rs59007384 variants or combined haplotypes deviating from Mendelian inheritance were estimated.
    RESULTS: Children with CP were more likely than expected to carry the APOEε3 allele (RR 7.5; CI: 0.99-53.7 for heterozygotes and 10.3; CI: 1.4-79.6 for homozygotes), and to have the haplotype of APOEε3 and rs59007384 G (RR 2.4; CI: 1-5.7 for heterozygotes, RR 3.7; CI: 1.4-9.5 for homozygotes) whereas the distribution was as expected for rs59007384 alone. In the subgroup analyses the findings were confined to children born preterm. Among siblings the distribution of these genes was as expected according to Mendelian inheritance.
    CONCLUSIONS: We speculate that children with APOEε2/APOEε4 alleles are more likely to die following cerebral injury in utero, resulting in a higher than expected proportion of children with CP carrying the APOEε3 allele.
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    文章类型: Case Reports
    在肽疫苗治疗后,晚期结肠癌患者获得了部分反应(PR)。一名61岁的妇女被转诊到我们医院接受肽疫苗治疗。她在附近的医院接受了乙状结肠切除术,最终发展到肺和盆腔淋巴结的多个转移,并伴有左肾积水。已插入输尿管支架置入导管治疗左肾积水,和口服阿片类药物用于缓解左骨盆区域的疼痛。三种肿瘤抗原衍生的肽(RNF43、TOMM34和KOC1)和两种人VEGFR衍生的肽(VEGFR1和VEGFR2)用作混合物。在第1、8、15和22天皮下接种肽混合物并以14天的间隔重复。患者治疗前血清癌胚抗原水平为28.9ng/mL(N<5ng/mL),开始治疗后迅速下降到正常范围。第5周的计算机断层扫描图像的评估显示了PR,如实体瘤标准中的反应评估标准所确定的。3个月后,停用口服阿片类药物。PR持续4个月,随后又稳定了4个月。没有观察到特别的副作用。通过免疫吸附斑点试验评估细胞毒性T淋巴细胞(CTL)反应,并且在六个疗程的每个末端识别出针对五种肽中的至少一种的阳性CTL应答。免疫疗法已被证明可以通过诱导活跃的抗肿瘤免疫反应来减缓肿瘤的生长;因此,很少观察到明显的肿瘤缩小。据我们所知,这是在晚期结肠癌患者中出现的首例PR报告.
    Partial response (PR) was obtained in a patient with advanced colon cancer following peptide vaccine therapy. A 61-year-old woman was referred to our hospital for peptide vaccine therapy. She had undergone sigmoidectomy at a nearby hospital and eventually developed multiple metastases to the lung and pelvic lymph nodes with left hydronephrosis. A ureteral stenting catheter had been inserted for left hydronephrosis, and oral opioids had been administered for relief of pain in the left pelvic region. Three tumor-antigen-derived peptides (RNF43, TOMM34, and KOC1) and two human VEGFR-derived peptides (VEGFR1 and VEGFR2) were used as a cocktail. The peptide cocktail was subcutaneously inoculated on days 1, 8, 15, and 22 and repeated at 14-day intervals. The patient\'s serum level of carcinoembryonic antigen was 28.9 ng/mL (N<5 ng/mL) before treatment, and it decreased promptly after the initiation of therapy to within a normal range. Evaluation of computed tomography images at week 5 revealed PR as determined by the Response Evaluation Criteria in Solid Tumor criteria. After month 3, the oral opioid was discontinued. The PR lasted for 4 months and was followed by stable disease for another 4 months. No particular adverse effects were observed. A cytotoxic T lymphocyte (CTL) response was evaluated by immunosorbent spot assay, and a positive CTL response was recognized against at least one of five peptides at each end of the six courses. Immunotherapy has been proven to slow tumor growth by inducing an active antitumor immune response; and therefore, significant tumor shrinkage is rarely observed. To our knowledge, this is the first case report of PR presented in a patient with advanced colon cancer.
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  • 文章类型: Journal Article
    The oxidative folding mechanism in the intermembrane space of human mitochondria underpins a disulfide relay system consisting of the import receptor Mia40 and the homodimeric FAD-dependent thiol oxidase ALR. The flavoprotein ALR receives two electrons per subunit from Mia40, which are then donated through one-electron reactions to two cytochrome c molecules, thus mediating a switch from two-electron to one-electron transfer. We dissect here the mechanism of the electron flux within ALR, characterizing at the atomic level the ALR intermediates that allow electrons to rapidly flow to cytochrome c. The intermediate critical for the electron-transfer process implies the formation of a specific inter-subunit disulfide which exclusively allows electron flow from Mia40 to FAD. This finding allows us to present a complete model for the electron-transfer pathway in ALR.
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  • 文章类型: Case Reports
    Mohr-Tranebjaerg syndrome is a rare X-linked condition characterized by the association of dystonia and progressive postlingual sensorineural hearing impairment. Here we report the clinical and genetic findings in a Spanish patient with MTS carrying a novel mutation in the DDP1 (deafness-dystonia peptide 1) gene, which encodes TIMM8a, a component of the mitochondrial protein translocation system. The phenotypic variability observed in patients with Mohr-Tranebjaerg syndrome suggests the involvement of modifier factors which may modulate the clinical manifestations of the syndrome.
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  • 文章类型: Case Reports
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