mitochondrial processing peptidase
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Upon their translocation into the mitochondrial matrix, the N-terminal pre-sequence of nuclear-encoded proteins undergoes cleavage by mitochondrial processing peptidases. Some proteins require more than a single processing step, which involves several peptidases. Down-regulation of the putative Trypanosoma brucei mitochondrial intermediate peptidase (MIP) homolog by RNAi renders the cells unable to grow after 48 hours of induction. Ablation of MIP results in the accumulation of the precursor of the trypanosomatid-specific trCOIV protein, the largest nuclear-encoded subunit of the cytochrome c oxidase complex in this flagellate. However, the trCOIV precursor of the same size accumulates also in trypanosomes in which either alpha or beta subunits of the mitochondrial processing peptidase (MPP) have been depleted. Using a chimeric protein that consists of the N-terminal sequence of a putative subunit of respiratory complex I fused to a yellow fluorescent protein, we assessed the accumulation of the precursor protein in trypanosomes, in which RNAi was induced against the alpha or beta subunits of MPP or MIP. The observed accumulation of precursors indicates MIP depletion affects the activity of the cannonical MPP, or at least one of its subunits.
- MeSH
- down regulace MeSH
- fluorescenční mikroskopie MeSH
- fylogeneze MeSH
- malá interferující RNA metabolismus MeSH
- metaloendopeptidasy antagonisté a inhibitory klasifikace genetika metabolismus MeSH
- mitochondrie enzymologie MeSH
- podjednotky proteinů antagonisté a inhibitory genetika metabolismus MeSH
- respirační komplex IV metabolismus MeSH
- RNA interference MeSH
- sekvence aminokyselin MeSH
- substrátová specifita MeSH
- Trypanosoma brucei brucei metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
An all atomic, non-restrained molecular dynamics (MD) simulation in explicit water was used to study in detail the structural features of the highly conserved glycine-rich loop (GRL) of the α-subunit of the yeast mitochondrial processing peptidase (MPP) and its importance for the tertiary and quaternary conformation of MPP. Wild-type and GRL-deleted MPP structures were studied using non-restrained MD simulations, both in the presence and the absence of a substrate in the peptidase active site. Targeted MD simulations were employed to study the mechanism of substrate translocation from the GRL to the active site. We demonstrate that the natural conformational flexibility of the GRL is crucial for the substrate translocation process from outside the enzyme towards the MPP active site. We show that the α-helical conformation of the substrate is important not only during its initial interaction with MPP (i.e. substrate recognition), but also later, at least during the first third of the substrate translocation trajectory. Further, we show that the substrate remains in contact with the GRL during the whole first half of the translocation trajectory and that hydrophobic interactions play a major role. Finally, we conclude that the GRL acts as a precisely balanced structural element, holding the MPP subunits in a partially closed conformation regardless the presence or absence of a substrate in the active site.
- MeSH
- časové faktory MeSH
- glycin chemie MeSH
- katalytická doména MeSH
- metaloendopeptidasy chemie metabolismus MeSH
- podjednotky proteinů chemie metabolismus MeSH
- Saccharomyces cerevisiae enzymologie MeSH
- sekundární struktura proteinů MeSH
- simulace molekulární dynamiky MeSH
- substrátová specifita MeSH
- výpočetní biologie * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mitochondrial processing peptidases are heterodimeric enzymes (alpha/betaMPP) that play an essential role in mitochondrial biogenesis by recognizing and cleaving the targeting presequences of nuclear-encoded mitochondrial proteins. The two subunits are paralogues that probably evolved by duplication of a gene for a monomeric metallopeptidase from the endosymbiotic ancestor of mitochondria. Here, we characterize the MPP-like proteins from two important human parasites that contain highly reduced versions of mitochondria, the mitosomes of Giardia intestinalis and the hydrogenosomes of Trichomonas vaginalis. Our biochemical characterization of recombinant proteins showed that, contrary to a recent report, the Trichomonas processing peptidase functions efficiently as an alpha/beta heterodimer. By contrast, and so far uniquely among eukaryotes, the Giardia processing peptidase functions as a monomer comprising a single betaMPP-like catalytic subunit. The structure and surface charge distribution of the Giardia processing peptidase predicted from a 3-D protein model appear to have co-evolved with the properties of Giardia mitosomal targeting sequences, which, unlike classic mitochondrial targeting signals, are typically short and impoverished in positively charged residues. The majority of hydrogenosomal presequences resemble those of mitosomes, but longer, positively charged mitochondrial-type presequences were also identified, consistent with the retention of the Trichomonas alphaMPP-like subunit. Our computational and experimental/functional analyses reveal that the divergent processing peptidases of Giardia mitosomes and Trichomonas hydrogenosomes evolved from the same ancestral heterodimeric alpha/betaMPP metallopeptidase as did the classic mitochondrial enzyme. The unique monomeric structure of the Giardia enzyme, and the co-evolving properties of the Giardia enzyme and substrate, provide a compelling example of the power of reductive evolution to shape parasite biology.
- MeSH
- down regulace genetika MeSH
- fylogeneze MeSH
- genová dávka MeSH
- Giardia lamblia genetika metabolismus ultrastruktura MeSH
- glycin fyziologie genetika chemie MeSH
- metaloendopeptidasy genetika chemie metabolismus MeSH
- mitochondrie metabolismus MeSH
- multimerizace proteinu MeSH
- organely metabolismus MeSH
- podjednotky proteinů genetika MeSH
- posttranslační úpravy proteinů genetika MeSH
- proteinové domény bohaté na prolin fyziologie genetika MeSH
- řízená evoluce molekul MeSH
- sekvence aminokyselin MeSH
- transport proteinů MeSH
- Trichomonas vaginalis genetika metabolismus ultrastruktura MeSH
- vodík metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Mitochondrial processing peptidase (MPP) consists of α and β subunits that catalyze the cleavage of N-terminal mitochondrial-targeting sequences (N-MTSs) and deliver preproteins to the mitochondria. In plants, both MPP subunits are associated with the respiratory complex bc1, which has been proposed to represent an ancestral form. Subsequent duplication of MPP subunits resulted in separate sets of genes encoding soluble MPP in the matrix and core proteins (cp1 and cp2) of the membrane-embedded bc1 complex. As only α-MPP was duplicated in Neurospora, its single β-MPP functions in both MPP and bc1 complexes. Herein, we investigated the MPP/core protein family and N-MTSs in the kinetoplastid Trypanosoma brucei, which is often considered one of the most ancient eukaryotes. Analysis of N-MTSs predicted in 336 mitochondrial proteins showed that trypanosomal N-MTSs were comparable with N-MTSs from other organisms. N-MTS cleavage is mediated by a standard heterodimeric MPP, which is present in the matrix of procyclic and bloodstream trypanosomes, and its expression is essential for the parasite. Distinct Genes encode cp1 and cp2, and in the bloodstream forms the expression of cp1 is downregulated along with the bc1 complex. Phylogenetic analysis revealed that all eukaryotic lineages include members with a Neurospora-type MPP/core protein family, whereas cp1 evolved independently in metazoans, some fungi and kinetoplastids. Evolution of cp1 allowed the independent regulation of respiration and protein import, which is essential for the procyclic and bloodstream forms of T. brucei. These results indicate that T. brucei possesses a highly derived MPP/core protein family that likely evolved in response to its complex life cycle and does not appear to have an ancient character proposed earlier for this eukaryote.
- MeSH
- Eukaryota genetika MeSH
- fylogeneze MeSH
- metaloendopeptidasy genetika MeSH
- mitochondriální proteiny genetika metabolismus MeSH
- mitochondrie genetika MeSH
- molekulární evoluce MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- Trypanosoma brucei brucei genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Trypanosoma brucei, the agent of human sleeping sickness and ruminant nagana, is the most genetically tractable representative of the domain Excavata. It is evolutionarily very distant from humans, with a last common ancestor over 1 billion years ago. Frataxin, a highly conserved small protein involved in iron-sulfur cluster synthesis, is present in both organisms, and its deficiency is responsible for Friedreich's ataxia in humans. We have found that T. brucei growth-inhibition phenotype caused by down-regulated frataxin is rescued by means of human frataxin. The rescue is fully dependent on the human frataxin being imported into the trypanosome mitochondrion. Processing of the imported protein by mitochondrial processing peptidase can be blocked by mutations in the signal peptide, as in human cells. Although in human cells frataxin must be processed to execute its function, the same protein in the T. brucei mitochondrion is functional even in the absence of processing. Our results illuminate remarkable conservation of the mechanisms of mitochondrial protein import and processing.
- MeSH
- buněčné linie MeSH
- cytosol metabolismus MeSH
- financování organizované MeSH
- geneticky modifikovaná zvířata MeSH
- lidé MeSH
- mitochondrie metabolismus MeSH
- molekulární sekvence - údaje MeSH
- proteiny obsahující železo a síru genetika metabolismus MeSH
- proteiny vázající železo genetika chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- Trypanosoma brucei brucei genetika metabolismus růst a vývoj MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
The mitochondrial rhomboid protease PARL regulates mitophagy by balancing intramembrane proteolysis of PINK1 and PGAM5. It has been implicated in the pathogenesis of Parkinson's disease, but its investigation as a possible therapeutic target is challenging in this context because genetic deficiency of PARL may result in compensatory mechanisms. To address this problem, we undertook a hitherto unavailable chemical biology strategy. We developed potent PARL-targeting ketoamide inhibitors and investigated the effects of acute PARL suppression on the processing status of PINK1 intermediates and on Parkin activation. This approach revealed that PARL inhibition leads to a robust activation of the PINK1/Parkin pathway without major secondary effects on mitochondrial properties, which demonstrates that the pharmacological blockage of PARL to boost PINK1/Parkin-dependent mitophagy is a feasible approach to examine novel therapeutic strategies for Parkinson's disease. More generally, this study showcases the power of ketoamide inhibitors for cell biological studies of rhomboid proteases.
- MeSH
- endopeptidasy MeSH
- lidé MeSH
- metaloproteasy genetika metabolismus MeSH
- mitochondriální proteiny metabolismus MeSH
- mitofagie MeSH
- Parkinsonova nemoc * farmakoterapie MeSH
- proteasy * MeSH
- proteinkinasy metabolismus MeSH
- ubikvitinligasy metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mitochondrial ATPases associated with diverse cellular activities (AAA) proteases are involved in the quality control and processing of inner-membrane proteins. Here we investigate the cellular activities of YME1L, the human orthologue of the Yme1 subunit of the yeast i-AAA complex, using stable short hairpin RNA knockdown and expression experiments. Human YME1L is shown to be an integral membrane protein that exposes its carboxy-terminus to the intermembrane space and exists in several complexes of 600-1100 kDa. The stable knockdown of YME1L in human embryonic kidney 293 cells led to impaired cell proliferation and apoptotic resistance, altered cristae morphology, diminished rotenone-sensitive respiration, and increased susceptibility to mitochondrial membrane protein carbonylation. Depletion of YME1L led to excessive accumulation of nonassembled respiratory chain subunits (Ndufb6, ND1, and Cox4) in the inner membrane. This was due to a lack of YME1L proteolytic activity, since the excessive accumulation of subunits was reversed by overexpression of wild-type YME1L but not a proteolytically inactive YME1L variant. Similarly, the expression of wild-type YME1L restored the lamellar cristae morphology of YME1L-deficient mitochondria. Our results demonstrate the importance of mitochondrial inner-membrane proteostasis to both mitochondrial and cellular function and integrity and reveal a novel role for YME1L in the proteolytic regulation of respiratory chain biogenesis.
- MeSH
- apoptóza MeSH
- genový knockdown MeSH
- GTP-fosfohydrolasy metabolismus MeSH
- lidé MeSH
- metaloendopeptidasy metabolismus MeSH
- mitochondriální membrány metabolismus MeSH
- mitochondrie metabolismus MeSH
- NADH, NADPH oxidoreduktasy metabolismus MeSH
- proliferace buněk MeSH
- proteasy závislé na ATP metabolismus MeSH
- proteasy metabolismus MeSH
- protein - isoformy metabolismus MeSH
- respirační komplex IV metabolismus MeSH
- Saccharomyces cerevisiae - proteiny metabolismus MeSH
- Saccharomyces cerevisiae cytologie metabolismus MeSH
- transport elektronů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The highly reduced mitochondria (mitosomes) of Giardia intestinalis are recently discovered organelles for which, it was suggested, iron-sulfur cluster assembly was their only conserved function. However, only an incomplete set of the components required for FeS cluster biogenesis was localized to the mitosomes. Via proteomic analysis of a mitosome-rich cellular fraction together with immunofluorescence microscopy, we identified a novel mitosomal protein homologous to monothiol glutaredoxins containing a CGFS motif at the active site. Sequence analysis revealed the presence of long nonconserved N-terminal extension of 77 amino acids, which was absent in the mature protein. Expression of the complete and N-terminally truncated forms of the glutaredoxin indicated that the extension is involved in glutaredoxin import into mitosomes. However, the mechanism of preprotein processing is unclear, as the mitosomal processing peptidase is unable to cleave this type of extension. The recombinant mature protein was shown to form a homodimeric structure, which binds a labile FeS cluster. The cluster is stabilized by glutathione and dithiothreitol. Phylogenetic analysis showed that giardial glutaredoxin is related to the mitochondrial monothiol glutaredoxins involved in FeS cluster assembly. The identification of a mitochondrial-type monothiol glutaredoxin in the mitosomes of G. intestinalis thus completes the mitosomal FeS cluster biosynthetic pathway and provides further evidence for the mitochondrial origin of these organelles.
