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Sökning: WFRF:(Amunts Alexey)

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  • [1]23Nästa
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1.
  • Aibara, Shintaro, et al. (författare)
  • Rapid Isolation of the Mitoribosome from HEK Cells
  • 2018
  • Ingår i: Journal of Visualized Experiments. - 1940-087X .- 1940-087X. ; :140
  • Tidskriftsartikel (refereegranskat)abstract
    • The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondria! genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart. Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only similar to 10(10) cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.
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2.
  • Aibara, Shintaro, et al. (författare)
  • Structural basis of mitochondrial translation
  • 2020
  • Ingår i: eLIFE. - 2050-084X. ; 9
  • Tidskriftsartikel (refereegranskat)abstract
    • Translation of mitochondrial messenger RNA (mt-mRNA) is performed by distinct mitoribosomes comprising at least 36 mitochondria-specific proteins. How these mitoribosomal proteins assist in the binding of mt-mRNA and to what extent they are involved in the translocation of transfer RNA (mt-tRNA) is unclear. To visualize the process of translation in human mitochondria, we report similar to 3.0 angstrom resolution structure of the human mitoribosome, including the L7/L12 stalk, and eight structures of its functional complexes with mt-mRNA, mt-tRNAs, recycling factor and additional trans factors. The study reveals a transacting protein module LRPPRC-SLIRP that delivers mt-mRNA to the mitoribosomal small subunit through a dedicated platform formed by the mitochondria-specific protein mS39. Mitoribosomal proteins of the large subunit mL40, mL48, and mL64 coordinate translocation of mt-tRNA. The comparison between those structures shows dynamic interactions between the mitoribosome and its ligands, suggesting a sequential mechanism of conformational changes.
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3.
  • Amunts, Alexey (författare)
  • The revolution evolution
  • 2022
  • Ingår i: Nature Plants. - 2055-0278. ; 8:1, s. 14-17
  • Tidskriftsartikel (övrigt vetenskapligt)
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4.
  • Bigalke, Janna M., et al. (författare)
  • Cryo-EM structure of the activated RET signaling complex reveals the importance of its cysteine-rich domain
  • 2019
  • Ingår i: Science Advances. - 2375-2548. ; 5:7
  • Tidskriftsartikel (refereegranskat)abstract
    • Signaling through the receptor tyrosine kinase RET is essential during normal development. Both gain- and loss-of-function mutations are involved in a variety of diseases, yet the molecular details of receptor activation have remained elusive. We have reconstituted the complete extracellular region of the RET signaling complex together with Neurturin (NRTN) and GFR alpha 2 and determined its structure at 5.7-angstrom resolution by cryo-EM. The proteins form an assembly through RET-GFR alpha 2 and RET-NRTN interfaces. Two key interaction points required for RET extracellular domain binding were observed: (i) the calcium-binding site in RET that contacts GFR alpha 2 domain 3 and (ii) the RET cysteine-rich domain interaction with NRTN. The structure highlights the importance of the RET cysteine-rich domain and allows proposition of a model to explain how complex formation leads to RET receptor dimerization and its activation. This provides a framework for targeting RET activity and for further exploration of mechanisms underlying neurological diseases.
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5.
  • Brown, Alan, et al. (författare)
  • Structures of the human mitochondrial ribosome in native states of assembly
  • 2017
  • Ingår i: Nature Structural & Molecular Biology. - 1545-9993 .- 1545-9985. ; 24:10, s. 866-869
  • Tidskriftsartikel (refereegranskat)abstract
    • Mammalian mitochondrial ribosomes (mitoribosomes) have less rRNA content and 36 additional proteins compared with the evolutionarily related bacterial ribosome. These differences make the assembly of mitoribosomes more complex than the assembly of bacterial ribosomes, but the molecular details of mitoribosomal biogenesis remain elusive. Here, we report the structures of two late-stage assembly intermediates of the human mitoribosomal large subunit (mt-LSU) isolated from a native pool within a human cell line and solved by cryo-EM to similar to 3-angstrom resolution. Comparison of the structures reveals insights into the timing of rRNA folding and protein incorporation during the final steps of ribosomal maturation and the evolutionary adaptations that are required to preserve biogenesis after the structural diversification of mitoribosomes. Furthermore, the structures redefine the ribosome silencing factor (RsfS) family as multifunctional biogenesis factors and identify two new assembly factors (L0R8F8 and mt-ACP) not previously implicated in mitoribosomal biogenesis.
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6.
  • Bugiardini, Enrico, et al. (författare)
  • MRPS25 mutations impair mitochondrial translation and cause encephalomyopathy
  • 2019
  • Ingår i: Human Molecular Genetics. - 0964-6906 .- 1460-2083. ; 28:16, s. 2711-2719
  • Tidskriftsartikel (refereegranskat)abstract
    • Mitochondrial disorders are clinically and genetically heterogeneous and are associated with a variety of disease mechanisms. Defects of mitochondrial protein synthesis account for the largest subgroup of disorders manifesting with impaired respiratory chain capacity; yet, only a few have been linked to dysfunction in the protein components of the mitochondrial ribosomes. Here, we report a subject presenting with dyskinetic cerebral palsy and partial agenesis of the corpus callosum, while histochemical and biochemical analyses of skeletal muscle revealed signs of mitochondrial myopathy. Using exome sequencing, we identified a homozygous variant c.215C>T in MRPS25, which encodes for a structural component of the 28S small subunit of the mitochondrial ribosome (mS25). The variant segregated with the disease and substitutes a highly conserved proline residue with leucine (p.P72L) that, based on the high-resolution structure of the 28S ribosome, is predicted to compromise inter-protein contacts and destabilize the small subunit. Concordant with the in silico analysis, patient's fibroblasts showed decreased levels of MRPS25 and other components of the 28S subunit. Moreover, assembled 28S subunits were scarce in the fibroblasts with mutant mS25 leading to impaired mitochondrial translation and decreased levels of multiple respiratory chain subunits. Crucially, these abnormalities were rescued by transgenic expression of wild-type MRPS25 in the mutant fibroblasts. Collectively, our data demonstrate the pathogenicity of the p.P72L variant and identify MRPS25 mutations as a new cause of mitochondrial translation defect.
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7.
  • Chen, Ming, et al. (författare)
  • Distinct structural modulation of photosystem I and lipid environment stabilizes its tetrameric assembly
  • 2020
  • Ingår i: Nature Plants. - 2055-026X .- 2055-0278. ; 6:3, s. 314-
  • Tidskriftsartikel (refereegranskat)abstract
    • Photosystem I (PSI) is able to form different oligomeric states across various species. To reveal the structural basis for PSI dimerization and tetramerization, we structurally investigated PSI from the cyanobacterium Anabaena. This revealed a disrupted trimerization domain due to lack of the terminal residues of PsaL in the lumen, which resulted in PSI dimers with loose connections between monomers and weaker energy-coupled chlorophylls than in the trimer. At the dimer surface, specific phospholipids, cofactors and interactions in combination facilitated recruitment of another dimer to form a tetramer. Taken together, the relaxed luminal connections and lipid specificity at the dimer interface account for membrane curvature. PSI tetramer assembly appears to increase the surface area of the thylakoid membrane, which would contribute to PSI crowding. Photosystem I from the cyanobacterium Anabaena has a disrupted trimerization domain resulting in dimers with loose connections between monomers. Phospholipids and cofactors at the dimer surface facilitate further dimerization to form a tetramer.
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8.
  • Desai, Nirupa, et al. (författare)
  • The structure of the yeast mitochondrial ribosome
  • 2017
  • Ingår i: Science. - 0036-8075 .- 1095-9203. ; 355:6324, s. 528-531
  • Tidskriftsartikel (refereegranskat)abstract
    • Mitochondria have specialized ribosomes (mitoribosomes) dedicated to the expression of the genetic information encoded by their genomes. Here, using electron cryomicroscopy, we have determined the structure of the 75-component yeast mitoribosome to an overall resolution of 3.3 angstroms. The mitoribosomal small subunit has been built de novo and includes 15S ribosomal RNA (rRNA) and 34 proteins, including 14 without homologs in the evolutionarily related bacterial ribosome. Yeast-specific rRNA and protein elements, including the acquisition of a putatively active enzyme, give the mitoribosome a distinct architecture compared to the mammalian mitoribosome. At an expanded messenger RNA channel exit, there is a binding platform for translational activators that regulate translation in yeast but not mammalian mitochondria. The structure provides insights into the evolution and species-specific specialization of mitochondrial translation.
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9.
  • Forsberg, Björn O., et al. (författare)
  • Cryo-EM reconstruction of the chlororibosome to 3.2 angstrom resolution within 24 h
  • 2017
  • Ingår i: IUCrJ. - 2052-2525. ; 4, s. 723-727
  • Tidskriftsartikel (refereegranskat)abstract
    • The introduction of direct detectors and the automation of data collection in cryo-EM have led to a surge in data, creating new opportunities for advancing computational processing. In particular, on-the-fly workflows that connect data collection with three-dimensional reconstruction would be valuable for more efficient use of cryo-EM and its application as a sample-screening tool. Here, accelerated on-the-fly analysis is reported with optimized organization of the data-processing tools, image acquisition and particle alignment that make it possible to reconstruct the three-dimensional density of the 70S chlororibosome to 3.2 angstrom resolution within 24 h of tissue harvesting. It is also shown that it is possible to achieve even faster processing at comparable quality by imposing some limits to data use, as illustrated by a 3.7 angstrom resolution map that was obtained in only 80 min on a desktop computer. These on-the-fly methods can be employed as an assessment of data quality from small samples and extended to high-throughput approaches.
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10.
  • Itoh, Yuzuru, et al. (författare)
  • Analysis of translating mitoribosome reveals functional characteristics of translation in mitochondria of fungi
  • 2020
  • Ingår i: Nature Communications. - 2041-1723 .- 2041-1723. ; 11:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Mitoribosomes are specialized protein synthesis machineries in mitochondria. However, how mRNA binds to its dedicated channel, and tRNA moves as the mitoribosomal subunit rotate with respect to each other is not understood. We report models of the translating fungal mitoribosome with mRNA, tRNA and nascent polypeptide, as well as an assembly intermediate. Nicotinamide adenine dinucleotide (NAD) is found in the central protuberance of the large subunit, and the ATPase inhibitory factor 1 (IF1) in the small subunit. The models of the active mitoribosome explain how mRNA binds through a dedicated protein platform on the small subunit, tRNA is translocated with the help of the protein mL108, bridging it with L1 stalk on the large subunit, and nascent polypeptide paths through a newly shaped exit tunnel involving a series of structural rearrangements. An assembly intermediate is modeled with the maturation factor Atp25, providing insight into the biogenesis of the mitoribosomal large subunit and translation regulation.
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  • Resultat 1-10 av 30
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