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41.
Heddy Soufari Florent Waltz Camila Parrot Stphanie Durrieu-Gaillard Anthony Bochler Lauriane Kuhn Marie Sissler Yaser Hashem 《Proceedings of the National Academy of Sciences of the United States of America》2020,117(47):29851
Kinetoplastids are unicellular eukaryotic parasites responsible for such human pathologies as Chagas disease, sleeping sickness, and leishmaniasis. They have a single large mitochondrion, essential for the parasite survival. In kinetoplastid mitochondria, most of the molecular machineries and gene expression processes have significantly diverged and specialized, with an extreme example being their mitochondrial ribosomes. These large complexes are in charge of translating the few essential mRNAs encoded by mitochondrial genomes. Structural studies performed in Trypanosoma brucei already highlighted the numerous peculiarities of these mitoribosomes and the maturation of their small subunit. However, several important aspects mainly related to the large subunit (LSU) remain elusive, such as the structure and maturation of its ribosomal RNA. Here we present a cryo-electron microscopy study of the protozoans Leishmania tarentolae and Trypanosoma cruzi mitoribosomes. For both species, we obtained the structure of their mature mitoribosomes, complete rRNA of the LSU, as well as previously unidentified ribosomal proteins. In addition, we introduce the structure of an LSU assembly intermediate in the presence of 16 identified maturation factors. These maturation factors act on both the intersubunit and the solvent sides of the LSU, where they refold and chemically modify the rRNA and prevent early translation before full maturation of the LSU.Kinetoplastids are unicellular eukaryotic parasites, causative agents of several human and livestock pathologies (1). They are potentially lethal, affecting more than 20 million people worldwide (1). Owing in part to their parasitic nature, they strongly diverged from other eukaryotic model species. Kinetoplastids evolved to live in and infect a large variety of eukaryotic organisms in very different molecular environments. Consequently, beyond the general similarities, kinetoplastid species have diverged evolutionarily from one another, and their protein sequence identity can be relatively low. They have a single large mitochondrion, a crucial component of their cellular architecture (2), where gene expression machineries have also largely diverged, notably their mitochondrial ribosomes (mitoribosomes) (3–10). These sophisticated RNA-protein complexes translate the few mRNAs still encoded by mitochondrial genomes. The mitoribosomes’ composition and structure diverged greatly from their bacterial ancestor, with the kinetoplastid mitoribosomes the most extreme case described to date, with highly reduced rRNAs and more than 80 supernumerary ribosomal proteins (r-proteins) compared with bacteria, completely reshaping the overall ribosome structure.Recent structural studies performed in Trypanosoma brucei have highlighted the particularities of this mitoribosome structure and composition, as well as the assembly processes of the small subunit (SSU) (3, 6). However, despite the very comprehensive structural characterization of the full T. brucei SSU and its maturation, several pivotal aspects related to the large subunit (LSU) have remained uncharacterized. For instance, a large portion of the LSU at the intersubunit side, including the whole rRNA peptidyl-transfer center (PTC) along with several r-proteins, were unresolved (3). Moreover, in contrast to the SSU, nearly nothing is known about LSU maturation and assembly. More generally, the maturation of the mitoribosomes in all eukaryotic species remains largely underexplored. Here we present a cryo-electron microscopy (EM) investigation of the full mature mitoribosomes from two different kinetoplastids, Leishmania tarentolae and Trypanosoma cruzi. In addition, we reveal the structure of an assembly intermediate of the LSU displaying unprecedented details on rRNA maturation in these very singular mitoribosomes, some of which likely can be generalized to the maturation of all rRNA.To obtain high-resolution reconstructions of the full kinetoplastid mitoribosomes, we purified mitochondrial vesicles from both L. tarentolae and T. cruzi and directly purified the mitoribosomes from the sucrose gradient (Methods) (SI Appendix, Fig. S1). All of our collected fractions were analyzed by nano-liquid chromatography tandem mass spectrometry (LC-MS/MS) (SI Appendix, Tables S1 and S2) to determine their proteomic composition. We collected micrographs from multiple vitrified samples corresponding to different sucrose gradient density peaks for both species, and following image processing, we obtained cryo-EM reconstructions of the full mitoribosomes, as well as of the dissociated SSU. Moreover, we also derived reconstructions of what appeared to be an assembly intermediate of L. tarentolae LSU. After extensive rounds of two-dimensional (2D) and three-dimensional (3D) classification and refinement we obtained the structure of L. tarentolae and T. cruzi complete and mature mitoribosomes at 3.9 Å and 6 Å, respectively (SI Appendix, Figs. S2–S4). Other notable features include the intersubunit contacts and two distinct rotational states in T. cruzi. Similarly to T. brucei (3), our cryo-EM analysis revealed a reconstruction of an early initiation complex from T. cruzi at 3.1 Å for the body and 3.2 Å for the head of the SSU (SI Appendix, Fig. S5). Further focused refinement on the LSU, SSU head, and SSU body generated reconstructions at 3.6, 3.8, and 4 Å, respectively, for L. tarentolae (SI Appendix, Figs. S2 and S3), and 3.7 and 4.5 Å for T. cruzi LSU and SSU, respectively (SI Appendix, Fig. S4). Combined, these reconstructions, along with the MS data allowed us to build nearly complete atomic models, with only few protein densities remaining unidentified. 相似文献
42.
Kai Mao Leon H. Chew Yuko Inoue-Aono Heesun Cheong Usha Nair Hana Popelka Calvin K. Yip Daniel J. Klionsky 《Proceedings of the National Academy of Sciences of the United States of America》2013,110(31):E2875-E2884
Macroautophagy (hereafter autophagy) functions in the nonselective clearance of cytoplasm. This process participates in many aspects of cell physiology, and is conserved in all eukaryotes. Autophagy begins with the organization of the phagophore assembly site (PAS), where most of the AuTophaGy-related (Atg) proteins are at least transiently localized. Autophagy occurs at a basal level and can be induced by various types of stress; the process must be tightly regulated because insufficient or excessive autophagy can be deleterious. A complex composed of Atg17-Atg31-Atg29 is vital for PAS organization and autophagy induction, implying a significant role in autophagy regulation. In this study, we demonstrate that Atg29 is a phosphorylated protein and that this modification is critical to its function; alanine substitution at the phosphorylation sites blocks its interaction with the scaffold protein Atg11 and its ability to facilitate assembly of the PAS. Atg29 has the characteristics of an intrinsically disordered protein, suggesting that it undergoes dynamic conformational changes on interaction with a binding partner(s). Finally, single-particle electron microscopy analysis of the Atg17-Atg31-Atg29 complex reveals an elongated structure with Atg29 located at the opposing ends.Autophagy is the major lysosome/vacuole-dependent cellular degradative pathway. During autophagy, cytoplasmic constituents including proteins, lipids, and even entire organelles are surrounded by the phagophore, the initial sequestering compartment. The phagophore then expands to form double-membrane vesicles, termed autophagosomes. The completed autophagosomes fuse with lysosomes/vacuoles allowing access of the cargo to the degradative enzymes within this organelle; the resulting breakdown products are released back into the cytosol as building blocks or catabolic substrates (1–3). Autophagy is not only critical for survival during nutrient deprivation but is also involved in various human pathophysiologies, including cancer and neurodegeneration (4).On autophagy induction, AuTophaGy-related (Atg) proteins accumulate at the phagophore assembly site (PAS) and initiate autophagosome formation. Among the 36 known Atg proteins, Atg1 is the only kinase, and it plays a particularly important role in autophagy induction by controlling the movement of other Atg proteins including Atg9 and Atg23 (5) and in the proper organization of the PAS (6, 7). Atg1 interacts with several proteins, including direct binding to Atg13 (which interacts with Atg17) and Atg11; the kinase activity of Atg1 is regulated in part by its binding to, and/or interaction with, some of these components (8, 9).Atg17 constitutively forms a stable protein complex with Atg29 and Atg31 in both growing and nitrogen starvation conditions (10), and Atg31 directly interacts with Atg17 and Atg29 to bridge these two proteins (11). When autophagy is initiated, the Atg17-Atg31-Atg29 complex is first targeted to the PAS and recruits other Atg proteins, including Atg1 and Atg13, highlighting the significance of the ternary complex (12, 13). Along these lines, a single deletion of the ATG17, ATG29, or ATG31 genes results in a dramatic decrease in autophagy activity (14–16).In this study, we examined the role of posttranslational modification of Atg29. We found that Atg29 is a phosphoprotein and that phosphorylation on the C-terminal domain is critical for autophagy activity; the N terminus of Atg29 contains the functional domain, whereas the C terminus plays a regulatory role. We continued and extended our study to include a structural and functional analysis of the Atg17-Atg31-Atg29 complex. Single-particle electron microscopy (EM) reveals that the recombinant Atg17-Atg31-Atg29 complex is present as an elongated S-shaped dimerized structure, with Atg17 forming the backbone. We further demonstrate that Atg29 has the characteristics of an intrinsically disordered protein (IDP), suggesting that the C-terminal half is flexible and capable of altering its conformation on binding to one or more interacting proteins. Finally, we determined that Atg11 is necessary and sufficient to recruit this complex to the PAS and that phosphorylation of Atg29 is required for its interaction with Atg11 and proper PAS localization. 相似文献
43.
I. J. van der Klei Meis van der Heide Richard J. S. Baerends Karl-Björn Rechinger Klaas Nicolay J. A. K. W. Kiel Marten Veenhuis 《Current genetics》1998,34(1):1-11
The Hansenula polymorpha per6-210 mutant is impaired in respect of growth on methanol (Mut–) and is characterized by aberrant peroxisome formation. The functionally complementing DNA fragment contains two open reading
frames. The first encodes dihydroxyacetone kinase (DAK), a cytosolic enzyme essential for formaldehyde assimilation; the second ORF codes for a novel protein (Pak1p). We have demonstrated
that per6-210 cells lack DAK activity, causing the Mut– phenotype, and have strongly reduced levels of Pak1p, resulting in peroxisomal defects. Sequence analysis revealed that per6-210 contains a mutation in the 3′ end of the DAK coding region, which overlaps with the promoter region of PAK1. Possibly this mutation also negatively affects PAK1 expression.
Received: 25 February / 12 May 1998 相似文献
44.
45.
46.
Elaine R. Frawley Robert G. Kranz 《Proceedings of the National Academy of Sciences of the United States of America》2009,106(25):10201-10206
Little is known about trafficking of heme from its sites of synthesis to sites of heme-protein assembly. We describe an integral membrane protein that allows trapping of endogenous heme to elucidate trafficking mechanisms. We show that CcsBA, a representative of a superfamily of integral membrane proteins involved in cytochrome c biosynthesis, exports and protects heme from oxidation. CcsBA has 10 transmembrane domains (TMDs) and reconstitutes cytochrome c synthesis in the Escherichia coli periplasm; thus, CcsBA is a cytochrome c synthetase. Purified CcsBA contains heme in an “external heme binding domain” for which two external histidines are shown to serve as axial ligands that protect the heme iron from oxidation. This is likely the active site of the synthetase. Furthermore, two conserved histidines in TMDs are required for heme to travel to the external heme binding domain. Remarkably, the function of CcsBA with mutations in these TMD histidines is corrected by exogenous imidazole, a result analogous to correction of heme binding by myoglobin when its proximal histidine is mutated. These data suggest that CcsBA has a heme binding site within the bilayer and that CcsBA is a heme channel. 相似文献
47.
Katsuya Saito Yukihiko Iizuka Shigeki Ohta Satoshi Takahashi Kenta Nakamura Hideyuki Saya Kazunari Yoshida Yutaka Kawakami Masahiro Toda 《Neuro-oncology》2014,16(12):1618-1629
Background
A cDNA library made from 2 glioma cell lines, U87MG and T98G, was screened by serological identification of antigens by recombinant cDNA expression (SEREX) using serum from a glioblastoma patient. Elongation factor Tu GTP binding domain containing protein 1 (EFTUD1), which is required for ribosome biogenesis, was identified. A cancer microarray database showed overexpression of EFTUD1 in gliomas, suggesting that EFTUD1 is a candidate molecular target for gliomas.Methods
EFTUD1 expression in glioma cell lines and glioma tissue was assessed by Western blot, quantitative PCR, and immunohistochemistry. The effect on ribosome biogenesis, cell growth, cell cycle, and induction of apoptosis and autophagy in glioma cells during the downregulation of EFTUD1 was investigated. To reveal the role of autophagy, the autophagy-blocker, chloroquine (CQ), was used in glioma cells downregulating EFTUD1. The effect of combining CQ with EFTUD1 inhibition in glioma cells was analyzed.Results
EFTUD1 expression in glioma cell lines and tissue was higher than in normal brain tissue. Downregulating EFTUD1 induced G1 cell-cycle arrest and apoptosis, leading to reduced glioma cell proliferation. The mechanism underlying this antitumor effect was impaired ribosome biogenesis via EFTUD1 inhibition. Additionally, protective autophagy was induced by glioma cells as an adaptive response to EFTUD1 inhibition. The antitumor effect induced by the combined treatment was significantly higher than that of either EFTUD1 inhibition or CQ alone.Conclusion
These results suggest that EFTUD1 represents a novel therapeutic target and that the combination of EFTUD1 inhibition with autophagy blockade may be effective in the treatment of gliomas. 相似文献48.
Rb intrinsically promotes erythropoiesis by coupling cell cycle exit with mitochondrial biogenesis 总被引:3,自引:0,他引:3
Regulation of the cell cycle is intimately linked to erythroid differentiation, yet how these processes are coupled is not well understood. To gain insight into this coordinate regulation, we examined the role that the retinoblastoma protein (Rb), a central regulator of the cell cycle, plays in erythropoiesis. We found that Rb serves a cell-intrinsic role and its absence causes ineffective erythropoiesis, with a differentiation block at the transition from early to late erythroblasts. Unexpectedly, in addition to a failure to properly exit the cell cycle, mitochondrial biogenesis fails to be up-regulated concomitantly, contributing to this differentiation block. The link between erythropoiesis and mitochondrial function was validated by inhibition of mitochondrial biogenesis. Erythropoiesis in the absence of Rb resembles the human myelodysplastic syndromes, where defects in cell cycle regulation and mitochondrial function frequently occur. Our work demonstrates how these seemingly disparate pathways play a role in coordinately regulating cellular differentiation. 相似文献
49.
50.
Campagnoli MF Ramenghi U Armiraglio M Quarello P Garelli E Carando A Avondo F Pavesi E Fribourg S Gleizes PE Loreni F Dianzani I 《Human mutation》2008,29(7):911-920
Diamond-Blackfan anemia (DBA) is an inherited disease characterized by pure erythroid aplasia. Thirty percent (30%) of patients display malformations, especially of the hands, face, heart, and urogenital tract. DBA has an autosomal dominant pattern of inheritance. De novo mutations are common and familial cases display wide clinical heterogeneity. Twenty-five percent (25%) of patients carry a mutation in the ribosomal protein (RP) S19 gene, whereas mutations in RPS24, RPS17, RPL35A, RPL11, and RPL5 are rare. These genes encode for structural proteins of the ribosome. A link between ribosomal functions and erythroid aplasia is apparent in DBA, but its etiology is not clear. Most authors agree that a defect in protein synthesis in a rapidly proliferating tissue, such as the erythroid bone marrow, may explain the defective erythropoiesis. A total of 77 RPS19 mutations have been described. Most are whole gene deletions, translocations, or truncating mutations (nonsense or frameshift), suggesting that haploinsufficiency is the basis of DBA pathology. A total of 22 missense mutations have also been described and several works have provided in vitro functional data for the mutant proteins. This review looks at the data on all these mutations, proposes a functional classification, and describes six new mutations. It is shown that patients with RPS19 mutations display a poorer response to steroids and a worse long-term prognosis compared to other DBA patients. 相似文献