bims-tricox Biomed News
on Translation, ribosomes and COX
Issue of 2022–07–24
thirteen papers selected by
Yash Verma, University of Delhi South Campus
  1. The accelerated evolution of human cytochrome c oxidase - Selection for reduced rate and proton pumping efficiency?
  2. Structural basis of mammalian complex IV inhibition by steroids.
  3. AIFM1 is a component of the mitochondrial disulfide relay that drives complex I assembly through efficient import of NDUFS5.
  4. Sequential rescue and repair of stalled and damaged ribosome by bacterial PrfH and RtcB.
  5. Selective footprinting of 40S and 80S ribosome subpopulations (Sel-TCP-seq) to study translation and its control.
  6. Purification of Mitochondrial Ribosomal Complexes from Trypanosoma cruzi and Leishmania tarentolae for Cryo-EM Analysis.
  7. Structural overview of the translocase of the mitochondrial outer membrane complex.
  8. Maize Shrek1 encodes a WD40 protein that regulates pre-rRNA processing in ribosome biogenesis.
  9. Modulating co-translational protein folding by rational design and ribosome engineering.
  10. Cryo-EM insights into tail-anchored membrane protein biogenesis in eukaryotes.
  11. Detergents and alternatives in cryo-EM studies of membrane proteins.
  12. A high-resolution map of human RNA translation.
  13. The amino acid sensor GCN2 suppresses Terminal Oligopyrimidine (TOP) mRNA translation via La-related Protein 1 (LARP1).
  1. Biochim Biophys Acta Bioenerg. 2022 Jul 15. pii: S0005-2728(22)00064-0. [Epub ahead of print] 148595
    The accelerated evolution of human cytochrome c oxidase - Selection for reduced rate and proton pumping efficiency?
    Hagai Rottenberg.
      The cytochrome c oxidase complex, complex VI (CIV), catalyzes the terminal step of the mitochondrial electron transport chain where the reduction of oxygen to water by cytochrome c is coupled to the generation of a protonmotive force that drive the synthesis of ATP. CIV evolution was greatly accelerated in humans and other anthropoid primates and appears to be driven by adaptive selection. However, it is not known if there are significant functional differences between the anthropoid primates CIV, and other mammals. Comparison of the high-resolution structures of bovine CIV, mouse CIV and human CIV shows structural differences that are associated with anthropoid-specific substitutions. Here I examine the possible effects of these substitutions in four CIV peptides that are known to affect proton pumping: the mtDNA-coded subunits I, II and III, and the nuclear-encoded subunit VIa2. I conclude that many of the anthropoid-specific substitutions could be expected to modulate the rate and/or the efficiency of proton pumping. These results are compatible with the previously proposed hypothesis that the accelerated evolution of CIV in anthropoid primates is driven by selection pressure to lower the mitochondrial protonmotive force and thus decrease the rate of superoxide generation by mitochondria.
    Keywords:  Anthropoid primates; Cytochrome c oxidase; Human; Proton pumping; Protonmotive force
    DOI:  https://doi.org/10.1016/j.bbabio.2022.148595
  2. Proc Natl Acad Sci U S A. 2022 Jul 26. 119(30): e2205228119
    Structural basis of mammalian complex IV inhibition by steroids.
    Justin M Di Trani, Agnes Moe, Daniel Riepl, Patricia Saura, Ville R I Kaila, Peter Brzezinski, John L Rubinstein.
      The mitochondrial electron transport chain maintains the proton motive force that powers adenosine triphosphate (ATP) synthesis. The energy for this process comes from oxidation of reduced nicotinamide adenine dinucleotide (NADH) and succinate, with the electrons from this oxidation passed via intermediate carriers to oxygen. Complex IV (CIV), the terminal oxidase, transfers electrons from the intermediate electron carrier cytochrome c to oxygen, contributing to the proton motive force in the process. Within CIV, protons move through the K and D pathways during turnover. The former is responsible for transferring two protons to the enzyme's catalytic site upon its reduction, where they eventually combine with oxygen and electrons to form water. CIV is the main site for respiratory regulation, and although previous studies showed that steroid binding can regulate CIV activity, little is known about how this regulation occurs. Here, we characterize the interaction between CIV and steroids using a combination of kinetic experiments, structure determination, and molecular simulations. We show that molecules with a sterol moiety, such as glyco-diosgenin and cholesteryl hemisuccinate, reversibly inhibit CIV. Flash photolysis experiments probing the rapid equilibration of electrons within CIV demonstrate that binding of these molecules inhibits proton uptake through the K pathway. Single particle cryogenic electron microscopy (cryo-EM) of CIV with glyco-diosgenin reveals a previously undescribed steroid binding site adjacent to the K pathway, and molecular simulations suggest that the steroid binding modulates the conformational dynamics of key residues and proton transfer kinetics within this pathway. The binding pose of the sterol group sheds light on possible structural gating mechanisms in the CIV catalytic cycle.
    Keywords:  complex IV; cryo-EM; electron transport chain; kinetics; molecular simulations
    DOI:  https://doi.org/10.1073/pnas.2205228119
  3. EMBO J. 2022 Jul 20. e110784
    AIFM1 is a component of the mitochondrial disulfide relay that drives complex I assembly through efficient import of NDUFS5.
    Silja Lucia Salscheider, Sarah Gerlich, Alfredo Cabrera-Orefice, Esra Peker, Robin Alexander Rothemann, Lena Maria Murschall, Yannik Finger, Karolina Szczepanowska, Zeinab Alsadat Ahmadi, Sergio Guerrero-Castillo, Alican Erdogan, Mark Becker, Muna Ali, Markus Habich, Carmelina Petrungaro, Nele Burdina, Guenter Schwarz, Merlin Klußmann, Ines Neundorf, David A Stroud, Michael T Ryan, Aleksandra Trifunovic, Ulrich Brandt, Jan Riemer.
      The mitochondrial intermembrane space protein AIFM1 has been reported to mediate the import of MIA40/CHCHD4, which forms the import receptor in the mitochondrial disulfide relay. Here, we demonstrate that AIFM1 and MIA40/CHCHD4 cooperate beyond this MIA40/CHCHD4 import. We show that AIFM1 and MIA40/CHCHD4 form a stable long-lived complex in vitro, in different cell lines, and in tissues. In HEK293 cells lacking AIFM1, levels of MIA40 are unchanged, but the protein is present in the monomeric form. Monomeric MIA40 neither efficiently interacts with nor mediates the import of specific substrates. The import defect is especially severe for NDUFS5, a subunit of complex I of the respiratory chain. As a consequence, NDUFS5 accumulates in the cytosol and undergoes rapid proteasomal degradation. Lack of mitochondrial NDUFS5 in turn results in stalling of complex I assembly. Collectively, we demonstrate that AIFM1 serves two overlapping functions: importing MIA40/CHCHD4 and constituting an integral part of the disulfide relay that ensures efficient interaction of MIA40/CHCHD4 with specific substrates.
    Keywords:  AIFM1; MIA40-CHCHD4; NDUFS5; complex I; mitochondrial disulfide relay
    DOI:  https://doi.org/10.15252/embj.2022110784
  4. Proc Natl Acad Sci U S A. 2022 Jul 19. 119(29): e2202464119
    Sequential rescue and repair of stalled and damaged ribosome by bacterial PrfH and RtcB.
    Yannan Tian, Fuxing Zeng, Adrika Raybarman, Shirin Fatma, Amy Carruthers, Qingrong Li, Raven H Huang.
      RtcB is involved in transfer RNA (tRNA) splicing in archaeal and eukaryotic organisms. However, most RtcBs are found in bacteria, whose tRNAs have no introns. Because tRNAs are the substrates of archaeal and eukaryotic RtcB, it is assumed that bacterial RtcBs are for repair of damaged tRNAs. Here, we show that a subset of bacterial RtcB, denoted RtcB2 herein, specifically repair ribosomal damage in the decoding center. To access the damage site for repair, however, the damaged 70S ribosome needs to be dismantled first, and this is accomplished by bacterial PrfH. Peptide-release assays revealed that PrfH is only active with the damaged 70S ribosome but not with the intact one. A 2.55-Å cryo-electron microscopy structure of PrfH in complex with the damaged 70S ribosome provides molecular insight into PrfH discriminating between the damaged and the intact ribosomes via specific recognition of the cleaved 3'-terminal nucleotide. RNA repair assays demonstrated that RtcB2 efficiently repairs the damaged 30S ribosomal subunit but not the damaged tRNAs. Cell-based assays showed that the RtcB2-PrfH pair reverse the damage inflicted by ribosome-specific ribotoxins in vivo. Thus, our combined biochemical, structural, and cell-based studies have uncovered a bacterial defense system specifically evolved to reverse the lethal ribosomal damage in the decoding center for cell survival.
    Keywords:  RNA repair; cryo-EM; ribosome rescue; ribotoxin
    DOI:  https://doi.org/10.1073/pnas.2202464119
  5. Nat Protoc. 2022 Jul 22.
    Selective footprinting of 40S and 80S ribosome subpopulations (Sel-TCP-seq) to study translation and its control.
    Susan Wagner, Jonathan Bohlen, Anna Herrmannova, Jan Jelínek, Thomas Preiss, Leoš Shivaya Valášek, Aurelio A Teleman.
      Multiple aspects of mRNA translation are subject to regulation. Here we present a ribosome footprinting protocol to determine the location and composition of 40S and 80S ribosome complexes on endogenous mRNAs transcriptome-wide in vivo in yeast and mammalian cells. We present an extension of the translation complex profiling (TCP-seq) protocol, originally developed in yeast, by including an immunoprecipitation step to assay the location of both 40S and 80S ribosome complexes containing proteins of interest. This yields information on where along mRNAs the ribosome-bound protein of interest joins the ribosome to act, and where it leaves again, thereby monitoring the sequential steps of translation and the roles of various translation factors therein. Rapid fixation of live cells ensures the integrity of all translation complexes bound to mRNA at native positions. Two procedures are described, differing mainly in the fixation conditions and the library preparation. Depending on the research question, either procedure offers advantages. Execution of a Sel-TCP-seq experiment takes 5-10 working days, and initial data analysis can be completed within 2 days.
    DOI:  https://doi.org/10.1038/s41596-022-00708-4
  6. Bio Protoc. 2022 May 20. 12(10): e4425
    Purification of Mitochondrial Ribosomal Complexes from Trypanosoma cruzi and Leishmania tarentolae for Cryo-EM Analysis.
    Stéphanie Durrieu-Gaillard, Marie Sissler, Yaser Hashem.
      Kinetoplastids are unicellular eukaryotic parasites responsible for human pathologies such as Chagas disease, sleeping sickness or Leishmaniasis, caused by Trypanosoma cruzi, Trypanosoma brucei, and various Leishmania spp., respectively. They harbor a single large mitochondrion that is essential for the survival of the parasite. Interestingly, most of the mitochondrial gene expression machineries and processes present significant differences from their nuclear and cytosolic counterparts. A striking example concerns their mitochondrial ribosomes, in charge of translating the few essential mRNAs encoded by mitochondrial genomes. Here, we present a detailed protocol including the specific procedures to isolate mitochondria from two species of kinetoplastids, T. cruzi and L. tarentolae, by differential centrifugations. Then, we detail the protocol to purify mitochondrial ribosomal complexes from these two species of parasites (including ribosomal maturating complexes) by a sucrose gradient approach. Finally, we describe how to prepare cryo-electron microscopy (cryo-EM) grids from these two sorts of samples. This protocol will be useful for further studies aiming at analyzing mitochondrial translation regulation.
    Keywords:  Cryo-EM; Kinetoplastids mitochondria; Leishmania tarentolae; Mitoribosome; Mitoribosome biogenesis; Sucrose density gradient; Trypanosoma cruzi
    DOI:  https://doi.org/10.21769/BioProtoc.4425
  7. Biophys Physicobiol. 2022 ;19 e190022
    Structural overview of the translocase of the mitochondrial outer membrane complex.
    Yuhei Araiso, Toshiya Endo.
      Most mitochondrial proteins are synthesized as precursor proteins (preproteins) in the cytosol and imported into mitochondria. The translocator of the outer membrane (TOM) complex functions as a main entry gate for the import of mitochondrial proteins. The TOM complex is a multi-subunit membrane protein complex composed of a β-barrel channel Tom40 and six single-pass membrane proteins. Recent cryo-EM studies have revealed high-resolution structures of the yeast and human TOM complexes, which enabled us to discuss the mechanism of protein import at an amino-acid residue level. The cryo-EM structures show that two Tom40 β-barrels are surrounded by two sets of small Tom subunits to form a dimeric structure. The intermembrane space (IMS) domains of Tom40, Tom22, and Tom7 form a binding site for presequence-containing preproteins in the middle of the dimer to achieve their efficient transfer of to the downstream translocase, the TIM23 complex. The N-terminal segment of Tom40 spans the channel from the cytosol to the IMS to interact with Tom5 at the periphery of the dimer, where downstream components of presequence-lacking preproteins are recruited. Structure-based biochemical analyses together with crosslinking experiments revealed that each Tom40 channel possesses two distinct paths and exit sites for protein translocation of different sets of mitochondrial preproteins. Here we summarize the current knowledge on the structural features, protein translocation mechanisms, and remaining questions for the TOM complexes, with particular emphasis on their determined cryo-EM structures. This article is an extended version of the Japanese article, Structural basis for protein translocation by the translocase of the outer mitochondrial membrane, published in SEIBUTSU BUTSURI Vol. 60, p. 280-283 (2020).
    Keywords:  Cryo-EM; TOM complex preprotein; mitochondria; protein translocation
    DOI:  https://doi.org/10.2142/biophysico.bppb-v19.0022
  8. Plant Cell. 2022 Jul 22. pii: koac216. [Epub ahead of print]
    Maize Shrek1 encodes a WD40 protein that regulates pre-rRNA processing in ribosome biogenesis.
    Hui Liu, Zhihui Xiu, Huanhuan Yang, Zhaoxing Ma, Dalin Yang, Hongqiu Wang, Bao-Cai Tan.
      Ribosome biogenesis is a fundamental and highly orchestrated process that involves hundreds of ribosome biogenesis factors. Despite advances that have been made in yeast, the molecular mechanism of ribosome biogenesis remains largely unknown in plants. We uncovered a WD40 protein, Shrunken and Embryo Defective Kernel 1 (SHREK1), and showed that it plays a crucial role in ribosome biogenesis and kernel development in maize (Zea mays). The shrek1 mutant shows an aborted embryo and underdeveloped endosperm and embryo-lethal in maize. SHREK1 localizes mainly to the nucleolus and accumulates to high levels in the seed. Depleting SHREK1 perturbs pre-rRNA processing and causes imbalanced profiles of mature rRNA and ribosome. The expression pattern of ribosomal-related genes is significantly altered in shrek1. Like its yeast (Saccharomyces cerevisiae) ortholog Periodic tryptophan protein 1 (PWP1), SHREK1 physically interacts with ribosomal protein ZmRPL7a, a transient component of the PWP1-subcomplex involved in pre-rRNA processing in yeast. Additionally, SHREK1 may assist in the A3 cleavage of the pre-rRNA in maize by interacting with the nucleolar protein ZmPOP4, a maize homolog of the yeast RNase MRP complex subunit. Overall, our work demonstrates a vital role of SHREK1 in pre-60S ribosome maturation, and reveals that impaired ribosome function accounts for the embryo lethality in shrek1.
    DOI:  https://doi.org/10.1093/plcell/koac216
  9. Nat Commun. 2022 Jul 22. 13(1): 4243
    Modulating co-translational protein folding by rational design and ribosome engineering.
    Minkoo Ahn, Tomasz Włodarski, Alkistis Mitropoulou, Sammy H S Chan, Haneesh Sidhu, Elena Plessa, Thomas A Becker, Nediljko Budisa, Christopher A Waudby, Roland Beckmann, Anaïs M E Cassaignau, Lisa D Cabrita, John Christodoulou.
      Co-translational folding is a fundamental process for the efficient biosynthesis of nascent polypeptides that emerge through the ribosome exit tunnel. To understand how this process is modulated by the shape and surface of the narrow tunnel, we have rationally engineered three exit tunnel protein loops (uL22, uL23 and uL24) of the 70S ribosome by CRISPR/Cas9 gene editing, and studied the co-translational folding of an immunoglobulin-like filamin domain (FLN5). Our thermodynamics measurements employing 19F/15N/methyl-TROSY NMR spectroscopy together with cryo-EM and molecular dynamics simulations reveal how the variations in the lengths of the loops present across species exert their distinct effects on the free energy of FLN5 folding. A concerted interplay of the uL23 and uL24 loops is sufficient to alter co-translational folding energetics, which we highlight by the opposite folding outcomes resulting from their extensions. These subtle modulations occur through a combination of the steric effects relating to the shape of the tunnel, the dynamic interactions between the ribosome surface and the unfolded nascent chain, and its altered exit pathway within the vestibule. These results illustrate the role of the exit tunnel structure in co-translational folding, and provide principles for how to remodel it to elicit a desired folding outcome.
    DOI:  https://doi.org/10.1038/s41467-022-31906-z
  10. Curr Opin Struct Biol. 2022 Jul 15. pii: S0959-440X(22)00107-5. [Epub ahead of print]75 102428
    Cryo-EM insights into tail-anchored membrane protein biogenesis in eukaryotes.
    Irmgard Sinning, Melanie A McDowell.
      Tail-anchored (TA) proteins are a biologically significant class of membrane proteins, which require specialised cellular pathways to insert their single C-terminal transmembrane domain into the correct membrane. Cryo-electron microscopy has recently provided new insights into the organelle-specific machineries for TA protein biogenesis. Structures of targeting and insertase complexes within the canonical guided entry of TA proteins (GET) pathway indicate how substrates are faithfully chaperoned into the endoplasmic reticulum (ER) membrane in metazoans. The core of the GET insertase is conserved within structures of the ER membrane protein complex (EMC), which acts in parallel to insert a different subset of TA proteins. Furthermore, structures of the dislocases Spf1 and Msp1 show how they remove mislocalised TA proteins from the ER and outer mitochondrial membranes respectively.
    DOI:  https://doi.org/10.1016/j.sbi.2022.102428
  11. Acta Biochim Biophys Sin (Shanghai). 2022 Jul 25.
    Detergents and alternatives in cryo-EM studies of membrane proteins.
    Shuo Li.
      Structure determination of membrane proteins has been a long-standing challenge to understand the molecular basis of life processes. Detergents are widely used to study the structure and function of membrane proteins by various experimental methods, and the application of membrane mimetics is also a prevalent trend in the field of cryo-EM analysis. This review focuses on the widely-used detergents and corresponding properties and structures, and also discusses the growing interests in membrane mimetic systems used in cryo-EM studies, providing insights into the role of detergent alternatives in structure determination.
    Keywords:  cryo-EM; detergent; membrane protein; micelle; nanodiscs
    DOI:  https://doi.org/10.3724/abbs.2022088
  12. Mol Cell. 2022 Jul 07. pii: S1097-2765(22)00606-2. [Epub ahead of print]
    A high-resolution map of human RNA translation.
    Sonia P Chothani, Eleonora Adami, Anissa A Widjaja, Sarah R Langley, Sivakumar Viswanathan, Chee Jian Pua, Nevin Tham Zhihao, Nathan Harmston, Giuseppe D'Agostino, Nicola Whiffin, Wang Mao, John F Ouyang, Wei Wen Lim, Shiqi Lim, Cheryl Q E Lee, Alexandra Grubman, Joseph Chen, J P Kovalik, Karl Tryggvason, Jose M Polo, Lena Ho, Stuart A Cook, Owen J L Rackham, Sebastian Schafer.
      Translated small open reading frames (smORFs) can have important regulatory roles and encode microproteins, yet their genome-wide identification has been challenging. We determined the ribosome locations across six primary human cell types and five tissues and detected 7,767 smORFs with translational profiles matching those of known proteins. The human genome was found to contain highly cell-type- and tissue-specific smORFs and a subset that encodes highly conserved amino acid sequences. Changes in the translational efficiency of upstream-encoded smORFs (uORFs) and the corresponding main ORFs predominantly occur in the same direction. Integration with 456 mass-spectrometry datasets confirms the presence of 603 small peptides at the protein level in humans and provides insights into the subcellular localization of these small proteins. This study provides a comprehensive atlas of high-confidence translated smORFs derived from primary human cells and tissues in order to provide a more complete understanding of the translated human genome.
    Keywords:  Ribo-seq; Ribosome profiling; dORFs; lncRNAs; smORFs; translation; uORFs; untranslated regions
    DOI:  https://doi.org/10.1016/j.molcel.2022.06.023
  13. J Biol Chem. 2022 Jul 18. pii: S0021-9258(22)00719-0. [Epub ahead of print] 102277
    The amino acid sensor GCN2 suppresses Terminal Oligopyrimidine (TOP) mRNA translation via La-related Protein 1 (LARP1).
    Zeenat Farooq, Fedho Kusuma, Phillip Burke, Catherine R Dufour, Duckgue Lee, Negar Tabatabaei, Phoenix Toboz, Ernest Radovani, Jack Greenblatt, Jalees Rehman, Jacob Class, Arkady Khoutorsky, Bruno D Fonseca, Justin M Richner, Eloi Mercier, Guillaume Bourque, Vincent Giguère, Arvind R Subramaniam, Jaeseok Han, Soroush Tahmasebi.
      La-related protein 1 (LARP1) has been identified as a key translational inhibitor of terminal oligopyrimidine (TOP) mRNAs downstream of the nutrient sensing protein kinase complex, mTORC1. LARP1 exerts this inhibitory effect on TOP mRNA translation by binding to the mRNA cap and the adjacent 5'TOP motif, resulting in the displacement of the cap-binding protein eIF4E from TOP mRNAs. However, the involvement of additional signaling pathway in regulating LARP1-mediated inhibition of TOP mRNA translation is not clear. In the present study, we identify a second nutrient sensing kinase GCN2 that converges on LARP1 to control TOP mRNA translation. Using chromatin-immunoprecipitation followed by massive parallel sequencing (ChIP-seq) analysis of activating transcription factor 4 (ATF4), an effector of GCN2 in nutrient stress conditions, in WT and GCN2 KO mouse embryonic fibroblasts (MEFs) we determined that LARP1 is a GCN2-dependent transcriptional target of ATF4. Moreover, we identified GCN1, a GCN2 activator, participates in a complex with LARP1 on stalled ribosomes, suggesting a role for GCN1 in LARP1-mediated translation inhibition in response to ribosome stalling. Therefore, our data suggest that the GCN2 pathway controls LARP1 activity via two mechanisms: ATF4-dependent transcriptional induction of LARP1 mRNA, and GCN1-mediated recruitment of LARP1 to stalled ribosomes.
    Keywords:  ATF4; GCN1; GCN2; LARP1; TOP mRNA translation; Translational control; amino acid deprivation; ribosome collision
    DOI:  https://doi.org/10.1016/j.jbc.2022.102277
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