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Toshifumi Inada
Accurate gene expression is a prerequisite for all cellular processes. Quality control machineries respond to errors during protein synthesis by refolding polypeptides or targeting them for degradation. As another layer of gene expression control, aberrant mRNAs can also be detected and eliminated by mRNA quality control systems while engaging the ribosome. In this review, I focus on recent studies on the cotranslational quality control mechanisms induced by abnormal translational elongation and termination, which result in the rapid degradation of aberrant polypeptides and mRNAs...
October 13, 2016: Trends in Biochemical Sciences
Atsuko Miki, Josephine Galipon, Satoshi Sawai, Toshifumi Inada, Kunihiro Ohta
Antisense RNA has emerged as a crucial regulator of opposite-strand protein-coding genes in the long noncoding RNA (lncRNA) category, but little is known about their dynamics and decay process in the context of a stress response. Antisense transcripts from the fission yeast fbp1 locus (fbp1-as) are expressed in glucose-rich conditions and anticorrelated with transcription of metabolic stress-induced lncRNA (mlonRNA) and mRNA on the sense strand during glucose starvation. Here, we investigate the localization and decay of antisense RNAs at fbp1 and other loci, and propose a model to explain the rapid switch between antisense and sense mlonRNA/mRNA transcription triggered by glucose starvation...
October 10, 2016: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Lena Thoring, Doreen A Wüstenhagen, Maria Borowiak, Marlitt Stech, Andrei Sonnabend, Stefan Kubick
Nowadays, biotechnological processes play a pivotal role in target protein production. In this context, Chinese Hamster Ovary (CHO) cells are one of the most prominent cell lines for the expression of recombinant proteins and revealed as a safe host for nearly 40 years. Nevertheless, the major bottleneck of common in vivo protein expression platforms becomes obvious when looking at the production of so called "difficult-to-express" proteins. This class of proteins comprises in particular several ion channels and multipass membrane proteins as well as cytotoxic proteins...
2016: PloS One
Kanghyun Lee, Ruchika Sharma, Om Kumar Shrestha, Craig A Bingman, Elizabeth A Craig
Ribosome-associated J protein-Hsp70 chaperones promote nascent-polypeptide folding and normal translational fidelity. The J protein Zuo1 is known to span the ribosomal subunits, but understanding of its function is limited. Here we present new structural and cross-linking data allowing more precise positioning of Saccharomyces cerevisiae Zuo1 near the 60S polypeptide-exit site and suggesting interactions of Zuo1 with the ribosomal protein eL31 and 25S rRNA helix 24. The junction between the 60S-interacting and subunit-spanning helices is a hinge that positions Zuo1 on the 40S yet accommodates subunit rotation...
September 26, 2016: Nature Structural & Molecular Biology
Karin Öjemalm, Takashi Higuchi, Patricia Lara, Erik Lindahl, Hiroaki Suga, Gunnar von Heijne
Cotranslational translocon-mediated insertion of membrane proteins into the endoplasmic reticulum is a key process in membrane protein biogenesis. Although the mechanism is understood in outline, quantitative data on the energetics of the process is scarce. Here, we have measured the effect on membrane integration efficiency of nonproteinogenic analogs of the positively charged amino acids arginine and lysine incorporated into model transmembrane segments. We provide estimates of the influence on the apparent free energy of membrane integration (ΔGapp) of "snorkeling" of charged amino acids toward the lipid-water interface, and of charge neutralization...
September 20, 2016: Proceedings of the National Academy of Sciences of the United States of America
Isotta Lorenzi, Silke Oeljeklaus, Christin Ronsör, Bettina Bareth, Bettina Warscheid, Peter Rehling, Sven Dennerlein
The three conserved core subunits of the cytochrome c oxidase are mitochondrial-encoded in close to all eukaryotes. The Cox2 subunit spans the inner membrane twice, exposing N- and C-terminus into the intermembrane space. For this the N-terminus is exported cotranslationally by Oxa1 and subsequently undergoes proteolytic maturation in Saccharomyces cerevisiae Little is known about the translocation of the C-terminus but Cox18 has been identified as a critical protein in this process. Here we find that the scaffold protein Cox20, which promotes processing of Cox2, is in complex with the ribosome-receptor Mba1 and translating mitochondrial ribosomes in a Cox2-dependent manner...
August 22, 2016: Molecular and Cellular Biology
Frédéric Frottin, Willy V Bienvenut, Jérôme Bignon, Eric Jacquet, Alvaro Sebastian Vaca Jacome, Alain Van Dorsselaer, Sarah Cianferani, Christine Carapito, Thierry Meinnel, Carmela Giglione
Fumagillin and its derivatives are therapeutically useful because they can decrease cancer progression. The specific molecular target of fumagillin is methionine aminopeptidase 2 (MetAP2), one of the two MetAPs present in the cytosol. MetAPs catalyze N-terminal methionine excision (NME), an essential pathway of cotranslational protein maturation. To date, it remains unclear the respective contribution of MetAP1 and MetAP2 to the NME process in vivo and why MetAP2 inhibition causes cell cycle arrest only in a subset of cells...
August 11, 2016: Oncotarget
Justin W Chartron, Katherine C L Hunt, Judith Frydman
Ribosome-associated factors must properly decode the limited information available in nascent polypeptides to direct them to their correct cellular fate. It is unclear how the low complexity information exposed by the nascent chain suffices for accurate recognition by the many factors competing for the limited surface near the ribosomal exit site. Questions remain even for the well-studied cotranslational targeting cycle to the endoplasmic reticulum, involving recognition of linear hydrophobic signal sequences or transmembrane domains by the signal recognition particle (SRP)...
August 11, 2016: Nature
Paul J Focke, Christopher Hein, Beate Hoffmann, Kimberly Matulef, Frank Bernhard, Volker Dötsch, Francis I Valiyaveetil
Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein...
August 2, 2016: Biochemistry
Albena Draycheva, Thomas Bornemann, Sergey Ryazanov, Nils-Alexander Lakomek, Wolfgang Wintermeyer
Proteins are inserted into the bacterial plasma membrane cotranslationally after translating ribosomes are targeted to the translocon in the membrane via the signal recognition particle (SRP) pathway. The targeting pathway involves an interaction between SRP and the SRP receptor, FtsY. Here we focus on the role of FtsY and its interaction with the translocon in controlling targeting. We show that in unbound FtsY the NG and A domains interact with one another. The interaction involves the membrane-targeting region at the junction between A and N domain...
October 2016: Molecular Microbiology
Ying Zhang, Thea Schäffer, Tina Wölfle, Edith Fitzke, Gerhard Thiel, Sabine Rospert
Targeting of transmembrane proteins to the endoplasmic reticulum (ER) proceeds via either the signal recognition particle (SRP) or the guided entry of tail-anchored proteins (GET) pathway, consisting of Get1 to -5 and Sgt2. While SRP cotranslationally targets membrane proteins containing one or multiple transmembrane domains, the GET pathway posttranslationally targets proteins containing a single C-terminal transmembrane domain termed the tail anchor. Here, we dissect the roles of the SRP and GET pathways in the sorting of homologous, two-membrane-spanning K(+) channel proteins termed Kcv, Kesv, and Kesv-VV...
September 15, 2016: Molecular and Cellular Biology
Martine A Collart
The Not4 RING E3 ligase is a subunit of the evolutionarily conserved Ccr4-Not complex. Originally identified in yeast by mutations that increase transcription, it was subsequently defined as an ubiquitin ligase. Substrates for this ligase were characterized in yeast and in metazoans. Interestingly, some substrates for this ligase are targeted for polyubiquitination and degradation, while others instead are stable monoubiquitinated proteins. The former are mostly involved in transcription, while the latter are a ribosomal protein and a ribosome-associated chaperone...
2013: ISRN molecular biology
Natalia Shcherbik, Tatiana A Chernova, Yury O Chernoff, Dimitri G Pestov
Cotranslational degradation of polypeptide nascent chains plays a critical role in quality control of protein synthesis and the rescue of stalled ribosomes. In eukaryotes, ribosome stalling triggers release of 60S subunits with attached nascent polypeptides, which undergo ubiquitination by the E3 ligase Ltn1 and proteasomal degradation facilitated by the ATPase Cdc48. However, the identity of factors acting upstream in this process is less clear. Here, we examined how the canonical release factors Sup45-Sup35 (eRF1-eRF3) and their paralogs Dom34-Hbs1 affect the total population of ubiquitinated nascent chains associated with yeast ribosomes...
August 19, 2016: Nucleic Acids Research
Fabio Trovato, Edward P O'Brien
Regulation of protein stability and function in vivo begins during protein synthesis, when the ribosome translates a messenger RNA into a nascent polypeptide. Cotranslational processes involving a nascent protein include folding, binding to other macromolecules, enzymatic modification, and secretion through membranes. Experiments have shown that the rate at which the ribosome adds amino acids to the elongating nascent chain influences the efficiency of these processes, with alterations to these rates possibly contributing to diseases, including some types of cancer...
July 5, 2016: Annual Review of Biophysics
E P Isakova, Y I Deryabina, O A Leonovich, M V Zylkova, Iu K Biriukova
No eukaryotic species has a system for homologous DNA recombination of the mitochondrial genome. We report on an integrative genetic systembased on the pQ-SRUS construct that allows the expression of the RecA recombinase from Bacillus subtilis and its transportation to mitochondria of Yarrowia lipolytica. The targeting of recombinant RecA to mitochondria is provided by leader sequences (5'-UTR and 3-UTR) derived from the SOD2 gene mRNA, which exhibit affinity to the outer mitochondrial membrane and provides cotranslational import of RecA to the inner space of mitochondria...
March 2016: Prikladnaia Biokhimiia i Mikrobiologiia
Anja O Paatero, Juho Kellosalo, Bryan M Dunyak, Jehad Almaliti, Jason E Gestwicki, William H Gerwick, Jack Taunton, Ville O Paavilainen
Apratoxin A is a cytotoxic natural product that prevents the biogenesis of secretory and membrane proteins. Biochemically, apratoxin A inhibits cotranslational translocation into the ER, but its cellular target and mechanism of action have remained controversial. Here, we demonstrate that apratoxin A prevents protein translocation by directly targeting Sec61α, the central subunit of the protein translocation channel. Mutagenesis and competitive photo-crosslinking studies indicate that apratoxin A binds to the Sec61 lateral gate in a manner that differs from cotransin, a substrate-selective Sec61 inhibitor...
May 19, 2016: Cell Chemical Biology
Kuan-Chun Huang, Zhihong Chen, Yimin Jiang, Sandeep Akare, Donna Kolber-Simonds, Krista Condon, Sergei Agoulnik, Karen Tendyke, Yongchun Shen, Kuo-Ming Wu, Steven Mathieu, Hyeong-Wook Choi, Xiaojie Zhu, Hajime Shimizu, Yoshihiko Kotake, William H Gerwick, Toshimitsu Uenaka, Mary Woodall-Jappe, Kenichi Nomoto
Apratoxin A is a natural product with potent antiproliferative activity against many human cancer cell lines. However, we and other investigators observed that it has a narrow therapeutic window in vivo Previous mechanistic studies have suggested its involvement in the secretory pathway as well as the process of chaperone-mediated autophagy. Still the link between the biologic activities of apratoxin A and its in vivo toxicity has remained largely unknown. A better understanding of this relationship is critically important for any further development of apratoxin A as an anticancer drug...
June 2016: Molecular Cancer Therapeutics
Ali J Vetter, Andrey L Karamyshev, Anna E Patrick, Henry Hudson, Philip J Thomas
The majority of cystic fibrosis (CF)-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to the misfolding, mistrafficking, and degradation of the mutant protein. Inhibition of degradation does not effectively increase the amount of trafficking competent CFTR, but typically leads to increased ER retention of misfolded forms. Thus, the initial off pathway steps occur early in the processing of the protein. To identify proteins that interact with these early forms of CFTR, in vitro crosslink experiments identified cotranslational partners of the nascent chain of the severe misfolded mutant, G85E CFTR...
2016: PloS One
Annika Deckert, Christopher A Waudby, Tomasz Wlodarski, Anne S Wentink, Xiaolin Wang, John P Kirkpatrick, Jack F S Paton, Carlo Camilloni, Predrag Kukic, Christopher M Dobson, Michele Vendruscolo, Lisa D Cabrita, John Christodoulou
The ribosome is increasingly becoming recognized as a key hub for integrating quality control processes associated with protein biosynthesis and cotranslational folding (CTF). The molecular mechanisms by which these processes take place, however, remain largely unknown, in particular in the case of intrinsically disordered proteins (IDPs). To address this question, we studied at a residue-specific level the structure and dynamics of ribosome-nascent chain complexes (RNCs) of α-synuclein (αSyn), an IDP associated with Parkinson's disease (PD)...
May 3, 2016: Proceedings of the National Academy of Sciences of the United States of America
Fang Liu, David K Jones, Willem J de Lange, Gail A Robertson
Oligomers of homomeric voltage-gated potassium channels associate early in biogenesis as the nascent proteins emerge from the polysome. Less is known about how proteins emerging from different polysomes associate to form hetero-oligomeric channels. Here, we report that alternate mRNA transcripts encoding human ether-à-go-go-related gene (hERG) 1a and 1b subunits, which assemble to produce ion channels mediating cardiac repolarization, are physically associated during translation. We show that shRNA specifically targeting either hERG 1a or 1b transcripts reduced levels of both transcripts, but only when they were coexpressed heterologously...
April 26, 2016: Proceedings of the National Academy of Sciences of the United States of America
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