scholarly journals Selective inhibition of human translation by a drug-like compound that traps terminated protein nascent chains on the ribosome

2020 ◽  
Author(s):  
Wenfei Li ◽  
Stacey Tsai-Lan Chang ◽  
Fred. R Ward ◽  
Jamie H. D. Cate
Keyword(s):  
Small Molecules ◽  
Stop Codon ◽  
Translation Termination ◽  
Selective Inhibition ◽  
Helical Conformation ◽  
28S Rrna ◽  
Inhibit Gene Expression ◽  
Nascent Chain ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel

AbstractMethods to directly inhibit gene expression using small molecules hold promise for the development of new therapeutics targeting proteins that have evaded previous attempts at drug discovery. Among these, small molecules including the drug-like compound PF-06446846 (PF846) selectively inhibit the synthesis of specific proteins, by stalling translation elongation 1–4. These molecules also inhibit translation termination 4 by an unknown mechanism. Using cryo-electron microscopy (cryo-EM) and biochemical approaches, we show that PF846 arrests translation at the stop codon by slowing hydrolysis of the protein nascent chain (NC) from peptidyl-site (P-site) tRNA by eukaryotic release factor 1 (eRF1). After NC hydrolysis from the P-site tRNA, PF846 traps the NC in the ribosome exit tunnel in a compact α-helical conformation that induces 28S rRNA nucleotide rearrangements propagating back to the ribosome peptidyl transferase center (PTC). Mutational analyses and human cell-based experiments elucidate the pivotal amino acids of the NC required for PF846-dependent termination arrest, all of which face the PF846 side of the ribosome exit tunnel. The structural and functional data support a model in which PF846 inhibits translation termination by inducing allosteric conformational rearrangements in the NC and PTC that suppress peptidyl-tRNA hydrolysis promoted by eRF1, and trap the NC in the ribosome exit tunnel. This unprecedented mechanism of action reveals new principles of translation termination and lays the foundation for new therapeutic strategies.

scholarly journals Selective inhibition of human translation termination by a drug-like compound

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Wenfei Li ◽  
Stacey Tsai-Lan Chang ◽  
Fred. R. Ward ◽  
Jamie H. D. Cate
Keyword(s):  
Small Molecules ◽  
Translation Termination ◽  
Selective Inhibition ◽  
Helical Conformation ◽  
Eukaryotic Translation ◽  
Inhibit Gene Expression ◽  
Nascent Chain ◽  
Specific Proteins ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel

Abstract Methods to directly inhibit gene expression using small molecules hold promise for the development of new therapeutics targeting proteins that have evaded previous attempts at drug discovery. Among these, small molecules including the drug-like compound PF-06446846 (PF846) selectively inhibit the synthesis of specific proteins, by stalling translation elongation. These molecules also inhibit translation termination by an unknown mechanism. Using cryo-electron microscopy (cryo-EM) and biochemical approaches, we show that PF846 inhibits translation termination by arresting the nascent chain (NC) in the ribosome exit tunnel. The arrested NC adopts a compact α-helical conformation that induces 28 S rRNA nucleotide rearrangements that suppress the peptidyl transferase center (PTC) catalytic activity stimulated by eukaryotic release factor 1 (eRF1). These data support a mechanism of action for a small molecule targeting translation that suppresses peptidyl-tRNA hydrolysis promoted by eRF1, revealing principles of eukaryotic translation termination and laying the foundation for new therapeutic strategies.

scholarly journals Structural basis for selective stalling of human ribosome nascent chain complexes by a drug-like molecule

2018 ◽  
Author(s):  
Wenfei Li ◽  
Fred R. Ward ◽  
Kim F. McClure ◽  
Stacey Tsai-Lan Chang ◽  
Elizabeth Montabana ◽  
...  
Keyword(s):  
Small Molecules ◽  
Translation Termination ◽  
Structural Basis ◽  
Small Subset ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Chain Complexes ◽  
Nascent Chain ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel

AbstractSmall molecules that target the ribosome generally have a global impact on protein synthesis. However, the drug-like molecule PF-06446846 (PF846) binds the human ribosome and selectively blocks the translation of a small subset of proteins by an unknown mechanism. In high-resolution cryo-electron microscopy (cryo-EM) structures of human ribosome nascent chain complexes stalled by PF846, PF846 binds in the ribosome exit tunnel in a newly-identified and eukaryotic-specific pocket formed by the 28S ribosomal RNA (rRNA), and redirects the path of the nascent polypeptide chain. PF846 arrests the translating ribosome in the rotated state that precedes mRNA and tRNA translocation, with peptidyl-tRNA occupying a mixture of A/A and hybrid A/P sites, in which the tRNA 3’-CCA end is improperly docked in the peptidyl transferase center. Using mRNA libraries, selections of PF846-dependent translation elongation stalling sequences reveal sequence preferences near the peptidyl transferase center, and uncover a newly-identified mechanism by which PF846 selectively blocks translation termination. These results illuminate how a small molecule selectively stalls the translation of the human ribosome, and provides a structural foundation for developing small molecules that inhibit the production of proteins of therapeutic interest.

scholarly journals Genome-wide effects of the antimicrobial peptide apidaecin on translation termination in bacteria

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Kyle Mangano ◽  
Tanja Florin ◽  
Xinhao Shao ◽  
Dorota Klepacki ◽  
Irina Chelysheva ◽  
...  
Keyword(s):  
Antimicrobial Peptide ◽  
Stop Codon ◽  
Translation Termination ◽  
Bacterial Cells ◽  
Release Factor ◽  
Translation Arrest ◽  
Genome Wide Analysis ◽  
Genome Wide ◽  
Exit Tunnel ◽  
Unique Mechanism

Biochemical studies suggested that the antimicrobial peptide apidaecin (Api) inhibits protein synthesis by binding in the nascent peptide exit tunnel and trapping the release factor associated with a terminating ribosome. The mode of Api action in bacterial cells had remained unknown. Here genome-wide analysis reveals that in bacteria, Api arrests translating ribosomes at stop codons and causes pronounced queuing of the trailing ribosomes. By sequestering the available release factors, Api promotes pervasive stop codon bypass, leading to the expression of proteins with C-terminal extensions. Api-mediated translation arrest leads to the futile activation of the ribosome rescue systems. Understanding the unique mechanism of Api action in living cells may facilitate the development of new medicines and research tools for genome exploration.

scholarly journals Genome-wide effects of the antimicrobial peptide apidaecin on translation termination

2020 ◽  
Author(s):  
Kyle Mangano ◽  
Tanja Florin ◽  
Xinhao Shao ◽  
Dorota Klepacki ◽  
Irina Chelysheva ◽  
...  
Keyword(s):  
Antimicrobial Peptide ◽  
Stop Codon ◽  
Translation Termination ◽  
Bacterial Cells ◽  
Release Factor ◽  
Translation Arrest ◽  
Genome Wide Analysis ◽  
Genome Wide ◽  
Exit Tunnel ◽  
Unique Mechanism

AbstractBiochemical studies suggested that the antimicrobial peptide apidaecin (Api) inhibits protein synthesis by binding in the nascent peptide exit tunnel and trapping the release factor associated with a terminating ribosome. The mode of Api action in bacterial cells had remained unknown. Here, genome-wide analysis revealed that Api arrests translating ribosomes at stop codons and causes pronounced queuing of the trailing ribosomes. By sequestering the available release factors, Api promotes pervasive stop codon bypass, leading to expression of proteins with C-terminal extensions. Api-mediated translation arrest leads to futile activation of the ribosome rescue systems. Understanding the unique mechanism of Api action in living cells may facilitate development of new medicines and research tools for genome exploration.

scholarly journals A compound that directly and selectively stalls PCSK9 translation

2016 ◽  
Author(s):  
Nathanael G. Lintner ◽  
Kim F. McClure ◽  
Donna Petersen ◽  
Allyn T. Londregan ◽  
David W. Piotrowski ◽  
...  
Keyword(s):  
Small Molecules ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Ribosome Profiling ◽  
Low Density ◽  
Low Density Lipoprotein Cholesterol ◽  
Pcsk9 Inhibitor ◽  
Cholesterol Levels ◽  
Nascent Chain ◽  
Exit Tunnel

AbstractProprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) plays a key role in regulating the levels of plasma low density lipoprotein cholesterol (LDL-C). Here we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation. The mechanism of action employed by PF-06446846 reveals a previously unexpected tunability of the human ribosome, which allows small molecules to specifically block translation of individual transcripts.One Sentence SummaryA small-molecule PCSK9 inhibitor targets the human ribosome and selectively prevents PCSK9 synthesis.

scholarly journals Folding of VemP into translation-arresting secondary structure is driven by the ribosome exit tunnel

2021 ◽  
Author(s):  
Michal H Kolar ◽  
Gabor Nagy ◽  
John Kunkel ◽  
Sara M Vaiana ◽  
Lars V Bock ◽  
...  
Keyword(s):  
Small Molecules ◽  
Protein Production ◽  
Driving Forces ◽  
Peptidyl Transferase ◽  
Helix Formation ◽  
Translational Arrest ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel ◽  
Α Helix ◽  
Dynamics Simulations

The ribosome is a fundamental biomolecular complex responsible for protein production in cells. Nascent proteins emerge from the ribosome through a tunnel, where they may interact with the tunnel walls or small molecules such as antibiotics. These interactions can cause translational arrest with notable physiologic consequences. Here, we studied the arrest caused by the regulatory peptide VemP, which is known to form an α-helix in the ribosome tunnel near the peptidyl transferase center under specific conditions. We used all-atom molecular dynamics simulations of the entire ribosome and circular dichroism spectroscopy to study the driving forces of helix formation and how VemP causes the translational arrest. To that aim, we compared VemP dynamics in the ribosome tunnel with its dynamics in solution. We show that the VemP sequence has a low helical propensity in water and that the propensity is higher in more hydrophobic solvents. We propose that helix formation within the ribosome is driven by the tunnel environment and that a portion of VemP acts as an anchor. This anchor might slow down VemP progression through the tunnel enabling the α-helix formation, which causes the elongation arrest.

scholarly journals Visualising nascent chain dynamics at the ribosome exit tunnel by cryo-electron microscopy

2019 ◽  
Author(s):  
Abid Javed ◽  
Tomasz Wlodarski ◽  
Anaïs. M.E. Cassaignau ◽  
Lisa D. Cabrita ◽  
John Christodoulou ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Active Component ◽  
Chain Dynamics ◽  
Cryo Electron Microscopy ◽  
Chain Complexes ◽  
Nascent Chain ◽  
Immunoglobulin Domain ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel

Ribosomes maintain a healthy cellular proteome by synthesising proteins. The nascent chain (NC), emerges into the cellular milieu via the ribosomal exit tunnel, which is an active component that regulates the NC passage. How the NC dynamics at the exit tunnel affect NC folding remains to be an important question, the answer on which has strong implications to medicine. Here, we report high-resolution cryo-EM maps of ribosome nascent-chain complexes (RNCs) displaying distinct steps during biosynthesis. These RNC structures reveal a range of pathways adopted by the NC. The most pronounced diversity in the NC trajectories were found in the vestibule region. Rearrangements of several ribosomal components further suggest that these elements may actively monitor the emerging NC during translation. The ribosome-NC contacts within the vestibule define these NC pathways and modulate position of a folded immunoglobulin domain outside the ribosome.

scholarly journals New insights into the interplay between the translation machinery and nonsense-mediated mRNA decay factors

2018 ◽  
Vol 46 (3) ◽  
pp. 503-512 ◽  
Author(s):  
Etienne Raimondeau ◽  
Joshua C. Bufton ◽  
Christiane Schaffitzel
Keyword(s):  
Mrna Decay ◽  
Stop Codon ◽  
Translation Termination ◽  
Premature Stop Codon ◽  
Molecular Function ◽  
Translation Machinery ◽  
New Findings ◽  
Nascent Chain ◽  
Nonsense Mediated Mrna Decay

Faulty mRNAs with a premature stop codon (PTC) are recognized and degraded by nonsense-mediated mRNA decay (NMD). Recognition of a nonsense mRNA depends on translation and on the presence of NMD-enhancing or the absence of NMD-inhibiting factors in the 3′-untranslated region. Our review summarizes our current understanding of the molecular function of the conserved NMD factors UPF3B and UPF1, and of the anti-NMD factor Poly(A)-binding protein, and their interactions with ribosomes translating PTC-containing mRNAs. Our recent discovery that UPF3B interferes with human translation termination and enhances ribosome dissociation in vitro, whereas UPF1 is inactive in these assays, suggests a re-interpretation of previous experiments and modification of prevalent NMD models. Moreover, we discuss recent work suggesting new functions of the key NMD factor UPF1 in ribosome recycling, inhibition of translation re-initiation and nascent chain ubiquitylation. These new findings suggest that the interplay of UPF proteins with the translation machinery is more intricate than previously appreciated, and that this interplay quality-controls the efficiency of termination, ribosome recycling and translation re-initiation.

scholarly journals Increased freedom of movement in the nascent chain results in dynamic changes in the structure of the SecM arrest motif

2019 ◽  
Vol 39 (1) ◽  
Author(s):  
Hazel A. Bracken ◽  
Cheryl A. Woolhead
Keyword(s):  
Point Mutations ◽  
Translation Arrest ◽  
E Coli ◽  
Freedom Of Movement ◽  
Conserved Sequence ◽  
C Terminus ◽  
Nascent Chain ◽  
Translational Arrest ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel

Abstract Ribosomes are responsible for the synthesis of all cellular proteins. Due to the diversity of sequence and properties, it was initially believed that translating nascent chains would travel unhindered through the ribosome exit tunnel, however a small but increasing number of proteins have been identified that interact with the exit tunnel to induce translational arrest, Escherichia coli (E. coli) secretion monitor (SecM) is one such stalling peptide. How and why these peptides interact with the exit tunnel is not fully understood, however key features required for stalling appear to be an essential peptide arrest motif at the C-terminus and compaction of the nascent chain within the exit tunnel upon stalling. Mutagenesis of the SecM arrest sequence has identified three conservative point mutations that can retain a degree of stalling in this highly conserved sequence. This level of stalling is further increased when coupled with mutation of a non-essential arrest motif residue P153A. Further analysis of these mutants by pegylation assays indicates that this increase in stalling activity during translation is due to the ability of the P153A mutation to reintroduce compaction of the nascent chain within the exit tunnel possibly due to the improved flexibility of the nascent chain provided by the removal of a restrictive proline residue. The data presented here suggest that arrest sequences may be more prevalent and less highly conserved than previously thought, and highlight the significance of the interactions between the nascent chain and the exit tunnel to affecting translation arrest.

scholarly journals A small single-domain protein folds through the same pathway on and off the ribosome

2018 ◽  
Vol 115 (48) ◽  
pp. 12206-12211 ◽  
Author(s):  
Emily J. Guinn ◽  
Pengfei Tian ◽  
Mia Shin ◽  
Robert B. Best ◽  
Susan Marqusee
Keyword(s):  
Point Mutations ◽  
Multidomain Proteins ◽  
Small Protein ◽  
Single Domain Protein ◽  
Folding Mechanism ◽  
Nascent Chain ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel ◽  
And Function

In vivo, proteins fold and function in a complex environment subject to many stresses that can modulate a protein’s energy landscape. One aspect of the environment pertinent to protein folding is the ribosome, since proteins have the opportunity to fold while still bound to the ribosome during translation. We use a combination of force and chemical denaturant (chemomechanical unfolding), as well as point mutations, to characterize the folding mechanism of the src SH3 domain both as a stalled ribosome nascent chain and free in solution. Our results indicate that src SH3 folds through the same pathway on and off the ribosome. Molecular simulations also indicate that the ribosome does not affect the folding pathway for this small protein. Taken together, we conclude that the ribosome does not alter the folding mechanism of this small protein. These results, if general, suggest the ribosome may exert a bigger influence on the folding of multidomain proteins or protein domains that can partially fold before the entire domain sequence is outside the ribosome exit tunnel.

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