RESUMO
In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not4 and deubiquitinated by Otu2 upon ribosomal subunit recycling. Despite its importance for translation efficiency the exact role and structural basis for this translational reset is poorly understood. Here, structural analysis by cryo-electron microscopy of native and reconstituted Otu2-bound ribosomal complexes reveals that Otu2 engages 40S subunits mainly between ribosome recycling and initiation stages. Otu2 binds to several sites on the intersubunit surface of the 40S that are not occupied by any other 40S-binding factors. This binding mode explains the discrimination against 80S ribosomes via the largely helical N-terminal domain of Otu2 as well as the specificity for mono-ubiquitinated eS7 on 40S. Collectively, this study reveals mechanistic insights into the Otu2-driven deubiquitination steps for translational reset during ribosome recycling/(re)initiation.
Assuntos
Proteínas Ribossômicas , Ribossomos , Microscopia Crioeletrônica , Biossíntese de Proteínas , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/metabolismoRESUMO
Translation of aberrant messenger RNAs can cause stalling of ribosomes resulting in ribosomal collisions. Collided ribosomes are specifically recognized to initiate stress responses and quality control pathways. Ribosome-associated quality control facilitates the degradation of incomplete translation products and requires dissociation of the stalled ribosomes. A central event is therefore the splitting of collided ribosomes by the ribosome quality control trigger complex, RQT, by an unknown mechanism. Here we show that RQT requires accessible mRNA and the presence of a neighboring ribosome. Cryogenic electron microscopy of RQT-ribosome complexes reveals that RQT engages the 40S subunit of the lead ribosome and can switch between two conformations. We propose that the Ski2-like helicase 1 (Slh1) subunit of RQT applies a pulling force on the mRNA, causing destabilizing conformational changes of the small ribosomal subunit, ultimately resulting in subunit dissociation. Our findings provide conceptual framework for a helicase-driven ribosomal splitting mechanism.
Assuntos
DNA Helicases , Ribossomos , Ubiquitinação , Ribossomos/metabolismo , DNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Biossíntese de ProteínasRESUMO
Ribosome collisions are recognized by E3 ubiquitin ligase Hel2/ZNF598, leading to RQC (ribosome-associated quality control) and to endonucleolytic cleavage and degradation of the mRNA termed NGD (no-go decay). NGD in yeast requires the Cue2 endonuclease and occurs in two modes, either coupled to RQC (NGDRQC+) or RQC uncoupled (NGDRQC-). This is mediated by an unknown mechanism of substrate recognition by Cue2. Here, we show that the ubiquitin binding activity of Cue2 is required for NGDRQC- but not for NGDRQC+, and that it involves the first two N-terminal Cue domains. In contrast, Trp122 of Cue2 is crucial for NGDRQC+. Moreover, Mbf1 is required for quality controls by preventing +1 ribosome frameshifting induced by a rare codon staller. We propose that in Cue2-dependent cleavage upstream of the collided ribosomes (NGDRQC-), polyubiquitination of eS7 is recognized by two N-terminal Cue domains of Cue2. In contrast, for the cleavage within collided ribosomes (NGDRQC+), the UBA domain, Trp122 and the interaction between Mbf1 and uS3 are critical.
Assuntos
Endonucleases , Proteínas de Saccharomyces cerevisiae , Biossíntese de Proteínas , Ribossomos/genética , Ribossomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Endonucleases/metabolismoRESUMO
Cells can respond to stalled ribosomes by sensing ribosome collisions and employing quality control pathways. How ribosome stalling is resolved without collisions, however, has remained elusive. Here, focusing on noncolliding stalling exhibited by decoding-defective ribosomes, we identified Fap1 as a stalling sensor triggering 18S nonfunctional rRNA decay via polyubiquitination of uS3. Ribosome profiling revealed an enrichment of Fap1 at the translation initiation site but also an association with elongating individual ribosomes. Cryo-EM structures of Fap1-bound ribosomes elucidated Fap1 probing the mRNA simultaneously at both the entry and exit channels suggesting an mRNA stasis sensing activity, and Fap1 sterically hinders the formation of canonical collided di-ribosomes. Our findings indicate that individual stalled ribosomes are the potential signal for ribosome dysfunction, leading to accelerated turnover of the ribosome itself.
Assuntos
Biossíntese de Proteínas , Ribossomos , Estabilidade de RNA , RNA Mensageiro/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Ribossomos/metabolismoRESUMO
Ribosome stalling at tandem CGA codons or poly(A) sequences activates quality controls for nascent polypeptides including ribosome-associated quality control (RQC) and no-go mRNA decay (NGD). In RQC pathway, Hel2-dependent uS10 ubiquitination and the RQC-trigger (RQT) complex are essential for subunit dissociation, and Ltn1-dependent ubiquitination of peptidyl-tRNA in the 60S subunit requires Rqc2. Here, we report that polytryptophan sequences induce Rqc2-independent RQC. More than 11 consecutive tryptophan residues induced RQC in a manner dependent on Hel2-mediated ribosome ubiquitination and the RQT complex. Polytryptophan sequence-mediated RQC was not coupled with CAT-tailing, and Rqc2 was not required for Ltn1-dependent degradation of the arrest products. Eight consecutive tryptophan residues located at the region proximal to the peptidyl transferase center in the ribosome tunnel inhibited CAT-tailing by tandem CGA codons. Polytryptophan sequences also induced Hel2-mediated canonical RQC-coupled NGD and RQC-uncoupled NGD outside the stalled ribosomes. We propose that poly-tryptophan sequences induce Rqc2-independent RQC, suggesting that CAT-tailing in the 60S subunit could be modulated by the polypeptide in the ribosome exit tunnel.
Assuntos
Peptídeos/metabolismo , Biossíntese de Proteínas , Proteínas de Ligação a RNA/fisiologia , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Códon , Peptídeos/química , Estabilidade de RNA , RNA Mensageiro/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Triptofano/metabolismo , UbiquitinaçãoRESUMO
Control of messenger RNA (mRNA) decay rate is intimately connected to translation elongation, but the spatial coordination of these events is poorly understood. The Ccr4-Not complex initiates mRNA decay through deadenylation and activation of decapping. We used a combination of cryo-electron microscopy, ribosome profiling, and mRNA stability assays to examine the recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site in Saccharomyces cerevisiae This interaction occurred when the ribosome lacked accommodated A-site transfer RNA, indicative of low codon optimality. Loss of the interaction resulted in the inability of the mRNA degradation machinery to sense codon optimality. Our findings elucidate a physical link between the Ccr4-Not complex and the ribosome and provide mechanistic insight into the coupling of decoding efficiency with mRNA stability.
Assuntos
Códon , Elongação Traducional da Cadeia Peptídica , Estabilidade de RNA , Proteínas Repressoras/metabolismo , Ribonucleases/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Microscopia Crioeletrônica , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Conformação Proteica em alfa-Hélice , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Ribonucleases/química , Ribonucleases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Ribosome-associated quality control (RQC) represents a rescue pathway in eukaryotic cells that is triggered upon translational stalling. Collided ribosomes are recognized for subsequent dissociation followed by degradation of nascent peptides. However, endogenous RQC-inducing sequences and the mechanism underlying the ubiquitin-dependent ribosome dissociation remain poorly understood. Here, we identified SDD1 messenger RNA from Saccharomyces cerevisiae as an endogenous RQC substrate and reveal the mechanism of its mRNA-dependent and nascent peptide-dependent translational stalling. In vitro translation of SDD1 mRNA enabled the reconstitution of Hel2-dependent polyubiquitination of collided disomes and, preferentially, trisomes. The distinct trisome architecture, visualized using cryo-EM, provides the structural basis for the more-efficient recognition by Hel2 compared with that of disomes. Subsequently, the Slh1 helicase subunit of the RQC trigger (RQT) complex preferentially dissociates the first stalled polyubiquitinated ribosome in an ATP-dependent manner. Together, these findings provide fundamental mechanistic insights into RQC and its physiological role in maintaining cellular protein homeostasis.
Assuntos
Proteínas de Ciclo Celular/ultraestrutura , Biossíntese de Proteínas , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Serina Endopeptidases/ultraestrutura , Ubiquitina-Proteína Ligases/ultraestrutura , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Peptídeos/química , Peptídeos/genética , RNA Mensageiro/genética , Ribossomos/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Serina Endopeptidases/química , Serina Endopeptidases/genética , Ubiquitina/química , Ubiquitina/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genéticaRESUMO
The proteasome is a major proteolytic machine that regulates cellular proteostasis through selective degradation of ubiquitylated proteins1,2. A number of ubiquitin-related molecules have recently been found to be involved in the regulation of biomolecular condensates or membraneless organelles, which arise by liquid-liquid phase separation of specific biomolecules, including stress granules, nuclear speckles and autophagosomes3-8, but it remains unclear whether the proteasome also participates in such regulation. Here we reveal that proteasome-containing nuclear foci form under acute hyperosmotic stress. These foci are transient structures that contain ubiquitylated proteins, p97 (also known as valosin-containing protein (VCP)) and multiple proteasome-interacting proteins, which collectively constitute a proteolytic centre. The major substrates for degradation by these foci were ribosomal proteins that failed to properly assemble. Notably, the proteasome foci exhibited properties of liquid droplets. RAD23B, a substrate-shuttling factor for the proteasome, and ubiquitylated proteins were necessary for formation of proteasome foci. In mechanistic terms, a liquid-liquid phase separation was triggered by multivalent interactions of two ubiquitin-associated domains of RAD23B and ubiquitin chains consisting of four or more ubiquitin molecules. Collectively, our results suggest that ubiquitin-chain-dependent phase separation induces the formation of a nuclear proteolytic compartment that promotes proteasomal degradation.
Assuntos
Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Estresse Fisiológico , Ubiquitinação , Linhagem Celular , Núcleo Celular/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Pressão Osmótica , Poliubiquitina/metabolismo , Proteólise , Proteostase , Proteínas Ribossômicas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína com Valosina/metabolismoRESUMO
18S non-functional rRNA decay (NRD) eliminates non-functional 18S rRNA with deleterious mutations in the decoding center. Dissociation of the non-functional 80S ribosome into 40S and 60S subunits is a prerequisite step for degradation of the non-functional 18S rRNA. However, the mechanisms by which the non-functional ribosome is recognized and dissociated into subunits remain elusive. Here, we report that the sequential ubiquitination of non-functional ribosomes is crucial for subunit dissociation. 18S NRD requires Mag2-mediated monoubiquitination followed by Hel2- and Rsp5-mediated K63-linked polyubiquitination of uS3 at the 212th lysine residue. Determination of the aberrant 18S rRNA levels in sucrose gradient fractions revealed that the subunit dissociation of stalled ribosomes requires sequential ubiquitination of uS3 by E3 ligases and ATPase activity of Slh1 (Rqt2), as well as Asc1 and Dom34. We propose that sequential uS3 ubiquitination of the non-functional 80S ribosome induces subunit dissociation by Slh1, leading to degradation of the non-functional 18S rRNA.
Assuntos
Estabilidade de RNA , RNA Fúngico/metabolismo , RNA Ribossômico 18S/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitinação , RNA Ribossômico 18S/genética , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Ribosome stalling triggers quality control pathways targeting the mRNA (NGD: no-go decay) and the nascent polypeptide (RQC: ribosome-associated quality control). RQC requires Hel2-dependent uS10 ubiquitination and the RQT complex in yeast. Here, we report that Hel2-dependent uS10 ubiquitination and Slh1/Rqt2 are crucial for RQC and NGD induction within a di-ribosome (disome) unit, which consists of the leading stalled ribosome and the following colliding ribosome. Hel2 preferentially ubiquitinated a disome over a monosome on a quality control inducing reporter mRNA in an in vitro translation reaction. Cryo-EM analysis of the disome unit revealed a distinct structural arrangement suitable for recognition and modification by Hel2. The absence of the RQT complex or uS10 ubiquitination resulted in the elimination of NGD within the disome unit. Instead, we observed Hel2-mediated cleavages upstream of the disome, governed by initial Not4-mediated monoubiquitination of eS7 and followed by Hel2-mediated K63-linked polyubiquitination. We propose that Hel2-mediated ribosome ubiquitination is required both for canonical NGD (NGDRQC+) and RQC coupled to the disome and that RQC-uncoupled NGD outside the disome (NGDRQC-) can occur in a Not4-dependent manner.
Assuntos
Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Microscopia Crioeletrônica , Estabilidade de RNA , RNA Mensageiro/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Accurate gene expression is a prerequisite for all cellular processes. Cells actively promote correct protein folding, which prevents the accumulation of abnormal and non-functional proteins. Translation elongation is the fundamental step in gene expression to ensure cellular functions, and abnormal translation arrest is recognized and removed by the quality controls. Recent studies demonstrated that ribosome plays crucial roles as a hub for gene regulation and quality controls. Ribosome-interacting factors are critical for the quality control mechanisms responding to abnormal translation arrest by targeting its products for degradation. Aberrant mRNAs are produced by errors in mRNA maturation steps and cause aberrant translation and are eliminated by the quality control system. In this review, we focus on recent progress on two quality controls, Ribosome-associated Quality Control (RQC) and No-Go Decay (NGD), for abnormal translational elongation. These quality controls recognize aberrant ribosome stalling and induce rapid degradation of aberrant polypeptides and mRNAs thereby maintaining protein homeostasis and preventing the protein aggregation.
RESUMO
Translation arrest by polybasic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control (RQC) system. Here we report that ubiquitination of the 40S ribosomal protein uS10 by the E3 ubiquitin ligase Hel2 (or RQT1) is required for RQC. We identify a RQC-trigger (RQT) subcomplex composed of the RNA helicase-family protein Slh1/Rqt2, the ubiquitin-binding protein Cue3/Rqt3, and yKR023W/Rqt4 that is required for RQC. The defects in RQC of the RQT mutants correlate with sensitivity to anisomycin, which stalls ribosome at the rotated form. Cryo-electron microscopy analysis reveals that Hel2-bound ribosome are dominantly the rotated form with hybrid tRNAs. Ribosome profiling reveals that ribosomes stalled at the rotated state with specific pairs of codons at P-A sites serve as RQC substrates. Rqt1 specifically ubiquitinates these arrested ribosomes to target them to the RQT complex, allowing subsequent RQC reactions including dissociation of the stalled ribosome into subunits.Several protein quality control mechanisms are in place to trigger the rapid degradation of aberrant polypeptides and mRNAs. Here the authors describe a mechanism of ribosome-mediated quality control that involves the ubiquitination of ribosomal proteins by the E3 ubiquitin ligase Hel2/RQT1.
Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , Ribossomos/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Células HEK293 , Humanos , Mutação , Biossíntese de Proteínas , Conformação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , UbiquitinaçãoRESUMO
Dom34-Hbs1 plays crucial roles in Nonstop Decay (NSD) and No-Go Decay (NGD). Here, we report a conserved function of human Pelota (hPelota) in mRNA quality control of nonstop mRNA. hPelota facilitated the expression of the nonstop products from GFP-Rz mRNA, which lacks a termination codon and a poly(A) tail, in exosome-defective mutant cells. hPelota promoted the dissociation of stalled ribosomes at the 3' end of GFP-Rz mRNA, and mutations in domain A diminished this activity. The hPelota-R45A mutant associated with ribosomes but was defective in peptide release. Finally, hPelota promoted the degradation of GFP-Rz mRNA and suppressed the sequential endonucleolytic cleavages caused by stalled ribosomes at the 3' end of mRNA in dom34∆ mutant cells.
Assuntos
Proteínas dos Microfilamentos/genética , Degradação do RNAm Mediada por Códon sem Sentido , Saccharomyces cerevisiae/genética , Animais , Proteínas de Ciclo Celular/genética , Códon de Terminação/genética , Drosophila/genética , Endonucleases , Endorribonucleases/genética , Exossomos/metabolismo , Células HEK293 , Humanos , Proteínas dos Microfilamentos/metabolismo , Mutação de Sentido Incorreto , Proteínas Nucleares , Ligação Proteica , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Dom34-Hbs1 stimulates degradation of aberrant mRNAs lacking termination codons by dissociating ribosomes stalled at the 3' ends, and plays crucial roles in Nonstop Decay (NSD) and No-Go Decay (NGD). In the dom34Δ mutant, nonstop mRNA is degraded by sequential endonucleolytic cleavages induced by a stalled ribosome at the 3' end. Here, we report that ribosome-associated Asc1/RACK1 is required for the endonucleolytic cleavage of nonstop mRNA by stalled ribosome at the 3' end of mRNA in dom34Δ mutant cells. Asc1/RACK1 facilitates degradation of truncated GFP-Rz mRNA in the absence of Dom34 and exosome-dependent decay. Asc1/RACK1 is required for the sequential endonucleolytic cleavages by the stalled ribosome in the dom34Δ mutant, depending on its ribosome-binding activity. The levels of peptidyl-tRNA derived from nonstop mRNA were elevated in dom34Δasc1Δ mutant cells, and overproduction of nonstop mRNA inhibited growth of mutant cells. E3 ubiquitin ligase Ltn1 degrades the arrest products from truncated GFP-Rz mRNA in dom34Δ and dom34Δasc1Δ mutant cells, and Asc1/RACK1 represses the levels of substrates for Ltn1-dependent degradation. These indicate that ribosome-associated Asc1/RACK1 facilitates endonucleolytic cleavage of nonstop mRNA by stalled ribosomes and represses the levels of aberrant products even in the absence of Dom34. We propose that Asc1/RACK1 acts as a fail-safe in quality control for nonstop mRNA.
Assuntos
Regiões 3' não Traduzidas , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Estabilidade de RNA , RNA Fúngico/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Proteínas de Ligação ao GTP/genética , Mutação , RNA Fúngico/genética , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/metabolismo , Ribossomos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The tRNA splicing endonuclease (Sen) complex is located on the mitochondrial outer membrane and splices precursor tRNAs in Saccharomyces cerevisiae. Here, we demonstrate that the Sen complex cleaves the mitochondria-localized mRNA encoding Cbp1 (cytochrome b mRNA processing 1). Endonucleolytic cleavage of this mRNA required two cis-elements: the mitochondrial targeting signal and the stem-loop 652-726-nt region. Mitochondrial localization of the Sen complex was required for cleavage of the CBP1 mRNA, and the Sen complex cleaved this mRNA directly in vitro. We propose that the Sen complex cleaves the CBP1 mRNA, which is co-translationally localized to mitochondria via its mitochondrial targeting signal.
Assuntos
Endorribonucleases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimologia , Sequência de Bases , Endorribonucleases/genética , Mitocôndrias/genética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Splicing de RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Quality control systems eliminate aberrant proteins derived from aberrant mRNAs. Two E3 ubiquitin ligases, Ltn1 and Not4, are involved in proteasomal protein degradation coupled to translation arrest. Here, we evaluated nonstop and translation arrest products degraded in a poly(A) tail-independent manner. Ltn1 was found to degrade aberrant nonstop polypeptides derived from nonstop mRNA lacking a termination codon, but not peptidyl-tRNA, even in the absence of the ribosome dissociation complex Dom34:Hbs1. The receptor for activated C kinase (RACK1/ASC1) was identified as a factor required for nascent peptide-dependent translation arrest as well as Ltn1-dependent protein degradation. Both Not4 and Ltn1 were involved in the degradation of various arrest products in a poly(A) tail-independent manner. Furthermore, carboxyl terminus-truncated degradation intermediates of arrest products were stabilized in a cdc48-3 mutant defective in unfolding or the disassembly related to proteasomal degradation. Thus, we propose that stalled ribosomes may be dissociated into subunits and that peptidyl-tRNA on the 60S subunit is ubiquitinated by Ltn1 and Cdc48 is required for the degradation following release from tRNA.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Endorribonucleases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/genética , Códon de Terminação , Endorribonucleases/genética , Proteínas de Ligação ao GTP/genética , Proteínas de Choque Térmico HSP70/genética , Fatores de Alongamento de Peptídeos/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Biossíntese de Proteínas/genética , Proteólise , Estabilidade de RNA , RNA Mensageiro/genética , Aminoacil-RNA de Transferência/metabolismo , Proteínas Repressoras , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Proteína com ValosinaRESUMO
We previously developed a novel ultrasound assessment system featuring wavelet transform to evaluate the material properties of articular cartilage. We aimed in this study to demonstrate the feasibility of quantitative evaluation of meniscus using ultrasound and to elucidate the relationships between its acoustic, mechanical, and biochemical properties. Meniscal disc specimens from mature pigs were assessed by ultrasound and compression testing, and their correlation was analyzed. A positive correlation was found between the ultrasound signal intensity and apparent Young's modulus (r=0.61). Subsequently, the porcine meniscal discs were treated with various enzymes and then characterized by ultrasound, by compression tests, by biochemical analyses, and by histology and immunohistochemistry. The signal intensity was decreased not by hyaluronidase but by collagenase treatment. Hyaluronidase-treated menisci showed a discrepancy between acoustic and mechanical properties, suggesting that the ultrasound reflection could not detect a reduction in proteoglycan content. Also, ultrasound signal intensity could only reflect superficial layers of the material. Several limitations exist at present, and further studies and improvements of the device are required. However, given the noninvasive nature and the requirement of only small equipment, this ultrasound assessment system will be an instrumental diagnostic tool for meniscal function in both research and clinical fields.
Assuntos
Cartilagem Articular/fisiologia , Meniscos Tibiais/fisiologia , Suínos , Animais , Cartilagem Articular/diagnóstico por imagem , Cartilagem Articular/efeitos dos fármacos , Colagenases/farmacologia , Força Compressiva/efeitos dos fármacos , Força Compressiva/fisiologia , Glicosaminoglicanos/metabolismo , Hialuronoglucosaminidase/farmacologia , Hidroxiprolina/metabolismo , Meniscos Tibiais/diagnóstico por imagem , Meniscos Tibiais/efeitos dos fármacos , Reologia/métodos , Ultrassom , Ultrassonografia , Água/metabolismoRESUMO
Effects of static magnetic field (SMF) on the vascularization in bone were evaluated using an ischemic bone model, where rat femoral artery was ligated. Magnetized and unmagnetized samarium-cobalt rods were implanted transcortically into the middle diaphysis of the ischemic femurs. Collateral circulation was evaluated by injection of microspheres into the abdominal aorta at the third week after ligation. It was found that the bone implanted with a magnetized rod showed a larger amount of trapped microspheres than that with an unmagnetized rod at the proximal and the distal region (P < 0.05 proximal region). There were no significant differences at the middle and the distal region. This tendency was similar to that of the bone mineral density in the SMF-exposed ischemic bone.
RESUMO
BACKGROUND: The current study aimed to determine whether the acoustic properties of living human cartilage during arthroscopy differ between damage from trauma and that from pigmented villonodular synovitis (PVNS). METHODS: Nine patients were evaluated with ultrasound during arthroscopy. As a quantitative index of cartilage quality, the percentage maximal magnitude (maximal magnitude of the measurement area divided by that of the intact cartilage; %MM) was selected. After ultrasound evaluation, the measurement points were divided into two groups on the basis of the etiologic findings (group T: cartilage damage from trauma and group P: cartilage damage from PVNS) and analyzed for the presence of significant differences in ultrasound analysis. RESULTS: In the ultrasound findings, the %MM values ranged from 34.4% to 92.3%. According to the etiologic differences, the mean %MM was 81.0% in group T and 39.3% in group P, and significantly higher in group T than in group P (p < 0.01). CONCLUSIONS: This study showed a correlation between the ultrasound results and the cartilage lesion etiology. Ultrasound evaluation may be useful for elucidating the process of articular cartilage degeneration with trauma and PVNS.
Assuntos
Doenças das Cartilagens/diagnóstico por imagem , Cartilagem Articular/diagnóstico por imagem , Tálus/diagnóstico por imagem , Adolescente , Adulto , Traumatismos do Tornozelo/complicações , Artroscopia , Doenças das Cartilagens/etiologia , Criança , Humanos , Pessoa de Meia-Idade , Sinovite Pigmentada Vilonodular/complicações , Sinovite Pigmentada Vilonodular/diagnóstico por imagem , UltrassonografiaRESUMO
Treatment of coronary artery stenosis with percutaneous coronary angioplasty and stenting is sometimes complicated by neointimal hyperplasia, possibly due to interaction of the stent with the arterial wall within a specific contact area. Therefore, we characterized the stress distribution at the contacts between the stent and the artery using mathematical and experimental modeling (an arterial cylinder model with a tube-like structure and an arterial stenosis model, consisting of a tube and plaque portion) and two kinds of link stents with different numbers of cells and links. First, the contact condition was investigated using a finite element method (FEM). Second, experimental visualization of the contact area between the stent and the artery models was performed. Comparison of the experimental results with the FEM analysis revealed that the contact area between the stent (with a high number of cells and links) and the artery model was distributed over the total surface of the stent. Further, values obtained from the experimental distribution and the calculated distribution were similar. These data indicate that experimental modeling and FEM analysis are useful methods for analyzing the relationship between stent structure and stent/wall stress distributions and may help guide the design of new stents.