Добірка наукової літератури з теми "Nascent polypeptide-Associated control"

The biogenesis of new polypeptides by ribosomes and their subsequent correct folding and localization to the appropriate cellular compartments are essential key processes to maintain protein homoeostasis. These complex mechanisms are governed by a repertoire of protein biogenesis factors that directly bind to the ribosome and chaperone nascent polypeptide chains as soon as they emerge from the ribosomal tunnel exit. This nascent chain ‘welcoming committee’ regulates multiple co-translational processes including protein modifications, folding, targeting and degradation. Acting at the front of the protein production line, these ribosome-associated protein biogenesis factors lead the way in the cellular proteostasis network to ensure proteome integrity. In this article, I focus on three different systems in eukaryotes that are critical for the maintenance of protein homoeostasis by controlling the birth, life and death of nascent polypeptide chains.

Nascent polypeptide-associated complex (NAC) was identified in eukaryotes as the first cytosolic factor that contacts the nascent polypeptide chain emerging from the ribosome. NAC is highly conserved from yeast to humans. Mutations in NAC cause severe embryonically lethal phenotypes in mice,Drosophila,andCaenorhabditis elegans.NAC was suggested to protect the nascent chain from inappropriate early interactions with cytosolic factors. Eukaryotic NAC is a heterodimer with two subunits sharing substantial homology with each other. All sequenced archaebacterial genomes exhibit only one gene homologous to the NAC subunits. Here we present the first archaebacterial NAC homolog. It forms a homodimer, and as eukaryotic NAC it is associated with ribosomes and contacts the emerging nascent chain on the ribosome. We present the first crystal structure of a NAC protein revealing two structural features: (i) a novel unique protein fold that mediates dimerization of the complex, and (ii) a ubiquitin-associated domain that suggests a yet unidentified role for NAC in the cellular protein quality control system via the ubiquitination pathway. Based on the presented structure we propose a model for the eukaryotic heterodimeric NAC domain.

The nascent polypeptide exit site of the ribosome is a crowded environment where multiple ribosome-associated protein biogenesis factors (RPBs) compete for the nascent polypeptide to influence their localization, folding, or quality control. Here we address how N-terminal methionine excision (NME), a ubiquitous process crucial for the maturation of over 50% of the bacterial proteome, occurs in a timely and selective manner in this crowded environment. In bacteria, NME is mediated by 2 essential enzymes, peptide deformylase (PDF) and methionine aminopeptidase (MAP). We show that the reaction of MAP on ribosome-bound nascent chains approaches diffusion-limited rates, allowing immediate methionine excision of optimal substrates after deformylation. Specificity is achieved by kinetic competition of NME with translation elongation and by regulation from other RPBs, which selectively narrow the processing time window for suboptimal substrates. A mathematical model derived from the data accurately predicts cotranslational NME efficiency in the cytosol. Our results demonstrate how a fundamental enzymatic activity is reshaped by its associated macromolecular environment to optimize both efficiency and selectivity, and provides a platform to study other cotranslational protein biogenesis pathways.

Secretory proteins are synthesized in a form of precursors with additional sequences at their N-terminal ends called signal peptides. The signal peptides are recognized co-translationally by signal recognition particle (SRP). This interaction leads to targeting to the endoplasmic reticulum (ER) membrane and translocation of the nascent chains into the ER lumen. It was demonstrated recently that in addition to a targeting function, SRP has a novel role in protection of secretory protein mRNAs from degradation. It was also found that the quality of secretory proteins is controlled by the recently discovered Regulation of Aberrant Protein Production (RAPP) pathway. RAPP monitors interactions of polypeptide nascent chains during their synthesis on the ribosomes and specifically degrades their mRNAs if these interactions are abolished due to mutations in the nascent chains or defects in the targeting factor. It was demonstrated that pathological RAPP activation is one of the molecular mechanisms of human diseases associated with defects in the secretory proteins. In this review, we discuss recent progress in understanding of translational control of secretory protein biogenesis on the ribosome and pathological consequences of its dysregulation in human diseases.

The nascent polypeptide–associated complex (NAC) interacts with newly synthesized proteins at the ribosomal tunnel exit and competes with the signal recognition particle (SRP) to prevent mistargeting of cytosolic and mitochondrial polypeptides to the endoplasmic reticulum (ER). How NAC antagonizes SRP and how this is overcome by ER targeting signals are unknown. Here, we found that NAC uses two domains with opposing effects to control SRP access. The core globular domain prevented SRP from binding to signal-less ribosomes, whereas a flexibly attached domain transiently captured SRP to permit scanning of nascent chains. The emergence of an ER-targeting signal destabilized NAC’s globular domain and facilitated SRP access to the nascent chain. These findings elucidate how NAC hands over the signal sequence to SRP and imparts specificity of protein localization.

ABSTRACT We report the characterization of clone 1.9.2, a gene expressed in mineralizing osteoblasts. Remarkably, clone 1.9.2 is the murine homolog of the alpha chain of the nascent polypeptide-associated complex (α-NAC). Based on sequence similarities between α-NAC/1.9.2 and transcriptional regulatory proteins and the fact that the heterodimerization partner of α-NAC was identified as the transcription factor BTF3b (B. Wiedmann, H. Sakai, T. A. Davis, and M. Wiedmann, Nature 370:434–440, 1994), we investigated a putative role for α-NAC/1.9.2 in transcriptional control. The α-NAC/1.9.2 protein potentiated by 10-fold the activity of the chimeric activator GAL4/VP-16 in vivo. The potentiation was shown to be mediated at the level of gene transcription, because α-NAC/1.9.2 increased GAL4/VP-16-mediated mRNA synthesis without affecting the half-life of the GAL4/VP-16 fusion protein. Moreover, the interaction of α-NAC/1.9.2 with a transcriptionally defective mutant of GAL4/VP-16 was severely compromised. Specific protein-protein interactions between α-NAC/1.9.2 and GAL4/VP-16 were demonstrated by gel retardation, affinity chromatography, and protein blotting assays, while interactions with TATA box-binding protein (TBP) were detected by immunoprecipitation, affinity chromatography, and protein blotting assays. Based on these interactions that define the coactivator class of proteins, we conclude that the α-NAC/1.9.2 gene product functions as a transcriptional coactivator.

The yeast Hsp70/40 system SSB–RAC (stress 70 B–ribosome-associated complex) binds to ribosomes and contacts nascent polypeptides to assist cotranslational folding. In this study, we demonstrate that nascent polypeptide–associated complex (NAC), another ribosome-tethered system, is functionally connected to SSB–RAC and the cytosolic Hsp70 network. Simultaneous deletions of genes encoding NAC and SSB caused conditional loss of cell viability under protein-folding stress conditions. Furthermore, NAC mutations revealed genetic interaction with a deletion of Sse1, a nucleotide exchange factor regulating the cytosolic Hsp70 network. Cells lacking SSB or Sse1 showed protein aggregation, which is enhanced by additional loss of NAC; however, these mutants differ in their potential client repertoire. Aggregation of ribosomal proteins and biogenesis factors accompanied by a pronounced deficiency in ribosomal particles and translating ribosomes only occurs in ssbΔ and nacΔssbΔ cells, suggesting that SSB and NAC control ribosome biogenesis. Thus, SSB–RAC and NAC assist protein folding and likewise have important functions for regulation of ribosome levels. These findings emphasize the concept that ribosome production is coordinated with the protein-folding capacity of ribosome-associated chaperones.

Due to numerous reasons, including faulty m RNA, insufficient availability of charged t RNA, genetic errors, ribosomes are failed to synthesize protein sometimes. All organisms develop their machinery to recognize stalled ribosomes. Stalled ribosomes, results in the production of a truncated polypeptide which can affect cells. So, they must be eliminated, by mechanisms known as Ribosome-associated protein quality control (RQC). E3 ubiquitin ligase Ltn1 in RQC promotes clearance of 60S subunit and targets aberrant nascent polypeptides for proteasomal degradation. In eukaryotes, RQC facilitates the ribosomal rescue, where staled m RNAs release and allow to degrade and ribosomal subunits are to be recycled for further use. Ribosome-associated protein quality control in yeast is accomplished by Hel2-dependent ubiquitination of uS10 and RQC-trigger (RQT) complex. RQC in a mammal is done by ZNF598-dependent ubiquitination of collided ribosomes, which also activates signal integrator 3, a component of the ASCC complex. Human RQT (h RQT) is made up of ASCC3, ASCC2, TRIP4, which are orthologs of RNA helicase Slh1, ubiquitin-binding protein Cue3, and ykR023W protein respectively. Ubiquitin-binding activity and ATPase activity of ASCC2 and ASCC3 respectively, are important for RQC. So, it is obvious that the h RQT complex recognizes the ubiquitinated defective ribosome and induces subunit dissociation for RQC. Biogenesis of new polypeptide, folding, correct localization are the fundamental processes to maintain proteostasis, which involve various factors directly attached with ribosomes and chaperones. Ribosome-associated protein biogenesis factors mediate the cellular proteostasis network to form integrity.

Protein folding is the process by which a polypeptide chain acquires its functional, native 3D structure. Protein misfolding, on the other hand, is a process in which protein fails to fold into its native functional conformation. This misfolding of proteins may lead to precipitation of a number of serious diseases such as Cystic Fibrosis (CF), Alzheimer’s Disease (AD), Parkinson’s Disease (PD), and Amyotrophic Lateral Sclerosis (ALS) etc. Protein Quality-control (PQC) systems, consisting of molecular chaperones, proteases and regulatory factors, help in protein folding and prevent its aggregation. At the same time, PQC systems also do sorting and removal of improperly folded polypeptides. Among the major types of PQC systems involved in protein homeostasis are cytosolic, Endoplasmic Reticulum (ER) and mitochondrial ones. The cytosol PQC system includes a large number of component chaperones, such as Nascent-polypeptide-associated Complex (NAC), Hsp40, Hsp70, prefoldin and T Complex Protein-1 (TCP-1) Ring Complex (TRiC). Protein misfolding diseases caused due to defective cytosolic PQC system include diseases involving keratin/collagen proteins, cardiomyopathies, phenylketonuria, PD and ALS. The components of PQC system of Endoplasmic Reticulum (ER) include Binding immunoglobulin Protein (BiP), Calnexin (CNX), Calreticulin (CRT), Glucose-regulated Protein GRP94, the thiol-disulphide oxidoreductases, Protein Disulphide Isomerase (PDI) and ERp57. ER-linked misfolding diseases include CF and Familial Neurohypophyseal Diabetes Insipidus (FNDI). The components of mitochondrial PQC system include mitochondrial chaperones such as the Hsp70, the Hsp60/Hsp10 and a set of proteases having AAA+ domains similar to the proteasome that are situated in the matrix or the inner membrane. Protein misfolding diseases caused due to defective mitochondrial PQC system include medium-chain acyl-CoA dehydrogenase (MCAD)/Short-chain Acyl-CoA Dehydrogenase (SCAD) deficiency diseases, hereditary spastic paraplegia. Among therapeutic approaches towards the treatment of various protein misfolding diseases, chaperones have been suggested as potential therapeutic molecules for target based treatment. Chaperones have been advantageous because of their efficient entry and distribution inside the cells, including specific cellular compartments, in therapeutic concentrations. Based on the chemical nature of the chaperones used for therapeutic purposes, molecular, chemical and pharmacological classes of chaperones have been discussed.

Protein segments with a high concentration of positively charged amino acid residues are often used in reporter constructs designed to activate ribosomal mRNA/protein decay pathways, such as those involving nonstop mRNA decay (NSD), no-go mRNA decay (NGD) and the ribosome quality control (RQC) complex. It has been proposed that the electrostatic interaction of the positively charged nascent peptide with the negatively charged ribosomal exit tunnel leads to translation arrest. When stalled long enough, the translation process is terminated with the degradation of the transcript and an incomplete protein. Although early experiments made a strong argument for this mechanism, other features associated with positively charged reporters, such as codon bias and mRNA and protein structure, have emerged as potent inducers of ribosome stalling. We carefully reviewed the published data on the protein and mRNA expression of artificial constructs with diverse compositions as assessed in different organisms. We concluded that, although polybasic sequences generally lead to lower translation efficiency, it appears that an aggravating factor, such as a nonoptimal codon composition, is necessary to cause translation termination events.

La traduction des ARNm et la production de protéines sont des phénomènes étroitement contrôlés dans la cellule. La séquence de l'ARNm et sa structure, auxquelles sont associées des protéines se liant à l'ARN, ainsi que la qualité de la protéine produite à partir de cet ARNm, évaluée notamment par la voie de contrôle qualité associée aux ribosomes, sont des éléments impliqués dans ce processus majeur pour la cellule. Dans ce domaine, le contrôle de la production de protéine EBNA1 (Epstein-Barr Nuclear Antigen 1) du virus d'Epstein -Barr est un exemple intéressant. La protéine EBNA1 est essentielle pour la survie du virus dans les cellules hôtes. Les niveaux cellulaires de protéine EBNA1 sont très faibles, bien que la protéine soit présente dans toutes les cellules infectées. Cette dernière est aussi extrêmement antigénique. Il est aujourd'hui admis que la quantité de protéine EBNA1 présente dans les cellules est suffisante pour assurer le maintien du virus dans la cellule, mais assez basse pour lui permettre d'échapper au système immunitaire de l'hôte. Un contrôle de sa production est nécessaire à cet équilibre. Des études précédentes ont montré que le domaine GAr (répétitions de glycine et alanine), présent dans la partie N-terminale de la protéine, déclenche un mécanisme conduisant à l'inhibition de l'initiation de la traduction de l'ARNm d'EBNA1 en cis, sans affecter la traduction des autres ARNm présents dans la cellule. L'équipe a montré précédemment que les structures G4s (G-quadruplex) peuvent être formées dans l'ARNm codant le GAr. De nombreuses études ont montré l'importance de ces structures secondaires de l'ARN dans la régulation de la traduction de l'ARNm d'EBNA1. La nucléoline, un facteur nucléaire, peut se lier aux G4s de l'ARNm du GAr. Cependant, il a aussi été montré que le peptide GAr, et non l'ARNm associé, est nécessaire au contrôle de la traduction de l'ARNm du GAr en cis. L'objectif principal de ma thèse est de mieux comprendre le mécanisme déclenché par l'ARNm et le polypeptide naissant conduisant au contrôle de la traduction de l'ARNm d'EBNA1 en cis. En accord avec le fait que les structures G4s de l'ARN sont extrêmement dynamiques, nous avons montré dans un premier temps que les fonctions associées au G4s de l'ARNm du GAr, à savoir la localisation de l'ARNm, sa traduction et sa capacité à se lier à certaines protéines, dépendent du contexte dans lesquelles ces structures se trouvent. Nous montrons ensuite que la traduction de l'ARNm d'EBNA1 est nécessaire à l'interaction nucléoline-ARNm, signifiant que la traduction de l'ARNm induit des changements dans les propriétés de l'ARNm. En parallèle, nous avons étudié le NACA, une sous-unité du complexe chaperon NAC (nascent polypeptide-associated complex). NACA se détache du ribosome lors de la synthèse du GAr et interagit avec le GAr. NACA est aussi capable de se lier aux ARN et est déterminant dans la suite des évènements liés à l'ARNm codant le GAr. Enfin, et de façon assez surprenante, les facteurs d'initiation de la traduction sont aussi des éléments clés dans l'inhibition de la traduction de l'ARNm d'EBNA1. Le facteur le plus impactant identifié jusqu'à maintenant est le facteur eIF4A1. Ces résultats indiquent que la séquence et structure de l'ARNm et le polypeptide naissant correspondant sont impliqués dans l'inhibition de l'initiation de la traduction de l'ARNm d'EBNA1. Cependant, cela n'enlève pas la possibilité que l'ARNm et le polypeptide naissant déclenchent chacun une voie d'inhibition de la synthèse d'EBNA1 distincte l'une de l'autre. Les virus utilisent des éléments déjà présents dans la cellule pour assurer leur maintien dans la cellule hôte. Ainsi, les principes de biologie cellulaire décrits ici peuvent apporter des indications importantes pour une meilleure compréhension d'autres pathologies en plus de celles liées au virus d'Epstein-Barr
MRNA translation and protein synthesis are tightly regulated events in the cell. Mechanisms describing these key cellular events involve the mRNA sequence and its structure with the association of RNA-binding protein to it, as well as the quality of the translation product encoded by the mRNA, assessed notably through ribosome-associated quality control. In this context, the Epstein-Barr virus EBNA1 (Epstein-Barr Nuclear Antigen 1) mRNA translation regulation is an interesting example. EBNA1 is known to be an essential protein for the virus survival in the host cells. Even though EBNA1 is present in every infected cell, its protein level is remarkably low. As EBNA1 is highly antigenic, it has been suggested that EBNA1 levels in the cells are low enough to escape the immune system of the host, but sufficient to maintain EBV infection. This balance requires a tightly controlled EBNA1 production. Further studies showed that the GAr (glycine-alanine repeat) domain, located in the N-terminal part of EBNA1, triggers an in cis mechanism leading to the inhibition of the translation initiation of its own mRNA, without affecting translation of other mRNAs in the cell. Thus, the GAr domain of EBNA1 is a unique tool to study selective mRNA translation control without affecting general protein synthesis. It was previously shown that RNA G4 (G-quadruplex) structures can be folded in the GAr-encoding mRNA. Numerous studies underlined the importance of these RNA structures in the regulation of EBNA1 mRNA translation, and the team previously showed that nucleolin can interact with these RNA G4 structures, interaction which can be competed by some G4 ligands. However, it was also formerly shown that the GAr peptide itself plays a role in controlling in cis the translation of EBNA1-encoding mRNA, rather than just the RNA sequence. The main focus of the study presented here is to shed light on how this translation event and the fate of the encoding mRNA are regulated in cis by the mRNA and the encoded nascent polypeptide. In line with the fact that RNA G4 structures are highly dynamic, we first showed that GAr RNA G4-associated functions, namely mRNA localisation, translation and ability to bind RNA-binding proteins, are dependent on the context they are in, i.e. their position in the mRNA, the structures in their surrounding or the factors binding the mRNA, such as G4 ligands. We next demonstrated that translation of the EBNA1 mRNA is necessary for nucleolin-binding to it, meaning that the translation event modifies some properties of the EBNA1 mRNA. In parallel, we showed that the NACA, a subunit of the NAC chaperone complex, is detached from the ribosome and interacts with the GAr polypeptide. Interestingly, the NACA is also an RNA binding protein in addition to its chaperone function, and is determinant for the future processing of the EBNA1 mRNA. Finally, and unexpectedly, we show that translation initiation factors are also key players in the downregulation of the EBNA1 mRNA translation, affecting also the mRNA nucleolin-binding capacity, the most effective translation initiation factor in the downregulation of EBNA1 mRNA translation identified so far being eIF4A1. These results support the idea that both the RNA sequence and structure and the corresponding nascent polypeptide are involved in the downregulation of EBNA1 mRNA translation. However, it does not rule out the possibility that both the RNA structure and the polypeptide sequence trigger also their own separated inhibitory pathway. As viruses use components already present in the cells to maintain themselves, the cellular biology elements brought out here can provide insights on many other pathologies in addition to EBV-associated diseases

The objectives of the BARD proposal were to determine the mechanisms of nonspecific cytotoxic cells (NCC) that are necessary to provide heightened innate resistance to infection and to identify the antigenic determinants in Streptococcus iniae that are best suited for vaccine development. Our central hypothesis was that anti-bacterial immunity in trout and tilapia can only be acquired by combining "innate" NCC responses with antibody responses to polysaccharide antigens. These Objectives were accomplished by experiments delineated by the following Specific Aims: Specific aim (SA) #1 (USA) "Clone and Identify the Apoptosis Regulatory Genes in NCC"; Specific aim #2 (USA)"Identify Regulatory Factors that Control NCC Responses to S. iniae"; Specific aim #3 (Israel) "Characterize the Biological Properties of the S. iniae Capsular Polysaccharide"; and Specific aim #4 (Israel) "Development of an Acellular Vaccine". Our model of S. iniae pathogenesis encompassed two approaches, identify apoptosis regulatory genes and proteins in tilapia that affected NCC activities (USA group) and determine the participation of S.iniae capsular polysaccharides as potential immunogens for the development of an acellular vaccine (Israel group). We previously established that it was possible to immunize tilapia and trout against experimental S. difficile/iniaeinfections. However these studies indicated that antibody responses in protected fish were short lived (3-4 months). Thus available vaccines were useful for short-term protection only. To address the issues of regulation of pathogenesis and immunogens of S. iniae, we have emphasized the role of the innate immune response regarding activation of NCC and mechanisms of invasiveness. Considerable progress was made toward accomplishing SA #1. We have cloned the cDNA of the following tilapia genes: cellular apoptosis susceptibility (CAS/AF547173»; tumor necrosis factor alpha (TNF / A Y 428948); and nascent polypeptide-associated complex alpha polypeptide (NACA/ A Y168640). Similar attempts were made to sequence the tilapia FasLgene/cDNA, however these experiments were not successful. Aim #2 was to "Identify Regulatory Factors that Control NCC Responses to S. iniae." To accomplish this, a new membrane receptor has been identified that may control innate responses (including apoptosis) of NCC to S. iniae. The receptor is a membrane protein on teleost NCC. This protein (NCC cationic antimicrobial protein-1/ncamp-1/AAQ99138) has been sequenced and the cDNA cloned (A Y324398). In recombinant form, ncamp-l kills S. iniae in vitro. Specific aim 3 ("Characterize the Biological Properties of the S.iniae Capsular Polysaccharide") utilized an in- vitro model using rainbow trout primary skin epithelial cell mono layers. These experiments demonstrated colonization into epithelial cells followed by a rapid decline of viable intracellular bacteria and translocation out of the cell. This pathogenesis model suggested that the bacterium escapes the endosome and translocates through the rainbow trout skin barrier to further invade and infect the host. Specific aim #4 ("Development of an Acellular Vaccine") was not specifically addressed. These studies demonstrated that several different apoptotic regulatory genes/proteins are expressed by tilapia NCC. These are the first studies demonstrating that such factors exist in tilapia. Because tilapia NCC bind to and are activated by S. iniae bacterial DNA, we predict that the apoptotic regulatory activity of S. iniae previously demonstrated by our group may be associated with innate antibacterial responses in tilapia.