RESUMO
Approximately 40% of the mammalian proteome undergoes N-terminal methionine excision and acetylation, mediated sequentially by methionine aminopeptidase (MetAP) and N-acetyltransferase A (NatA), respectively1. Both modifications are strictly cotranslational and essential in higher eukaryotic organisms1. The interaction, activity and regulation of these enzymes on translating ribosomes are poorly understood. Here we perform biochemical, structural and in vivo studies to demonstrate that the nascent polypeptide-associated complex2,3 (NAC) orchestrates the action of these enzymes. NAC assembles a multienzyme complex with MetAP1 and NatA early during translation and pre-positions the active sites of both enzymes for timely sequential processing of the nascent protein. NAC further releases the inhibitory interactions from the NatA regulatory protein huntingtin yeast two-hybrid protein K4,5 (HYPK) to activate NatA on the ribosome, enforcing cotranslational N-terminal acetylation. Our results provide a mechanistic model for the cotranslational processing of proteins in eukaryotic cells.
RESUMO
Ribosome biogenesis is a fundamental multi-step cellular process that culminates in the formation of ribosomal subunits, whose production and modification are regulated by numerous biogenesis factors. In this study, we analyze physiologic prokaryotic ribosome biogenesis by isolating bona fide pre-50S subunits from an Escherichia coli strain with the biogenesis factor ObgE, affinity tagged at its native gene locus. Our integrative structural approach reveals a network of interacting biogenesis factors consisting of YjgA, RluD, RsfS, and ObgE on the immature pre-50S subunit. In addition, our study provides mechanistic insight into how the GTPase ObgE, in concert with other biogenesis factors, facilitates the maturation of the 50S functional core and reveals both conserved and divergent evolutionary features of ribosome biogenesis between prokaryotes and eukaryotes.
Assuntos
Proteínas de Escherichia coli , Evolução Molecular , Loci Gênicos , Hidroliases , Proteínas Monoméricas de Ligação ao GTP , Subunidades Ribossômicas Maiores de Bactérias , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Hidroliases/química , Hidroliases/genética , Hidroliases/metabolismo , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/genética , Subunidades Ribossômicas Maiores de Bactérias/metabolismoRESUMO
Ribosomes interact with a variety of different protein biogenesis factors that guide newly synthesized proteins to their native 3D shapes and cellular localization. Depending on the type of translated substrate, a distinct set of cotranslational factors must interact with the ribosome in a timely and coordinated manner to ensure proper protein biogenesis. While cytonuclear proteins require cotranslational maturation and folding factors, secretory proteins must be maintained in an unfolded state and processed cotranslationally by transport and membrane translocation factors. Here we explore the specific cotranslational processing steps for cytonuclear, secretory, and membrane proteins in eukaryotes and then discuss how the nascent polypeptide-associated complex (NAC) cotranslationally sorts these proteins into the correct protein biogenesis pathway.
Assuntos
Biossíntese de Proteínas , Ribossomos , Ribossomos/metabolismo , Transporte Proteico , Proteínas de Membrana/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Macrolide antibiotics are thought to clog up the ribosomal tunnel and thereby block general protein synthesis. By using a combination of elegant in vivo and in vitro approaches, Kannan et al. show that the inhibitory action of these drugs on bacterial protein synthesis is selective rather than global.
RESUMO
Cotranslational processing of newly synthesized proteins is fundamental for correct protein maturation. Protein biogenesis factors are thought to bind nascent polypeptides not before they exit the ribosomal tunnel. Here, we identify a nascent chain recognition mechanism deep inside the ribosomal tunnel by an essential eukaryotic cytosolic chaperone. The nascent polypeptide-associated complex (NAC) inserts the N-terminal tail of its ß subunit (N-ßNAC) into the ribosomal tunnel to sense substrates directly upon synthesis close to the peptidyl-transferase center. N-ßNAC escorts the growing polypeptide to the cytosol and relocates to an alternate binding site on the ribosomal surface. Using C. elegans as an in vivo model, we demonstrate that the tunnel-probing activity of NAC is essential for organismal viability and critical to regulate endoplasmic reticulum (ER) protein transport by controlling ribosome-Sec61 translocon interactions. Thus, eukaryotic protein maturation relies on the early sampling of nascent chains inside the ribosomal tunnel.
Assuntos
Proteínas de Caenorhabditis elegans/biossíntese , Caenorhabditis elegans/metabolismo , Retículo Endoplasmático/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Canais de Translocação SEC/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Retículo Endoplasmático/genética , Humanos , Ribossomos/genética , Canais de Translocação SEC/genética , Saccharomyces cerevisiaeRESUMO
The nascent polypeptide-associated complex (NAC) is a conserved ribosome-associated protein biogenesis factor. Whether NAC exerts chaperone activity and whether this function is restricted to de novo protein synthesis is unknown. Here, we demonstrate that NAC directly exerts chaperone activity toward structurally diverse model substrates including polyglutamine (PolyQ) proteins, firefly luciferase, and Aß40. Strikingly, we identified the positively charged ribosome-binding domain in the N terminus of the ßNAC subunit (N-ßNAC) as a major chaperone entity of NAC. N-ßNAC by itself suppressed aggregation of PolyQ-expanded proteins in vitro, and the positive charge of this domain was critical for this activity. Moreover, we found that NAC also exerts a ribosome-independent chaperone function in vivo. Consistently, we found that a substantial fraction of NAC is non-ribosomal bound in higher eukaryotes. In sum, NAC is a potent suppressor of aggregation and proteotoxicity of mutant PolyQ-expanded proteins associated with human diseases like Huntington's disease and spinocerebellar ataxias.
Assuntos
Peptídeos beta-Amiloides/genética , Chaperonas Moleculares/genética , Agregação Patológica de Proteínas/genética , Peptídeos beta-Amiloides/química , Sítios de Ligação/genética , Humanos , Doença de Huntington/genética , Doença de Huntington/patologia , Luciferases/química , Luciferases/genética , Chaperonas Moleculares/química , Peptídeos/química , Peptídeos/genética , Ligação Proteica/genética , Biossíntese de Proteínas/genética , Domínios Proteicos/genética , Dobramento de Proteína , Ribossomos/genética , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologiaRESUMO
Hammerhead ribozyme-based RNA switches have been proven to be powerful tools for conditional gene regulation in various organisms. We present neomycin-dependent hammerhead ribozymes (HHR) that influence gene expression in a ligand- and dose-dependent manner in S. cerevisiae. We utilized a novel design of fusing the aptamer domain to the HHR enabling for the first time the identification of genetic ON- and OFF-switches within the same library. For this purpose a neomycin aptamer was fused to stem 1 of a type 3 hammerhead ribozyme via an addressable three-way junction that shows high flexibility at the connection site. An in vivo screening approach identified sequences that allow to induce or repress gene expression 2- to 3-fold in response to neomycin addition. The ribozyme switches operate at neomycin concentrations that show no toxic effect on cell growth and pose powerful genetic tools to study and modulate cellular function in yeast.
Assuntos
Regulação Fúngica da Expressão Gênica , Neomicina/farmacologia , RNA Catalítico/biossíntese , RNA Catalítico/genética , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Antibacterianos/farmacologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Saccharomyces cerevisiaeRESUMO
How nascent polypeptides emerging from ribosomes fold into functional structures is poorly understood. Here, we monitor disulfide bond formation, protease resistance, and enzymatic activity in nascent polypeptides to show that in close proximity to the ribosome, conformational space and kinetics of folding are restricted. Folding constraints decrease incrementally with distance from the ribosome surface. Upon ribosome binding, the chaperone Trigger Factor counters folding also of longer nascent chains, to extents varying between different chain segments. Trigger Factor even binds and unfolds pre-existing folded structures, the unfolding activity being limited by the thermodynamic stability of nascent chains. Folding retardation and unfolding activities are not shared by the DnaK chaperone assisting later folding steps. These ribosome- and Trigger Factor-specific activities together constitute an efficient mechanism to prevent or even revert premature folding, effectively limiting misfolded intermediates during protein synthesis.
Assuntos
Proteínas de Escherichia coli/metabolismo , Peptidilprolil Isomerase/metabolismo , Dobramento de Proteína , Ribossomos/metabolismo , Proteínas de Bactérias , Dissulfetos/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Modelos Biológicos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase/química , Conformação Proteica , Estrutura Terciária de Proteína , Ribonucleases/química , Ribonucleases/metabolismo , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
As newly synthesized polypeptides emerge from the ribosome, it is crucial that they fold correctly. To prevent premature aggregation, nascent chains interact with chaperones that facilitate folding or prevent misfolding until protein synthesis is complete. Nascent polypeptide-associated complex (NAC) is a ribosome-associated chaperone that is important for protein homeostasis. However, how NAC binds its substrates remains unclear. Using native electrospray ionization MS (ESI-MS), limited proteolysis, NMR, and cross-linking, we analyzed the conformational properties of NAC from Caenorhabditis elegans and studied its ability to bind proteins in different conformational states. Our results revealed that NAC adopts an array of compact and expanded conformations and binds weakly to client proteins that are unfolded, folded, or intrinsically disordered, suggestive of broad substrate compatibility. Of note, we found that this weak binding retards aggregation of the intrinsically disordered protein α-synuclein both in vitro and in vivo These findings provide critical insights into the structure and function of NAC. Specifically, they reveal the ability of NAC to exploit its conformational plasticity to bind a repertoire of substrates with unrelated sequences and structures, independently of actively translating ribosomes.
Assuntos
Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/metabolismo , Chaperonas Moleculares/química , Peptídeos/metabolismo , Biossíntese de Proteínas , Sinucleínas/química , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Cristalografia por Raios X , Chaperonas Moleculares/metabolismo , Peptídeos/química , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Sinucleínas/metabolismoRESUMO
Translation of aberrant or problematic mRNAs can cause ribosome stalling which leads to the production of truncated or defective proteins. Therefore, cells evolved cotranslational quality control mechanisms that eliminate these transcripts and target arrested nascent polypeptides for proteasomal degradation. Here we show that Not4, which is part of the multifunctional Ccr4-Not complex in yeast, associates with polysomes and contributes to the negative regulation of protein synthesis. Not4 is involved in translational repression of transcripts that cause transient ribosome stalling. The absence of Not4 affected global translational repression upon nutrient withdrawal, enhanced the expression of arrested nascent polypeptides and caused constitutive protein folding stress and aggregation. Similar defects were observed in cells with impaired mRNA decapping protein function and in cells lacking the mRNA decapping activator and translational repressor Dhh1. The results suggest a role for Not4 together with components of the decapping machinery in the regulation of protein expression on the mRNA level and emphasize the importance of translational repression for the maintenance of proteome integrity.
Assuntos
Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Regulação Fúngica da Expressão Gênica , Homeostase , Polilisina/metabolismo , Polirribossomos/genética , Polirribossomos/metabolismo , Proteínas de Ligação ao Cap de RNA/genética , Proteínas de Ligação ao Cap de RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas Repressoras , Ribonucleases/genética , Ribonucleases/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Polyglutamine (polyQ) diseases, including Huntington's disease, result from the aggregation of an abnormally expanded polyQ repeat in the affected protein. The length of the polyQ repeat is essential for the disease's onset; however, the molecular mechanism of polyQ aggregation is still poorly understood. Controlled conditions and initiation of the aggregation process are prerequisites for the detection of transient intermediate states. We present an attenuated total reflection Fourier-transform infrared spectroscopic approach combined with protein immobilization to study polyQ aggregation dependent on the polyQ length. PolyQ proteins were engineered mimicking the mammalian N-terminus fragment of the Huntingtin protein and containing a polyQ sequence with the number of glutamines below (Q11), close to (Q38), and above (Q56) the disease threshold. A monolayer of the polyQ construct was chemically immobilized on the internal reflection element of the attenuated total reflection cell, and the aggregation was initiated via enzymatic cleavage. Structural changes of the polyQ sequence were monitored by time-resolved infrared difference spectroscopy. We observed faster aggregation kinetics for the longer sequences, and furthermore, we could distinguish ß-structured intermediates for the different constructs, allowing us to propose aggregation mechanisms dependent on the repeat length. Q11 forms a ß-structured aggregate by intermolecular interaction of stretched monomers, whereas Q38 and Q56 undergo conformational changes to various ß-structured intermediates, including intramolecular ß-sheets.
Assuntos
Peptídeos/química , Agregados Proteicos , Sequências Repetitivas de Aminoácidos , Sequência de Aminoácidos , Modelos Moleculares , Conformação ProteicaRESUMO
The adaptation of protein synthesis to environmental and physiological challenges is essential for cell viability. Here, we show that translation is tightly linked to the protein-folding environment of the cell through the functional properties of the ribosome bound chaperone NAC (nascent polypeptide-associated complex). Under non-stress conditions, NAC associates with ribosomes to promote translation and protein folding. When proteostasis is imbalanced, NAC relocalizes from a ribosome-associated state to protein aggregates in its role as a chaperone. This results in a functional depletion of NAC from the ribosome that diminishes translational capacity and the flux of nascent proteins. Depletion of NAC from polysomes and re-localisation to protein aggregates is observed during ageing, in response to heat shock and upon expression of the highly aggregation-prone polyglutamine-expansion proteins and Aß-peptide. These results demonstrate that NAC has a central role as a proteostasis sensor to provide the cell with a regulatory feedback mechanism in which translational activity is also controlled by the folding state of the cellular proteome and the cellular response to stress.
Assuntos
Envelhecimento/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Chaperonas Moleculares/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Resposta ao Choque Térmico , Chaperonas Moleculares/genética , Peptídeos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismoRESUMO
De novo protein folding is delicate and error-prone and requires the guidance of molecular chaperones. Besides cytosolic and organelle-specific chaperones, cells have evolved ribosome-associated chaperones that support early folding events and prevent misfolding and aggregation. This class of chaperones includes the bacterial trigger factor (TF), the archaeal and eukaryotic nascent polypeptide-associated complex (NAC) and specialized eukaryotic heat shock protein (Hsp) 70/40 chaperones. This review focuses on the cellular activities of ribosome-associated chaperones and highlights new findings indicating additional functions beyond de novo folding. These activities include the assembly of oligomeric complexes, such as ribosomes, modulation of translation and targeting of proteins.
Assuntos
Chaperonas Moleculares/metabolismo , Ribossomos/metabolismo , Animais , Humanos , Modelos Biológicos , Chaperonas Moleculares/genética , Dobramento de Proteína , Proteínas/química , Proteínas/genética , Proteínas/metabolismoRESUMO
While the structure of mature ribosomes is analyzed in atomic detail considerably less is known about their assembly process in living cells. This is mainly due to technical and conceptual hurdles. To analyze ribosome assembly in vivo, we designed and engineered an Escherichiacoli strain--using chromosomal gene knock-in techniques--that harbors large and small ribosomal subunits labeled with the fluorescent proteins EGFP and mCherry, respectively. A thorough characterization of this reporter strain revealed that its growth properties and translation apparatus were wild-type like. Alterations in the ratio of EGFP over mCherry fluorescence are supposed to indicate ribosome assembly defects. To provide proof of principle, subunit specific assembly defects were provoked and could be identified by both manual and fully automated fluorometric in vivo assays. This is to our knowledge the first methodology that directly detects ribosome assembly defects in vivo in a high-throughput compatible format. Screening of knock-out collections and small molecule libraries will allow identification of new ribosome assembly factors and possible inhibitors.
Assuntos
Escherichia coli/genética , Fluorometria/métodos , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Engenharia Celular , Cloranfenicol/farmacologia , Eritromicina/farmacologia , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Corantes Fluorescentes , Técnicas de Inativação de Genes , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Microscopia de Fluorescência , Inibidores da Síntese de Proteínas/farmacologia , Proteína Ribossômica L3 , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/química , Ribossomos/química , Proteína Vermelha FluorescenteRESUMO
BACKGROUND: Ribosomes and functional complexes of them have been analyzed at the atomic level. Far less is known about the dynamic assembly and degradation events that define the half-life of ribosomes and guarantee their quality control. RESULTS: We developed a system that allows visualization of intact ribosomal subunits and assembly intermediates (i.e. assembly landscapes) by convenient fluorescence-based analysis. To this end, we labeled the early assembly ribosomal proteins L1 and S15 with the fluorescent proteins mAzami green and mCherry, respectively, using chromosomal gene insertion. The reporter strain harbors fluorescently labeled ribosomal subunits that operate wild type-like, as shown by biochemical and growth assays. Using genetic and chemical perturbations by depleting genes encoding the ribosomal proteins L3 and S17, respectively, or using ribosome-targeting antibiotics, we provoked ribosomal subunit assembly defects. These defects were readily identified by fluorometric analysis after sucrose density centrifugation in unprecedented resolution. CONCLUSION: This strategy is useful to monitor and characterize subunit specific assembly defects caused by ribosome-targeting drugs that are currently used and to characterize new molecules that affect ribosome assembly and thereby constitute new classes of antibacterial agents.
Assuntos
Proteínas de Escherichia coli/genética , Fluorometria/métodos , Proteínas Ribossômicas/genética , Ribossomos/química , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Técnicas de Inativação de Genes , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Mutagênese Insercional , Multimerização Proteica/efeitos dos fármacos , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Proteína Vermelha FluorescenteRESUMO
The folding of proteins in living cells may start during their synthesis when the polypeptides emerge gradually at the ribosomal exit tunnel. However, our current understanding of cotranslational folding processes at the atomic level is limited. We employed NMR spectroscopy to monitor the conformation of the SH3 domain from alpha-spectrin at sequential stages of elongation via in vivo ribosome-arrested (15)N,(13)C-labeled nascent polypeptides. These nascent chains exposed either the entire SH3 domain or C-terminally truncated segments thereof, thus providing snapshots of the translation process. We show that nascent SH3 polypeptides remain unstructured during elongation but fold into a compact, native-like beta-sheet assembly when the entire sequence information is available. Moreover, the ribosome neither imposes major conformational constraints nor significantly interacts with exposed unfolded nascent SH3 domain moieties. Our data provide evidence for a domainwise folding of the SH3 domain on ribosomes without significant population of folding intermediates. The domain follows a thermodynamically favorable pathway in which sequential folding units are stabilized, thus avoiding kinetic traps during the process of cotranslational folding.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Peptídeos/química , Dobramento de Proteína , Estrutura Terciária de Proteína , Espectrina/química , Isótopos de Carbono , Isótopos de Nitrogênio , Ribossomos/metabolismo , Espectrina/metabolismo , TermodinâmicaRESUMO
The translocon-associated protein (TRAP) complex resides in the endoplasmic reticulum (ER) membrane and interacts with the Sec translocon and the ribosome to facilitate biogenesis of secretory and membrane proteins. TRAP plays a key role in the secretion of many hormones, including insulin. Here we reveal the molecular architecture of the mammalian TRAP complex and how it engages the translating ribosome associated with Sec61 translocon on the ER membrane. The TRAP complex is anchored to the ribosome via a long tether and its position is further stabilized by a finger-like loop. This positions a cradle-like lumenal domain of TRAP below the translocon for interactions with translocated nascent chains. Our structure-guided TRAP mutations in Caenorhabditis elegans lead to growth deficits associated with increased ER stress and defects in protein hormone secretion. These findings elucidate the molecular basis of the TRAP complex in the biogenesis and translocation of proteins at the ER.
Assuntos
Retículo Endoplasmático , Glicoproteínas de Membrana , Animais , Glicoproteínas de Membrana/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Canais de Translocação SEC/metabolismo , Transporte Proteico , Mamíferos/metabolismoRESUMO
N-terminal methionine excision from newly synthesized proteins, catalyzed cotranslationally by methionine aminopeptidases (METAPs), is an essential and universally conserved process that plays a key role in cell homeostasis and protein biogenesis. However, how METAPs interact with ribosomes and how their cleavage specificity is ensured is unknown. We discovered that in eukaryotes the nascent polypeptide-associated complex (NAC) controls ribosome binding of METAP1. NAC recruits METAP1 using a long, flexible tail and provides a platform for the formation of an active methionine excision complex at the ribosomal tunnel exit. This mode of interaction ensures the efficient excision of methionine from cytosolic proteins, whereas proteins targeted to the endoplasmic reticulum are spared. Our results suggest a broader mechanism for how access of protein biogenesis factors to translating ribosomes is controlled.
Assuntos
Metionina , Metionil Aminopeptidases , Biossíntese de Proteínas , Metionina/metabolismo , Metionil Aminopeptidases/metabolismo , Ribossomos/metabolismo , Humanos , AnimaisRESUMO
Ribosome-associated chaperone Trigger Factor (TF) initiates folding of newly synthesized proteins in bacteria. Here, we pinpoint by site-specific crosslinking the sequence of molecular interactions of Escherichia coli TF and nascent chains during translation. Furthermore, we provide the first full-length structure of TF associated with ribosome-nascent chain complexes by using cryo-electron microscopy. In its active state, TF arches over the ribosomal exit tunnel accepting nascent chains in a protective void. The growing nascent chain initially follows a predefined path through the entire interior of TF in an unfolded conformation, and even after folding into a domain it remains accommodated inside the protective cavity of ribosome-bound TF. The adaptability to accept nascent chains of different length and folding states may explain how TF is able to assist co-translational folding of all kinds of nascent polypeptides during ongoing synthesis. Moreover, we suggest a model of how TF's chaperoning function can be coordinated with the co-translational processing and membrane targeting of nascent polypeptides by other ribosome-associated factors.
Assuntos
Proteínas de Escherichia coli/química , Chaperonas Moleculares/química , Peptidilprolil Isomerase/química , Biossíntese de Proteínas , Ribossomos/química , Sequência de Aminoácidos , Reagentes de Ligações Cruzadas/química , Microscopia Crioeletrônica , Peptídeos/química , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: Covalent linkage of the ubiquitin-like protein ISG15 interferes with viral infection and USP18 is the major protease which specifically removes ISG15 from target proteins. Thus, boosting ISG15 modification by protease inhibition of USP18 might represent a new strategy to interfere with viral replication. However, so far no heterologous expression system was available to yield sufficient amounts of catalytically active protein for high-throughput based inhibitor screens. RESULTS: High-level heterologous expression of USP18 was achieved by applying a chaperone-based fusion system in E. coli. Pure protein was obtained in a single-step on IMAC via a His6-tag. The USP18 fusion protein exhibited enzymatic activity towards cell derived ISG15 conjugated substrates and efficiently hydrolyzed ISG15-AMC. Specificity towards ISG15 was shown by covalent adduct formation with ISG15 vinyl sulfone but not with ubiquitin vinyl sulfone. CONCLUSION: The results presented here show that a chaperone fusion system can provide high yields of proteins that are difficult to express. The USP18 protein obtained here is suited to setup high-throughput small molecule inhibitor screens and forms the basis for detailed biochemical and structural characterization.