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1.
EMBO J ; 2024 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-39322754

RESUMO

RNA stability, important for eukaryotic gene expression, is thought to depend on deadenylation rates, with shortened poly(A) tails triggering decapping and 5' to 3' degradation. In contrast to this view, recent large-scale studies indicate that the most unstable mRNAs have, on average, long poly(A) tails. To clarify the role of deadenylation in mRNA decay, we first modeled mRNA poly(A) tail kinetics and mRNA stability in yeast. Independent of deadenylation rates, differences in mRNA decapping rates alone were sufficient to explain current large-scale results. To test the hypothesis that deadenylation and decapping are uncoupled, we used rapid depletion of decapping and deadenylation enzymes and measured changes in mRNA levels, poly(A) length and stability, both transcriptome-wide and with individual reporters. These experiments revealed that perturbations in poly(A) tail length did not correlate with variations in mRNA stability. Thus, while deadenylation may be critical for specific regulatory mechanisms, our results suggest that for most yeast mRNAs, it is not critical for mRNA decapping and degradation.

2.
EMBO J ; 40(1): e105179, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33289941

RESUMO

In eukaryotic translation, termination and ribosome recycling phases are linked to subsequent initiation of a new round of translation by persistence of several factors at ribosomal sub-complexes. These comprise/include the large eIF3 complex, eIF3j (Hcr1 in yeast) and the ATP-binding cassette protein ABCE1 (Rli1 in yeast). The ATPase is mainly active as a recycling factor, but it can remain bound to the dissociated 40S subunit until formation of the next 43S pre-initiation complexes. However, its functional role and native architectural context remains largely enigmatic. Here, we present an architectural inventory of native yeast and human ABCE1-containing pre-initiation complexes by cryo-EM. We found that ABCE1 was mostly associated with early 43S, but also with later 48S phases of initiation. It adopted a novel hybrid conformation of its nucleotide-binding domains, while interacting with the N-terminus of eIF3j. Further, eIF3j occupied the mRNA entry channel via its ultimate C-terminus providing a structural explanation for its antagonistic role with respect to mRNA binding. Overall, the native human samples provide a near-complete molecular picture of the architecture and sophisticated interaction network of the 43S-bound eIF3 complex and the eIF2 ternary complex containing the initiator tRNA.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Células HEK293 , Humanos , Ligação Proteica/fisiologia , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
3.
EMBO J ; 38(14): e100640, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31304628

RESUMO

The Ski2-Ski3-Ski8 (SKI) complex assists the RNA exosome during the 3' to 5' degradation of cytoplasmic transcripts. Previous reports showed that the SKI complex is involved in the 3' to 5' degradation of mRNAs, including 3' untranslated regions (UTRs) and devoid of ribosomes. Paradoxically, we recently showed that the SKI complex directly interacts with ribosomes during the co-translational mRNA decay and that this interaction is necessary for its RNA degradation promoting activity. Here, we characterised a new SKI-associated factor, Ska1, that associates with a subpopulation of the SKI complex. We showed that Ska1 is specifically involved in the degradation of long 3'UTR-containing mRNAs, poorly translated mRNAs as well as other RNA regions not associated with ribosomes, such as cytoplasmic lncRNAs. We further show that the overexpression of SKA1 antagonises the SKI-ribosome association. We propose that the Ska1-SKI complex assists the cytoplasmic exosome in the absence of direct association of the SKI complex with ribosomes.


Assuntos
Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Citoplasma/genética , Estabilidade de RNA , RNA Fúngico/química , RNA Longo não Codificante/química , RNA Mensageiro/química , Saccharomyces cerevisiae/metabolismo
4.
EMBO J ; 37(21)2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30275269

RESUMO

Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway involved in many cellular pathways and crucial for telomere maintenance and embryo development. Core NMD factors Upf1, Upf2 and Upf3 are conserved from yeast to mammals, but a universal NMD model is lacking. We used affinity purification coupled with mass spectrometry and an improved data analysis protocol to characterize the composition and dynamics of yeast NMD complexes in yeast (112 experiments). Unexpectedly, we identified two distinct complexes associated with Upf1: Upf1-23 (Upf1, Upf2, Upf3) and Upf1-decappingUpf1-decapping contained the mRNA decapping enzyme, together with Nmd4 and Ebs1, two proteins that globally affected NMD and were critical for RNA degradation mediated by the Upf1 C-terminal helicase region. The fact that Nmd4 association with RNA was partially dependent on Upf1-23 components and the similarity between Nmd4/Ebs1 and mammalian Smg5-7 proteins suggest that NMD operates through conserved, successive Upf1-23 and Upf1-decapping complexes. This model can be extended to accommodate steps that are missing in yeast, to serve for further mechanistic studies of NMD in eukaryotes.


Assuntos
Modelos Biológicos , Complexos Multiproteicos/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , RNA Helicases/metabolismo , RNA Fúngico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Complexos Multiproteicos/genética , RNA Helicases/genética , RNA Fúngico/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
5.
Nucleic Acids Res ; 48(10): 5670-5683, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32329775

RESUMO

Human CWC27 is an uncharacterized splicing factor and mutations in its gene are linked to retinal degeneration and other developmental defects. We identify the splicing factor CWC22 as the major CWC27 partner. Both CWC27 and CWC22 are present in published Bact spliceosome structures, but no interacting domains are visible. Here, the structure of a CWC27/CWC22 heterodimer bound to the exon junction complex (EJC) core component eIF4A3 is solved at 3Å-resolution. According to spliceosomal structures, the EJC is recruited in the C complex, once CWC27 has left. Our 3D structure of the eIF4A3/CWC22/CWC27 complex is compatible with the Bact spliceosome structure but not with that of the C complex, where a CWC27 loop would clash with the EJC core subunit Y14. A CWC27/CWC22 building block might thus form an intermediate landing platform for eIF4A3 onto the Bact complex prior to its conversion into C complex. Knock-down of either CWC27 or CWC22 in immortalized retinal pigment epithelial cells affects numerous common genes, indicating that these proteins cooperate, targeting the same pathways. As the most up-regulated genes encode factors involved in inflammation, our findings suggest a possible link to the retinal degeneration associated with CWC27 deficiencies.


Assuntos
Ciclofilinas/química , Fator de Iniciação 4A em Eucariotos/química , Proteínas de Ligação a RNA/química , Spliceossomos/química , Linhagem Celular , Ciclofilinas/genética , Ciclofilinas/metabolismo , Fator de Iniciação 4A em Eucariotos/metabolismo , Éxons , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Inflamação/genética , Modelos Moleculares , Domínios Proteicos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Spliceossomos/metabolismo
6.
J Biol Chem ; 291(23): 12245-53, 2016 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-27129255

RESUMO

Protein homeostasis is maintained by quality control mechanisms that detect and eliminate deficient translation products. Cytosolic defective proteins can arise from translation of aberrant mRNAs lacking a termination codon (NonStop) or containing a sequence that blocks translation elongation (No-Go), which results in translational arrest. Stalled ribosomes are dissociated, aberrant mRNAs are degraded by the cytoplasmic exosome, and the nascent peptides remaining in stalled 60S exit tunnels are detected by the ribosome-bound quality control complex (RQC) composed of Ltn1, Rqc1, Rqc2, and Cdc48. Whereas Ltn1 polyubiquitylates these nascent peptides, Rqc2 directs the addition of C-terminal alanine-threonine tails (CAT-tails), and a Cdc48 hexamer is recruited to extract the nascent peptides, which are addressed to the proteasome for degradation. Although the functions of most RQC components have been described, the role of Rqc1 in this quality control process remains undetermined. In this article we show that the absence of Rqc1 or Ltn1 results in the aggregation of aberrant proteins, a phenomenon that requires CAT-tail addition to the nascent peptides by Rqc2. Our results suggest that aberrant CAT-tailed protein aggregation results from a defect in Cdc48 recruitment to stalled 60S particles, a process that requires both Rqc1 and Ltn1. These protein aggregates contain Ltn1-dependent polyubiquitin chains and are degraded by the proteasome. Finally, aggregate characterization by proteomics revealed that they contain specific chaperones including Sis1, Sgt2, Ssa1/2, and Hsp82, suggesting that these protein aggregates may be addressed to aggresome-like structures when the RQC complex fails to deliver aberrant nascent peptides to the proteasome for degradation.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Alanina/química , Alanina/genética , Alanina/metabolismo , Western Blotting , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Microscopia de Fluorescência , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Agregados Proteicos , Biossíntese de Proteínas/genética , Proteólise , Proteômica/métodos , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Treonina/química , Treonina/genética , Treonina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Proteína com Valosina
7.
Mol Cell Proteomics ; 13(7): 1769-86, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24741115

RESUMO

Cilia and flagella are complex organelles made of hundreds of proteins of highly variable structures and functions. Here we report the purification of intact flagella from the procyclic stage of Trypanosoma brucei using mechanical shearing. Structural preservation was confirmed by transmission electron microscopy that showed that flagella still contained typical elements such as the membrane, the axoneme, the paraflagellar rod, and the intraflagellar transport particles. It also revealed that flagella severed below the basal body, and were not contaminated by other cytoskeletal structures such as the flagellar pocket collar or the adhesion zone filament. Mass spectrometry analysis identified a total of 751 proteins with high confidence, including 88% of known flagellar components. Comparison with the cell debris fraction revealed that more than half of the flagellum markers were enriched in flagella and this enrichment criterion was taken into account to identify 212 proteins not previously reported to be associated to flagella. Nine of these were experimentally validated including a 14-3-3 protein not yet reported to be associated to flagella and eight novel proteins termed FLAM (FLAgellar Member). Remarkably, they localized to five different subdomains of the flagellum. For example, FLAM6 is restricted to the proximal half of the axoneme, no matter its length. In contrast, FLAM8 is progressively accumulating at the distal tip of growing flagella and half of it still needs to be added after cell division. A combination of RNA interference and Fluorescence Recovery After Photobleaching approaches demonstrated very different dynamics from one protein to the other, but also according to the stage of construction and the age of the flagellum. Structural proteins are added to the distal tip of the elongating flagellum and exhibit slow turnover whereas membrane proteins such as the arginine kinase show rapid turnover without a detectible polarity.


Assuntos
Flagelos/metabolismo , Proteínas de Membrana/análise , Proteínas de Protozoários/análise , Trypanosoma brucei brucei/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Perfilação da Expressão Gênica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Proteômica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Interferência de RNA , RNA Interferente Pequeno
8.
Proc Natl Acad Sci U S A ; 110(13): 5046-51, 2013 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-23479637

RESUMO

Ribosome stalling on eukaryotic mRNAs triggers cotranslational RNA and protein degradation through conserved mechanisms. For example, mRNAs lacking a stop codon are degraded by the exosome in association with its cofactor, the SKI complex, whereas the corresponding aberrant nascent polypeptides are ubiquitinated by the E3 ligases Ltn1 and Not4 and become proteasome substrates. How translation arrest is linked with polypeptide degradation is still unclear. Genetic screens with SKI and LTN1 mutants allowed us to identify translation-associated element 2 (Tae2) and ribosome quality control 1 (Rqc1), two factors that we found associated, together with Ltn1 and the AAA-ATPase Cdc48, to 60S ribosomal subunits. Translation-associated element 2 (Tae2), Rqc1, and Cdc48 were all required for degradation of polypeptides synthesized from Non-Stop mRNAs (Non-Stop protein decay; NSPD). Both Ltn1 and Rqc1 were essential for the recruitment of Cdc48 to 60S particles. Polysome gradient analyses of mutant strains revealed unique intermediates of this pathway, showing that the polyubiquitination of Non-Stop peptides is a progressive process. We propose that ubiquitination of the nascent peptide starts on the 80S and continues on the 60S, on which Cdc48 is recruited to escort the substrate for proteasomal degradation.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Biossíntese de Proteínas/fisiologia , Proteólise , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitinação/fisiologia , Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Ligação a RNA , Proteínas Repressoras , Subunidades Ribossômicas Maiores de Eucariotos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteína com Valosina
10.
Nucleic Acids Res ; 41(1): 288-301, 2013 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-23093592

RESUMO

Protein complexes directing messenger RNA (mRNA) degradation are present in all kingdoms of life. In Escherichia coli, mRNA degradation is performed by an RNA degradosome organized by the major ribonuclease RNase E. In bacteria lacking RNase E, the existence of a functional RNA degradosome is still an open question. Here, we report that in the bacterial pathogen Helicobacter pylori, RNA degradation is directed by a minimal RNA degradosome consisting of Hp-RNase J and the only DExD-box RNA helicase of H. pylori, RhpA. We show that the protein complex promotes faster degradation of double-stranded RNA in vitro in comparison with Hp-RNase J alone. The ATPase activity of RhpA is stimulated in the presence of Hp-RNase J, demonstrating that the catalytic capacity of both partners is enhanced upon interaction. Remarkably, both proteins are associated with translating ribosomes and not with individual 30S and 50S subunits. Moreover, Hp-RNase J is not recruited to ribosomes to perform rRNA maturation. Together, our findings imply that in H. pylori, the mRNA-degrading machinery is associated with the translation apparatus, a situation till now thought to be restricted to eukaryotes and archaea.


Assuntos
Endorribonucleases/metabolismo , Helicobacter pylori/enzimologia , Complexos Multienzimáticos/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/enzimologia , Adenosina Trifosfatases/metabolismo , Bacillus subtilis/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endorribonucleases/genética , Endorribonucleases/isolamento & purificação , Helicobacter pylori/genética , Helicobacter pylori/crescimento & desenvolvimento , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Biossíntese de Proteínas , RNA Helicases/isolamento & purificação , RNA de Cadeia Dupla/metabolismo , RNA Ribossômico/metabolismo
11.
Nucleic Acids Res ; 41(20): 9461-70, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23945946

RESUMO

Ribosome biogenesis requires >300 assembly factors in Saccharomyces cerevisiae. Ribosome assembly factors Imp3, Mrt4, Rlp7 and Rlp24 have sequence similarity to ribosomal proteins S9, P0, L7 and L24, suggesting that these pre-ribosomal factors could be placeholders that prevent premature assembly of the corresponding ribosomal proteins to nascent ribosomes. However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing that the two proteins can bind simultaneously to pre-ribosomal particles. Cross-linking and cDNA analysis experiments showed that Rlp7 binds to the ITS2 region of 27S pre-rRNAs, at two sites, in helix III and in a region adjacent to the pre-rRNA processing sites C1 and E. However, L7 binds to mature 25S and 5S rRNAs and cross-linked predominantly to helix ES7(L)b within 25S rRNA. Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different positions on the same pre-60S particles. Our results also suggest that Rlp7 facilitates the formation of the hairpin structure of ITS2 during 60S ribosomal subunit maturation.


Assuntos
RNA Ribossômico/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Sítios de Ligação , Dados de Sequência Molecular , Precursores de RNA/química , Precursores de RNA/metabolismo , RNA Ribossômico/química , RNA Ribossômico 5S/química , RNA Ribossômico 5S/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/química
12.
EMBO J ; 29(9): 1585-99, 2010 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-20360685

RESUMO

Programmed necrosis induced by DNA alkylating agents, such as MNNG, is a caspase-independent mode of cell death mediated by apoptosis-inducing factor (AIF). After poly(ADP-ribose) polymerase 1, calpain, and Bax activation, AIF moves from the mitochondria to the nucleus where it induces chromatinolysis and cell death. The mechanisms underlying the nuclear action of AIF are, however, largely unknown. We show here that, through its C-terminal proline-rich binding domain (PBD, residues 543-559), AIF associates in the nucleus with histone H2AX. This interaction regulates chromatinolysis and programmed necrosis by generating an active DNA-degrading complex with cyclophilin A (CypA). Deletion or directed mutagenesis in the AIF C-terminal PBD abolishes AIF/H2AX interaction and AIF-mediated chromatinolysis. H2AX genetic ablation or CypA downregulation confers resistance to programmed necrosis. AIF fails to induce chromatinolysis in H2AX or CypA-deficient nuclei. We also establish that H2AX is phosphorylated at Ser139 after MNNG treatment and that this phosphorylation is critical for caspase-independent programmed necrosis. Overall, our data shed new light in the mechanisms regulating programmed necrosis, elucidate a key nuclear partner of AIF, and uncover an AIF apoptogenic motif.


Assuntos
Fator de Indução de Apoptose/metabolismo , Caspases/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Necrose/metabolismo , Animais , Fator de Indução de Apoptose/química , Calpaína/metabolismo , Linhagem Celular , Ciclofilina A/genética , Ciclofilina A/metabolismo , Dano ao DNA , Regulação para Baixo , Fibroblastos/citologia , Fibroblastos/metabolismo , Deleção de Genes , Histonas/química , Histonas/genética , Metilnitronitrosoguanidina/farmacologia , Camundongos , Modelos Moleculares , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína X Associada a bcl-2/metabolismo
13.
Proc Natl Acad Sci U S A ; 107(18): 8381-6, 2010 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-20404152

RESUMO

Leishmania is exposed to a sudden increase in environmental temperature during the infectious cycle that triggers stage differentiation and adapts the parasite phenotype to intracellular survival in the mammalian host. The absence of classical promoter-dependent mechanisms of gene regulation and constitutive expression of most of the heat-shock proteins (HSPs) in these human pathogens raise important unresolved questions as to regulation of the heat-shock response and stage-specific functions of Leishmania HSPs. Here we used a gel-based quantitative approach to assess the Leishmania donovani phosphoproteome and revealed that 38% of the proteins showed significant stage-specific differences, with a strong focus of amastigote-specific phosphoproteins on chaperone function. We identified STI1/HOP-containing chaperone complexes that interact with ribosomal client proteins in an amastigote-specific manner. Genetic analysis of STI1/HOP phosphorylation sites in conditional sti1(-/-) null mutant parasites revealed two phosphoserine residues essential for parasite viability. Phosphorylation of the major Leishmania chaperones at the pathogenic stage suggests that these proteins may be promising drug targets via inhibition of their respective protein kinases.


Assuntos
Proteínas de Choque Térmico/metabolismo , Leishmania donovani/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Leishmania donovani/crescimento & desenvolvimento , Dados de Sequência Molecular , Fosfoproteínas/química , Fosforilação , Ligação Proteica , Proteoma/química , Proteínas de Protozoários/química , Alinhamento de Sequência
14.
MicroPubl Biol ; 20232023.
Artigo em Inglês | MEDLINE | ID: mdl-37746059

RESUMO

mRNA degradation is one of the main steps of gene expression, and a key player is the 5'-3' exonuclease Xrn1. In Saccharomyces cerevisiae , it was previously shown, by a microscopy approach, that Xrn1 is located to different cellular compartments, depending on physiological state. During exponential growth, Xrn1 is distributed in the cytoplasm, while it co-localizes with eisosomes after the post-diauxic shift (PDS). Here, we biochemically characterize the Xrn1-associated complexes in different cellular states. We demonstrate that, after PDS, Xrn1 but not the decapping nor Lsm1-7/Pat1 complexes associates with eisosomal proteins, strengthening the model that sequestration of Xrn1 in eisosomes preserves mRNAs from degradation during PDS.

15.
PLoS One ; 18(11): e0293228, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38011112

RESUMO

Translation initiation is a complex and highly regulated process that represents an important mechanism, controlling gene expression. eIF2A was proposed as an alternative initiation factor, however, its role and biological targets remain to be discovered. To further gain insight into the function of eIF2A in Saccharomyces cerevisiae, we identified mRNAs associated with the eIF2A complex and showed that 24% of the most enriched mRNAs encode proteins related to cell wall biogenesis and maintenance. In agreement with this result, we showed that an eIF2A deletion sensitized cells to cell wall damage induced by calcofluor white. eIF2A overexpression led to a growth defect, correlated with decreased synthesis of several cell wall proteins. In contrast, no changes were observed in the transcriptome, suggesting that eIF2A controls the expression of cell wall-related proteins at a translational level. The biochemical characterization of the eIF2A complex revealed that it strongly interacts with the RNA binding protein, Ssd1, which is a negative translational regulator, controlling the expression of cell wall-related genes. Interestingly, eIF2A and Ssd1 bind several common mRNA targets and we found that the binding of eIF2A to some targets was mediated by Ssd1. Surprisingly, we further showed that eIF2A is physically and functionally associated with the exonuclease Xrn1 and other mRNA degradation factors, suggesting an additional level of regulation. Altogether, our results highlight new aspects of this complex and redundant fine-tuned regulation of proteins expression related to the cell wall, a structure required to maintain cell shape and rigidity, providing protection against harmful environmental stress.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , RNA Mensageiro/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Expressão Gênica , Regulação Fúngica da Expressão Gênica
16.
Indian J Microbiol ; 52(4): 660-5, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24293727

RESUMO

Vibrio parahaemolyticus and V. alginolyticus, marine foodborne pathogens, were treated with UVC-radiation (240 J/m(2)) to evaluate alterations in their outer membrane protein profiles. Outer membrane protein patterns of UVC-irradiated bacteria were found altered when analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Altered proteins were identified by mass spectrometry (MS and MS/MS) and analysis revealed that OmpW, OmpA, Long-chain fatty acid transport protein, Outer membrane receptor protein, Putative uncharacterized protein VP0167, Maltoporin (lamB), Polar flagellin B/D, Agglutination protein Peptidoglycan-associated lipoprotein and MltA-interacting protein MipA were appeared, thereby they can be considered as UVC-stress proteins in some vibrios. In addition, expression of OmpK decreased to non-detectable level. Furthermore, we observed a decrease or an increase in the expression level of other outer membrane proteins.

17.
Methods Mol Biol ; 2477: 225-236, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35524120

RESUMO

Multiple protein complexes are fundamental parts of living systems. Identification of the components of these complexes and characterization of the molecular mechanisms that allow their formation, function, and regulation can be done by affinity purification of proteins and associated factors followed by mass spectrometry of peptides. Speed and specificity for the isolation of complexes from whole cell extracts improved over time, together with the reliable identification and quantification of proteins by mass spectrometry. Relative quantification of proteins in such samples can now be done to characterize even relatively nonabundant complexes. We describe here our experience with proteins fused with the Z domain, derived from staphylococcal protein A, and IgG affinity purification for the analysis of protein complexes involved in RNA metabolism in the budding yeast Saccharomyces cerevisiae. We illustrate the use of enrichment calculations for proteins in purified samples as a way to robust identification of protein partners. While the protocols presented here are specific for yeast, their principles can be applied to the study of protein complexes in any other organism.


Assuntos
Proteínas , Saccharomyces cerevisiae , Cromatografia de Afinidade/métodos , Espectrometria de Massas/métodos , Peptídeos/química , Proteínas/metabolismo , Saccharomyces cerevisiae/genética
18.
J Biol Chem ; 285(20): 14898-14908, 2010 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-20167604

RESUMO

Interleukin (IL)-7 is a central cytokine that controls homeostasis of the CD4 T lymphocyte pool. Here we show on human primary cells that IL-7 binds to preassembled receptors made up of proprietary chain IL-7Ralpha and the common chain gammac shared with IL-2, -4, -9, -15, and -21 receptors. Upon IL-7 binding, both chains are driven in cholesterol- and sphingomyelin-rich rafts where associated signaling proteins Jak1, Jak3, STAT1, -3, and -5 are found to be phosphorylated. Meanwhile the IL-7.IL-7R complex interacts with the cytoskeleton that halts its diffusion as measured by single molecule fluorescence autocorrelated spectroscopy monitored by microimaging. Comparative immunoprecipitations of IL-7Ralpha signaling complex from non-stimulated and IL-7-stimulated cells confirmed recruitment of proteins such as STATs, but many others were also identified by mass spectrometry from two-dimensional gels. Among recruited proteins, two-thirds are involved in cytoskeleton and raft formation. Thus, early events leading to IL-7 signal transduction involve its receptor compartmentalization into membrane nanodomains and cytoskeleton recruitment.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Compartimento Celular , Interleucina-7/metabolismo , Receptores de Interleucina-7/metabolismo , Transdução de Sinais , Sítios de Ligação , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoprecipitação , Ativação Linfocitária , Espectrometria de Massas , Espectrometria de Fluorescência , Ultracentrifugação
19.
Proteomics ; 10(21): 3868-83, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20960452

RESUMO

Human pathogenic protozoa of the genus Leishmania undergo various developmental transitions during the infectious cycle that are triggered by changes in the host environment. How these parasites sense, transduce, and respond to these signals is only poorly understood. Here we used phosphoproteomic approaches to monitor signaling events in L. donovani axenic amastigotes, which may be important for intracellular parasite survival. LC-ESI-MS/MS analysis of IMAC-enriched phosphoprotein extracts identified 445 putative phosphoproteins in two independent biological experiments. Functional enrichment analysis allowed us to gain insight into parasite pathways that are regulated by protein phosphorylation and revealed significant enrichment in our data set of proteins whose biological functions are associated with protein turn-over, stress response, and signal transduction. LC-ESI-MS/MS analysis of TiO(2)-enriched phosphopeptides confirmed these results and identified 157 unique phosphopeptides covering 181 unique phosphorylation sites in 126 distinct proteins. Investigation of phosphorylation site conservation across related trypanosomatids and higher eukaryotes by multiple sequence alignment and cluster analysis revealed L. donovani-specific phosphoresidues in highly conserved proteins that share significant sequence homology to orthologs of the human host. These unique phosphorylation sites reveal important differences between host and parasite biology and post-translational protein regulation, which may be exploited for the design of novel anti-parasitic interventions.


Assuntos
Cromatografia Líquida/métodos , Leishmania/química , Fosfoproteínas/química , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Sequência de Aminoácidos , Animais , Células Cultivadas , Análise por Conglomerados , Bases de Dados de Proteínas , Eletroforese em Gel Bidimensional , Proteínas Fúngicas , Humanos , Leishmania/metabolismo , Estágios do Ciclo de Vida , Camundongos , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Alinhamento de Sequência , Espectrometria de Massas em Tandem/métodos
20.
Mol Cell Biol ; 27(8): 2897-909, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17308036

RESUMO

Ribosome biogenesis is driven by a large number of preribosomal factors that associate with and dissociate from the preribosomal particles along the maturation pathway. We have previously shown that budding yeast Mak11, whose homologues in other eukaryotes were described as modulating a p21-activated protein kinase function, accumulates in Rlp24-associated pre-60S complexes when their maturation is impeded in Saccharomyces cerevisiae. The functional inactivation of WD40 repeat protein Mak11 interfered with the 60S rRNA maturation, led to a cell cycle delay in G(1), and blocked green fluorescent protein-tagged Rpl25 in the nucleoli of yeast cells, indicating an early role of Mak11 in ribosome assembly. Surprisingly, Mak11 inactivation also led to a dramatic destabilization of Rlp24. The suppression of the thermosensitive phenotype of a mak11 mutant by RLP24 overexpression and a direct in vitro interaction between Rlp24 and Mak11 suggest that Mak11 acts as an Rlp24 cofactor during early steps of 60S ribosomal subunit assembly. Moreover, we found that Skb15, the Mak11 homologue in Schizosaccharomyces pombe, also associated with preribosomes and affected 60S biogenesis in fission yeast. It is thus likely that the previously observed phenotypes for MAK11 homologues in other eukaryotes are secondary to the main function of these proteins in ribosome formation.


Assuntos
Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Homologia de Sequência , Sequência de Aminoácidos , Nucléolo Celular/metabolismo , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Precursores de RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Schizosaccharomyces pombe/química , Quinases Ativadas por p21
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