Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 27
Filtrar
1.
Proc Natl Acad Sci U S A ; 120(3): e2210300120, 2023 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-36634142

RESUMO

Rhizogenic Agrobacterium strains comprise biotrophic pathogens that cause hairy root disease (HRD) on hydroponically grown Solanaceae and Cucurbitaceae crops, besides being widely explored agents for the creation of hairy root cultures for the sustainable production of plant-specialized metabolites. Hairy root formation is mediated through the expression of genes encoded on the T-DNA of the root-inducing (Ri) plasmid, of which several, including root oncogenic locus B (rolB), play a major role in hairy root development. Despite decades of research, the exact molecular function of the proteins encoded by the rol genes remains enigmatic. Here, by means of TurboID-mediated proximity labeling in tomato (Solanum lycopersicum) hairy roots, we identified the repressor proteins TOPLESS (TPL) and Novel Interactor of JAZ (NINJA) as direct interactors of RolB. Although these interactions allow RolB to act as a transcriptional repressor, our data hint at another in planta function of the RolB oncoprotein. Hence, by a series of plant bioassays, transcriptomic and DNA-binding site enrichment analyses, we conclude that RolB can mitigate the TPL functioning so that it leads to a specific and partial reprogramming of phytohormone signaling, immunity, growth, and developmental processes. Our data support a model in which RolB manipulates host transcription, at least in part, through interaction with TPL, to facilitate hairy root development. Thereby, we provide important mechanistic insights into this renowned oncoprotein in HRD.


Assuntos
Agrobacterium , Proteínas Repressoras , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Agrobacterium/genética , Agrobacterium/metabolismo , Plasmídeos , Produtos Agrícolas/genética , Imunidade Vegetal , Raízes de Plantas/metabolismo
2.
J Bacteriol ; 206(4): e0001424, 2024 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-38470120

RESUMO

In bacteria, cell poles function as subcellular compartments where proteins localize during specific lifecycle stages, orchestrated by polar "hub" proteins. Whereas most described bacteria inherit an "old" pole from the mother cell and a "new" pole from cell division, generating cell asymmetry at birth, non-binary division poses challenges for establishing cell polarity, particularly for daughter cells inheriting only new poles. We investigated polarity dynamics in the obligate predatory bacterium Bdellovibrio bacteriovorus, proliferating through filamentous growth followed by non-binary division within prey bacteria. Monitoring the subcellular localization of two proteins known as polar hubs in other species, RomR and DivIVA, revealed RomR as an early polarity marker in B. bacteriovorus. RomR already marks the future anterior poles of the progeny during the predator's growth phase, during a precise period closely following the onset of divisome assembly and the end of chromosome segregation. In contrast to RomR's stable unipolar localization in the progeny, DivIVA exhibits a dynamic pole-to-pole localization. This behavior changes shortly before the division of the elongated predator cell, where DivIVA accumulates at all septa and both poles. In vivo protein interaction networks for DivIVA and RomR, mapped through endogenous miniTurbo-based proximity labeling, further underscore their distinct roles in cell polarization and reinforce the importance of the anterior "invasive" cell pole in prey-predator interactions. Our work also emphasizes the precise spatiotemporal order of cellular processes underlying B. bacteriovorus proliferation, offering insights into the subcellular organization of bacteria with filamentous growth and non-binary division.IMPORTANCEIn bacteria, cell poles are crucial areas where "hub" proteins orchestrate lifecycle events through interactions with multiple partners at specific times. While most bacteria exhibit one "old" and one "new" pole, inherited from the previous division event, setting polar identity poses challenges in bacteria with non-binary division. This study explores polar proteins in the predatory bacterium Bdellovibrio bacteriovorus, which undergoes filamentous growth followed by non-binary division inside another bacterium. Our research reveals distinct localization dynamics of the polar proteins RomR and DivIVA, highlighting RomR as an early "hub" marking polar identity in the filamentous mother cell. Using miniTurbo-based proximity labeling, we uncovered their unique protein networks. Overall, our work provides new insights into the cell polarity in non-binary dividing bacteria.


Assuntos
Proteínas de Bactérias , Bdellovibrio bacteriovorus , Recém-Nascido , Humanos , Proteínas de Bactérias/genética , Bactérias/metabolismo , Divisão Celular , Polaridade Celular
3.
J Biol Chem ; 299(2): 102824, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36567016

RESUMO

N-terminal acetylation is a conserved protein modification among eukaryotes. The yeast Saccharomyces cerevisiae is a valuable model system for studying this modification. The bulk of protein N-terminal acetylation in S. cerevisiae is catalyzed by the N-terminal acetyltransferases NatA, NatB, and NatC. Thus far, proteome-wide identification of the in vivo protein substrates of yeast NatA and NatB has been performed by N-terminomics. Here, we used S. cerevisiae deleted for the NatC catalytic subunit Naa30 and identified 57 yeast NatC substrates by N-terminal combined fractional diagonal chromatography analysis. Interestingly, in addition to the canonical N-termini starting with ML, MI, MF, and MW, yeast NatC substrates also included MY, MK, MM, MA, MV, and MS. However, for some of these substrate types, such as MY, MK, MV, and MS, we also uncovered (residual) non-NatC NAT activity, most likely due to the previously established redundancy between yeast NatC and NatE/Naa50. Thus, we have revealed a complex interplay between different NATs in targeting methionine-starting N-termini in yeast. Furthermore, our results showed that ectopic expression of human NAA30 rescued known NatC phenotypes in naa30Δ yeast, as well as partially restored the yeast NatC Nt-acetylome. Thus, we demonstrate an evolutionary conservation of NatC from yeast to human thereby underpinning future disease models to study pathogenic NAA30 variants. Overall, this work offers increased biochemical and functional insights into NatC-mediated N-terminal acetylation and provides a basis for future work to pinpoint the specific molecular mechanisms that link the lack of NatC-mediated N-terminal acetylation to phenotypes of NatC deletion yeast.


Assuntos
Acetiltransferases N-Terminal , Saccharomyces cerevisiae , Humanos , Acetilação , Cromatografia Líquida , Sequência Conservada , Teste de Complementação Genética , Metionina/metabolismo , Acetiltransferase N-Terminal C/genética , Acetiltransferase N-Terminal C/metabolismo , Acetiltransferase N-Terminal E , Acetiltransferases N-Terminal/deficiência , Acetiltransferases N-Terminal/genética , Acetiltransferases N-Terminal/metabolismo , Fenótipo , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato
4.
Int J Mol Sci ; 22(7)2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33916271

RESUMO

The evolutionary conserved N-alpha acetyltransferase Naa40p is among the most selective N-terminal acetyltransferases (NATs) identified to date. Here we identified a conserved N-terminally truncated Naa40p proteoform named Naa40p25 or short Naa40p (Naa40S). Intriguingly, although upon ectopic expression in yeast, both Naa40p proteoforms were capable of restoring N-terminal acetylation of the characterized yeast histone H2A Naa40p substrate, the Naa40p histone H4 substrate remained N-terminally free in human haploid cells specifically deleted for canonical Naa40p27 or 237 amino acid long Naa40p (Naa40L), but expressing Naa40S. Interestingly, human Naa40L and Naa40S displayed differential expression and subcellular localization patterns by exhibiting a principal nuclear and cytoplasmic localization, respectively. Furthermore, Naa40L was shown to be N-terminally myristoylated and to interact with N-myristoyltransferase 1 (NMT1), implicating NMT1 in steering Naa40L nuclear import. Differential interactomics data obtained by biotin-dependent proximity labeling (BioID) further hints to context-dependent roles of Naa40p proteoforms. More specifically, with Naa40S representing the main co-translationally acting actor, the interactome of Naa40L was enriched for nucleolar proteins implicated in ribosome biogenesis and the assembly of ribonucleoprotein particles, overall indicating a proteoform-specific segregation of previously reported Naa40p activities. Finally, the yeast histone variant H2A.Z and the transcriptionally regulatory protein Lge1 were identified as novel Naa40p substrates, expanding the restricted substrate repertoire of Naa40p with two additional members and further confirming Lge1 as being the first redundant yNatA and yNatD substrate identified to date.


Assuntos
Acetiltransferase N-Terminal D/metabolismo , Histonas/metabolismo , Humanos , Isoformas de Proteínas , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição
5.
Mol Cell Proteomics ; 17(4): 694-708, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29317475

RESUMO

Excision of the N-terminal initiator methionine (iMet) residue from nascent peptide chains is an essential and omnipresent protein modification carried out by methionine aminopeptidases (MetAPs) that accounts for a major source of N-terminal proteoform diversity. Although MetAP2 is known to be implicated in processes such as angiogenesis and proliferation in mammals, the physiological role of MetAP1 is much less clear. In this report we studied the omics-wide effects of human MetAP1 deletion and general MetAP inhibition. The levels of iMet retention are inversely correlated with cellular proliferation rates. Further, despite the increased MetAP2 expression on MetAP1 deletion, MetAP2 was unable to restore processing of Met-Ser-, Met-Pro-, and Met-Ala- starting N termini as inferred from the iMet retention profiles observed, indicating a higher activity of MetAP1 over these N termini. Proteome and transcriptome expression profiling point to differential expression of proteins implicated in lipid metabolism, cytoskeleton organization, cell proliferation and protein synthesis upon perturbation of MetAP activity.


Assuntos
Aminopeptidases/genética , Proteoma , Linhagem Celular , Mutação da Fase de Leitura , Glicoproteínas/genética , Humanos , Metionil Aminopeptidases , Proteômica
6.
Mol Cell Proteomics ; 17(12): 2309-2323, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30150368

RESUMO

N-terminal acetylation (Nt-acetylation) is a highly abundant protein modification in eukaryotes and impacts a wide range of cellular processes, including protein quality control and stress tolerance. Despite its prevalence, the mechanisms regulating Nt-acetylation are still nebulous. Here, we present the first global study of Nt-acetylation in yeast cells as they progress to stationary phase in response to nutrient starvation. Surprisingly, we found that yeast cells maintain their global Nt-acetylation levels upon nutrient depletion, despite a marked decrease in acetyl-CoA levels. We further observed two distinct sets of protein N termini that display differential and opposing Nt-acetylation behavior upon nutrient starvation, indicating a dynamic process. The first protein cluster was enriched for annotated N termini showing increased Nt-acetylation in stationary phase compared with exponential growth phase. The second protein cluster was conversely enriched for alternative nonannotated N termini (i.e. N termini indicative of shorter N-terminal proteoforms) and, like histones, showed reduced acetylation levels in stationary phase when acetyl-CoA levels were low. Notably, the degree of Nt-acetylation of Pcl8, a negative regulator of glycogen biosynthesis and two components of the pre-ribosome complex (Rsa3 and Rpl7a) increased during starvation. Moreover, the steady-state levels of these proteins were regulated both by starvation and NatA activity. In summary, this study represents the first comprehensive analysis of metabolic regulation of Nt-acetylation and reveals that specific, rather than global, Nt-acetylation events are subject to metabolic regulation.


Assuntos
Acetilcoenzima A/metabolismo , Saccharomyces cerevisiae/enzimologia , Acetilação , Acetiltransferases/metabolismo , Análise de Variância , Células Cultivadas , Distribuição de Qui-Quadrado , Ciclinas/metabolismo , Histonas/metabolismo , Acetiltransferases N-Terminal/metabolismo , Proteoma/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas em Tandem
7.
Mol Cell Proteomics ; 16(6): 1064-1080, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28432195

RESUMO

Proteogenomics is an emerging research field yet lacking a uniform method of analysis. Proteogenomic studies in which N-terminal proteomics and ribosome profiling are combined, suggest that a high number of protein start sites are currently missing in genome annotations. We constructed a proteogenomic pipeline specific for the analysis of N-terminal proteomics data, with the aim of discovering novel translational start sites outside annotated protein coding regions. In summary, unidentified MS/MS spectra were matched to a specific N-terminal peptide library encompassing protein N termini encoded in the Arabidopsis thaliana genome. After a stringent false discovery rate filtering, 117 protein N termini compliant with N-terminal methionine excision specificity and indicative of translation initiation were found. These include N-terminal protein extensions and translation from transposable elements and pseudogenes. Gene prediction provided supporting protein-coding models for approximately half of the protein N termini. Besides the prediction of functional domains (partially) contained within the newly predicted ORFs, further supporting evidence of translation was found in the recently released Araport11 genome re-annotation of Arabidopsis and computational translations of sequences stored in public repositories. Most interestingly, complementary evidence by ribosome profiling was found for 23 protein N termini. Finally, by analyzing protein N-terminal peptides, an in silico analysis demonstrates the applicability of our N-terminal proteogenomics strategy in revealing protein-coding potential in species with well- and poorly-annotated genomes.


Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Biossíntese de Proteínas/genética , Genoma de Planta , Biblioteca de Peptídeos , Peptídeos/genética , Peptídeos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteogenômica
8.
Nucleic Acids Res ; 45(13): 7997-8013, 2017 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-28541577

RESUMO

Alternative translation initiation mechanisms such as leaky scanning and reinitiation potentiate the polycistronic nature of human transcripts. By allowing for reprogrammed translation, these mechanisms can mediate biological responses to stimuli. We combined proteomics with ribosome profiling and mRNA sequencing to identify the biological targets of translation control triggered by the eukaryotic translation initiation factor 1 (eIF1), a protein implicated in the stringency of start codon selection. We quantified expression changes of over 4000 proteins and 10 000 actively translated transcripts, leading to the identification of 245 transcripts undergoing translational control mediated by upstream open reading frames (uORFs) upon eIF1 deprivation. Here, the stringency of start codon selection and preference for an optimal nucleotide context were largely diminished leading to translational upregulation of uORFs with suboptimal start. Interestingly, genes affected by eIF1 deprivation were implicated in energy production and sensing of metabolic stress.


Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Iniciação Traducional da Cadeia Peptídica , Linhagem Celular , Códon de Iniciação , Metabolismo Energético/genética , Fatores de Iniciação em Eucariotos/antagonistas & inibidores , Fatores de Iniciação em Eucariotos/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Conformação de Ácido Nucleico , Fases de Leitura Aberta , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Estresse Fisiológico/genética
9.
Nucleic Acids Res ; 45(20): e168, 2017 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-28977509

RESUMO

Prokaryotic genome annotation is highly dependent on automated methods, as manual curation cannot keep up with the exponential growth of sequenced genomes. Current automated methods depend heavily on sequence composition and often underestimate the complexity of the proteome. We developed RibosomeE Profiling Assisted (re-)AnnotaTION (REPARATION), a de novo machine learning algorithm that takes advantage of experimental protein synthesis evidence from ribosome profiling (Ribo-seq) to delineate translated open reading frames (ORFs) in bacteria, independent of genome annotation (https://github.com/Biobix/REPARATION). REPARATION evaluates all possible ORFs in the genome and estimates minimum thresholds based on a growth curve model to screen for spurious ORFs. We applied REPARATION to three annotated bacterial species to obtain a more comprehensive mapping of their translation landscape in support of experimental data. In all cases, we identified hundreds of novel (small) ORFs including variants of previously annotated ORFs and >70% of all (variants of) annotated protein coding ORFs were predicted by REPARATION to be translated. Our predictions are supported by matching mass spectrometry proteomics data, sequence composition and conservation analysis. REPARATION is unique in that it makes use of experimental translation evidence to intrinsically perform a de novo ORF delineation in bacterial genomes irrespective of the sequence features linked to open reading frames.


Assuntos
Bacillus subtilis/genética , Biologia Computacional/métodos , Escherichia coli K12/genética , Genoma Bacteriano/genética , Anotação de Sequência Molecular/métodos , Salmonella typhimurium/genética , Algoritmos , Mapeamento Cromossômico , Aprendizado de Máquina , Fases de Leitura Aberta/genética , Ribossomos/genética
10.
Mol Cell Proteomics ; 15(11): 3361-3372, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27694331

RESUMO

N-terminal acetylation (Nt-acetylation) by N-terminal acetyltransferases (NATs) is one of the most common protein modifications in eukaryotes. The NatC complex represents one of three major NATs of which the substrate profile remains largely unexplored. Here, we defined the in vivo human NatC Nt-acetylome on a proteome-wide scale by combining knockdown of its catalytic subunit Naa30 with positional proteomics. We identified 46 human NatC substrates, expanding our current knowledge on the substrate repertoire of NatC which now includes proteins harboring Met-Leu, Met-Ile, Met-Phe, Met-Trp, Met-Val, Met-Met, Met-His and Met-Lys N termini. Upon Naa30 depletion the expression levels of several organellar proteins were found reduced, in particular mitochondrial proteins, some of which were found to be NatC substrates. Interestingly, knockdown of Naa30 induced the loss of mitochondrial membrane potential and fragmentation of mitochondria. In conclusion, NatC Nt-acetylates a large variety of proteins and is essential for mitochondrial integrity and function.


Assuntos
Proteínas Mitocondriais/metabolismo , Acetiltransferase N-Terminal C/genética , Acetiltransferase N-Terminal C/metabolismo , Proteômica/métodos , Acetilação , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Ligação Proteica , Mapas de Interação de Proteínas , Especificidade por Substrato
11.
BMC Biol ; 15(1): 76, 2017 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-28854918

RESUMO

BACKGROUND: While methods for annotation of genes are increasingly reliable, the exact identification of translation initiation sites remains a challenging problem. Since the N-termini of proteins often contain regulatory and targeting information, developing a robust method for start site identification is crucial. Ribosome profiling reads show distinct patterns of read length distributions around translation initiation sites. These patterns are typically lost in standard ribosome profiling analysis pipelines, when reads from footprints are adjusted to determine the specific codon being translated. RESULTS: Utilising these signatures in combination with nucleotide sequence information, we build a model capable of predicting translation initiation sites and demonstrate its high accuracy using N-terminal proteomics. Applying this to prokaryotic translatomes, we re-annotate translation initiation sites and provide evidence of N-terminal truncations and extensions of previously annotated coding sequences. These re-annotations are supported by the presence of structural and sequence-based features next to N-terminal peptide evidence. Finally, our model identifies 61 novel genes previously undiscovered in the Salmonella enterica genome. CONCLUSIONS: Signatures within ribosome profiling read length distributions can be used in combination with nucleotide sequence information to provide accurate genome-wide identification of translation initiation sites.


Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Processamento de Proteína Pós-Traducional , Ribossomos/metabolismo
12.
Hum Mol Genet ; 24(7): 1956-76, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25489052

RESUMO

The X-linked lethal Ogden syndrome was the first reported human genetic disorder associated with a mutation in an N-terminal acetyltransferase (NAT) gene. The affected males harbor an Ser37Pro (S37P) mutation in the gene encoding Naa10, the catalytic subunit of NatA, the major human NAT involved in the co-translational acetylation of proteins. Structural models and molecular dynamics simulations of the human NatA and its S37P mutant highlight differences in regions involved in catalysis and at the interface between Naa10 and the auxiliary subunit hNaa15. Biochemical data further demonstrate a reduced catalytic capacity and an impaired interaction between hNaa10 S37P and Naa15 as well as Naa50 (NatE), another interactor of the NatA complex. N-Terminal acetylome analyses revealed a decreased acetylation of a subset of NatA and NatE substrates in Ogden syndrome cells, supporting the genetic findings and our hypothesis regarding reduced Nt-acetylation of a subset of NatA/NatE-type substrates as one etiology for Ogden syndrome. Furthermore, Ogden syndrome fibroblasts display abnormal cell migration and proliferation capacity, possibly linked to a perturbed retinoblastoma pathway. N-Terminal acetylation clearly plays a role in Ogden syndrome, thus revealing the in vivo importance of N-terminal acetylation in human physiology and disease.


Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Proteínas/metabolismo , Acetilação , Acetiltransferases/química , Acetiltransferases/genética , Acetiltransferases/metabolismo , Motivos de Aminoácidos , Domínio Catalítico , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/enzimologia , Doenças Genéticas Ligadas ao Cromossomo X/genética , Humanos , Masculino , Mutação , Linhagem , Proteínas/química , Proteínas/genética
13.
Mol Cell Proteomics ; 12(10): 2921-34, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23788529

RESUMO

Granzymes are structurally related serine proteases involved in cell death and immunity. To date four out of five human granzymes have assigned orthologs in mice; however for granzyme H, no murine ortholog has been suggested and its role in cytotoxicity remains controversial. Here, we demonstrate that, as is the case for granzyme C, human granzyme H is an inefficient cytotoxin that together with their similar pattern of GrB divergence and functional similarity strongly hint to their orthologous relationship. Besides analyzing the substrate specificity profile of granzyme H by substrate phage display, substrate cleavage susceptibility of human granzyme H and mouse granzyme C was assessed on a proteome-wide level. The extended specificity profiles of granzymes C and H (i.e. beyond cleavage positions P4-P4') match those previously observed for granzyme B. We demonstrate conservation of these extended specificity profiles among various granzymes as granzyme B cleavage susceptibility of an otherwise granzyme H/C specific cleavage site can simply be conferred by altering the P1-residue to aspartate, the preferred P1-residue of granzyme B. Our results thus indicate a conserved, but hitherto underappreciated specificity-determining role of extended protease-substrate contacts in steering cleavage susceptibility.


Assuntos
Granzimas/metabolismo , Animais , Linhagem Celular , Granzimas/genética , Humanos , Células K562 , Camundongos , Proteômica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
14.
Proteomics ; 14(17-18): 1990-8, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24737740

RESUMO

The oxidation of free and protein-bound methionine into methionine sulfoxide is a frequently occurring modification caused by ROS. Most organisms express methionine sulfoxide reductases (MSR enzymes) to repair this potentially damaging modification. Humans express three different MSRB enzymes which reside in different cellular compartments. In this study, we have explored the specificity of the human MSRB enzymes both by in silico modeling and by experiments on oxidized peptides. We found that MSRB1 is the least specific MSRB enzyme, which is in agreement with the observation that MSRB1 is the only MSRB enzyme found in the cytosol and the nucleus, and therefore requires a broad specificity to reduce all possible substrates. MSRB2 and MSRB3, which are both found in mitochondria, are more specific but because of their co-occurrence they can likely repair all possible substrates.


Assuntos
Simulação por Computador , Metionina Sulfóxido Redutases/química , Metionina Sulfóxido Redutases/metabolismo , Modelos Moleculares , Humanos , Metionina/química , Metionina/metabolismo , Oxirredução , Especificidade por Substrato
15.
BMC Biochem ; 15: 21, 2014 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-25208769

RESUMO

BACKGROUND: Previous screening of the substrate repertoires and substrate specificity profiles of granzymes resulted in long substrate lists highly likely containing bystander substrates. Here, a recently developed degradomics technology that allows distinguishing efficiently from less efficiently cleaved substrates was applied to study the degradome of mouse granzyme B (mGrB). RESULTS: In vitro kinetic degradome analysis resulted in the identification of 37 mGrB cleavage events, 9 of which could be assigned as efficiently targeted ones. Previously, cleavage at the IEAD75 tetrapeptide motif of Bid was shown to be efficiently and exclusively targeted by human granzyme B (hGrB) and thus not by mGrB. Strikingly, and despite holding an identical P4-P1 human Bid (hBid) cleavage motif, mGrB was shown to efficiently cleave the BCL2/adenovirus E1B 19 kDa protein-interacting protein 2 or BNIP-2 at IEAD28. Like Bid, BNIP-2 represents a pro-apoptotic Bcl-2 protein family member and a potential regulator of GrB induced cell death. Next, in vitro analyses demonstrated the increased efficiency of human and mouse BNIP-2 cleavage by mGrB as compared to hGrB indicative for differing Bid/BNIP-2 substrate traits beyond the P4-P1 IEAD cleavage motif influencing cleavage efficiency. Murinisation of differential primed site residues in hBNIP-2 revealed that, although all contributing, a single mutation at the P3' position was found to significantly increase the mGrB/hGrB cleavage ratio, whereas mutating the P1' position from I29 > T yielded a 4-fold increase in mGrB cleavage efficiency. Finally, mutagenesis analyses revealed the composite BNIP-2 precursor patterns to be the result of alternative translation initiation at near-cognate start sites within the 5' leader sequence (5'UTR) of BNIP-2. CONCLUSIONS: Despite their high sequence similarity, and previously explained by their distinct tetrapeptide specificities observed, the substrate repertoires of mouse and human granzymes B only partially overlap. Here, we show that the substrate sequence context beyond the P4-P1 positions can influence orthologous granzyme B cleavage efficiencies to an unmatched extent. More specifically, in BNIP-2, the identical and hGrB optimal IEAD tetrapeptide substrate motif is targeted highly efficiently by mGrB, while this tetrapeptide motif is refractory towards mGrB cleavage in Bid.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Granzimas/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Animais , Cromatografia Líquida , Humanos , Camundongos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Espectrometria de Massas em Tandem
16.
Mol Cell Proteomics ; 11(8): 255-71, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22448045

RESUMO

Vertebrate nonmuscle cells express two actin isoforms: cytoplasmic ß- and γ-actin. Because of the presence and localized translation of ß-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates ß-actin in gene regulation. Cell migration without ß-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking ß-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, ß-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of ß-actin knockout cells. This also explains why reintroducing ß-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in ß-actin knockout cells based on increased Rho-ROCK signaling and increased TGFß production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of ß-actin knockout cells indicating that other actins compensate for ß-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but ß-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation.


Assuntos
Actinas/metabolismo , Movimento Celular/fisiologia , Fibroblastos/metabolismo , Proteômica/métodos , Actinas/genética , Amidas/farmacologia , Animais , Western Blotting , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Adesão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Knockout , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Pseudópodes/genética , Pseudópodes/metabolismo , Pseudópodes/fisiologia , Piridinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo
17.
Mol Cell Proteomics ; 10(5): M110.006866, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21406390

RESUMO

We here present a new method to measure the degree of protein-bound methionine sulfoxide formation at a proteome-wide scale. In human Jurkat cells that were stressed with hydrogen peroxide, over 2000 oxidation-sensitive methionines in more than 1600 different proteins were mapped and their extent of oxidation was quantified. Meta-analysis of the sequences surrounding the oxidized methionine residues revealed a high preference for neighboring polar residues. Using synthetic methionine sulfoxide containing peptides designed according to the observed sequence preferences in the oxidized Jurkat proteome, we discovered that the substrate specificity of the cellular methionine sulfoxide reductases is a major determinant for the steady-state of methionine oxidation. This was supported by a structural modeling of the MsrA catalytic center. Finally, we applied our method onto a serum proteome from a mouse sepsis model and identified 35 in vivo methionine oxidation events in 27 different proteins.


Assuntos
Metionina/análogos & derivados , Proteoma/química , Motivos de Aminoácidos , Animais , Domínio Catalítico , Cromatografia Líquida de Alta Pressão/métodos , Feminino , Humanos , Peróxido de Hidrogênio/farmacologia , Células Jurkat , Metanálise como Assunto , Metionina/química , Metionina/metabolismo , Metionina Sulfóxido Redutases/química , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Oxidantes/farmacologia , Oxirredução , Estresse Oxidativo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Proteoma/metabolismo , Infecções por Salmonella/sangue , Salmonella enteritidis , Choque Séptico/sangue
18.
Methods Mol Biol ; 2690: 311-334, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37450157

RESUMO

Mapping protein-protein interactions is crucial to understand protein function. Recent advances in proximity-dependent biotinylation (BioID) coupled to mass spectrometry (MS) allow the characterization of protein complexes in diverse plant models. Here, we describe the use of BioID in hairy root cultures of tomato and provide detailed information on how to analyze the data obtained by MS.


Assuntos
Mapeamento de Interação de Proteínas , Proteínas , Biotinilação , Catálise , Mapeamento de Interação de Proteínas/métodos
19.
Cell Death Dis ; 14(4): 282, 2023 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-37080966

RESUMO

Citrobacter rodentium is an enteropathogen that causes intestinal inflammatory responses in mice reminiscent of the pathology provoked by enteropathogenic and enterohemorrhagic Escherichia coli infections in humans. C. rodentium expresses various virulence factors that target specific signaling proteins involved in executing apoptotic, necroptotic and pyroptotic cell death, suggesting that each of these distinct cell death modes performs essential host defense functions that the pathogen aims to disturb. However, the relative contributions of apoptosis, necroptosis and pyroptosis in protecting the host against C. rodentium have not been elucidated. Here we used mice with single or combined deficiencies in essential signaling proteins controlling apoptotic, necroptotic or pyroptotic cell death to reveal the roles of these cell death modes in host defense against C. rodentium. Gastrointestinal C. rodentium infections in mice lacking GSDMD and/or MLKL showed that both pyroptosis and necroptosis were dispensable for pathogen clearance. In contrast, while RIPK3-deficient mice showed normal C. rodentium clearance, mice with combined caspase-8 and RIPK3 deficiencies failed to clear intestinal pathogen loads. Although this demonstrated a crucial role for caspase-8 signaling in establishing intestinal host defense, Casp8-/-Ripk3-/- mice remained capable of preventing systemic pathogen persistence. This systemic host defense relied on inflammasome signaling, as Casp8-/-Ripk3-/- mice with combined caspase-1 and -11 deletion succumbed to C. rodentium infection. Interestingly, although it is known that C. rodentium can activate the non-canonical caspase-11 inflammasome, selectively disabling canonical inflammasome signaling by single caspase-1 deletion sufficed to render Casp8-/-Ripk3-/- mice vulnerable to C. rodentium-induced lethality. Moreover, Casp8-/-Ripk3-/- mice lacking GSDMD survived a C. rodentium infection, suggesting that pyroptosis was not crucial for the protective functions of canonical inflammasomes in these mice. Taken together, our mouse genetic experiments revealed an essential cooperation between caspase-8 signaling and GSDMD-independent canonical inflammasome signaling to establish intestinal and systemic host defense against gastrointestinal C. rodentium infection.


Assuntos
Citrobacter rodentium , Inflamassomos , Animais , Humanos , Camundongos , Caspase 1/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Caspases/metabolismo , Citrobacter rodentium/metabolismo , Gasderminas , Inflamassomos/metabolismo , Camundongos Endogâmicos C57BL
20.
Microlife ; 3: uqac005, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-37223358

RESUMO

Genomic studies of bacteria have long pointed toward widespread prevalence of small open reading frames (sORFs) encoding for short proteins, <100 amino acids in length. Despite the mounting genomic evidence of their robust expression, relatively little progress has been made in their mass spectrometry-based detection and various blanket statements have been used to explain this observed discrepancy. In this study, we provide a large-scale riboproteogenomics investigation of the challenging nature of proteomic detection of such small proteins as informed by conditional translation data. A panel of physiochemical properties alongside recently developed mass spectrometry detectability metrics was interrogated to provide a comprehensive evidence-based assessment of sORF-encoded polypeptide (SEP) detectability. Moreover, a large-scale proteomics and translatomics compendium of proteins produced by Salmonella Typhimurium (S. Typhimurium), a model human pathogen, across a panel of growth conditions is presented and used in support of our in silico SEP detectability analysis. This integrative approach is used to provide a data-driven census of small proteins expressed by S. Typhimurium across growth phases and infection-relevant conditions. Taken together, our study pinpoints current limitations in proteomics-based detection of novel small proteins currently missing from bacterial genome annotations.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA