RESUMO
Proteasome is essential for cell survival, and proteasome inhibition induces proteasomal gene transcription via the activated endoplasmic-reticulum-associated transcription factor nuclear factor erythroid 2-like 1 (Nrf1/NFE2L1). Nrf1 activation requires proteolytic cleavage by DDI2 and N-glycan removal by NGLY1. We previously showed that Nrf1 ubiquitination by SKP1-CUL1-F-box (SCF)FBS2/FBXO6, an N-glycan-recognizing E3 ubiquitin ligase, impairs its activation, although the molecular mechanism remained elusive. Here, we show that SCFFBS2 cooperates with the RING-between-RING (RBR)-type E3 ligase ARIH1 to ubiquitinate Nrf1 through oxyester bonds in human cells. Endo-ß-N-acetylglucosaminidase (ENGASE) generates asparagine-linked N-acetyl glucosamine (N-GlcNAc) residues from N-glycans, and N-GlcNAc residues on Nrf1 served as acceptor sites for SCFFBS2-ARIH1-mediated ubiquitination. We reconstituted the polyubiquitination of N-GlcNAc and serine/threonine residues on glycopeptides and found that the RBR-specific E2 enzyme UBE2L3 is required for the assembly of atypical ubiquitin chains on Nrf1. The atypical ubiquitin chains inhibited DDI2-mediated activation. The present results identify an unconventional ubiquitination pathway that inhibits Nrf1 activation.
RESUMO
K63-linked ubiquitin chains attached to plasma membrane proteins serve as tags for endocytosis and endosome-to-lysosome sorting. USP8 is an essential deubiquitinase for the maintenance of endosomal functions. Prolonged depletion of USP8 leads to cell death, but the major effects on cellular signaling pathways are poorly understood. Here, we show that USP8 depletion causes aberrant accumulation of K63-linked ubiquitin chains on endosomes and induces immune and stress responses. Upon USP8 depletion, two different decoders for K63-linked ubiquitin chains, TAB2/3 and p62, were recruited to endosomes and activated the TAK1-NF-κB and Keap1-Nrf2 pathways, respectively. Oxidative stress, an environmental stimulus that potentially suppresses USP8 activity, induced accumulation of K63-linked ubiquitin chains on endosomes, recruitment of TAB2, and expression of the inflammatory cytokine. The results demonstrate that USP8 is a gatekeeper of misdirected ubiquitin signals and inhibits immune and stress response pathways by removing K63-linked ubiquitin chains from endosomes.
Assuntos
Fator 2 Relacionado a NF-E2 , NF-kappa B , Ubiquitina Tiolesterase , Endossomos/genética , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/genética , NF-kappa B/genética , Ubiquitina/genética , Humanos , Ubiquitina Tiolesterase/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genéticaRESUMO
The HECT-type ubiquitin E3 ligases including ITCH regulate many aspects of cellular function through ubiquitinating various substrates. These ligases are known to be allosterically autoinhibited and to require an activator protein to fully achieve the ubiquitination of their substrates. Here we demonstrate that FAM189A2, a downregulated gene in breast cancer, encodes a new type of ITCH activator. FAM189A2 is a transmembrane protein harboring PPxY motifs, and the motifs mediate its association with and ubiquitination by ITCH. FAM189A2 also associates with Epsin and accumulates in early and late endosomes along with ITCH. Intriguingly, FAM189A2 facilitates the association of a chemokine receptor CXCR4 with ITCH and enhances ITCH-mediated ubiquitination of CXCR4. FAM189A2-knockout prohibits CXCL12-induced endocytosis of CXCR4, thereby enhancing the effects of CXCL12 on the chemotaxis and mammosphere formation of breast cancer cells. In comparison to other activators or adaptors known in the previous studies, FAM189A2 is a unique activator for ITCH to desensitize CXCR4 activity, and we here propose that FAM189A2 be renamed as ENdosomal TRansmembrane binding with EPsin (ENTREP).
Assuntos
Neoplasias da Mama , Proteínas Repressoras , Ubiquitina-Proteína Ligases , Neoplasias da Mama/genética , Quimiocina CXCL12 , Feminino , Técnicas de Inativação de Genes , Humanos , Receptores CXCR4 , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
The 26S proteasome is a 2.5-MDa protease complex responsible for the selective and ATP-dependent degradation of ubiquitylated proteins in eukaryotic cells. Proteasome-mediated protein degradation accounts for ~70% of all cellular proteolysis under basal conditions, and thereby any dysfunction can lead to drastic changes in cell homeostasis. A major function of ubiquitylation is to target proteins for proteasomal degradation. Accompanied by deciphering the structural diversity of ubiquitin chains with eight linkages and chain lengths, the ubiquitin code for proteasomal degradation has been expanding beyond the best-characterized Lys48-linked ubiquitin chains. Whereas polyubiquitylated proteins can be directly recognized by the proteasome, in several cases, these proteins need to be extracted or segregated by the conserved ATPases associated with diverse cellular activities (AAA)-family ATPase p97/valosin-containing protein (VCP) complex and escorted to the proteasome by ubiquitin-like (UBL)-ubiquitin associated (UBA) proteins; these are called substrate-shuttling factors. Furthermore, proteasomes are highly mobile and are appropriately spatiotemporally regulated in response to different cellular environments and stresses. In this review, we highlight an emerging key link between p97, shuttling factors, and proteasome for efficient proteasomal degradation. We also present evidence that proteasome-containing nuclear foci form by liquid-liquid phase separation under acute hyperosmotic stress.
RESUMO
The proteasome is a major proteolytic machine that regulates cellular proteostasis through selective degradation of ubiquitylated proteins1,2. A number of ubiquitin-related molecules have recently been found to be involved in the regulation of biomolecular condensates or membraneless organelles, which arise by liquid-liquid phase separation of specific biomolecules, including stress granules, nuclear speckles and autophagosomes3-8, but it remains unclear whether the proteasome also participates in such regulation. Here we reveal that proteasome-containing nuclear foci form under acute hyperosmotic stress. These foci are transient structures that contain ubiquitylated proteins, p97 (also known as valosin-containing protein (VCP)) and multiple proteasome-interacting proteins, which collectively constitute a proteolytic centre. The major substrates for degradation by these foci were ribosomal proteins that failed to properly assemble. Notably, the proteasome foci exhibited properties of liquid droplets. RAD23B, a substrate-shuttling factor for the proteasome, and ubiquitylated proteins were necessary for formation of proteasome foci. In mechanistic terms, a liquid-liquid phase separation was triggered by multivalent interactions of two ubiquitin-associated domains of RAD23B and ubiquitin chains consisting of four or more ubiquitin molecules. Collectively, our results suggest that ubiquitin-chain-dependent phase separation induces the formation of a nuclear proteolytic compartment that promotes proteasomal degradation.
Assuntos
Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Estresse Fisiológico , Ubiquitinação , Linhagem Celular , Núcleo Celular/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Pressão Osmótica , Poliubiquitina/metabolismo , Proteólise , Proteostase , Proteínas Ribossômicas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína com Valosina/metabolismoRESUMO
Npl4 is likely to be the most upstream factor recognizing Lys48-linked polyubiquitylated substrates in the proteasomal degradation pathway in yeast. Along with Ufd1, Npl4 forms a heterodimer (UN), and functions as a cofactor for the Cdc48 ATPase. Here, we report the crystal structures of yeast Npl4 in complex with Lys48-linked diubiquitin and with the Npl4-binding motif of Ufd1. The distal and proximal ubiquitin moieties of Lys48-linked diubiquitin primarily interact with the C-terminal helix and N-terminal loop of the Npl4 C-terminal domain (CTD), respectively. Mutational analysis suggests that the CTD contributes to linkage selectivity and initial binding of ubiquitin chains. Ufd1 occupies a hydrophobic groove of the Mpr1/Pad1 N-terminal (MPN) domain of Npl4, which corresponds to the catalytic groove of the MPN domain of JAB1/MPN/Mov34 metalloenzyme (JAMM)-family deubiquitylating enzyme. This study provides important structural insights into the polyubiquitin chain recognition by the Cdc48-UN complex and its assembly.
Assuntos
Proteínas de Transporte Nucleocitoplasmático/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Ubiquitina/ultraestrutura , Proteínas de Transporte Vesicular/ultraestrutura , Cristalografia por Raios X , Proteínas de Transporte Nucleocitoplasmático/isolamento & purificação , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Proteína com Valosina/metabolismo , Proteínas de Transporte Vesicular/isolamento & purificação , Proteínas de Transporte Vesicular/metabolismoRESUMO
To understand the biological roles of different ubiquitin chains, it is important to determine the types of ubiquitin linkages, the lengths of the polymers, and the combinations of ubiquitin chains attached to substrates. In this chapter, we describe a mass spectrometry-based quantification method of ubiquitin chains, named Ub-AQUA/PRM (ubiquitin-absolute quantification/parallel reaction monitoring), for direct and highly sensitive measurement of the stoichiometry of all eight ubiquitin-ubiquitin linkage types simultaneously. We also show a method to quantify the K48/K63 branched ubiquitin chain, a recently identified ubiquitin signal with a complex topology. In addition, we describe a method to measure ubiquitin chain length of ubiquitylated substrates using a chain protector and limited digestion of ubiquitin chains, named Ub-ProT (ubiquitin chain protection from trypsinization). These strategies will contribute to our knowledge of polymeric ubiquitin signals and permit investigation of new mechanisms concerning the ubiquitin code.
Assuntos
Peptídeos/análise , Ubiquitina/análise , Sequência de Aminoácidos , Animais , Humanos , Espectrometria de Massas/métodos , Proteômica/métodos , UbiquitinaçãoRESUMO
Ubiquitination is a transient posttranslational modification; polyubiquitin chains are removed from proteins by deubiquitinating enzymes (DUBs) and many ubiquitinated proteins are degraded by the proteasome. Exogenously expressed trypsin-resistant tandem ubiquitin-binding entity (TR-TUBE) protects polyubiquitin chains from DUBs and inhibits proteasomal degradation in cells. TR-TUBE effectively binds to substrates ubiquitinated by an exogenously expressed ubiquitin ligase, and enables detection of the specific activity of a given ubiquitin ligase and isolation of its substrates. In this chapter, we describe methods for the detection of ubiquitin ligase activity as well as the identification of substrates of a given ubiquitin ligase using two enrichment tools, TR-TUBE and anti-diGly antibody, coupled with mass spectrometry (MS).
Assuntos
Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo , Animais , Enzimas Desubiquitinantes/metabolismo , Ensaios Enzimáticos/métodos , Humanos , Poliubiquitina/metabolismo , Especificidade por Substrato , Tripsina/metabolismo , UbiquitinaçãoRESUMO
Protein ubiquitylation regulates diverse cellular processes via distinct ubiquitin chains that differ by linkage type and length. However, a comprehensive method for measuring these properties has not been developed. Here we describe a method for assessing the length of substrate-attached polyubiquitin chains, "ubiquitin chain protection from trypsinization (Ub-ProT)." Using Ub-ProT, we found that most ubiquitylated substrates in yeast-soluble lysate are attached to chains of up to seven ubiquitin molecules. Inactivation of the ubiquitin-selective chaperone Cdc48 caused a dramatic increase in chain lengths on substrate proteins, suggesting that Cdc48 complex terminates chain elongation by substrate extraction. In mammalian cells, we found that ligand-activated epidermal growth factor receptor (EGFR) is rapidly modified with K63-linked tetra- to hexa-ubiquitin chains following EGF treatment in human cells. Thus, the Ub-ProT method can contribute to our understanding of mechanisms regulating physiological ubiquitin chain lengths and composition.
Assuntos
Receptores ErbB/metabolismo , Poliubiquitina/análise , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitinação , Proteína com Valosina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Células HeLa , Humanos , Leupeptinas/farmacologia , Poliubiquitina/química , Poliubiquitina/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/química , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo , Proteína com Valosina/genéticaRESUMO
Different polyubiquitin chain linkages direct substrates toward distinct cellular pathways. K63-linked ubiquitylation is known to regulate proteasome-independent events such as signal transduction, but its function in the context of heterogeneous ubiquitin chains remains unclear. Here, we report that K63 ubiquitylation plays a critical role in proteasome-mediated substrate degradation by serving as a "seed" for K48/K63 branched ubiquitin chains. Quantitative analysis revealed that K48/K63 branched linkages preferentially associate with proteasomes in cells. We found that ITCH-dependent K63 ubiquitylation of the proapoptotic regulator TXNIP triggered subsequent assembly of K48/K63 branched chains by recruiting ubiquitin-interacting ligases such as UBR5, leading to TXNIP degradation. These results reveal a role for K63 chains as a substrate-specific mark for proteasomal degradation involved in regulating cell fate. Our findings provide insight into how cellular interpretation of the ubiquitin code is altered by combinations of ubiquitin linkages.
Assuntos
Proteínas de Transporte/metabolismo , Lisina/metabolismo , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Células HeLa , Humanos , Proteólise , Transdução de SinaisRESUMO
[Purpose] The fingernails allow for increased sensory perception at the finger pulp, and contribute to the accurate picking up of small objects. The purpose of the present study was to clarify the effect of fingernail length on hand dexterity using subjects' own fingernails. [Subjects and Methods] The hand sizes and fingernail configurations of 38 young healthy volunteers (eighteen males and twenty females) were measured. The effect of fingernail length (0 and 2â mm) on hand dexterity also was investigated using the simple test for evaluating hand function. [Results] The hand and finger sizes as well as fingernail widths were significantly larger in males than in females. The time taken for each subtest of the simple test for evaluating hand function was generally shorter at a fingernail length of 2â mm than at 0 mm, and it was significantly shorter for a number of subtests. There was little significant difference in the time taken for the subtests between genders. [Conclusion] It was clear that a fingernail length of 2â mm had an advantageous effect on hand dexterity, with little gender difference observed. These findings suggest that the fingernail lengths of the subjects should be standardized when evaluating changes in their hand dexterity with time.
RESUMO
Translation arrest by polybasic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control (RQC) system. Here we report that ubiquitination of the 40S ribosomal protein uS10 by the E3 ubiquitin ligase Hel2 (or RQT1) is required for RQC. We identify a RQC-trigger (RQT) subcomplex composed of the RNA helicase-family protein Slh1/Rqt2, the ubiquitin-binding protein Cue3/Rqt3, and yKR023W/Rqt4 that is required for RQC. The defects in RQC of the RQT mutants correlate with sensitivity to anisomycin, which stalls ribosome at the rotated form. Cryo-electron microscopy analysis reveals that Hel2-bound ribosome are dominantly the rotated form with hybrid tRNAs. Ribosome profiling reveals that ribosomes stalled at the rotated state with specific pairs of codons at P-A sites serve as RQC substrates. Rqt1 specifically ubiquitinates these arrested ribosomes to target them to the RQT complex, allowing subsequent RQC reactions including dissociation of the stalled ribosome into subunits.Several protein quality control mechanisms are in place to trigger the rapid degradation of aberrant polypeptides and mRNAs. Here the authors describe a mechanism of ribosome-mediated quality control that involves the ubiquitination of ribosomal proteins by the E3 ubiquitin ligase Hel2/RQT1.
Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , Ribossomos/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Células HEK293 , Humanos , Mutação , Biossíntese de Proteínas , Conformação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , UbiquitinaçãoRESUMO
Ubiquitin-binding domain (UBD) proteins regulate numerous cellular processes, but their specificities toward ubiquitin chain types in cells remain obscure. Here, we perform a quantitative proteomic analysis of ubiquitin linkage-type selectivity of 14 UBD proteins and the proteasome in yeast. We find that K48-linked chains are directed to proteasomal degradation through selectivity of the Cdc48 cofactor Npl4. Mutating Cdc48 results in decreased selectivity, and lacking Rad23/Dsk2 abolishes interactions between ubiquitylated substrates and the proteasome. Among them, only Npl4 has K48 chain specificity in vitro. Thus, the Cdc48 complex functions as a K48 linkage-specifying factor upstream of Rad23/Dsk2 for proteasomal degradation. On the other hand, K63 chains are utilized in endocytic pathways, whereas both K48 and K63 chains are found in the MVB and autophagic pathways. Collectively, our results provide an overall picture of the ubiquitin network via UBD proteins and identify the Cdc48-Rad23/Dsk2 axis as a major route to the proteasome.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Ubiquitina/metabolismo , Ubiquitinação , Ubiquitinas/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteassoma/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteólise , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Tempo , Ubiquitinação/efeitos dos fármacos , Ubiquitinas/genética , Proteína com ValosinaRESUMO
Ubiquitylation is an essential post-translational modification (PTM) of proteins with diverse cellular functions. Polyubiquitin chains with different topologies have different cellular roles, and are referred to as a 'ubiquitin code'. Recent studies have begun to reveal that more complex ubiquitin architectures function as important signals in several biological pathways. These include PTMs of ubiquitin itself, such as acetylated ubiquitin and phospho-ubiquitin. Moreover, important roles for heterogeneous polyubiquitin chains, such as mixed or branched chains, have been reported, which significantly increase the diversity of the ubiquitin code. In this review, we describe mass spectrometry-based methods to characterize the ubiquitin signal. We also describe recent advances in our understanding of complex ubiquitin architectures, including our own findings concerning ubiquitin acetylation and branching within polyubiquitin chains.
Assuntos
Processamento de Proteína Pós-Traducional , Transdução de Sinais , Ubiquitina/metabolismo , Ubiquitinação , Acetilação , Humanos , Espectrometria de Massas/métodos , Modelos Biológicos , Fosforilação , PoliubiquitinaRESUMO
The identification of substrates for ubiquitin ligases has remained challenging, because most substrates are either immediately degraded by the proteasome or processed by deubiquitinating enzymes (DUBs) to remove polyubiquitin. Although a methodology that enables detection of ubiquitinated proteins using ubiquitin Lys-ε-Gly-Gly (diGly) remnant antibodies and MS has been developed, it is still insufficient for identification and characterization of the ubiquitin-modified proteome in cells overexpressing a particular ubiquitin ligase. Here, we show that exogenously expressed trypsin-resistant tandem ubiquitin-binding entity(ies) (TR-TUBE) protect polyubiquitin chains on substrates from DUBs and circumvent proteasome-mediated degradation in cells. TR-TUBE effectively associated with substrates ubiquitinated by an exogenously overexpressed ubiquitin ligase, allowing detection of the specific activity of the ubiquitin ligase and isolation of its substrates. Although the diGly antibody enabled effective identification of ubiquitinated proteins in cells, overexpression of an ubiquitin ligase and treatment with a proteasome inhibitor did not increase the level of diGly peptides specific for the ligase relative to the background level of diGly peptides, probably due to deubiquitination. By contrast, in TR-TUBE-expressing cells, the level of substrate-derived diGly peptides produced by the overexpressed ubiquitin ligase was significantly elevated. We developed a method for identifying the substrates of specific ubiquitin ligases using two enrichment strategies, TR-TUBE and diGly remnant antibodies, coupled with MS. Using this method, we identified target substrates of FBXO21, an uncharacterized F-box protein.
Assuntos
Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação , Sequência de Aminoácidos , Anticorpos/imunologia , Sequência de Bases , Proteínas F-Box/genética , Proteínas F-Box/imunologia , Proteínas F-Box/metabolismo , Glicina/genética , Glicina/metabolismo , Células HEK293 , Humanos , Immunoblotting/métodos , Imunoprecipitação/métodos , Lisina/genética , Lisina/metabolismo , Dados de Sequência Molecular , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Oligopeptídeos/metabolismo , Ligação Proteica/imunologia , Proteoma/genética , Proteoma/imunologia , Proteoma/metabolismo , Proteômica , Reprodutibilidade dos Testes , Especificidade por Substrato , Espectrometria de Massas em Tandem/métodos , Tripsina/genética , Tripsina/metabolismo , Ubiquitina/genética , Ubiquitina/imunologia , Ubiquitina-Proteína Ligases/genética , Proteínas Ubiquitinadas/genéticaRESUMO
Ubiquitin is known to be one of the most soluble and stably folded intracellular proteins, but it is often found in inclusion bodies associated with various diseases including neurodegenerative disorders and cancer. To gain insight into this contradictory behaviour, we have examined the physicochemical properties of ubiquitin and its polymeric chains that lead to aggregate formation. We find that the folding stability of ubiquitin chains unexpectedly decreases with increasing chain length, resulting in the formation of amyloid-like fibrils. Furthermore, when expressed in cells, polyubiquitin chains covalently linked to EGFP also form aggregates depending on chain length. Notably, these aggregates are selectively degraded by autophagy. We propose a novel model in which the physical and chemical instability of polyubiquitin chains drives the formation of fibrils, which then serve as an initiation signal for autophagy.
Assuntos
Poliubiquitina/química , Poliubiquitina/metabolismo , Animais , Autofagia , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Escherichia coli/metabolismo , Imuno-Histoquímica , Camundongos , Microscopia Eletrônica de Transmissão , Poliubiquitina/ultraestrutura , Células Sf9 , Espectrometria de Fluorescência , Ubiquitina/química , Ubiquitina/metabolismoRESUMO
Ubiquitylation is a versatile post-translational modification (PTM). The diversity of ubiquitylation topologies, which encompasses different chain lengths and linkages, underlies its widespread cellular roles. Here, we show that endogenous ubiquitin is acetylated at lysine (K)-6 (AcK6) or K48. Acetylated ubiquitin does not affect substrate monoubiquitylation, but inhibits K11-, K48-, and K63-linked polyubiquitin chain elongation by several E2 enzymes in vitro. In cells, AcK6-mimetic ubiquitin stabilizes the monoubiquitylation of histone H2B-which we identify as an endogenous substrate of acetylated ubiquitin-and of artificial ubiquitin fusion degradation substrates. These results characterize a mechanism whereby ubiquitin, itself a PTM, is subject to another PTM to modulate mono- and polyubiquitylation, thus adding a new regulatory layer to ubiquitin biology.
Assuntos
Poliubiquitina/metabolismo , Ubiquitina/metabolismo , Acetilação , Humanos , Processamento de Proteína Pós-Traducional , UbiquitinaçãoRESUMO
PINK1 (PTEN induced putative kinase 1) and PARKIN (also known as PARK2) have been identified as the causal genes responsible for hereditary recessive early-onset Parkinsonism. PINK1 is a Ser/Thr kinase that specifically accumulates on depolarized mitochondria, whereas parkin is an E3 ubiquitin ligase that catalyses ubiquitin transfer to mitochondrial substrates. PINK1 acts as an upstream factor for parkin and is essential both for the activation of latent E3 parkin activity and for recruiting parkin onto depolarized mitochondria. Recently, mechanistic insights into mitochondrial quality control mediated by PINK1 and parkin have been revealed, and PINK1-dependent phosphorylation of parkin has been reported. However, the requirement of PINK1 for parkin activation was not bypassed by phosphomimetic parkin mutation, and how PINK1 accelerates the E3 activity of parkin on damaged mitochondria is still obscure. Here we report that ubiquitin is the genuine substrate of PINK1. PINK1 phosphorylated ubiquitin at Ser 65 both in vitro and in cells, and a Ser 65 phosphopeptide derived from endogenous ubiquitin was only detected in cells in the presence of PINK1 and following a decrease in mitochondrial membrane potential. Unexpectedly, phosphomimetic ubiquitin bypassed PINK1-dependent activation of a phosphomimetic parkin mutant in cells. Furthermore, phosphomimetic ubiquitin accelerates discharge of the thioester conjugate formed by UBCH7 (also known as UBE2L3) and ubiquitin (UBCH7â¼ubiquitin) in the presence of parkin in vitro, indicating that it acts allosterically. The phosphorylation-dependent interaction between ubiquitin and parkin suggests that phosphorylated ubiquitin unlocks autoinhibition of the catalytic cysteine. Our results show that PINK1-dependent phosphorylation of both parkin and ubiquitin is sufficient for full activation of parkin E3 activity. These findings demonstrate that phosphorylated ubiquitin is a parkin activator.
Assuntos
Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Animais , Ativação Enzimática , Fibroblastos , Células HeLa , Humanos , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias/metabolismo , Mutação/genética , Doença de Parkinson , Fosforilação , Fosfosserina/metabolismo , Ubiquitina/química , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
The 26S proteasome is a 2.5-MDa multisubunit protease complex that degrades polyubiquitylated proteins. Although its functions and structure have been extensively characterized, little is known about its dynamics in living cells. Here, we investigate the absolute concentration, spatio-temporal dynamics and complex formation of the proteasome in living cells using fluorescence correlation spectroscopy. We find that the 26S proteasome complex is highly mobile, and that almost all proteasome subunits throughout the cell are stably incorporated into 26S proteasomes. The interaction between 19S and 20S particles is stable even in an importin-α mutant, suggesting that the 26S proteasome is assembled in the cytoplasm. Furthermore, a genetically stabilized 26S proteasome mutant is able to enter the nucleus. These results suggest that the 26S proteasome completes its assembly process in the cytoplasm and translocates into the nucleus through the nuclear pore complex as a holoenzyme.
Assuntos
Citoplasma/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Microscopia Crioeletrônica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Mutação , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Fatores de Tempo , Imagem com Lapso de Tempo/métodos , Proteína Vermelha FluorescenteRESUMO
Gene expression is controlled by alterations in the epigenome, including DNA methylation and histone modification. Recently, it was reported that 5-methylcytosine (5mC) is converted to 5-hydroxymethylcytosine (5hmC) by proteins in the ten-eleven translocation (TET) family. This conversion is believed to be part of the mechanism by which methylated DNA is demethylated. Moreover, histones undergo modifications such as phosphorylation and acetylation. In addition, modification with O-linked-N-acetylglucosamine (O-GlcNAc) by O-GlcNAc transferase (OGT) was recently identified as a novel histone modification. Herein, we focused on TET3, the regulation of which is still unclear. We attempted to elucidate the mechanism of its regulation by biochemical approaches. First, we conducted mass spectrometric analysis in combination with affinity purification of FLAG-TET3, which identified OGT as an important partner of TET3. Co-immunoprecipitation assays using a series of deletion mutants showed that the C-terminal H domain of TET3 was required for its interaction with OGT. Furthermore, we showed that TET3 is GlcNAcylated by OGT, although the GlcNAcylation did not affect the global hydroxylation of methylcytosine by TET3. Moreover, we showed that TET3 enhanced its localization to chromatin through the stabilization of OGT protein. Taken together, we showed a novel function of TET3 that likely supports the function of OGT.