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1.
Structure ; 30(9): 1221-1223, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36055221

RESUMO

Capturing the enzymatic activity of RBR ligases in molecular detail is challenging due to their inherent dynamic behavior. In this issue of Structure, Reiter and colleagues tackle this problem using a multidisciplinary approach. They show that activation of the ubiquitin ligase HHARI by phosphorylation induces a major conformational rearrangement, which reveals an unexpected substrate binding site.


Assuntos
Ligases , Ubiquitina-Proteína Ligases , Ligases/metabolismo , Modelos Moleculares , Conformação Proteica , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química
2.
Annu Rev Microbiol ; 76: 211-233, 2022 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-36075095

RESUMO

Ubiquitination is a posttranslational modification that regulates a multitude of cellular functions. Pathogens, such as bacteria and viruses, have evolved sophisticated mechanisms that evade or counteract ubiquitin-dependent host responses, or even exploit the ubiquitin system to their own advantage. This is largely done by numerous pathogen virulence factors that encode E3 ligases and deubiquitinases, which are often used as weapons in pathogen-host cell interactions. Moreover, upon pathogen attack, host cellular signaling networks undergo major ubiquitin-dependent changes to protect the host cell, including coordination of innate immunity, remodeling of cellular organelles, reorganization of the cytoskeleton, and reprogramming of metabolic pathways to restrict growth of the pathogen. Here we provide mechanistic insights into ubiquitin regulation of host-pathogen interactions and how it affects bacterial and viral pathogenesis and the organization and response of the host cell.


Assuntos
Interações Hospedeiro-Patógeno , Ubiquitina , Bactérias/metabolismo , Imunidade Inata , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Fatores de Virulência/metabolismo
3.
Int J Mol Sci ; 23(17)2022 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-36077009

RESUMO

Chloroplasts are ancient organelles responsible for photosynthesis and various biosynthetic functions essential to most life on Earth. Many of these functions require tightly controlled regulatory processes to maintain homeostasis at the protein level. One such regulatory mechanism is the ubiquitin-proteasome system whose fundamental role is increasingly emerging in chloroplasts. In particular, the role of E3 ubiquitin ligases as determinants in the ubiquitination and degradation of specific intra-chloroplast proteins. Here, we highlight recent advances in understanding the roles of plant E3 ubiquitin ligases SP1, COP1, PUB4, CHIP, and TT3.1 as well as the ubiquitin-dependent segregase CDC48 in chloroplast function.


Assuntos
Cloroplastos , Ubiquitina-Proteína Ligases , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
4.
Int J Mol Sci ; 23(17)2022 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-36077022

RESUMO

DNA damage in early-stage embryos impacts development and is a risk factor for segregation of altered genomes. DNA damage response (DDR) encompasses a sophisticated network of proteins involved in sensing, signaling, and repairing damage. DDR is regulated by reversible post-translational modifications including acetylation, methylation, phosphorylation, ubiquitylation, and SUMOylation. While important regulators of these processes have been characterized in somatic cells, their roles in early-stage embryos remain broadly unknown. The objective of this study was to explore how ubiquitylation and SUMOylation are involved in the regulation of early development in porcine embryos by assessing the mRNA profile of genes encoding ubiquitination (UBs), deubiquitination (DUBs), SUMOylation (SUMOs) or deSUMOylation (deSUMOs) enzymes in oocyte and embryos at different stages of development, and to evaluate if the induction of DNA damage at different stages of embryo development would alter the mRNA abundance of these genes. Pig embryos were produced by in vitro fertilization and DNA damage was induced by ultraviolet (UV) light exposure for 10 s on days 2, 4 or 7 of development. The relative mRNA abundance of most UBs, DUBs, SUMOs, and deSUMOs was higher in oocytes and early-stage embryos than in blastocysts. Transcript levels for UBs (RNF20, RNF40, RNF114, RNF169, CUL5, DCAF2, DECAF13, and DDB1), DUBs (USP16), and SUMOs (CBX4, UBA2 and UBC9), were upregulated in early-stage embryos (D2 and/or D4) compared to oocytes and blastocysts. In response to UV-induced DNA damage, transcript levels of several UBs, DUBs, SUMOs, and deSUMOs decreased in D2 and D4 embryos, but increased in blastocysts. These findings revealed that transcript levels of genes encoding for important UBs, DUBs, SUMOs, and deSUMOs are regulated during early embryo development and are modulated in response to induced DNA damage. This study has also identified candidate genes controlling post-translational modifications that may have relevant roles in the regulation of normal embryo development, repair of damaged DNA, and preservation of genome stability in the pig embryo.


Assuntos
Blastocisto , Ubiquitina , Animais , Blastocisto/metabolismo , Dano ao DNA , Desenvolvimento Embrionário/genética , Oócitos/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Suínos , Ubiquitina/metabolismo
5.
Nat Commun ; 13(1): 5435, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36114200

RESUMO

Covalent attachment of ubiquitin (Ub) to proteins is a highly versatile posttranslational modification. Moreover, Ub is not only a modifier but itself is modified by phosphorylation and lysine acetylation. However, the functional consequences of Ub acetylation are poorly understood. By generation and comprehensive characterization of all seven possible mono-acetylated Ub variants, we show that each acetylation site has a particular impact on Ub structure. This is reflected in selective usage of the acetylated variants by different E3 ligases and overlapping but distinct interactomes, linking different acetylated variants to different cellular pathways. Notably, not only electrostatic but also steric effects contribute to acetylation-induced changes in Ub structure and, thus, function. Finally, we provide evidence that p300 acts as a position-specific Ub acetyltransferase and HDAC6 as a general Ub deacetylase. Our findings provide intimate insights into the structural and functional consequences of Ub acetylation and highlight the general importance of Ub acetylation.


Assuntos
Lisina , Ubiquitina , Acetilação , Acetiltransferases/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Eletricidade Estática , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
6.
Proc Natl Acad Sci U S A ; 119(38): e2205691119, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36095189

RESUMO

The human Mixed Lineage Leukemia-1 (MLL1) complex methylates histone H3K4 to promote transcription and is stimulated by monoubiquitination of histone H2B. Recent structures of the MLL1-WRAD core complex, which comprises the MLL1 methyltransferase, WDR5, RbBp5, Ash2L, and DPY-30, have revealed variability in the docking of MLL1-WRAD on nucleosomes. In addition, portions of the Ash2L structure and the position of DPY30 remain ambiguous. We used an integrated approach combining cryoelectron microscopy (cryo-EM) and mass spectrometry cross-linking to determine a structure of the MLL1-WRAD complex bound to ubiquitinated nucleosomes. The resulting model contains the Ash2L intrinsically disordered region (IDR), SPRY insertion region, Sdc1-DPY30 interacting region (SDI-motif), and the DPY30 dimer. We also resolved three additional states of MLL1-WRAD lacking one or more subunits, which may reflect different steps in the assembly of MLL1-WRAD. The docking of subunits in all four states differs from structures of MLL1-WRAD bound to unmodified nucleosomes, suggesting that H2B-ubiquitin favors assembly of the active complex. Our results provide a more complete picture of MLL1-WRAD and the role of ubiquitin in promoting formation of the active methyltransferase complex.


Assuntos
Leucemia , Nucleossomos , Microscopia Crioeletrônica , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Ubiquitina/metabolismo
7.
Int J Mol Sci ; 23(17)2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36077334

RESUMO

Protein ubiquitination is a precisely controlled enzymatic cascade reaction belonging to the post-translational modification of proteins. In this process, E3 ligases catalyze the binding of ubiquitin (Ub) to protein substrates and define specificity. The neuronally expressed developmentally down-regulated 4 (NEDD4) subfamily, belonging to the homology to E6APC terminus (HECT) class of E3 ligases, has recently emerged as an essential determinant of multiple cellular processes in different tissues, including bone and tooth. Here, we place special emphasis on the regulatory role of the NEDD4 subfamily in the molecular and cell biology of osteogenesis. We elucidate in detail the specific roles, downstream substrates, and upstream regulatory mechanisms of the NEDD4 subfamily. Further, we provide an overview of the involvement of E3 ligases and deubiquitinases in the development, repair, and regeneration of another mineralized tissue-tooth.


Assuntos
Ubiquitina-Proteína Ligases , Ubiquitina , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Estrutura Terciária de Proteína , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
8.
Cells ; 11(17)2022 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-36078118

RESUMO

The modification of proteins by small ubiquitin-related modifier (SUMO) molecules, SUMOylation, is a key post-translational modification involved in a variety of biological processes, such as chromosome organization, DNA replication and repair, transcription, nuclear transport, and cell signaling transduction. In recent years, emerging evidence has shown that SUMOylation regulates the development and homeostasis of the skeletal system, with its dysregulation causing skeletal diseases, suggesting that SUMOylation pathways may serve as a promising therapeutic target. In this review, we summarize the current understanding of the molecular mechanisms by which SUMOylation pathways regulate skeletal cells in physiological and disease contexts.


Assuntos
Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina , Sumoilação , Homeostase , Processamento de Proteína Pós-Traducional , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitina/metabolismo
9.
Cell Rep ; 40(11): 111349, 2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-36103833

RESUMO

Macroautophagy is a bulk degradation system in which double membrane-bound structures called autophagosomes to deliver cytosolic materials to lysosomes. Autophagy promotes cellular homeostasis by selectively recognizing and sequestering specific targets, such as damaged organelles, protein aggregates, and invading bacteria, termed selective autophagy. We previously reported a type of selective autophagy, lysophagy, which helps clear damaged lysosomes. Damaged lysosomes become ubiquitinated and recruit autophagic machinery. Proteomic studies using transfection reagent-coated beads and further evaluations reveal that a CUL4A-DDB1-WDFY1 E3 ubiquitin ligase complex is essential to initiate lysophagy and clear damaged lysosomes. Moreover, we show that LAMP2 is ubiquitinated by the CUL4A E3 ligase complex as a substrate on damaged lysosomes. These results reveal how cells selectively tag damaged lysosomes to initiate autophagy for the clearance of lysosomes.


Assuntos
Macroautofagia , Proteômica , Lisossomos/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
10.
Cell Mol Life Sci ; 79(9): 484, 2022 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-35974206

RESUMO

Ubiquitin is a small, globular protein that is conjugated to other proteins as a posttranslational event. A palette of small, folded domains recognizes and binds ubiquitin to translate and effectuate this posttranslational signal. Recent computational studies have suggested that protein regions can recognize ubiquitin via a process of folding upon binding. Using peptide binding arrays, bioinformatics, and NMR spectroscopy, we have uncovered a disordered ubiquitin-binding motif that likely remains disordered when bound and thus expands the palette of ubiquitin-binding proteins. We term this motif Disordered Ubiquitin-Binding Motif (DisUBM) and find it to be present in many proteins with known or predicted functions in degradation and transcription. We decompose the determinants of the motif showing it to rely on features of aromatic and negatively charged residues, and less so on distinct sequence positions in line with its disordered nature. We show that the affinity of the motif is low and moldable by the surrounding disordered chain, allowing for an enhanced interaction surface with ubiquitin, whereby the affinity increases ~ tenfold. Further affinity optimization using peptide arrays pushed the affinity into the low micromolar range, but compromised context dependence. Finally, we find that DisUBMs can emerge from unbiased screening of randomized peptide libraries, featuring in de novo cyclic peptides selected to bind ubiquitin chains. We suggest that naturally occurring DisUBMs can recognize ubiquitin as a posttranslational signal to act as affinity enhancers in IDPs that bind to folded and ubiquitylated binding partners.


Assuntos
Proteínas Intrinsicamente Desordenadas , Proteínas , Sequência de Aminoácidos , Proteínas Intrinsicamente Desordenadas/química , Peptídeos/metabolismo , Ligação Proteica , Proteínas/metabolismo , Ubiquitina/metabolismo
11.
Essays Biochem ; 66(2): 155-168, 2022 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-35920279

RESUMO

The response to abiotic and biotic stresses in plants and crops is considered a multifaceted process. Due to their sessile nature, plants have evolved unique mechanisms to ensure that developmental plasticity remains during their life cycle. Among these mechanisms, post-translational modifications (PTMs) are crucial components of adaptive responses in plants and transduce environmental stimuli into cellular signalling through the modulation of proteins. SUMOylation is an emerging PTM that has received recent attention due to its dynamic role in protein modification and has quickly been considered a significant component of adaptive mechanisms in plants during stress with great potential for agricultural improvement programs. In the present review, we outline the concept that small ubiquitin-like modifier (SUMO)-mediated response in plants and crops to abiotic and biotic stresses is a multifaceted process with each component of the SUMO cycle facilitating tolerance to several different environmental stresses. We also highlight the clear increase in SUMO genes in crops when compared with Arabidopsis thaliana. The SUMO system is understudied in crops, given the importance of SUMO for stress responses, and for some SUMO genes, the apparent expansion provides new avenues to discover SUMO-conjugated targets that could regulate beneficial agronomical traits.


Assuntos
Arabidopsis , Ubiquitina , Arabidopsis/genética , Arabidopsis/metabolismo , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Estresse Fisiológico , Sumoilação , Ubiquitina/metabolismo
12.
Cells ; 11(15)2022 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-35954242

RESUMO

TAR DNA-binding protein 43 (TDP-43) is a predominant component of inclusions in the brains and spines of patients with amyotrophic lateral sclerosis (ALS). The progressive accumulation of inclusions leads to proteinopathy in neurons. We have previously shown that Met1(M1)-linked linear ubiquitin, which is specifically generated by the linear ubiquitin chain assembly complex (LUBAC), is colocalized with TDP-43 inclusions in neurons from optineurin-associated familial and sporadic ALS patients, and affects NF-κB activation and apoptosis. To examine the effects of LUBAC-mediated linear ubiquitination on TDP-43 proteinopathies, we performed cell biological analyses using full-length and truncated forms of the ALS-associated Ala315→Thr (A315T) mutant of TDP-43 in Neuro2a cells. The truncated A315T mutants of TDP-43, which lack a nuclear localization signal, efficiently generated cytoplasmic aggregates that were colocalized with multiple ubiquitin chains such as M1-, Lys(K)48-, and K63-chains. Genetic ablation of HOIP or treatment with a LUBAC inhibitor, HOIPIN-8, suppressed the cytoplasmic aggregation of A315T mutants of TDP-43. Moreover, the enhanced TNF-α-mediated NF-κB activity by truncated TDP-43 mutants was eliminated in the presence of HOIPIN-8. These results suggest that multiple ubiquitinations of TDP-43 including M1-ubiquitin affect protein aggregation and inflammatory responses in vitro, and therefore, LUBAC inhibition ameliorates TDP-43 proteinopathy.


Assuntos
Esclerose Amiotrófica Lateral , Proteínas de Ligação a DNA/metabolismo , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Hidrocarbonetos Aromáticos , NF-kappa B/metabolismo , Ubiquitina/metabolismo , Ubiquitinação
13.
Cell Rep ; 40(7): 111195, 2022 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-35977480

RESUMO

ATG9A is a highly conserved membrane protein required for autophagy initiation. It is trafficked from the trans-Golgi network (TGN) to the phagophore to act as a membrane source for autophagosome expansion. Here, we show that ATG9A is not just a passenger protein in the TGN but rather works in concert with GRASP55, a stacking factor for Golgi structure, to organize Golgi dynamics and integrity. Upon heat stress, the E3 ubiquitin ligase MARCH9 is promoted to ubiquitinate ATG9A in the form of K63 conjugation, and the nondegradable ubiquitinated ATG9A disperses from the Golgi apparatus to the cytoplasm more intensely, accompanied by inhibiting GRASP55 oligomerization, further resulting in Golgi fragmentation. Knockout of ATG9A or MARCH9 largely prevents Golgi fragmentation and protects Golgi functions under heat and other Golgi stresses. Our results reveal a noncanonical function of ATG9A for Golgi dynamics and suggest the pathway for sensing Golgi stress via the MARCH9/ATG9A axis.


Assuntos
Autofagossomos , Complexo de Golgi , Autofagossomos/metabolismo , Autofagia , Proteínas Relacionadas à Autofagia/metabolismo , Complexo de Golgi/metabolismo , Transporte Proteico , Ubiquitina/metabolismo , Rede trans-Golgi/metabolismo
14.
J Cell Biol ; 221(9)2022 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-35938958

RESUMO

The BRCA1-A complex contains matching lysine-63 ubiquitin (K63-Ub) binding and deubiquitylating activities. How these functionalities are coordinated to effectively respond to DNA damage remains unknown. We generated Brcc36 deubiquitylating enzyme (DUB) inactive mice to address this gap in knowledge in a physiologic system. DUB inactivation impaired BRCA1-A complex damage localization and repair activities while causing early lethality when combined with Brca2 mutation. Damage response dysfunction in DUB-inactive cells corresponded to increased K63-Ub on RAP80 and BRCC36. Chemical cross-linking coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and cryogenic-electron microscopy (cryo-EM) analyses of isolated BRCA1-A complexes demonstrated the RAP80 ubiquitin interaction motifs are occupied by ubiquitin exclusively in the DUB-inactive complex, linking auto-inhibition by internal K63-Ub chains to loss of damage site ubiquitin recognition. These findings identify RAP80 and BRCC36 as autologous DUB substrates in the BRCA1-A complex, thus explaining the evolution of matching ubiquitin-binding and hydrolysis activities within a single macromolecular assembly.


Assuntos
Proteína BRCA1 , Dano ao DNA , Proteínas de Ligação a DNA , Enzimas Desubiquitinantes , Chaperonas de Histonas , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Cromatografia Líquida , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/metabolismo , Células HeLa , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Humanos , Camundongos , Espectrometria de Massas em Tandem , Ubiquitina/metabolismo
15.
Sci Rep ; 12(1): 13160, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35915203

RESUMO

The cell cycle is tightly regulated by protein phosphorylation and ubiquitylation events. During mitosis, the multi-subunit cullin-RING E3 ubiquitin ligase APC/c functions as a molecular switch which signals for one cell to divide into two daughter cells, through the ubiquitylation and proteasomal degradation of mitotic cyclins. The contributions of other E3 ligase families during cell cycle progression remain less well understood. Similarly, the roles of ubiquitin chain types beyond homotypic K48 chains in S-phase or branched K11/K48 chains during mitosis, also remain to be fully determined. Our recent findings that HECTD1 ubiquitin ligase activity assembles branched K29/K48 ubiquitin linkages prompted us to evaluate HECTD1 function during the cell cycle. We used transient knockdown and genetic knockout to show that HECTD1 depletion in HEK293T and HeLa cells decreases cell number and we established that this is mediated through loss of ubiquitin ligase activity. Interestingly, we found that HECTD1 depletion increases the proportion of cells with aligned chromosomes (Prometa/Metaphase) and we confirmed this molecularly using phospho-Histone H3 (Ser28) as a marker of mitosis. Time-lapse microscopy of NEBD to anaphase onset established that HECTD1-depleted cells take on average longer to go through mitosis. In line with this data, HECTD1 depletion reduced the activity of the Spindle Assembly Checkpoint, and BUB3, a component of the Mitosis Checkpoint Complex, was identified as novel HECTD1 interactor. BUB3, BUBR1 or MAD2 protein levels remained unchanged in HECTD1-depleted cells. Overall, this study reveals a novel putative role for HECTD1 during mitosis and warrants further work to elucidate the mechanisms involved.


Assuntos
Proteínas de Ciclo Celular , Ubiquitina-Proteína Ligases , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Células HEK293 , Células HeLa , Humanos , Mitose , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética
16.
Nat Commun ; 13(1): 4462, 2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35915093

RESUMO

Defects in cellular proteostasis and mitochondrial function drive many aspects of infertility, cancer, and other age-related diseases. All of these conditions rely on quiescent cells, such as oocytes and adult stem cells, that reduce their activity and remain dormant as part of their roles in tissue homeostasis, reproduction, and even cancer recurrence. Using a multi-organism approach, we show that dynamic shifts in the ubiquitin proteasome system drive mitochondrial remodeling during cellular quiescence. In contrast to the commonly held view that the ubiquitin-proteasome system (UPS) is primarily regulated by substrate ubiquitination, we find that increasing proteasome number and their recruitment to mitochondria support mitochondrial respiratory quiescence (MRQ). GSK3 triggers proteasome recruitment to the mitochondria by phosphorylating outer membrane proteins, such as VDAC, and suppressing mitochondrial fatty acid oxidation. This work defines a process that couples dynamic regulation of UPS activity to coordinated shifts in mitochondrial metabolism in fungi, Drosophila, and mammals during quiescence.


Assuntos
Complexo de Endopeptidases do Proteassoma , Ubiquitina , Animais , Quinase 3 da Glicogênio Sintase/metabolismo , Mamíferos/metabolismo , Mitocôndrias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Ubiquitinação
17.
PLoS Pathog ; 18(8): e1010744, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35914008

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA virus with the capacity to establish life-long latent infection. During latent infection, the viral genome persists as a circular episome that associates with cellular histones and exists as a nonintegrated minichromosome in the nucleus of infected cells. Chromatin structure and epigenetic programming are required for the proper control of viral gene expression and stable maintenance of viral DNA. However, there is still limited knowledge regarding how the host regulates the chromatin structure and maintenance of episomal DNA. Here, we found that the cellular protein structural maintenance of chromosome (SMC) complex SMC5/6 recognizes and associates with the KSHV genome to inhibit its replication. The SMC5/6 complex can bind to the KSHV genome and suppress KSHV gene transcription by condensing the viral chromatin and creating a repressive chromatin structure. Correspondingly, KSHV employs an antagonistic strategy by utilizing the viral protein RTA to degrade the SMC5/6 complex and antagonize the inhibitory effect of this complex on viral gene transcription. Interestingly, this antagonistic mechanism of RTA is evolutionarily conserved among γ-herpesviruses. Our work suggests that the SMC5/6 complex is a new host factor that restricts KSHV replication.


Assuntos
Herpesvirus Humano 8 , Proteínas Imediatamente Precoces , Infecção Latente , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/fisiologia , Humanos , Proteínas Imediatamente Precoces/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transativadores , Ubiquitina/metabolismo , Latência Viral/genética , Replicação Viral/genética
18.
Nat Commun ; 13(1): 4672, 2022 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-35945250

RESUMO

Linear (Met1-linked) ubiquitination is involved inflammatory and innate immune signaling. Previous studies have characterized enzymes regulating the addition and removal of this modification in mammalian systems. However, only a few plant-derived deubiquitinases targeting Met1-linked ubiquitin chains have been reported and their mechanism of action remains elusive. Here, using a dehydroalanine-bearing Met1-diubiquitin suicide probe, we discover OTUB1 from Oryza sativa (OsOTUB1) as a Met1-linked ubiquitin chain-targeting deubiquitinase. By solving crystal structures of apo OsOTUB1 and an OsOTUB1/Met1-diubiquitin complex, we find that Met1 activity is conferred by Met1-specific motifs in the S1' pocket of OsOTUB1. Large-scale sequence alignments and hydrolysis experiments provide evidence that these motifs are a general determinant of Met1 activity in the OTUB subfamily across species. Analysis of the species distribution of OTUBs capable of hydrolysing Met1-linked ubiquitin chains shows that this activity is conserved in green plants (Viridiplantae) and does not exist in metazoans, providing insights into the evolutionary differentiation between primitive plants and animals.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Oryza , Transdução de Sinais , Animais , Humanos , Mamíferos/metabolismo , Oryza/genética , Oryza/metabolismo , Ubiquitina/metabolismo , Ubiquitinação
19.
Nat Commun ; 13(1): 4789, 2022 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-35970836

RESUMO

The covalent modification of target proteins with ubiquitin or ubiquitin-like modifiers is initiated by E1 activating enzymes, which typically transfer a single modifier onto cognate conjugating enzymes. UBA6 is an unusual E1 since it activates two highly distinct modifiers, ubiquitin and FAT10. Here, we report crystal structures of UBA6 in complex with either ATP or FAT10. In the UBA6-FAT10 complex, the C-terminal domain of FAT10 binds to where ubiquitin resides in the UBA1-ubiquitin complex, however, a switch element ensures the alternate recruitment of either modifier. Simultaneously, the N-terminal domain of FAT10 interacts with the 3-helix bundle of UBA6. Site-directed mutagenesis identifies residues permitting the selective activation of either ubiquitin or FAT10. These results pave the way for studies investigating the activation of either modifier by UBA6 in physiological and pathophysiological settings.


Assuntos
Ubiquitina , Ubiquitinas , Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitinas/metabolismo
20.
Nat Commun ; 13(1): 4880, 2022 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-35986001

RESUMO

The E1 enzyme Uba6 initiates signal transduction by activating ubiquitin and the ubiquitin-like protein FAT10 in a two-step process involving sequential catalysis of adenylation and thioester bond formation. To gain mechanistic insights into these processes, we determined the crystal structure of a human Uba6/ubiquitin complex. Two distinct architectures of the complex are observed: one in which Uba6 adopts an open conformation with the active site configured for catalysis of adenylation, and a second drastically different closed conformation in which the adenylation active site is disassembled and reconfigured for catalysis of thioester bond formation. Surprisingly, an inositol hexakisphosphate (InsP6) molecule binds to a previously unidentified allosteric site on Uba6. Our structural, biochemical, and biophysical data indicate that InsP6 allosterically inhibits Uba6 activity by altering interconversion of the open and closed conformations of Uba6 while also enhancing its stability. In addition to revealing the molecular mechanisms of catalysis by Uba6 and allosteric regulation of its activities, our structures provide a framework for developing Uba6-specific inhibitors and raise the possibility of allosteric regulation of other E1s by naturally occurring cellular metabolites.


Assuntos
Enzimas Ativadoras de Ubiquitina , Ubiquitina , Catálise , Domínio Catalítico , Humanos , Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitinas/metabolismo
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