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1.
Science ; 372(6549): eabf6548, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34739333

RESUMO

Stress granules are dynamic, reversible condensates composed of RNA and protein that assemble in eukaryotic cells in response to a variety of stressors and are normally disassembled after stress is removed. The composition and assembly of stress granules is well understood, but little is known about the mechanisms that govern disassembly. Impaired disassembly has been implicated in some diseases including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy. Using cultured human cells, we found that stress granule disassembly was context-dependent: Specifically in the setting of heat shock, disassembly required ubiquitination of G3BP1, the central protein within the stress granule RNA-protein network. We found that ubiquitinated G3BP1 interacted with the endoplasmic reticulum­associated protein FAF2, which engaged the ubiquitin-dependent segregase p97/VCP (valosin-containing protein). Thus, targeting of G3BP1 weakened the stress granule­specific interaction network, resulting in granule disassembly.


Assuntos
Proteínas Sanguíneas/metabolismo , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Resposta ao Choque Térmico , Proteínas de Membrana/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas Ubiquitinadas/metabolismo , Proteína com Valosina/metabolismo , Autofagia , Linhagem Celular Tumoral , DNA Helicases/química , DNA Helicases/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Membranas Intracelulares/metabolismo , Mutação , Proteínas de Ligação a Poli-ADP-Ribose/química , Proteínas de Ligação a Poli-ADP-Ribose/genética , Poliubiquitina/metabolismo , Domínios Proteicos , Proteólise , RNA Helicases/química , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/química , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas Ubiquitinadas/química , Ubiquitinação
2.
Immunity ; 54(10): 2218-2230.e5, 2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34644557

RESUMO

The RNA sensor MDA5 recruits the signaling adaptor MAVS to initiate type I interferon signaling and downstream antiviral responses, a process that requires K63-linked polyubiquitin chains. Here, we examined the mechanisms whereby K63-polyUb chain regulate MDA5 activation. Only long unanchored K63-polyUbn (n ≥ 8) could mediate tetramerization of the caspase activation and recruitment domains of MDA5 (MDA5CARDs). Cryoelectron microscopy structures of a polyUb13-bound MDA5CARDs tetramer and a polyUb11-bound MDA5CARDs-MAVSCARD assembly revealed a tower-like formation, wherein eight Ubs tethered along the outer rim of the helical shell, bridging MDA5CARDs and MAVSCARD tetramers into proximity. ATP binding and hydrolysis promoted the stabilization of RNA-bound MDA5 prior to MAVS activation via allosteric effects on CARDs-polyUb complex. Abundant ATP prevented basal activation of apo MDA5. Our findings reveal the ordered assembly of a MDA5 signaling complex competent to recruit and activate MAVS and highlight differences with RIG-I in terms of CARD orientation and Ub sensing that suggest different abilities to induce antiviral responses.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/química , Microscopia Crioeletrônica , Células HEK293 , Humanos , Imunidade Inata/fisiologia , Helicase IFIH1 Induzida por Interferon/química , Helicase IFIH1 Induzida por Interferon/ultraestrutura , Poliubiquitina/química , Poliubiquitina/metabolismo , Ligação Proteica
3.
Nat Cell Biol ; 23(9): 978-991, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34497368

RESUMO

The extracellular-signal-regulated kinases ERK1 and ERK2 (hereafter ERK1/2) represent the foremost mitogenic pathway in mammalian cells, and their dysregulation drives tumorigenesis and confers therapeutic resistance. ERK1/2 are known to be activated by MAPK/ERK kinase (MEK)-mediated phosphorylation. Here, we show that ERK1/2 are also modified by lysine-63 (K63)-linked polyubiquitin chains. We identify the tripartite motif-containing protein TRIM15 as a ubiquitin ligase and the tumour suppressor CYLD as a deubiquitinase of ERK1/2. TRIM15 and CYLD regulate ERK ubiquitination at defined lysine residues through mutually exclusive interactions as well as opposing activities. K63-linked polyubiquitination enhances ERK interaction with and activation by MEK. Downregulation of TRIM15 inhibits the growth of both drug-responsive and drug-resistant melanomas. Moreover, high TRIM15 expression and low CYLD expression are associated with poor prognosis of patients with melanoma. These findings define a role of K63-linked polyubiquitination in the ERK signalling pathway and suggest a potential target for cancer therapy.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Enzima Desubiquitinante CYLD/metabolismo , Lisina/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Poliubiquitina/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Genes Supressores de Tumor/fisiologia , Humanos , Fosforilação/fisiologia , Transdução de Sinais/fisiologia , Ubiquitina/metabolismo
4.
Biophys J ; 120(16): 3355-3362, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34242591

RESUMO

TAK1-binding protein 2 (TAB2) has generally been considered to bind specifically to K63-linked polyubiquitin chains via its C-terminal Npl4 zinc-finger (NZF) domain. However, a recent study showed that the NZF domain of TAB2 (TAB2-NZF) could also interact with K6-linked polyubiquitin chains. Here, we report the crystal structure of TAB2-NZF in complex with K6-linked diubiquitin (K6-Ub2) at 1.99-Å resolution. TAB2-NZF simultaneously interacts with the distal and proximal ubiquitin moieties of K6-Ub2. By comparing the structures of TAB2-NZF in complex with K6-Ub2 and with K63-linked diubiquitin (K63-Ub2), we reveal that the binding mechanism of TAB2-NZF with K6-Ub2 is similar to that with K63-Ub2, except for the flexible C-terminal region of the distal ubiquitin. Therefore, we conclude that the C-terminal flexibility of the distal ubiquitin contributes to the dual specificity of TAB2-NZF toward K6- and K63-linked ubiquitin chains. This study provides important insights into the functions of K6-linked ubiquitin chains, which are currently unclear.


Assuntos
Poliubiquitina , Dedos de Zinco , Modelos Moleculares , Poliubiquitina/metabolismo , Ligação Proteica , Ubiquitina/metabolismo
5.
Mol Cell ; 81(15): 3187-3204.e7, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34157307

RESUMO

OTULIN coordinates with LUBAC to edit linear polyubiquitin chains in embryonic development, autoimmunity, and inflammatory diseases. However, the mechanism by which angiogenesis, especially that of endothelial cells (ECs), is regulated by linear ubiquitination remains unclear. Here, we reveal that constitutive or EC-specific deletion of Otulin resulted in arteriovenous malformations and embryonic lethality. LUBAC conjugates linear ubiquitin chains onto Activin receptor-like kinase 1 (ALK1), which is responsible for angiogenesis defects, inhibiting ALK1 enzyme activity and Smad1/5 activation. Conversely, OTULIN deubiquitinates ALK1 to promote Smad1/5 activation. Consistently, embryonic survival of Otulin-deficient mice was prolonged by BMP9 pretreatment or EC-specific ALK1Q200D (constitutively active) knockin. Moreover, mutant ALK1 from type 2 hereditary hemorrhagic telangiectasia (HHT2) patients exhibited excessive linear ubiquitination and increased HOIP binding. As such, a HOIP inhibitor restricted the excessive angiogenesis of ECs derived from ALK1G309S-expressing HHT2 patients. These results show that OTULIN and LUBAC govern ALK1 activity to balance EC angiogenesis.


Assuntos
Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Endopeptidases/genética , Complexos Multiproteicos/metabolismo , Neovascularização Patológica/genética , Poliubiquitina/metabolismo , Adulto , Animais , Endopeptidases/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Fator 2 de Diferenciação de Crescimento/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos Mutantes , Mutação , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Fisiológica/genética , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad5/genética , Proteína Smad5/metabolismo , Telangiectasia Hemorrágica Hereditária , Ubiquitina-Proteína Ligases/metabolismo
6.
Plant Cell Physiol ; 62(6): 1044-1057, 2021 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-34086919

RESUMO

Ubiquitination, one of the most frequently occurring post-translational modifications, is essential for regulating diverse cellular processes in plants during abiotic stress. The E3 ubiquitin (Ub) ligase Arabidopsis thaliana really interesting new gene (RING) zinc finger 1 (AtRZF1) mutation is known to enhance drought tolerance in A. thaliana seedlings. To further investigate the function of AtRZF1 in osmotic stress, we isolated Ub-associated protein 1 (AtUAP1) which interacts with AtRZF1 using a yeast two-hybrid system. AtUAP1, a Ub-associated motif containing protein, increased the amount of Ub-conjugated AtRZF1. Moreover, AtUAP1 RNA interference lines were more tolerant to osmotic stress than wild type, whereas AtUAP1-overexpressing (OX) transgenic lines showed sensitive responses, including cotyledon greening, water loss, proline accumulation and changes in stress-related genes expression, indicating that AtUAP1 could negatively regulate dehydration-mediated signaling. In addition, AtUAP1-green fluorescent protein fusion protein was observed in the nuclei of root cells of transgenic seedlings. Genetic studies showed that the AtRZF1 mutation could rescue the sensitive phenotype of AtUAP1-OX lines in response to osmotic stress, suggesting that AtRZF1 was epistatic to AtUAP1 in dehydration signaling. Taken together, our findings describe a new component in the AtRZF1 ubiquitination pathway which controls the dehydration response in A. thaliana.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Desidratação , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/genética , Sítios de Ligação , Regulação da Expressão Gênica de Plantas , Pressão Osmótica , Plantas Geneticamente Modificadas , Poliubiquitina/metabolismo , Domínios Proteicos , Mapas de Interação de Proteínas , Técnicas do Sistema de Duplo-Híbrido , Ubiquitinação
7.
Nat Commun ; 12(1): 2674, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976226

RESUMO

The transcription coactivator YAP plays a vital role in Hippo pathway for organ-size control and tissue homeostasis. Recent studies have demonstrated YAP is closely related to immune disorders and inflammatory diseases, but the underlying mechanisms remain less defined. Here, we find that YAP promotes the activation of NLRP3 inflammasome, an intracellular multi-protein complex that orchestrates host immune responses to infections or sterile injuries. YAP deficiency in myeloid cells significantly attenuates LPS-induced systemic inflammation and monosodium urate (MSU) crystals-induced peritonitis. Mechanistically, YAP physically interacts with NLRP3 and maintains the stability of NLRP3 through blocking the association between NLRP3 and the E3 ligase ß-TrCP1, the latter increases the proteasomal degradation of NLRP3 via K27-linked ubiquitination at lys380. Together, these findings establish a role of YAP in the activation of NLRP3 inflammasome, and provide potential therapeutic target to treat the NLRP3 inflammasome-related diseases.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Poliubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células Cultivadas , Células HEK293 , Humanos , Inflamassomos/genética , Lisina/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Interferência de RNA , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas Contendo Repetições de beta-Transducina/genética , Proteínas Contendo Repetições de beta-Transducina/metabolismo
8.
J Biol Chem ; 297(1): 100811, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34023381

RESUMO

A20 is a potent anti-inflammatory protein that mediates both inflammation and ubiquitination in mammals, but the related mechanisms are not clear. In this study, we performed mass spectrometry (MS) screening, gene ontology (GO) analysis, and coimmunoprecipitation (co-IP) in a lipopolysaccharide (LPS)-induced inflammatory cell model to identify novel A20-interacting proteins. We confirmed that the E3 ubiquitin ligase Nrdp1, also known as ring finger protein 41 (RNF41), interacted with A20 in LPS-stimulated cells. Further co-IP analysis demonstrated that when A20 was knocked out, degradation-inducing K48-linked ubiquitination of inflammatory effector MyD88 was decreased, but protein interaction-mediating K63-linked ubiquitination of another inflammatory effector TBK1 was increased. Moreover, western blot experiments showed that A20 inhibition induced an increase in levels of MyD88 and phosphorylation of downstream effector proteins as well as of TBK1 and a downstream effector, while Nrdp1 inhibition induced an increase in MyD88 but a decrease in TBK1 levels. When A20 and Nrdp1 were coinhibited, no further change in MyD88 was observed, but TBK1 levels were significantly decreased compared with those upon A20 inhibition alone. Gain- and loss-of-function analyses revealed that the ZnF4 domain of A20 is required for Nrdp1 polyubiquitination. Upon LPS stimulation, the inhibition of Nrdp1 alone increased the secretion of IL-6 and TNF-α but decreased IFN-ß secretion, as observed in other studies, suggesting that Nrdp1 preferentially promotes the production of IFN-ß. Taken together, these results demonstrated that A20/Nrdp1 interaction is important for A20 anti-inflammation, thus revealing a novel mechanism for the anti-inflammatory effects of A20.


Assuntos
Inflamação/metabolismo , Lisina/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Poliubiquitina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais , Ativação Enzimática , Inflamação/patologia , Interferons/metabolismo , Macrófagos/metabolismo , Camundongos , Modelos Biológicos , Ligação Proteica , Domínios Proteicos , Proteólise , Células RAW 264.7 , Transdução de Sinais , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/química
9.
Biochem Biophys Res Commun ; 559: 203-209, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33951500

RESUMO

Optineurin produces intracellular multi-functions involving autophagy, vesicular trafficking, and negative regulation of inflammation signaling through interaction with various proteins such as ATG8/LC3, Rab8, and polyubiquitin. Optineurin is a component of cytoplasmic inclusion bodies (IBs) in motor neurons from amyotrophic lateral sclerosis (ALS), and its mutation E478G, has been identified in patients with ALS. However, the mechanism by which polyubiquitin binding modulates the interaction partners of OPTN and ALS-associated IB formation is still unclear. To address this issue, we analyzed the interaction of Optineurin with Rab8 and LC3 in the absence and presence of linear polyubiquitin chains using fluorescence cross-correlation spectroscopy and IB formation efficiency of the E478G mutant of Optineurin during Rab8 depletion using fluorescence microscopy. Here, we hypothesize that linear polyubiquitin binding to Optineurin dynamically induces LC3 association and Rab8 dissociation, likely through a conformational change of Optineurin, and the dynamic conformational change may prevent the aggregate formation of mutant Optineurin.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Poliubiquitina/metabolismo , Esclerose Amiotrófica Lateral/genética , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proteínas de Membrana Transportadoras/genética , Camundongos , Modelos Biológicos , Mutação/genética , Agregados Proteicos , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Transporte Proteico , Proteínas rab de Ligação ao GTP/metabolismo
10.
Plant Cell ; 33(2): 420-438, 2021 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-33866370

RESUMO

Plants take up and translocate nutrients through transporters. In Arabidopsis thaliana, the borate exporter BOR1 acts as a key transporter under boron (B) limitation in the soil. Upon sufficient-B supply, BOR1 undergoes ubiquitination and is transported to the vacuole for degradation, to avoid overaccumulation of B. However, the mechanisms underlying B-sensing and ubiquitination of BOR1 are unknown. In this study, we confirmed the lysine-590 residue in the C-terminal cytosolic region of BOR1 as the direct ubiquitination site and showed that BOR1 undergoes K63-linked polyubiquitination. A forward genetic screen identified that amino acid residues located in vicinity of the substrate-binding pocket of BOR1 are essential for the vacuolar sorting. BOR1 variants that lack B-transport activity showed a significant reduction of polyubiquitination and subsequent vacuolar sorting. Coexpression of wild-type (WT) and a transport-defective variant of BOR1 in the same cells showed degradation of the WT but not the variant upon sufficient-B supply. These findings suggest that polyubiquitination of BOR1 relies on its conformational transition during the transport cycle. We propose a model in which BOR1, as a B transceptor, directly senses the B concentration and promotes its own polyubiquitination and vacuolar sorting for quick and precise maintenance of B homeostasis.


Assuntos
Antiporters/metabolismo , Proteínas de Arabidopsis/metabolismo , Boro/farmacologia , Proteólise/efeitos dos fármacos , Ubiquitinação , Sequência de Aminoácidos , Substituição de Aminoácidos , Antiporters/química , Proteínas de Arabidopsis/química , Sítios de Ligação , Testes Genéticos , Proteínas de Fluorescência Verde/metabolismo , Lisina/metabolismo , Modelos Biológicos , Poliubiquitina/metabolismo , Transporte Proteico/efeitos dos fármacos , Prótons , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Ubiquitinação/efeitos dos fármacos , Vacúolos/metabolismo
11.
Nat Commun ; 12(1): 2155, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846325

RESUMO

Cryptochromes (CRYs) are photoreceptors or components of the molecular clock in various evolutionary lineages, and they are commonly regulated by polyubiquitination and proteolysis. Multiple E3 ubiquitin ligases regulate CRYs in animal models, and previous genetics study also suggest existence of multiple E3 ubiquitin ligases for plant CRYs. However, only one E3 ligase, Cul4COP1/SPAs, has been reported for plant CRYs so far. Here we show that Cul3LRBs is the second E3 ligase of CRY2 in Arabidopsis. We demonstrate the blue light-specific and CRY-dependent activity of LRBs (Light-Response Bric-a-Brack/Tramtrack/Broad 1, 2 & 3) in blue-light regulation of hypocotyl elongation. LRBs physically interact with photoexcited and phosphorylated CRY2, at the CCE domain of CRY2, to facilitate polyubiquitination and degradation of CRY2 in response to blue light. We propose that Cul4COP1/SPAs and Cul3LRBs E3 ligases interact with CRY2 via different structure elements to regulate the abundance of CRY2 photoreceptor under different light conditions, facilitating optimal photoresponses of plants grown in nature.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Criptocromos/metabolismo , Fotorreceptores de Plantas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Criptocromos/química , Criptocromos/genética , Células HEK293 , Humanos , Luz , Modelos Biológicos , Mutação/genética , Fosforilação/efeitos da radiação , Poliubiquitina/metabolismo , Ligação Proteica/efeitos da radiação , Proteólise/efeitos da radiação , Plântula/efeitos da radiação , Ubiquitinação/efeitos da radiação
12.
J Immunol ; 206(10): 2376-2385, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33893171

RESUMO

NLRP3 inflammasome plays an important role in innate immune system through recognizing pathogenic microorganisms and danger-associated molecules. Deubiquitination of NLRP3 has been shown to be essential for its activation, yet the functions of Ubc13, the K63-linked specific ubiquitin-conjugating enzyme E2, in NLRP3 inflammasome activation are not known. In this study, we found that in mouse macrophages, Ubc13 knockdown or knockout dramatically impaired NLRP3 inflammasome activation. Catalytic activity is required for Ubc13 to control NLRP3 activation, and Ubc13 pharmacological inhibitor significantly attenuates NLRP3 inflammasome activation. Mechanistically, Ubc13 associates with NLRP3 and promotes its K63-linked polyubiquitination. Through mass spectrum and biochemical analysis, we identified lysine 565 and lysine 687 as theK63-linked polyubiquitination sites of NLRP3. Collectively, our data suggest that Ubc13 potentiates NLRP3 inflammasome activation via promoting site-specific K63-linked ubiquitination of NLRP3. Our study sheds light on mechanisms of NLRP3 inflammasome activation and identifies that targeting Ubc13 could be an effective therapeutic strategy for treating aberrant NLRP3 inflammasome activation-induced pathogenesis.


Assuntos
Inflamassomos/metabolismo , Lisina/metabolismo , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Poliubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/deficiência , Ubiquitinação/genética , Animais , Células HEK293 , Humanos , Inflamassomos/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Transgênicos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Ligação Proteica , Transfecção , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitinação/efeitos dos fármacos
13.
Biochem Biophys Res Commun ; 550: 184-190, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33706102

RESUMO

Linear ubiquitination is an atypic ubiquitination process that directly connects the N- and C-termini of ubiquitin and is catalyzed by HOIL-1-interacting protein (HOIP). It is involved in the immune response or apoptosis by activating the nuclear factor-κB pathway and is associated with polyglucosan body myopathy 1, an autosomal recessive disorder with progressive muscle weakness and cardiomyopathy. However, little is currently known regarding the function of linear ubiquitination in muscles. Here, we investigated the role of linear ubiquitin E3 ligase (LUBEL), a DrosophilaHOIP ortholog, in the development and aging of muscles. The muscles of the flies with down-regulation of LUBEL or its downstream factors, kenny and Relish, developed normally, and there were no obvious abnormalities in function in young flies. However, the locomotor activity of the LUBEL RNAi flies was reduced compared to age-matched control, while LUBEL RNAi did not affect the increased mitochondrial fusion or myofiber disorganization during aging. Interestingly, the accumulation of polyubiquitinated protein aggregation during aging decreased in muscles by silencing LUBEL, kenny, or Relish. Meanwhile, the levels of autophagy and global translation, which are implicated in the maintenance of proteostasis, did not change due to LUBEL down-regulation. In conclusion, we propose a new role of linear ubiquitination in proteostasis in the muscle aging.


Assuntos
Envelhecimento/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Músculos/metabolismo , Proteostase , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Autofagia , Regulação para Baixo , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimologia , Inativação Gênica , Quinase I-kappa B/deficiência , Quinase I-kappa B/metabolismo , Locomoção , Masculino , Força Muscular , Músculos/enzimologia , NF-kappa B/metabolismo , Poliubiquitina/metabolismo , Agregados Proteicos , Biossíntese de Proteínas , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
14.
Mol Cells ; 44(2): 101-115, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33658435

RESUMO

The INO80 chromatin remodeling complex has roles in many essential cellular processes, including DNA replication. However, the mechanisms that regulate INO80 in these processes remain largely unknown. We previously reported that the stability of Ino80, the catalytic ATPase subunit of INO80, is regulated by the ubiquitin proteasome system and that BRCA1-associated protein-1 (BAP1), a nuclear deubiquitinase with tumor suppressor activity, stabilizes Ino80 via deubiquitination and promotes replication fork progression. However, the E3 ubiquitin ligase that targets Ino80 for proteasomal degradation was unknown. Here, we identified the C-terminus of Hsp70-interacting protein (CHIP), the E3 ubiquitin ligase that functions in cooperation with Hsp70, as an Ino80-interacting protein. CHIP polyubiquitinates Ino80 in a manner dependent on Hsp70. Contrary to our expectation that CHIP degrades Ino80, CHIP instead stabilizes Ino80 by extending its halflife. The data suggest that CHIP stabilizes Ino80 by inhibiting degradative ubiquitination. We also show that CHIP works together with BAP1 to enhance the stabilization of Ino80, leading to its chromatin binding. Interestingly, both depletion and overexpression of CHIP compromise replication fork progression with little effect on fork stalling, as similarly observed for BAP1 and Ino80, indicating that an optimal cellular level of Ino80 is important for replication fork speed but not for replication stress suppression. This work therefore idenitifes CHIP as an E3 ubiquitin ligase that stabilizes Ino80 via nondegradative ubiquitination and suggests that CHIP and BAP1 act in concert to regulate Ino80 ubiquitination to fine-tune its stability for efficient DNA replication.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Sítios de Ligação , Cromatina/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP70/metabolismo , Células HT29 , Humanos , Poliubiquitina/metabolismo , Ligação Proteica , Estabilidade Proteica
15.
Mol Cell ; 81(7): 1411-1424.e7, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33567268

RESUMO

Targeted protein degradation is an emerging therapeutic paradigm. Small-molecule degraders such as proteolysis-targeting chimeras (PROTACs) induce the degradation of neo-substrates by hijacking E3 ubiquitin ligases. Although ubiquitylation of endogenous substrates has been extensively studied, the mechanism underlying forced degradation of neo-substrates is less well understood. We found that the ubiquitin ligase TRIP12 promotes PROTAC-induced and CRL2VHL-mediated degradation of BRD4 but is dispensable for the degradation of the endogenous CRL2VHL substrate HIF-1α. TRIP12 associates with BRD4 via CRL2VHL and specifically assembles K29-linked ubiquitin chains, facilitating the formation of K29/K48-branched ubiquitin chains and accelerating the assembly of K48 linkage by CRL2VHL. Consequently, TRIP12 promotes the PROTAC-induced apoptotic response. TRIP12 also supports the efficiency of other degraders that target CRABP2 or TRIM24 or recruit CRBN. These observations define TRIP12 and K29/K48-branched ubiquitin chains as accelerators of PROTAC-directed targeted protein degradation, revealing a cooperative mechanism of branched ubiquitin chain assembly unique to the degradation of neo-substrates.


Assuntos
Proteínas de Transporte/metabolismo , Poliubiquitina/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células HCT116 , Células HEK293 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Poliubiquitina/genética , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética
16.
Biochemistry ; 60(8): 573-583, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33616406

RESUMO

Polyubiquitin is a multifunctional protein tag formed by the covalent conjugation of ubiquitin molecules. Due to the high rigidity of the ubiquitin fold, the ubiquitin moieties in a polyubiquitin chain appear to be structurally equivalent to each other. It is therefore unclear how a specific ubiquitin moiety in a chain may be preferentially recognized by some proteins, such as the kinase PINK1. Here we show that there is structural dynamic heterogeneity in the two ubiquitin moieties of K48-linked diubiquitin by NMR spectroscopic analyses. Our analyses capture subunit-asymmetric structural fluctuations that are not directly related to the closed-to-open transition of the two ubiquitin moieties in diubiquitin. Strikingly, these newly identified heterogeneous structural fluctuations may be linked to an increase in susceptibility to phosphorylation by PINK1. Coupled with the fact that there are almost no differences in static tertiary structure among ubiquitin moieties in a chain, the observed subunit-specific structural fluctuations may be an important factor that distinguishes individual ubiquitin moieties in a chain, thereby aiding both efficiency and specificity in post-translational modifications.


Assuntos
Poliubiquitina/química , Proteínas Quinases/química , Processamento de Proteína Pós-Traducional , Humanos , Modelos Moleculares , Fosforilação , Poliubiquitina/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Quinases/metabolismo
17.
J Biol Chem ; 296: 100450, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33617881

RESUMO

Proteasome-mediated substrate degradation is an essential process that relies on the coordinated actions of ubiquitin (Ub), shuttle proteins containing Ub-like (UBL) domains, and the proteasome. Proteinaceous substrates are tagged with polyUb and shuttle proteins, and these signals are then recognized by the proteasome, which subsequently degrades the substrate. To date, three proteasomal receptors have been identified, as well as multiple shuttle proteins and numerous types of polyUb chains that signal for degradation. While the components of this pathway are well-known, our understanding of their interplay is unclear-especially in the context of Rpn1, the largest proteasomal subunit. Here, using nuclear magnetic resonance (NMR) spectroscopy in combination with competition assays, we show that Rpn1 associates with UBL-containing proteins and polyUb chains, while exhibiting a preference for shuttle protein Rad23. Rpn1 appears to contain multiple Ub/UBL-binding sites, theoretically as many as one for each of its hallmark proteasome/cyclosome repeats. Remarkably, we also find that binding sites on Rpn1 can be shared among Ub and UBL species, while proteasomal receptors Rpn1 and Rpn10 can compete with each other for binding of shuttle protein Dsk2. Taken together, our results rule out the possibility of exclusive recognition sites on Rpn1 for individual Ub/UBL signals and further emphasize the complexity of the redundancy-laden proteasomal degradation pathway.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitinas/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Espectroscopia de Ressonância Magnética/métodos , Proteínas de Membrana/metabolismo , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Ligação Proteica , Proteólise , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Ubiquitina/metabolismo
18.
Mol Biol Rep ; 48(3): 2163-2171, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33620660

RESUMO

Gestational choriocarcinoma is aggressive trophoblastic disease. The development, progression and the cure of this disease is not well-established. p97/Valosin containing protein has been shown to play critical roles in many cellular processes. In various cancers, higher expression of p97/VCP has been reported and targeting of p97/VCP with its spesific inhibitors or siRNA's (siVCP) in cancer therapy was suggested. However, no study is avaible about the expression and function of p97/VCP in gestational choriocarcinoma. Hence, the aim of the study was to evaluate effects of p97/VCP inhibitor, DBeQ and siVCP on choriocarcinoma cells. We use human placental choriocarcinoma cell line (Jeg3) as model to find out the effects of DBeQ and VCP siRNA's (siVCP) on apoptotic and autophagic pathway by immunflouroscence staining, Western blotting, qPCR and flow-cytometry. p97/VCP siRNA's and DBeQ induced accumulation of autophagic proteins, LC3II and p62 in the cytoplasm of Jeg3 cells detected. Concurrently, Jeg3 cells treated with DBeQ and siVCP demonstrated G0/G1 cell cycle arrest, accompanied by accumulation of poly-ubiquitinated proteins. Moreover, disruption of p97/VCP by siRNA and DBeQ inhibited cancer cell growth managing the caspases-3 and -7. Our results show that inhibition of p97/VCP activity with DBeQ and depletion of p97/VCP expression with siRNA in Jeg3 cells induce caspase activation, inhibits cell proliferation and leads to a defect in autophagosome maturation, thus providing potential target for the prevention and treatment of choriocarcinoma.


Assuntos
Apoptose , Autofagossomos/metabolismo , Pontos de Checagem do Ciclo Celular , Coriocarcinoma/metabolismo , Coriocarcinoma/patologia , Proteína com Valosina/metabolismo , Apoptose/efeitos dos fármacos , Autofagossomos/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Poliubiquitina/metabolismo , Quinazolinas/farmacologia , RNA Interferente Pequeno/metabolismo , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Proteína Sequestossoma-1/metabolismo , Ubiquitinação/efeitos dos fármacos
19.
BMB Rep ; 54(4): 189-195, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33612153

RESUMO

Ubiquitin (Ub) is one of the proteins that are highly conserved from yeast to humans. It is an essential core unit of the welldefined post-translational modification, called ubiquitination, which is involved in a variety of biological processes. In metazoans, Ub is encoded by two monoubiquitin genes and two polyubiquitin genes, in which a single Ub is fused to a ribosomal protein or Ub coding units are arranged in tandem repeats. In mice, polyubiquitin genes (Ubb and Ubc) play a pivotal role to meet the requirement of cellular Ub pools during embryonic development. In addition, expression levels of polyubiquitin genes are increased to adapt to environmental stimuli such as oxidative, heat-shock, and proteotoxic stress. Several researchers have reported about the perturbation of Ub pools through genetic alteration or exogenous Ub delivery using diverse model systems. To study Ub pool changes in a physiologically relevant manner, changing Ub pools via the regulation of endogenous polyubiquitin gene expression has recently been introduced. Furthermore, to understand the regulation of polyubiquitin gene expression more precisely, cis-acting elements and trans-acting factors, which are regulatory components of polyubiquitin genes, have been analyzed. In this review, we discuss how the role of polyubiquitin genes has been studied during the past decade, especially focusing on their regulation. [BMB Reports 2021; 54(4): 189-195].


Assuntos
Poliubiquitina/metabolismo , Animais , Regulação da Expressão Gênica/genética , Humanos , Poliubiquitina/genética , Ubiquitinação
20.
Neurobiol Learn Mem ; 179: 107386, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33476748

RESUMO

The destabilization/reconsolidation process can be triggered by memory recall, allowing consolidated memories to be modified. We have previously reported that stress prior to fear conditioning induces memories that exhibit resistance to the engagement of some molecular events associated with the destabilization/reconsolidation process. Here, we evaluated whether stress could affect the expression of Lys-48 polyubiquitinated proteins within the basolateral amygdala complex, a phenomenon crucially linked to memory destabilization. As expected, a post-recall increase of Lys-48 polyubiquitinated proteins in control animals was observed; however, this phenomenon was prevented by stress exposure before fear conditioning. On the other hand, pre-recall administration of D-cycloserine -a positive modulator of NMDA sites capable of reverting memory resistance to pharmacological interference-, facilitated the increase of Lys-48 polyubiquitinated proteins in stressed animals. In conclusion, the protein polyubiquitination-dependent destabilization is impaired after the recall of stress-induced resistant memories, with D-cycloserine restoring such molecular event. Hence, the present report contributes to further characterize the neurobiological events associated with stress-induced memory resistance as well as to corroborate the connection between glutamatergic signaling, protein degradation and memory destabilization in stress-induced resistant memories.


Assuntos
Complexo Nuclear Basolateral da Amígdala/metabolismo , Condicionamento Clássico/fisiologia , Medo , Consolidação da Memória/fisiologia , Rememoração Mental/fisiologia , Estresse Psicológico/metabolismo , Animais , Complexo Nuclear Basolateral da Amígdala/efeitos dos fármacos , Condicionamento Clássico/efeitos dos fármacos , Ciclosserina/farmacologia , Masculino , Memória/efeitos dos fármacos , Memória/fisiologia , Consolidação da Memória/efeitos dos fármacos , Rememoração Mental/efeitos dos fármacos , Poliubiquitina/metabolismo , Ratos , Ubiquitinação/efeitos dos fármacos
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