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1.
Cell ; 178(2): 316-329.e18, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31257023

RESUMO

Approximately 30% of human lung cancers acquire mutations in either Keap1 or Nfe2l2, resulting in the stabilization of Nrf2, the Nfe2l2 gene product, which controls oxidative homeostasis. Here, we show that heme triggers the degradation of Bach1, a pro-metastatic transcription factor, by promoting its interaction with the ubiquitin ligase Fbxo22. Nrf2 accumulation in lung cancers causes the stabilization of Bach1 by inducing Ho1, the enzyme catabolizing heme. In mouse models of lung cancers, loss of Keap1 or Fbxo22 induces metastasis in a Bach1-dependent manner. Pharmacological inhibition of Ho1 suppresses metastasis in a Fbxo22-dependent manner. Human metastatic lung cancer display high levels of Ho1 and Bach1. Bach1 transcriptional signature is associated with poor survival and metastasis in lung cancer patients. We propose that Nrf2 activates a metastatic program by inhibiting the heme- and Fbxo22-mediated degradation of Bach1, and that Ho1 inhibitors represent an effective therapeutic strategy to prevent lung cancer metastasis.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Neoplasias Pulmonares/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linhagem Celular Tumoral , Movimento Celular , Proteínas F-Box/antagonistas & inibidores , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Feminino , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Estimativa de Kaplan-Meier , Proteína 1 Associada a ECH Semelhante a Kelch/antagonistas & inibidores , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Metástase Neoplásica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Ativação Transcricional
2.
Mol Cell ; 83(22): 4123-4140.e12, 2023 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-37848033

RESUMO

Purinosomes serve as metabolons to enhance de novo purine synthesis (DNPS) efficiency through compartmentalizing DNPS enzymes during stressed conditions. However, the mechanism underpinning purinosome assembly and its pathophysiological functions remains elusive. Here, we show that K6-polyubiquitination of the DNPS enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) by cullin-5/ankyrin repeat and SOCS box containing 11 (Cul5/ASB11)-based ubiquitin ligase plays a driving role in purinosome assembly. Upon several purinosome-inducing cues, ASB11 is upregulated by relieving the H3K9me3/HP1α-mediated transcriptional silencing, thus stimulating PAICS polyubiquitination. The polyubiquitinated PAICS recruits ubiquitin-associated protein 2 (UBAP2), a ubiquitin-binding protein with multiple stretches of intrinsically disordered regions, thereby inducing phase separation to trigger purinosome assembly for enhancing DNPS pathway flux. In human melanoma, ASB11 is highly expressed to facilitate a constitutive purinosome formation to which melanoma cells are addicted for supporting their proliferation, viability, and tumorigenesis in a xenograft model. Our study identifies a driving mechanism for purinosome assembly in response to cellular stresses and uncovers the impact of purinosome formation on human malignancies.


Assuntos
Ligases , Melanoma , Humanos , Células HeLa , Ubiquitinação , Ubiquitinas
3.
EMBO Rep ; 25(2): 646-671, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38177922

RESUMO

The dorsoventral gradient of BMP signaling plays an essential role in embryonic patterning. Zinc Finger SWIM-Type Containing 4 (zswim4) is expressed in the Spemann-Mangold organizer at the onset of Xenopus gastrulation and is then enriched in the developing neuroectoderm at the mid-gastrula stages. Knockdown or knockout of zswim4 causes ventralization. Overexpression of zswim4 decreases, whereas knockdown of zswim4 increases the expression levels of ventrolateral mesoderm marker genes. Mechanistically, ZSWIM4 attenuates the BMP signal by reducing the protein stability of SMAD1 in the nucleus. Stable isotope labeling by amino acids in cell culture (SILAC) identifies Elongin B (ELOB) and Elongin C (ELOC) as the interaction partners of ZSWIM4. Accordingly, ZSWIM4 forms a complex with the Cul2-RING ubiquitin ligase and ELOB and ELOC, promoting the ubiquitination and degradation of SMAD1 in the nucleus. Our study identifies a novel mechanism that restricts BMP signaling in the nucleus.


Assuntos
Proteínas Morfogenéticas Ósseas , Proteínas de Transporte , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Organizadores Embrionários/metabolismo , Xenopus laevis/metabolismo , Padronização Corporal/fisiologia , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Regulação da Expressão Gênica no Desenvolvimento
4.
Biochem Biophys Res Commun ; 646: 30-35, 2023 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-36701892

RESUMO

In targeted protein degradation, immunomodulatory drugs (IMiDs) or cereblon (CRBN) E3 ligase modulatory drugs (CELMoDs) recruit neo-substrate proteins to the E3 ubiquitin ligase receptor CRBN for ubiquitination and subsequent proteasomal degradation. While the structural basis of this mechanism is generally understood, we have only recently described the recognition mode of the natural CRBN degron. In this communication, we reveal that the IMiD- or CELMoD-mediated binding of neo-substrates closely mimics the recognition of natural degrons. In crystal structures, we identify a conserved binding mode for natural degron peptides with an elaborate hydrogen bonding network involving the backbone of each of the six C-terminal degron residues, without the involvement of side chains. In a structural comparison, we show that neo-substrates recruited by IMiDs or CELMoDs emulate every single hydrogen bond of this network and thereby explain the origins of the largely sequence-independent recognition of neo-substrates. Our results imply that the V388I substitution in CRBN does not impair natural degron recognition and complete the structural basis for the rational design of CRBN effectors.


Assuntos
Agentes de Imunomodulação , Peptídeo Hidrolases , Peptídeo Hidrolases/metabolismo , Ubiquitinação , Ubiquitina-Proteína Ligases/metabolismo , Proteólise
5.
Cell Mol Life Sci ; 79(2): 86, 2022 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-35066747

RESUMO

Deubiquitinylases (DUBs) are central regulators of the ubiquitin system involved in protein regulation and cell signalling and are important for a variety of physiological processes. Most DUBs are cysteine proteases, and few other proteases are metalloproteases of the JAB1/MPN +/MOV34 protease family (JAMM). STAM-binding protein like 1 (STAMBPL1), a member of the JAMM family, cleaves ubiquitin bonds and has a function in regulating cell survival, Tax-mediated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and epithelial-mesenchymal transition. However, the molecular mechanism by which STAMBPL1 influences cell survival is not well defined, especially with regard to its deubiquitinylation function. Here, we show that reactive oxygen species (ROS) induced by chemotherapeutic agents or the human microbial pathogen Helicobacter pylori can induce cullin 1-RING ubiquitin ligase (CRL1) and 26S proteasome-dependent degradation STAMBPL1. Interestingly, STAMBPL1 has a direct interaction with the constitutive photomorphogenic 9 (COP9 or CSN) signalosome subunits CSN5 and CSN6. The interaction with the CSN is required for the stabilisation and function of the STAMBPL1 protein. In addition, STAMBPL1 deubiquitinylates the anti-apoptotic protein Survivin and thus ameliorates cell survival. In summary, our data reveal a previously unknown mechanism by which the deubiquitinylase STAMBPL1 and the E3 ligase CRL1 balance the level of Survivin degradation and thereby determine apoptotic cell death. In response to genotoxic stress, the degradation of STAMBPL1 augments apoptotic cell death. This new mechanism may be useful to develop therapeutic strategies targeting STAMBPL1 in tumours that have high STAMBPL1 and Survivin protein levels.


Assuntos
Apoptose , Complexo do Signalossomo COP9/metabolismo , Helicobacter pylori/fisiologia , Peptídeo Hidrolases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular , Doxorrubicina/farmacologia , Humanos , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Survivina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
6.
Bioessays ; 43(7): e2100057, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33857330

RESUMO

Deciphering how DCAFs (DDB1-CUL4 Associated Factors) modulate a broad spectrum of cellular processes, including cell cycle progression and maintenance of genomic integrity is critical to better understand cellular homeostasis and diseases. Cells contain more than 100 DCAFs that associate with the Cullin-Ring Ubiquitin Ligase 4 (CRL4) complex that target specific protein substrates for degradation. DCAFs are thought to act as substrate receptors that dictate the specificity of the ubiquitination machinery ("catalytic DCAFs"). However, recent studies have suggested that some DCAFs might play a different role by targeting CRL4 complexes to distinct cellular compartments ("structural DCAFs"). Once localized to their correct cellular domains, these CRLs dissociate from the structural DCAFs prior to their association with other, substrate-specific catalytic DCAFs. Thus, we propose that DCAF switches can provide a mechanistic basis for the degradation of proteins that regulate cell growth and proliferation at precise points in space and time.


Assuntos
Proteínas de Ligação a DNA , Ubiquitina-Proteína Ligases , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
7.
Proc Natl Acad Sci U S A ; 117(14): 7950-7960, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32193347

RESUMO

Intracellular pathogen infection leads to proteotoxic stress in host organisms. Previously we described a physiological program in the nematode Caenorhabditis elegans called the intracellular pathogen response (IPR), which promotes resistance to proteotoxic stress and appears to be distinct from canonical proteostasis pathways. The IPR is controlled by PALS-22 and PALS-25, proteins of unknown biochemical function, which regulate expression of genes induced by natural intracellular pathogens. We previously showed that PALS-22 and PALS-25 regulate the mRNA expression of the predicted ubiquitin ligase component cullin cul-6, which promotes thermotolerance in pals-22 mutants. However, it was unclear whether CUL-6 acted alone, or together with other cullin-ring ubiquitin ligase components, which comprise a greatly expanded gene family in C. elegans Here we use coimmunoprecipitation studies paired with genetic analysis to define the cullin-RING ligase components that act together with CUL-6 to promote thermotolerance. First, we identify a previously uncharacterized RING domain protein in the TRIM family we named RCS-1, which acts as a core component with CUL-6 to promote thermotolerance. Next, we show that the Skp-related proteins SKR-3, SKR-4, and SKR-5 act redundantly to promote thermotolerance with CUL-6. Finally, we screened F-box proteins that coimmunoprecipitate with CUL-6 and find that FBXA-158 and FBXA-75 promote thermotolerance. In summary, we have defined the three core components and two F-box adaptors of a cullin-RING ligase complex that promotes thermotolerance as part of the IPR in C. elegans, which adds to our understanding of how organisms cope with proteotoxic stress.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/imunologia , Proteínas Culina/metabolismo , Proteínas F-Box/metabolismo , Microsporídios/imunologia , Termotolerância/imunologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/microbiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/imunologia , Proteínas Culina/genética , Proteínas Culina/imunologia , Proteínas F-Box/imunologia , Interações Hospedeiro-Patógeno/imunologia , Modelos Animais , Proteostase/imunologia
8.
EMBO J ; 36(3): 260-273, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28007894

RESUMO

The F-box protein FBXW7 is the substrate-recruiting subunit of an SCF ubiquitin ligase and a major tumor-suppressor protein that is altered in several human malignancies. Loss of function of FBXW7 results in the stabilization of numerous proteins that orchestrate cell proliferation and survival. Little is known about proteins that directly regulate the function of this protein. In the current work, we have mapped the interactome of the enigmatic pseudophosphatase STYX We reasoned that a catalytically inactive phosphatase might have adopted novel mechanisms of action. The STYX interactome contained several F-box proteins, including FBXW7. We show that STYX binds to the F-box domain of FBXW7 and disables its recruitment into the SCF complex. Therefore, STYX acts as a direct inhibitor of FBXW7, affecting the cellular levels of its substrates. Furthermore, we find that levels of STYX and FBXW7 are anti-correlated in breast cancer patients, which affects disease prognosis. We propose the STYX-FBXW7 interaction as a promising drug target for future investigations.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas F-Box/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Ligases SKP Culina F-Box/antagonistas & inibidores , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Proteína 7 com Repetições F-Box-WD , Humanos
9.
Adv Exp Med Biol ; 1217: 61-78, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31898222

RESUMO

The cullin-RING ubiquitin ligases comprise the largest subfamily of ubiquitin ligases. They control ubiquitylation and degradation of a large number of protein substrates in eukaryotes. p97 is an ATPase domain-containing protein segregase. It plays essential roles in post-ubiquitylational events in the ubiquitin-proteasome pathway. Together with its cofactors, p97 collaborates with ubiquitin ligases to extract ubiquitylated substrates and deliver them to the proteasome for proteolysis. Here we review the structure, functions, and mechanisms of p97 in cellular protein degradation in coordination with its cofactors and the cullin-RING ubiquitin ligases.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Culina/metabolismo , Proteínas Nucleares/metabolismo , Proteólise , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação
10.
Biochim Biophys Acta Mol Cell Res ; 1864(8): 1405-1412, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28499918

RESUMO

Cullin 3 (Cul3) belongs to the family of cullins (Cul1-7) providing the scaffold for cullin-RING ubiquitin (Ub) ligases (CRLs), which are activated by neddylation and represent essential E3 ligases of the Ub proteasome system. During adipogenic differentiation neddylated Cul3 accumulates in LiSa-2 preadipocytes. Downregulation of Cul3 and inhibition of neddylation by MLN4924 blocks the formation of lipid droplets (LDs), the lipid storage organelles and markers of adipogenesis. Neddylation of Cul3 coincides with an increase of Rab18, a GTPase associated with LDs. Immunoprecipitation and confocal fluorescence microscopy revealed physical association of Cul3 and Rab18 at the membrane of LDs. RhoA, a suppressor of adipogenesis decreased during differentiation. Our results in LiSa-2 cells, but also mouse embryonic fibroblasts revealed a connection between Cul3, Rab18 and RhoA. Downregulation of Cul3 led to a marked increase in RhoA protein expression after 6days of LiSa-2 cell differentiation, suggesting that Cul3 is involved in the regulation of RhoA stability.


Assuntos
Adipócitos/metabolismo , Adipogenia/genética , Proteínas Culina/genética , Processamento de Proteína Pós-Traducional , Proteínas rab de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/genética , Adipócitos/citologia , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Proteínas Culina/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Gotículas Lipídicas , Camundongos , Proteína NEDD8 , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma , Proteólise , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ubiquitinas/genética , Ubiquitinas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
11.
Am J Physiol Renal Physiol ; 315(4): F1006-F1018, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29897280

RESUMO

Familial hyperkalemic hypertension is caused by mutations in with-no-lysine kinases (WNKs) or in proteins that mediate their degradation, kelch-like 3 (KLHL3) and cullin 3 (CUL3). Although the mechanisms by which WNK and KLHL3 mutations cause the disease are now clear, the effects of the disease-causing CUL3Δ403-459 mutation remain controversial. Possible mechanisms, including hyperneddylation, altered ubiquitin ligase activity, decreased association with the COP9 signalosome (CSN), and increased association with and degradation of KLHL3 have all been postulated. Here, we systematically evaluated the effects of Cul3Δ403-459 using cultured kidney cells. We first identified that the catalytically active CSN subunit jun activation domain-binding protein-1 (JAB1) does not associate with the deleted Cul3 4-helix bundle domain but instead with the adjacent α/ß1 domain, suggesting that altered protein folding underlies the impaired binding. Inhibition of deneddylation with JAB1 siRNA increased Cul3 neddylation and decreased KLHL3 abundance, similar to the Cul3 mutant. We next determined that KLHL3 degradation has both ubiquitin ligase-dependent and -independent components. Proteasomal KLHL3 degradation was enhanced by Cul3Δ403-459; however, autophagic degradation was also upregulated by this Cul3 mutant. Finally, to evaluate whether deficient substrate adaptor was responsible for the disease, we restored KLHL3 to wild-type (WT) Cul3 levels. In the absence of WT Cul3, WNK4 was not degraded, demonstrating that Cul3Δ403-459 itself cannot degrade WNK4; conversely, when WT Cul3 was present, as in diseased humans, WNK4 degradation was restored. In conclusion, deletion of exon 9 from Cul3 generates a protein that is itself ubiquitin-ligase defective but also capable of enhanced autophagocytic KLHL3 degradation, thereby exerting dominant-negative effects on the WT allele.


Assuntos
Complexo do Signalossomo COP9/metabolismo , Proteínas Culina/metabolismo , Hipertensão/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/metabolismo , Proteínas Culina/genética , Rim/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Mutação/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitinação/genética , Ubiquitinação/fisiologia
12.
Cell Rep ; 43(6): 114279, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38795346

RESUMO

Heat shock can be a lethal stressor. Previously, we described a CUL-6/cullin-ring ubiquitin ligase complex in the nematode Caenorhabditis elegans that is induced by intracellular intestinal infection and proteotoxic stress and that promotes improved survival upon heat shock (thermotolerance). Here, we show that CUL-6 promotes thermotolerance by targeting the heat shock protein HSP-90 for degradation. We show that CUL-6-mediated lowering of HSP-90 protein levels, specifically in the intestine, improves thermotolerance. Furthermore, we show that lysosomal function is required for CUL-6-mediated promotion of thermotolerance and that CUL-6 directs HSP-90 to lysosome-related organelles upon heat shock. Altogether, these results indicate that a CUL-6 ubiquitin ligase promotes organismal survival upon heat shock by promoting HSP-90 degradation in intestinal lysosomes. Thus, HSP-90, a protein commonly associated with protection against heat shock and promoting degradation of other proteins, is itself degraded to protect against heat shock.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Proteínas de Choque Térmico HSP90 , Lisossomos , Termotolerância , Animais , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Culina/metabolismo , Resposta ao Choque Térmico , Proteínas de Choque Térmico HSP90/metabolismo , Intestinos , Lisossomos/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo
13.
Methods Mol Biol ; 2581: 31-42, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36413308

RESUMO

CRL (Cullin-Ring ubiquitin ligases) are the major class of plant E3 ubiquitin ligases. Immunoprecipitation-based methods are useful techniques for revealing interactions among Cullin-Ring Ligase (CRL) subunits or between CRLs and other proteins, as well as for detecting poly-ubiquitin modifications of the CRLs themselves. Here, we describe two immunoprecipitation (IP) procedures suitable for CRLs in Arabidopsis: (1) a procedure for IP analysis of CRL subunits and their interactors and a second procedure for in vivo ubiquitination analysis of the CRLs. Both protocols can be divided into two major steps: (1) preparation of cell extracts without disruption of protein interactions and (2) affinity purification of the protein complexes and subsequent detection. We provide a thorough description of all the steps, as well as advice on how to choose proper buffers for these analyses. We also suggest a series of negative controls that can be used to verify the specificity of the procedure.


Assuntos
Arabidopsis , Arabidopsis/metabolismo , Plântula/metabolismo , Proteínas Culina/metabolismo , Ubiquitina/metabolismo , Imunoprecipitação
14.
Viruses ; 13(8)2021 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-34452475

RESUMO

Human cytomegalovirus causes diseases in individuals with insufficient immunity. Cytomegaloviruses exploit the ubiquitin proteasome pathway to manipulate the proteome of infected cells. The proteasome degrades ubiquitinated proteins. The family of cullin RING ubiquitin ligases (CRL) regulates the stability of numerous important proteins. If the cullin within the CRL is modified with Nedd8 ("neddylated"), the CRL is enzymatically active, while CRLs lacking Nedd8 modifications are inactive. The Nedd8-activating enzyme (NAE) is indispensable for neddylation. By binding to NAE and inhibiting neddylation, the drug MLN4924 (pevonedistat) causes CRL inactivation and stabilization of CRL target proteins. We showed that MLN4924 elicits potent antiviral activity against cytomegaloviruses, suggesting that NAE might be a druggable host dependency factor (HDF). However, MLN4924 is a nucleoside analog related to AMP, and the antiviral activity of MLN4924 may have been influenced by off-target effects in addition to NAE inhibition. To test if NAE is indeed an HDF, we assessed the novel NAE inhibitor TAS4464 and observed potent antiviral activity against mouse and human cytomegalovirus. Additionally, we raised an MLN4924-resistant cell clone and showed that MLN4924 as well as TAS4464 lose their antiviral activity in these cells. Our results indicate that NAE, the neddylation process, and CRLs are druggable HDFs of cytomegaloviruses.


Assuntos
Antivirais/farmacologia , Citomegalovirus/efeitos dos fármacos , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Muromegalovirus/efeitos dos fármacos , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Enzimas Ativadoras de Ubiquitina/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proteínas Culina/metabolismo , Ciclopentanos/metabolismo , Citomegalovirus/patogenicidade , Humanos , Camundongos , Muromegalovirus/patogenicidade , Proteína NEDD8/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Pirróis/farmacologia , Enzimas Ativadoras de Ubiquitina/genética , Ubiquitina-Proteína Ligases/metabolismo
15.
Mol Cells ; 43(11): 935-944, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-33168788

RESUMO

Aryl hydrocarbon receptor nuclear translocator (ARNT) plays an essential role in maintaining cellular homeostasis in response to environmental stress. Under conditions of hypoxia or xenobiotic exposure, ARNT regulates the subset of genes involved in adaptive responses, by forming heterodimers with hypoxia-inducible transcription factors (HIF1α and HIF2α) or aryl hydrocarbon receptor (AhR). Here, we have shown that ARNT interacts with DDB1 and CUL4-associated factor 15 (DCAF15), and the aryl sulfonamides, indisulam and E7820, induce its proteasomal degradation through Cullin-RING finger ligase 4 containing DCAF15 (CRL4DCAF15) E3 ligase. Moreover, the two known neo-substrates of aryl sulfonamide, RNA-binding motif protein 39 (RBM39) and RNA-binding motif protein 23 (RBM23), are not required for ARNT degradation. In line with this finding, aryl sulfonamides inhibited the transcriptional activities of HIFs and AhR associated with ARNT. Our results collectively support novel regulatory roles of aryl sulfonamides in both hypoxic and xenobiotic responses.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Sulfonamidas/uso terapêutico , Ubiquitina-Proteína Ligases/metabolismo , Animais , Humanos , Sulfonamidas/farmacologia , Transfecção
16.
Genes Genomics ; 41(2): 167-174, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30267325

RESUMO

Beta-transducin repeat containing protein 1 (ß-TrCP1) is a versatile F-box protein that is responsible for substrate recognition of SCFß-TrCP1 ubiquitin ligase. In human cells, two major alternatively spliced isoforms (b and f) of ß-TrCP1 were found. Recently, we identified that CENP-W interacts with the ß-TrCP1 and regulates the cellular distribution of ß-TrCP1. In this study, we examined whether CENP-W, a new kinetochore component, may differentially regulate the two major isoforms of human ß-TrCP1 (b and f), especially in the cytoplasmic-nuclear shuttling of ß-TrCP1. An in vivo binding assay was performed to examine whether CENP-W binds differently to the two isoforms of ß-TrCP1. EGFP-conjugated ß-TrCP1 isoforms were co-transfected with NLS-defective mutant CENP-W and their cellular distribution were observed using a fluorescence microscopy. Although CENP-W interacts with both b and f isoforms, it has a greater affinity for the b isoform rather than f isoform. Moreover, CENP-W effectively regulates the nuclear-cytoplasmic shuttling of these two ß-TrCP1 isoforms, but with a slight preference towards the b isoform. The Elongin C-binding motif existing in the b isoform may be involved in their specific association. CENP-W showed a higher affinity toward the ß-TrCP1 b isoform, and translocated isoform b more efficiently than isoform f, which may allow a fine regulation of of ß-TrCP1 in the cells.


Assuntos
Processamento Alternativo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Sítios de Ligação , Células HEK293 , Células HeLa , Humanos , Ligação Proteica , Isoformas de Proteínas/metabolismo , Proteínas Contendo Repetições de beta-Transducina/química
17.
mBio ; 10(3)2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31213557

RESUMO

E3 cullin-RING ubiquitin ligase (CRL) complexes recognize specific substrates and are activated by covalent modification with ubiquitin-like Nedd8. Deneddylation inactivates CRLs and allows Cand1/A to bind and exchange substrate recognition subunits. Human as well as most fungi possess a single gene for the receptor exchange factor Cand1, which is split and rearranged in aspergilli into two genes for separate proteins. Aspergillus nidulans CandA-N blocks the neddylation site, and CandA-C inhibits the interaction to the adaptor/substrate receptor subunits similar to the respective N-terminal and C-terminal parts of single Cand1. The pathogen Aspergillus fumigatus and related species express a CandA-C with a 190-amino-acid N-terminal extension domain encoded by an additional exon. This extension corresponds in most aspergilli, including A. nidulans, to a gene directly upstream of candA-C encoding a 20-kDa protein without human counterpart. This protein was named CandA-C1, because it is also required for the cellular deneddylation/neddylation cycle and can form a trimeric nuclear complex with CandA-C and CandA-N. CandA-C and CandA-N are required for asexual and sexual development and control a distinct secondary metabolism. CandA-C1 and the corresponding domain of A. fumigatus control spore germination, vegetative growth, and the repression of additional secondary metabolites. This suggests that the dissection of the conserved Cand1-encoding gene within the genome of aspergilli was possible because it allowed the integration of a fungus-specific protein required for growth into the CandA complex in two different gene set versions, which might provide an advantage in evolution.IMPORTANCEAspergillus species are important for biotechnological applications, like the production of citric acid or antibacterial agents. Aspergilli can cause food contamination or invasive aspergillosis to immunocompromised humans or animals. Specific treatment is difficult due to limited drug targets and emerging resistances. The CandA complex regulates, as a receptor exchange factor, the activity and substrate variability of the ubiquitin labeling machinery for 26S proteasome-mediated protein degradation. Only Aspergillus species encode at least two proteins that form a CandA complex. This study shows that Aspergillus species had to integrate a third component into the CandA receptor exchange factor complex that is unique to aspergilli and required for vegetative growth, sexual reproduction, and activation of the ubiquitin labeling machinery. These features have interesting implications for the evolution of protein complexes and could make CandA-C1 an interesting candidate for target-specific drug design to control fungal growth without affecting the human ubiquitin-proteasome system.


Assuntos
Aspergillus nidulans/enzimologia , Aspergillus nidulans/genética , Proteínas Culina/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ubiquitina-Proteína Ligases/genética , Complexos Multiproteicos , Ubiquitina/metabolismo
18.
Cell Rep ; 23(11): 3381-3391.e4, 2018 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-29898406

RESUMO

Although much is known about how chromosome segregation is coupled to cell division, how intracellular organelles partition during mitotic division is poorly understood. We report that the phosphorylation-dependent degradation of the ARFGEF GBF1 regulates organelle trafficking during cell division. We show that, in mitosis, GBF1 is phosphorylated on Ser292 and Ser297 by casein kinase-2 allowing recognition by the F-box protein ßTrCP. GBF1 interaction with ßTrCP recruits GBF1 to the SCFßTrCP ubiquitin ligase complex, triggering its degradation. Phosphorylation and degradation of GBF1 occur along microtubules at the intercellular bridge of telophase cells and are required for Golgi membrane positioning and postmitotic Golgi reformation. Indeed, expression of a non-degradable GBF1 mutant inhibits the transport of the Golgi cluster adjacent to the midbody toward the Golgi twin positioned next to the centrosome and results in defective Golgi reassembly and cytokinesis failure. These findings define a mechanism that controls postmitotic Golgi reassembly and inheritance.


Assuntos
Citocinese , Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Caseína Quinase II/metabolismo , Linhagem Celular Tumoral , Centrossomo/metabolismo , Citocinese/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Humanos , Microscopia Confocal , Mitose , Mutagênese , Nocodazol/farmacologia , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Imagem com Lapso de Tempo , Proteínas Contendo Repetições de beta-Transducina/antagonistas & inibidores , Proteínas Contendo Repetições de beta-Transducina/genética , Proteínas Contendo Repetições de beta-Transducina/metabolismo
19.
Mol Cell Oncol ; 4(2): e1277293, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28401182

RESUMO

Mitosis inhibitors, which include antimicrotubule drugs, are chemotherapy agents that induce the arrest and apoptosis of mitotic cells. Mitotic slippage, in which mitotically arrested cells exit mitosis, limits the effectiveness of mitosis inhibitors. We have discovered that the CRL2ZYG11A/B ubiquitin ligase promotes mitotic slippage. The combination of antimicrotubule drugs and a CRL2ZYG11A/B inhibitor prevents mitotic slippage to increase antimitotic efficacy.

20.
Methods Mol Biol ; 1450: 11-21, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27424742

RESUMO

CRL (Cullin-RING ubiquitin ligase) is the major class of plant E3 ubiquitin ligases. Immunoprecipitation-based methods are useful techniques for revealing interactions among Cullin-RING Ligase (CRL) subunits or between CRLs and other proteins, as well as for detecting poly-ubiquitin modifications of the CRLs themselves. Here, we describe two immunoprecipitation (IP) procedures suitable for CRLs in Arabidopsis: a procedure for IP analysis of CRL subunits and their interactors and a second procedure for in vivo ubiquitination analysis of the CRLs. Both protocols can be divided into two major steps: (1) preparation of cell extracts without disruption of protein interactions and (2) affinity purification of the protein complexes and subsequent detection. We provide a thorough description of all the steps, as well as advice on how to choose proper buffers for these analyses. We also suggest a series of negative controls that can be used to verify the specificity of the procedure.


Assuntos
Proteínas Culina/isolamento & purificação , Biologia Molecular/métodos , Ubiquitina-Proteína Ligases/química , Arabidopsis/enzimologia , Proteínas Culina/química , Plântula/química , Plântula/enzimologia , Ubiquitinação
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