RESUMEN
Multiple sclerosis, and its murine model experimental autoimmune encephalomyelitis (EAE), is a neurodegenerative autoimmune disease of the CNS characterized by T cell influx and demyelination. Similar to other autoimmune diseases, therapies can alleviate symptoms but often come with side effects, necessitating the exploration of new treatments. We recently demonstrated that the Cullin-RING E3 ubiquitin ligase 4b (CRL4b) aided in maintaining genome stability in proliferating T cells. In this study, we examined whether CRL4b was required for T cells to expand and drive EAE. Mice lacking Cul4b (Cullin 4b) in T cells had reduced EAE symptoms and decreased inflammation during the peak of the disease. Significantly fewer CD4+ and CD8+ T cells were found in the CNS, particularly among the CD4+ T cell population producing IL-17A, IFN-γ, GM-CSF, and TNF-α. Additionally, Cul4b-deficient CD4+ T cells cultured in vitro with their wild-type counterparts were less likely to expand and differentiate into IL-17A- or IFN-γ-producing effector cells. When wild-type CD4+ T cells were activated in vitro in the presence of the recently developed CRL4 inhibitor KH-4-43, they exhibited increased apoptosis and DNA damage. Treatment of mice with KH-4-43 following EAE induction resulted in stabilized clinical scores and significantly reduced numbers of T cells and innate immune cells in the CNS compared with control mice. Furthermore, KH-4-43 treatment resulted in elevated expression of p21 and cyclin E2 in T cells. These studies support that therapeutic inhibition of CRL4 and/or CRL4-related pathways could be used to treat autoimmune disease.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Ratones , Animales , Interleucina-17/metabolismo , Proteínas Cullin/metabolismo , Linfocitos T CD4-Positivos , Ratones Endogámicos C57BLRESUMEN
Cullin (CUL)-RING (Really Interesting New Gene) E3 ubiquitin (Ub) ligases (CRLs) are the largest E3 family. The E3 CRL core ligase is a subcomplex formed by the CUL C-terminal domain bound with the ROC1/RBX1 RING finger protein, which acts as a hub that mediates and organizes multiple interactions with E2, Ub, Nedd8, and the ARIH family protein, thereby resulting in Ub transfer to the E3-bound substrate. This report describes the modulation of CRL-dependent ubiquitination by small molecule compounds including KH-4-43, #33, and suramin, which target the CRL core ligases. We show that both KH-4-43 and #33 inhibit the ubiquitination of CK1α by CRL4CRBN. However, either compound's inhibitory effect on this reaction is significantly reduced when a neddylated form of CRL4CRBN is used. On the other hand, both #33 and KH-4-43 inhibit the ubiquitination of ß-catenin by CRL1ß-TrCP and Nedd8-CRL1ß-TrCP almost equally. Thus, neddylation of CRL1ß-TrCP does not negatively impact the sensitivity to inhibition by #33 and KH-4-43. These findings suggest that the effects of neddylation to alter the sensitivity of CRL inhibition by KH-4-43/#33 is dependent upon the specific CRL type. Suramin, a compound that targets CUL's basic canyon, can effectively inhibit CRL1/4-dependent ubiquitination regardless of neddylation status, in contrast to the results observed with KH-4-43/#33. This observed differential drug sensitivity of KH-4-43/#33 appears to echo CUL-specific Nedd8 effects on CRLs as revealed by recent high-resolution structural biology efforts. The highly diversified CRL core ligase structures may provide opportunities for specific targeting by small molecule modulators.
Asunto(s)
Ligandos , Ubiquitina-Proteína Ligasas , Ubiquitinación , Animales , Humanos , Ratones , beta Catenina/metabolismo , Proteínas con Repetición de beta-Transducina/metabolismo , Proteínas Cullin/metabolismo , Suramina/farmacología , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/efectos de los fármacos , Proteína NEDD8/metabolismoRESUMEN
Ubiquitination often generates lysine 48-linked polyubiquitin chains that signal proteolytic destruction of the protein target. A significant subset of ubiquitination proceeds by a priming/extending mechanism, in which a substrate is first monoubiquitinated with a priming E2-conjugating enzyme or a set of E3 ARIH/E2 enzymes specific for priming. This is then followed by ubiquitin (Ub) chain extension catalyzed by an E2 enzyme capable of elongation. This report provides further insights into the priming/extending mechanism. We employed reconstituted ubiquitination systems of substrates CK1α (casein kinase 1α) and ß-catenin by Cullin-RING E3 Ub ligases (CRLs) CRL4CRBN and CRL1ßTrCP, respectively, in the presence of priming E2 UbcH5c and elongating E2 Cdc34b (cell division cycle 34b). We have established a new "apyrase chase" strategy that uncouples priming from chain elongation, which allows accurate measurement of the decay rates of the ubiquitinated substrate with a defined chain length. Our work has revealed highly robust turnover of monoubiquitinated ß-catenin that empowers efficient polyubiquitination. The results of competition experiments suggest that the interactions between the ubiquitinated ß-catenin and CRL1ßTrCP are highly dynamic. Moreover, ubiquitination of the Ub-modified ß-catenin appeared more resistant to inhibition by competitors than the unmodified substrate, suggesting tighter binding with CRL1ßTrCP. These findings support a role for conjugated Ub in enhancing interactions with E3.
Asunto(s)
Ubiquitina , Ubiquitinación , beta Catenina , beta Catenina/metabolismo , Proteínas con Repetición de beta-Transducina/metabolismo , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Cullin-RING (really intersting new gene) E3 ubiquitin ligases (CRLs) are the largest E3 family and direct numerous protein substrates for proteasomal degradation, thereby impacting a myriad of physiological and pathological processes including cancer. To date, there are no reported small-molecule inhibitors of the catalytic activity of CRLs. Here, we describe high-throughput screening and medicinal chemistry optimization efforts that led to the identification of two compounds, 33-11 and KH-4-43, which inhibit E3 CRL4 and exhibit antitumor potential. These compounds bind to CRL4's core catalytic complex, inhibit CRL4-mediated ubiquitination, and cause stabilization of CRL4's substrate CDT1 in cells. Treatment with 33-11 or KH-4-43 in a panel of 36 tumor cell lines revealed cytotoxicity. The antitumor activity was validated by the ability of the compounds to suppress the growth of human tumor xenografts in mice. Mechanistically, the compounds' cytotoxicity was linked to aberrant accumulation of CDT1 that is known to trigger apoptosis. Moreover, a subset of tumor cells was found to express cullin4 proteins at levels as much as 70-fold lower than those in other tumor lines. The low-cullin4-expressing tumor cells appeared to exhibit increased sensitivity to 33-11/KH-4-43, raising a provocative hypothesis for the role of low E3 abundance as a cancer vulnerability.
Asunto(s)
Antineoplásicos/farmacología , Biomarcadores de Tumor/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Animales , Antineoplásicos/química , Apoptosis , Biomarcadores de Tumor/genética , Proliferación Celular , Inhibidores Enzimáticos/química , Femenino , Humanos , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Tumorales Cultivadas , Ubiquitina/metabolismo , Ubiquitinación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Post-translational modification of protein by ubiquitin (Ub) alters the stability, subcellular location, or function of the target protein, thereby impacting numerous biological processes and directly contributing to myriad cellular defects or disease states, such as cancer. Tracking substrate ubiquitination by fluorescence provides opportunities for advanced reaction dynamics studies and for translational research including drug discovery. However, fluorescence-based techniques in ubiquitination studies remain underexplored at least partly because of challenges associated with Ub chain complexity and requirement for additional substrate modification. Here we describe a general strategy, FRET diubiquitination, to track substrate ubiquitination by fluorescence. This platform produces a uniform di-Ub product depending on specific interactions between a substrate and its cognate E3 Ub ligase. The diubiquitination creates proximity between the Ub-linked donor and acceptor fluorophores, respectively, enabling energy transfer to yield a distinct fluorescent signal. FRET diubiquitination relies on Ub-substrate fusion, which can be implemented using either one of the two validated strategies. Method 1 is the use of recombinant substrate-Ub fusion, applicable to all substrate peptides that can bind to E3. Method 2 is a chemoenzymatic ligation approach that employs synthetic chemistry to fuse Ub with a substrate peptide containing desired modification. Taken together, our new FRET-based diubiquitination system provides a timely technology of potential to advance both basic research and translation sciences.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Inhibidor NF-kappaB alfa/metabolismo , Procesamiento Proteico-Postraduccional , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , beta Catenina/metabolismo , Secuencia de Aminoácidos , Colorantes Fluorescentes/química , Humanos , Inhibidor NF-kappaB alfa/genética , Péptidos/genética , Péptidos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor de Células Madre/genética , Factor de Células Madre/metabolismo , Ubiquitina/genética , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , beta Catenina/genéticaRESUMEN
Human homolog of mouse double minute 2 (HDM2) is an oncogene frequently overexpressed in cancers with poor prognosis, but mechanisms of controlling its abundance remain elusive. In an unbiased biochemical search, we discovered Skp1-Cullin 1-FBXO22-ROC1 (SCFFBXO22) as the most dominating HDM2 E3 ubiquitin ligase from human proteome. The results of protein decay rate analysis, ubiquitination, siRNA-mediated silencing, and coimmunoprecipitation experiments support a hypothesis that FBXO22 targets cellular HDM2 for ubiquitin-dependent degradation. In human breast cancer cells, FBXO22 knockdown (KD) increased cell invasiveness, which was driven by elevated levels of HDM2. Moreover, mouse 4T1 breast tumor model studies revealed that FBXO22 KD led to a significant increase of breast tumor cell metastasis to the lung. Finally, low FBXO22 expression is correlated with worse survival and high HDM2 expression in human breast cancer. Altogether, these findings suggest that SCFFBXO22 targets HDM2 for degradation and possesses inhibitory effects against breast cancer tumor cell invasion and metastasis.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Movimiento Celular/fisiología , Proteínas F-Box/metabolismo , Invasividad Neoplásica/patología , Metástasis de la Neoplasia/patología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Línea Celular Tumoral , Femenino , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Procesos Neoplásicos , ARN Interferente Pequeño/metabolismo , Transfección/métodos , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/fisiologíaRESUMEN
The COP9 signalosome (CSN) is an evolutionarily conserved multisubunit protein complex, which controls protein degradation through deneddylation and inactivation of cullin-RING ubiquitin E3 ligases (CRLs). Recently, the CSN complex has been linked to the NF-κB signaling pathway due to its association with the IKK complex. However, how the CSN complex is regulated in this signaling pathway remains unclear. Here, we have carried out biochemical experiments and confirmed the interaction between the CSN and IKK complexes. In addition, we have determined that overexpression of IKKα or IKKß leads to enhanced phosphorylation of CSN5, the catalytic subunit for CSN deneddylase activity. Mutational analyses have revealed that phosphorylation at serine 201 and threonine 205 of CSN5 impairs CSN-mediated deneddylation activity in vitro. Interestingly, TNF-α treatment not only enhances the interaction between CSN and IKK but also induces an IKK-dependent phosphorylation of CSN5 at serine 201, linking CSN to TNF-α signaling through IKK. Moreover, TNF-α treatment affects the CSN interaction network globally, especially the associations of CSN with the proteasome complex, eukaryotic translation initiation factor complex, and CRL components. Collectively, our results provide new insights into IKK-mediated regulation of CSN associated with the NF-κB signaling pathway.
Asunto(s)
FN-kappa B , Transducción de Señal , Complejo del Señalosoma COP9/genética , Complejo del Señalosoma COP9/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , FosforilaciónRESUMEN
CRL7Fbxw8 is an E3 ubiquitin ligase complex, containing cullin7 (CUL7) as a scaffold, the F-box protein Fbxw8 as a substrate receptor, the Skp1 adaptor, and the ROC1/Rbx1 RING finger protein for working with E2 enzyme to facilitate ubiquitin transfer. This chapter provides an update on studies linking CRL7Fbxw8 to hereditary human growth retardation disease, as at least 64 cul7 germ line mutations were found in patients with autosomal recessive 3-M syndrome. CRL7Fbxw8 interacts with two additional 3-M associated proteins OBSL1 and CCDC8, leading to subcellular localization of the E3 complex to regions including plasma membrane, centrosome, and Golgi. At least ten mammalian cellular proteins were identified or implicated as CRL7Fbxw8 substrates. Discussion focuses on the possible impact of CRL7Fbxw8-mediated proteolytic or non-proteolytic pathways in growth control and cancer.
Asunto(s)
Proteínas Cullin/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Animales , Proteínas Portadoras/metabolismo , Proliferación Celular , Proteínas del Citoesqueleto/metabolismo , Proteínas F-Box/metabolismo , Humanos , Proteolisis , Ubiquitina/metabolismoRESUMEN
Cullin-RING E3 ubiquitin ligases (CRL) control a myriad of biological processes by directing numerous protein substrates for proteasomal degradation. Key to CRL activity is the recruitment of the E2 ubiquitin-conjugating enzyme Cdc34 through electrostatic interactions between E3's cullin conserved basic canyon and the acidic C terminus of the E2 enzyme. This report demonstrates that a small-molecule compound, suramin, can inhibit CRL activity by disrupting its ability to recruit Cdc34. Suramin, an antitrypansomal drug that also possesses antitumor activity, was identified here through a fluorescence-based high-throughput screen as an inhibitor of ubiquitination. Suramin was shown to target cullin 1's conserved basic canyon and to block its binding to Cdc34. Suramin inhibits the activity of a variety of CRL complexes containing cullin 2, 3, and 4A. When introduced into cells, suramin induced accumulation of CRL substrates. These observations help develop a strategy of regulating ubiquitination by targeting an E2-E3 interface through small-molecule modulators.
Asunto(s)
Ligasas/antagonistas & inhibidores , Suramina/farmacología , Relación Estructura-ActividadRESUMEN
We describe a mechanistic model of polyubiquitination by the SCF(beta TrCP2) E3 ubiquitin (Ub) ligase using human I kappaB alpha as a substrate. Biochemical reconstitution experiments revealed that the polyubiquitination of I kappaB alpha began with the action of the UbcH5 E2 Ub-conjugating enzyme, transferring a single Ub to I kappaB alpha K21/K22 rapidly and efficiently. Subsequently, the Cdc34 E2 functioned in the formation of polyubiquitin chains. It was determined that a Ub fused at I kappaB alpha K21 acts as a receptor, directing Cdc34 for rapid and efficient K48-linked Ub chain synthesis that depends on SCF(beta TrCP2) and the substrate's N terminus. The I kappaB alpha-linked fusion Ub appears to mediate direct contacts with Cdc34 and the SCF's RING subcomplex. Taken together, these results suggest a role for the multifaceted interactions between the I kappaB alpha K21/K22-linked receptor Ub, the SCF's RING complex, and Cdc34 approximately S approximately Ub in establishing the optimal orientation of the receptor Ub to drive conjugation.
Asunto(s)
Proteínas Ligasas SKP Cullina F-box/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitinación , Ciclosoma-Complejo Promotor de la Anafase , Biocatálisis , Línea Celular Tumoral , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , ARN Interferente Pequeño/genética , Especificidad por Sustrato , Enzimas Ubiquitina-Conjugadoras/genética , Complejos de Ubiquitina-Proteína Ligasa/genéticaRESUMEN
Simian virus 40 (SV40) large tumor antigen (LT) triggers oncogenic transformation by inhibition of key tumor suppressor proteins, including p53 and members of the retinoblastoma family. In addition, SV40 transformation requires binding of LT to Cullin 7 (CUL7), a core component of Cullin-RING E3 ubiquitin ligase 7 (CRL7). However, the pathomechanistic effects of LT-CUL7 interaction are mostly unknown. Here we report both in vitro and in vivo experimental evidence that SV40 LT suppresses the ubiquitin ligase function of CRL7. We show that SV40 LT, but not a CUL7 binding-deficient mutant (LT(Δ69-83)), impaired 26S proteasome-dependent proteolysis of the CRL7 target protein insulin receptor substrate 1 (IRS1), a component of the insulin and insulin-like growth factor 1 signaling pathway. SV40 LT expression resulted in the accumulation and prolonged half-life of IRS1. In vitro, purified SV40 LT reduced CRL7-dependent IRS1 ubiquitination in a concentration-dependent manner. Expression of SV40 LT, or depletion of CUL7 by RNA interference, resulted in the enhanced activation of IRS1 downstream signaling pathways phosphatidylinositol-3-kinase/AKT and Erk mitogen-activated pathway kinase, as well as up-regulation of the downstream target gene c-fos. Finally, SV40 LT-positive carcinoma of carcinoembryonic antigen 424/SV40 LT transgenic mice displayed elevated IRS1 protein levels and activation of downstream signaling. Taken together, these data suggest that SV40 LT protects IRS1 from CRL7-mediated degradation, thereby sustaining high levels of promitogenic IRS1 downstream signaling pathways.
Asunto(s)
Antígenos Virales de Tumores/metabolismo , Proteínas Cullin/antagonistas & inhibidores , Proteínas Sustrato del Receptor de Insulina/metabolismo , Transducción de Señal/fisiología , Virus 40 de los Simios/química , Análisis de Varianza , Animales , Proteínas Cullin/metabolismo , Electroforesis en Gel de Poliacrilamida , Células HEK293 , Humanos , Inmunoprecipitación , Ratones , Ratones Transgénicos , Microscopía , Microscopía Fluorescente , Proteolisis , Interferencia de ARN , Virus 40 de los Simios/metabolismo , Ubiquitina/metabolismoRESUMEN
Lysine 48 (K48)-polyubiquitination is the predominant mechanism for mediating selective protein degradation, but the underlying molecular basis of selecting ubiquitin (Ub) K48 for linkage-specific chain synthesis remains elusive. Here, we present biochemical, structural, and cell-based evidence demonstrating a pivotal role for the Ub Y59-E51 loop in supporting K48-polyubiquitination. This loop is established by a hydrogen bond between Ub Y59's hydroxyl group and the backbone amide of Ub E51, as substantiated by NMR spectroscopic analysis. Loop residues Y59 and R54 are specifically required for the receptor activity enabling K48 to attack the donor Ub-E2 thiol ester in reconstituted ubiquitination catalyzed by Skp1-Cullin1-F-box (SCF)(ßTrCP) E3 ligase and Cdc34 E2-conjugating enzyme. When introduced into mammalian cells, loop-disruptive mutant Ub(R54A/Y59A) diminished the production of K48-polyubiquitin chains. Importantly, conditional replacement of human endogenous Ub by Ub(R54A/Y59A) or Ub(K48R) yielded profound apoptosis at a similar extent, underscoring the global impact of the Ub Y59-E51 loop in cellular K48-polyubiquitination. Finally, disulfide cross-linking revealed interactions between the donor Ub-bound Cdc34 acidic loop and the Ub K48 site, as well as residues within the Y59-E51 loop, suggesting a mechanism in which the Ub Y59-E51 loop helps recruit the E2 acidic loop that aligns the receptor Ub K48 to the donor Ub for catalysis.
Asunto(s)
Lisina/metabolismo , Poliubiquitina/metabolismo , Ubiquitina/metabolismo , Ubiquitinación , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Apoptosis/genética , Biocatálisis , Línea Celular Tumoral , Células HEK293 , Humanos , Enlace de Hidrógeno , Immunoblotting , Lisina/química , Lisina/genética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Poliubiquitina/genética , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Interferencia de ARN , Proteínas Ligasas SKP Cullina F-box/química , Proteínas Ligasas SKP Cullina F-box/metabolismo , Ubiquitina/química , Ubiquitina/genética , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
Recent genetic studies have documented a pivotal growth-regulatory role played by the Cullin 7 (CUL7) E3 ubiquitin ligase complex containing the Fbw8-substrate-targeting subunit, Skp1, and the ROC1 RING finger protein. In this report, we identified insulin receptor substrate 1 (IRS-1), a critical mediator of the insulin/insulin-like growth factor 1 signaling, as a proteolytic target of the CUL7 E3 ligase in a manner that depends on mammalian target of rapamycin and the p70 S6 kinase activities. Interestingly, while embryonic fibroblasts of Cul7-/- mice were found to accumulate IRS-1 and exhibit increased activation of IRS-1's downstream Akt and MEK/ERK pathways, these null cells grew poorly and displayed phenotypes reminiscent of those associated with oncogene-induced senescence. Taken together, our findings demonstrate a key role for the CUL7 E3 in targeting IRS-1 for degradation, a process that may contribute to the regulation of cellular senescence.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Cullin/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Línea Celular , Senescencia Celular , Proteínas Cullin/genética , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Humanos , Proteínas Sustrato del Receptor de Insulina , Ratones , Ratones Noqueados , Fenotipo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , Serina-Treonina Quinasas TORRESUMEN
We have explored the mechanisms of polyubiquitin chain assembly with reconstituted ubiquitination of IκBα and ß-catenin by the Skp1-cullin 1-ßTrCP F-box protein (SCF(ßTrCP)) E3 ubiquitin (Ub) ligase complex. Competition experiments revealed that SCF(ßTrCP) formed a complex with IκBα and that the Nedd8 modified E3-substrate platform engaged in dynamic interactions with the Cdc34 E2 Ub conjugating enzyme for chain elongation. Using "elongation intermediates" containing ß-catenin linked with Ub chains of defined length, it was observed that a Lys-48-Ub chain of a length greater than four, but not its Lys-63 linkage counterparts, slowed the rate of additional Ub conjugation. Thus, the Ub chain length and linkage impact kinetic rates of chain elongation. Given that Lys-48-tetra-Ub is packed into compact conformations due to extensive intrachain interactions between Ub subunits, this topology may limit the accessibility of SCF(ßTrCP)/Cdc34 to the distal Ub Lys-48 and result in slowed elongation.
Asunto(s)
Lisina/metabolismo , Extensión de la Cadena Peptídica de Translación , Poliubiquitina/biosíntesis , Ubiquitinación , Células HEK293 , Humanos , Proteínas I-kappa B/metabolismo , Lisina/genética , Proteína NEDD8 , Inhibidor NF-kappaB alfa , Poliubiquitina/genética , Proteínas Ligasas SKP Cullina F-box/metabolismo , Ubiquitinas/metabolismo , beta Catenina/metabolismoRESUMEN
Intrauterine growth retardation is caused by maternal, fetal or placental factors that result in impaired endovascular trophoblast invasion and reduced placental perfusion. Although various causes of intrauterine growth retardation have been identified, most cases remain unexplained. Studying 29 families with 3-M syndrome (OMIM 273750), an autosomal recessive condition characterized by severe pre- and postnatal growth retardation, we first mapped the underlying gene to chromosome 6p21.1 and then identified 25 distinct mutations in the gene cullin 7 (CUL7). CUL7 assembles an E3 ubiquitin ligase complex containing Skp1, Fbx29 (also called Fbw8) and ROC1 and promotes ubiquitination. Using deletion analysis, we found that CUL7 uses its central region to interact with the Skp1-Fbx29 heterodimer. Functional studies indicated that the 3-M-associated CUL7 nonsense and missense mutations R1445X and H1464P, respectively, render CUL7 deficient in recruiting ROC1. These results suggest that impaired ubiquitination may have a role in the pathogenesis of intrauterine growth retardation in humans.
Asunto(s)
Cromosomas Humanos Par 6/genética , Proteínas Cullin/genética , Retardo del Crecimiento Fetal/genética , Proteínas Portadoras/metabolismo , Niño , Mapeo Cromosómico , Codón sin Sentido , Análisis Mutacional de ADN , Femenino , Homocigoto , Humanos , Masculino , Mutación Missense , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Eliminación de Secuencia , SíndromeRESUMEN
BACKGROUND: Negative feedback regulation of insulin signaling involves ubiquitin-dependent degradation of insulin receptor substrate 1 (IRS1). RESULTS: Cullin-RING E3 ubiquitin ligase 7 (CRL7) mediates the ubiquitination of IRS1 in hyperphosphorylated form. CONCLUSION: Multisite IRS1 phosphorylation triggers interactions with CRL7 for ubiquitin modification. SIGNIFICANCE: Insulin signaling is self-restrained when its downstream effector kinases are hyperactivated to trigger the negative feedback inhibition. Hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) and its effector kinase S6 kinase 1 (S6K1) is known to trigger multisite seryl phosphorylation of insulin receptor substrate 1 (IRS1), leading to its ubiquitination and degradation. This negative feedback inhibition functions to restrain PI3K activity and plays critical roles in the pathogenesis of cancer and type II diabetes. Recent work has implicated a role for cullin-RING E3 ubiquitin ligase 7 (CRL7) in targeting IRS1 for mTORC1/S6K1-dependent degradation. In the present study we have employed both cell-based degradation and reconstituted ubiquitination approaches to define molecular features associated with IRS1 critical for CRL7-mediated ubiquitination and degradation. We have mapped IRS1 degradation signal sequence to its N-terminal 574 amino acid residues, of which the integrity of Ser-307/Ser-312 and Ser-527, each constituting a S6K1 phosphorylation consensus site, was indispensible for supporting CRL7-forced degradation. In vitro, S6K1 was able to support the ubiquitination of bacterially expressed IRS1 N-terminal fragment by CRL7 but at low levels. In contrast, CRL7 supported efficient ubiquitination of IRS1 N-terminal fragment in hyperphosphorylated form, which was isolated from infected insect cells, suggesting requirement of additional phosphorylation by kinases yet to be identified. Finally, removal of IRS1 amino acids 1-260 led to substantial reduction of ubiquitination efficiency, suggesting a role for this region in mediating productive interactions with CRL7. The requirement of multisite phosphorylation and the N terminus of IRS1 for its turnover may ensure that complete IRS1 degradation occurs only when mTORC1 and S6K1 reach exceedingly high levels.
Asunto(s)
Proteínas Sustrato del Receptor de Insulina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Humanos , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina/genética , Fosforilación , Proteolisis , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
RING E3 ligases are proteins that must selectively recruit an E2-conjugating enzyme and facilitate ubiquitin transfer to a substrate. It is not clear how a RING E3 ligase differentiates a naked E2 enzyme from the E2â¼ubiquitin-conjugated form or how this is altered upon ubiquitin transfer. RING-box protein 1 (Rbx1/ROC1) is a key protein found in the Skp1/Cullin-1/F-box (SCF) E3 ubiquitin ligase complex that functions with the E2 ubiquitin conjugating enzyme CDC34. The solution structure of Rbx1/ROC1 revealed a globular RING domain (residues 40-108) stabilized by three structural zinc ions (root mean square deviation 0.30 ± 0.04 Å) along with a disordered N terminus (residues 12-39). Titration data showed that Rbx1/ROC1 preferentially recruits CDC34 in its ubiquitin-conjugated form and favors this interaction by 50-fold compared with unconjugated CDC34. Furthermore, NMR and biochemical assays identified residues in helix α2 of Rbx1/ROC1 that are essential for binding and activating CDC34â¼ubiquitin for ubiquitylation. Taken together, this work provides the first direct structural and biochemical evidence showing that polyubiquitylation by the RING E3 ligase Rbx1/ROC1 requires the preferential recruitment of an E2â¼ubiquitin complex and subsequent release of the unconjugated E2 protein upon ubiquitin transfer to a substrate or ubiquitin chain.
Asunto(s)
Proteínas Portadoras/química , Complejos de Ubiquitina-Proteína Ligasa/química , Ubiquitina-Proteína Ligasas/química , Ciclosoma-Complejo Promotor de la Anafase , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Humanos , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Ubiquitina/química , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras , Complejos de Ubiquitina-Proteína Ligasa/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Conjugation of Nedd8 to a cullin protein, termed neddylation, is an evolutionarily conserved process that functions to activate the cullin-RING family E3 ubiquitin ligases, leading to increased proteasomal degradation of a wide range of substrate proteins. Recent emerging evidence demonstrates that cellular neddylation requires the action of Dcn1, which, in humans, consists of five homologues designated as hDCNL1-5. Here we revealed a previously unknown mechanism that regulates hDCNL1. In cultured mammalian cells ectopically expressed hDCNL1 was mono-ubiquitinated predominantly at K143, K149, and K171. Using a classical chromatographic purification strategy, we identified Nedd4-1 as an E3 ligase that can catalyze mono-ubiquitination of hDCNL1 in a reconstituted ubiquitination system. In addition, the hDCNL1 N-terminal ubiquitin-binding domain is necessary and sufficient to mediate mono-ubiquitination. Finally, fluorescence microscopic and subcellular fractionation analyses revealed a role for mono-ubiquitination in driving nuclear export of hDCNL1. Taken together, these results suggest a mono-ubiquitination-mediated mechanism that governs nuclear-cytoplasmic trafficking of hDCNL1, thereby regulating hDCNL1-dependent activation of the cullin-RING E3 ubiquitin ligases in selected cellular compartments.
Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/fisiología , Transporte Activo de Núcleo Celular/fisiología , Núcleo Celular/genética , Citoplasma/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ubiquitina-Proteína Ligasas Nedd4 , Estructura Terciaria de Proteína , Proteínas , Proteínas Proto-Oncogénicas/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Cdc34 is an E2 ubiquitin-conjugating enzyme that functions in conjunction with SCF (Skp1.Cullin 1.F-box) E3 ubiquitin ligase to catalyze covalent attachment of polyubiquitin chains to a target protein. Here we identified direct interactions between the human Cdc34 C terminus and ubiquitin using NMR chemical shift perturbation assays. The ubiquitin binding activity was mapped to two separate Cdc34 C-terminal motifs (UBS1 and UBS2) that comprise residues 206-215 and 216-225, respectively. UBS1 and UBS2 bind to ubiquitin in the proximity of ubiquitin Lys(48) and C-terminal tail, both of which are key sites for conjugation. When bound to ubiquitin in one orientation, the Cdc34 UBS1 aromatic residues (Phe(206), Tyr(207), Tyr(210), and Tyr(211)) are probably positioned in the vicinity of ubiquitin C-terminal residue Val(70). Replacement of UBS1 aromatic residues by glycine or of ubiquitin Val(70) by alanine decreased UBS1-ubiquitin affinity interactions. UBS1 appeared to support the function of Cdc34 in vivo because human Cdc34(1-215) but not Cdc34(1-200) was able to complement the growth defect by yeast Cdc34 mutant strain. Finally, reconstituted IkappaBalpha ubiquitination analysis revealed a role for each adjacent pair of UBS1 aromatic residues (Phe(206)/Tyr(207), Tyr(210)/Tyr(211)) in conjugation, with Tyr(210) exhibiting the most pronounced catalytic function. Intriguingly, Cdc34 Tyr(210) was required for the transfer of the donor ubiquitin to a receptor lysine on either IkappaBalpha or a ubiquitin in a manner that depended on the neddylated RING sub-complex of the SCF. Taken together, our results identified a new ubiquitin binding activity within the human Cdc34 C terminus that contributes to SCF-dependent ubiquitination.
Asunto(s)
Factor de Células Madre/química , Complejos de Ubiquitina-Proteína Ligasa/química , Ubiquitina/química , Secuencia de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Hongos/metabolismo , Prueba de Complementación Genética , Glicina/química , Humanos , Proteínas I-kappa B/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Datos de Secuencia Molecular , Mutación , Inhibidor NF-kappaB alfa , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Tirosina/química , Enzimas Ubiquitina-ConjugadorasRESUMEN
The resident prokaryotic microflora of the mammalian intestine influences diverse homeostatic functions of the gut, including regulation of cellular growth and immune responses; however, it is unknown how commensal prokaryotic organisms mechanistically influence eukaryotic signaling networks. We have shown that bacterial coculture with intestinal epithelial cells modulates ubiquitin-mediated degradation of important signaling intermediates, including beta-catenin and the NF-kappaB inhibitor IkappaB-alpha. Ubiquitination of these proteins as well as others is catalyzed by the SCF(betaTrCP) ubiquitin ligase, which itself requires regulated modification of the cullin-1 subunit by the ubiquitin-like protein NEDD8. Here we show that epithelia contacted by enteric commensal bacteria in vitro and in vivo rapidly generate reactive oxygen species (ROS). Bacterially induced ROS causes oxidative inactivation of the catalytic cysteine residue of Ubc12, the NEDD8-conjugating enzyme, resulting in complete but transient loss of cullin-1 neddylation and consequent effects on NF-kappaB and beta-catenin signaling. Our results demonstrate that commensal bacteria directly modulate a critical control point of the ubiquitin-proteasome system, and suggest how enteric commensal bacterial flora influences the regulatory pathways of the mammalian intestinal epithelia.