RESUMEN
Mitochondrial outer membrane permeabilisation (MOMP) is often essential for apoptosis, by enabling cytochrome c release that leads to caspase activation and rapid cell death. Recently, MOMP has been shown to be inherently pro-inflammatory with emerging cellular roles, including its ability to elicit anti-tumour immunity. Nonetheless, how MOMP triggers inflammation and how the cell regulates this remains poorly defined. We find that upon MOMP, many proteins localised either to inner or outer mitochondrial membranes are ubiquitylated in a promiscuous manner. This extensive ubiquitylation serves to recruit the essential adaptor molecule NEMO, leading to the activation of pro-inflammatory NF-κB signalling. We show that disruption of mitochondrial outer membrane integrity through different means leads to the engagement of a similar pro-inflammatory signalling platform. Therefore, mitochondrial integrity directly controls inflammation, such that permeabilised mitochondria initiate NF-κB signalling.
Asunto(s)
FN-kappa B , Ubiquitina , Humanos , FN-kappa B/genética , FN-kappa B/metabolismo , Ubiquitina/metabolismo , Membranas Mitocondriales/metabolismo , Mitocondrias/metabolismo , Apoptosis/fisiología , Inflamación/metabolismoRESUMEN
Receptor tyrosine kinase KIT is frequently activated in acute myeloid leukemia (AML). While high PRL2 (PTP4A2) expression is correlated with activation of SCF/KIT signaling in AML, the underlying mechanisms are not fully understood. We discovered that inhibition of PRL2 significantly reduces the burden of oncogenic KIT-driven leukemia and extends leukemic mice survival. PRL2 enhances oncogenic KIT signaling in leukemia cells, promoting their proliferation and survival. We found that PRL2 dephosphorylates CBL at tyrosine 371 and inhibits its activity toward KIT, leading to decreased KIT ubiquitination and enhanced AKT and ERK signaling in leukemia cells. IMPLICATIONS: Our studies uncover a novel mechanism that fine-tunes oncogenic KIT signaling in leukemia cells and will likely identify PRL2 as a novel therapeutic target in AML with KIT mutations.
Asunto(s)
Leucemia Mieloide Aguda , Monoéster Fosfórico Hidrolasas , Animales , Ratones , Leucemia Mieloide Aguda/genética , Mutación , Monoéster Fosfórico Hidrolasas/genética , Fosforilación , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Transducción de Señal/genéticaRESUMEN
Dysregulation of the PI3K/AKT pathway is a common occurrence in high-grade serous ovarian carcinoma (HGSOC), with the loss of the tumour suppressor PTEN in HGSOC being associated with poor prognosis. The cellular mechanisms of how PTEN loss contributes to HGSOC are largely unknown. We here utilise time-lapse imaging of HGSOC spheroids coupled to a machine learning approach to classify the phenotype of PTEN loss. PTEN deficiency induces PI(3,4,5)P3 -rich and -dependent membrane protrusions into the extracellular matrix (ECM), resulting in a collective invasion phenotype. We identify the small GTPase ARF6 as a crucial vulnerability of HGSOC cells upon PTEN loss. Through a functional proteomic CRISPR screen of ARF6 interactors, we identify the ARF GTPase-activating protein (GAP) AGAP1 and the ECM receptor ß1-integrin (ITGB1) as key ARF6 interactors in HGSOC regulating PTEN loss-associated invasion. ARF6 functions to promote invasion by controlling the recycling of internalised, active ß1-integrin to maintain invasive activity into the ECM. The expression of the CYTH2-ARF6-AGAP1 complex in HGSOC patients is inversely associated with outcome, allowing the identification of patient groups with improved versus poor outcome. ARF6 may represent a therapeutic vulnerability in PTEN-depleted HGSOC.
Asunto(s)
Proteínas de Unión al GTP Monoméricas , Neoplasias Ováricas , Humanos , Femenino , Integrinas/metabolismo , Proteómica , Fosfatidilinositol 3-Quinasas/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Proteínas de Unión al GTP Monoméricas/metabolismo , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismoRESUMEN
Acute myeloid leukemia (AML) is an aggressive blood cancer with poor prognosis. FMS-like tyrosine kinase receptor-3 (FLT3) is one of the major oncogenic receptor tyrosine kinases aberrantly activated in AML. Although protein tyrosine phosphatase PRL2 is highly expressed in some subtypes of AML compared with normal human hematopoietic stem and progenitor cells, the mechanisms by which PRL2 promotes leukemogenesis are largely unknown. We discovered that genetic and pharmacological inhibition of PRL2 significantly reduce the burden of FLT3-internal tandem duplications-driven leukemia and extend the survival of leukemic mice. Furthermore, we found that PRL2 enhances oncogenic FLT3 signaling in leukemia cells, promoting their proliferation and survival. Mechanistically, PRL2 dephosphorylates the E3 ubiquitin ligase CBL at tyrosine 371 and attenuates CBL-mediated ubiquitination and degradation of FLT3, leading to enhanced FLT3 signaling in leukemia cells. Thus, our study reveals that PRL2 enhances oncogenic FLT3 signaling in leukemia cells through dephosphorylation of CBL and will likely establish PRL2 as a novel druggable target for AML.
Asunto(s)
Leucemia Mieloide Aguda , Ubiquitina-Proteína Ligasas , Humanos , Animales , Ratones , Ubiquitina-Proteína Ligasas/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Transducción de Señal/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogénicas c-cbl/genética , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismo , MutaciónRESUMEN
Glutamine synthetase (GS) activity is conserved from prokaryotes to humans, where the ATP-dependent production of glutamine from glutamate and ammonia is essential for neurotransmission and ammonia detoxification. Here, we show that mammalian GS uses glutamate and methylamine to produce a methylated glutamine analog, N5-methylglutamine. Untargeted metabolomics revealed that liver-specific GS deletion and its pharmacological inhibition in mice suppress hepatic and circulating levels of N5-methylglutamine. This alternative activity of GS was confirmed in human recombinant enzyme and cells, where a pathogenic mutation in the active site (R324C) promoted the synthesis of N5-methylglutamine over glutamine. N5-methylglutamine is detected in the circulation, and its levels are sustained by the microbiome, as demonstrated by using germ-free mice. Finally, we show that urine levels of N5-methylglutamine correlate with tumor burden and GS expression in a ß-catenin-driven model of liver cancer, highlighting the translational potential of this uncharacterized metabolite.
Asunto(s)
Glutamina , Neoplasias , Humanos , Ratones , Animales , Glutamina/metabolismo , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Amoníaco , Ácido Glutámico/metabolismo , Hígado/metabolismo , Neoplasias/metabolismo , Homeostasis , MamíferosRESUMEN
ARF tumor suppressor protein is a key regulator of the MDM2-p53 signaling axis. ARF interferes with MDM2-mediated ubiquitination and degradation of p53 by sequestering MDM2 in the nucleolus and preventing MDM2-p53 interaction and nuclear export of p53. Moreover, ARF also directly inhibits MDM2 ubiquitin ligase (E3) activity, but the mechanism remains elusive. Here, we apply nuclear magnetic resonance and biochemical analyses to uncover the mechanism of ARF-mediated inhibition of MDM2 E3 activity. We show that MDM2 acidic and zinc finger domains (AD-ZnF) form a weak intramolecular interaction with the RING domain, where the binding site overlaps with the E2â¼ubiquitin binding surface and thereby partially reduces MDM2 E3 activity. Binding of human N-terminal 32 residues of p14ARF to the acidic domain of MDM2 strengthens the AD-ZnF-RING domain interaction. Furthermore, the N-terminal RxFxV motifs of p14ARF participate directly in the MDM2 RING domain interaction. This bivalent binding mode of p14ARF to MDM2 acidic and RING domains restricts E2â¼ubiquitin recruitment and massively hinders MDM2 E3 activity. These findings elucidate the mechanism by which ARF inhibits MDM2 E3 activity.
Asunto(s)
Proteínas Proto-Oncogénicas c-mdm2 , Proteína p14ARF Supresora de Tumor , Ubiquitina-Proteína Ligasas , Humanos , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/química , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p14ARF Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Ubiquitin (Ub) chain types govern distinct biological processes. K48-linked polyUb chains target substrates for proteasomal degradation, but the mechanism of Ub chain synthesis remains elusive due to the transient nature of Ub handover. Here, we present the structure of a chemically trapped complex of the E2 UBE2K covalently linked to donor Ub and acceptor K48-linked di-Ub, primed for K48-linked Ub chain synthesis by a RING E3. The structure reveals the basis for acceptor Ub recognition by UBE2K active site residues and the C-terminal Ub-associated (UBA) domain, to impart K48-linked Ub specificity and catalysis. Furthermore, the structure unveils multiple Ub-binding surfaces on the UBA domain that allow distinct binding modes for K48- and K63-linked Ub chains. This multivalent Ub-binding feature serves to recruit UBE2K to ubiquitinated substrates to overcome weak acceptor Ub affinity and thereby promote chain elongation. These findings elucidate the mechanism of processive K48-linked polyUb chain formation by UBE2K.
Asunto(s)
Poliubiquitina , Ubiquitina , Poliubiquitina/metabolismo , Unión Proteica , Dominios Proteicos , Ubiquitina/química , Enzimas Ubiquitina-Conjugadoras/metabolismo , UbiquitinaciónRESUMEN
Casitas B-lineage lymphoma (CBL) is a ubiquitin ligase (E3) that becomes activated upon Tyr371-phosphorylation and targets receptor protein tyrosine kinases for ubiquitin-mediated degradation. Deregulation of CBL and its E3 activity is observed in myeloproliferative neoplasms and other cancers, including breast, colon, and prostate cancer. Here, we explore the oncogenic mechanism of E3-inactive CBL mutants identified in myeloproliferative neoplasms. We show that these mutants bind strongly to CIN85 under normal growth conditions and alter the CBL interactome. Lack of E3 activity deregulates CIN85 endosomal trafficking, leading to an altered transcriptome that amplifies signaling events to promote oncogenesis. Disruption of CBL mutant interactions with EGFR or CIN85 reduces oncogenic transformation. Given the importance of the CBL-CIN85 interaction in breast cancers, we examined the expression levels of CIN85, CBL, and the status of Tyr371-phosphorylated CBL (pCBL) in human breast cancer tissue microarrays. Interestingly, pCBL shows an inverse correlation with both CIN85 and CBL, suggesting that high expression of inactivated CBL could coordinate with CIN85 for breast cancer progression. Inhibition of the CBL-CIN85 interaction with a proline-rich peptide of CBL that binds CIN85 reduced the proliferation of MDA-MB-231 cells. Together, these results provide a rationale for exploring the potential of targeting the EGFR-CBL-CIN85 axis in CBL-inactivated mutant cancers.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Linfoma de Células B/genética , Proteínas Proto-Oncogénicas c-cbl/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/genética , Receptores ErbB/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Linfoma de Células B/patología , Mutación/genética , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patología , Unión Proteica , Proteolisis , Análisis de Matrices Tisulares , Ubiquitina/genéticaRESUMEN
As a key regulator of the tumour suppressor protein p53, MDM2 is involved in various types of cancer and has thus been an attractive drug target. So far, small molecule design has primarily focussed on the N-terminal p53-binding domain although on-target toxicity effects have been reported. Targeting the catalytic RING domain of MDM2 resembles an alternative approach to drug MDM2 with the idea to prevent MDM2-mediated ubiquitination of p53 while retaining MDM2's ability to bind p53. The design of RING inhibitors has been limited by the extensive aggregation tendency of the RING domain, making it challenging to undertake co-crystallization attempts with potential inhibitors. Here we compare the purification profiles of the MDM2 RING domain from several species and show that the MDM2 RING domain of other species than human is much less prone to aggregate although the overall structure of the RING domain is conserved. Through sequence comparison and mutagenesis analyses, we identify a single point mutation, G443T, which greatly enhances the dimeric fraction of human MDM2 RING domain during purification. Neither does the mutation alter the structure of the RING domain, nor does it affect E2(UbcH5B)-Ub binding and activity. Hence, MDM2-G443T facilitates studies involving binding partners that would be hampered by the low solubility of the wild-type RING domain. Furthermore, it will be valuable for the development of MDM2 RING inhibitors.
Asunto(s)
Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-mdm2/química , Proteína p53 Supresora de Tumor/química , Enzimas Ubiquitina-Conjugadoras/química , Ubiquitina/química , Secuencia de Aminoácidos , Animales , Biocatálisis , Dominio Catalítico , Secuencia Conservada , Cristalografía por Rayos X , Expresión Génica , Humanos , Mamíferos , Modelos Moleculares , Agregado de Proteínas , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación , Xenopus , Pez CebraRESUMEN
The p53 tumor suppressor protein is a potent activator of proliferative arrest and cell death. In normal cells, this pathway is restrained by p53 protein degradation mediated by the E3-ubiquitin ligase activity of MDM2. Oncogenic stress releases p53 from MDM2 control, so activating the p53 response. However, many tumors that retain wild-type p53 inappropriately maintain the MDM2-p53 regulatory loop in order to continuously suppress p53 activity. We have shown previously that single point mutations in the human MDM2 RING finger domain prevent the interaction of MDM2 with the E2/ubiquitin complex, resulting in the loss of MDM2's E3 activity without preventing p53 binding. Here, we show that an analogous mouse MDM2 mutant (MDM2 I438K) restrains p53 sufficiently for normal growth but exhibits an enhanced stress response in vitro. In vivo, constitutive expression of MDM2 I438K leads to embryonic lethality that is rescued by p53 deletion, suggesting MDM2 I438K is not able to adequately control p53 function through development. However, the switch to I438K expression is tolerated in adult mice, sparing normal cells but allowing for an enhanced p53 response to DNA damage. Viewed as a proof of principle model for therapeutic development, our findings support an approach that would inhibit MDM2 E3 activity without preventing MDM2/p53 binding as a promising avenue for development of compounds to activate p53 in tumors with reduced on-target toxicities.
Asunto(s)
Desarrollo Embrionario/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Animales , Antineoplásicos Hormonales/farmacología , Proliferación Celular/genética , Células Cultivadas , Embrión de Mamíferos/enzimología , Activación Enzimática/efectos de los fármacos , Femenino , Masculino , Ratones , Mutación , Tamoxifeno/farmacologíaRESUMEN
Cross-talk between ubiquitination and ADP-ribosylation regulates spatiotemporal recruitment of key players in many signaling pathways. The DELTEX family ubiquitin ligases (DTX1 to DTX4 and DTX3L) are characterized by a RING domain followed by a C-terminal domain (DTC) of hitherto unknown function. Here, we use two label-free mass spectrometry techniques to investigate the interactome and ubiquitinated substrates of human DTX2 and identify a large proportion of proteins associated with the DNA damage repair pathway. We show that DTX2-catalyzed ubiquitination of these interacting proteins requires PARP1/2-mediated ADP-ribosylation and depends on the DTC domain. Using a combination of structural, biochemical, and cell-based techniques, we show that the DTX2 DTC domain harbors an ADP-ribose-binding pocket and recruits poly-ADP-ribose (PAR)-modified proteins for ubiquitination. This PAR-binding property of DTC domain is conserved across the DELTEX family E3s. These findings uncover a new ADP-ribose-binding domain that facilitates PAR-dependent ubiquitination.
Asunto(s)
Poli Adenosina Difosfato Ribosa , Ubiquitina-Proteína Ligasas , Adenosina Difosfato/metabolismo , Humanos , Poli Adenosina Difosfato Ribosa/química , Poli Adenosina Difosfato Ribosa/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Cellular cross-talk between ubiquitination and other posttranslational modifications contributes to the regulation of numerous processes. One example is ADP-ribosylation of the carboxyl terminus of ubiquitin by the E3 DTX3L/ADP-ribosyltransferase PARP9 heterodimer, but the mechanism remains elusive. Here, we show that independently of PARP9, the conserved carboxyl-terminal RING and DTC (Deltex carboxyl-terminal) domains of DTX3L and other human Deltex proteins (DTX1 to DTX4) catalyze ADP-ribosylation of ubiquitin's Gly76 Structural studies reveal a hitherto unknown function of the DTC domain in binding NAD+ Deltex RING domain recruits E2 thioesterified with ubiquitin and juxtaposes it with NAD+ bound to the DTC domain to facilitate ADP-ribosylation of ubiquitin. This ubiquitin modification prevents its activation but is reversed by the linkage nonspecific deubiquitinases. Our study provides mechanistic insights into ADP-ribosylation of ubiquitin by Deltex E3s and will enable future studies directed at understanding the increasingly complex network of ubiquitin cross-talk.
Asunto(s)
Ubiquitina-Proteína Ligasas , Ubiquitina , ADP-Ribosilación , Humanos , NAD/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Phosphorylation of MDM2 by ATM upon DNA damage is an important mechanism for deregulating MDM2, thereby leading to p53 activation. ATM phosphorylates multiple residues near the RING domain of MDM2, but the underlying molecular basis for deregulation remains elusive. Here we show that Ser429 phosphorylation selectively enhances the ubiquitin ligase activity of MDM2 homodimer but not MDM2-MDMX heterodimer. A crystal structure of phospho-Ser429 (pS429)-MDM2 bound to E2-ubiquitin reveals a unique 310-helical feature present in MDM2 homodimer that allows pS429 to stabilize the closed E2-ubiquitin conformation and thereby enhancing ubiquitin transfer. In cells Ser429 phosphorylation increases MDM2 autoubiquitination and degradation upon DNA damage, whereas S429A substitution protects MDM2 from auto-degradation. Our results demonstrate that Ser429 phosphorylation serves as a switch to boost the activity of MDM2 homodimer and promote its self-destruction to enable rapid p53 stabilization and resolve a long-standing controversy surrounding MDM2 auto-degradation in response to DNA damage.
Asunto(s)
Daño del ADN , Proteínas Proto-Oncogénicas c-mdm2/química , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Dominios RING Finger , Secuencia de Aminoácidos , Línea Celular Tumoral , Humanos , Modelos Moleculares , Fosforilación , Fosfoserina/metabolismo , Unión Proteica , Multimerización de Proteína , Relación Estructura-Actividad , Ubiquitina/metabolismoRESUMEN
Ubiquitin (Ub) is a small protein that post-translationally modifies a variety of substrates in eukaryotic cells to modulate substrate function. The ability of Ub to interact with numerous protein domains makes Ub an attractive scaffold for engineering ubiquitin variants (UbVs) with high target specificity. Previously, we identified a UbV that formed a non-covalent stable dimer via a ß-strand exchange, and in the current work we identified and characterized the minimal substitutions in the primary sequence of Ub required to form a higher ordered complex. Using solution angle scattering and X-ray crystallography, we show that a single substitution of residue Gly10 to either Ala or Val is sufficient to convert Ub from a monomer to a dimer. We also investigate contributions to dimer formation by the residues in the surrounding sequence. These results can be used to develop next-generation phage-display libraries of UbVs to engineer new interfaces for protein recognition.
Asunto(s)
Sustitución de Aminoácidos , Ubiquitina/química , Ubiquitina/genética , Cristalografía por Rayos X , Modelos Moleculares , Multimerización de Proteína , Estructura Secundaria de Proteína , UbiquitinaciónRESUMEN
Ubiquitin (Ub)-conjugating enzymes and Ub ligases control protein degradation and regulate many cellular processes in eukaryotes. Cellular inhibitor of apoptosis protein-1 (cIAP1) plays a central role in apoptosis and tumor necrosis factor signaling. It harbors a C-terminal RING domain that homodimerizes to recruit E2â¼Ub (where â¼ denotes a thioester bond) complex to catalyze Ub transfer. Noncovalent Ub binding to the backside of the E2 Ub-conjugating enzyme UbcH5 has previously been shown to enhance RING domain activity, but the molecular basis for this enhancement is unclear. To investigate how dimeric cIAP1 RING activates E2â¼Ub for Ub transfer and what role noncovalently bound Ub has in Ub transfer, here we determined the crystal structure of the cIAP1 RING dimer bound to both UbcH5B covalently linked to Ub (UbcH5B-Ub) and a noncovalent Ub to 1.7 Å resolution. The structure along with biochemical analyses revealed that the cIAP1 RING domain interacts with UbcH5B-Ub and thereby promotes the formation of a closed UbcH5B-Ub conformation that primes the thioester bond for Ub transfer. We observed that the noncovalent Ub binds to the backside of UbcH5B and abuts UbcH5B's α1ß1-loop, which, in turn, stabilizes the closed UbcH5B-Ub conformation. Our results disclose the mechanism by which cIAP1 RING dimer activates UbcH5Bâ¼Ub and indicate that noncovalent Ub binding further stabilizes the cIAP1-UbcH5Bâ¼Ub complex in the active conformation to stimulate Ub transfer.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/química , Proteínas Inhibidoras de la Apoptosis/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , UbiquitinaciónRESUMEN
RING and U-box ubiquitin ligases promote ubiquitin (Ub) transfer by priming Ub-conjugated E2 in a closed conformation to optimize the thioester bond for nucleophilic attack by substrate lysine. Here, we describe a single-turnover lysine discharge assay for direct assessment of the activity of any RING/U-box E3-E2~Ub complex.
Asunto(s)
Bioensayo , Lisina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Arabidopsis/metabolismo , Bioensayo/métodos , Activación Enzimática , Marcaje Isotópico , Especificidad por Sustrato , UbiquitinaciónRESUMEN
The histone chaperone complex facilitates chromatin transcription (FACT) plays important roles in DNA repair, replication, and transcription. In the formation of this complex, structure-specific recognition protein-1 (SSRP1) heterodimerizes with suppressor of Ty 16 (SPT16). SSRP1 also has SPT16-independent functions, but how SSRP1 functions alone remains elusive. Here, using analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS) techniques, we characterized human SSRP1 and that from the amoeba Dictyostelium discoideum and show that both orthologs form an elongated homodimer in solution. We found that substitutions in the SSRP1 pleckstrin homology domain known to bind SPT16 also disrupt SSRP1 homodimerization. Moreover, AUC and SAXS analyses revealed that SSRP1 homodimerization and heterodimerization with SPT16 (resulting in FACT) involve the same SSRP1 surface, namely the PH2 region, and that the FACT complex contains only one molecule of SSRP1. These observations suggest that SSRP1 homo- and heterodimerization might be mutually exclusive. Moreover, isothermal titration calorimetry analyses disclosed that SSRP1 binds both histones H2A-H2B and H3-H4 and that disruption of SSRP1 homodimerization decreases its histone-binding affinity. Together, our results provide evidence for regulation of SSRP1 by homodimerization and suggest a potential role for homodimerization in facilitating SPT16-independent functions of SSRP1.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/química , Proteínas del Grupo de Alta Movilidad/metabolismo , Histonas/metabolismo , Multimerización de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Factores de Elongación Transcripcional/química , Factores de Elongación Transcripcional/metabolismo , Secuencia de Aminoácidos , Dictyostelium , Humanos , Unión Proteica , Dominios Proteicos , Estructura Cuaternaria de ProteínaRESUMEN
RING and U-box E3 ubiquitin ligases regulate diverse eukaryotic processes and have been implicated in numerous diseases, but targeting these enzymes remains a major challenge. We report the development of three ubiquitin variants (UbVs), each binding selectively to the RING or U-box domain of a distinct E3 ligase: monomeric UBE4B, phosphorylated active CBL, or dimeric XIAP. Structural and biochemical analyses revealed that UbVs specifically inhibited the activity of UBE4B or phosphorylated CBL by blocking the E2â¼Ub binding site. Surprisingly, the UbV selective for dimeric XIAP formed a dimer to stimulate E3 activity by stabilizing the closed E2â¼Ub conformation. We further verified the inhibitory and stimulatory functions of UbVs in cells. Our work provides a general strategy to inhibit or activate RING/U-box E3 ligases and provides a resource for the research community to modulate these enzymes.
Asunto(s)
Descubrimiento de Drogas/métodos , Activadores de Enzimas , Inhibidores Enzimáticos , Multimerización de Proteína/efectos de los fármacos , Proteínas Supresoras de Tumor , Complejos de Ubiquitina-Proteína Ligasa , Proteína Inhibidora de la Apoptosis Ligada a X , Activadores de Enzimas/química , Activadores de Enzimas/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Células HEK293 , Células HeLa , Humanos , Proteínas Supresoras de Tumor/agonistas , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/antagonistas & inhibidores , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina-Proteína Ligasas , Proteína Inhibidora de la Apoptosis Ligada a X/agonistas , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
MDM2-MDMX complexes bind the p53 tumor-suppressor protein, inhibiting p53's transcriptional activity and targeting p53 for proteasomal degradation. Inhibitors that disrupt binding between p53 and MDM2 efficiently activate a p53 response, but their use in the treatment of cancers that retain wild-type p53 may be limited by on-target toxicities due to p53 activation in normal tissue. Guided by a novel crystal structure of the MDM2-MDMX-E2(UbcH5B)-ubiquitin complex, we designed MDM2 mutants that prevent E2-ubiquitin binding without altering the RING-domain structure. These mutants lack MDM2's E3 activity but retain the ability to limit p53's transcriptional activity and allow cell proliferation. Cells expressing these mutants respond more quickly to cellular stress than cells expressing wild-type MDM2, but basal p53 control is maintained. Targeting the MDM2 E3-ligase activity could therefore widen the therapeutic window of p53 activation in tumors.