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1.
Nat Chem Biol ; 2024 Jul 04.
Artículo en Inglés | MEDLINE | ID: mdl-38965384

RESUMEN

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain-of-function mutation p.E1099K, resulting in growth suppression, apoptosis and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 to recruit the SCFFBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCFFBXO22. Overall, we present a potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a new FBXO22-recruitment strategy for TPD.

2.
Nucleic Acids Res ; 50(6): 3505-3522, 2022 04 08.
Artículo en Inglés | MEDLINE | ID: mdl-35244724

RESUMEN

Despite MYC dysregulation in most human cancers, strategies to target this potent oncogenic driver remain an urgent unmet need. Recent evidence shows the PP1 phosphatase and its regulatory subunit PNUTS control MYC phosphorylation, chromatin occupancy, and stability, however the molecular basis remains unclear. Here we demonstrate that MYC interacts directly with PNUTS through the MYC homology Box 0 (MB0), a highly conserved region recently shown to be important for MYC oncogenic activity. By NMR we identified a distinct peptide motif within MB0 that interacts with PNUTS residues 1-148, a functional unit, here termed PNUTS amino-terminal domain (PAD). Using NMR spectroscopy we determined the solution structure of PAD, and characterised its MYC-binding patch. Point mutations of residues at the MYC-PNUTS interface significantly weaken their interaction both in vitro and in vivo, leading to elevated MYC phosphorylation. These data demonstrate that the MB0 region of MYC directly interacts with the PAD of PNUTS, which provides new insight into the control mechanisms of MYC as a regulator of gene transcription and a pervasive cancer driver.


Asunto(s)
Cromatina , Proteínas Nucleares , Proteínas de Unión al ADN/genética , Humanos , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/genética , Proteína Fosfatasa 1/metabolismo , Proteínas de Unión al ARN/genética
3.
J Biol Chem ; 294(8): 2593-2603, 2019 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-30591587

RESUMEN

Carboxysomes are compartments in bacterial cells that promote efficient carbon fixation by sequestering RubisCO and carbonic anhydrase within a protein shell that impedes CO2 escape. The key to assembling this protein complex is CcmM, a multidomain protein whose C-terminal region is required for RubisCO recruitment. This CcmM region is built as a series of copies (generally 3-5) of a small domain, CcmMS, joined by unstructured linkers. CcmMS domains have weak, but significant, sequence identity to RubisCO's small subunit, RbcS, suggesting that CcmM binds RubisCO by displacing RbcS. We report here the 1.35-Å structure of the first Thermosynechococcus elongatus CcmMS domain, revealing that it adopts a compact, well-defined structure that resembles that of RbcS. CcmMS, however, lacked key RbcS RubisCO-binding determinants, most notably an extended N-terminal loop. Nevertheless, individual CcmMS domains are able to bind RubisCO in vitro with 1.16 µm affinity. Two or four linked CcmMS domains did not exhibit dramatic increases in this affinity, implying that short, disordered linkers may frustrate successive CcmMS domains attempting to simultaneously bind a single RubisCO oligomer. Size-exclusion chromatography-coupled right-angled light scattering (SEC-RALS) and native MS experiments indicated that multiple CcmMS domains can bind a single RubisCO holoenzyme and, moreover, that RbcS is not released from these complexes. CcmMS bound equally tightly to a RubisCO variant in which the α/ß domain of RbcS was deleted, suggesting that CcmMS binds RubisCO independently of its RbcS subunit. We propose that, instead, the electropositive CcmMS may bind to an extended electronegative pocket between RbcL dimers.


Asunto(s)
Proteínas Bacterianas/química , Cianobacterias/enzimología , Ribulosa-Bifosfato Carboxilasa/química , Proteínas Bacterianas/genética , Sitios de Unión , Cristalografía por Rayos X , Cianobacterias/genética , Dominios Proteicos , Ribulosa-Bifosfato Carboxilasa/genética
4.
bioRxiv ; 2023 Nov 30.
Artículo en Inglés | MEDLINE | ID: mdl-37961297

RESUMEN

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here, we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain of function mutation p.E1099K, resulting in growth suppression, apoptosis, and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 in a covalent and reversible manner to recruit the SCF FBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCF FBXO22 . Overall, we present a highly potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a novel FBXO22-dependent TPD strategy.

5.
Methods Mol Biol ; 2318: 45-67, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34019286

RESUMEN

By identifying MYC protein-protein interactors, we aim to gain a deeper mechanistic understanding of MYC as a regulator of gene transcription and potent oncoprotein. This information can then be used to devise strategies for disrupting critical MYC protein-protein interactions to inhibit MYC-driven tumorigenesis. In this chapter, we discuss four techniques to identify and validate MYC-interacting partners. First, we highlight BioID, a powerful discovery method used to identify high-confidence proximal interactors in living cells. We also discuss bioinformatic prioritization strategies for the BioID-derived MYC-proximal complexes. Next, we discuss how protein interactions can be validated using techniques such as in vivo-in vitro pull-down assays and the proximity ligation assay (PLA). We conclude with an overview of biolayer interferometry (BLI), a quantitative method used to characterize direct interactions between two proteins in vitro. Overall, we highlight the principles of each assay and provide methodology necessary to conduct these experiments and adapt them to the study of interactors of additional proteins of interest.


Asunto(s)
Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/aislamiento & purificación , Secuencia de Aminoácidos/genética , Sitios de Unión , Biología Computacional/métodos , ADN/química , ADN/genética , Dimerización , Genes myc/genética , Genes myc/fisiología , Humanos , Unión Proteica , Dominios Proteicos , Mapeo de Interacción de Proteínas/métodos , Proteínas Proto-Oncogénicas c-myc/metabolismo
6.
Nat Rev Cancer ; 21(9): 579-591, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34188192

RESUMEN

The transcription factor and oncoprotein MYC is a potent driver of many human cancers and can regulate numerous biological activities that contribute to tumorigenesis. How a single transcription factor can regulate such a diverse set of biological programmes is central to the understanding of MYC function in cancer. In this Perspective, we highlight how multiple proteins that interact with MYC enable MYC to regulate several central control points of gene transcription. These include promoter binding, epigenetic modifications, initiation, elongation and post-transcriptional processes. Evidence shows that a combination of multiple protein interactions enables MYC to function as a potent oncoprotein, working together in a 'coalition model', as presented here. Moreover, as MYC depends on its protein interactome for function, we discuss recent research that emphasizes an unprecedented opportunity to target protein interactors to directly impede MYC oncogenesis.


Asunto(s)
Neoplasias/metabolismo , Proteína Oncogénica p55(v-myc)/metabolismo , Transcripción Genética , Animales , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/genética , Proteína Oncogénica p55(v-myc)/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
7.
J Med Chem ; 64(2): 1139-1169, 2021 01 28.
Artículo en Inglés | MEDLINE | ID: mdl-33444025

RESUMEN

The essential eukaryotic chaperone Hsp90 regulates the form and function of diverse client proteins, many of which govern thermotolerance, virulence, and drug resistance in fungal species. However, use of Hsp90 inhibitors as antifungal therapeutics has been precluded by human host toxicities and suppression of immune responses. We recently described resorcylate aminopyrazoles (RAPs) as the first class of Hsp90 inhibitors capable of discriminating between fungal (Cryptococcus neoformans, Candida albicans) and human isoforms of Hsp90 in biochemical assays. Here, we report an iterative structure-property optimization toward RAPs capable of inhibiting C. neoformans growth in culture. In addition, we report the first X-ray crystal structures of C. neoformans Hsp90 nucleotide binding domain (NBD), as the apoprotein and in complexes with the non-species-selective Hsp90 inhibitor NVP-AUY922 and three RAPs revealing unique ligand-induced conformational rearrangements, which reaffirm the hypothesis that intrinsic differences in protein flexibility can confer selective inhibition of fungal versus human Hsp90 isoforms.


Asunto(s)
Antifúngicos/farmacología , Cryptococcus neoformans/efectos de los fármacos , Hongos/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Pirazoles/farmacología , Animales , Antifúngicos/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Microsomas Hepáticos/metabolismo , Unión Proteica , Pirazoles/química , Especificidad de la Especie , Relación Estructura-Actividad
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