RESUMO
Sorafenib is a multikinase inhibitor indicated for first-line treatment of unresectable hepatocellular carcinoma. Despite its widespread use in the clinic, the existing knowledge of sorafenib mode-of-action remains incomplete. To build upon the current understanding, we used the Cellular Thermal Shift Assay (CETSA) coupled to Mass Spectrometry (CETSA-MS) to monitor compound binding to its target proteins in the cellular context on a proteome-wide scale. Among the potential sorafenib targets, we identified aldehyde dehydrogenase 2 (ALDH2), an enzyme that plays a major role in alcohol metabolism. We validated the interaction of sorafenib with ALDH2 by orthogonal methods using pure recombinant protein, proving that this interaction is not mediated by other cellular components. Moreover, we showed that sorafenib inhibits ALDH2 activity, supporting a functional role for this interaction. Finally, we were able to demonstrate that both ALDH2 protein expression and activity were reduced in sorafenib-resistant cells compared to the parental cell line. Overall, our study allowed the identification of ALDH2 as a novel sorafenib target and sheds light on its potential role in both hepatocellular carcinoma and sorafenib resistance condition.
Assuntos
Aldeído-Desidrogenase Mitocondrial , Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteoma , Sorafenibe , Sorafenibe/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/metabolismo , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Aldeído-Desidrogenase Mitocondrial/genética , Aldeído-Desidrogenase Mitocondrial/metabolismo , Linhagem Celular Tumoral , Inibidores de Proteínas Quinases/farmacologia , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Ligação Proteica/efeitos dos fármacosRESUMO
Plant homeodomain (PHD) zinc fingers are histone reader domains that are often associated with human diseases. Despite this, they constitute a poorly targeted class of readers, suggesting low ligandability. Here, we describe a successful fragment-based campaign targeting PHD fingers from the proteins BAZ2A and BAZ2B as model systems. We validated a pool of in silico fragments both biophysically and structurally and solved the first crystal structures of PHD zinc fingers in complex with fragments bound to an anchoring pocket at the histone binding site. The best-validated hits were found to displace a histone H3 tail peptide in competition assays. This work identifies new chemical scaffolds that provide suitable starting points for future ligand optimization using structure-guided approaches. The demonstrated ligandability of the PHD reader domains could pave the way for the development of chemical probes to drug this family of epigenetic readers.
Assuntos
Proteínas de Homeodomínio/química , Fragmentos de Peptídeos/química , Proteínas de Plantas/química , Dedos de Zinco , Sítios de Ligação , Proteínas Cromossômicas não Histona/química , Cristalografia por Raios X , Histonas/metabolismo , Humanos , LigantesRESUMO
Efforts to develop inhibitors, activators, and effectors of biological reactions using small molecule libraries are often hampered by interference compounds, artifacts, and false positives that permeate the pool of initial hits. Here, we report the discovery of a promising initial hit compound targeting the Fanconi anemia ubiquitin-conjugating enzyme Ube2T and describe its biophysical and biochemical characterization. Analysis of the co-crystal structure led to the identification of a contaminating zinc ion as solely responsible for the observed effects. Zinc binding to the active site cysteine induces a domain swap in Ube2T that leads to cyclic trimerization organized in an open-ended linear assembly. Our study serves as a cautionary tale for screening small molecule libraries and provides insights into the structural plasticity of ubiquitin-conjugating enzymes.
Assuntos
Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/efeitos dos fármacos , Compostos de Zinco/química , Compostos de Zinco/farmacologia , Domínio Catalítico , Cristalografia por Raios X , Cisteína , Ensaios de Triagem em Larga Escala , Modelos Moleculares , Dobramento de Proteína , Estrutura Secundária de Proteína/efeitos dos fármacos , Bibliotecas de Moléculas PequenasRESUMO
Ube2T is the E2 ubiquitin-conjugating enzyme of the Fanconi anemia DNA repair pathway and it is overexpressed in several cancers, representing an attractive target for the development of inhibitors. Despite the extensive efforts in targeting the ubiquitin system, very few E2 binders have currently been discovered. Herein we report the identification of a new allosteric pocket on Ube2T through a fragment screening using biophysical methods. Several fragments binding to this site inhibit ubiquitin conjugation in vitro.
Assuntos
Anemia de Fanconi/enzimologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Regulação Alostérica , Humanos , UbiquitinaçãoRESUMO
The plant homeodomain (PHD) fingers are among the largest family of epigenetic domains, first characterized as readers of methylated H3K4. Readout of histone post-translational modifications by PHDs has been the subject of intense investigation; however, less is known about the recognition of secondary structure features within the histone tail itself. We solved the crystal structure of the PHD finger of the bromodomain adjacent to zinc finger 2A [BAZ2A, also known as TIP5 (TTF-I/interacting protein 5)] in complex with unmodified N-terminal histone H3 tail. The peptide is bound in a helical folded-back conformation after K4, induced by an acidic patch on the protein surface that prevents peptide binding in an extended conformation. Structural bioinformatics analyses identify a conserved Asp/Glu residue that we name 'acidic wall', found to be mutually exclusive with the conserved Trp for K4Me recognition. Neutralization or inversion of the charges at the acidic wall patch in BAZ2A, and homologous BAZ2B, weakened H3 binding. We identify simple mutations on H3 that strikingly enhance or reduce binding, as a result of their stabilization or destabilization of H3 helicity. Our work unravels the structural basis for binding of the helical H3 tail by PHD fingers and suggests that molecular recognition of secondary structure motifs within histone tails could represent an additional layer of regulation in epigenetic processes.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Histonas/metabolismo , Modelos Moleculares , Proteínas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Sequência Conservada , Cristalografia por Raios X , Histonas/química , Histonas/genética , Humanos , Cinética , Ligantes , Simulação de Dinâmica Molecular , Mutação , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteínas/química , Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fatores Genéricos de TranscriçãoRESUMO
RNA polymerase-binding protein A (RbpA), encoded by Rv2050, is specific to the actinomycetes, where it is highly conserved. In the pathogen Mycobacterium tuberculosis, RbpA is essential for growth and survival. RbpA binds to the ß subunit of the RNA polymerase where it activates transcription by unknown mechanisms, and it may also influence the response of M. tuberculosis to the current frontline anti-tuberculosis drug rifampicin. Here we report the solution structure of RbpA and identify the principle sigma factor σ(A) and the stress-induced σ(B) as interaction partners. The protein has a central ordered domain with a conserved hydrophobic surface that may be a potential protein interaction site. The N and C termini are highly dynamic and are involved in the interaction with the sigma factors. RbpA forms a tight complex with the N-terminal domain of σ(B) via its N- and C-terminal regions. The interaction with sigma factors may explain how RbpA stabilizes sigma subunit binding to the core RNA polymerase and thereby promotes initiation complex formation. RbpA could therefore influence the competition between principal and alternative sigma factors and hence the transcription profile of the cell.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Mutação , Mycobacterium tuberculosis/metabolismo , Fator sigma/metabolismo , Transativadores/química , Actinobacteria/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Teste de Complementação Genética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Fenótipo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Transativadores/genética , Transcrição Gênica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Mycobacterium tuberculosis is an extremely well adapted intracellular human pathogen that is exposed to multiple DNA damaging chemical assaults originating from the host defence mechanisms. As a consequence, this bacterium is thought to possess highly efficient DNA repair machineries, the nucleotide excision repair (NER) system amongst these. Although NER is of central importance to DNA repair in M. tuberculosis, our understanding of the processes in this species is limited. The conserved UvrABC endonuclease represents the multi-enzymatic core in bacterial NER, where the UvrA ATPase provides the DNA lesion-sensing function. The herein reported genetic analysis demonstrates that M. tuberculosis UvrA is important for the repair of nitrosative and oxidative DNA damage. Moreover, our biochemical and structural characterization of recombinant M. tuberculosis UvrA contributes new insights into its mechanism of action. In particular, the structural investigation reveals an unprecedented conformation of the UvrB-binding domain that we propose to be of functional relevance. Taken together, our data suggest UvrA as a potential target for the development of novel anti-tubercular agents and provide a biochemical framework for the identification of small-molecule inhibitors interfering with the NER activity in M. tuberculosis.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Mycobacterium tuberculosis/enzimologia , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Modelos Moleculares , MutaçãoRESUMO
DPP10 is a transmembrane glycosylated protein belonging to the family of dipeptidyl aminopeptidase-like proteins (DPPLs). DPPLs are auxiliary subunits involved in the regulation of voltage-gated Kv4 channels, key determinants of cardiac and neuronal excitability. Although it is known that DPPLs are needed to generate native-like currents in heterologous expression systems, the molecular basis of this involvement are still poorly defined. In this study, we investigated the functional relevance of DPP10 glycosylation in modulating Kv4.3 channel activities. Using transfected Chinese hamster ovary (CHO) cells to reconstitute Kv4 complex, we show that the pharmacological inhibition of DPP10 glycosylation by tunicamycin and neuraminidase affects transient outward potassium current (I (to)) kinetics. Tunicamycin completely blocked DPP10 glycosylation and reduced DPP10 cell surface expression. The accelerating effects of DPP10 on Kv4.3 current kinetics, i.e. on inactivation and recovery from inactivation, were abolished. Neuraminidase produced different effects on current kinetics than tunicamycin, i.e., shifted the voltage dependence to more negative potentials. The effects of tunicamycin on the native I (to) currents of human atrial myocytes expressing DPP10 were similar to those of the KV4.3/KChIP2/DPP10 complex in CHO cells. Our results suggest that N-linked glycosylation of DPP10 plays an important role in modulating Kv4 channel activities.