RESUMEN
Bacteria use an array of sigma factors to regulate gene expression during different stages of their life cycles. Full-length, atomic-level structures of sigma factors have been challenging to obtain experimentally as a result of their many regions of intrinsic disorder. AlphaFold has now supplied plausible full-length models for most sigma factors. Here we discuss the current understanding of the structures and functions of sigma factors in the model organism, Bacillus subtilis, and present an X-ray crystal structure of a region of B. subtilis SigE, a sigma factor that plays a critical role in the developmental process of spore formation.
RESUMEN
Global changes in bacterial gene expression can be orchestrated by the coordinated activation/deactivation of alternative sigma (σ) factor subunits of RNA polymerase. Sigma factors themselves are regulated in myriad ways, including via anti-sigma factors. Here, we have determined the solution structure of anti-sigma factor CsfB, responsible for inhibition of two alternative sigma factors, σG and σE, during spore formation by Bacillus subtilis. CsfB assembles into a symmetrical homodimer, with each monomer bound to a single Zn2+ ion via a treble-clef zinc finger fold. Directed mutagenesis indicates that dimer formation is critical for CsfB-mediated inhibition of both σG and σE, and we have characterized these interactions in vitro. This work represents an advance in our understanding of how CsfB mediates inhibition of two alternative sigma factors to drive developmental gene expression in a bacterium.
Asunto(s)
Bacillus subtilis/química , Regulación Bacteriana de la Expresión Génica , Proteínas Represoras/química , Factor sigma/química , Esporas Bacterianas/química , Zinc/química , Secuencia de Aminoácidos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Sitios de Unión , Cationes Bivalentes , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factor sigma/antagonistas & inhibidores , Factor sigma/genética , Factor sigma/metabolismo , Esporas Bacterianas/genética , Esporas Bacterianas/metabolismo , Zinc/metabolismoRESUMEN
Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.
Asunto(s)
Piperidinas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Peptidasa Específica de Ubiquitina 7/antagonistas & inhibidores , Animales , Apoenzimas/antagonistas & inhibidores , Apoenzimas/química , Apoenzimas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Femenino , Humanos , Ratones , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/patología , Piperidinas/síntesis química , Proteínas Proto-Oncogénicas c-mdm2/química , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Pirazoles/síntesis química , Pirimidinas/síntesis química , Especificidad por Sustrato , Transcripción Genética/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Peptidasa Específica de Ubiquitina 7/química , Peptidasa Específica de Ubiquitina 7/metabolismo , Ubiquitinación/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Small glutamine-rich tetratricopeptide repeat-containing protein 2 (Sgt2) is a multi-module co-chaperone involved in several protein quality control pathways. The TPR domain of Sgt2 and several other proteins, including SGTA, Hop, and CHIP, is a highly conserved motif known to form transient complexes with molecular chaperones such as Hsp70 and Hsp90. In this work, we present the first high resolution crystal structures of Sgt2_TPR alone and in complex with a C-terminal peptide PTVEEVD from heat shock protein, Ssa1. Using nuclear magnetic resonance spectroscopy and isothermal titration calorimetry, we demonstrate that Sgt2_TPR interacts with peptides corresponding to the C-termini of Ssa1, Hsc82, and Ybr137wp with similar binding modes and affinities.
RESUMEN
The fate of secretory and membrane proteins that mislocalize to the cytosol is decided by a collaboration between cochaperone SGTA (small, glutamine-rich, tetratricopeptide repeat protein alpha) and the BAG6 complex, whose operation relies on multiple transient and subtly discriminated interactions with diverse binding partners. These include chaperones, membrane-targeting proteins and ubiquitination enzymes. Recently a direct interaction was discovered between SGTA and the proteasome, mediated by the intrinsic proteasomal ubiquitin receptor Rpn13. Here, we structurally and biophysically characterize this binding and identify a region of the Rpn13 C-terminal domain that is necessary and sufficient to facilitate it. We show that the contact occurs through a carboxylate clamp-mediated molecular recognition event with the TPR domain of SGTA, and provide evidence that the interaction can mediate the association of Rpn13 and SGTA in a cellular context.
Asunto(s)
Proteínas Portadoras/química , Glicoproteínas de Membrana/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares , Unión Proteica , Dominios ProteicosRESUMEN
RNF126 is an E3 ubiquitin ligase that collaborates with the BAG6 sortase complex to ubiquitinate hydrophobic substrates in the cytoplasm that are destined for proteasomal recycling. Composed of a trimeric complex of BAG6, TRC35 and UBL4A the BAG6 sortase is also associated with SGTA, a co-chaperone from which it can obtain hydrophobic substrates. Here we solve the solution structure of the RNF126 zinc finger domain in complex with the BAG6 UBL domain. We also characterise an interaction between RNF126 and UBL4A and analyse the competition between SGTA and RNF126 for the N-terminal BAG6 binding site. This work sheds light on the sorting mechanism of the BAG6 complex and its accessory proteins which, together, decide the fate of stray hydrophobic proteins in the aqueous cytoplasm.
Asunto(s)
Complejos Multiproteicos/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Sitios de Unión , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Células HeLa , Humanos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Complejos Multiproteicos/química , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Ubiquitinas/química , Ubiquitinas/metabolismo , Dedos de ZincRESUMEN
A weak screening hit with suboptimal physicochemical properties was optimized against PFKFB3 kinase using critical structure-guided insights. The resulting compounds demonstrated high selectivity over related PFKFB isoforms and modulation of the target in a cellular context. A selected example demonstrated exposure in animals following oral dosing. Examples from this series may serve as useful probes to understand the emerging biology of this metabolic target.