RESUMEN
The post-translational modification of proteins with polyubiquitin regulates virtually all aspects of cell biology. Eight distinct chain linkage types co-exist in polyubiquitin and are independently regulated in cells. This 'ubiquitin code' determines the fate of the modified protein. Deubiquitinating enzymes of the ovarian tumour (OTU) family regulate cellular signalling by targeting distinct linkage types within polyubiquitin, and understanding their mechanisms of linkage specificity gives fundamental insights into the ubiquitin system. Here we reveal how the deubiquitinase Cezanne (also known as OTUD7B) specifically targets Lys11-linked polyubiquitin. Crystal structures of Cezanne alone and in complex with monoubiquitin and Lys11-linked diubiquitin, in combination with hydrogen-deuterium exchange mass spectrometry, enable us to reconstruct the enzymatic cycle in great detail. An intricate mechanism of ubiquitin-assisted conformational changes activates the enzyme, and while all chain types interact with the enzymatic S1 site, only Lys11-linked chains can bind productively across the active site and stimulate catalytic turnover. Our work highlights the plasticity of deubiquitinases and indicates that new conformational states can occur when a true substrate, such as diubiquitin, is bound at the active site.
Asunto(s)
Enzimas Desubicuitinizantes/metabolismo , Endopeptidasas/metabolismo , Lisina/metabolismo , Poliubiquitina/metabolismo , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Enzimas Desubicuitinizantes/química , Enzimas Desubicuitinizantes/genética , Medición de Intercambio de Deuterio , Endopeptidasas/química , Endopeptidasas/genética , Activación Enzimática , Humanos , Espectrometría de Masas , Modelos Moleculares , Unión Proteica , Conformación Proteica , Especificidad por Sustrato , Ubiquitinación , Ubiquitinas/metabolismoRESUMEN
Cruciferous vegetables such as broccoli and kale have well documented chemopreventative and anticancer effects that are attributed to the presence of isothiocyanates (ITCs). ITCs modulate the levels of many oncogenic proteins, but the molecular mechanisms of ITC action are not understood. We previously reported that phenethyl isothiocyanate (PEITC) inhibits two deubiquitinases (DUBs), USP9x and UCH37. DUBs regulate many cellular processes and DUB dysregulation is linked to the pathogenesis of human diseases including cancer, neurodegeneration, and inflammation. Using SILAC assisted quantitative mass spectrometry, here we identify 9 new PEITC-DUB targets: USP1, USP3, USP10, USP11, USP16, USP22, USP40, USP48 and VCPIP1. Seven of these PEITC-sensitive DUBs have well-recognized roles in DNA repair or chromatin remodeling. PEITC both inhibits USP1 and increases its ubiquitination and degradation, thus decreasing USP1 activity by two mechanisms. The loss of USP1 activity increases the level of mono-ubiquitinated DNA clamp PCNA, impairing DNA repair. Both the inhibition/degradation of USP1 and the increase in mono-ubiquitinated PCNA are new activities for PEITC that can explain the previously recognized ability of ITCs to enhance cancer cell sensitivity to cisplatin treatment. Our work also demonstrates that PEITC reduces the mono-ubiquityl histones H2A and H2B. Understanding the mechanism of action of ITCs should facilitate their use as therapeutic agents.
RESUMEN
Post-translational modification of proteins with ubiquitin (Ub) and Ub chains controls numerous biochemical events. Although it has been proven that all Ub-Ub linkages are formed in cells, studies have been limited for a long time to K48 and K63 chains as these can be generated biochemically. Access to the remaining (atypical) Ub-Ub chain types has been hampered by a lack of specific E2 enzymes. In this chapter we present a solution to this problem by using a native chemical ligation approach to obtain all other (i.e. K6, K11, K27, K29 and K33) diubiquitin chains.