RESUMO
Many multicellular organisms specify germ cells during early embryogenesis by the inheritance of ribonucleoprotein (RNP) granules known as germplasm. However, the role of complex interactions of RNP granules during germ cell specification remains elusive. This study characterizes the interaction of RNP granules, Buc, and zebrafish Vasa (zfVasa) during germ cell specification. We identify a novel zfVasa-binding motif (Buc-VBM) in Buc and a Buc-binding motif (zfVasa-BBM) in zfVasa. Moreover, we show that Buc and zfVasa directly bind in vitro and that this interaction is independent of the RNA. Our circular dichroism spectroscopy data reveal that the intrinsically disordered Buc-VBM peptide forms alpha-helices in the presence of the solvent trifluoroethanol. Intriguingly, we further demonstrate that Buc-VBM enhances zfVasa ATPase activity, thereby annotating the first biochemical function of Buc as a zfVasa ATPase activator. Collectively, these results propose a model in which the activity of zfVasa is a central regulator of primordial germ cell (PGC) formation and is tightly controlled by the germplasm organizer Buc.
Assuntos
RNA Helicases DEAD-box/genética , Ribonucleoproteínas/genética , Proteínas de Peixe-Zebra/genética , Adenosina Trifosfatases/genética , Animais , Citoplasma , Células Germinativas/crescimento & desenvolvimento , Células Germinativas/metabolismo , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Ligação Proteica/genética , RNA/genética , Peixe-Zebra/genéticaRESUMO
CRM1 is a nuclear export receptor that has been intensively targeted over the last decade for the development of antitumor and antiviral drugs. Structural analysis of several inhibitor compounds bound to CRM1 revealed that their mechanism of action relies on the covalent modification of a critical cysteine residue (Cys528 in the human receptor) located in the nuclear export signal-binding cleft. This study presents the crystal structure of human CRM1, covalently modified by 2-mercaptoethanol on Cys528, in complex with RanGTP at 2.58â Å resolution. The results demonstrate that buffer components can interfere with the characterization of cysteine-dependent inhibitor compounds.
Assuntos
Cisteína/química , Carioferinas/química , Carioferinas/metabolismo , Mercaptoetanol/química , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Sinais de Exportação Nuclear , Proteína ran de Ligação ao GTP/química , Proteína Exportina 1RESUMO
The receptor CRM1 is responsible for the nuclear export of many tumor-suppressor proteins and viral ribonucleoproteins. This renders CRM1 an interesting target for therapeutic intervention in diverse cancer types and viral diseases. Structural studies of Saccharomyces cerevisiae CRM1 (ScCRM1) complexes with inhibitors defined the molecular basis for CRM1 inhibition. Nevertheless, no structural information is available for inhibitors bound to human CRM1 (HsCRM1). Here, we present the structure of the natural inhibitor Leptomycin B bound to the HsCRM1-RanGTP complex. Despite high sequence conservation and structural similarity in the NES-binding cleft region, ScCRM1 exhibits 16-fold lower binding affinity than HsCRM1 toward PKI-NES and significant differences in affinities toward potential CRM1 inhibitors. In contrast to HsCRM1, competition assays revealed that a human adapted mutant ScCRM1-T539C does not bind all inhibitors tested. Taken together, our data indicate the importance of using HsCRM1 for molecular analysis and development of novel antitumor and antiviral drugs.