RESUMO
We demonstrate how RNA binding protein FOX-1 functions as a dose-dependent X-signal element to communicate X-chromosome number and thereby determine nematode sex. FOX-1, an RNA recognition motif protein, triggers hermaphrodite development in XX embryos by causing non-productive alternative pre-mRNA splicing of xol-1, the master sex-determination switch gene that triggers male development in XO embryos. RNA binding experiments together with genome editing demonstrate that FOX-1 binds to multiple GCAUG and GCACG motifs in a xol-1 intron, causing intron retention or partial exon deletion, thereby eliminating male-determining XOL-1 protein. Transforming all motifs to GCAUG or GCACG permits accurate alternative splicing, demonstrating efficacy of both motifs. Mutating subsets of both motifs partially alleviates non-productive splicing. Mutating all motifs blocks it, as does transforming them to low-affinity GCUUG motifs. Combining multiple high-affinity binding sites with the twofold change in FOX-1 concentration between XX and XO embryos achieves dose-sensitivity in splicing regulation to determine sex.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Proteínas de Ligação a RNA/fisiologia , Cromossomo X/genética , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Feminino , Íntrons/genética , Íntrons/fisiologia , Masculino , Proteínas de Ligação a RNA/metabolismo , Processos de Determinação SexualRESUMO
With structural guidance, tropane-derived HTS hits were modified to optimize for HSP90 inhibition and a desirable in vivo profile. Through an iterative SAR development process 12i (XL888) was discovered and shown to reduce HSP90 client protein content in PD studies. Furthermore, efficacy experiments performed in a NCI-N87 mouse xenograft model demonstrated tumor regression in some dosing regimens.
Assuntos
Antineoplásicos/química , Antineoplásicos/uso terapêutico , Compostos Azabicíclicos/química , Compostos Azabicíclicos/uso terapêutico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Ácidos Ftálicos/química , Ácidos Ftálicos/uso terapêutico , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Compostos Azabicíclicos/farmacocinética , Compostos Azabicíclicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Descoberta de Drogas , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos , Modelos Moleculares , Neoplasias/metabolismo , Neoplasias/patologia , Ácidos Ftálicos/farmacocinética , Ácidos Ftálicos/farmacologiaRESUMO
Mutations that inactivate the retinoblastoma (Rb) pathway are common in human tumors. Such mutations promote tumor growth by deregulating the G1 cell cycle checkpoint. However, uncontrolled cell cycle progression can also produce new liabilities for cell survival. To uncover such liabilities in Rb mutant cells, we performed a clonal screen in the Drosophila eye to identify second-site mutations that eliminate Rbf(-) cells, but allow Rbf(+) cells to survive. Here we report the identification of a mutation in a novel highly conserved peptidyl prolyl isomerase (PPIase) that selectively eliminates Rbf(-) cells from the Drosophila eye.
Assuntos
Drosophila melanogaster/embriologia , Olho/embriologia , Peptidilprolil Isomerase/genética , Proteína do Retinoblastoma/genética , Sequência de Aminoácidos , Animais , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Olho/enzimologia , Dados de Sequência Molecular , MutaçãoRESUMO
Presenilins are components of the gamma-secretase protein complex that mediates intramembranous cleavage of betaAPP and Notch proteins. A C. elegans genetic screen revealed two genes, aph-1 and pen-2, encoding multipass transmembrane proteins, that interact strongly with sel-12/presenilin and aph-2/nicastrin. Human aph-1 and pen-2 partially rescue the C. elegans mutant phenotypes, demonstrating conserved functions. The human genes must be provided together to rescue the mutant phenotypes, and the inclusion of presenilin-1 improves rescue, suggesting that they interact closely with each other and with presenilin. RNAi-mediated inactivation of aph-1, pen-2, or nicastrin in cultured Drosophila cells reduces gamma-secretase cleavage of betaAPP and Notch substrates and reduces the levels of processed presenilin. aph-1 and pen-2, like nicastrin, are required for the activity and accumulation of gamma-secretase.