RESUMO
The bare lymphocyte syndrome, a severe combined immunodeficiency due to loss of major histocompatibility complex (MHC) class II gene expression, is caused by inherited mutations in the genes encoding the heterotrimeric transcription factor RFX (RFX-B, RFX5, and RFXAP) and the class II transactivator CIITA. Mutagenesis of the RFX genes was performed, and the properties of the proteins were analyzed with regard to transactivation, DNA binding, and protein-protein interactions. The results identified specific domains within each of the three RFX subunits that were necessary for RFX complex formation, including the ankyrin repeats of RFX-B. DNA binding was dependent on RFX complex formation, and transactivation was dependent on a region of RFX5. RFX5 was found to interact with CIITA, and this interaction was dependent on a proline-rich domain within RFX5. Thus, these studies have defined the protein domains required for the functional regulation of MHC class II genes.
Assuntos
Proteínas Nucleares , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/metabolismo , Animais , Anquirinas/metabolismo , Células COS , Linhagem Celular , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Genes MHC da Classe II/genética , Genes Reporter , Glutationa Transferase/metabolismo , Humanos , Mutagênese , Plasmídeos/metabolismo , Testes de Precipitina , Prolina/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição de Fator Regulador X , Transativadores/química , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , TransfecçãoRESUMO
UDP-galactose-4-epimerase (GALE) is a highly conserved enzyme that catalyzes the interconversion of UDP-galactose and UDP-glucose. Impairment of this enzyme in humans results in one of two clinically distinct forms of epimerase-deficiency galactosemia-one benign, the other severe. The molecular and biochemical distinction between these disorders remains unknown. To enable structural and functional studies of both wild-type and patient-derived alleles of human GALE (hGALE), we have developed and applied a null-background yeast expression system for the human enzyme. We have demonstrated that wild-type hGALE sequences phenotypically complement a yeast gal10 deletion, and we have biochemically characterized the wild-type human enzyme isolated from these cells. Furthermore, we have expressed and characterized two mutant alleles, L183P-hGALE and N34S-hGALE, both derived from a patient with no detectable GALE activity in red blood cells but with approximately 14% activity in cultured lymphoblasts. Analyses of crude extracts of yeast expressing L183P-hGALE demonstrated 4% wild-type activity and 6% wild-type abundance. Extracts of yeast expressing N34S-hGALE demonstrated approximately 70% wild-type activity and normal abundance. However, yeast coexpressing both L183P-hGALE and N34S-hGALE exhibited only approximately 7% wild-type levels of activity, thereby confirming the functional impact of both substitutions and raising the intriguing possibility that some form of dominant-negative interaction may exist between the mutant alleles found in this patient. The results reported here establish the utility of the yeast-based hGALE-expression system and set the stage for more-detailed studies of this important enzyme and its role in epimerase-deficiency galactosemia.
