RESUMO
Loss of galactose-1 phosphate uridylyltransferase (GALT) activity in humans results in Classic Galactosemia, and the GalT-deficient (GalT-/-) mouse mimics the patient condition. GalT-/- ovaries display elevated endoplasmic reticulum (ER) stress marker, BiP, and downregulated canonical phosphatidylinositol 3-kinase (Pi3k)/protein kinase B (Akt) growth/pro-survival signaling. Numbers of primordial follicles are reduced in the mutants, recapitulating the accelerated ovarian aging seen in human patients. We previously found that oral administration of the compound Salubrinal (an eIF2α phosphatase inhibitor), resulted in reduction of ovarian BiP expression, rescued Pi3k/Akt signaling, and a doubling of primordial follicles in GalT-/- adults. Here, we further characterized galactosemic stress in GalT-/- mice versus wild-type (WT) controls, and examined whether Salubrinal treatment improved broader reproductive parameters. We assessed the expression levels of factors of the unfolded protein response (UPR), and found that BiP, phospho-Perk, and phospho-eIF2α were all elevated in GalT-/- ovaries. However, neither IKK activation (NFκB pathway) nor alternative Xbp1 splicing downstream of ER membrane protein Ire1α activation was induced, suggesting an Xbp1-independent UPR in galactosemic stress. Moreover, Salubrinal treatment significantly increased the number of ovulated eggs in mutant animals after gonadotrophic superovulation. Salubrinal treatment also normalized estrus cycle stage lengths and resulted in significantly larger litter sizes than vehicle-treated mutants. Overall, we show that Salubrinal protects against galactosemia-induced primordial follicle loss in a fashion that includes suppressing the de-phosphorylation of eIF2α, and that intervention in this way significantly improves and extends ovarian function, fertility, and fecundity.
Assuntos
Cinamatos/uso terapêutico , Fator de Iniciação 2 em Eucariotos/metabolismo , Galactosemias/complicações , Infertilidade Feminina/tratamento farmacológico , Infertilidade Feminina/etiologia , Tioureia/análogos & derivados , Animais , Cinamatos/farmacologia , Modelos Animais de Doenças , Feminino , Fertilidade/efeitos dos fármacos , Galactosemias/metabolismo , Infertilidade Feminina/metabolismo , Camundongos , Ovário/efeitos dos fármacos , Ovário/metabolismo , Fosforilação/efeitos dos fármacos , Tioureia/farmacologia , Tioureia/uso terapêutico , Resposta a Proteínas não Dobradas/efeitos dos fármacosAssuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Coenzimas , Metaloproteínas/biossíntese , Trifosfato de Adenosina/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Cristalografia por Raios X , Metaloproteínas/metabolismo , Modelos Moleculares , Cofatores de Molibdênio , Fosfatos/metabolismo , Ligação Proteica , Conformação Proteica , Pteridinas/metabolismo , Ubiquitina/química , Ubiquitina/metabolismoAssuntos
Guanilato Ciclase/fisiologia , Receptores do Fator Natriurético Atrial/fisiologia , Cristalografia por Raios X , Guanilato Ciclase/química , Guanilato Ciclase/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Conformação Proteica , Receptores do Fator Natriurético Atrial/química , Receptores do Fator Natriurético Atrial/metabolismo , Transdução de SinaisAssuntos
Bioquímica/métodos , Proteoma/metabolismo , Anticorpos/genética , Anticorpos/imunologia , Lipossomos/química , Lipossomos/metabolismo , Fases de Leitura Aberta/genética , Fosfatidilinositóis/metabolismo , Ligação Proteica , Proteoma/genética , Proteoma/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , LevedurasAssuntos
Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Proteínas de Xenopus , Proteínas de Peixe-Zebra , Animais , Sítios de Ligação , Dimerização , Humanos , Camundongos , Modelos Moleculares , Conformação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Superfície Celular/genética , Transdução de Sinais , Proteínas WntRESUMO
Stability parameters for individual residues in Thermus thermophilus cysteine-free RNase H were determined by native state hydrogen exchange, thus providing a unique comparison of regional thermodynamics between thermophilic and mesophilic homologues. The general distribution of stability in the thermophilic protein is similar to that of its mesophilic homologue, with a proportional increase in stability for almost all residues. As a consequence, the residue-specific stabilities of the two proteins are remarkably similar under conditions where their global stabilities are the same. These results indicate that T. thermophilus RNase H is stabilized in a delocalized fashion, preserving a finely tuned balance of stabilizing interactions throughout the structure. Therefore, although protein stability can be altered by single amino acid substitution, evolution for optimal function may require more subtle and delocalized mechanisms.
Assuntos
Ribonuclease H/metabolismo , Thermus thermophilus/enzimologia , Deutério , Transferência de Energia , Estabilidade Enzimática , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonuclease H/química , Ribonuclease H/genéticaRESUMO
The mechanisms by which thermophilic proteins attain their increased thermostability remain unclear, as usually the sequence and structure of these proteins are very similar to those of their mesophilic homologues. To gain insight into the basis of thermostability, we have determined protein stability curves describing the temperature dependence of the free energy of unfolding for two ribonucleases H, one from the mesophile Escherichia coli and one from the thermophile Thermus thermophilus. The circular dichroism signal was monitored as a function of temperature and guanidinium chloride concentration, and the resulting free energies of unfolding were fit to the Gibbs-Helmholtz equation to obtain a set of thermodynamic parameters for these proteins. Although the maximal stabilities for these proteins occur at similar temperatures, the heat capacity of unfolding for T. thermophilus RNase H is lower, resulting in a smaller temperature dependence of the free energy of unfolding and therefore a higher thermal melting temperature. In addition, the stabilities of these proteins are similar at the optimal growth temperatures for their respective organisms, suggesting that a balance of thermodynamic stability and flexibility is important for function.