making a difference in human health through collaborative scientific discovery
Every year in the U.S., approximately 1,300 babies are born with spina bifida, a serious developmental defect characterized by failure of the neural tube to close. Maternal supplementation with the vitamin folic acid around the time of conception has been shown to decrease the incidence of neural tube defects by up to 70%. Even so, little is known about the mechanism of this preventive effect and what form(s) of folate is protective in vivo. In order to begin to address this, we generated a mouse model for methionine synthase reductase (Mtrr) deficiency. Mtrr is required to support the catalytic activity of methionine synthase, which converts homocysteine to methionine using methyltetrahy-drofolate as a methyl donor with concurrent return of tetrahydrofolate to the active folate pool. Mothers homozygous for a common polymorphism in Mtrr are at higher risk of neural tube defects in offspring.
Our mouse model has a gene trap insertion in the Mtrr gene. In the homozygous Mtrrgt/gt mouse, partial restoration of wild type mRNA occurs by splicing around the gene trap, and as a result, homozygotes are hypomorphic for methionine synthase reductase. The gene trap results in decreased expression of wild type Mtrr mRNA and reduces methionine synthase activity in all tissues analyzed. Mtrrgt/gt mice have increased plasma homocysteine and decreased plasma methionine, and male Mtrrgt/gt mice exhibit growth retardation. The folate pools from kidney show altered distributions with increased levels of methyltetrahydrofolate and decreased levels of methylene-tetrahydrofolate and tetrahydrofolate. Unexpectedly, Mtrrgt/gt mice have significantly increased S-adenosylmethionine and decreased S-adenosylhomocysteine in liver. Analysis of the expression of methionine synthase reductase in the Mtrr+/gt E9.5 embryo indicated high expression in the developing brain and neural tube. We anticipate that this mouse model will be useful in dissecting the roles of different folate derivatives in the pathologies associated with neural tube defects.
Target of rapamycin (TOR) is an evolutionally conserved protein kinase in eukaryotes and a central cell growth controller. TOR exists in two distinct complexes, termed TORC1 and TORC2. Mammalian TORC2 has recently been shown to possess kinase activity towards the C-terminal hydrophobic site of Akt/PKB. Here, we report that Sin1 is an essential component of TORC2 but not of TORC1, and functions similarly to Rictor, the defining member of TORC2, in complex formation and kinase activity. Knockdown of Sin1 decreases Akt phosphorylation in both Drosophila and mammalian cells, and diminishes Akt function in vivo. It also disrupts the interaction between Rictor and mTOR. Furthermore, Sin1 is required for TORC2 kinase activity in vitro. Disruption of the Rictor gene in mice results in embryonic lethality and ablates Akt phosphorylation. These data demonstrate that Sin1 together with Rictor are key components of mTORC2 and play an essential role in Akt phosphorylation and signaling.
Cellular survival is dependent upon the cell’s ability to maintain a homeostasis between biogenesis and degradation. Autophagy is an evolutionarily conserved mechanism by which cells rid themselves of superfluous or damaged proteins and organelles. Nearly thirty gene products that function in at least one of the various types of autophagy have been identified in yeast. These gene products are called AuTophaGy-related, or Atg proteins. Despite our knowledge of the proteins required for these pathways, the molecular mechanisms of autophagy remain largely unknown. One particular aspect of the pathway that is unclear is the membrane source for newly forming autophagosomes. Using the baker’s yeast, Saccharomyces cerevisiae, as a model system to study autophagy, the Klionsky laboratory has identified two transmembrane proteins, Atg9 and Atg27, and one peripheral membrane protein, Atg23, that exhibit a localization pattern that is unique among Atg proteins. Whereas most Atg proteins localize at a single punctate structure (the pre-autophagosomal structure, or PAS), these proteins reside in multiple punctate structures, including the PAS (the organizing center for newly forming autophagosomes) and discrete regions on the mitochondrial membrane. Moreover, these proteins cycle between the two locations. Atg9, Atg27 and Atg23 all interact with one another, and each of the three proteins requires the other two for proper cycling. We believe that these proteins may, in part, mark the membrane source for newly forming autophagosomes, and in fact may contribute to the expanding membrane.