Asparagine synthetase (ASNS) catalyzes the synthesis of asparagine (Asn) from aspartate. Given ASNS expression in most human tissues, Asn is not a dietary essential amino acid, but insufficient ASNS activity causes tissues to become Asn-dependent. A recently identified disease, termed Asparagine Synthetase Deficiency (ASNSD), is associated with mutations in the ASNS gene. Newborns with this disease exhibit severe microcephaly that continues as progressive brain atrophy, intractable epileptic seizures, suppressed development, and shortened lifespan. While exome sequencing of newborns with these symptoms has already identified about 30-40 children, unfortunately, there is no convenient, reproducible assay for ASNS enzymatic activity. Also, the human ASNS crystal structure has not been published, forcing the use of molecular modeling based on the E. coli AS-B structure to predict the possible consequences of ASNSD- associated mutations. Consequently, an ?association? between ASNS mutations and disease exists, but conclusive evidence that ASNS mutations account for the phenotype and the degree to which specific ASNS variants function within cells remains to be established. Thus, there are critical gaps in our knowledge of ASNS and ASNSD that are addressed as follows.
Aim I hypothesis: mutations within the human ASNS gene cause changes in protein conformation that lead to reduced enzyme activity. In parallel, we will develop a reproducible ASNS enzymatic assay and crystallize the human ASNS wild type protein and specific ASNSD-linked variants identified in afflicted children.
Aim II hypothesis: ASNS AA variants expressed by ASNSD children lead to suppression of cell proliferation. We will test whether expression of specific ASNSD-associated AA variants in an ASNS-null cell support proliferation in Asn-free medium. We will also test whether ectopic expression of wild-type ASNS protein in fibroblasts from ASNSD children complements the disease phenotype and rescues cell growth in the absence of Asn. The proposed studies are of high reward because the novel information gained will positively impact the investigation, diagnosis, and treatment monitoring of ASNSD children for the following reasons. 1) Development of a direct and reproducible ASNS enzyme assay will yield much improved methods of diagnosis and treatment monitoring for ASNSD children. 2) Structure-function studies will firmly establish the consequences of specific ASNS mutations on protein conformation and enzyme activity. 3) The effect of ASNSD mutations on cell growth has yet to be subjected to the expected classic characterization of an inborn error of metabolism. For example, complementation of ASNSD-derived fibroblasts with wild type ASNS to document rescue and, conversely, expression of ASNS AA variants in an ASNS-null cell to show lack of growth relative to wild type enzyme. 4) Ectopic expression experiments will also reveal the impact of specific ASNSD-associated AA variants on cell function and metabolism, as well as proliferation.
Asparagine is an amino acid required to synthesize proteins, but is not considered a dietary ?essential? amino acid because most cells in the human body have asparagine synthetase (ASNS), an enzyme which synthesizes asparagine from aspartic acid. However, if ASNS is absent or defective in its activity, the deficient cells, tissue, or organ become dependent on dietary asparagine to survive. The proposed research will characterize novel structure- function relationships for mutations in the human ASNS gene which are thought to cause the recently discovered disease Asparagine Synthetase Deficiency (ASNSD), a devastating childhood disease that leads to premature death.
