Investigations of Mammalian Aminopropyltransferases

DESCRIPTION (provided by applicant): Polyamines are ubiquitous cellular components that play many roles in cellular physiology and in normal and neoplastic growth. There is good evidence that the polyamine biosynthetic pathway is a valid target for drug design. The planned experiments focus on studies of the synthesis, function and regulation of spermine levels in mammals. They follow up on several major findings made during the last period of support. These include: determination of the crystal structure of an aminopropyltransferase with an associated multisubstrate analog inhibitor;the finding that a rare X-linked human syndrome (Snyder-Robinson syndrome-SRS) is due to a mutation reducing activity of the spermine synthase gene;our observation that CAG-SpmS transgenic mice that greatly overexpress spermine synthase have only modest alterations in spermine, indicating that compensatory mechanism must prevent its accumulation;the findings that male Gy mice, which have an X-chromosomal deletion in the SpmS gene, have no spermine despite the presence of spermine in their diets;and preliminary results indicating that restoring a source of spermine by breeding female Gy carrier mice with CAG-SpmS mice restores normal growth and fertility to these mice despite the unregulated nature of spermine synthase expression. There are four interrelated specific aims: (1) To understand the mechanism of action and substrate specificity of the aminopropyltransferase enzymes. These studies will use structural data, which allows predictions to be made concerning the aminopropyltransferase reaction and tested by site-directed mutagenesis. They will determine the mechanism of aminopropyl transfer from decarboxylated S-adenosyl-methionine, compare this process to methyl transfer from S-adenosylmethionine and determine the molecular basis of the strikingly exclusive substrate specificities of spermidine and spermine synthases. (2) To understand the regulatory mechanisms in vivo that control spermine levels and the consequences of overproduction or underproduction of spermine. These studies will use CAG-SpmS mice and crosses of them with mice that overexpress other enzymes of polyamine metabolism to evaluate the regulatory factors the control spermine levels and the effects of overriding these factors. (3) To examine the uptake of spermine from exogenous sources and devise procedures to facilitate such uptake and thus restore normal polyamine levels and function in Gy mice and SRS cells. (4) To investigate the regulation of spermine content and the unique function(s) of spermine in cultured cells using cells derived from Gy mice, and SRS cells that have a greatly reduced spermine synthase level due to a mutation in a splice site.