P2-030: Elucidating function and proteolytic properties of families of aspartic intramembrane proteases

Abstract

Regulated intramembrane proteolysis (RIP) is a key mechanism in the pathogenesis of Alzheimer's Disease (AD). Presenilins are putative aspartic proteases required for the RIP cleavage of Amyloid Precursor Protein and other type I proteins including Notch receptors that are critical in early development. We and others previously found a novel family of genes related to presenilins and termed IMPAS (or IMP/SPP/SPPL/PSH). The aim of this study was to elucidate functional properties of IMPAS proteins. We applied mammalian cell culture proteolytic assays and soil nematode C.elegans model. Three IMPAS genes in C.elegans (Ce–imp–1, Ce–imp–2, Ce–imp–3) and five paralogous human genes genes (IMP1–IMP5) were cloned and studied. Based on original alignment comparisons we predicted that human hIMP1/SPP and Ce–imp–2 are structural and functional orthologs. Inhibition of Ce–imp–2 gene by dsRNAi or in knock–out mutant strains resulted in slow growth, uncoordinated movement, reduced brood size and larval death due to the incomplete shedding of cuticle (ICS) in the animals. The progeny of heterozygous deletion mutant Ce–imp–2 was analyzed by single worm PCR. The data demonstrated that homozygous mutants showed development pathology of late embryo death and ISC defects that is unrelated to Notch defects described for presenilin (sel–12, hop–1) mutants. This specific phenotype mimics features observed in worms with low function of megalin gene or on cholesterol diet. Inhibition of other IMPAS homologues in C.elegans (Ce–imp–1, Ce–imp–3) did not result in any obvious phenotype. We revealed that in co–expressing experiments with PS1 both human IMP1 and Ce–imp–2 were capable to cleave C–terminal part of PS1 in transmembrane domain. The data indicate that Ce–imp–2 is likely orthologous gene to hIMP1/SPP with similar proteolytic functions. The IMP1/Ce–imp–2–induced and PS1–induced proteolytic cleavage assays were used to delineate essential domains and amino–acid signatures important for enzymatic or conformation properties critical for proteolytic functions of two types of intramembrane proteases (IMPAS and presenilin families).