Leissring, Malcolm A.

Insulin-degrading enzyme (IDE) is a ubiquitous zinc-metalloprotease that hydrolyzes several pathophysiologically relevant peptides, including insulin and the amyloid β-protein (Aβ). IDE is inhibited irreversibly by compounds that covalently modify cysteine residues, a mechanism that could be operative in the etiology of type 2 diabetes mellitus (DM2) or Alzheimer's disease (AD). However, despite prior investigation, the molecular basis underlying the sensitivity of IDE to thiol-alkylating agents has not been elucidated. To address this topic, we conducted a comprehensive mutational analysis of the 13 cysteine residues within IDE. Our analysis implicates C178, C812, and C819 as the principal residues conferring thiol sensitivity. The involvement of C812 and C819, residues quite distant from the catalytic zinc atom, provides functional evidence that the active site of IDE comprises two separate domains that are operational only in close apposition. Structural analysis and other evidence predict that alkylation of C812 and C819 disrupts substrate binding, whereas alkylation of C178 interferes with the apposition of active-site domains and subtly repositions zinc-binding residues. Unexpectedly, alkylation of C590 was found to activate hydrolysis of Aβ significantly, while having no effect on insulin, demonstrating that chemical modulation of IDE can be both bidirectional and highly substrate selective. Our findings resolve a long-standing riddle about the basic enzymology of IDE with important implications for the etiology of DM2 and AD. Moreover, this work uncovers key details about the mechanistic basis of the unusual substrate selectivity of IDE that may aid the development of pharmacological agents or IDE mutants with therapeutic value.

Inclusion body myositis (IBM), the most common age-related muscle disease in the elderly population, is an incurable disorder leading to severe disability. Sporadic IBM has an unknown etiology, although affected muscle fibers are characterized by many of the pathobiochemical alterations traditionally associated with neurodegenerative brain disorders such as Alzheimer's disease. Accumulation of the amyloid-β peptide, which is derived from proteolysis of the larger amyloid-β precursor protein (βAPP), seems to be an early pathological event in Alzheimer's disease and also in IBM, where in the latter, it predominantly occurs intracellularly within affected myofibers. To elucidate the possible role of βAPP mismetabolism in the pathogenesis of IBM, transgenic mice were derived in which we selectively targeted βAPP overexpression to skeletal muscle by using the muscle creatine kinase promoter. Here we report that older (>10 months) transgenic mice exhibit intracellular immunoreactivity to βAPP and its proteolytic derivatives in skeletal muscle. In this transgenic model, selective overexpression of βAPP leads to the development of a subset of other histopathological and clinical features characteristic of IBM, including centric nuclei, inflammation, and deficiencies in motor performance. These results are consistent with a pathogenic role for βAPP mismetabolism in human IBM.

Presenilins mediate an unusual intramembranous proteolytic activity known as γ-secretase, two substrates of which are the Notch receptor (Notch) and the β-amyloid precursor protein (APP). γ-Secretase-mediated cleavage of APP, like that of Notch, yields an intracellular fragment [ A PP i ntra c ellular d omain (AICD)] that forms a transcriptively active complex. We now demonstrate a functional role for AICD in regulating phosphoinositide-mediated calcium signaling. Genetic ablation of the presenilins or pharmacological inhibition of γ-secretase activity (and thereby AICD production) attenuated calcium signaling in a dose-dependent and reversible manner through a mechanism involving the modulation of endoplasmic reticulum calcium stores. Cells lacking APP (and hence AICD) exhibited similar calcium signaling deficits, and—notably—these disturbances could be reversed by transfection with APP constructs containing an intact AICD, but not by constructs lacking this domain. Our findings indicate that the AICD regulates phosphoinositide-mediated calcium signaling through a γ-secretase-dependent signaling pathway, suggesting that the intramembranous proteolysis of APP may play a signaling role analogous to that of Notch.