Regulated Intramembrane Proteolysis and Signal Transduction

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Gamma-secretase

Gamma-secretase was originally defined as a membrane-bound protease that cleaves the transmembrane region of APP to generate the C-termini of Aâ peptides, which are believed to play a central role in neuropathogenesis of Alzheimer's Disease (AD). Recently, Notch has also been identified a substrate of g-secretase. After ligands of the Delta/Serrate/Jagged family bind to the Notch receptor, 2 proteolytic events are initiated for signal transduction of Notch: cleavages by TACE in the extracellular domain then by g-secretase cleavage within the transmembrane domain. Following cleavage by gamma-secretase, the Notch intracellular domain (NICD) that contains the transcriptional activator motif translocates to the nucleus to activate transcription of target genes.

The Notch signaling pathway plays an essential role in cell fate decisions during embryogenesis, hematopoiesis, and neuronal stem cell differentiation. Abnormal Notch signaling contributes to cancer development. Human Notch1 was originally identified through the analysis of a subset of T cell acute lymphoblastic leukemias (T-ALL). Constitutively active forms of Notch have been shown to contribute to oncogenesis in both animal and in vitro model systems. Furthermore, the activation of Notch signaling has been linked with B-cell chronic lymphocytic leukemia, Hodgkin's Disease and anaplastic large cell lymphoma, prostate cancers and Ras-associated malignancies.

In addition, gamma-secretase activity has also been associated with regulated intramembrane proteolysis of ErbB-4, CD44, the low-density lipoprotein-receptor-related protein (LRP), E-cadherin, and nectin1a. ErbB-4 is a member of the epidermal growth factor receptor family that regulates cell proliferation and differentiation. ErbB-4 has also been implicated in malignancies. CD44 is a widely distributed cell surface adhesion molecule and is implicated in tumor cell growth and metastasis. Moreover, the gamma-secretase/Notch signal pathway is a model system to study RIP-mediated signal transduction; and gamma-secretase represents an extraordinary class of enzymes that catalyze substrate hydrolysis within transmembrane domains.

Recognition of gamma-secretase as an important therapeutic target and as an unusual catalyst for signal transduction has driven gamma-secretase to the forefront in both basic and translational research. Development of an appropriate assay in which a substrate with a transmembrane domain is cleaved by gamma-secretase was a key step in decoding the molecular mechanism of gamma-secretase. We invented the first cell-free gamma-secretase assay. This novel assay, which uses a recombinant protein substrate, allowed characterization of biochemical properties of gamma-secretase that could not be determined using cell-based assays. The cell-free gamma-secretase assay was exploited to generate direct biochemical evidence showing that gamma-secretase activity arises from an aspartyl protease within a presenilin-containing macromolecular complex.

Moreover, we used transition-state analogs to develop active-sited directed photoaffinity inhibitors of gamma-secretase and found that these photoreactive inhibitors specifically label presenilins. These biochemical studies provided compelling evidence that presenilins contain the active site of gamma-secretase (See Figure).

Rhomboid -- Putative Serine Protease

Rhomboid (Rho) is a multiple-transmembrane domain protein and a key trigger of epidermal growth factor (EGF) receptor activation in Drosophila, and as such, controls cell differentiation, proliferation, and survival. A family of Rho-like proteins is conserved from bacteria to human. Interestingly, EGF hyperactivity is strongly implicated in human cancer. The principal activating ligand of the fly EGF receptor is Spitz -- a protein that is synthesized with a single transmembrane domain and one extracellular EGF domain. It is proposed that biological activity of Spitz is linked to proteolytic release of its EGF domain. Two molecules, Rho and Star, have been implicated in the production of active ligand. Recent studies indicated that Rho is a novel intramembrane serine protease that directly cleaves Spitz.

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An overview of these projects and possible links to other signal pathways is portrayed in the figure to the immediate right. Our areas of interest include:

• isolating and identifying the gamma-secretase complex and a putative Rho serine protease;
• developing mechanism-based inhibitors to probe the active site of these proteases;
• exploring the structure basis of the extraordinary catalysis and substrate specificity;
• delineating these intramembrane protease-mediated signal transduction pathways using chemical and genetic approaches;
• elucidating the molecular mechanism of pathogenesis of human diseases associated with these pathways and developing effective therapies.

Unraveling the intricacies of these enigmatic intramembrane proteases is a formidable challenge. However, decoding these extraordinary reactions will provide novel insights into the molecular basis of signaling and thereby facilitate the discovery of novel therapeutic agents.