The research focus of the laboratory is geared toward studying the mechanisms and treatments of Alzheimer's disease (AD) and related disorders. Our studies are directed toward two general areas: the role of presenilins (PS) and associated proteins in AD, and the role of protein misfolding and the ubiquitin-proteasome system in neurodegenerative disorders.

Role of Presenilins in Alzheimer's disease
The majority of familial cases of AD (FAD) are due to inheritance of mutations in the genes encoding the two highly homologous proteins, PS1 and PS2. Our studies focus on the function of PS proteins and on how AD-associated mutations in these proteins cause disease. A primary focus of these studies is directed toward two PS-associated proteins, ubiquilin and calmyrin, which we have found modulate PS functions. In earlier studies we showed that overexpression of wild type (wt) PS2 induces apoptosis and that the FAD-associated PS2(N141I) mutation increases apoptosis. We also showed that overexpression of presenilins in dividing cells causes cell cycle arrest, and that the arrest is potentiated by the FAD PS1 and PS2 mutations. These studies suggest possible mechanisms involved in AD and potential strategies to halt or prevent disease.

Functional Studies of Ubiquilin
We first identified human ubiquilin-1 as a PS-interacting protein. We found it was involved in regulating PS protein levels. Database searches indicate that ubiquilin is a highly conserved protein of about 66 kDa in size, and is found in all eukaryotes examined. There are three human ubiquilin genes, which are differentially expressed. Ubiquilin proteins (also called PLIC) contain a ubiquitin-like domain (UBL) at their N-termini, a central more variable domain containing chaperonin-binding motifs, with a ubiquitin-associated domain (UBA) at their C-termini. Several observations, both from our laboratory as well as others, suggest that ubiquilin is involved in regulating protein stability, either directly or indirectly, by functioning as a molecular chaperone (i.e., ubiquilin binds several molecular chaperones including HSP70-like proteins and high levels of ubiquilin protect cells against certain types of stress). Our studies are directed toward a detailed understanding of the function(s) of ubiquilin in cells and organisms. We are using molecular and cell biologic approaches to dissect its function, including expression of wt and mutant ubiquilin proteins, RNA interference, and in vitro and biophysical approaches. In addition we are building a global picture of the network of ubiquilin interactors in cells. Toward this goal, we are characterizing the properties of novel ubiquilin interactors that we recently identified. Ubiquilin has been shown to associate with protein aggregates in several neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease and Huntington's disease. Studies are underway to understand the involvement of ubiquilin in protein aggregation and toxicity in these disorders. Finally, we have also initiated studies to examine the effects of loss of function of ubiquilin in mouse and C. elegans.

Functional Studies of Calmyrin
The second major focus of the laboratory is directed toward studies of the second PS interactor we identified, which we named calmyrin (also called CIB1 and KIP). Calmyrin is a small 191-amino acid protein that shares highest homology with the regulatory subunit of protein phosphatase 2B (calcineurin). However, the function of calmyrin is not known, although some reports suggest that it may regulate activities of some of the proteins it binds, but exactly how is not known. Although calmyrin has four putative EF-hands, we found that only EF-hands 3 and 4, located in the C-terminal half of the molecule, bind calcium. Using circular dichroism, UV fluorescence spectroscopy, and NMR spectroscopy, we found that calcium binding triggers a large conformational change in calmyrin, suggesting that calmyrin functions as a calcium sensor. We further found that EF-hands 3 and 4 have to be intact for calmyrin interaction with PS2 loop sequences, suggesting that calcium binding is important for the interaction. We also demonstrated that calmyrin is myristoylated at its N-terminus, and that this fatty acid modification is important for the stability and the dynamic targeting of the protein within cells. Our studies have also indicated that coexpression of calmyrin and PS2 induces additive apoptosis compared to cell death induced by overexpression of each protein individually. We believe that calmyrin interaction with PS2 is important because it links PS to calcium signaling pathways. Studies are underway to determine the function of calmyrin and its role in cell signaling using approaches similar to those described for ubiquilin.