RESEARCH in the CREAMER LAB
The primary focus of the Creamer laboratory is on the function and regulation of the serine/threonine phosphatase calcineurin (CaN). CaN is ubiquitously expressed and highly conserved in all eukaryotes. As an integral component of several signaling pathways, this enzyme dephosphorylates, and thereby regulates, a number of important proteins such as the NFAT family of transcription activators, the microtubule-associated tau, and nitric oxide synthase. CaN plays central roles in neuronal signaling, cardiac growth and immune system activation. Dysregulation of CaN contributes to a number of disorders including Alzheimer’s disease, Down syndrome, mental retardation, cardiac hypertrophy, and autoimmune diseases.
CaN activity is tightly regulated by a number of other proteins. Calmodulin (CaM) binds to and activates CaN. Other proteins, including Rcan1, CHP1 and cabin/cain, are endogenous inhibitors of CaN. Yet others, such as AKAP79, serve to localize CaN to specific regions within a cell. Breakdowns in any of these regulatory systems can lead to pathologies associated with the diseases listed above. For example, Rcan1 is overexpressed as a consequence of the trisomy underlying Down syndrome. This in turn leads to inappropriate inhibition of CaN. Notably, the CaN regulatory mechanisms are poorly understood at the molecular/structural level.
Ongoing CaN projects
1) Upon an increase in cellular calcium levels, CaM binds to and activates CaN. CaM binds to a regulatory domain within CaN which leads to a conformational change that in turn ejects an autoinhibitory domain from CaN’s active site. The regulatory domain of CaN appears to be devoid of structure prior to CaM binding. In other words this domain is intrinsically disordered. Upon CaM binding the CaN regulatory domain gains structure. We are currently studying this disorder-to-order transition and how it leads to CaN activity.
2) Rcan1 (regulator of calcineurin 1) is an endogenous inhibitor of CaN that has been associated with Down syndrome and Alzheimer’s disease. This protein is known to bind to the catalytic domain of CaN, blocking substrate binding. Rcan1 possesses two domains: a domain of unknown function that is thought to be structured, and the CaN binding domain which appears to be completely unstructured. We therefore have an enzyme with an intrinsically disordered regulatory domain being inhibited by a protein with an intrinsically disordered inhibitory domain. Rcan1 binding to CaN is a potential drug target in Alzheimer’s and Down syndrome, but little is known regarding the details of the interactions between these proteins. We are studying this system in order to gain an understanding of the structural details of these interactions.
3) The microtubule-binding and stabilizing protein tau is a known substrate of CaN. CaN dephosphorylates residues on tau that are believed to regulate binding to microtubules. This process goes awry in Alzheimer's disease, potentially a result of the over-expression of Rcan1. We are studying the interactions between tau and CaN, and the effects upon these of Rcan1, in relation to Alzheimer's.
The Creamer lab employes a variety of biophysical approaches to tease apart interactions between proteins. Our primary approaches involvesteady-state and time-resolved fluorescence techniques (FRET, anisotropy, etc.). These are mostly carried out using an ISS K2 multifrequency cross-correlation phase and modulation fluorometer. We also employ biolayer interferometry and circular dichroism (CD) spectroscopy, as well as bright-field and fluorescence microscopy. The biochemical approaches used include enzyme activity assays. We work with collaborators in order to utilize other approaches such as NMR spectrometry, X-ray crystallography, and a variety of computational methods.