Recent studies suggest that small soluble aggregates (oligomers) of human islet amyloid polypeptide (hIAPP), that form before amyloid deposits develop in the pancreas, are cytotoxic and may be critical players in the etiology of Type-2 Diabetes. Due to the heterogeneity and transient nature of these hIAPP oligomers, their detailed characterization by traditional techniques has been challenging. In this R21 we will apply single molecule spectroscopy (SMS) approaches to gain deeper insight into these toxic species along the following specific aims:
Aim 1 : We will use SMS to follow the time evolution of hIAPP oligomers in solution and to identify which ones develop into ordered structures and/or insoluble amyloid deposit. We will use SMS of fluorescently labeled IAPP to follow the formation of oligomers as a function of time and to examine the basis for the apparent increased propensity for aggregation of NAPP compared to cat (c) and rat(r) IAPP. The specific hypothesis to be tested is that soluble aggregates form by multiple reaction pathways and that hIAPP more readily forms an initial nucleus (relative to clAPP and rIAPP) thus facilitating peptide aggregation into amyloid.
Aim 2 : To study the mechanism of hIAPP oligomer/protofibril formation on the surface of membrane liposomes and to determine the size of the aggregates leading to pore formation and membrane permeabilization as well as the size and specificity (if any) of the pore. Membrane binding of pre- fibril structures has been suggested as the origin of cytotoxicity of hIAPP. We will initiate SMS experiments to test the hypothesis that membranes facilitate the formation of hIAPP oligomers leading to integration on the membrane surface of well-defined oligomeric structures or protofibrils, and that some of these species lead to membrane permeabilization.
Aim 3. To directly observe and characterize the micellar structures formed by hIAPP, to determine their size distribution and critical micelle concentration (CMC) values and to test whether these structures can explain the observed differences in amyloidogenicity between h-, r-, and clAPP. Our previous work revealed that the micelles serve to buffer the concentration of free monomeric peptide and thereby set the lag time for aggregation. The hypothesis to be tested is that the mutations in c- and rIAPP lead to lower CMC values (hence to correspondingly lower concentrations of monomeric peptide) thus retarding IAPP aggregation. This may explain the lower incidence of diabetes in these animals. ? ? ?
| Brender, Jeffrey R; Lee, Edgar L; Hartman, Kevin et al. (2011) Biphasic effects of insulin on islet amyloid polypeptide membrane disruption. Biophys J 100:685-692 |
| Brender, Jeffrey R; Lee, Edgar L; Cavitt, Marchello A et al. (2008) Amyloid fiber formation and membrane disruption are separate processes localized in two distinct regions of IAPP, the type-2-diabetes-related peptide. J Am Chem Soc 130:6424-9 |
