Graduate Seminar: Georges Belfort

Institute Professor, Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute

Insight into the Kinetics of Oligomer Formation During Amyloidosis

Abstract

 

 

Although the critical causative proteins for more than 20 amyloid diseases have been identified, the process and mechanism by which these proteins induce disease are unknown.  What is known is that these proteins are converted to cross-b-sheet rich fibril structures called amyloid fibrils.  For over 100 years, the presence of these fibrils in brain tissue has been associated with disease (i.e. Alzheimer’s).  Recently, however, new evidence has implicated dissolved oligomers or precursors to fibrils in the form of circular annular structures as the possible toxic agents.  As a result, there is a great need to study the thermodynamic, kinetic and toxic properties of the oligomer-fiber transition.  We chose human insulin as a model amyloid protein for this in vitro study because it is an amyloid protein, it forms fibrils in ~3 h at pH 1.6 and 65 C, exibits the usual sigmoidal fibril growth curve, and has been widely used by others.

We are interested mainly in both the kinetics and the structural conversion process of native insulin to fibrils and the dissolution of preformed fibrils in order to isolate, purify and identify the toxic species.  Hence, we probe the so-called nucleation process for converting a native folded protein to a b-sheet rich amyloid fibril and the reverse process.  Using a rigorous mechanistic reaction model that incorporates the physical chemistry of nucleation and fibril growth dynamics, we show that the rate constants for nucleation were ~10 million times smaller than those for fibril growth (Lee et al. 2007).  Seeding with different fibril lengths, L, provides a uniform insulin fibril growth rate, k_app = 0.86 min^-1, independent of L, with a lag time to initiate fibril growth proportional to L^-3/2.  Tagging washed insulin fibrils and monomers with different fluorescent dyes, and using an inverted fluorescent microscope to image the dyes, we show for the first time that a majority of the elongated fibrils propagated along only one end of the seed, with the remaining fibrils having bidirectional elongation or no elongation (Heldt et al. 2010).  During lag phase and prior to the formation of fibrils, we provide consistent evidence of the composition of the insulin nucleus that comprised three dimers or six monomers (Nayak et al. 2009) and it was likely asymmetric polytetrahedron rather than symmetric octahedron (Meng et al. 2010).  Cooling during the lag phase prior to the onset of fibril formation indicated that the oligomers changed linearly with time and that fibril growth was slowed at the expense of producing more nuclei (Sorci et al. 2009).  Recently, we have isolated toxic and non-toxic oligomers from fibril dissolution experiments (Heldt et al. 2011).  Also, using the final fibril length distribution, we back-calculate for the first time the initial nuclei concentration to be in the range of 20-200 pM (Sorci et al. 2010).  This very low concentration of monomers, dimers and trimers could explain the difficulty in isolating and blocking oligomers or nuclei toxicity and the long onset time for amyloid diseases (Pease et al. 2010).

References

Heldt, C.L., Kurouski,D., Sorci, M., Grafeld, E., Lednev, I.K. and Belfort, G (2011) Isolating toxic insulin amyloid oligomers that lack b-sheets and have wide pH stability, Biophysical J. 100 (11):2792-800.

Heldt, C. L., S. Zhang and G. Belfort (2010). "Asymmetric Amyloid Fibril Elongation: A New Perspective on a Symmetric World." Proteins: Structure, Function and Bioinformatics 79, 92-98.

Lee, C. C., A. Nayak, A. Sethuraman, G. Belfort and G. J. McRae (2007). "A three-stage kinetic model of amyloid fibrillation." Biophysical Journal 92(10): 3448-3458.

Meng, G., N. Arkus, M. P. Brenner and V. N. Manoharan (2010). "The free-energy landscape of clusters of attractive hard spheres." Science 327(5965): 560-3.

Nayak, A., M. Sorci, S. Krueger and G. Belfort (2009). "A universal pathway for amyloid nucleus and precursor formation for insulin." Proteins-Structure Function and Bioinformatics 74(3): 556-565. 

Pease L, F., Sorci, M., Guha, S., Tsai, D. H., Zachariah, M. R., Tarlov, M. J., Belfort, G. (2010) Biophysical J. 99 (12) 3979-85. 

Sorci, M., R. A. Grassucci, I. Hahn, J. Frank and G. Belfort (2009). "Time-dependent insulin oligomer reaction pathway prior to fibril formation: Cooling and seeding." Proteins-Structure Function and Bioinformatics 77(1): 62-73.

Sorci, M., W. Silkworth, T. Gehan and G. Belfort (2011). "Estimating insulin nuclei concentration from amyloid fibrils: A detection challenge." PLoS ONE, 6 (5) e20072.

Bio

Dr. Georges Belfort: Endowed chair Russell Sage Professor of Chemical and Biological Engineering at RPI. He received his BS degree in CHME at the University of Cape Town and PhD in Engineering from UC Irvine. He has broad research interests include mass transfer and membrane filtration, protein misfolding and kinetics, single molecule force spectroscopy, and bioseparations. He has received the two major awards in the US on Separations (ACS (1995) and AIChE (2000)), the ACS Murphree Award in Industrial and Engineering Chemistry (2008), and is one of the "100 Chemical Engineers of the Modern Era" as part of the AIChE Centennial Celebration in 2008. He was elected a member of the US National Academy of Engineering, February 2003.