Amyloid structure & assembly


 

Aggregate structure. The protein aggregates associated with Alzheimer's Disease, Huntington's Disease, and other diseases tend to be highly ordered structures possessing, so far as we can establish, the same degree of packing specificity normally found in globular proteins. The structure of the amyloid fibril is both intriguing and mysterious. These assemblies can form from a number of different proteins which differ widely in amino acid sequence, size, and secondary structural features of the native state. The structure of amyloid is obscure because its polydispersity, size, insolubility, and inability to form diffraction quality crystals renders ineffective the normal techniques for solving protein structure. We use standard techniques like CD, FTIR, and EM (in the lab of John Dunlap at UT); to study the gross morphology of these aggregates. We attempt to get at finer structural points by adapting other methods previously applied to globular proteins. For example, we are mapping the secondary structural features of Ab amyloid fibrils using hydrogen-deuterium exchange as assessed by mass spectrometry. We have also used limited proteolysis of fibrils to determine which parts of the Ab peptide are intimately involved in the b-sheet network of the fibril. We are also characterizing a unique set of monoclonal antibodies that have the ability to bind to a conformational epitope presesnt on amyloid fibrils. On-going projects include the use of scanning mutagenesis techniques coupled with fibril formation assays to map the tolerances of each residue position of the peptide for various structural changes. In collaboration with Dr. Ying Xu at Oak Ridge National Labs, we are building and testing models of fibril structure based on this and other experimental data.

Fibril assembly and inhibition. We are interested in fibril assembly mechanism, kinetics and thermodynamics. From a basic, theoretical point of view, very little is known about the basis of fibril stability. The observation that many proteins that normally fold into stable, monomeric structures can nonetheless form amyloid fibrils suggests that the rules governing fibril packing and stability may be somewhat relaxed compared to the rules for globular proteins. On the other hand, amino acid replacements in amyloidogenic peptides can significantly alter fibril formation. We want to understand these constraints as another way to study fibril structure. From a practical point of view, learning about the "hot spots" for fibril stabilization should tell us what places to target in inhibitor design. We have developed and optimized a number of assays for following fibril formation that we routinely use to assess the kinetics and thermodynamics of fibril formation and to screen for inhibitors, both in the Alzheimer's Ab system and in the Huntington's Disease polyglutamine system.

The basis of aggregate cytotoxicity. When bacteria produce inclusion bodies filled with densely packed, misfolded recombinant proteins, they often suffer very little from the presence of these abnormal structures outside of perhaps a slight diminution in growth rate. Particular protein aggregates can have devastating consequences, however, to human biology. We are interested in the mechanisms by which cells die or become dystrophic in response to aggregates. The mechanism we are particularly interested in has been called the "recruitment" or "sequestration" mechanism. The idea is that the loss of the major amyloid component (Ab, for example); is not the problem, but rather it is the loss to the local environment of other proteins when these molecules become entrapped in the growing amyloid deposit. Particularly good evidence for this mechanism exists in the polyglutamine system, where cellular proteins containing short polyglutamine sequences can be observed to co-aggregate in the cell with the expanded polyglutamine-containing protein forms inclusions. We are studying polyglutamine aggregation in vitro to develop the biophysical underpinnings of this mechanism, and are also studying what happens when polyglutamine aggregates made in vitro are introduced into cells.


 
Education

PhD 1973, University of California, Berkeley 

 

Postdoctoral Training


Max Planck Institute for Experimental Medicine in Goettingen, Germany
Yale University


Department of Structural Biology
University of Pittsburgh
Biomedical Science Tower 3
3501 5th Avenue
Pittsburgh, PA 15260

Phone: (412) 383-5271
Fax: (412) 648-9008

E-mail: rwetzel@pitt.edu 

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