Approaches to medicine are rapidly changing as we begin to comprehend human disease at the most fundamental molecular level. Much of this change is heralded by a more quantitative and mechanistic understanding of the alterations of molecular structure and dynamics that produce disease. Recent years have brought a dramatic increase in the number of known associations between human disease and abnormalities in protein dynamics and structure. In particular, the number of diseases known to be associated with protein aggregation has increased several-fold. Since protein structure and dynamics are intimately related to protein cellular function, abnormalities in protein folding dynamics and structural stability often adversely affect cell life. Understanding protein folding, misfolding and aggregation will be vital to understanding human diseases, ranging from various forms of cancer to neurodegenerative diseases, and will facilitate the development of therapeutic strategies to combat these diseases.
We are holding an InterAction Meeting session at Bryn Mawr College, Philadelphia on October 12th on "Protein Folding, Misfolding & Aggregation: Applications to Disease". The purpose of the meeting is to bring together world-class researchers working on both theoretical and experimental fronts of the protein folding field, and have them present and discuss recent and new results in cutting-edge research areas relevant to Alzheimer’s, Huntington’s Disease, other neurodegenerative diseases and Renal Failure. The emphasis of the meeting activity is on communication and open discussion, which we hope will lead to new ideas and collaborations towards progress and innovation in the disease areas discussed. A significant amount of time at the meeting will be devoted to question time and panel and group discussions towards this goal.
A description of the session and invited speaker presentations follows:
eCheminfo InterAction Meeting Session, Philadelphia, 12 October 2005
Protein Folding, Misfolding & Aggregation: Applications to Disease
chaired by Nikolay V. Dokholyan (University of North Carolina at Chapel Hill),
eCheminfo and InnovationWell Autumn 2005 InterAction Meetings, 11-12 October 2005, Philadelphia, USA
Program Updates loaded in the Program Areas on http://echeminfo.com/ and http://innovationwell.net/
Invited Speakers and Discussion Leaders:
David Teplow (David Geffen School of Medicine at UCLA), Ron Wetzel (University of Tenessee Medical Center), Michael Thorpe (Arizona State University), Feng Ding (University of North Carolina), Andrew Miranker (Yale University)
Presentation Abstracts:
Amyloid beta-protein oligomerization and Alzheimer’s disease
David B. Teplow, Department of Neurology, David Geffen School of Medicine at UCLA, 635
Charles E. Young Drive South (Room 445), Los Angeles, CA 90095-7334, USA
A seminal etiologic component of many neurodegenerative diseases is the abnormal folding and assembly of proteins. This process produces a variety of monomeric, oligomeric, and polymeric structures. In Alzheimer’s disease (AD), work by anatomists in the 19th century first implicated amyloid fibrils in disease causation. Amyloid fibrils are protein homopolymers with large aspect ratios, diameters of ca.10 nm, and common core structural organization. Amyloid fibrils also form in other neurodegenerative diseases, including the transmissible spongiform encephalopathies, Parkinson’s disease, amyotrophic lateral sclerosis, and familial amyloid polyneuropathy. The wide occurrence and obvious clinical linkage of amyloid formation to neurodegeneration has stimulated intense study of amyloidogenesis. The amyloid hypothesis, which argues the primacy of amyloid fibrils in the neuropathogenesis of AD, was one of the first results of these investigations. However, a broad and increasingly compelling body of recent work now supports a revision of the hypothesis. Specifically, the primacy of fibrils in AD pathogenesis has been supplanted by the primacy of low-order oligomers.
In AD, fibrils are formed by the amyloid beta-protein (Abeta). Understanding, in molecular detail, the folding and oligomerization of the Abeta monomer has been complicated by the facts that the peptide has no stable native fold, displays a complex folding topography, and populates biologically-relevant conformational states transitorily. Nevertheless, new experimental and computational approaches have provided the means to identify and characterize novel assembly intermediates, including short, flexible, fibril-like polymers termed “protofibrils” and small pentamer/hexamer units termed “paranuclei.”
Protofibrils are neurotoxic and their formation appears to be a general feature of the assembly of many different amyloidogenic proteins. Paranuclei-like assemblies have been found in AD patients and may be the proximate neurotoxins in AD. I will discuss recent results of studies of Abeta folding and assembly and the implications for understanding and treating AD.
The assembly and structure of amyloid-like polyglutamine aggregates associated with Huntington’s Disease
Ronald Wetzel, University of Tenessee Medical Center
In Huntington’s disease (HD) and seven other expanded CAG repeat diseases, the mutational increase in length of a polyglutamine (polyGln) segment of various disease-specific proteins above a pathological repeat length of about 35 residues is associated with a sharp increase in disease risk. Additional lengthening of the polyglutamine leads to earlier disease onset. Since these polyglutamine disease proteins vary in size, structure, sub-cellular localization and function, the expanded polyglutamine sequence itself must be the driving force of disease. To arrive at the basis for polyglutamine pathogenesis, we are studying how repeat length influences the physical properties of polyglutamine sequences, and in particular their abilities to form amyloid-like aggregates similar to those found in HD neurons.
Cells treated with nuclear-targeted aggregates of polyglutamine peptides are very effectively killed in an apoptosis-related mechanism. Cells pretreated with a peptide-based elongation inhibitor are protected. Small aggregation foci can be identified in 10-20 year old archival HD brain tissue using a staining procedure based on aggregate elongation. All this is consistent with toxicity mechanisms depending on the ability of polyglutamine sequences to aggregate. Learning more about the details of polyglutamine folding and aggregation is therefore critically important.
The solution conformation of monomeric polyglutamine appears to depend little on repeat length, suggesting that a repeat length dependent conformational change is not responsible for toxicity. In contrast, aggregation kinetics are sharply dependent on repeat length. Polyglutamine makes amyloid-like aggregates by a classical nucleation-dependent polymerization mechanism. Surprisingly, we found the critical nucleus to be one, suggesting that the energetically unfavorable nucleation event in this system is not oligomerization, but rather folding of the monomer into an aggregation-competent structure. Longer polyGln sequences aggregate more efficiently because their nucleation equilibrium constant is more favorable. The nucleation equilibrium constant for a Q47 repeat sequence is in the range of 10-9, illustrating the unfavorable nature of nucleus formation. The interplay between nucleus formation and subsequent elongation events is illustrated by the ability of short polyGln peptides to enhance the overall nucleation and aggregation of a long polyGln sequence. This underscores the potential importance of the entire polyglutamine protein population in controlling the cell’s susceptibility to an expanded polyGln protein.
It should now be feasible to begin to probe how additional features of the various disease proteins and their environments produce disease-specific effects. Other examples of important variables now being studied include the polyGln flanking sequences of huntingtin, and the presence of certain molecular chaperones in the cellular environment. It should also be possible to develop small molecule inhibitors of polyglutamine aggregation as potential therapeutics.
Catalytic origins of protein misfolding in end-stage renal failure
Andrew Miranker, Dept. of Molecular Biophysics and Biochemistry Office, Yale University Lab, 266 Whitney Avenue / Bass 318 PO Box 208114, New Haven, CT 06520-8114, USA
Amyloid fibers are long, unbranched and insoluble homo-assemblies of proteins. The self-assembly of such structures occurs in a number of human diseases such as Alzheimer's, as part of biological function such as in melanin deposition, and as a scaffold for design as in the formation of conductive wires. The process of fiber formation is complex with nucleation dependent kinetics giving rise to cytotoxic intermediates resulting in a product which is macroscopic. These properties are the result, in part, of a transient and heterogeneous assembly mechanism making structural insight particularly challenging. b-2-microglobulin (b2m) is the conserved, 99 amino acid globular protein required for the correct folding and cell surface expression of class I major histocompatability complex. In patients with end-stage renal disease treated by hemodialysis, b2m undergoes transitions resulting in its deposition as amyloid principally in the liver and joint spaces. Our recent analyses of b2m amyloid formation have enabled us to identify the existence of a monomeric and native-like intermediate on the pathway of fiber formation. This state, is catalytically accessed by the presence of transition metal cation. This intermediate rapidly assembles into discrete oligomeric states which display little additional oligomerization on the timescale of their own formation (<1hr). Amyloid fiber formation progresses from these intermediate states but on much longer timescales. The native-like structure and discrete oligomeric size of these amyloid intermediates suggests that this protein forms fibers by structural domain swapping. As transition metal cation effects are reported in many other amyloidoses, e.g. prion, Parkinson's, and Alzheimer's, elucidation of the mechanism of b2m amyloid formation enables us to define general mechanisms for divalent ion associated amyloidosis.
Direct observation of protein folding, misfolding and prion-like conformational infectivity
Feng Ding, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, 303 Mary Ellen Jones, CB#7260, Chapel Hill, NC 27599, USA
Protein conformational transition from alpha-helices to beta-sheets precedes aggregation of proteins implicated in many diseases, including Alzheimer’s and prion diseases. Direct characterization of such transition is often hindered by the complicated nature of the interaction network among amino acids. A recently engineered small protein-like peptide with a simple amino acid composition features a temperature-driven alpha-helix to beta-sheets conformational change. Here, we study the conformational transition of this peptide by molecular dynamics simulations. We find a critical temperature, below which the peptide folds into an alpha-helical coiled-coil state, and above which the peptide misfolds into beta-rich structures with a high propensity to aggregate. The structures adopted by this peptide during low temperature simulations have a backbone root mean square deviation less than 2 Å from the crystal structure. At high temperatures, this peptide adopts an amyloid-like structure, which is mainly comprised of coiled anti-parallel beta-sheets with the cross-beta signature of amyloid fibrils. Strikingly, we directly observe “infective” conformational conversions, where an alpha-helix is converted into a beta-strand by proximate stable beta-sheets with exposed hydrophobic surfaces and unsaturated hydrogen bonds. Our study suggests a possible molecular mechanism of the seeded aggregation process as proposed by Prusiner for the infectivity of prions.
Flexibility and Mobility in Biomolecules
M.F. Thorpe, B.M. Hespenheide, S. Menor and S. Wells, Bateman Physical Sciences PSF 359
Arizona State University, Tempe, AZ 85287-1504, USA
We present a novel approach to the calculation of flexibility and mobility in proteins, protein complexes and other large macromolecular complexes like virus capsids. Rather than using conventional molecular dynamics, we use the constraint approach of Lagrange, incorporating covalent bonds, hydrogen bonds, and tethers for hydrophobic interactions. The rigid clusters, including the core, are identified as well as the flexible joints between them. This is used as the basis for dynamics, using Monte Carlo approaches that maintain all the original constraints, as well as van der Waals excluded volumes.
In our original work [1, 2], we focused on ring closure and added the side groups later. This was successful in exploring the available conformational space and also in exploring directed trajectories between known distinct protein structures.
In more recent work, we have used ghost templates attached to the rigid regions to guide a protein through the allowed conformational space. The generation of a new protein conformation requires about 100 millsecs CPU time on a single processor [3].
We show that such techniques can be used on a single X-ray crystallographic structure to generate an ensemble of structures remarkably similar to those observed in NMR. We also show how this approach can be used to generate multiple protein complexes for use in ligand docking studies, as well as exploring the allowed conformations of protein-ligand complexes. We discuss applications to virtual screening and the drug discovery process.
Acknowledgement: Work supported by NSF and NIH.
References:
[1] Ming Lei, Leslie A. Kuhn, Maria I. Zavodszky and M.F. Thorpe Sampling Protein Conformations and Pathways, Journal of Computational Chemistry 25, 1133–1148 (2004).
[2] Maria I. Zavodszky, Ming Lei, M.F. Thorpe, Anthony R. Day and Leslie A. Kuhn Modeling Correlated Main-Chain Motions in Proteins for Flexible Molecular Recognition, Proteins: Structure, Function and Bioinformatics 57, 243–261 (2004).
[3] Stephen Wells, Brandon Hespenheide, Scott Menor and M.F. Thorpe Constrained Geometrical Simulation of Diffusive Motions in Proteins (to be published in Physical Biology).
Barry Hardy
eCheminfo Community of Practice Manager
Douglas Connect
Comments