Ronald B. Wetzel - US grants

DESCRIPTION (From the Applicant's Abstract): Amyloid fibrils are found associated with a growing number of human diseases, including several neurodegenerative diseases, the most prevalent of which is Alzheimer's Disease (AD). In each of these diseases, the protein that is the main component of the amyloid fibril is different. Thus, amyloid is a characteristic type of aggregate structure, rather than a particular protein molecule. Indeed, there is some speculation that this amyloid type of aggregate structure may be a protein folding motif that can be accessed by many protein sequences under the right circumstances. Because of its relevance to both human disease and fundamental understanding of protein folding, it is particularly important to know in more detail the folded structure of the amyloid fibril. Amyloid fibrils do not lend themselves to standard techniques for determining protein structure, with the result that we know very little about this structure at the molecular level beyond the fact that these aggregates are rich in beta sheet secondary structure. We have chosen to work on the amyloid associated with AD, composed of the peptide A-beta. In the research proposed here we will use the technique of hydrogen-deuterium exchange (HX) to map the secondary structure of A-beta in the fibril. Hydrogen bonds, such as are found between polypeptide backbone amide hydrogens in beta sheet, protect amide hydrogens from exchange while most other amide hydrogens freely exchange. The specific aims of this application are to (1) Develop methods for collecting HX data on A-beta incorporated into amyloid fibrils, using both mass spectrometry (MS) and nuclear magnetic resonance (NMR) to quantify levels of exchange, and use these methods to map the protected and exposed amide hydrogens of A-beta in the fibril; (2) carry out similar exchange studies on other A-beta aggregates, like protofibrils, that are implicated in both amyloid assembly and in Abeta aggregate toxicity; (3) use computational methods in concert with the HX data to build, test, and refine models of protofilament and fibril structure; and (4) conduct exchange experiments on other aggregates of A-beta, including fibrils made in vitro from A-beta fragments as well as neuritic plaque cores isolated from human AD brain material. These studies will give us a closer look at the structure of the amyloid fibril, which will allow us to better understand how fibrils grow and how they disrupt human cells and tissue. An improved knowledge of structure will also improved our ability to identify and design therapeutic molecules for inhibiting the growth and toxicity of A-beta amyloid and other aggregates.

DESCRIPTION (Adapted from applicant's abstract): In the expanded CAG repeat diseases, such as Huntington's Disease, abnormally long polyglutamine sequences have been implicated as the principle causative factor of the disease. The mechanism by which these molecules promote neurodegeneration is not clear, but a large body of evidence suggests that the mechanism has something to do with the enhanced ability of lengthened polyglutamine sequences to self-associate into oligomers or aggregates. While there is an imperfect correlation of disease physiology with the appearance of large aggregates in cellular nuclei, there is also a lot of indirect evidence, such as the widely reported involvement of cellular chaperones, that a misfolding / aggregation process is involved in these diseases. One limitation in our ability to progress more deeply into the disease mechanism is our ignorance of the details of the process and products of polyglutamine aggregation. For example, improved correlation of disease development with polyglutamine aggregation might be obtained if it were possible to distinguish different sizes and types of aggregates. There is significant information on the behavior of polyglutamine sequences from cellular expression experiments, but, owing to the very poor solubility properties of these molecules, there is much less mechanistic and structural information on defined polyglutamine sequences at the in vitro chemical level. This application is based on recent advances in this laboratory in the ability to solubilize these molecules and control their aggregation in vitro. A series of chemically synthesized polyglutamine sequences of different lengths will be developed and used to pursue the following aims. The length dependence of the kinetics and thermodynamics of the aggregation process will be characterized. The length dependence of aggregate structure will also be characterized. Mutational analysis o f the polyglutamine sequence will be conducted in order to better define the unique role of glutamine in aggregation and to better understand aggregate structure at the atomic level. Finally, the abilities of a series of molecular chaperones to retard and reverse the aggregation process will be characterized in vitro. The work should lead to a better understanding of the role of polyglutamine self-association in the disease mechanism and the nature of the toxic entity in turn; this should stimulate new work on the therapeutic intervention.

DESCRIPTION (Provided by applicant): Amyloid fibrils and other ordered aggregation products of the Alzheimer's plaque peptide A-beta have been implicated by genetic, pathological, and cell culture studies to have a role in the development of Alzheimer's disease. Strategies targeting the growth or toxicity of these aggregates are formally possible but technically difficult owing in part to our ignorance of both fibril structure and of fibril assembly pathways and energetics. In this grant application we propose to implement new strategies to obtain information on structure and assembly, which will contribute, for example, to both the design and testing of inhibitors of fibril formation. We will utilize scanning mutagenesis linked to a number of in vitro assays for fibril growth to dissect the roles of key amino acid residue positions of A-beta in fibril formation and stability. We will use surface plasmon resonance (SPR) to study details of the kinetics of fibril assembly and disassembly with both wild type and mutant A-beta sequences.

[unreadable] DESCRIPTION (provided by applicant): A number of neurological and other diseases are associated with the formation of protein aggregates called amyloid fibrils. Although the protein component of amyloid is different for different diseases, the three-dimensional shape of the amyloid seems to be the same in all cases. It has proven exceedingly difficult to get detailed structural information on amyloid, compromising our efforts to understand and devise treatments for Alzheimer's disease (AD), Parkinson's disease, Huntington's disease, and an array of other devastating conditions. We have developed antibody molecules that have the unique ability to bind to amyloid fibrils, regardless of the amino acid sequence of the constituent protein. Here we propose to develop these conformational antibodies as tools to improve our understanding of the amyloid structure and how it develops in human diseases. The specific aims are to (1) develop additional antibodies and antibody fragments, (2) structurally characterize these antibodies as a novel window onto the structure of amyloid fibrils, (3) characterize the fundamental basis of the antibody-amyloid interaction, and (4) develop the antibodies into tests for detecting amyloid in tissue and serum, as well as to monitor the emergence of the amyloid motif during in vitro amyloidogenesis. The tools involved in this work will include hybridoma and phage display technology, recombinant expression, mutagenesis, protein modeling and crystallography, immunochemical binding assays, and in vitro amyloid fibril assembly reactions. Since recently described vaccine approaches to amyloid diseases such as AD depend on the generation of antibodies, characterizing the structural basis of the anti-amyloid reaction is important. It is also important to understand the structure of amyloid and how these fibrils grow, as a means toward developing agents that will compromise fibril cytotoxicity and fibril growth. Finally, as a ubiquitous alternative folding pattern of protein polymers, amyloid and its structure is of fundamental importance to our understanding of the molecular basis of life.

DESCRIPTION (provided by applicant): Nine (9) human genetic neurodegenerative diseases, including Huntington's disease, are caused by the expansion of CAG repeat sequences in the coding regions of different proteins. These proteins differ in size, structure, sub-cellular localization, and, where known, function. Since their only common feature is the polyglutamine (polyGIn) sequence encoded by the CAG repeat, and since for all but 1 of these proteins the repeat length threshold for disease risk is in the same 30-40 length range, neurotoxicity is almost certainly triggered by some property of the expanded polyGIn sequence. This proposal builds on and continues the threads of the previous grant by investigating in more detail the biophysical properties of expanded polyGIn sequences, focusing in particular on the formation of amyloid-like aggregates. In recent work we showed that nucleation of polyGIn aggregation involves a highly unfavorable folding reaction within monomeric polyGIn; The first aim of this proposal is to investigate in greater detail the elongation and nucleation phases of the aggregation reaction, and their responses to changes in solution conditions. In very recent work we showed that the nucleation reaction, and hence the overall aggressiveness of aggregation, is greatly influenced by benign-repeat length polyGIn peptides in the environment; The second aim of this proposal is to investigate how local sequence context and mutational breaks in the polyGIn sequence, as found in Gin-rich sequences in the human genome, impact aggregation ability. In other recent work we showed that the molecular chaperone Hdj2, previously shown to protect organisms against polyGIn toxicity, inhibits the polyGIn aggregation process in vitro; The third aim of the new proposal is to determine structurally how Hdj2 recognizes and neutralizes aggregation-prone polyGIn sequences. These studies will continue our long-term goal of determining how polyGIn sequences form toxic aggregates, and how both sequence and cellular contexts modulate their intrinsic aggregation propensity, thus ultimately determining disease risk.

DESCRIPTION (provided by applicant): The immediate cause of Huntington's disease is a triplet expansion in the DNA leading to an inherited gene for the protein huntingtin (htt) with an equivalent expansion of a polyglutamine (polyQ) repeat. How this mutated protein sequence triggers the molecular and cellular events leading to HD pathology continues to be debated. There is now general agreement that the large htt containing inclusions observed in HD brains on autopsy, and in cellular and animal models of HD, are probably not the toxic species. However, no evidence as been presented against the possible involvement of smaller aggregates not visible in fluorescence microscopy. This is significant since the only widely accepted known consequence of polyQ expansion to the behavior of polyQ disease proteins like htt is an enhancement of protein aggregation. A number of screening assays for small molecules capable of blocking aggregation have been conducted leading to compounds that block aggregation and suppress toxicity in animal models. One such compound, riluzole, failed to induce a response in human clinical trials, but then it also failed in a animal model preclinical trial, perhaps because of insufficient concentration in the brain. Recently our group has elucidated a new aggregation mechanism for the exon1 fragment of huntingtin containing the polyQ sequence, which depends on a triggering protein unfolding event in the exon1 N-terminus just before the polyQ. Characterization of exon1 aggregation in vitro led to the discovery that there are two aggregation pathways competing for exon1 molecules, each of which produces different aggregation intermediates and/or products. Using a new staining method specific for amyloid-like aggregates of polyQ, we identified a new aggregate in mammalian cells producing exon1 that have been thought to only produce large inclusions of exon1. In this application we propose to develop new high throughput screening assays for identifying aggregation inhibitors in large libraries of small molecules. The premise of this proposal is that we do not know which of the known huntingtin exon1 aggregated species is the most toxic and most likely to contribute to HD. We propose two new screening assays based on our preliminary data. One focuses on finding molecules that will prevent, in vitro, the protein misfolding event in the exon1 N-terminus that we believe triggers a portion of htt exon1 aggregation. The other focuses on preventing the formation in mammalian cell culture of the amyloid-like aggregates of exon1 that are different from the large inclusions previously focused on in screening and molecular mechanism studies. We propose to develop these assays and fine-tune them to a state ready for high throughput screening. We will also develop a variety of secondary screening assays that will be required to eliminate false positives from future high throughput screens. We believe that these assays could lead to the discovery of new classes of inhibitors capable of slowing the progression of HD. PUBLIC HEALTH RELEVANCE: This research project is immediately relevant to human health in that it proposes to develop new screening assays for drug discovery of potential Huntington's disease therapeutics. If successful, these assays could very quickly be consigned to high throughput screens to identify new candidate molecules.

DESCRIPTION (provided by applicant): In spite of our knowledge of the Huntington's disease gene and of the disease mutations, and in spite of an enormous amount of creative work, the twin goals of a molecular level understanding of the disease mechanism, and the molecular therapy one hopes would emerge from this understanding, are not in reach. One difficulty holding the field back has been the unusual nature and unknown function of the disease gene product, a seemingly poorly behaved protein of over 3500 amino acids and no proven function. The full length Huntingtin protein has been very challenging to make and to work with, while the more manageable N- terminal fragment, exon1, though accessible by recombinant synthesis, is also poorly behaved in solution, especially when it contains a polyglutamine sequence in the pathological repeat length range of 37 or more. During previous terms of this grant, our group has taken a reductionist approach to this problem, working with peptide fragments that are accessible by chemical synthesis. Over the last funding period we moved from studies on simple polyglutamine (polyQ) sequences to studies of how the N and C-terminal flanking sequences of the polyQ in exon1 influence the solution conformation and aggregation of polyQ. These studies revealed a powerful and mechanistically complex accelerating effect by the 17 amino acid sequence on the N-terminal side of polyQ in exon1 (httNT) on polyQ aggregation along with a complete change in aggregation mechanism and the structures of the aggregated products. As part of our work to understand this effect, we developed the ability to produce chemically accessible analogs of htt N-terminal fragments (NTFs) related to exon1 that appear to behave similarly to authentic recombinant exon1, but at the same time are more manageable and amenable to introduction of small, well-defined probes for such techniques as NMR and FRET. In other recent work, we developed a staining method specific for amyloid forms of htt NTFs and polyQ in tissue and cells that has allowed us to visualize different aggregate structures in the cellular context. We also discovered, and engineered for cellular delivery, peptide-based inhibitors of different aggregation pathways of htt NTFs. In this renewal application, we propose to exploit these tools to reach a new understanding of the molecular events that are triggered by expanded polyQ in htt. The current wisdom in the HD field is that htt must be proteolytically processed to an N-terminal fragment as an early step in the disease mechanism. The physical state that NTF takes when it becomes toxic remains a mystery. This toxic physical state may be a misfolded form of the monomeric fragment, a short-lived aggregation intermediate, or a stable aggregate such as an amyloid fibril. In this proposal we will focus on structural analysis of monomers and stable aggregates, using chemically synthesized and recombinant htt NTFs including exon1, with biophysical tools such as NMR, fluorescence resonance energy transfer, hydrogen-deuterium exchange, and mutational analysis. We will also conduct a detailed study of the polyQ repeat length effect on the structure of the monomeric htt NTF as well as its aggregation propensity, and compare both to the repeat length dependence of HD risk. We will also explore the toxicities of these physical states by testing in vitro-produced materials and by probing the effect of inhibitors on htt-producing cells. PUBLIC HEALTH RELEVANCE: Huntington's disease is one of a family of genetic disorders leading to progressive neurodegeneration manifesting in loss of neuromuscular control, development of cognitive impairment and psychiatric conditions, and ending in death. Although there are some therapies that can provide comfort for particular symptoms, there are no known therapies that provide a cure or attack the central disease mechanism. Our project is aimed at improving knowledge about how the genetic mutation in Huntington's disease generates disease symptoms by focusing on the abnormal physical and cellular behavior of the protein Huntingtin that is altered by the mutation. Improved understanding of such molecular mechanisms of disease can be a powerful first step in developing effective treatments.

DESCRIPTION (provided by applicant): This proposal seeks funding to support and extend an interdisciplinary predoctoral program that prepares students with strong backgrounds in quantitative physical sciences to conceptualize, conduct, and direct independent research in molecular biophysics and structural biology. This Molecular Biophysics and Structural Biology (MBSB) program places a strong emphasis on interdisciplinary, collaborative research in an environment where the practical human health consequences of research are appreciated and contribute to guiding and driving basic research. The training faculty consists of 40 independent investigators spread over 14 departments within the School of Medicine and the Faculty of Arts and Sciences at the University of Pittsburgh and within Carnegie Mellon University. The training faculty comprises a large group of senior members who have strong records in graduate training, funding, and publication, and a smaller contingent of junior faculty who bring new ideas and technical expertise to the group. The research programs of training faculty emphasize not only characterization of biomolecular phenomena but also development of new methodologies. At every stage of the curriculum, emphasis is placed on the relationship between physical methods and biological understanding. The research is concentrated in core areas at the challenging forefront of biomolecular science, including membrane proteins, protein-protein interactions and oligomeric complexes, nucleic acid structure and protein-nucleic acid interactions, poorly structured protein elements, protein misfolding and aggregation, and small molecule ligand - protein interactions. The training program includes a first year core curriculum featuring an introduction to biophysical instrumentation and methods, how these are used to address specific molecular questions, an introduction to theory and simulation of biomolecular behavior, and courses on statistical analysis and computer programming. Student's abilities to synthesize and creatively utilize what they have learned in the classroom are tested in a qualifying exam that requires them to construct and defend a research proposal not related to their own laboratory work. After passing this exam students move on to develop and pursue their thesis project. Students are required to participate in a weekly journal/data club and to attend a Molecular Biophysics seminar series featuring invited local and national speakers. Students are also encouraged to participate in several local, annual science meetings. This application proposes special features new to the MBSB program including incentives to develop co-mentored, collaborative thesis projects and an annual mini-symposium, with significant student participation, set within the MBSB graduate program. Students are also encouraged to take advantage of the rich environment of the School of Medicine to attend seminars in molecular aspects of human disease. We are requesting funds to support six traineeships with the goal to train a new generation of molecular biophysicists who will be leaders in future efforts to understand normal and abnormal human biology at the physical and molecular level.

DESCRIPTION (provided by applicant): The aggregation of peptides and proteins into highly organized aggregates such as amyloid fibrils is a very important and fundamental process in biology. Most proteins suffer some degree of aggregation after they are synthesized in the cell, and a variety of cellular mechanisms have evolved to deal with the consequences of aggregation, which range from rendering protein synthesis inefficient to creating toxic species. In some cases amyloid fibril formation is beneficial and necessary for a particular biological function, such as the bacterial adhesins that support biofilm growth, and the secretory storage granules used by mammals for storage and release of protein hormones like insulin. In many other cases amyloid fibril formation is detrimental, being associated with serious human diseases as well as with normal aging. Protein aggregation also plays important roles in biotechnology, in the recombinant synthesis, purification and formulation of protein therapeutic agents. For all of these reasons it is important to come to a better mechanistic understanding of the protein aggregation process, and in particular the events that initiate aggregation, a process termed nucleation. Many cases of spontaneous amyloid fibril formation involve highly complex nucleation mechanisms with multiple molecular species in high flux, greatly complicating nucleation analysis. In other cases, however, nucleation is more simple, and in fact more resembles the classical model of nucleated growth polymerization from basic polymer chemistry. This grant application proposes a series of experiments to unravel the nucleation mechanism of a series of modified polyglutamine (polyQ) molecules, which are an important place to begin to study nucleation because they fall into the second, more simple class of nucleation mechanisms. The basic approach will be to design polyQ sequences containing various mutations that are predicted to have defined conformational effects on the solution structure of the protein, and to carry out detailed nucleation kinetics analyses on these proteins. These analyses will yield important parameters such as the thermodynamic driving force for nucleus formation, and the thermodynamics associated with simple fibril growth (elongation). These experimentally determined values for the energetics of amyloid nucleation and growth will then be compared with expectations based on the known conformational tendencies introduced into the test polyQ molecules by the added mutations. In this way, certain hypotheses for the structural nature of the aggregation nucleus will be tested. Success in this project will provide important clues as to how amyloid formation is nucleated that should have implications over broad areas of biology, such as biological systems affected by amyloid diseases, as well as related protein aggregation processes that occur in normal cell biology and in normal aging. Since it is likely that conformational rearrangements are important even in the more complex type of amyloid nucleation reactions, the results may open the way to new approaches to the study of the more complex and currently largely impenetrable class of nucleation mechanisms.