Protein Aggregation and Protein Deposition
Diseases
(Parkinson's disease, Light chain amyloidosis, Insulin
Fibrillation, Leptin aggregation)
The aggregation of proteins is of critical importance in a wide variety of biomedical situations, ranging from abnormal disease states (such as Alzheimer's and Parkinson's disease) to the production (e. g. inclusion bodies), stability and delivery of protein drugs.
We are interested in the molecular mechanism of formation of protein aggregates, both amorphous and fibrillar, and the conformation of the protein molecules in the aggregated state. The systems we study cover both in vivo (e. g. amyloid, inclusion bodies) and in vitro aggregates (e. g. amyloid, refolding aggregates). We use a wide range of techniques in these investigations, including FTIR, predominantly in the attenuated total reflectance mode, atomic force microscopy, electron microscopy, fluorescence anisotropy, light scattering, small-angle X-ray scattering, circular dichroism, and Thioflavin T fluorescence assays).
Our basic hypothesis is that partially-folded intermediates are the immediate precursors for aggregation (see scheme 1). Thus conditions which populate partially-folded intermediates (either from the native state or during folding) will tend to favor aggregation.
Scheme 1
The aggregating systems we are studying range from simple peptides and isolated partially-folded intermediates, to small proteins such as a-synuclein, the variable domain of immunoglobulin light chains, insulin, leptin, a-lactalbumin, phosphodiesterase g, prothymosin a and apo-lipoprotein E4.
The immunoglobulin VL domains are responsible for several lethal protein deposition diseases, including light-chain (AL or light chain amyloidosis, light-chain deposition disease (LCDD) and myeloma (cast) nephropathy). The protein deposits result in renal, heart and other organ failure. We are interested in answering the question of why some immunoglobulin light-chains form amyloid and related deposits and others do not. To do this we are performing a systematic comparison of the properties of two VL domains, one that is amyloidogenic (SMA) and one that is not (LEN). These investigations are aimed at uncovering differences in the stability of these proteins, the properties of partially-unfolded intermediates of these VLs, their propensity to aggregate, the kinetics of formation of aggregates and the structure of the aggregates formed under different conditions. The results of these investigations should allow us to prove, or disprove, our hypothesis that the underlying molecular mechanisms for protein aggregation in these diseases is the presence of partially-unfolded intermediates which act as the soluble precursors to the protein deposits. We are also investigating potential therapeutic agents that might be effective at inhibiting amyloid formation. For example, we are using a combinatorial library of peptides using a phage display system to find specific peptides that inhibit the fibrillation process. The similarity in properties of amyloid in different amyloidoses suggests that principles learnt in this study should be of relevance to other amyloid diseases.
Recently, a-synuclein has been identified as a major component of Lewy bodies, the intracellular inclusions that are a pathological hallmark of Parkinson's disease (PD). Our goals are to test the hypothesis that a critical step in Parkinson's disease is the aggregation of a-synuclein, which leads to the formation of Lewy Bodies and subsequently to neuronal death. Specifically we are investigating the molecular basis for a-synuclein aggregation and potential inhibitors of a-synuclein aggregation. Our preliminary results have revealed a number of factors that lead to a conformational change in a-synuclein at neutral pH, and also to aggregation and fibril formation. We plan a systematic characterization of the biophysical properties of a-synuclein to determine if there is a correlation between its conformation and its propensity to aggregate, with both wild-type and mutant a-synucleins. In preliminary experiments we have found that various factors associated with PD, for example, metal ions and pesticides, enhance the aggregation of a-synuclein. Details of the aggregation process are being studied to elucidate the molecular mechanism of aggregation and fibril formation. We are screening a series of peptides and small molecules for inhibitory effects on a-synuclein as potential inhibitors that may lay the groundwork for future therapeutic approaches. Techniques used in these studies include various biophysical/biochemical methods, such as attenuated total reflectance FTIR to analyze the conformational state of aggregated a-synuclein, atomic force and electron microscopy to image the aggregates, and kinetic methods to monitor the rate of formation of fibrils.
Insulin is a major hormone involved in carbohydrate, lipid and protein energy metabolism, and is critical for the control of blood glucose level. In Type I diabetes the effective levels of insulin are insufficient for normal health, and patients must use frequent injections or infusions of insulin. Several tons of insulin are produced annually for use by diabetics. The tendency for insulin to aggregate and form fibrils leads to serious problems, both in the production of the protein, and its storage and delivery. It is also a problem in new delivery systems currently under development. The molecular mechanism of insulin aggregation remains substantially unknown. Our major goal is to understand the molecular basis for the aggregation of insulin. This knowledge will be useful in developing methods to minimize insulin fibrillation, and in determining the generality of such aggregation among other potential protein drugs. Surprisingly, there is very limited knowledge about the details of the molecular events involved in the aggregation of insulin. Through a systematic investigation of the process we hope to learn the detailed mechanism whereby insulin aggregates. Based on our current understanding of protein aggregation our starting hypothesis is that aggregation arises from a partially-folded intermediate. Current experiments include demonstration of the presence of such an intermediate and its correlation with aggregation. The detailed mechanism of aggregation is being probed by determining the regions of the molecule involved in the intermolecular interactions leading to aggregation using various insulin analogs (point mutants), as well as the role of soluble oligomers. From an understanding of the underlying basis of insulin aggregation we hope to be able to develop methods to circumvent the problem.
Leptin is a naturally occurring hormone involved in metabolic regulation,and a potential anti-obesity drug. Leptin is a member of a large family of cytokines which contain a four-helix bundle structural motif. Clinically, leptin is administered through subcutaneous injection at high doses, however, at neutral pH, human leptin has limited solubility, although at lower pHs it has a very high solubility. When a solution of leptin at low pH and high concentration is injected, it leads to irreversible protein precipitation and a significant response by the host defensive system. In vitro experiments show similar effects in that a solution of leptin at high concentration in acid exhibits irreversible precipitation when adjusted to neutral pH. Our goals are to understand the molecular basis for the irreversible aggregation of leptin and to develop methods to ameliorate it.