Muscular dystrophy (MD) refers to a group of degenerative muscle diseases that cause progressive muscle weakness. MD affects all types of muscles. For example, decreased function of heart muscles causes heart diseases that include cardiomyopathy and congestive heart failure. At present there is no cure available for MD, although certain palliative treatments are available to ease the pain associated with MD. Duchenne MD (DMD) and Becker MD (BMD) are two prominent types of MD, which are caused by the deficiency of a vital muscle protein known as dystrophin. These dystrophin-related diseases physically weaken patients to a state of immobility, and often cause death at an early age. Dystrophin stabilizes the sarcolemma membrane against the mechanical forces associated with muscle contraction and stretch. Mutations in dystrophin trigger the disease. Although dystrophin was identified as a key molecular player in MD 25 years ago, little is known about the biophysical mechanisms that trigger the disease at the fundamental protein level.
Utrophin, the closest homologue of dystrophin (60% sequence similarity), has been shown to compensate the loss of functional dystrophin in animal studies, but its exact biological function is not known. It binds to actin, protects actin against its depolymerization, and interacts with dystrophin-related proteins. Utrophin is confined specifically to the sarcolemma in fetal and regenerating muscle cells. After down-regulation at birth, it is only found in the neuromuscular junctions in adult muscle cells to aid in optimal synapse transmission and to play a stabilizing role at these junctions.
In this project, we are trying to understand the biophysical and structural principles of how these two important proteins function and the effect thereon of disease-causing mutations.
Relevant Publications
S.M. Singh, N. Kongari, J. Cabello-Villegas, and K.M.G. Mallela, Mutations in dystrophin that trigger muscular dystrophy decrease protein stability and lead to cross-ß aggregates, Proceedings of the National Academy of Sciences of the United States of America, 107 (2010) 15069-15074.
Approximately one-third of pharmaceutical formulations are of multi-dose. Multi-dose protein formulations are desirable for reasons of economics, patient compliance, and safety. Because of the risk of microbial growth after the first dose has been removed from the product vial, multi-dose formulations require an effective antimicrobial preservative (AP). However, APs have been shown to cause protein aggregation and the underlying mechanisms are less understood. Such knowledge is required for developing approaches to inhibit AP-induced protein aggregation, and for choosing the right AP that causes less protein aggregation yet offers the desired antimicrobial function. In this project, we are studying the effect of various phenolic APs such as benzyl alcohol and m-cresol on protein aggregation using a model protein cytochrome c.
Alcohols form a major class of APs. Besides therapeutic proteins, alcohols were recently shown to aggregate disease-related and non-disease-related proteins. Alcohols have been traditionally used as “co-solvents” to probe the effect of various physical parameters of solvents on protein stability and folding, but the mechanisms of alcohol-induced protein aggregation were not understood. Also, alcohol effects on partial protein unfolding and function were never examined. This is important because protein function is often controlled by its partial rather than global unfolding. In this project, we are studying how the physical properties of alcohols affect partial protein unfolding and protein aggregation.
Relevant Publications
S.M. Singh, R.L. Hutchings, and K.M.G. Mallela, Mechanisms of m-cresol induced protein aggregation studied using a model protein cytochrome c, Journal of Pharmaceutical Sciences, In Press (2011).
S.M. Singh, J. Cabello-Villegas, R.L. Hutchings, and K.M.G. Mallela, Role of partial protein unfolding in alcohol-induced protein aggregation, Proteins: Structure, Function, and Bioinformatics, 78 (2010) 2625-2637.
Detailed structural information from hydrogen exchange and related experiments indicated the following three physical principles underlying protein folding pathways: (1) Proteins are made up of small cooperative unfolding/folding submolecular units known as foldons. (2) Proteins construct these foldon pieces to progressively build their final native states using the sequential stabilization principle where prior structure guides and templates the subsequent foldons. (3) Optional misfolding errors, which are ubiquitous, can corrupt different naturally occurring on-pathway intermediates and cause intermediates to accumulate by inserting error-repair barriers at different points along the pathway. The first two principles dictate that the folding pathway of a protein is predetermined by its component foldon substructure, and the order of steps is set by the way the foldon units are organized in the native structure. The third principle determines whether the pathway appears to be kinetically 2-state or multi-state or heterogeneous. Preliminary evidence shows that proteins may use the same foldons to control their function. For example, in the case of cytochrome c, the process that triggers the intracellular apoptotic pathway is the unfolding of its least stable or most flexible foldon. In this project, we will examine the generality of these principles in folding, misfolding, and function of other proteins.
Relevant Publications
S. Bédard†, M.M.G. Krishna†, L. Mayne, and S.W. Englander, Protein folding: Independent unrelated pathways or predetermined pathway with optional errors, Proceedings of the National Academy of Sciences of the United States of America, 105 (2008) 7182-7187. († Equal contribution)
S.W. Englander, L. Mayne, and M.M.G. Krishna, Protein folding and misfolding: mechanism and principles, Quarterly Reviews of Biophysics, 40 (2007) 287-326.
M.M.G. Krishna and S.W. Englander, A unified mechanism for protein folding: Predetermined pathways with optional barriers, Protein Science, 16 (2007) 449-464.