Project 1: Structure-function-disease analysis of dystrophin and utrophin in muscular dystrophy
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. 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 30 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 for 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, the effect thereon of disease-causing mutations, and whether we can develop new therapies based on the fundamental understanding of structure-function of dystrophin and utrophin.
- S.M. Singh, S. Bandi, and K.M.G. Mallela, The N-terminal flanking region modulates the actin binding affinity of the utrophin tandem calponin-homology domain, Biochemistry, 56 (2017) 2627-2636. PDF
- S.M. Singh, S. Bandi, and K.M.G. Mallela, The N- and C-terminal domains differentially contribute to the structure and function of dystrophin and utrophin tandem calponin-homology domains, Biochemistry, 54 (2015) 6942-6950. PDF
- S. Bandi, S.M. Singh, and K.M.G. Mallela, Interdomain linker determines primarily the structural stability of dystrophin and utrophin tandem calponin-homology domains rather than their actin-binding affinity, Biochemistry, 54 (2015) 5480-5488. PDF
- S.M. Singh, S. Bandi, D.D. Shah, G. Armstrong, and K.M.G. Mallela, Missense mutation Lys18Asn in dystrophin that triggers X-linked dilated cardiomyopathy decreases protein stability, increases protein unfolding, and perturbs protein structure, but does not affect protein function, PLoS One, 9 (2014) e110439. PDF
- S. Bandi, S.M. Singh, and ;K.M.G. Mallela, The C-terminal domain of the utrophin tandem calponin-homology domain appears to be thermodynamically and kinetically more stable than the full-length protein, Biochemistry, 53 (2014) 2209-2211. PDF
Project 2: Mechanisms of excipient interactions with pharmaceutical proteins
Excipients play a major role in formulating a drug substance into a drug product. These include antimicrobial preservatives such as benzyl alcohol to prevent the accidental growth of microbes in protein formulations, aggregation suppressors such as polysorbates, reactive oxygen scavengers such as methionine, surface deadsorbents such as silicone oil, and others. In principle, excipients should be inert substances that should merely serve as the vehicle or medium for a drug or active substance, but in reality, these can interact with protein drugs causing unwanted protein destabilization and aggregation. In this project, we are trying to understand the fundamental biophysical and structural mechanisms by which excipients interact with pharmaceutical proteins using a suite of biophysical techniques that include far-UV and near-UV circular dichroism, fluorescence, isothermal titration calorimetry, differential scanning calorimetry, and 2D NMR. This work is being done as part of our Center for Pharmaceutical Biotechnology, and please contact us for any future collaborations.
- D. Shah, J. Zhang, H. Maity, and K.M.G. Mallela, Effect of photo-degradation on the structure, stability, aggregation, and function of an IgG1 monoclonal antibody, International Journal of Pharmaceutics, 547 (2018) 438-449. PDF
- S.M. Singh, S. Bandi, D.N.M. Jones, and K.M.G. Mallela, Effect of polysorbate 20 and polysorbate 80 on the higher-order structure of a monoclonal antibody and its Fab and Fc fragments probed using 2D nuclear magnetic resonance spectroscopy, Journal of Pharmaceutical Sciences, 106 (2017) 3486-3498. PDF
- R.L. Bis, S.M. Singh, J. Cabello-Villegas, and K.M.G. Mallela, Role of benzyl alcohol in the unfolding and aggregation of interferon alpha-2a, Journal of Pharmaceutical Sciences, 104 (2015) 407-415. PDF
- R.L. Bis and K.M.G. Mallela, Antimicrobial preservatives induce aggregation of interferon alpha-2a: The order in which preservatives induce protein aggregation is independent of the protein, International Journal of Pharmaceutics, 472 (2014) 356-361. PDF
- R.L. Bis, T.M. Stauffer, S.M. Singh, T.B. Lavoie, and K.M.G. Mallela, High yield soluble bacterial expression and streamlined purification of recombinant human interferon alpha-2a, Protein Expression and Purification, 99 (2014) 138-146. PDF