A key step in cellular signaling pathways is the reversible phosphorylation of proteins. Metabolism, cell growth and differentiation, proliferation and apoptosis, cytoskeletal rearrangement and angiogenesis, are controlled by reversible phosphorylation. This process is mediated by the action of protein kinases that catalyze the transfer of a phosphate group from ATP or GTP to lipids or proteins. The phosphate acceptor in proteins is the hydroxyl group of either a serine, threonine, or tyrosine residue. The acceptor residues can be on the same protein kinase, as in the case of autophosphorylation, or on different protein targets. The human kinome contains 518 protein kinases and approximately 20 lipid kinases that comprise 1.7% of human genes. The pivotal role of kinases in disease pathophysiology is evident by the observation that kinases mutation lead to alteration in cellular signalling then leading to different pathologies, ranging from diabetes, to cancer, cardiovascular and neurodegenerative or immunological disorders. Particularly, in cancer tissues, has been reported the expressions of several single residue variants (SRV) of kinases caused by non synonymous single nucleotide polymorphisms (nsSNPs) that occurr in the DNA coding region and encode a change in the amino acid sequence. The single amino acid substitution can potentially affect the protein structure-function relationships in different ways, such as changes in protein function, stability, flexibility and interaction with other proteins, nucleic acids, and other molecules. The aim of this project is the study of some natural SRVs of MAPKs, in particular MAPK1. In this study we will select some natural SRV of MAPK1 that are expressed in cancer tissues and reported in cancer databases. These variants will be characterized to investigate the effect of single amino acid substitution on MAPK1 thermal and thermodynamic stability and structure in solution.
In the post-genomic era, how human genetic and somatic variations are associated with diseases and how mechanisms form the basis of the relationship between genotype and phenotype are still open questions. Available data on polymorphisms in the human genome are expanding rapidly, though, knowledge of the molecular mechanisms of many genetic diseases is lagging, due to the laborious and time consuming nature of experimental studies. Experimental analysis of the impact of nonsynonymous single nucleotide polymorphisms (nsSNPs) on protein structure, function and stability studies require mutagenesis, protein expression and purification followed by thermal and chemical unfolding: the entire process is therefore costly and time consuming. Biophysical and stability studies of protein variants help when analyzing the effect of variations on protein structure and function, however, information is available for only few proteins. There is need to solve the 3D structure of natural variant to explore, at the atomic level, the consequences of the amino acid substitution derived from single nucleotide polymorphism [1]. Structural analysis of nsSNPs in the human DNA sequences may also help to predict personal response to certain drugs, susceptibility to environmental factors, and risk of developing particular diseases.
The aim of this project is the study of single residue variants, expressed in cancer tissues, of MAPK1, a kinase involved in cell signalling. The protein encoded by this gene is a member of the MAP kinase family. MAP kinases, also known as extracellular signal-regulated kinases (ERKs), act as an integration point for multiple biochemical signals, and are involved in a wide variety of cellular processes such as proliferation, differentiation, transcription regulation and development. The activation of this kinase requires its phosphorylation by upstream kinases. Upon activation, this kinase translocates to the nucleus of the stimulated cells, where it phosphorylates nuclear targets [2].
Deviation from the strict control of MAPK signaling pathways has been implicated in the development of many human diseases including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and various types of cancers. In particular, the ERK signaling pathway plays a key role in several steps of tumorigenesis including cancer cell proliferation, migration, and invasion. Accordingly, MAPKs are important clinical targets with a number of kinase inhibitors in
clinical use or undergoing clinical trials [3].
A detailed understanding of the changes of the investigated gene products at the molecular level to assess how genetic variations impact the protein folding, structure, function and interactions is required to develop new therapeutic strategies, particularly in the search of small molecules able to selectively interact with the variants, which is an essential preliminary step to personalized medicine, and help to identify new potential therapeutic targets. Precision medicine aims at classifying individuals into subpopulations that differ in their susceptibility to a particular disease, in the biology and/or prognosis of those diseases they may develop, or in their response to a specific treatment. The structural analysis of protein variants expressed in cancer tissues may help in understanding the molecular basis of the disease and, since individuals carrying variants may respond differently to drugs, it may provide information for personalized drugs tailored to the individual variant.
References
1. Bhattacharya, R et al., PLoS One 12 2017; e0171355
2. Kim E.K., Eui-Ju Choi E.J., Biochimica et Biophysica Acta (BBA) Volume 1802, Issue 4, 2010, Pages 396-405, ISSN 0925-4439, https://doi.org/10.1016/j.bbadis.2009.12.009.
3. Duong-Ly K.C. et al. Curr Protoc Pharmacol. 2013; Chapter 2: Unit2.9. doi:10.1002/0471141755.ph0209s60