Biological processes, metabolism, cell growth and differentiation, apoptosis and angiogenesis are controlled by reversible phosphorylation of proteins.
Protein kinases catalyze the transfer of a phosphate group from ATP or GTP to the hydroxyl group of either a serine, threonine, or tyrosine protein residue, and protein phosphatases remove the phosphate group. Addition and/or removal of the phosphate group may affect the function of the protein target by modifying its structure, stability, substrate or ligand affinity, and activity. Dysregulation of the protein kinases signaling is observed in many diseases like cardiovascular and neurodegenerative diseases or in immunological disorders, in diabetes and cancer. MAPK3 (ERK1) is a component of the complex MAPK signaling involved in oncogenesis, tumor progression and drug resistance. In cancer tissues has been reported the expressions of several somatic non-synonymous single nucleotide variants (nsSNVs) of MAPK3 carrying a single amino acid substitution in the sequence. A 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, or drugs and other molecules. The aim
of this project is the selection of some natural nsSNVs of MAPK3 expressed in cancer tissues and reported in cancer databases. These variants will be characterized to investigate the effect of a single amino acid substitution on MAPK3 activity, 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 single amino acid substitution 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 missense variants 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 of MAPK3, a kinase involved in cell signalling, expressed in cancer tissues. The protein encoded by this gene is a member of the MAPK family. MAPK (mitogen-activated protein 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 MAPK 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