Polymorphisms in the genome are responsible in humans for phenotypic differences and susceptibility to genetic disease. A significant fraction of molecular functional diversity in the human population is attributable to effects on protein function caused by non synonymous single nucleotide polymorphisms (nsSNPs). nsSNPs occur in the DNA coding region and induce a change in the amino acid sequence of the protein, namely single residue variation (SRV). Several investigations have addressed the effect of nsSNPs on protein stability, functions and interactions. SRV associated to nsSNPs may be related to variation of the kinetic parameters of enzymes, the DNA-binding properties of proteins that regulate transcription, the signal transduction activities of receptors, and the architectural roles of structural proteins. This project focuses on natural variants of the Peroxisome Proliferator-Activated Receptor gamma (PPARgamma), a nuclear receptor that belongs to the superfamily of ligand inducible transcription factors, involved in several biological processes. PPARgamma is expressed in the adipose tissue and plays a key role in the regulation of lipid metabolism in mature adipocytes and macrophages. In addition, PPARgamma has been reported to be involved in several processes related to cellular differentiation and development and to carcinogenesis. In this study we will select nsSNPs resulting in PPARgamma variants, reported in databases such as COSMIC and OMIM, that are expressed in cancer tissues or that are related to alteration of metabolic control. The impact of PPARgamma nsSNPs on important pathways for the cell, such as cellular differentiation, apoptosis and propensity to tumorigenicity, will be analyzed and compared with the effect of the mutation on the protein structure and stability. The aim of the present study is to give insight the signaling network which orchestrates the regulation of cancer by PPARgamma variants activation in human cell lines.
In the biomedical context, analysis of nsSNPs in the human DNA sequences may help to understand personal susceptibility to environmental factors and risk of developing particular diseases. When a nsSNP causes an amino acid change (a missense mutation) there may be an effect on the structure and function of the encoded protein. Loss of function may lead to disease. In the last decades there was an increment of the publication number about the effect of nsSNPs and protein stability but knowledge of the impact of variations on the biological pathways is lagging, so experimental studies are required.
The number of studies about the structural and thermodynamic characterization of protein variants is small as compared to the large number of nsSNPs that are apparently associated to diseases [1] and only few data are available regarding the impact of missense mutations induced by nsSNPS on cellular pathways. Indeed, the disruption of cellular pathways may result in the development of several diseases and pathological states.
The aim of this project is the study of nsSNPs of PPARgamma LBD expressed in cancer tissues [2] and related to metabolic disorder [3]. In a previous study, we have reported that, when compared to the wild type protein, most of these variants display changes in protein dynamics, thermal and thermodynamic stability and in tertiary contacts that affect structural elements crucially important for PPARgamma functioning [4]. In the present project, we will investigate the effect of above mentioned SRVs on specific signaling pathways normally regulated by PPARgamma. Since PPARgamma is involved in lipid metabolism and is highly expressed in adipose tissue, we will use mouse pre-adipocyte 3T3L1 cell line that will be transfected with pCDNA plasmids harboring PPARgamma wild type gene and compared with those harboring the single amino acid substitutions of SNPs. Adipocyte maturation will be evaluated. Given that PPARgamma is also related to carcinogenesis, we will express nsSNPs PPARgamma variants in various human tumor cell lines in order to evaluate the effects of the chosen PPARgamma mutations on oncogenic pathways.
PPARgamma modifies its cellular target if variations are present and different concentrations or different type of ligands, used like PPARgamma agonists, can be modulated in order to avoid side effects according to the different response to drugs of individuals affected by nsSNP.
Given the remarkable progress over the past few years in characterization of human gene sequences and in additional new molecular technologies, there is particular interest in the potential for using individual molecular biomarkers to direct patient specific decisions on the management of diseases such as diabetes mellitus and cancer. Technological advances in genetics, genomics, proteomics, and metabolomics make possible the efficient analysis of thousands of genes, proteins, and metabolites, thus offering new opportunities for identifying genetic factors and gene products that are linked to different subtypes of diseases. SNP variations in the DNA sequences of humans may help to predict an individual's response to certain drugs or therapy, susceptibility to environmental factors, and risk of developing particular diseases.
SNPs are therefore important at all levels of drug research and development, from investigation of the molecular basis of a disease through the search for and evaluation of new targets to the clinical testing and regulatory approval of new drugs. When knowledge of the distribution and effects of SNPs does not lead to the discovery of new medicines, it may help to diagnostic possibilities that permit better targeted and safer use of existing forms of treatment. A detailed investigation at the molecular and cellular level of the protein variants produced by nsSNP may provide useful information for the development of 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. These findings emphasize the importance of studying human genetic variation as a guide to combating diseases.
Since we might expect that some nsSNPs will make the output of the pathway more or less efficient, it is necessary to test how these variants can modulate the efficiency of the cellular pathways of the protein. Research over the next few years will likely uncover this gap in the study of nsSNP variants and it will make treatment of diseases more successful.
References
1) Kucukkal TG et al. Curr. Opin. Struct. Biol. 2015; 32: 18
2) Forbes SA et al. Nucleic Acids Res 2011; 39, D945
3) Chan KH, et al. J. Clin. Endocrinol. Metab. 2013, 98, E600¿E604
4) Petrosino M, et al. Int. J. Mol. Sci. 2017, 18, 361; doi:10.3390/ijms18020361