ROLE OF RHO GTPASES IN CELLULAR FUNCTIONS AND DISEASE BIOLOGY

Abstract

Leukocyte migration is a central process in physiological and pathological conditions, including sepsis. The systematic interplay between several molecules such as integrin, chemokine receptors, GTPases and GPCRs regulate various events of cell migration. Migration, localization and functional dynamics of innate immune cells have been key areas of research in host response mediated disease pathologies such as sepsis. The formation of leading (lamellipodia) and trailing edge extension (filopodia) are controlled by small monomeric Gprotein of Rho family GTPases that function as molecular switches between inactive GDPbound and active GTP-bound states. Rho family proteins are small monomeric G-proteins associated with diverse cellular functions, including cytoskeleton reorganization, gene transcription, cellular motility, cell cycle progression, and other functions. The Rho proteins Rac1, Cdc42, and RhoA extensively studied Rho proteins in innate immune cell motility, but the role of RhoG is not well explored in innate immune cell functions. During the process of cell migration, activating integrins by binding to ECM proteins could regulate Rho GTPases via forming FAK- Src complexes associated with leading and trailing edge cytoskeletal rearrangement in cells. Rho GTPase, such as Rac1, is also known to regulate integrin localization. In order to study the role of Rho GTPases, initially, I learnt the computational tools such as MODELLER, and PyMOL for homology modelling and electrostatic surface charge calculation, respectively, for shortlisting the molecules which are not well studied, I also learnt protein modelling and calculation of electrostatic charges on the interacting and extracellular domains of proteins expressed on immune cells such as macrophages under various changing conditions of tissue microenvironment such as temperature and pH using Delphi and H++ server. As described in the first part of my doctoral dissertation, by implicating such computational modelling tools, I demonstrated that inflammatory or M1 macrophages show the overall cumulative charges of extracellular domains of -12 due to the presence of surface proteins such as CD16, CD80, CD120b, CD89, MHCII, CD197, PD-L1 and CD64 whereas M2 polarized or immunosuppressive macrophage showed relatively more negative cumulative electrostatic potentials − 16 on their surface which was attributed to the expression of surface molecules such as CD1a, CD1b, PD-L2 and CD200R. These data were validated by Chakraborty et al. during similar studies, and the computationally calculated values correlated with the zeta potential of macrophages and monocytes under inflammatory and polarized conditions (Chakraborty, Dipankar, et al. 2020). The second part of my doctoral work focused on understanding the role of RhoG in innate immune cell functions, including neutrophils and monocytes. With homology modelling and sequence analysis of various unexplored Rho GTPases with well studies Rho GTPases such as Rac-1, my work demonstrated that RhoG could be an important Rho GTPase that may have a potential role in immune cell migration and function and, to date very limited information is available on the functions of RhoG in immune cell migration and function. The expression dynamics of RhoG at transcript and proteins are not well understood with respect to inflammatory monocytes and neutrophil. Our data shows that along with Rac-1 GTPase, RhoG transcripts were also upregulated in human neutrophils and monocytes upon stimulation with Lipopolysaccharide (LPS) and chemokines fMLP and MCP-1 in vitro. The presence of extracellular matrix protein such as fibronectin altered the expression of RhoG mRNA in the immune cells activated with LPS. Similarly, blocking antibodies against integrin β1 reduced the expression of RhoG transcripts in vitro. Inflammatory cells isolated from the bone marrow of endotoxemic animals also demonstrated higher levels of RhoG expression along with Rac1 transcript. Thus, the results from this study provide insight into designing therapeutics targeting inflammatory and tissue-damaging inflammatory cells for systemic inflammatory diseases. The third part of my work focused on screening RhoG-based inhibitors and mutants G12V RhoG and Q61L RhoG using the library of a known inhibitor of Rac1. Rho family GTPases serve as molecular switches in numerous cellular processes, and their overexpression is involved in disease conditions. Experimental mutations in RhoG (i.e., RhoGG12V and RhoGQ61L) are shown to dysregulate cell migration. Thus, targeting upstream activators of RhoG, such as guanine nucleotide exchange factors (GEFs), may be an important strategy for inhibiting RhoG activation. In the current study, we have modelled the 3D structure of RhoG with greater accuracy, as confirmed through PROCHECK, ProSA, and Verify3D. Our results indicate that 90.4% of residues are in the Ramachandran plots favoured region, with the Zscore of –6.46, and 87.96% of residues had an average 3D–1D score ≥0.2. Further, we have evaluated and binding dynamics of ten Rac1 inhibitors to investigate their potential to inhibit RhoG by targeting GEFs binding grooves. To this end, the binding energy of the docked complexes of the wild-type (WT) RhoG and its mutant proteins with inhibitor molecules was calculated using the MM/PBSA method. Our docking studies showed that macrolide1 binds efficiently with the GEF site of WT RhoG and the mutants mentioned above. However, an extensive analysis using MD simulations (200 ns) showed that the Rac1-based inhibitor, EHop- 016, and NSC23766 might bind with greater affinity to GEF sites of mutants and WT RhoG. Thus, the results from the study indicate that Rac1 inhibitors have the potential for use as therapeutics in conditions involving dysregulation of RhoG.