Asian Research Thesis Index

Abstract

Cell proliferation is regulated by controlling the abundance of specific proteins due to surrounding positive and negative signals. Ubiquitin-proteasome system is a principal mechanism for regulating protein turnover through various cellular processes. In mammals, ubiquitination regulates phase transitions throughout cell cycle by mediating the activity of cell cycle regulatory proteins via principal E3 ubiquitin ligase; SCF (SKP, Cullin, F-box containing complex). The F-box protein βTrCP1 (F-box/WD repeatcontaining protein 1A) contributes to SCF complex by binding with multiple target proteins and bringing them in close to E2-protein. Despite the widely updated contemporary range of βTrCP1-specific substrates, their list is undoubtedly incomplete.The current interest in the identification and characterization of βTrCP1 substrates necessitates a promising approach with broad structural constraints of WD40 potential binding sites. Here, we employed in silico integrative approach to identify putative novel substrates of βTrCP1. Through screened degradation motif (DSGXXS) for entire human proteome and comparative substrate binding analysis of βTrCP1, we identified 344 substrates, sharing high sequence similarity with the consensus motif. Subsequent filtering on the basis of functional annotation and clustering resulted in the isolation of hits having clear roles in various cancer types. These substrates were phosphorylated at the Ser residues (Ser14 and Ser18) of conserved motif. A comprehensive and thorough analysis of βTrCP1-phosphopeptide association indicated residual contributions located at the upper face of β-propeller. Evidently, upon binding to phosphopeptides, central channel of βTrCP1 attains a more open conformation to assist substrate binding. To elaborate the oncogenic role of βTrCP1, SKP1 (S-Phase Kinase Associated Protein 1)-βTrCP1-CDH6 (Cadherin 6) ternary complex was docked against CUL1-RBX1 (Cullin1 and RING-box protein 1 complex) and the acquired model exactly resembled with the previously characterized SKP1-βTrCP1-β-catenin model. Overall, a deeper understanding of substrate targeting mechanisms coupled with structural knowledge of βTrCP1 and associated proteins will be useful in designing novel targets for cancer therapeutics.  Abstract  Evaluation of Structure-based Functional diversity of E3-ligases in parallel to their carcinogenic potential characterization through small molecule inhibitors xiv  Controlled ubiquitin (Ub)-mediated protein degradation is essential for various cellular processes. Glioma-associated oncogene (GLI) family regulates the transcriptional events of the sonic hedgehog pathway genes that are implicated in almost one fourth of human tumors. GLI3 phosphorylation by Ser/Thr kinases is a primary factor for their transcriptional activity that incurs the formation of both GLI3 repressor and activator forms. GLI3 processing is triggered in an Ub-dependent manner via SCFβTrCP1 complex; however, structural characterization, mode of action based on sequence of phosphorylation signatures and induced conformational readjustments remain elusive. Here, through structural assessment and molecular dynamics simulation assays, we investigated relative binding configuration of GLI3 phosphopeptides against βTrCP1. A comprehensive and thorough analysis demarcated GLI3 presence in the binding cleft shared by inter-bladed binding grooves of β-propeller. Our results revealed the involvement of all seven WD40 repeats of βTrCP1 in GLI3 interaction. Conversely, GLI3 phosphorylation pattern at primary protein kinase A (PKA) sites and secondary casein kinase 1 (CK1) or glycogen synthase kinase 3 (GSK3) sites was carefully evaluated. Our results indicated that GLI3 processing depends on the 19 phosphorylation sites (849, 852, 855, 856, 860, 861, 864, 865, 868, 872, 873, 876, 877, 880, 899, 903, 906, 907 and 910 positions) by a cascade of PKA, GSK3β and CSKI kinases. The presence of a sequential phosphorylation in the binding induction of GLI3 and βTrCP1 may be a hallmark to authenticate GLI3 processing. We speculate that mechanistic information of the individual residual contributions through structure-guided approaches may be pivotal for the rational design of specific and more potent inhibitors against activated GLI3 with a special emphasis on the anticancer activity.  The critical role of βTrCP1 in cancer development makes it a discerning target for the development of small drug like molecules. Currently, no inhibitor exists that is able to target its substrate binding site. Through molecular docking and dynamics simulation assays, we explored the comparative binding pattern of βTrCP1-WD40 domain with ACV (acyclovir) and its phospho-derivatives (ACVMP, ACVDP and ACVTP). Consequently, through principal component analysis, βTrCP1-ACVTP was found to be more stable complex by obscuring a reduced conformational space than other systems. Thus based on the residual contributions and hydrogen bonding pattern, ACVTP was  Abstract  Evaluation of Structure-based Functional diversity of E3-ligases in parallel to their carcinogenic potential characterization through small molecule inhibitors xv  considered as a noteworthy inhibitor that demarcated binding in the cleft formed by βTrCP1-WD40 specific β-propeller. The outcomes of this study may provide a platform for the rational design of specific and potent inhibitor against βTrCP1, with special emphasis on anticancer activity. Cullin-RING E3 ubiquitin ligases (CRLs) are attractive therapeutic targets as they regulate diverse biological processes important for cancer cell survival by conferring substrate selectivity for ubiquitination and degradation. Given the complexity of CRL complexes, steps toward the structure-based design of small-molecule inhibitors to modulate their activity have remained elusive. In this study, we explored the structural assembly and interaction details of closely related CUL scaffolds (CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5 and CUL7) with RBX1 to screen potent small molecules against CRLs. The RING-Box (RBX1 and RBX2) proteins heterodimerize with CULs and dynamically facilitate the ubiquitination process. The docked complexes of conserved CUL C-terminal domains exhibited a common RBX1 binding pattern through the incorporation of intermolecular β-sheet and α/β core, stabilized by hydrophobic contacts. The comparative binding pattern analysis of CUL-RBX1 interfaces revealed a unique structural motif (VLYRLWLN) that directs the binding of RBX1 N-terminal β-strand. Through reinvigorating the subtle structural dynamics of bound complexes and application of structure-based drug design approaches, we proposed a set of inhibitors which could be further optimized to target CRL activity. One reference compound (C64) was extensively characterized for selective binding at the RBX1-binding grooves/VLYRLWLN of CUL1-7. We speculate that mechanistic information of the individual residual contributions through structure-guided approaches could be pivotal for the rational design of more promising and active drug candidates against CRLs. Another E3-ligase JADE1 (Jade Family PHD Finger 1) is a candidate of Renal cancer that is the major cause of mortality.JADE1 is stabilized by von Hippel-Lindau (pVHL) interaction through its plant homeodomains. JADE1 acts as a renal tumor suppressor that promotes β-catenin ubiquitination and degradation by inhibiting canonical WNT signalling. Current study focuses on the structural characterization of reported missense mutations in pVHL through in silico approaches. The predicted 3-dimensional structures  Abstract  Evaluation of Structure-based Functional diversity of E3-ligases in parallel to their carcinogenic potential characterization through small molecule inhibitors xvi  of pVHLWT, pVHLY98H, pVHLY112H, pVHLL118P and pVHLR167W were subjected to binding analysis against JADE1 through molecular docking and simulation assays. In all cases, JADE1 binding was observed at the β-domain, except pVHLL118P that exhibited binding with JADE1 through its α-domain. Our results signify that JADE1 stabilization is induced by pVHL α-domain, while β-domain is required for JADE1 binding. pVHL binding was mediated through β1 and β2-strands against the concave surface of the JADE1-PHD domain. The pVHL-JADE1 complex was evaluated to scrutinize the βcatenin-binding interface, which suggested the contribution of both α and β-domains of pVHL in β-catenin binding. The eleven-residue (Tyr30-Thr40) β-catenin segment exhibited association in a bipartite manner with pVHL-JADE1 complex. The presented model depicts a pVHL-JADE1 interface for the cooperative regulation of β-catenin binding. We propose that reduced JADE1 stabilization in case of pVHLL118P and pVHLR167W may correlate with the increased activity of β-catenin that may lead to renal cancer through β-catenin de-repression. Overall, β-catenin targeting mechanism coupled with the structural knowledge of JADE1-pVHL complex will provide better understanding of renal cancer pathogenesis." xml:lang="en_US

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