PROTEINOPATHIES, PROTEOTOXICITY AND TRIAGING IN NEURODEGENERATIVE DISORDERS

Abstract

Lysine-based post-translational modifications (PTMs) such as acylation, acetylation, deamination, methylation, SUMOylation, and ubiquitination have proven to be major regulators of gene expression, chromatin structure, protein stability, protein-protein interaction, protein degradation, and cellular localization. However, besides all the PTMs, ubiquitination stands as the second most common PTM after phosphorylation that is involved in the etiology of neurodegenerative diseases (NDDs) namely, Alzheimer’s disease (AD) and Parkinson’s disease (PD). NDDs are characterized by the accumulation of misfolded protein aggregates in the brain that lead to disease-related gene mutation and irregular protein homeostasis. The ubiquitin-proteasome system (UPS) is in charge of degrading these misfolded proteins, which involve an interplay of E1, E2, E3, and deubiquitinase enzymes. Impaired UPS has been commonly observed in NDDs and E3 ligases are the key members of the UPS, thus, dysfunction of the same can accelerate the neurodegeneration process. Therefore, this study explores the impact of point mutation on BRCA1, BARD1, RNF8, and RNF168. All these E3 ligases are involved in the DNA repair mechanism following a complex ubiquitination cascade. The first study concludes BRCA1, a potential E3 ligase common in both AD and PD, and RING domain mutation at sites K32 and K45 possibly disturbs its interaction with its E2, Ube2k.BRCA1 is mostly found in a heterodimeric state, tightly bonded with BARD1. BRCA1 operates its DNA repair mechanism primarily in the nucleus and any DNA repair deficiencies result in neurodegeneration, particularly AD. Therefore, the second study investigates the impact of BARD1 mutation on the binding affinity of BRCA1 with UbcH5c, H2A, and Tau. The result concludes, L44A mutation significantly destabilizes BARD1 and its interaction with BRCA1, although no significant deviations were observed in BRCA1-BARD1 vii (L44A) docked with UbcH5c. However, destabilization of BRCA1-BARD1(L44A) with H2A and stabilization with Tau are noted, underscoring the importance of the RING domain mutation of BARD1 on BRCA1 and its interactions with UbcH5c, H2A, and Tau. However, RNF8 and RNF168 are crucial E3 ligases that are recruited first to the site of DNA damage. RNF8 initiates the conjugation of ubiquitin with H2A and H2AX, subsequently, RNF168 is employed to amplify the K-63-linked ubiquitin chain. These two E3 ligases are necessary for the downstream signaling where 53BP1 and BRCA1 are recruited. RNF168 directs 53BP1 involvement in non-homologous end joining (NHEJ) but is unable to save BRCA1 which is involved in homologous recombination (HR); therefore, RNF8 plays a huge role in the recruitment of BRCA1. Thus, the third and fourth studies are on assessing the impact of mutagenesis of RNF8 and RNF168 on its binding with E2 conjugating enzyme, UBE2N and UbcH5c, respectively. The RING domain of RNF8 was taken and its interacting sites with UBE2N were carefully studied. The result depicted that I405, S407, E408, E429, R433, P438, I439, and R441 could be possible sites that affect the binding of RNF8 with UBE2N and thereby hampering its E3 ligase activity. Likewise, mutagenesis on RNF168 at C31G and C50G stood as potential sites that could hamper its E3 ligase activity. However, this study was further continued where warheads of PROTACs were screened and docked with these mutants so that they could be marked for degradation by UPS. The inclusive result inferred that Warhead 4 (Ligand 4) (UniProt ID: Q86U86) and Warhead 13 (Ligand 11) (UniProt ID: P51531) were potential ligands that represented better binding and stability than the wild-type structure. C31G with warhead 11 showed exceptional binding and stability. Overall, the study tries to identify potential mutants that can affect the binding affinity of E3 ligases, BRAC1, BARD1, RNF8, and RNF168 with their corresponding E2-conjugating enzymes, disturbing its E3 ligase activity.