Dissertations, Theses, and Capstone Projects

Date of Degree

2-2024

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Shaneen M. Singh

Committee Members

Amy Ikui

Sanjai Kumar Pathak

Diana Murray

Phillip Staniczenko

Subject Categories

Biochemistry, Biophysics, and Structural Biology | Bioinformatics | Cancer Biology | Cell Biology

Keywords

cell cycle, cancer, bioinformatics, Cdc6, NEK10

Abstract

The examination of the cell cycle carries significant implications for the biology, health, and overall existence of all living things. These implications span from the development and growth of these organisms to the aging process and cancer, as well as the potential of stem cell therapies to repair diseases and injuries. Numerous proteins of the cell cycle are essential for cellular division and proliferation and are widely conserved over the course of evolution. In this work, we aimed to investigate the molecular processes of protein-protein interactions in cell cycle proteins, centering on two key players: Cdc6 in budding yeast and NEK10 in humans. In the first part of our study, we investigated Cdc6, an essential replication factor that binds origin recognition complex (ORC) and together these proteins work as a helicase loader. Regulation of Cdc6 is required for proper cell division and is carried out by interacting with proteins such as Cdc4 and Clb2. The lack of structural information was addressed by generation of robust 3D models. Docking algorithms were uses to analyze Cdc6s’s protein-protein interactions. Our results indicate that the Cdc6-Cdc4 interaction displays a dynamic shift between S-phase and mitosis, and that Cdc6’s Intrinsically Disordered Region (IDR) seems to undergo a disorder-to-structure transition upon binding to Clb2. Though numerous papers emphasize the importance of Cdc6’s LxF motif in its binding to Clb2, we revealed that a glutamate residue near the LxF motif appears to play a key role in Clb2 interactions with its substrates. This finding was further extended by mutation studies, calculation of free energy using molecular dynamics, and comparison of Cdc6-Clb2 with other Clb2 interactions.

In the second part of our study, we explored NEK10, a member of the NEK family of proteins. NEK10 participates in ciliogenesis, DNA Damage Repair (DDR), and mitochondrial homeostasis. Studies show NEK10’s participation in various disorders, such bronchiectasis syndrome, breast cancer, and lung cancer. However, NEK10 is largely understudied. To address the lack of structural information for NEK10 we modeled and characterized the full-length NEK10 protein as well as for its homolog in Caenorhabditis elegans (C. elegans), NEKL-4. In addition, we have delineated a comprehensive NEK10 interactome, choosing to focus on two NEK10 interactions with cancer-causing proteins: (1) Mitogen-activated protein kinase kinase kinase 1 (MAP3K1) and (2) Heat Shock Factor Binding Protein 1 (HSPB1). Our findings place both NEK10 – MAP3K1 and NEK10 – HSPB1 interactions in the context of ciliogenesis and provide valuable insights into the underlying mechanism and functional significance of NEK10 in human ciliopathies and cancer, laying the groundwork for delving into the detailed molecular mechanisms of NEK10 interactions with other proteins to further investigate its potential as a therapeutic target.

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