Date Approved

4-2023

Document Type

Dissertation

Degree Name

PhD in Molecular Cell Biology and Neuroscience

Department

Molecular Biology

College

Graduate School of Biomedical Sciences

First Advisor

Randy Strich, PhD

Committee Member 1

Brian Weiser

Committee Member 2

Mikhail Anikin, PhD

Committee Member 3

Natalia Shcherbik, PhD

Committee Member 4

Kai-Ti Chang, PhD

Subject(s)

Cyclin C; DNA Damage; Apoptosis; Mitochondrial Dynamics; Transcription, Genetic; Humans; Yeasts

Disciplines

Cell Biology | Fungi | Laboratory and Basic Science Research | Medicine and Health Sciences | Molecular Biology | Molecular Genetics

Abstract

For normal cellular function, exogenous signals must be interpreted and careful coordination must take place to ensure desired fates are achieved. Mitochondria are key regulatory nodes of cellular fate, undergoing fission/fusion cycles depending on the needs of the cell, and help mediate cell death fates. The CKM or Cdk8 kinase module, is composed of cyclin C (CC), Cdk8, Med12/12L, and Med13/13L. The CKM controls RNA polymerase II, acting as a regulator of stress-response and growth-control genes. Following stress, CC translocates to the mitochondria and interacts with both fission and iRCD apoptotic mediators. We hypothesize that CC represents a key mediator, linking transcription to mitochondrial fission and RCD. A more in-depth analysis of the roles of CC and the protein interactions that mediate them encompasses the focus of this dissertation.

To mediate individual functions, CC uses distinct binding partners. We revealed the presence two separable/discrete cyclin box domains. To determine the residues mediating these functions, rigid body protein-protein docking simulations were performed using human cyclin C, Drp1, and Bax. These analyses revealed specific residues which support distinct functions of the CB1 and CB2 domains. Results indicate that modeled Bax-interacting residues are concentrated to the first half of the CB2 domain, while Drp1-interacting residues span the entirety of the CB2 domain. Interestingly, we determined that CC contains a unique BH2-like domain, normally only found in Bcl-2 protein family members, which appears to mediate interactions with Bax.

Results from human protein modeling simulations were then applied to yeast homologous proteins. As presented here, yeast studies have confirmed residues that mediate interaction between CC and fission machinery. The results support the model that CB1 and CB2 are distinct, mediating independent functionalities. We suggest a model that CC possesses three distinct interaction domains and acts to bridge fission and apoptotic machinery, either in a mutually exclusive or trimeric manner. In conclusion, CC is shown to mediate each of its unique functions through distinct interacting residues and interfaces. With CC implicated in many human disorders, this will serve as a tool to study disease pathogeneses and treatments, taking into account unique interfaces governing the tripartite functions.

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