Year: 2024
Type: dissertation
Abstract: Autophagy is a fundamental process of cellular quality control and maintenance that is used to protect cells during natural, healthy aging. Defects in autophagy are implicated in a host of pathologies of aging, notably neurodegenerative diseases. The Rubicon Homology (RH) proteins are a class of autophagy-regulating proteins defined by their RAB7A binding RH domains, consisting of the proteins Rubicon, Pacer, and PLEKHM1. These proteins bidirectionally regulate autophagy, as determined by the phosphorylation state of RAB7A.Here, I demonstrate a mitochondrial autophagy (mitophagy) specific form of regulation for RH family proteins. Activation of PINK1 and Parkin in response to mitochondrial damage initiates a response that includes phosphorylation of RAB7A at Ser72. Rubicon is a RAB7A binding negative regulator of autophagy. The structure of the Rubicon:RAB7A complex suggests that phosphorylation of RAB7A at Ser72 would block Rubicon binding. Indeed, in vitro phosphorylation of RAB7A by TBK1 abrogates Rubicon:RAB7A binding. Pacer, a positive regulator of autophagy, has an RH domain with a basic triad predicted to bind an introduced phosphate. Consistent with this, Pacer-RH binds to phosho-RAB7A but not to unphosphorylated RAB7A. In cells, mitochondrial depolarization reduces Rubicon:RAB7A colocalization whilst recruiting Pacer to phospho-RAB7A–positive puncta. Pacer knockout reduces Parkin mitophagy with little effect on bulk autophagy or Parkin-independent mitophagy. Rescue of Parkin-dependent mitophagy requires the intact pRAB7A phosphate-binding basic triad of Pacer. Together these structural and functional data support a model in which the TBK1-dependent phosphorylation of RAB7A serves as a switch, promoting mitophagy by relieving Rubicon inhibition and favoring Pacer activation.The stability of the Rubicon protein has recently been established as a natural regulator of autophagy flux in a variety of tissues (adipose, hepatic, cardiac, and neuronal), where cells will stabilize or degrade Rubicon as a means of inhibiting or promoting autophagy flux respectively. To develop a novel pharmacological inducer of autophagy, I sought to mimic this form of regulation by creating a small molecule heterobifunctional degrader of Rubicon that colocalizes Rubicon with the E3 ligase Cereblon, inducing ubiquitinylation and subsequent degradation.
Author: Tudorica, Dan Alexandru
Institution: University of California, Berkeley
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7071318664