Intrinsically Disordered Protein Regions Encoded by the Diabetes Gene CLEC16A Regulate Mitophagy

Abstract

CLEC16A controls mitochondrial health and is associated with nearly 20 human diseases including diabetes. Unhealthy mitochondria are found in pancreatic β-cells in all types of diabetes, and CLEC16A maintains mitochondrial health in β-cells by clearing damaged mitochondria through mitophagy. However, important functional and structural domains of CLEC16A are unknown. To identify regions critical to CLEC16A function, I focused on a disease-associated CLEC16A variant that lacks both an internal and C terminal region.

Interestingly, the regions disrupted in the CLEC16A disease variant are predicted to overlap with intrinsically disordered protein regions (IDPRs). IDPRs are defined by lack of a fixed 3-dimensional structure. While IDPRs account for almost half of the human proteome and are implicated in human disease, their molecular functions and role in disease remain unclear. My thesis research investigates the structure and function of the putative IDPRs in CLEC16A, and evaluates their role in mitophagy, pancreatic β-cell function, and protection against diabetes.

I experimentally validated that CLEC16A has two intrinsically disordered regions that are disrupted in the CLEC16A disease variant using carbon-detect nuclear magnetic resonance and circular dichroism spectroscopy. Deleting the CLEC16A C terminal IDPR impaired mitochondrial function and mitophagy in pancreatic β-cells in a genetic mouse model. Loss of the CLEC16A C terminal IDPR in vivo impaired β-cell insulin secretion and ultimately caused glucose intolerance. Deleting the CLEC16A C terminal IDPR increased CLEC16A ubiquitination and degradation, which consequently reduced assembly of the mitophagy-regulatory complex comprised of CLEC16A, Nrdp1, and USP8. Interestingly, the ability of the C terminal IDPR to stabilize CLEC16A depends on the proline bias of the IDPR, but not on the amino acid sequence order or charge within the IDPR.

While the C terminal IDPR stabilized CLEC16A, I found that the internal IDPR oppositely controlled CLEC16A turnover. Disrupting the CLEC16A internal IDPR increased CLEC16A stability, observed by mutagenesis and cell-based studies. The CLEC16A internal IDPR depends on the position of its lysine residues to control CLEC16A turnover, which is in part driven by ubiquitination of the IDPR lysine residues by Nrdp1. Disrupting the CLEC16A internal IDPR impaired assembly of the CLEC16A-Nrdp1-USP8 mitophagy complex, due to inability of CLEC16A to bind and ubiquitinate Nrdp1. Together, this work identifies the internal CLEC16A IDPR to regulate reciprocal CLEC16A/Nrdp1 interactions.

My thesis research implicates a disease-associated genetic variant that disrupts CLEC16A IDPRs as a novel contributor to diabetes. This work clarifies how IDPRs within CLEC16A regulate mitophagy and pancreatic β-cell function from a structural, mechanistic, and physiological perspective. In defining key functional regions of CLEC16A, this research provides avenues for future therapeutics to promote CLEC16A function and mitochondrial health in diabetes or other mitochondria-related human diseases.