Disruption of CAD Oligomerization by Pathogenic Variants

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• CAD is a multi-enzymatic protein crucial for de novo pyrimidine nucleotide biosynthesis.
• CAD forms large hexamers, but their structure and function remain incompletely understood.
• Defects in CAD lead to a severe, difficult-to-diagnose neurometabolic disorder.

Purpose of the Study:

• To develop a cellular assay for identifying defective CAD variants.
• To characterize the functional impact of pathogenic missense mutations in CAD's DHO and ATC domains.
• To elucidate the role of CAD oligomerization in its function and the pathogenesis of associated disorders.

Main Methods:

• Development of a cellular functional assay for CAD variants.
• Biochemical characterization of five pathogenic missense mutations.
• Analysis of protein oligomerization (DHO dimers, ATC trimers).
• Integration of crystal structures and AlphaFold predictions for hexamer modeling.

Main Results:

• All five characterized mutations impaired CAD enzymatic activities.
• Two mutations significantly disrupted the formation of DHO dimers and ATC trimers.
• Modeled hexameric CAD structure highlights the DHO and ATC domains' importance in assembly.
• Correct CAD oligomerization into a supramolecular complex is essential for nucleotide synthesis.

Conclusions:

• Pathogenic mutations affecting CAD assembly into functional hexamers are linked to neurometabolic disorders.
• Understanding CAD structure and assembly provides insights into its stability and function.
• The study reveals the critical role of supramolecular complex formation for CAD's enzymatic activity.