Recombinant Danio rerio NEDD8-activating enzyme E1 catalytic subunit (uba3)

Basic Research Questions

• What is the function of UBA3 in the NEDD8 pathway?

  UBA3 serves as the catalytic subunit of the dimeric UBA3-NAE1 E1 enzyme that initiates the neddylation cascade. This post-translational modification process involves NEDD8 conjugation onto target proteins. Specifically, UBA3 activates NEDD8 through a multi-step process: first adenylating NEDD8's C-terminal glycine residue with ATP, then linking this residue to the side chain of the catalytic cysteine (forming a NEDD8-UBA3 thioester and releasing AMP), and finally transferring NEDD8 to the catalytic cysteine of UBE2M . This process is crucial for the regulation of cullin-based E3 ubiquitin ligase activity and subsequent protein degradation pathways.

• Why is zebrafish (Danio rerio) used as a model organism for UBA3 research?

  Zebrafish serves as an excellent model organism for UBA3 research for several reasons:

  • Genetic similarity: Zebrafish shares 70% of its genes with humans and possesses more than 84% of the genes that cause genetic disease in humans

  • Transparent embryos: Allow for direct visualization of developmental processes and protein interactions

  • Rapid development: Most major organs form within 24 hours, enabling efficient experimental timelines

  • External fertilization: Facilitates embryo manipulation and treatment

  • High reproductive capacity: Can produce up to 300 embryos every 2-3 days, enabling large-scale studies

  These characteristics make zebrafish particularly valuable for studying the developmental roles of the NEDD8 pathway and UBA3's function in vertebrate systems.

• What expression systems are commonly used for producing recombinant Danio rerio UBA3?

  Several expression systems are utilized for recombinant Danio rerio UBA3 production, each with specific advantages:

  Expression System	Advantages	Applications	Representative Products
  Yeast	Post-translational modifications, high yield	Raw material for antibody production, enzymatic assays	UBA3 (AA 1-462) with His tag
  E. coli	Cost-effective, high yield, simplicity	Structural studies, in vitro assays	Various UBA3 constructs with purification tags
  Mammalian cells (HEK-293)	Native folding, complex modifications	Interaction studies, cell-based assays	UBA3 with Myc-DYKDDDDK Tag

  The selection depends on experimental requirements, with yeast-expressed UBA3 being particularly common for zebrafish UBA3 production .

• What are the key structural characteristics of recombinant Danio rerio UBA3?

  Recombinant Danio rerio UBA3 (AA 1-462) has several notable structural features:

  • Molecular weight: Approximately 53 kDa

  • Active site: Contains a critical catalytic cysteine residue essential for thioester bond formation with NEDD8

  • ATP binding domain: Facilitates adenylation of NEDD8

  • NAE1 binding interface: Enables formation of the functional heterodimeric E1 enzyme

  • The protein sequence contains NEDD8 recognition motifs and domains required for E2 enzyme recruitment

  Crystal structure studies of the human ortholog indicate that mutations near the catalytic site (such as the I310N and Y352H mutations identified in resistant cell lines) can affect substrate binding and enzyme function .