Recombinant Drosophila mojavensis Ubiquitin carboxyl-terminal hydrolase 36 (Usp36), partial

Introduction

Recombinant Drosophila mojavensis Ubiquitin carboxyl-terminal hydrolase 36 (Usp36), partial, is a truncated form of the deubiquitinating enzyme Usp36 derived from Drosophila mojavensis, a subspecies of fruit flies. This enzyme belongs to the ubiquitin-specific protease (USP) family, which plays critical roles in regulating protein ubiquitination and stability. While direct experimental data on D. mojavensis Usp36 is limited, studies on closely related species (D. melanogaster and D. grimshawi) provide insights into its structure, function, and applications.

Structure and Biochemical Features

Usp36 enzymes, including those from Drosophila, typically consist of a catalytic USP domain (~200–300 residues) responsible for cleaving ubiquitin chains, followed by disordered regions that mediate substrate interactions. The recombinant D. grimshawi Usp36 partial protein (CSB-EP025727DLL-B) expresses a 1–1240 amino acid fragment with a molecular weight of ~140 kDa (calculated from sequence) . Structural studies on D. melanogaster Usp36 reveal a catalytic site (C181 and H439 residues) that facilitates ubiquitin cleavage via a conserved mechanism involving a thioester intermediate .

Production and Purification Methods

High-yield production of Usp36 recombinant proteins is typically achieved using heterologous systems like Pichia pastoris or E. coli. For D. melanogaster Usp36, expression in P. pastoris under methanol-inducible promoters yields up to 210 mg/L of secreted enzyme, purified via nickel-affinity chromatography (Ni-NTA) . A similar approach might apply to D. mojavensis Usp36, though species-specific codon optimization may enhance yields.

Enzymatic Activity and Applications

Usp36 enzymes are specialized for cleaving ubiquitin fusion proteins, enabling the recovery of bioactive peptides (e.g., magainin) without residual tags. D. melanogaster Usp36 efficiently processes ubiquitin–magainin fusions, yielding antimicrobial peptides with potent activity against E. coli . The enzyme’s substrate specificity is mediated by its recognition of the ubiquitin carboxyl terminus, enabling precise cleavage at Gly76 .

Functional Roles in Cellular Regulation

Usp36 homologs regulate cell growth and ubiquitination of key proteins. In D. melanogaster, the nucleolar isoform (dUSP36-D) stabilizes the oncogene dMyc by removing ubiquitin chains, promoting proliferation . Mouse studies reveal that Usp36 is essential for embryonic development, particularly in ribosomal RNA processing and translation . These findings suggest conserved roles for Usp36 in growth regulation across species.

Research Challenges and Future Directions

• Species-Specific Data: Direct studies on D. mojavensis Usp36 are absent, necessitating extrapolation from related species.

• Therapeutic Potential: Targeting Usp36’s deubiquitinating activity could modulate oncogenic pathways or enhance ribosomal biogenesis.

• Structural Studies: Crystallographic analysis of D. mojavensis Usp36 bound to ubiquitin or Fubi (a ubiquitin-like protein) would clarify substrate recognition mechanisms.