title: Characterizing substrate selectivity of ubiquitin C-terminal hydrolase-L3 using engineered α-linked ubiquitin substrates.
abstract: The ubiquitin-proteasome system (UPS) is highly complex and entails the concerted actions of many enzymes that function to ubiquitinate proteins targeted to the proteasome as well as enzymes that remove and recycle ubiquitin for additional rounds of proteolysis. Ubiquitin C-terminal hydrolase-L3 (UCH-L3) is a human cytosolic deubiquitinase whose precise biological function is not known. It is believed to hydrolyze small peptides or chemical adducts from the C-terminus of ubiquitin that may be remnant from proteasomal processing. In addition, UCH-L3 is a highly effective biotechnological tool that is used to produce small or unstable peptides/proteins recalcitrant to production in Escherichia coli expression systems. Previous research, which explored the substrate selectivity of UCH-L3, demonstrated a substrate size limitation for proteins/peptides expressed as α-linked C-terminal fusions to ubiquitin and also suggested that an additional substrate property may affect UCH-L3 hydrolysis [ Larsen , C. N. et al. (1998) Biochemistry 37 , 3358 - 3368 ]. Using a series of engineered protein substrates, which are similar in size yet differ in secondary structure, we demonstrate that thermal stability is a key factor that significantly affects UCH-L3 hydrolysis. In addition, we show that the thermal stabilities of the engineered substrates are not altered by fusion to ubiquitin and offer a possible mechanism as to how ubiquitin affects the structural and unfolding properties of natural in vivo targets.
authors.affiliation: Department of Chemistry and Biochemistry, San Diego State University , 5500 Campanile Drive, San Diego, California 92182-1030, United States.
authors.forename: Mario F
authors.initials: MF
authors.lastname: Navarro
authors.forename: Lisa
authors.initials: L
authors.lastname: Carmody
authors.forename: Octavio
authors.initials: O
authors.lastname: Romo-Fewell
authors.forename: Melissa E
authors.initials: ME
authors.lastname: Lokensgard
authors.forename: John J
authors.initials: JJ
authors.lastname: Love
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase
chemical_list: D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; C509806:UCHL3 protein, human; D043222:Ubiquitin Thiolesterase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex