Divergent N-terminal sequences of a deubiquitinating enzyme modulate substrate specificity

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Ubiquitin-specific processing proteases (UBPs) possess a conserved catalytic core domain.
• UBPs feature divergent N-terminal sequences influencing subcellular localization.
• Two testis-specific UBP isoforms, UBP-t1 and UBP-t2, were previously identified with distinct N termini.

Purpose of the Study:

• To investigate the impact of N-terminal extensions on the biochemical functions of UBPs.
• To determine if N-terminal domains modulate the enzymatic activity and specificity of the UBP core.
• To compare the isopeptidase activity of UBP-t1, UBP-t2, and the UBP core domain.

Main Methods:

• Expression of UBP-t1, UBP-t2, and UBP core in Escherichia coli.
• Assays to measure the cleavage of branched triubiquitin and linear diubiquitin.
• Analysis of ubiquitin removal from testis ubiquitinated proteins.

Main Results:

• All three UBP isoforms demonstrated isopeptidase activity, cleaving branched triubiquitin significantly faster than linear diubiquitin.
• N-terminal extensions of UBP-t1 and UBP-t2 inhibited the generation of free ubiquitin from peptide-linked substrates.
• The N-terminal extension of UBP-t2 enhanced the cleavage of branched triubiquitin, while the UBP core domain showed faster ubiquitin removal from testis proteins compared to the full-length isoforms.

Conclusions:

• UBP enzymes possess a catalytic core domain whose activity and specificity are modulated by N-terminal extensions.
• Divergent N termini play a crucial role in regulating UBP localization and function.
• These findings highlight the functional diversity within the UBP family, driven by N-terminal variations.