Profiling Substrate Specificity of the SUMO Protease Ulp1 by the YESS–PSSC System to Advance the Conserved Mechanism for Substrate Cleavage

Abstract

SUMO modification is a vital post-translational regulation process in eukaryotes, in which the SUMO protease is responsible for the maturation of the SUMO precursor and the deconjugation of the SUMO protein from modified proteins by accurately cleaving behind the C-terminal Gly–Gly motif. To promote the understanding of the high specificity of the SUMO protease against the SUMO protein as well as to clarify whether the conserved Gly–Gly motif is strictly required for the processing of the SUMO precursor, we systematically profiled the specificity of the S. cerevisiae SUMO protease (Ulp1) on Smt3 at the P2–P1↓P1’ (Gly–Gly↓Ala) position using the YESS–PSSC system. Our results demonstrated that Ulp1 was able to cleave Gly–Gly↓ motif-mutated substrates, indicating that the diglycine motif is not strictly required for Ulp1 cleavage. A structural-modeling analysis indicated that it is the special tapered active pocket of Ulp1 conferred the selectivity of small residues at the P1–P2 position of Smt3, such as Gly, Ala, Ser and Cys, and only which can smoothly deliver the scissile bond into the active site for cleavage. Meanwhile, the P1’ position Ala of Smt3 was found to play a vital role in maintaining Ulp1’s precise cleavage after the Gly–Gly motif and replacing Ala with Gly in this position could expand Ulp1 inclusivity against the P1 and P2 position residues of Smt3. All in all, our studies advanced the traditional knowledge of the SUMO protein, which may provide potential directions for the drug discovery of abnormal SUMOylation-related diseases.

Targeting the I7L Protease: A Rational Design for Anti-Monkeypox Drugs?

“…Furthermore, based on our structural analysis, it can be noticed how peptides with higher cleavage rates have better electrostatic complementarities with the binding groove, especially because of the presence of the negatively charged residues in positions P7–P8, which have been previously linked with substrate specificity [ 24 ], and which interacts with an electropositive patch formed by basic residues, such as R124, R172, and R196 ( Figure S14, Supplementary Materials ). Consequently, it is possible to hypothesize that the faster cleavage rates of AGA sequences compared to AGS [ 41 ] may not be related to the P1′ residue, coherently with the tolerated variability in P2–P1′ residues across substrate sequences within the CE clan [ 42 ], yet might be instead related to ancillary residues located before and after the proteolytic cut site, which confer an increased affinity towards the binding groove.…”

Peptidases: Role and Function in Health and Disease

“…The first [12] is focused on the role of legumain in the regulation of biological processes and in the pathogenesis of various malignant and nonmalignant diseases, including cancer, bone remodeling, cardiovascular and cerebrovascular diseases, fibrosis, aging and senescence, and neurodegenerative diseases. The second [13] describes SUMO modification as one of post-translational regulation processes in eukaryotes. In this process, SUMO protease is responsible for the maturation of the SUMO precursor and the deconjugation of the SUMO protein from modified proteins by cleaving behind the C-terminal Gly-Gly motif.…”