In‐Plate Chemical Synthesis of Isopeptide‐Linked SUMOylated Peptide Fluorescence Polarization Reagents for High‐Throughput Screening of SENP Preferences. (7th December 2022)

• Record Type: Journal Article
• Authors: Huppelschoten, Yara; Mukhopadhyay, Rishov; Buchardt, Jens; Nielsen, Thomas E.; Vertegaal, Alfred C. O.; Ovaa, Huib; van der Heden van Noort, Gerbrand J.
• Is Part Of: Chembiochem. Volume 24:Number 2(2023)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2022-12-07
• Subjects: assay development -- chemical protein synthesis -- fluorescence polarisation -- proteases -- solid phase peptide synthesis; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.202200601
• Languages: English
• ISSNs: 1439-4227

Abstract

Abstract: Small ubiquitin‐like modifiers (SUMOs) are conjugated to protein substrates in cells to regulate their function. The attachment of SUMO family members SUMO1‐3 to substrate proteins is reversed by specific isopeptidases called SENPs (sentrin‐specific protease). Whereas SENPs are SUMO‐isoform or linkage type specific, comprehensive analysis is missing. Furthermore, the underlying mechanism of SENP linkage specificity remains unclear. We present a high‐throughput synthesis of 83 isopeptide‐linked SUMO‐based fluorescence polarization reagents to study enzyme preferences. The assay reagents were synthesized via a native chemical ligation‐desulfurization protocol between 11‐mer peptides containing a γ‐thiolysine and a SUMO3 thioester. Subsequently, five recombinantly expressed SENPs were screened using these assay reagents to reveal their deconjugation activity and substrate preferences. In general, we observed that SENP1 is the most active and nonselective SENP while SENP6 and SENP7 show the least activity. Furthermore, SENPs differentially process peptides derived from SUMO1‐3, who form a minimalistic representation of diSUMO chains. To validate our findings, five distinct isopeptide‐linked diSUMO chains were chemically synthesized and proteolysis was monitored using a gel‐based read‐out. Abstract : In‐plate chemical synthesis of a set of fluorescent SUMO3‐peptide conjugates using a native chemical ligation‐desulfurization strategy is described. These fluorescence polarization reagents were used to screen and reveal the activity and substrate preferences of five SUMO‐proteases (SENPs) in a high‐throughput manner.