SUMO targeting of a stress-tolerant Ulp1 SUMO protease

Abstract

SUMO proteases of the SENP/Ulp family are master regulators of both sumoylation and desumoylation and regulate SUMO homeostasis in eukaryotic cells. SUMO conjugates rapidly increase in response to cellular stress, including nutrient starvation, hypoxia, osmotic stress, DNA damage, heat shock, and other proteotoxic stressors. Nevertheless, little is known about the regulation and targeting of SUMO proteases during stress. To this end we have undertaken a detailed comparison of the SUMO-binding activity of the budding yeast protein Ulp1 (ScUlp1) and its ortholog in the thermotolerant yeast Kluyveromyces marxianus, KmUlp1. We find that the catalytic UD domains of both ScUlp1 and KmUlp1 show a high degree of sequence conservation, complement a ulp1Δ mutant in vivo, and process a SUMO precursor in vitro. Next, to compare the SUMO-trapping features of both SUMO proteases we produced catalytically inactive recombinant fragments of the UD domains of ScUlp1 and KmUlp1, termed ScUTAG and KmUTAG respectively. Both ScUTAG and KmUTAG were able to efficiently bind a variety of purified SUMO isoforms and bound immobilized SUMO1 with nanomolar affinity. However, KmUTAG showed a greatly enhanced ability to bind SUMO and SUMO-modified proteins in the presence of oxidative, temperature and other stressors that induce protein misfolding. We also investigated whether a SUMO-interacting motif (SIM) in the UD domain of KmULP1 that is not conserved in ScUlp1 may contribute to the SUMO-binding properties of KmUTAG. In summary, our data reveal important details about how SUMO proteases target and bind their sumoylated substrates, especially under stress conditions. We also show that the robust pan-SUMO binding features of KmUTAG can be exploited to detect and study SUMO-modified proteins in cell culture systems.

Ginkgolic Acid Rescues Lens Epithelial Cells from Injury Caused by Redox Regulated-Aberrant Sumoylation Signaling by Reviving Prdx6 and Sp1 Expression and Activities

“…Our group and others have reported that Senp1 regulates the activity of many transcription factors, including specificity protein1 (Sp1) in vivo and in vitro [ 9 , 13 , 14 , 17 , 29 ]. The Senp family regulates Sumo homeostasis in eukaryotic cells [ 30 ]. Dysregulation or imbalance between the Sumoylation and deSumoylation processes has been implicated in a wide variety of degenerative diseases [ 4 ].…”

Detection of Rapidly Accumulating Stress-Induced SUMO in Prostate Cancer Cells by a Fluorescent SUMO Biosensor

“…Next, the cells were incubated with 0.1M glycine-HCL (pH 2.0) for 10 seconds. The pH was immediately neutralized with 500μL of 10X SPB (500mM Tris-HCL, pH 8.0 2% NP-40, 1.5M NaCl) (Peek et al, 2018)…”

“…We recently developed a novel SUMO-trapping protein from a thermotolerant yeast, Kluyveromyces marxianus (Km) (Peek et al, 2018). Specifically, we generated a catalytically inactive recombinant SUMO-trapping fragment of the UD domain of Ulp1 (the yeast SUMO protease), termed KmUTAG.…”

“…Inactivation of these SUMO proteases invariably results in an accumulation of SUMO-conjugated proteins as well as SUMO chains. Recent research from our lab indicates that the S. cerevisiae SUMO protease Ulp1 is unable to bind SUMO in the presence of extremely low levels of hydrogen peroxide [1.8mM], underscoring a potential role of SUMO proteases as redox sensors (Peek et al, 2018). One notable exception, the mammalian SUMO protease SENP6 is not inactivated by heat stress and becomes recruited to chromatin (Pinto et al, 2012; Seifert et al, 2015).…”

“…KmUTAG can efficiently bind to a variety of purified SUMO isoforms and immobilized SUMO1 with nanomolar affinity (Kd∼12.8nM). Moreover, KmUTAG has high affinity for SUMO and SUMO-modified proteins even in the presence of oxidative stressors (170mM H 2 O 2 ), reducing agents (5mM TCEP Tris(2-carboxyethyl)phosphine hydrochloride), denaturants (up to 2M UREA), and temperature stress (42°C) that induce protein misfolding (Peek et al, 2018).…”