In vivo localization and identification of SUMOylated proteins in the brain of His ₆ -HA-SUMO1 knock-in mice

Abstract

SUMOylation, an essential posttranslational protein modification, is involved in many eukaryotic cellular signaling pathways. The identification of SUMOylated proteins is difficult, because SUMOylation sites in proteins are hard to predict, SUMOylated protein states are transient in vivo and labile in vitro, only a small substrate fraction is SUMOylated in vivo, and identification tools for natively SUMOylated proteins are rare. To solve these problems, we generated knock-in mice expressing His 6 -HA-SUMO1. By anti-HA immunostaining, we show that SUMO1 conjugates in neurons are only detectable in nuclei and annulate lamellae. By anti-HA affinity purification, we identified several hundred candidate SUMO1 substrates, of which we validated Smchd1, Ctip2, TIF1γ, and Zbtb20 as novel substrates. The knock-in mouse represents an excellent mammalian model for studies on SUMO1 localization and screens for SUMO1 conjugates in vivo.synapse | affinity purification S UMOylation is a conserved posttranslational protein modification in eukaryotes, akin to ubiquitylation, and can affect the localization, interactions, function, or stability of substrates (1). SUMOylation processes participate in many cellular signaling pathways, where they intersect with other posttranslational regulatory processes such as phosphorylation, ubiquitylation, or acetylation. Consequently, many cellular processes, from nuclear transport to neuronal synaptic transmission, are controlled by SUMOylation (2), and key SUMOylation substrates or altered SUMOylation are involved in many diseases, from cancer to neurological disorders (3).Reflecting the prominent nuclear role of SUMOylation, most known SUMOylation substrates are nuclear proteins (1, 2). However, SUMOylation appears to play a much more pervasive regulatory role in cells; recent studies have shown SUMOylation of ion channels, membrane-bound receptors, solute carriers, and mitochondrial or neuronal scaffolding and signaling proteins (4-6), leading to the notion that SUMOylation is a core regulatory process in all cellular subcompartments. This triggered substantial activities to develop tools for the discovery and validation of SUMOylation substrates in cells, which has proven difficult.Mammalian genomes contain four SUMO genes, encoding SUMO1, SUMO2, SUMO3 (7, 8), and SUMO4, of which SUMO4 is poorly characterized (9). SUMO2 and SUMO3 are almost identical, whereas SUMO1 is 50% homologous to SUMO2 and SUMO3. The 3D structure of SUMOs is similar to that of ubiquitin (10), and like ubiquitylation, SUMOylation involves an E1 activating enzyme, E2 conjugating enzymes, and E3 ligases (1,2,7,8).SUMOs are conjugated to lysine residues, often within a ϕKxD/E motif (ϕ, hydrophobic residue; x, any amino acid) (1), but in general SUMO acceptor lysines cannot be predicted, which prevents the identification of SUMO substrates by bioinformatics (1). Further, SUMOylated states of proteins are transient in vivo and labile in vitro because of isopeptidases that revert SUMOylation (1), and usually only a small f...

SUMO1‐conjugation is altered during normal aging but not by increased amyloid burden

“…The SUMO1 KI mouse line has been established as useful tool to study bona fide SUMO1 substrates (Daniel et al., 2017; Tirard & Brose, 2016; Tirard et al., 2012). Here, we assessed SUMO1 conjugation during alterations of proteostasis, as observed during aging and the development of AD‐like pathology.…”

“…Therefore, it is possible that increased amyloid burden alters the subcellular localization of SUMO1 conjugates in extranuclear compartments in general and at synapses in particular. Using biochemical methods and immunostaining, we demonstrated previously that SUMO1 conjugates are mainly localized to the cell nucleus in adult mice aged 8–12 weeks (Daniel et al., 2017; Tirard et al., 2012). Here, we determined whether aging and/or amyloid pathology alters the subcellular distribution of SUMO1 substrates in vivo, for example , by increasing the amount of extranuclear SUMO1 targets, particularly with regard to synapses.…”

“…At first, we performed subcellular fractionation of aged (>36 weeks) KI/AD and KI/WT mouse brains (Figure 2a). Western blot analysis of the various subcellular fractions revealed that the majority of SUMO1 conjugates remained predominantly in the nuclear P1 fraction in both KI/WT and KI/AD mice (Figure 2a, bracket), similar to material from 8‐week‐old animals (Daniel et al., 2017; Tirard et al., 2012). A weak anti‐HA signal was observed in S2 fractions but not in subsequent fractions—in particular not in the synaptic plasma membrane (SPM) fraction—indicating that aging and amyloid burden do not alter the overall distribution of SUMO1 substrates in the brains of aged mice.…”

“…In that context, the use of the His 6 ‐HA‐SUMO1 knock‐in (KI) mouse line in combination with high‐affinity antibodies against the HA tag has proven to be a powerful tool for the identification and localization of endogenous SUMO1 conjugates in vivo , as well as their enrichment by affinity purification (Daniel et al., 2017; Tirard & Brose, 2016; Tirard et al., 2012). The addition of the His 6 ‐HA tag after the start codon of the endogenous Sumo1 locus does not alter the overall pattern of SUMO1 conjugation as visualized by Western blot, the localization of SUMO1 substrates in vivo, nor the global pool of SUMO1 substrates as identified by mass spectrometry (Becker et al., 2013; Daniel et al., 2017; Tirard et al., 2012).…”

“…In that context, the use of the His 6 ‐HA‐SUMO1 knock‐in (KI) mouse line in combination with high‐affinity antibodies against the HA tag has proven to be a powerful tool for the identification and localization of endogenous SUMO1 conjugates in vivo , as well as their enrichment by affinity purification (Daniel et al., 2017; Tirard & Brose, 2016; Tirard et al., 2012). The addition of the His 6 ‐HA tag after the start codon of the endogenous Sumo1 locus does not alter the overall pattern of SUMO1 conjugation as visualized by Western blot, the localization of SUMO1 substrates in vivo, nor the global pool of SUMO1 substrates as identified by mass spectrometry (Becker et al., 2013; Daniel et al., 2017; Tirard et al., 2012). Indeed, lysine acceptor site mutation within SUMO peptides or addition of small tags has been widely used in the SUMO proteomics field with no obvious changes in global SUMOylation capacity (Hendriks & Vertegaal, 2016; Matic et al., 2010), and particularly, the replacement of SUMO by tagged variants is well tolerated in all model organisms tested so far (Kaminsky et al., 2009; Miller, Barrett‐Wilt, Hua & Vierstra, 2010; Panse, Hardeland, Werner, Kuster & Hurt, 2004).…”