Small ubiquitin-like modifier (SUMO) modification of E1 Cys domain inhibits E1 Cys domain enzymatic activity

Although it is well established that ubiquitin-like modifications are tightly regulated, it has been unclear how their E1 activities are controlled. In this study, we found that the SAE2 subunit of the small ubiquitin-like modifier (SUMO) E1 is autoSUMOylated at residue Lys-236, and SUMOylation was catalyzed by Ubc9 at several additional Lys residues surrounding the catalytic Cys-173 of SAE2. AutoSUMOylation of SAE2 did not affect SUMO adenylation or formation of E1·SUMO thioester, but did significantly inhibit the transfer of SUMO from E1 to E2 and overall SUMO conjugations to target proteins due to the altered interaction between E1 and E2. Upon heat shock, SUMOylation of SAE2 was reduced, which corresponded with an increase in global SUMOylation, suggesting that SUMOylation of the Cys domain of SAE2 is a mechanism for "storing" a pool of E1 that can be quickly activated in response to environmental changes. This study is the first to show how E1 activity is controlled by post-translational modifications, and similar regulation likely exists across the homologous E1s of ubiquitin-like modifications.     

Thyroid Hormone Receptor Isoform-specific Modification by Small Ubiquitin-like Modifier (SUMO) Modulates Thyroid Hormone-dependent Gene Regulation

Thyroid hormone receptor (TR) α and β mediate thyroid hormone action at target tissues. TR isoforms have specific roles in development and in adult tissues. The mechanisms underlying TR isoform-specific action, however, are not well understood. We demonstrate that posttranslational modification of TR by conjugation of small SUMO to TRα and TRβ plays an important role in triiodothyronine (T3) action and TR isoform specificity. TRα was sumoylated at lysines 283 and 389, and TRβ at lysines 50, 146, and 443. Sumoylation of TRβ was ligand-dependent, and sumoylation of TRα was ligand-independent. TRα-SUMO conjugation utilized the E3 ligase PIASxβ and TRβ-SUMO conjugation utilized predominantly PIAS1. SUMO1 and SUMO3 conjugation to TR was important for T3-dependent gene regulation, as demonstrated in transient transfection assay and studies of endogenous gene regulation. The functional role of SUMO1 and SUMO3 in T3 induction in transient expression assays was closely matched to the pattern of TR and cofactor recruitment to thyroid hormone response elements (TREs) as determined by ChIP assays. SUMO1 was required for the T3-induced recruitment of the co-activator CREB-binding protein (CBP) and release of nuclear receptor co-repressor (NCoR) on a TRE but had no significant effect on TR DNA binding. SUMO1 was required for T3-mediated recruitment of NCoR and release of CBP from the TSHβ-negative TRE. SUMO3 was required for T3-stimulated TR binding to the TSHβ-negative TRE and recruitment of NCoR. These findings demonstrate that conjugation of SUMO to TR has a TR-isoform preference and is important for T3-dependent gene induction and repression.     

Identification of Biochemically Distinct Properties of the Small Ubiquitin-related Modifier (SUMO) Conjugation Pathway in Plasmodium falciparum

Small ubiquitin-related modifiers (SUMOs) are post-translationally conjugated to other proteins and are thereby essential regulators of a wide range of cellular processes. Sumoylation, and enzymes of the sumoylation pathway, are conserved in the malaria causing parasite, Plasmodium falciparum. However, the specific functions of sumoylation in P. falciparum, and the degree of functional conservation between enzymes of the human and P. falciparum sumoylation pathways, have not been characterized. Here, we demonstrate that sumoylation levels peak during midstages of the intra-erythrocyte developmental cycle, concomitant with hemoglobin consumption and elevated oxidative stress. In vitro studies revealed that P. falciparum E1- and E2-conjugating enzymes interact effectively to recognize and modify RanGAP1, a model mammalian SUMO substrate. However, in heterologous reactions, P. falciparum E1 and E2 enzymes failed to interact with cognate human E2 and E1 partners, respectively, to modify RanGAP1. Structural analysis, binding studies, and functional assays revealed divergent amino acid residues within the E1-E2 binding interface that define organism-specific enzyme interactions. Our studies identify sumoylation as a potentially important regulator of oxidative stress response during the P. falciparum intra-erythrocyte developmental cycle, and define E1 and E2 interactions as a promising target for development of parasite-specific inhibitors of sumoylation and parasite replication.     

Modulation of transcriptional corepressor activity of prospero-related homeobox protein (Prox1) by SUMO modification

Prospero-related homeobox protein (Prox1) plays essential roles in the development of many tissues and organs. In the present study, we show that Prox1 is modified by the small ubiquitin-like protein SUMO-1 in cultured cells. Mutation analysis identified at least four potential sumoylation sites within the repression domain of Prox1. Our data indicate that sumoylation of Prox1 reduces its interaction with HDAC3 and as a result downregulates its corepressor activity. These findings suggest that sumoylation may serve as a novel mechanism for the regulation of Prox1’s corepressor activity.

Structured summary

MINT-6787569:

PROX1 (uniprotkb:Q92786) physically interacts (MI:0218) with HDAC3 (uniprotkb:O15379) by anti tag coimmunoprecipitation (MI:0007)

MINT-6787767:

PROX1 (uniprotkb:Q92786) physically interacts (MI:0218) with SUMO-1 (uniprotkb:P63165) by anti tag coimmunoprecipitation (MI:0007)

     

Small Ubiquitin-like Modifier (SUMO) Isoforms and Conjugation-independent Function in DNA Double-strand Break Repair Pathways

Small ubiquitin-like modifier (SUMO) proteins act in DNA double-strand break (DSB) repair, but the pathway specificity of the three major isoforms has not been defined. In experiments in which we depleted the endogenous SUMO protein by RNAi, we found that SUMO1 functioned in all subpathways of either homologous recombination (HR) or non-homologous end joining (NHEJ), whereas SUMO2/3 was required for the major NHEJ pathway, called conservative NHEJ, but dispensable in other DSB repair pathways. To our surprise, we found that depletion of UBC9, the unique SUMO E2 enzyme, had no effect in HR or alternative NHEJ (Alt-NHEJ) but was required for conservative NHEJ. Consistent with this result, both non-conjugatable mutant and wild-type SUMO1 proteins functioned similarly in HR and Alt-NHEJ. These results detail the functional roles of specific SUMO isoforms in DSB repair in mammalian cells and reveal that SUMO1 functions in HR or Alt-NHEJ as a free protein and not as a protein conjugate.     

Dual Regulatory Functions of SUFU and Targetome of GLI2 in SHH Subgroup Medulloblastoma

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E2-mediated Small Ubiquitin-like Modifier (SUMO) Modification of Thymine DNA Glycosylase Is Efficient but Not Selective for the Enzyme-Product Complex

Thymine DNA glycosylase (TDG) initiates the repair of G·T mismatches that arise by deamination of 5-methylcytosine (mC), and it excises 5-formylcytosine and 5-carboxylcytosine, oxidized forms of mC. TDG functions in active DNA demethylation and is essential for embryonic development. TDG forms a tight enzyme-product complex with abasic DNA, which severely impedes enzymatic turnover. Modification of TDG by small ubiquitin-like modifier (SUMO) proteins weakens its binding to abasic DNA. It was proposed that sumoylation of product-bound TDG regulates product release, with SUMO conjugation and deconjugation needed for each catalytic cycle, but this model remains unsubstantiated. We examined the efficiency and specificity of TDG sumoylation using in vitro assays with purified E1 and E2 enzymes, finding that TDG is modified efficiently by SUMO-1 and SUMO-2. Remarkably, we observed similar modification rates for free TDG and TDG bound to abasic or undamaged DNA. To examine the conjugation step directly, we determined modification rates (k_obs) using preformed E2∼SUMO-1 thioester. The hyperbolic dependence of k_obs on TDG concentration gives k_max = 1.6 min⁻¹ and K_1/2 = 0.55 μm, suggesting that E2∼SUMO-1 has higher affinity for TDG than for the SUMO targets RanGAP1 and p53 (peptide). Whereas sumoylation substantially weakens TDG binding to DNA, TDG∼SUMO-1 still binds relatively tightly to AP-DNA (K_d ∼50 nm). Although E2∼SUMO-1 exhibits no specificity for product-bound TDG, the relatively high conjugation efficiency raises the possibility that E2-mediated sumoylation could stimulate product release in vivo. This and other implications for the biological role and mechanism of TDG sumoylation are discussed.     

The small ubiquitin-like modifier (SUMO) protein modification system in Arabidopsis. Accumulation of SUMO1 and -2 conjugates is increased by stress

Small ubiquitin-like modifier (SUMO) is a member of the superfamily of ubiquitin-like polypeptides that become covalently attached to various intracellular target proteins as a way to alter their function, location, and/or half-life. Here we show that the SUMO conjugation system operates in plants through a characterization of the Arabidopsis SUMO pathway. An eight-gene family encoding the SUMO tag was discovered as were genes encoding the various enzymes required for SUMO processing, ligation, and release. A diverse array of conjugates could be detected, some of which appear to be SUMO isoform-specific. The levels of SUMO1 and -2 conjugates but not SUMO3 conjugates increased substantially following exposure of seedlings to stress conditions, including heat shock, H(2)O(2), ethanol, and the amino acid analog canavanine. The heat-induced accumulation could be detected within 2 min from the start of a temperature upshift, suggesting that SUMO1/2 conjugation is one of the early plant responses to heat stress. Overexpression of SUMO2 enhanced both the steady state levels of SUMO2 conjugates under normal growth conditions and the subsequent heat shock-induced accumulation. This accumulation was dampened in an Arabidopsis line engineered for increased thermotolerance by overexpressing the cytosolic isoform of the HSP70 chaperonin. Taken together, the SUMO conjugation system appears to be a complex and functionally heterogeneous pathway for protein modification in plants with initial data indicating that one important function may be in stress protection and/or repair.