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. 2008;3(12):e4032.
doi: 10.1371/journal.pone.0004032. Epub 2008 Dec 24.

Human polycomb 2 protein is a SUMO E3 ligase and alleviates substrate-induced inhibition of cystathionine beta-synthase sumoylation

Nitish Agrawal  1 , Ruma Banerjee
Affiliations

"VSports最新版本" Human polycomb 2 protein is a SUMO E3 ligase and alleviates substrate-induced inhibition of cystathionine beta-synthase sumoylation

Nitish Agrawal et al. PLoS One. 2008.

Abstract

Human cystathionine beta-synthase (CBS) catalyzes the first irreversible step in the transsulfuration pathway and commits homocysteine to the synthesis of cysteine. Mutations in CBS are the most common cause of severe hereditary hyperhomocysteinemia. A yeast two-hybrid approach to screen for proteins that interact with CBS had previously identified several components of the sumoylation pathway and resulted in the demonstration that CBS is a substrate for sumoylation. In this study, we demonstrate that sumoylation of CBS is enhanced in the presence of human polycomb group protein 2 (hPc2), an interacting partner that was identified in the initial yeast two-hybrid screen. When the substrates for CBS, homocysteine and serine for cystathionine generation and homocysteine and cysteine for H(2)S generation, are added to the sumoylation mixture, they inhibit the sumoylation reaction, but only in the absence of hPc2. Similarly, the product of the CBS reaction, cystathionine, inhibits sumoylation in the absence of hPc2. Sumoylation in turn decreases CBS activity by approximately 28% in the absence of hPc2 and by 70% in its presence. Based on these results, we conclude that hPc2 serves as a SUMO E3 ligase for CBS, increasing the efficiency of sumoylation. We also demonstrate that gamma-cystathionase, the second enzyme in the transsulfuration pathway is a substrate for sumoylation under in vitro conditions VSports手机版. We speculate that the role of this modification may be for nuclear localization of the cysteine-generating pathway under conditions where nuclear glutathione demand is high. .

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effect of hPc2 on sumoylation of human CBS.
A. The in vitro sumoylation reaction was performed as described under Methods and SUMO-1 antibody was used to detect the presence of sumoylated CBS. Lanes 1 and 2 show sumoylation reactions that were conducted in the absence (lane 1) and presence (lane 2) of hPc2. B and C. Equal loading controls for CBS in the two reaction mixtures. The CBS concentration prior to the start of the sumoylation reaction was identical in both lanes as detected by Coomassie blue staining (B) or by Western blot analysis using CBS antibody (C).
Figure 2
Figure 2. Effect of substrates on the sumoylation of human CBS.
Western blot analysis using CBS-antibody of sumoylation reaction mixtures containing the substrates for CBS in the absence (A) or presence (B) of the allosteric activator, S-adenosylmethionine (AdoMet). (C) The effect of hPc2 on sumoylation of human CBS in the presence of substrates.
Figure 3
Figure 3. Effect of the H2S generating substrates, cysteine and homocysteine, and the CBS reaction product, cystathionine, on sumoylation in the presence and absence of hPc2.
The Western blot was detected using SUMO-1 antibody.
Figure 4
Figure 4. Human CSE is a target of in vitro sumoylation.
Sumoylation of CSE was conducted as described under Methods in the presence or absence of 5 mM cystathionine. The Western blot was detected using SUMO antibody. The band with a molecular mass of ∼16 kDa represents SUMO-1 present in the reaction mixture.
Figure 5
Figure 5. Structures of human CBS and CSE showing locations of potential SUMO modification sites.
A. Locations of K211 and K269 in the canonical and noncanonical SUMO modification sites respectively in human CBS (PDB 1M54). The two subunits of CBS are shown in grey and blue and the heme and PLP cofactors are shown in red and yellow respectively. K211 and K269 are shown in one of the two subunits in navy and cyan respectively. B. Locations of K361 (blue), K330 (cyan) and K260 (red) in one of the four subunits of human CSE (PDB file 2NMP). The PLP cofactor is shown in yellow in ball and stick representation.
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References (VSports)

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