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Chemical Science
Edge Article
antibody-based approaches in this study, the substrate scope of primary alcohols of glycerol were rst protected with tert-
this new PTM remains insufficiently explored, which has butyldimethylsilyl (TBS) ethers 1 (93% yield). The remaining
limited the understanding of its regulatory mechanisms and secondary alcohol was then transformed into ketone 2 by mild
cellular functions. Here, we report the development of an Swern oxidation (92% yield). For the synthesis of the homo-
alkyne-functionalized chemical reporter, HMGAM-yne, for propargyl alcohol derivative 3 via the Grignard reaction, prop-
protein HMGylation. We demonstrate that HMGAM-yne can be argylmagnesium bromide was prepared in the presence of
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readily incorporated into proteins through metabolic labeling, mercury chloride at 0 C to avoid its rearrangement into alle-
enabling the visualization of HMGylation substrates. Pull-down nylmagnesium bromide. Subsequent dropwise addition of the
experiments using this reporter not only led to the enrichment obtained Grignard reagent to the solution of ketone 2 led to the
of known HMGylated proteins in mitochondria, but also the formation of 3 (80% yield). Aer the deprotection of the two TBS
identication of multiple nuclear proteins, including histones, groups, the resulting diol 4 was converted to its corresponding
as the novel substrates of HMG-K.
dinitrile 6 via a tosylate intermediate 5 (94%, 79%, and 71%
yield for each step, respectively). The following hydrolysis of the
dinitrile 6 afforded the dicarboxylate 7 (30% yield), which was
further masked by AM esters to give the desired HMGAM-yne
(80% yield).
Results and discussion
Based on our previous work on the chemical reporter for lysine
malonylation,5 we designed a 3-hydroxyl-3-methylglutaric acid
(HMG) analogue (HMG-yne, Fig. 1b) as the reporter for lysine
HMGylation. In the HMG-yne, the original methyl group was
armed with a terminal alkyne, which could mediate bio-
orthogonal conjugation with uorescent or affinity tags for the
detection and enrichment of HMGylated proteins. At the
physiological pH, the two carboxylates of the HMG are nega-
tively charged and thus limit its permeability across the cell
membrane. Therefore, we masked the two carboxylates with the
acetoxymethyl (AM) group in our chemical reporter (Fig. 1c). We
expected that this uncharged reporter could readily enter the
cells. The cleavage of the AM esters by nonspecic cellular
esterase should rapidly release the carboxylate form of the
reporter inside the cells for metabolic labeling (Fig. 1c).
We rst examined whether HMGAM-yne could be metabol-
ically incorporated into cellular proteins. To this end, HeLa S3
cells were treated with HMGAM-yne. Aer metabolic labeling,
the whole-cell lysates were subjected to a Cu(I)-catalyzed azide–
alkyne cycloaddition (CuAAC or ‘click’ chemistry) to conjugate
the reporter-labeled proteins to a uorescent dye (rhodamine).
The labeled proteins were then resolved by SDS-PAGE and
visualized by in-gel uorescent scanning. As shown in Fig. 2a,
a diverse spectrum of proteins was labeled with HMGAM-yne.
The pattern of the proteins labeled with HMGAM-yne did not
resemble those obtained by other tested chemical reporters for
lysine acetylation (4-pentynoate3) and even malonylation
(MalAM-yne5) (Fig. 2b). Importantly, the addition of HMG,
a precursor of the HMG-K donor, largely impeded the labeling
of proteins with HMGAM-yne (Fig. 2c), but not that with the
acetylation or malonylation reporters (Fig. 2d), indicating that
HMGAM-yne likely labeled HMGylation substrates. To further
assess the ability of the reporter to identify HMG-K substrates,
we used HMGAM-yne to enrich known HMGylated proteins.
Aer metabolic labeling in HeLa S3 cells, the HMGAM-yne
labeled proteins were conjugated to biotin and isolated using
high-capacity streptavidin beads. CPS1 and MDH2, two known
mitochondrial HMG-K substrates,11 were indeed selectively
enriched by the reporter (Fig. 2e and S1†). Consistent with the
uorescent labeling experiment (Fig. 2c), the enrichment of
these HMG-K substrates by HMGAM-yne was largely impaired
by addition of HMG (Fig. 2e and S1†).
The synthesis of HMGAM-yne followed an eight-step route by
using glycerol as a starting material (Scheme 1). The two
Like most PTMs, lysine HMGylation is a reversible process.
While the addition of this modication is currently known as
a non-enzymatic process, human sirtuin 4 (Sirt4) has been
identied as a de-HMGylase.12,13 However, given the promiscuity
of sirtuin proteins toward diverse acylations, it is possible that
other members of the sirtuin family might also contribute to the
regulation of cellular HMGylation. To test this hypothesis, we
incubated a histone H3 peptide carrying an HMG mark at Lys9
(H3K9hmg and Note S1†), with human Sirt1, Sirt2, Sirt3, Sirt5
and Sirt6, respectively. The enzymatic reactions were then
monitored by liquid chromatography-mass spectrometry
(LC-MS). Among the ve sirtuins tested, Sirt5 showed robust
activity to catalyze the removal of HMG on H3K9 (Fig. 3). Sirt5
was recently reported to serve as the ‘erasers’ for acylations
Scheme 1 Synthetic route of HMGAM-yne. TBS ¼ tert-butyldime-
thylsilyl, THF ¼ tetrahydrofuran, r.t. ¼ room temperature, DMSO ¼
dimethyl sulfoxide, Et ¼ ethyl, DCM ¼ dichloromethane, Ts ¼ tosyl, Me
¼ methyl, Py ¼ pyridine, DIEA ¼ N,N-diisopropylethylamine, and DMF
¼ N,N-dimethylformamide.
Chem. Sci.
This journal is © The Royal Society of Chemistry 2018