A QUICk look at O-GlcNAc dynamicsLance Wells

In addition to expanding the chemical tools for exploring O-GlcNAc protein modifications, an innovative chemical biology approach has yielded new insights into the dynamic nature of this post-translational modification in the rodent brain.

Intracellular post-translational modifica-tions, particularly protein phosphorylation, are critical modulators of mammalian sig-nal transduction pathways. To be regulatory, a post-translational modification needs to be both inducible and dynamic. Regulatory post-translational modifications are typically widespread, occurring on multiple compo-nents in various signaling pathways, which allows attenuation of the signal and cross-talk between different signaling cascades. Achieving this complexity requires a cellular system to modify particular sites on given proteins in response to a specific stimulus without alter-ing the same modification on other proteins. For phosphorylation, this is accomplished in mammalian cells by producing literally hun-dreds of different protein kinases—the largest enzyme family in the human genome. In this issue of Nature Chemical Biology, Khidekel et al. reveal that the O-linked β-N-acetylglucos-amine (O-GlcNAc) modification of proteins, which like phosphorylation modifies serines and threonines of intracellular proteins, is not only dynamic but also differentially regulated: the stoichiometry of certain O-GlcNAc sites is extensively altered by a given stimulus, whereas other sites are not significantly affected1.

Khidekel et al. recently developed a GlcNAc-specific labeling strategy2 that borrows on the concept of analog-sensitive variants from the kinase community3. This chemoenzymatic approach relies on specifically modifying proteins containing a terminal GlcNAc moiety by using a β-1,4-galactosyltransferase that has been engineered to transfer a ketone-contain-ing galactose to the C4 hydroxyl of a GlcNAc

acceptor. The ketone functionality can then be tagged with an aminooxy biotin derivative for purposes of enrichment and identifica-tion (Fig. 1). In the paper presented here, the authors add an additional step to their work-flow that allows for relative quantitative mass spectrometry–based analysis. Using a method developed by Hsu et al.4, peptides are labeled using isotope tagging of primary amines via dimethyl (light/heavy) groups. The authors have termed their combined approach quan-titative isotopic and chemoenzymatic tagging (QUIC-Tag) (Fig. 1).

Combining this labeling approach with the power of electron-transfer dissociation (ETD)—a relatively new mass spectrometry fragmentation approach for identifying labile post-translational modifications that was pio-neered by coauthor J. Coons5—permits site mapping for multiple O-GlcNAc modifica-tions. The addition of the ETD approach is an important component, as it overcomes one of the main limitations of the original chemoen-zymatic approach: relying on collision-assisted dissociation (CAD) to map labile modifica-tions. In CAD, the weakest bond (that is, the bond to the serine/threonine post-transla-tional modification) cleaves first. Alternatively, ETD has proven robust in fragmenting post-translationally modified proteins while retain-ing the modification on the hydroxyl amino acid. Though other quantitative site-mapping strategies exist for O-GlcNAc, such as BEMAD (β-elimination followed by Michael addition with DTT)6, the QUIC-Tag approach com-bined with ETD seems to now offer the best strategy in terms of enrichment, specificity, quantification and site mapping.

Importantly, Khidekel et al. apply their newly developed method to biological systems: cul-tured cortical neurons and in vivo–stimulated rodent cerebral cortex. In these systems, the true power of developing chemical biology approaches is revealed—the uncovering of

new biological phenomena. Although multiple investigators have demonstrated that O-GlcNAc levels can be globally elevated or decreased in response to various stimuli7, this new method reveals for the first time that whereas certain sites of modification may undergo tremendous changes in occupancy in response to a particu-lar perturbation or stimulus, other sites remain virtually unchanged. This differential regula-tion illustrates an important way in which O-GlcNAc behaves, in a fashion analogous to that of phosphorylation, like a regulatory post-translational modification.

Like all good scientific work, this new dis-covery leaves those of us in the O-GlcNAc field with many more questions to answer and avenues to explore. It seems that evolu-tion overcame the problem of how to differ-entially phosphorylate proteins by generating hundreds of different protein kinases with different donor specificity, expression, local-ization and regulatory-associated proteins. However, for O-GlcNAc, there is only one apparent animal O-GlcNAc transferase. Thus, how is O-GlcNAc modification being differ-entially regulated? Perhaps instead of taking the route of gene duplication and diversifica-tion (as for phosphorylation), the O-GlcNAc modification is controlled much as mRNA expression is regulated. Like O-GlcNAc trans-ferase, only one RNA polymerase II exists and is responsible for mRNA transcription, but expression is exquisitely regulated by protein-protein associations, localization, substrate availability and other post-translational modifications. More experimental data will need to be acquired before the regulation of O-GlcNAc modification can be addressed. However, aided by advancements such as those presented here by Khidekel et al., we are well on our way to a better understanding of the biology of this small carbohydrate modi-fication of nuclear and cytosolic proteins. One final comment: just as phosphorylation

Lance Wells is in the Complex Carbohydrate Research Center and the Department of Biochemistry and Molecular Biology, University of Georgia, 315 Riverbend Road, Athens, Georgia 30602-4172, USA.e-mail: lwells@ccrc.uga.edu

NATURE CHEMICAL BIOLOGY VOLUME 3 NUMBER 6 JUNE 2007 303

N E W S A N D V I E W S

was not fully appreciated until the work of Krebs and Fischer defined the first of many clear functions for phosphate modification on a protein8, O-GlcNAc cannot be classified as a key regulatory modification until we dis-cover the ‘smoking gun’—evidence that O-GlcNAc modification at a specific site on a given protein alters its biological properties.

The QUIC-Tag method gives the community a valuable tool for conducting this hunt.

COMPETING INTERESTS STATEMENTThe author declares no competing financial interests.

1. Khidekel, N. et al. Nat. Chem. Biol. 3, 339–348 (2007).

2. Khidekel, N., Ficarro, S.B., Peters, E.C. & Hsieh-Wilson, L.C. Proc. Natl. Acad. Sci. USA 101, 13132–13137

(2004).3. Bishop, A.C. et al. Nature 407, 395–401 (2000).4. Hsu, J.L., Huang, S.Y., Chow, N.H. & Chen, S.H. Anal.

Chem. 75, 6843–6852 (2003).5. Mikesh, L.M. et al. Biochim. Biophys. Acta 1764,

1811–1822 (2006).6. Vosseller, K. et al. Proteomics 5, 388–398 (2005).7. Zachara, N.E. & Hart, G.W. Biochim. Biophys. Acta

1761, 599–617 (2006).8. Fischer, E.H. & Krebs, E.G.J. Biol. Chem. 216, 121–

132 (1955).

Figure 1 The QUIC-Tag strategy for quantitative analysis of O-GlcNAc. O-GlcNAc–modified proteins (blue squares) from two samples are specifically labeled with a ketone-containing galactose (yellow circles), which is then further reacted with a biotin moiety (green hexagon). After tryptic digestion, light (purple) or heavy (red) methyl groups are added to the amino groups of the peptides, and the O-GlcNAc–modified peptides are enriched via avidin. Both identification (most robustly performed by ETD fragmentation) and relative quantification can then be achieved.

Stimulating the cell’s appetite for itselfAnne Simonsen & Harald Stenmark

New inducers of autophagy—the process by which cells use lysosomes to degrade portions of their cytoplasm—are lead compounds for new drugs targeting neurodegenerative protein aggregation diseases.

Induction of a process that augments the cell’s capacity to degrade intracellular pro-tein aggregates is a goal in therapy of neu-rodegenerative diseases such as Parkinson’s, Alzheimer’s and Huntington’s disease. These diseases are characterized by accumulation of intracellular protein aggregates in nerve cells,

which ultimately cause cell death and ensu-ing loss of brain functions1,2. On p. 331 of this issue, Sarkar et al.3 describe a set of new neuro-protective compounds that stimulate the cell’s digestion of protein aggregates.

One of the major cellular pathways for scavenging intracellular protein aggregates is autophagy (literally, “self-eating”)2. Autophagy is a bulk degradation process that involves the sequestration of portions of cytoplasm by a double-membrane autophagosome, followed by digestion of the sequestered material when the autophagosome fuses with a lysosome full of hydrolytic enzymes (Fig. 1)4. Recently,

researchers found that loss of autophagy causes neurodegeneration even in the absence of any disease-associated mutant proteins5,6, which suggests that the continuous clearance of cellular proteins through basal autophagy prevents their accumulation, and in turn pre-vents neurodegeneration. Experiments in fly and mouse models have provided proof of principle that stimulation of autophagy can prevent and even reverse neurodegenerative disease7.

The compound that has been used for such studies, the immunosuppressant rapamycin, stimulates autophagy and aggregate digestion

Anne Simonsen and Harald Stenmark are in the Centre for Cancer Biomedicine, University of Oslo, and the Department of Biochemistry, the Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.e-mail: stenmark@ulrik.uio.no

304 VOLUME 3 NUMBER 6 JUNE 2007 NATURE CHEMICAL BIOLOGY

N E W S A N D V I E W S