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COMMENTARY

Nutrient-driven O-GlcNAc cycling – think globally but act locally

Katryn R. Harwood* and John A. Hanover*

ABSTRACT

Proper cellular functioning requires that cellular machinery behave

in a spatiotemporally regulated manner in response to global

changes in nutrient availability. Mounting evidence suggests that

one way this is achieved is through the establishment of physically

defined gradients of O-GlcNAcylation (O-linked addition of N-

acetylglucosamine to serine and threonine residues) and O-

GlcNAc turnover. Because O-GlcNAcylation levels are dependent

on the nutrient-responsive hexosamine signaling pathway, this

modification is uniquely poised to inform upon the nutritive state of

an organism. The enzymes responsible for O-GlcNAc addition and

removal are encoded by a single pair of genes: both the O-GlcNAc

transferase (OGT) and the O-GlcNAcase (OGA, also known as

MGEA5) genes are alternatively spliced, producing protein variants

that are targeted to discrete cellular locations where they must

selectively recognize hundreds of protein substrates. Recent

reports suggest that in addition to their catalytic functions, OGT

and OGA use their multifunctional domains to anchor O-GlcNAc

cycling to discrete intracellular sites, thus allowing them to establish

gradients of deacetylase, kinase and phosphatase signaling

activities. The localized signaling gradients established by targeted

O-GlcNAc cycling influence many important cellular processes,

including lipid droplet remodeling, mitochondrial functioning,

epigenetic control of gene expression and proteostasis. As such,

the tethering of the enzymes of O-GlcNAc cycling appears to play a

role in ensuring proper spatiotemporal responses to global alterations

in nutrient supply.

KEY WORDS: O-GlcNAc, O-GlcNAcylation, Metabolism

Post-translational modification, Signaling, Lipid droplets,

Mitochondria, Epigenetics, Nuclear pores

IntroductionHundreds of proteins are modified at the hydroxyl group of their

serine and threonine residues by N-acetylglucosamine. This

modification has been termed O-GlcNAcylation (Torres and

Hart, 1984). The dynamic addition and removal of O-GlcNAc is

governed by a single pair of enzymes, O-GlcNAc transferase

(OGT) and b-N-acetylglucosaminidase C (O-GlcNAcase; OGA),

respectively (Kreppel et al., 1997; Lubas et al., 1997). Both OGT

and OGA are expressed in all tissue types, where they are

necessary for either vertebrate development (OGT) or for the

transition from the fetal stage to adulthood (OGA) (Shafi et al.,

2000; Yang et al., 2012). Deregulation of O-GlcNAc cycling

has been implicated in a variety of disease states, including

cardiovascular disease, neurodegeneration, cancer and diabetes

(Bond and Hanover, 2013; Hart et al., 2011; Ngoh et al., 2010;Zachara, 2012).

O-GlcNAc metabolism is widely believed to be part of a

nutrient-sensing pathway termed the hexosamine biosyntheticpathway (HBP). This pathway is sensitive to carbohydrate, aminoacid, fatty acid and ATP levels (Slawson et al., 2010) (Fig. 1).

The substrate of OGT, uridine diphosphate N-acetylglucosamine(UDP-GlcNAc), is the final product of the HBP, making O-GlcNAc addition ideal to reflect the nutritional state of the

cell. Specifically, key components that feed into UDP-GlcNAcsynthesis are derived from nutrients, such as the amino acidglutamine, acetyl-coenzyme A (acetyl-CoA), glucose and uridine.In this regard, the enzymes of the HBP join the signaling proteins

AMP activated protein kinase (AMPK) and mammalian target ofrapamycin (mTOR), which act as sensors of energy charge andamino acid levels, respectively.

O-GlcNAcylation is often considered to be analogous to proteinphosphorylation. However, unlike protein phosphorylation,whereby phosphate is added to and removed from proteins

by many different kinases and phosphatases, protein O-GlcNAcylation is performed by the products of a single pair ofhuman genes (Nolte and Muller, 2002; Shafi et al., 2000).Hundreds of proteins with a range of functions have been shown

to be O-GlcNAcylated (Copeland et al., 2008), suggesting thatmany pathways exist by which specific substrates are modified toensure proper downstream cellular responses. The specific pattern

of protein O-GlcNAcylation in the cell is determined in part by O-GlcNAc levels, the subcellular localization of OGT and OGA,and the different interaction partners of OGT and OGA either at

specific cellular locations, during specific times, or in specific celltypes. Because disease states are associated with deregulated O-GlcNAc cycling (Bond and Hanover, 2013; Hart et al., 2011; Ngoh

et al., 2010; Zachara, 2012), the factors that ensure that OGT andOGA are able to recognize their substrates and achieve substratespecificity must be tightly regulated.

Owing to its nutrient dependence, the presence or absence of an

O-GlcNAc modification on a protein could initiate a cascadethat is intended to ‘direct’ the cell to augment processes that areassociated with nutrient conservation, such as a reduction in cell

proliferation and attenuation of nutrient storage and cell growth.The question remains as to how a balance is achieved between theapparently competing functions of OGA and OGT to modulate

highly specific spatiotemporal responses. One possibility is thatunder certain conditions, such as those outlined in Fig. 2A, O-GlcNAcylation of proteins might be influenced in a globalmanner (Fig. 2A). Concurrently, a more fine-tuned mechanism

might govern protein O-GlcNAcylation to ensure both global andlocal cellular response to changes in UDP-GlcNAc levels, whichwould be important both under conditions of very low or

very high UDP-GlcNAc, or under conditions of UDP-GlcNAchomeostasis. Based on recent findings in the field, we proposethat this could be achieved by interaction of OGA and OGT with

specific binding partners that tether them to precise intracellular

Laboratory of Cell and Molecular Biology, NIDDK, National Institutes of Health,Bethesda MD 20892-0851, USA.

*Authors for correspondence (Kate.Harwood@nih.gov; jah@helix.nih.gov)

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 1857–1867 doi:10.1242/jcs.113233

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Fig. 1. Generation of the OGT substrate, UDP-GlcNAc – an overview of the hexosamine biosynthetic pathway. The hexosamine biosynthetic pathway(HBP) integrates many extracellular physiological inputs, including nutritional intake, with the metabolism of key carbohydrate, amino acid, nucleotide and fattyacid components, to produce the OGT substrate UDP-GlcNAc. O-GlcNAc cycling, the dynamic addition to and removal of O-GlcNAc on serine or threonineresidues of a plethora of OGT and OGA targets, allows for the regulation of important downstream cellular processes in a nutrient-dependent manner. Theenzymes involved are given in brackets.

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locations or organelles to allow them to evoke localizedresponses, as has been reported in the past for other enzymesystems (Gorlich et al., 1996; Griffin et al., 2011; Izaurralde et al.,

1997; Pratt et al., 2005). This local gradient of OGA or OGTmight therefore establish a threshold of activity: enzyme activityis ‘on’ when the target proteins are close to the tethered enzyme

or ‘off’ with increasing distance, allowing for sensitive andspecific responses to UDP-GlcNAc levels (Fig. 2B). Such amechanism would allow the cell to translate information about

the nutritional state into highly specialized changes in cellularresponses. Interestingly, a study using fluorescence resonance

energy transfer (FRET) to detect changes in the spatiotemporaldynamics of the O-GlcNAc modification in response to signal-inducing stimuli suggested that O-GlcNAc cycling does in factexhibit compartment-specific dynamics (Carrillo et al., 2011).

Specifically, Carrillo and colleagues observed a rapid increase inthe O-GlcNAcylation of a FRET-based sensor specifically atthe plasma membrane and nucleus, but not elsewhere in the

cytoplasm, following serum-stimulated signal transduction inCos7 cells.

In this Commentary, we will present in detail the localization

and properties of the different isoforms of OGT and OGA, as wellas the specific interactions they undergo with a wide variety ofcellular components. This information can provide insight into

how the cell is able to translate changes in nutritional status intodistinct metabolic responses as a means to maintain cellularhomeostasis. Furthermore, we will examine the mechanisms bywhich the modulation of protein O-GlcNAcylation alters cellular

metabolism and touch upon how the deregulation of thesesignaling pathways, either genetically or through alterednutritional conditions, might result in certain disease states.

Domain structure and localization of OGT and OGA isoformsO-GlcNAc transferaseIn mammals, the gene coding for OGT is found on the Xchromosome (Shafi et al., 2000), and several alternatively splicedtranscripts encoding distinct OGT isoforms have been identified,

including isoforms that localize to the nucleocytoplasm (ncOGT)and to the mitochondria (mOGT) (Love et al., 2003), as well as ashort isoform (sOGT) that shows nucleocytoplasmic localization(Hanover et al., 2003; Nolte and Muller, 2002; Shafi et al., 2000).

More recently, a genetically unrelated enzyme with O-GlcNActransferase activity, extracellular OGT (eOGT), has also beendescribed (Sakaidani et al., 2012; Sakaidani et al., 2011). The

ncOGT isoform is ,116 kDa and is encoded by all 23 exonsof the OGT gene (Hanover et al., 2003), whereas mOGT is

Fig. 2. Global and local responses to changes in UDP-GlcNAclevels. (A) The cell can respond to certain extracellular physiological inputswith global changes in intracellular UDP-GlcNAc levels and subsequentchanges in total protein O-GlcNAcylation levels to modulate properdownstream cellular responses. (B) The tethering of OGA or OGT atspecific cellular locations could establish local gradients of protein O-GlcNAcylation, and thus potentially introduce gradients of protein activity(illustrated by the gray ‘cloud’) that allow for highly specific spatiotemporalresponses to the overall UDP-GlcNAc levels, with consequentcellular responses.

Fig. 3. OGT and OGA isoforms and domain structure. (A) The threesplice isoforms of OGT, ncOGT, mOGT and sOGT, possess variable N-termini but identical catalytic domains. (B) The two splice isoforms of OGA,OGA-L and OGA-S, differ at their C-termini. OGA-L possesses a putativeHAT domain, whereas OGA-S lacks this domain and instead contains aunique 15-amino-acid long C-terminal extension.

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,103 kDa and is encoded by exons 5 to 23 (Love et al., 2003),and sOGT is ,78 kDa and encoded by exons 10 to 23 (Hanover

et al., 2003; Nolte and Muller, 2002; Shafi et al., 2000) (Fig. 3A).The newly described eOGT utilizes UDP-GlcNAc but performsits function in the endomembrane system; it has been lessextensively characterized than the three OGT isoforms, to which

it exhibits no apparent homology (Sakaidani et al., 2011). Assuch, our discussion of OGT will focus on the similarities anddifferences between ncOGT, mOGT and sOGT.

In general, the structural domains of the three differentiallyspliced OGT isoforms can be divided into regions that include avariable N-termini and tetratricopeptide repeat (TPR) domain, a

linker region and a multidomain catalytic region (Kreppel et al.,1997; Lazarus et al., 2011; Lubas et al., 1997) (Fig. 3A). Eachisoform possesses an identical catalytic domain, and variation

between the isoforms occurs primarily in the length and sequenceof their unique N-termini. For example, mOGT possesses amitochondrial-targeting sequence in its variable N-terminal regionupstream of the TPR domain (Love et al., 2003). The isoforms also

differ in their TPR domains, and contain between 2.5 and 12.5 TPRmotifs, consisting of 34 amino acid repeats each, that are thought tohave a role in protein–protein interactions (Goebl and Yanagida,

1991; Iyer et al., 2003; Liu et al., 1999). Specifically, ncOGTpossesses 12.5 TPR motifs, whereas mOGT has 9.5 motifs andsOGT only 2.5 motifs (Hanover et al., 2003). The TPR domain of

ncOGT has been crystallized and exists as a superhelix with anamphipathic center groove that is lined with Asn residues (Blatchand Lassle, 1999; Jınek et al., 2004). The TPR domain bears some

resemblance to the importin-a family of nuclear transport receptors(Jınek et al., 2004). The characteristics of this groove might have arole in determining substrate specificity for a variety of OGTsubstrates, although a specific substrate sequence motif is not

apparent (Blatch and Lassle, 1999; Hanover et al., 2003; Iyer et al.,2003; Lazarus et al., 2006). Consequently, the differences in thelength of the TPR domains in each OGT isoform has been

suggested to modulate the substrate specificity among the differentisoforms (Lubas and Hanover, 2000).

As mentioned above, the catalytic domain of all three OGT

isoforms is identical, and several crystal structures of OGTvariants that are in a complex with UDP alone, with UDP andpeptide substrate or with sugar donor analogs have been solved(reviewed in Vocadlo, 2012). These structures have demonstrated

that when in a complex with UDP alone, OGT adopts a ‘closed’conformation, with interactions between TPR10 and 11 and ahelix of the catalytic domain acting as a ‘latch’ to stabilize this

conformation (Lazarus et al., 2011). In the structure of OGT incomplex with both UDP and peptide substrate, a wide cleftappears to be formed through a hinge-like rotation between

TPR12 and 13, opening the active site for peptide substratebinding (Lazarus et al., 2011). Based on these crystal structuresand kinetic experiments, Lazarus and co-workers proposed a

sequential bi-bi kinetic mechanism for OGT action wherebyUDP-GlcNAc binds prior to the protein substrate, although theexact mechanism of glycosyl transfer, including the identity ofthe catalytic residue, remains to be determined (Lazarus et al.,

2011; Schimpl et al., 2012b). To examine the ternary complexwith both UDP and modified peptide substrate bound, crystalstructures were solved using a very slowly hydrolyzed UDP-

GlcNAc analog, UDP-5SGlcNAc, in which the endocyclicoxygen atom is replaced by sulfur (Lazarus et al., 2012). Thesestructures suggest that OGT promotes catalysis using an

electrophilic migration mechanism (Lazarus et al., 2012). This

ternary complex also demonstrates that the substrate interactioninterface is large (Lazarus et al., 2012), suggesting that the

binding of different protein substrates is likely to cause globalstructural changes in the active center of OGT, which arereflected in the wide range of affinities observed for UDP-GlcNAc (from 1 mm to over 20 mm) (Shen et al., 2012).

In addition, the cell cycle regulator host cell factor 1 (HCF-1)is an evolutionarily conserved epigenetic regulator that undergoesan unusual proteolytic processing event to generate two stably

associated fragments that regulate different stages of the cellcycle (Hanover, 2011; Mazars et al., 2010). Interestingly, recentreports have demonstrated that OGT is involved in the proteolytic

maturation of HFC-1, with cleavage of HCF-1 being carried outin the same active site where protein glycosylation occurs andwith UPD-GlcNAc used as a co-substrate during the cleavage

reaction (Capotosti et al., 2011; Daou et al., 2011; Hanover, 2011;Lazarus et al., 2013). It is not yet clear if there are other substratesthat might be cleaved by OGT.

O-GlcNAcaseFirst identified as meningioma-expressed antigen 5 (MGEA5),the gene encoding OGA is located on chromosome 10 in humans

(Comtesse et al., 2001; Heckel et al., 1998). Alternative genesplicing yields two isoforms, a long isoform (OGA-L) and a shortisoform (OGA-S). OGA-L is found predominantly in the

cytoplasm (Comtesse et al., 2001), whereas OGA-S exhibitsnuclear (Comtesse et al., 2001) and lipid-droplet-associatedlocalization (Keembiyehetty et al., 2011). Both isoforms

possess an identical N-terminal hyaluronidase domain, butdiffer at their C-termini (Fig. 3B). The OGA-L isoformcontains a C-terminal extension with similarity to knownhistone acetyl transferase (HAT) domains (Comtesse et al.,

2001; Schultz and Pils, 2002), whereas OGA-S has a unique 15-amino-acid-long C-terminal extension. Despite the similarity ofthe OGA-L C-terminus to known HAT domains, it is unclear

whether the protein functions as a HAT (Butkinaree et al., 2008;He et al., 2014; Toleman et al., 2004). OGA-L is encoded by 16exons, and produces a 916-amino-acid protein product (Gao et al.,

2001). OGA-S is a splice variant of the OGA gene wherein the 59

splice site of intron 11 is skipped, leading to an extended exon 10that utilizes an alternate stop codon relative to OGA-L (Comtesseet al., 2001). As a result, OGA-S is produced as a truncated 677-

amino-acid species. In OGA-L, a linker region between the N-and C-terminal domains contains a caspase 3 cleavage site that iscleaved during apoptosis (Butkinaree et al., 2008). In in vitro

experiments, OGA-S exhibits lower enzyme activity than OGA-L(Kim et al., 2006; Macauley and Vocadlo, 2009), although it isunclear whether this reflects their relative activities in an intact

cell.Because efforts to crystallize human OGA have not yet been

successful, work to understand the catalytic mechanism of OGA

has been based on structural studies of bacterial homologs(Dennis et al., 2006; He et al., 2010; Rao et al., 2006) and onmechanistic studies that use the human variant and syntheticOGA substrates and inhibitors (Cetinbas et al., 2006; Greig

et al., 2009; Macauley et al., 2005a; Macauley et al., 2005b;Whitworth et al., 2007). These studies suggest that the catalyticmechanism in human OGA-L requires two key catalytic

aspartate residues, Asp174 and Asp175, and proceeds via atwo-step substrate-assisted mechanism. Additional studies havefocused on determining the factors that are important for

substrate recognition by OGA, including determination of the

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residues of OGA involved and the features of the substrate thatcontribute to catalysis (Schimpl et al., 2012a; Schimpl et al.,

2010; Shen et al., 2012). The results of these studies suggest thatit is either contacts between OGA and the sugar on the proteinsubstrate that modulate OGA recognition (Shen et al., 2012), orcontacts between OGA and both the sugar and the peptide

backbone of the protein substrate (Schimpl et al., 2012a). Theindividual amino acid side chains appear not to play a role insubstrate binding and recognition.

The spatiotemporal effects of targeted O-GlcNAc cyclingOverall, it is likely that the unique characteristics of the various

OGA and OGT isoforms provide a means for the differentialrecognition of specific protein substrates in a highly regulatedmanner. Further, the specific localization patterns and physical

properties of these isoforms are likely to allow them to interactwith specific protein substrates in a nutritionally regulatedmanner. Identifying these interactions might provide insight

into how the cell is able to communicate its nutritional statusand elicit appropriate responses in order to maintain cellular

homeostasis. Moreover, they might offer an explanation for thedisease states that arise when homeostasis cannot be achievedowing to dietary or genetic factors. Specific examples of targetedO-GlcNAc cycling and the subsequent cellular response are

discussed below.

Lipid droplet remodeling – OGA-S, perilipin-2 andthe proteasomeLipid droplets are cellular organelles responsible for the storageand hydrolysis of neutral lipids. Lipid droplets were originally

considered to be inert organelles; however, more recently, they haveemerged as key players in various aspects of cellular homeostasis,including lipid and energy metabolism and beyond (reviewed in Guo

et al., 2009). Furthermore, their study has garnered interest becauseexcessive lipid storage in lipid droplets can be linked to thedevelopment of obesity, diabetes and atherosclerosis. A better

Fig. 4. O-GlcNAc cycling and localized cellular remodeling. (A) Lipid droplet remodeling. The targeted localization of OGA-S by perilipin-2 at the surface oflipid droplets results in the removal of O-GlcNAc from the proteasome and its subsequent activation. Proteasomal activation at the surface of lipid dropletsincreases the degradation of surface localized proteins, resulting in lipid droplet remodeling. (B) Mitochondrial trafficking. OGT binds to TRAKs and has a role inthe trafficking of mitochondria along actin or microtubule networks, potentially either directly through TRAK binding, via TRAK O-GlcNAcylation, or both.(C) Mitochondrial fission and fusion. OGT O-GlcNAcylates DRP1 and OPA1 (indicated by the yellow star), which results in increased mitochondrial fission.Overexpression of OGA reverses this effect and promotes mitochondrial fusion. (D) Cytokinesis. During cytokinesis, both OGTand OGA are found in a transientcomplex with both Aurora B and the phosphatase PP1. It has been shown that the localization of OGT in this complex is dependent on Aurora B. These pairs ofenzymes with opposing activities somehow coordinate their activities in a presumably complex manner to ensure proper protein partitioning into daughter cellsand completion of cytokinesis. (E) Nuclear pore remodeling. O-GlcNAcylation of Nup98 by OGT (yellow star) can relax the ‘sieve’-like structure of the nuclearpore, allowing the passage of larger molecules not permitted prior to Nup98 O-GlcNAcylation.

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understanding of the complex and dynamic mechanism by whichlipid droplets are formed and remodeled will provide further insight

into the regulation of lipid and energy metabolism.Interestingly, OGA-S has been shown to be targeted to the

surface of nascent lipid droplets, where it colocalizes withperilipin-2, a protein known to be actively involved in lipid

droplet formation (Fig. 4A) (Bickel et al., 2009; Brasaemle,2007; Londos et al., 1999). In addition, it is known that theactivity of the 26S subunit of the proteasome is inhibited by O-

GlcNAc modification (Zhang et al., 2007); this is interestingbecause proteasomal activity is required at the surface of lipiddroplets for lipid droplet remodeling (Ohsaki et al., 2006).

Furthermore, the activity of OGA-S is enhanced by lipidaccumulation (Keembiyehetty et al., 2011). Therefore, a highconcentration of OGA-S on the surface of lipid droplets could

lead to the removal of the O-GlcNAc modification from theproteasome in the immediate area surrounding the lipid droplet,resulting in a local increase in proteasomal activity and proteinremodeling on the lipid droplet surface. Moreover, it has been

demonstrated that reduction of OGA-S levels results in thestabilization of proteins that are associated with lipid droplets(Keembiyehetty et al., 2011), suggesting that there might be a

complex feedback system between proteasomal activity and thelevel of OGA-S that could have a role in regulating lipid dropletmaturation. Potentially, this is a mechanism by which the cell

regulates lipid storage and mobilization, with OGA-S uniquelysuitable to link carbohydrate and lipid metabolism.

Mitochondrial trafficking – OGT, Miro, the TRAKs and kinesinsMitochondria are trafficked along both actin and microtubulenetworks (Ligon and Steward, 2000a; Ligon and Steward, 2000b;Morris and Hollenbeck, 1995; Rube and van der Bliek, 2004),

and the regulation of mitochondrial trafficking is particularlyimportant during embryonic development and for the regulationof apoptosis, as well as in neurons, where mitochondria often

need to be transported over long distances (Detmer and Chan,2007). In Drosophila, mitochondrial trafficking is regulated by aprotein complex containing the mitochondrial Rho GTPase Miro

and the kinesin-binding protein Milton (Guo et al., 2005; Stowerset al., 2002). This complex also includes the kinesin-bindingadaptor proteins TRAK1 (OIP106 in humans) and TRAK2(OIP98 in humans, also known as Grif-1) (Brickley and

Stephenson, 2011), which, interestingly, have been found tobind to OGT (Iyer et al., 2003). It is therefore possible that TRAKO-GlcNAcylation either modulates their function directly, allows

the TRAKs to recruit OGT to Miro or kinesins, or alters boththe function and recruitment roles of the TRAKs (Fig. 4B).Interestingly, using co-immunoprecipitation experiments, Iyer

et al. (Iyer et al., 2003) have shown that OIP106 interacts withboth OGT and RNA polymerase II (RNA Pol II) in vivo andforms a RNA-Pol-II–OIP106–OGT ternary complex. The authors

suggest that, in addition to a role in localized regulation ofmitochondrial trafficking through its interaction with OGT,OIP106 might also target OGT to transcriptional complexeswhere it could modify transcriptional machinery components (i.e.

RNA Pol II and transcription factors). As the levels of UDP-GlcNAc are influenced by input from a number of nutritionalpathways (Fig. 1), including those that are necessary for

mitochondrial function, it is possible that the O-GlcNAcylationstate of regulatory proteins might communicate nutritional status(i.e. the availability of mitochondrial substrates, to modulate

mitochondrial mobility) (Wells et al., 2003). Because

mitochondrial trafficking is particularly important in neurons(Schwarz, 2013), its deregulation could result in neurological

disease. It would be particularly interesting to address whetherOGT activity is involved in modulating mitochondrial transportthrough O-GlcNAcylation of Miro, the TRAKs or other kinesins,as this could provide a link between nutritional status and the

development of neurological pathologies (MacAskill and Kittler,2010).

Mitochondrial fission versus fusion – O-GlcNAcylation, OPA1and DRP1In addition to proper trafficking, the regulated and dynamic

remodeling of mitochondria is also essential for appropriatemitochondrial function (Kane and Youle, 2010; Westermann,2010). One protein that plays a prominent role in mitochondrial

fission is the GTPase dynamin-related protein 1 (DRP1, also knownas DNM1L) (Chang and Blackstone, 2010). Although primarilya cytoplasmic protein (Smirnova et al., 2001), Gawlowskiand colleagues have demonstrated that, in cardiac cells,

DRP1 translocates to the mitochondria upon O-GlcNAcylation byOGT (Gawlowski et al., 2012). Furthermore, they have shown thatO-GlcNAcylation increases the amount of DRP1 that is in an active

GTP-bound state. Following the O-GlcNAcylation andmitochondrial translocation of DRP1, Gawlowski and colleaguesobserved increased mitochondrial fragmentation with reduced

membrane potential. In addition, they found that high glucoselevels both increase the O-GlcNAcylation of optical atrophy 1(OPA1), a mitochondrial fusion-related protein, and decrease either

OPA1 expression or protein stability in neonatal cardiac myocytes,resulting in the same phenotype as observed upon increasedDRP1 O-GlcNAcylation. Furthermore, they determined thatoverexpression of OGA was able to restore proper mitochondrial

structure, suggesting that the localized activity of OGT or OGAresults in the subsequent fragmentation (fission) or restoration(fusion) of mitochondria, respectively (Fig. 4C). Perhaps this is a

mechanism by which the cell communicates its nutritive state forthe maintenance of proper mitochondrial function, for example inpreparation for cellular division, in which a localized fragmentation

or fission of mitochondria is required to ensure that mitochondriaare allocated properly, or to help maximize the capacity foroxidative phosphorylation, through fusion, when the cell is underlow nutrient conditions. As such, the role of O-GlcNAc cycling in

mitochondrial trafficking and fission–fusion could have particularlyimportant implications in the development of mitochondrialdysfunction observed in diabetes.

Cell division and cytokinesis – O-GlcNAc cycling, Aurora Band PP1During cell division, many proteins need to be targeted to distinctcellular locations to accurately regulate mitotic progression andensure that each daughter cell receives the appropriate complement

of cellular material and a complete set of chromosomes. One suchprotein is Aurora B, a kinase essential for normal mitoticprogression (Carmena and Earnshaw, 2003), that exhibits cell-cycle-dependent localization and is found to be targeted to the

midbody during cytokinesis, where its activity is necessary for thesuccessful completion of cytokinesis. Disruption of either AuroraB expression or activity has been shown to increase polyploidy in

cells (Adams et al., 2001; Nguyen et al., 2005; Ota et al., 2002;Tatsuka et al., 1998). Interestingly, both OGT and OGA are alsofound at the midbody during cytokinesis in a transient complex

with Aurora B and protein phosphatase 1 (PP1) (Fig. 4D) (Slawson

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et al., 2008), a phosphatase whose activity opposes that of AuroraB (Emanuele et al., 2008; Goto et al., 2006; Sugiyama et al., 2002).

Furthermore, Aurora B activity appears to be necessary for therecruitment of OGT to the midbody, as its localization is lost uponreduction of Aurora B activity (Slawson et al., 2008). It appearsthat the combined activities of the members of this complex

regulate the post-translational modification status of mitoticsubstrates, such as that of the cytoskeletal intermediate filamentprotein vimentin, in a highly localized manner (Slawson et al.,

2008). It is possible that the pattern of modifications achieved bythese pairs of enzymes plays a role in ensuring the propersegregation of the substrate proteins into the daughter cells, in the

case of vimentin potentially by inducing the disassembly ofvimentin filaments (Izawa and Inagaki, 2006). It is not yet clearhow a balance between the activities of these pairs of enzymes with

opposing actions is achieved to accomplish this task.

Nuclear pore structure and permeability – O-GlcNAcylationand Nup98Nuclear pore complexes (NPCs) are important for controlling thepassage of material from the cytoplasm into the nucleus and arecomposed of nucleoporins (Nups). A common motif in the Nups

is the phenylalanine-glycine (FG) motif that consists of up to 50repetitive FG sequences (Starr and Hanover, 1991). It has beensuggested that the FG–FG contacts between Nups creates a

‘sieve’-like structure in the pore cavity (Ribbeck and Gorlich,2001) through which small molecules can easily pass into thenucleus, but which excludes species larger than 5 nm (Mohr

et al., 2009). Nuclear transport receptors (NTRs) provide a meansfor import and export of molecules larger than this cutoff.

Interestingly, the FG-repeat domains of the Nups are also bindingsites for NTRs, further emphasizing the importance of the repeats

in creating the NPC barrier. It has also been suggested that FG-repeat domains function in modulating nuclear entry and exit, andthat the binding of NTRs to Nup FG-repeats replaces FG–FGcontacts, resulting in a local disruption of the sieve structure and

subsequent nuclear entrance of molecules with higher molecularmass (Frey and Gorlich, 2007; Frey et al., 2006; Labokha et al.,2013). Interestingly, it has been shown that a subset of FG-

containing Nups are modified by O-GlcNAc (Hanover et al.,1987), and one of these is Nup98 (Powers et al., 1995). Recently,using Nup98-derived hydrogels, Labokha and co-workers

demonstrated that O-GlcNAc modification of Nup98 relievesthe strict sieving property of the hydrogel, thereby allowingpassage of larger molecules that could not be accommodated

prior to Nup98 O-GlcNAcylation (Fig. 4E) (Labokha et al.,2013). Because other Nups are also known to be O-GlcNAcylated, it would be particularly interesting to determinewhether the targeted or localized addition and removal of the O-

GlcNAc moiety allows the cell to dynamically regulate thepermeability of specific nuclear pore complexes in response tonutritional cues, which could have a profound effect on cellular

homeostasis.

Transcriptional repression through interaction of OGTwith Sin3The packaging of DNA into chromatin is an essential mechanismby which transcription is regulated. The transcription of specific

genes can be up- or down-regulated through post-translationalmodification of histones that alter both protein interactions and

Fig. 5. O-GlcNAc cycling andtranscriptional regulation. Theproteins Sin3 (shown on the left) andTETs (shown on the right) might interactwith OGTat targeted locations to repressor activate, respectively, thetranscription of a specific subset ofgenes in response to nutritional cues.Simultaneously, the putative histoneacetyl transferase (HAT) domain of OGAmight regulate its binding to specifichistone tail marks, allowing OGA, inconjunction with histone acetylation, toactivate gene transcription.

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chromatin structure (Kouzarides, 2007). In addition, it hasbeen proposed that a complex ‘histone code’ exists: specific

combinations of multiple histone modifications result in theactivation or repression of particular genes, thus allowing for thefine-tuning of gene expression in order to appropriately respondto a variety of environmental and nutritional cues (Jenuwein and

Allis, 2001; Strahl and Allis, 2000; Turner, 2000).One of these histone modifications is the acetylation of specific

lysine residues, which alters chromatin structure and thus the

state of activation of specific genes (Kuo and Allis, 1998).Consequently, enzymes that either acetylate or deacetylatehistones, HATs and histone deacetylases (HDACs), respectively,

play an integral role in determining gene expression profiles. Ingeneral, HDACs can be found in a diverse array of corepressorcomplexes that allow for the repression of distinct subsets of genes

(Burke and Baniahmad, 2000; Ng and Bird, 2000). One of thesecomplexes is the Sin3-containing HDAC corepressor complex.Furthermore, Sin3 has been shown to interact physically with andbe O-GlcNAcylated by OGT (Fig. 5), and this interaction results in

the repression of genes through a pathway that might beindependent of HDAC activity (Yang et al., 2002). Potentially,the localized activity of Sin3 and OGT, together with the O-

GlcNAc modification, could act synergistically to repress specificsubsets of regulatory genes, as has been seen with, for example, theretinoblastoma tumor suppressor protein (Siddiqui et al., 2003).

In addition, as noted above, OGA-L has a predicted HATdomain that is similar to that of the histone acetyltransferaseGCN5. Despite the debate over the ability of OGA to act as a

HAT (Butkinaree et al., 2008; He et al., 2014), owing to thissimilarity, it is possible that this domain could facilitate theassociation of OGA-L with histone proteins. Potentially, aspecific pattern of histone modification could recruit OGA at

certain locations and times, for example, cell cycle stages, toremove the O-GlcNAc modification and, in conjunction withhistone acetylation, activate gene transcription (Fig. 5).

Transcriptional activation through interaction of OGT withTET proteinsAs mentioned above, post-translational modification of histones isone way in which transcriptional activity can be regulated. Anothermechanism of regulating transcriptional activity is through directmodification of DNA itself. It is known that DNA methylation on

cytosine by DNA methyltransferases, termed 5-methylcytosine or5-mC, generally results in gene repression (Cedar and Bergman,2009; Suzuki and Bird, 2008). Proteins from the ten-eleven

translocation (TET) family (TET1, TET2 and TET3) catalyze theconversion of 5-mC to cytosine 5-hydroxymethylation or 5-hmC,and much work is being done to better understand the roles of this

mark (Ito et al., 2010; Kriaucionis and Heintz, 2009; Tahiliani et al.,2009). Recently, it has been shown that OGT interacts physicallywith and is recruited to chromatin by the TET proteins (Chen et al.,

2013; Deplus et al., 2013b; Vella et al., 2013) (Fig. 5). Theinteraction of OGT with the TETs plays a complicated role inregulating transcription, which is just beginning to be understood,although it appears that these interactions are important for TET-

dependent gene activation. Specifically, it has been shown thatOGT positively regulates TET1 protein levels and hmC levels onTET1 target genes (Shi et al., 2013). Furthermore, OGT and TET1

are found together at promoters near CpG-rich transcription startsites genome wide, where low levels of DNA methylation areobserved (Vella et al., 2013). This might mean TET1 and OGT

together play a role in regulating CpG island methylation.

Interestingly, TET2 levels positively influence histone 2B (H2B)Ser112 O-GlcNAcylation levels, independently of TET2 enzymatic

activity (Chen et al., 2013). This suggests OGT recruitment byTET2 can increase O-GlcNAcylation of specific chromatinsubstrates, influencing the transcriptional activation of specificsubsets of genes. OGT and TET2 and TET3 have also been found to

localize to promoters where they appear to play a role in H3K4me3enrichment by promoting binding of the SET1/COMPASS H3K4methyltransferase SETD1A, and subsequent transcriptional

activation (Deplus et al., 2013a). The results of these studies areparticularly interesting considering that under other conditions, forexample, while interacting with the Sin3 HDAC complex, OGT

results in transcriptional repression of a distinct subset of genes.Thus, it is possible that changes in the nutritional status cansimultaneously translate into the up- and down-regulation of

distinct subsets of genes to ensure appropriate transcriptionalprofiles are achieved (Fig. 5).

Conclusions and perspectivesAlthough, in this Commentary, we have explored a few examples ofthe localization-dependent effects of O-GlcNAc cycling, the O-GlcNAc field is in its infancy and much remains to be discovered.

As such, many more interaction partners responsible for OGA andOGT tethering and their functional relevance in the cell will likely beuncovered in the future. For example, although it has not yet been

demonstrated directly that the protein myosin phosphatase targetsubunit 1 (MYPT1), a regulatory subunit of PP1, can recruit OGT toa particular cellular location, there are a number of observations that

suggest that localized interactions between MYPT1 and OGT mightbe involved in ensuring that events take place accurately and at thecorrect cellular location during mitosis and cytokinesis, as has beendemonstrated for Aurora B: (1) MYPT1 plays an integral and highly

spatiotemporal role in mitosis and cytokinesis, (2) disruptions in O-GlcNAc cycling also disrupt mitosis and cytokinesis (Sakabe andHart, 2010), and (3) MYPT1 interacts with OGT (Cheung et al.,

2008). A similar mechanism in targeting OGA or OGT might alsotake places during Polycomb-mediated repression, the specializedsilencing of genes that must be tightly regulated in a spatiotemporal

manner. In fact, it has been shown in both Drosophila and mouseembryonic stem cells that OGT plays an essential role in Polycomb-mediated repression that is crucial for proper development andpluripotency (Gambetta et al., 2009; Myers et al., 2011; Sinclair

et al., 2009). However, although it has been shown that OGT O-GlcNAcylates the Polycomb group member Polyhomeotic inDrosophila (Gambetta et al., 2009), the molecular details of its

role in Polycomb-mediated repression remain incompletely definedand might involve a similar targeting mechanism to that describedabove for the other cellular processes that require localized OGT and

OGA activity. Further efforts to elucidate the interaction partnersthat target OGT and OGA to particular locations for specific cellularfunctions will be pivotal to fully elucidate the pathways that

communicate between lipid, amino acid, nucleotide and sugarmetabolism. This research is anticipated to provide crucial insightsthat will allow us to both better understand the various disease statesthat can arise from such metabolic defects.

Competing interestsThe authors declare no competing interests.

FundingThe work of our laboratory is funded from the intramural program of the NationalInstitutes of Diabetes and Digestive and Kidney Diseases at the NationalInstitutes of Health. Deposited in PMC for release after 12 months.

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