O‐GlcNAcylation

O‐GlcNAcylation

O-GlcNAcylation uses the substrate UDP-GlcNAc, the final product of nutrient flux through the hexosamine biosynthetic pathway (HBP) which integrates amino acid, carbohydrate, fatty acid, nucleotide, and energy metabolism.

The HBP fluctuates with cellular metabolism and may be dramatically altered under physiological and pathological conditions. The extent of O-GlcNAcylation can reflect metabolic dynamics in the cell. (R4)

Impact on proteins

Target Protein	Impact
NRF1	Protects from ubiquitination, increases activity
KEAP1 / NRF2	O‐GlcNAcylation of KEAP1 is required for the efficient ubiquitination and degradation of NRF2
GS	Reduces Glycogen Synthase activity by 60%
CSE	Increases production of H2S by CSE (CTH)

Factors affecting O-GlcNAcylation

Factor	Impact
GSK3b	Increases activity of OGT by phosphorylating it
Glucose deprivation	Increases expression of OGT and lowers expression of OGA

OGT

Three isoforms of OGT

Alternative splicing results in three variants of OGT, namely nucleocytoplasmic OGT (ncOGT), mitochondrial OGT(mOGT) and short form OGT(sOGT).

The longest OGT isoform, ncOGT, is mostly localized in the nucleus but is able to shuttle toward the cytoplasm and plasma membrane in response to signaling cues (R4)

One prime example is the recruitment of OGT from the nucleus toward the plasma membrane upon prolonged insulin activation and PIP3 production.

OGT is also found to alter its nuclear localization upon acute AMPK activation. The mechanism underlying how OGT can be localized in both nucleus and cytosol has recently been elucidated where three amino acids (DFP; residues 451-453) in OGT is able to act as a nuclear localization signal. In addition to the DFP sequence, OGlcNAcylation of the TPR domain of OGT is required for its direct nuclear translocation.

The ability to translocate between different cellular locations places OGT at a unique position in coordinating signaling activities within different cellular compartments. Two isoforms of OGA have been identified and characterized. The long isoform of OGA resides mainly within the cytosol, whereas the short isoform (sOGA) localizes at the nucleus and lipid droplets. (R4)

OGT binds PIP3

OGT is identified to bind to phosphatidylinositol-3,4,5-trisphosphate (PIP3) while not possessing the pleckstrin-homology domain like PDK1 and AKT, suggesting different binding affinity toward PIP3 production.

This allows OGT to moderate insulinmediated signaling transduction within 30 min of activation. After insulin signaling activation, OGT will be recruited to the plasma membrane where it O-GlcNAcylates and attenuates the multiple components of the insulin signaling pathway. (R4)

OGA

TBD

OGT / OGA complex localization

Within the nucleus and particularly at sites of transcription, the two O-GlcNAc cycling enzymes are often found within the same complex. Paradoxically, although OGT is mostly nuclear, it is excluded from the nucleolus, and O-GlcNAcase, which is mostly cytosolic, is highly enriched within the nucleolus. (R2)

Observations

Improves KEAP1’s function to degrade NRF2

we identified KEAP1 (also known as KLHL19), the primary negative regulator of NRF2, as a direct substrate of OGT.

We show that O‐GlcNAcylation of KEAP1 at serine 104 is required for the efficient ubiquitination and degradation of NRF2. Interestingly, O‐GlcNAc levels and NRF2 activation co‐vary in response to glucose fluctuations, indicating that KEAP1 O‐GlcNAcylation links nutrient sensing to downstream stress resistance. (R1)

• Transcriptomics show that O‐GlcNAc transferase (OGT) inhibition activates the NRF2 pathway.
• O‐GlcNAcylation of the KEAP1 adaptor is required to restrain NRF2 via ubiquitin‐dependent proteolysis.
• Ser104 O‐GlcNAcylation of KEAP1 promotes its productive interaction with CUL3 ubiquitin ligase.
• Glucose deprivation reduces KEAP1 O‐GlcNAcylation and activates the NRF2 transcription factor. (R1)

Note to self: reduced O‐GlcNAcylation of KEAP1 increases NRF2 activation.

Binding to CSE and activating H2S production

The tandem ETD spectrum unambiguously identified Ser138 (S138) as the only O-GlcNAc site of CSE. A comparison of the Homo sapiens CSE with other vertebrate orthologues revealed high conservation of S138 site in mammals. … These data confirm that the enhanced H2S production by O-GlcNAcylated CSE inhibits trophoblast syncytialization.

The next question to be addressed is how H2S affects trophoblast syncytialization. A study in prostate cells revealed that H2S impeded AR dimerization through posttranslational S-sulfhydration on AR. … It remains unclear how the O-GlcNAc modification on CSE affects its enzymatic activity. The protein stability of CSE seems not affected by O-GlcNAcylation based on our observation that TMG treatment only increased CSE O-GlcNAcylation but had little effect on its protein level.

Lys212 is a vital catalytic residue of CSE, and CSE binds to the cofactor pyridoxal 5′-phosphate (PLP) by a covalent bond at Lys212 to accelerate proton transfer in the α,γ-elimination reaction of L-cystathionine. The Ser138 site is located in the N-terminal region of CSE that binds to PLP.

We thus predict that O-GlcNAcylation of Ser138 residue may lead to structural changes that facilitate the binding of CSE-Lys212 to PLP. (R6)

Reduced O-GlcNAcylation is associated with Alzheimer’s disease

Impaired O-GlcNAcylation appears to be most strongly associated with the pathogenesis of Alzheimer’s disease (AD). Several lines of evidence support a link between decreased O-GlcNAcylation and AD:

1. Key AD-related proteins are O-GlcNAcylated and this modification regulates their functions:

• Tau, which forms neurofibrillary tangles in AD, is O-GlcNAcylated. Reduced O-GlcNAcylation of tau is associated with increased phosphorylation and aggregation into toxic tangles[6][10][12].
• The amyloid precursor protein (APP) that generates amyloid-beta (Aβ) peptides is also O-GlcNAcylated. Impaired O-GlcNAcylation may alter APP processing to promote the amyloidogenic pathway and Aβ accumulation[10][12].

2. O-GlcNAcylation is decreased in AD brains:

• Post-mortem studies found reduced global O-GlcNAcylation levels in AD brains compared to controls[6][10][11].
• Tau aggregates in AD lack O-GlcNAc, suggesting a deficiency of this modification may enable tangle formation[8][12].

3. Increasing O-GlcNAcylation is protective in AD models:

• Pharmacologically elevating O-GlcNAc levels reduces pathological aggregation of tau and Aβ in cell and animal models of AD[6][8][10].
• Boosting O-GlcNAcylation protects neurons against Aβ and tau toxicity and improves cognitive function in AD mice[6][10][11].

4. Impaired brain glucose metabolism may drive O-GlcNAc deficits in AD:

• Glucose hypometabolism occurs early in AD and the biosynthesis of O-GlcNAc is coupled to glucose availability[8][9].
• Disrupted glucose utilization in the AD brain could thus lead to impaired protein O-GlcNAcylation[8][9][12].

Mechanistically, deficient O-GlcNAcylation may contribute to AD pathogenesis by enabling the hyperphosphorylation and aggregation of tau, promoting amyloidogenic APP processing, dysregulating signaling pathways like ERK, disrupting synaptic function, and increasing neuronal vulnerability to stress[6][7][10][11].

While most research has focused on AD, some studies suggest altered O-GlcNAcylation may also play a role in other neurodegenerative conditions like Parkinson’s and Huntington’s diseases, potentially by regulating the aggregation and toxicity of α-synuclein and huntingtin proteins respectively[4][6][8].

In summary, impaired protein O-GlcNAcylation, likely resulting from brain glucose dysregulation, appears to be an important pathogenic factor in AD that enables the development of multiple disease hallmarks. Pharmacological inhibition of O-GlcNAc removal to restore levels of this modification has shown promise as a potential disease-modifying therapeutic strategy for AD in preclinical studies[8][10][12]. However, more research is needed to fully define the mechanisms involved and establish the therapeutic potential of targeting impaired O-GlcNAcylation in AD and related neurodegenerative disorders.

Citations: [1] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6568225/ [2] https://www.nature.com/articles/nrm.2017.22 [3] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8639716/ [4] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8639716/ [5] https://www.frontiersin.org/articles/10.3389/fnagi.2023.1155630/full [6] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8639716/ [7] https://www.jbc.org/article/S0021-9258%2823%2902439-0/fulltext [8] https://pubs.rsc.org/en/content/articlelanding/2014/cs/c4cs00038b [9] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9700112/ [10] https://pubmed.ncbi.nlm.nih.gov/31894464/ [11] https://www.nature.com/articles/s12276-021-00709-5 [12] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263855/

Glucose deprivation increases OGT and lowers OGA expression

Increased O-GlcNAc modification is not mediated by increased UDP-GlcNAc, the rate-limiting substrate for O-GlcNAcylation.

Rather, the mRNA for nucleocytoplasmic O-linked N-acetylglucosaminyltransferase (OGT) increases 3.4-fold within 6 h of glucose deprivation.

Within 12 h, OGT protein increases 1.7-fold (p = 0.01) compared with normal glucose-treated cells. In addition, 12-h glucose deprivation leads to a 49% decrease in O-GlcNAcase protein levels. (R3)

Numerous studies have reported a global increase in cellular O-GlcNAcylation in response to nutrient starvation, a condition in which low UDP-GlcNAc levels are expected. (R4)

Glycogen Synthase activity is reduced by O-GlcNAcylation

O-GlcNAcylation of glycogen synthase is significantly increased with glucose deprivation, and this O-GlcNAc increase contributes to a 60% decrease (p = 0.004) in glycogen synthase activity (R3)

Fatty Acids Oxidation is mediated by O-GlcNAcylation

AMP-activated protein kinase is activated in adipocytes with elevated HBP flux, resulting in O-GlcNAc-mediated elevation of fatty acid oxidation (R3)

GSK3b increases OGT activity

GSK3β, has been shown to phosphorylate OGT at serine 3 or 4, which leads to increased OGT activity and potential reciprocal regulation. (R4)

Hypoxia has impact on O-GlcNAcylation

Global O-GlcNAc levels often show drastic changes in response to cellular stress including heat shock, hypoxia and nutrient deprivation. (R4)

Insufficient O-GlcNAcylation

Hypo-O-GlcNAcylation usually arises from low UDP-GlcNAc levels or a dramatic imbalance between OGT/OGA expression and activity.

Given the observation that some of OGT’s protein substrates are constitutively modified at physiological UDPGlcNAc levels while some vary widely, it can be envisioned that the cells would preferentially feed O-GlcNAc moieties toward protein residues that are required for essential structural and functional integrity of the protein.

At this point, dynamic O-GlcNAc signaling may dampen its amplitude and achieve limited effectiveness. In the event of persistent hypoO-GlcNAcylation, proteins would then be prone to structural changes without the protective effects from O-GlcNAcylation, thus manifesting states that may have deleterious effects for the cell.

NRF1 is regulated by OGT

Through screening for the putative regulators of Nrf1a, we found that Nrf1a interacts with HCF1 and OGT. We show that O-GlcNAc modification by OGT leads to decreased ubiquitination and stabilization of Nrf1a, and HCF1 and OGT enhances transcriptional activation by Nrf1a. (R5)

Conclusion: O-GlcNAcylation of NRF1 protects it from degradation and leads to increased activity.