Dissecting OGT's TPR domain to identify determinants of cellular function.

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that glycosylates myriad intracellular proteins and cleaves the transcriptional coregulator Host Cell Factor 1 to regulate cell cycle processes. Via these catalytic activities as well as noncatalytic protein-protein interactions, OGT maintains cell homeostasis. OGT's tetratricopeptide repeat (TPR) domain is important in substrate recognition, but there is little information on how changing the TPR domain impacts its cellular functions. Here, we investigate how altering OGT's TPR domain impacts cell growth after the endogenous enzyme is deleted. We find that disrupting the TPR residues required for OGT dimerization leads to faster cell growth, whereas truncating the TPR domain slows cell growth. We also find that OGT requires eight of its 13 TPRs to sustain cell viability. OGT-8, like the nonviable shorter OGT variants, is mislocalized and has reduced Ser/Thr glycosylation activity; moreover, its interactions with most of wild-type OGT's binding partners are broadly attenuated. Therefore, although OGT's five N-terminal TPRs are not essential for cell viability, they are required for proper subcellular localization and for mediating many of OGT's protein-protein interactions. Because the viable OGT truncation variant we have identified preserves OGT's essential functions, it may facilitate their identification.

Mammalian cell proliferation requires noncatalytic functions of O-GlcNAc transferase.

O-GlcNAc transferase (OGT), found in the nucleus and cytoplasm of all mammalian cell types, is essential for cell proliferation. Why OGT is required for cell growth is not known. OGT performs two enzymatic reactions in the same active site. In one, it glycosylates thousands of different proteins, and in the other, it proteolytically cleaves another essential protein involved in gene expression. Deconvoluting OGT's myriad cellular roles has been challenging because genetic deletion is lethal; complementation methods have not been established. Here, we developed approaches to replace endogenous OGT with separation-of-function variants to investigate the importance of OGT's enzymatic activities for cell viability. Using genetic complementation, we found that OGT's glycosyltransferase function is required for cell growth but its protease function is dispensable. We next used complementation to construct a cell line with degron-tagged wild-type OGT. When OGT was degraded to very low levels, cells stopped proliferating but remained viable. Adding back catalytically inactive OGT rescued growth. Therefore, OGT has an essential noncatalytic role that is necessary for cell proliferation. By developing a method to quantify how OGT's catalytic and noncatalytic activities affect protein abundance, we found that OGT's noncatalytic functions often affect different proteins from its catalytic functions. Proteins involved in oxidative phosphorylation and the actin cytoskeleton were especially impacted by the noncatalytic functions. We conclude that OGT integrates both catalytic and noncatalytic functions to control cell physiology.

Inhibition of O-GlcNAc Transferase Renders Prostate Cancer Cells Dependent on CDK9.

O-GlcNAc transferase (OGT) is a nutrient-sensitive glycosyltransferase that is overexpressed in prostate cancer, the most common cancer in males. We recently developed a specific and potent inhibitor targeting this enzyme, and here, we report a synthetic lethality screen using this compound. Our screen identified pan-cyclin-dependent kinase (CDK) inhibitor AT7519 as lethal in combination with OGT inhibition. Follow-up chemical and genetic approaches identified CDK9 as the major target for synthetic lethality with OGT inhibition in prostate cancer cells. OGT expression is regulated through retention of the fourth intron in the gene and CDK9 inhibition blunted this regulatory mechanism. CDK9 phosphorylates carboxy-terminal domain (CTD) of RNA Polymerase II to promote transcription elongation. We show that OGT inhibition augments effects of CDK9 inhibitors on CTD phosphorylation and general transcription. Finally, the combined inhibition of both OGT and CDK9 blocked growth of organoids derived from patients with metastatic prostate cancer, but had minimal effects on normal prostate spheroids. We report a novel synthetic lethal interaction between inhibitors of OGT and CDK9 that specifically kills prostate cancer cells, but not normal cells. Our study highlights the potential of combining OGT inhibitors with other treatments to exploit cancer-specific vulnerabilities. IMPLICATIONS: The primary contribution of OGT to cell proliferation is unknown, and in this study, we used a compound screen to indicate that OGT and CDK9 collaborate to sustain a cancer cell-specific pro-proliferative program. A better understanding of how OGT and CDK9 cross-talk will refine our understanding of this novel synthetic lethal interaction.

Aspartate Residues Far from the Active Site Drive O-GlcNAc Transferase Substrate Selection.

O-GlcNAc is an abundant post-translational modification found on nuclear and cytoplasmic proteins in all metazoans. This modification regulates a wide variety of cellular processes, and elevated O-GlcNAc levels have been implicated in cancer progression. A single essential enzyme, O-GlcNAc transferase (OGT), is responsible for all nucleocytoplasmic O-GlcNAcylation. Understanding how this enzyme chooses its substrates is critical for understanding, and potentially manipulating, its functions. Here we use protein microarray technology and proteome-wide glycosylation profiling to show that conserved aspartate residues in the tetratricopeptide repeat (TPR) lumen of OGT drive substrate selection. Changing these residues to alanines alters substrate selectivity and unexpectedly increases rates of protein glycosylation. Our findings support a model where sites of glycosylation for many OGT substrates are determined by TPR domain contacts to substrate side chains five to fifteen residues C-terminal to the glycosite. In addition to guiding design of inhibitors that target OGT's TPR domain, this information will inform efforts to engineer substrates to explore biological functions.

HCF-1 Regulates De Novo Lipogenesis through a Nutrient-Sensitive Complex with ChREBP.

Carbohydrate response element binding protein (ChREBP) is a key transcriptional regulator of de novo lipogenesis (DNL) in response to carbohydrates and in hepatic steatosis. Mechanisms underlying nutrient modulation of ChREBP are under active investigation. Here we identify host cell factor 1 (HCF-1) as a previously unknown ChREBP-interacting protein that is enriched in liver biopsies of nonalcoholic steatohepatitis (NASH) patients. Biochemical and genetic studies show that HCF-1 is O-GlcNAcylated in response to glucose as a prerequisite for its binding to ChREBP and subsequent recruitment of OGT, ChREBP O-GlcNAcylation, and activation. The HCF-1:ChREBP complex resides at lipogenic gene promoters, where HCF-1 regulates H3K4 trimethylation to prime recruitment of the Jumonji C domain-containing histone demethylase PHF2 for epigenetic activation of these promoters. Overall, these findings define HCF-1's interaction with ChREBP as a previously unappreciated mechanism whereby glucose signals are both relayed to ChREBP and transmitted for epigenetic regulation of lipogenic genes.

O-GlcNAc Transferase Recognizes Protein Substrates Using an Asparagine Ladder in the Tetratricopeptide Repeat (TPR) Superhelix.

The essential mammalian enzyme O-GlcNAc Transferase (OGT) is uniquely responsible for transferring N-acetylglucosamine to over a thousand nuclear and cytoplasmic proteins, yet there is no known consensus sequence and it remains unclear how OGT recognizes its substrates. To address this question, we developed a protein microarray assay that chemoenzymatically labels de novo sites of glycosylation with biotin, allowing us to simultaneously assess OGT activity across >6000 human proteins. With this assay we examined the contribution to substrate selection of a conserved asparagine ladder within the lumen of OGT's superhelical tetratricopeptide repeat (TPR) domain. When five asparagines were mutated, OGT retained significant activity against short peptides, but showed limited limited glycosylation of protein substrates on the microarray. O-GlcNAcylation of protein substrates in cell extracts was also greatly attenuated. We conclude that OGT recognizes the majority of its substrates by binding them to the asparagine ladder in the TPR lumen proximal to the catalytic domain.

Uromodulin p.Cys147Trp mutation drives kidney disease by activating ER stress and apoptosis.

Uromodulin-associated kidney disease (UAKD) is caused by mutations in the uromodulin (UMOD) gene that result in a misfolded form of UMOD protein, which is normally secreted by nephrons. In UAKD patients, mutant UMOD is poorly secreted and accumulates in the ER of distal kidney epithelium, but its role in disease progression is largely unknown. Here, we modeled UMOD accumulation in mice by expressing the murine equivalent of the human UMOD p.Cys148Trp point mutation (UmodC147W/+ mice). Like affected humans, these UmodC147W/+ mice developed spontaneous and progressive kidney disease with organ failure over 24 weeks. Analysis of diseased kidneys and purified UMOD-producing cells revealed early activation of the PKR-like ER kinase/activating transcription factor 4 (PERK/ATF4) ER stress pathway, innate immune mediators, and increased apoptotic signaling, including caspase-3 activation. Unexpectedly, we also detected autophagy deficiency. Human cells expressing UMOD p.Cys147Trp recapitulated the findings in UmodC147W/+ mice, and autophagy activation with mTOR inhibitors stimulated the intracellular removal of aggregated mutant UMOD. Human cells producing mutant UMOD were susceptible to TNF-α- and TRAIL-mediated apoptosis due to increased expression of the ER stress mediator tribbles-3. Blocking TNF-α in vivo with the soluble recombinant fusion protein TNFR:Fc slowed disease progression in UmodC147W/+ mice by reducing active caspase-3, thereby preventing tubule cell death and loss of epithelial function. These findings reveal a targetable mechanism for disease processes involved in UAKD.

The Biochemistry of O-GlcNAc Transferase: Which Functions Make It Essential in Mammalian Cells?

O-linked N-acetylglucosamine transferase (OGT) is found in all metazoans and plays an important role in development but at the single-cell level is only essential in dividing mammalian cells. Postmitotic mammalian cells and cells of invertebrates such as Caenorhabditis elegans and Drosophila can survive without copies of OGT. Why OGT is required in dividing mammalian cells but not in other cells remains unknown. OGT has multiple biochemical activities. Beyond its well-known role in adding ß-O-GlcNAc to serine and threonine residues of nuclear and cytoplasmic proteins, OGT also acts as a protease in the maturation of the cell cycle regulator host cell factor 1 (HCF-1) and serves as an integral member of several protein complexes, many of them linked to gene expression. In this review, we summarize current understanding of the mechanisms underlying OGT's biochemical activities and address whether known functions of OGT could be related to its essential role in dividing mammalian cells.

Stereochemically versatile synthesis of the C1-C12 fragment of tedanolide C.

A flexible synthesis of the C1-C12 fragment of Tedanolide C has been accomplished in eight steps from 2-methyl-2,4-pentadienal. Asymmetric hydroformylation of a 1,3-diene allows for the late-stage generation of either C10 epimer with complete catalyst control. Diastereoselective addition of an isobutyryl ß-ketoester dianion to an α,ß-disubstituted chiral aldehyde sets the C5 stereochemistry while installing the geminal dimethyl unit. Differential protection of a syn-1,3-diol is performed as a highly efficient single-pot operation.