Integration of Sugar Metabolism and Proteoglycan Synthesis by UDP-glucose Dehydrogenase

Abstract

Regulation of proteoglycan and glycosaminoglycan synthesis is critical throughout development, and to maintain normal adult functions in wound healing and the immune system, among others. It has become increasingly clear that these processes are also under tight metabolic control and that availability of carbohydrate and amino acid metabolite precursors has a role in the control of proteoglycan and glycosaminoglycan turnover. The enzyme uridine diphosphate (UDP)-glucose dehydrogenase (UGDH) produces UDP-glucuronate, an essential precursor for new glycosaminoglycan synthesis that is tightly controlled at multiple levels. Here, we review the cellular mechanisms that regulate UGDH expression, discuss the structural features of the enzyme, and use the structures to provide a context for recent studies that link post-translational modifications and allosteric modulators of UGDH to its function in downstream pathways:

Introduction

Synthesis of proteoglycans (PGs) is a complex multistep process that occurs primarily in the lumen of the Golgi apparatus, using uridine diphosphate (UDP)-esterified sugars and amino sugars provided from the cytosol via nucleotide sugar transporters (NST).¹ Once in the lumen, nucleotide sugars may be further modified and/or added directly to the core protein of a nascent PG. Initiation of each chain requires UDP-xylose, which is the decarboxylated product of UDP-glucuronate, another essential precursor of the growing chain.² Many of the nucleotide sugars can be supplied by more than one metabolic route in the cell. However, UDP-glucuronate, and ultimately UDP-xylose, are dependent upon UDP-glucose dehydrogenase (UGDH), a single essential gene product that is ubiquitously distributed but is most highly expressed in specific tissues such as the liver, kidney, prostate, and mammary.³

UGDH is a cytosolic enzyme that catalyzes the oxidation of UDP-glucose to UDP-glucuronate, supplying this metabolite for three immediate downstream fates (Fig. 1). In addition to the synthesis of PGs, UDP-glucuronate is also a requisite substrate for glucuronidation, which is a key phase 2 detoxification pathway in the ER lumen, and for production of the extracellular matrix glycosaminoglycan (GAG) component hyaluronan (HA). Each of these pathways is essential for normal cellular function, and the dysfunction of any of the three is associated with multiple pathological outcomes. Because of this, UGDH has been the focus of drug development efforts, and these aspects of its biology have been reviewed elsewhere.⁴ In this review, we will provide an overview of cellular mechanisms that regulate UGDH expression, discuss the molecular features of the enzyme, and provide a context for recent studies relating structural characterizations of UGDH to its cellular function in downstream pathways. An understanding of the complex allosteric interactions occurring within the enzyme reveals insights to mechanisms that underlie its role as a sensory node in metabolite distribution.

Figure 1.

Subcellular distribution of UDP-glucuronate and downstream pathways. UDP-glucose dehydrogenase (UGDH) catalyzes the cytosolic NAD-dependent oxidation of UDP-glucose to UDP-glucuronate (UDP-glcA), which is a critical upstream precursor for three possible processes: (1) hyaluronan synthesis at the plasma membrane; (2) UDP-xylose and proteoglycan synthesis in the Golgi; and (3) hormone (e.g., DHT) glucuronidation in the ER. UDP-glcA is transported into the lumens of ER and Golgi by specific antiporters of the SLC35 class that concurrently export UMP, the leaving group released by UDP-sugar consumption. UDP-glcA is converted to UDP-xylose in the ER and Golgi by UDP-xylose synthase (UXS1). Abbreviations: DHT, dihydrotestosterone; UDP, uridine diphosphate; UMP, uridine monophosphate; PM, plasma membrane.

Regulation and Function of UGDH in Development and Disease

As the sole route for synthesis of an essential nucleotide sugar precursor, UGDH has a critical role in synthesis of GAGs and PGs. Significant advances in understanding how GAG polymers and the PG core proteins impact development has resulted from studies of UGDH loss of function in multiple diverse organisms. In flies, worms, and plants, which do not express HA synthases, the UDP-glucuronate product is directed to the assembly of chondroitin sulfate and heparan sulfate PGs. In plants, UGDH loss causes severe cell wall composition defects that stunt root development.⁵ Lack of UGDH in Drosophila results in a phenotype called sugarless, where core proteins are expressed without GAG chains. Consequently, the temporally specific gradients of Wnt, fibroblast growth factor (FGF), and TGFβ required for appropriate wing development do not form.⁶ This mutant revealed the critical role of the GAG chains in binding and limiting the diffusion of developmental growth and differentiation factors. Similarly, in C. elegans, morphogenesis defects arise during vulval differentiation due to the absence and/or GAG-deficiency of PGs that control cell division,^7–9 and gastrulation stalls in UGDH null mice from loss of the FGF gradient.¹⁰ In organisms that produce HA, UGDH deletions or mutants with reduced activity have been analyzed to reveal the unique roles of HA and PGs in development. For example, in Xenopus, UGDH deletion results in aborted gastrulation, which is compounded by GAG insufficiency and the loss of HA production to physically drive organ formation.¹¹ Zebrafish UGDH mutants have faulty cardiac valve development due to the absence of HA production needed for formation of cardiac cushion and signaling of endothelial-mesenchymal transition.¹² UGDH has more recently been shown to provide HA that is mechanically essential for ear development, and demonstrated the role of GAG-mediated gradients in the initiation of mammalian left-right asymmetry.¹³

Basal UGDH expression requires the transcription factor Sp1.¹⁴ Numerous factors known to impact HA or PG synthesis have been shown to do so via Sp1-mediated control of UGDH levels. Examination of the human UGDH basal promoter revealed multiple CG and CT-rich motifs, which transcription factors of the Sp1 family are frequently found to recognize. These putative Sp1 binding sites have been functionally implicated in TGFβ-mediated stimulation of UGDH expression in fibroblasts and HepG2 cells,¹⁴ as well as Hela cells¹⁵ and primary articular surface cells.¹⁶ Dynamic regulation of UGDH expression both temporally and in a tissue-specific context has been found to result from the differential expression of Sp1,¹⁷ and its negative co-regulators Sp3 and c-Krox,^18,19 both of which can compete with Sp1 to reduce its occupancy at the requisite consensus activation sequences, thereby reducing UGDH transcription in response to HA and PG repressive factors. In particular, TGF-β treatment stimulated the secretion of sulfated GAGs and HA from primary articular surface cells in a manner dependent on p38MAPK signaling, and on the elevation of UGDH.¹⁶ In a later study, inhibiting the activation of p38MAPK in primary articular chondrocytes was shown to reduce Sp1 transcription while increasing Sp3 and c-Krox, leading to reduced chromatin immunoprecipitation of Sp1 at the UGDH promoter and a significant diminution of HA and sulfated GAGs.¹⁹

Transcriptional regulation of UGDH can also occur through androgen and estrogen receptor-mediated activation via their respective response elements in the distal UGDH promoter. In hormone-responsive tissues such as the prostate, UGDH expression is increased by the presence of hormone receptor ligands (e.g., DHT), and its upregulation stimulates androgen glucuronidation and/or PG production,^20,21 while in articular chondrocytes, 17β-estradiol up-regulates UGDH expression to support biosynthesis of PGs.²² Recently, Kruppel-like factors KLF2²³ and KLF4²⁴ have also been defined as critical regulators of UGDH transcription in cardiac development and in tumor progression, respectively, in the latter of which the methylation of abundant CpG elements in the UGDH promoter generates higher affinity recognition sites for KLF4.

In general, the negative regulation of UGDH corresponds with reduced accumulation of HA and sulfated GAGs in many cell and tissue types. Examples of negative regulators include hypoxia, where a complete mechanism was not defined but occurred with reduced Sp1 expression;¹⁴ some xenobiotics that may act as ligands for the PPARα/RXR transcriptional heterodimer through a functionally defined PPRE suppressive element in the UGDH promoter;^25,26 and IL-1β, a pro-inflammatory cytokine that activates SAP/JNK signaling and increases Sp3 and c-Krox, leading to impaired chondrogenesis and contributing to osteoarthritis.¹⁹

Additional mechanisms that lead to reduced UGDH expression have been revealed by investigating congenital developmental defects linked to UGDH deficiency. Missense mutations in UGDH that interfere with quaternary assembly of the enzyme result in impaired catalytic activity and reduced stability of UGDH expression, ultimately manifesting in conditions such as cardiac valve malformation,²⁷ global developmental delay,²⁸ and epileptic encephalopathy.²⁹ Loss-of-function variants of UGDH in these studies were reported to impair the binding of substrate and cofactor, and/or to disrupt hexameric assembly and stability of the enzyme as discussed in the next section. Patients showed a general deficiency in HA and sulfated GAGs, particularly in specific areas of the brain, that underscored the essential demand for the UGDH product in tissue morphogenesis.

Structure, Mechanism and Allosteric Control of UGDH

The catalytic mechanism of UGDH has been studied extensively and has been summarized in recent reviews.^4,30 However, the importance of oligomerization and conformational dynamics has only recently begun to be appreciated. The eukaryotic UGDH apoenzyme exists in a dynamic equilibrium between dimeric, tetrameric, and hexameric states,^31,32 which are sustained by an intricate network of non-covalent bonds. UGDH is maximally functional as a homohexameric assembly, consisting of a trimer of dimers (Fig. 2). Targeted disruption of critical subunit–subunit interactions by site-directed mutagenesis generates dimeric forms of the enzyme with diminished activity. The epsilon amino group of Lys 94 in the active hexamer forms a hydrogen bond with the backbone carbonyl oxygen of Met 98 and a salt bridge with the side chain carboxylate of Glu 360 in an adjacent subunit. Substitution of this lysine with a glutamate shifts the oligomeric equilibrium nearly exclusively to the dimeric state, significantly increases the K_m values for NAD⁺ and UDP-glucose, and dramatically decreases k_cat.³³ Proximal to this subunit–subunit interface, an ordered water molecule bridges the interface by forming hydrogen bonds with the hydroxyl groups of Thr 325 and Thr 327 in one subunit and the carboxylate of Asp 105 and the hydroxyl group of Tyr 96 in a neighboring subunit, thereby further stabilizing the hexameric assembly. Replacement of Thr 325 with an aspartate disrupts this hydrogen bond network, resulting in a dimeric enzyme. Although the impact is not as dramatic as the K94E substitution, the T325D mutation reduces catalytic activity more than 5-fold with modest impact on K_m values,³¹ reinforcing the conclusion that the hexameric form of the enzyme is the catalytically relevant oligomeric state. In a later study, a triple point mutant was created within that secondary structure element, F323T/N324T/T325D, and was confirmed to have similar loss of activity in the dimeric enzyme.³⁴ Conversely, one group used unnatural amino acid incorporation at position 468 (Fig. 3C) and photoactivated cross-linking to generate a fixed, obligate hexameric species,³⁵ while another group engineered an obligate hexamer of UGDH by deletion of residue 132 to restrict movement of a critical active site loop.³⁶ In both cases, the constrained hexameric species had compromised enzymatic activity, further supporting the notion that dynamic conformational switching at the dimer–dimer interface is necessary for optimal UGDH activity.

Figure 2.

Hexameric assembly of human UDP-glucose dehydrogenase (UGDH) subunits. The abortive ternary complex of the substrate, UDP-glucose, and the reduced cofactor, NADH (2Q3E⁴²), and the feedback inhibited complex of UDP-xylose and NAD⁺ (3PTZ³²) are shown in ribbon representation and illustrate the active (E) and inhibited (E^Ω) sub-state conformations of the enzyme, respectively. The enzyme can be described as a trimer of dimers, and individual dimers are illustrated in dark/light pairs of red, green, and blue. The α6 helix of the allosteric switch is highlighted in pink, and the anchor point of the 30-residue intrinsically disordered C-terminal tail, ID-tail, is represented as a black sphere. Ligands are shown in space-filling representation with oxygen colored in red, nitrogen in blue, carbon in gray, and phosphorus in orange, and sites of observed clinical mutations are highlighted in yellow. Formation of the active complex, E, or the UDP-xylose inhibited complex, E^Ω, leads to defined movement of the active site Thr 131 loop, which translates through the allosteric switch via the α6 helix to the dimer/dimer interface, leading to the distinct conformations observed. Abbreviation: UDP, uridine diphosphate.

Figure 3.

Potential contributions of amino acid side chains at positions corresponding to the clinically observed point mutations near the allosteric switch and ID-tail. The models are colored as in Fig. 2, and relevant atoms within hydrogen bond distance are illustrated as solid black lines. (A) Thr 131 of the allosteric switch, highlighted in pink, participates in an extensive hydrogen bond network at the active site of the enzyme which positions the α6 helix optimally for the formation of the E sub-state. (B) UDP-xylose lacks the C5’-hydroxymethyl substituent of UDP-glucose, allowing the Thr 131 loop to move deeper into the active site, altering the position of the α6 helix at the dimer/dimer interface. Arg141 and Glu217 are particularly relevant clinically as discussed in the text. (C) The ID-tail encompasses residues 465–494 and modulates the energetic landscape of the enzyme, pushing the conformational equilibrium to favor the formation of the E^Ω sub-state. (Please note that the illustrated subunit cannot be modeled beyond Lys 464.) As illustrated in the panel, there are numerous residues, including Arg 317, Arg 443, and His 449, that can form favorable hydrogen bonds that likely anchor the base of the ID-tail. Abbreviations: UDP, uridine diphosphate; ID, intrinsically disordered C-terminal tail.

Hexamer formation is required for maximal activity, but conformational dynamics also contribute to catalytic function and its regulation. Hexameric human UGDH has been proposed to sample three distinct sub-states: an active hexamer (E), an inactive hexamer (E*), and a UDP-xylose inhibited hexamer (E^Ω). This model is supported by mutagenesis studies, coupled with detailed kinetic and biophysical characterizations.^{32–34,36–41} Briefly, the key pivot in each of these three sub-states is the placement of the loop containing Thr 131, and its impact on the position of the sequence adjacent α6 helix (Fig. 3A and B). In the active hexamer with substrate and cofactor present, the backbone amide of Thr 131 forms a hydrogen bond to the 3’-hydroxyl group of the nicotinamide ribose and the side chain hydroxyl group forms a hydrogen bond to an ordered water molecule (Fig. 3A). The water can hydrogen bond with the C6’-hydroxyl group of UDP-glucose, the active site thiol of Cys 276, and the 2’-hydroxyl group of the nicotinamide ribose (Fig. 3A). In the inhibited hexamer (E^Ω), Thr131 moves deeper into the active site due to the absence of the hydroxymethyl substituent at the C5 position of xylose, and displaces the nicotinamide ring of the cofactor (Fig. 3B).³² The loop containing Thr 131 adopts an intermediate position in the inactive hexamer (E*), leading to partial occlusion of the cofactor and substrate binding sites (not shown).³⁴ These movements of the Thr 131 loop require structural rearrangements in the protein core and produce shifts in the positioning of the α6 helix, which impacts the dimer–dimer interactions and contributes to the cooperativity observed in ligand binding. Under physiological conditions, the net impact is that the predominant E* sub-state favors binding of UDP-xylose over UDP-glucose, making the enzyme more responsive to changes in metabolite levels.³⁴ Such dynamic conformational changes and strong consequence on quaternary assembly are consistent with potential for complex allosteric regulation.

A number of clinically observed point mutations have been identified that likely impact the dynamics of the allosteric switch [Fig. 3A and B]. A heterozygous substitution at position 141 (R141C), which is located in helix 6 of the allosteric switch, was previously characterized and found to dramatically reduce the stability of the enzyme, compromising high-order oligomerization, despite having relatively modest impacts on steady-state kinetic parameters.²⁷ In the holo complex (Fig. 3A), Arg 141 is pointed away from the active site and forms numerous structural hydrogen bonds. In contrast, Arg 141 samples two distinct conformations in the UDP-xylose inhibited structure, one comparable to the holo enzyme (3 subunits) and one less constrained conformation (3 subunits; Fig. 3B), that orients toward the Thr 131 loop. It may be informative to re-evaluate the contributions of this residue with respect to UDP-xylose inhibition, given its likely involvement in positioning of the allosteric switch. A recent study of UGDH mutations associated with recessive epileptic encephalopathy²⁹ identified numerous residues likely to be important for protein stability and enzymatic function (Figs. 2 and 3, yellow residues). Similar to Arg 141, Glu 217 forms hydrogen bonds that are likely critical to the positioning of the allosteric switch (Fig. 3B) and its replacement with an aspartate residue would be predicted to impact the dynamics of the allosteric switch and the overall stability of the enzyme.

An additional level of regulation of UGDH activity resides in the intrinsically disordered C-terminal tail (ID-tail; residues 465–494). Both the predicted location of the ID-tail and the α6 helix of the allosteric switch are located near the dimer–dimer interface (Fig. 2). Initially regarded as inconsequential since its removal had little impact on steady state kinetic parameters,⁴² the ID-tail has been demonstrated to be an integral component of the allosteric regulation of the enzyme by altering its energetic landscape.⁴⁰ Electron density for this stretch of amino acids has not been observed in the numerous human UGDH structures determined, but its removal results in an approximately 10-fold increase in the K_i for UDP-xylose⁴⁰ in the intact hexamer, with a similar increase observed in an engineered dimer, suggesting that hexamer assembly is not essential for the entropic contributions of the ID-tail. Importantly, neither the sequence nor the overall charge is essential to confer wild-type kinetic parameters. Instead, the length of the ID-tail strongly correlates with the relative affinity for UDP-xylose, suggesting that the entropic force produced by the ID-tail shifts the conformational equilibrium toward the E^Ω sub-state, favoring inhibitor binding.

As shown in Figs. 2 and 3C, numerous clinically observed mutations (yellow) cluster around the anchor point (black spheres) of the intrinsically disordered C-terminal tail. Mutations arising in this region are becoming increasingly reported and occur only in the heterozygous condition, suggesting the dimer–dimer interface is a hotspot for non-lethal substitutions that result in significantly reduced UGDH functionality. For example, an arginine substitution at position 317 to a glutamine (R317Q) has been associated with recessive developmental epileptic encephalopathy²⁹ and developmental delay and axial hypotonia.²⁸ Arg 317 forms a salt-bridge with Glu 460 and this interaction likely contributes to the positioning of the ID-tail. The helix containing Arg 317 is also immediately upstream of Thr 325, which is critical to efficient hexamer formation.³¹ A glutamine substitution would remove the positive charge of the guanidinium group, disrupting a critical salt bridge with the negatively charged Glu 460, potentially disrupting the subunit–subunit interface. Although the net outcome is predicted to be similar, the structural interpretations provided in Alhamoudi et al.²⁸ should be viewed with skepticism.

Sampling of the described UGDH sub-states differentially exposes residues on the surface of the enzyme, which may impact yet to be determined protein–protein interactions. Similarly, the ID-tail may mediate formation of novel complexes. Although the length of the ID-tail has been shown to be the major determinant when examining the impact on UDP-xylose binding, there is significant sequence conservation in this region.⁴⁰ Residues in the ID-tail may be bound by other proteins to moderate its entropic contributions or to direct the enzyme’s subcellular localization. Interestingly, tyrosine 473 in the ID-tail has recently been shown to be phosphorylated (discussed below⁴³), suggesting that the ID-tail may make additional contributions to UGDH regulation.

Post-translational Regulation of UGDH and PG Production

UGDH activity is sensitive to multiple layers of regulation in the cellular context that are more clearly informed by the structural and biophysical data. Of note are cellular impacts of substrate and cofactor availability, product inhibition and product removal, downstream rate-limiting pathway activities, and post-translational modifications. The substrate, UDP-glucose, derives almost exclusively in mammalian systems from cellular glucose uptake following the uridine triphosphate (UTP)-dependent conversion of glucose-6-phosphate, via glucose-1-phosphate and UDP-glucose pyrophosphorylase in the Leloir pathway. As discussed above, presence of both substrate and cofactor coordinately activates UGDH in a concentration-dependent manner, and the buildup of cytosolic UDP-glucuronate or its downstream product, UDP-xylose, inhibits UGDH. UDP-xylose is produced from UDP-glucuronate in the lumen of the ER and Golgi by the transmembrane enzyme UDP-xylose synthase (UXS1, Fig. 1). UDP-xylose removal by deletion of UXS1 can increase cellular UGDH activity by an order of magnitude or more,⁴⁴ and the potency of UDP-xylose as an inhibitor of UGDH in vitro has long been established.⁴⁵ Structural studies suggest UDP-xylose works through an atypical allosteric mechanism in which UDP-xylose binds at the UDP-glucose binding site, inducing a conformational change that reduces affinity for substrate, strengthens dimer–dimer associations, and stabilizes the inactive E^Ω sub-state.^32,46 Although both the UDP-glucuronate and NADH products of UGDH can inhibit the enzyme, the inhibition by NADH may be more physiologically significant, given its strong potency in vitro.⁴⁵

Expression levels of UGDH impact the localization and flux of UDP-sugars, but UGDH activity can also be modulated by the flux of its products through downstream pathways. UDP-glucuronate is transported into the ER and Golgi via nucleotide sugar transporters (NST), which are gradient-driven antiporters that co-transport uridine monophosphate (UMP) back to the cytosol. It is probable that different transporters have differing rates of nucleotide sugar antiport that promote the activity of UGDH by facilitating inhibitory product removal. SLC35B1 transports UDP-glucuronate to the ER lumen in HepG2 liver adenocarcinoma cells, which stimulates glucuronidation in the presence of xenobiotic substrates.⁴⁷ Use of UDP-glucuronate in this pathway can promote activation of UGDH both by limiting product inhibition and by direct competition with UDP-xylose production, and is dependent on glucose availability. Flux of UDP-glucuronate through PG synthesis and ultimate production of complex glycans requires an array of these antiporters. Additional antiporters such as SLC35A3 are specific to the transport of UDP-N-acetylglucosamine into the Golgi and are required for the elongation of glycan polymers.⁴⁸ Like UGDH deletion, the loss of NST activity is associated with multiple developmental disorders.^49,50 Both SLC35A3 and SLC35D1 loss are responsible for severe and/or lethal skeletal dysplasia.^51,52 A missense mutation in SLC35D1 was described recently in a patient with the skeletal disorder known as Schneckenbecken-like dysplasia.⁵² Structural modeling of the mutant transporter predicted impaired binding and/or release of the UDP-glucuronate by residues expected to occur within the channel, and in vitro kinetic assays using lipid-reconstituted enzyme vesicles revealed that the mutant retained only 2–4% of the activity of the wildtype enzyme.⁵² The impact on flux of UDP-glucuronate through other pathways was not characterized, but it would be interesting to determine whether UGDH activity was also altered by loss of this NST.

Metabolite regulation of UGDH has rapid and dynamic impacts on cellular processes. For example, knocking down UGDH expression or pharmacological scavenging of UDP-glucuronate through the glucuronidation pathway by 4-methylumbelliferone is sufficient to inhibit cellular invasion and colony formation in a breast cancer model, and the authors suggested that metabolic reprogramming to support epithelial-mesenchymal transition may require the shift to glucose metabolism because remodeling of the extracellular matrix involves upregulation of HA production.⁵³ In contrast, another study found that knocking down UGDH also reduced the migration of lung adenocarcinoma cells, but feeding back UDP-glucuronate and HA was insufficient to recover this effect,⁵⁴ suggesting it is not dependent on the UGDH product. Such apparently contrasting results may be partially reconciled by the finding that genetically and pharmacologically reducing HA precursor pools, UDP-N-acetylglucosamine and UDP-glucuronate, stimulates HA synthase 3 (HAS3) endocytosis, whereas excess UDP-N-acetylglucosamine caused vesicle recycling to the plasma membrane, promoted HAS3-positive extracellular vesicle shedding, and increased metastatic potential.⁵⁵ Furthermore, upregulation of UGDH has been shown to increase PG production at the loss of glucuronide output, suggesting there is a coordinated process involving UGDH that drives intracellular priority in use of UDP-sugar pools.^20,21 Since high levels of UGDH activity have the potential to impact dynamic flux of NAD⁺/NADH ratios, it is also relevant to consider that UGDH impacts may be partially achieved through processes involving these redox cofactors. This is underscored by a recent report linking activation of the NAD⁺-dependent deacetylase, SIRT1, to the suppression of HA production via the transcriptional regulation of HAS2 (Caon et al.⁵⁶ and reviewed in Caon et al.⁵⁷).

Additional Roles for UGDH

The clinical significance of UGDH expression in the context of prostate cancer biopsies was previously established by quantitative immunofluorescence and revealed the value of UGDH as a field effect biomarker.⁵⁸ Subsequently, UGDH was found elevated in epithelial-mesenchymal transition of invasive breast cancer, where its role is hypothesized to be in support of metabolic reprogramming that fuels HA production.⁵³ Two recent studies have now reported the detection of UGDH protein in the nucleus,^43,54 opening the possibility for previously unappreciated functions in signal transduction and transcriptional regulation. The quantification of UGDH in the nucleus was associated with poorly differentiated cells, larger tumors, and overall reduced survival in lung adenocarcinoma patients.⁴³ Mechanistically, upon extracellular stimulation with epidermal growth factor (EGF), UGDH was found to interact with the cytosolic-nuclear translocating Hu antigen R (HuR), an RNA stabilizing protein. Importantly, this interaction was dependent on the phosphorylation of UGDH at tyrosine 473. As discussed above, this residue is located within the ID-tail, so its phosphorylation presumably alters the conformational dynamic among UGDH subunits to expose alternative interacting motifs. Once bound, the UGDH-HuR complex could be detected within the nucleus, where UGDH may relieve inhibition of HuR by UDP-glucose, converting it to UDP-glucuronate and thereby allowing HuR to stabilize SNAI1 mRNA.⁵⁴ As a result, increased Snail expression activated epithelial-mesenchymal transition by initiating the transcriptional switch from E-cadherin to N-cadherin, and promoted lung metastasis in mice. It is important to note that the studies quantifying nuclear UGDH emphasize a strong level of nuclear expression that has not been previously observed in other studies. The polyclonal antibodies used for the studies were commercially available but a rigorous validation was not included in the manuscript.

The notion that UGDH-mediated adjustment of the nucleotide sugar equilibrium could impact gene expression via RNA binding proteins inside the cell (e.g., as shown for HuR and SNAI1) raises the obvious possibility that UGDH activity could also adjust UDP-glucose to influence extracellular signaling. UDP-glucose is released to the extracellular space through the constitutive Golgi secretory pathway. Many cell types express purinergic receptors such as P2Y14, which is a G-protein-coupled receptor that specifically recognizes UDP and UDP-glucose as ligands, acting via inhibitory G proteins to suppress cAMP production (reviewed in Lazarowski and Harden⁵⁹). Inhibition of UGDH would be expected to increase UDP-glucose signaling through P2Y14, the effect of which is microenvironment dependent, but includes the induction of osteoclast formation in conjunction with RANKL,⁶⁰ stimulation of macrophages in the acute inflammatory response through STAT1 phosphorylation and potentiation of RARβ signaling,⁶¹ and aggravation of ischemic acute kidney injury in cardiac surgery patients.⁶² UDP-glucose increases are also known to provide a substrate for the enzyme UDP-glucose ceramide glucosyltransferase, which is a component of sphingolipid metabolism that mitigates ceramide toxicity normally, but promotes therapeutic resistance in chronic myeloid leukemia⁶³ and alters metabolic fuel dependence in breast cancer.⁶⁴

In conclusion, increased numbers of associations have been reported between the function of UGDH, its inter-subunit contacts, and its heterologous protein–protein interactions, which supports a role for UGDH in prioritizing metabolite distribution among its critical downstream pathways. Novel post-translational modifications of UGDH in response to extracellular signals that trigger motility and proliferation are an insight into nuances of the mechanisms that link HA and PGs to cellular processes. It is clear that UGDH post-translational modifications and protein–protein interactions are part of transient and reversible metabolic reprogramming events that support such processes in response to microenvironment conditions. The recent biophysical insights about atomic and molecular level structure perturbations and their impacts on UGDH function in support of PG production, leading to sustained development of tissues or tumors, provide essential links in the cascade underlying metabolic precursor provision and response to metabolic demand.