Structural basis for 2′-phosphate incorporation into glycogen by glycogen synthase

Glycogen is a glucose polymer that contains minor amounts of covalently attached phosphate. Hyperphosphorylation is deleterious to glycogen structure and can lead to Lafora disease. Recently, it was demonstrated that glycogen synthase catalyzes glucose–phosphate transfer in addition to its characteristic glucose transfer reaction. Glucose-1,2-cyclic-phosphate (GCP) was proposed to be formed from UDP-Glc breakdown and subsequently transferred, thus providing a source of phosphate found in glycogen. To gain further insight into the molecular basis for glucose–phosphate transfer, two structures of yeast glycogen synthase were determined; a 3.0-Å resolution structure of the complex with UMP/GCP and a 2.8-Å resolution structure of the complex with UDP/glucose. Structural superposition of the complexes revealed that the bound ligands and most active site residues are positioned similarly, consistent with the use of a common transfer mechanism for both reactions. The N-terminal domain of the UDP⋅glucose complex was found to be 13.3° more closed compared with a UDP complex. However, the UMP⋅GCP complex was 4.8° less closed than the glucose complex, which may explain the low efficiency of GCP transfer. Modeling of either α- or β-glucose or a mixture of both anomers can account for the observed electron density of the UDP⋅glucose complex. NMR studies of UDP-Glc hydrolysis by yeast glycogen synthase were used to verify the stereochemistry of the product, and they also showed synchronous GCP accumulation. The similarities in the active sites of glycogen synthase and glycogen phosphorylase support the idea of a common catalytic mechanism in GT-B enzymes independent of the specific reaction catalyzed.Branched glucose polymers, glycogen and starch, are used by nearly all living organisms as an osmotically neutral means of energy storage. These polymers are constructed through the formation of α-1,4-glycosidic bonds and branching points, which use α-1,6-glycosidic linkages (1, 2). Glycogen is found in most animal, fungi, bacteria, and archaea, whereas photosynthetic eukaryotes or their nonphotosynthetic derivatives (such as apicomplexa parasites) use starch (2). Although primarily a glucose reservoir, glycogen also contains minor amounts of both glucosamine and phosphate (3). In animals and yeast, glycogen biosynthesis requires the action of three enzymes: glycogenin, glycogen synthase, and the branching enzyme. Most fungal and animal glycogen synthases use uridine diphosphoglucose (UDP-Glc) as the glucose donor, whereas bacterial, some parasitic, and plant glycogen/starch synthases use adenosine diphosphoglucose (ADP-Glc). Glycogen breakdown is catalyzed by glycogen phosphorylase which uses the cofactor pyridoxal 5′-phosphate (PLP) in combination with inorganic phosphate to phosphorolytically cleave glycogen and generate glucose-1-phosphate.Glycogen synthase (GS) is classified as a glycosyltransferase (GT), a large superfamily of enzymes that transfer a sugar residue from an activated sugar donor to an acceptor molecule (4). To date, GTs have been grouped into more than 90 families (5) (www.cazy.org) based on sequence similarity. The nucleotide sugar-dependent GTs have been shown to adopt either GT-A or GT-B folds which consist of two associated domains, one of which contains a dinucleotide fold responsible for donor nucleotide recognition (4). The GT-A fold consists of two tightly associated domains, resulting in some describing it as a single-domain fold, whereas the GT-B fold is composed of two structurally distinct dinucleotide folds characteristically separated by a deep interdomain cleft (6). GS enzymes have been classified as GT-B enzymes that are further subdivided into two families, GT3 and GT5 (7). The bacterial, archaeal, and plant glycogen/starch synthase enzymes are grouped into the GT5 family, whereas the mammalian, yeast, and fungal enzymes are grouped into the GT3 family. Although it catalyzes the breakdown of glycogen, glycogen phosphorylase is structurally classified as a GT-B enzyme and is placed in the GT35 family. Functionally, the GTs are classified as either retaining or inverting, in reference to the stereochemistry at the anomeric carbon of the substrates and products; both GS and phosphorylase are retaining-type GTs. Although the catalytic mechanism of inverting GTs is generally well understood, the mechanism used by the retaining GTs remains elusive.Glycogen is known to contain minor amounts of covalently attached phosphate, and excessive accumulation of covalent phosphate is thought to underlie Lafora disease, a devastating form of myoclonus epilepsy that is ultimately fatal (8–10). Where the phosphate is located on the glucose residues with glycogen is an active area of investigation, as is the mechanism by which it is introduced. Tagliabracci et al. recently showed that GS not only catalyzed the transfer of glucose but also incorporated the β-phosphate of UDP-Glc at a frequency of 1 phosphate per ∼10,000 glucoses, with release of UMP rather than UDP (11). This study also revealed that the phosphate in glycogen was present as C2- and C3-phosphomonoesters (11). It was proposed that the presence of phosphomonoesters in glycogen was due to the ability of GS to use either glucose-1,2-cyclic phosphate (GCP) or glucose-1,3-cyclic phosphate, which can be formed from the breakdown of UDP-Glc (12, 13), as donors in the active site (11). By this mechanism, GS could use the same catalytic mechanism regardless of whether UDP-Glc or the cyclic phosphates were present in the active site. No physiological role has yet been associated with phosphate in glycogen, although the presence of excessive phosphate is known to be detrimental to glycogen structure (14). In contrast, another report has refuted the ability of glycogen synthase to catalyze this incorporation and identified, in addition to the aforementioned 2′ and 3′ phosphate, glucose residues modified at the 6′ position with covalent phosphate (15). The authors suggested that the radioactive phosphate we observed in glycogen elongated by glycogen synthase was, in fact, due to the retention of nonspecifically bound β-³²P-UDP in the glycogen (15). However, laforin phosphatase was able to remove the phosphate from this labeled glycogen (11), and there is no evidence to suggest that Laforin can catalyze the removal of phosphate groups from UDP. Our laboratory continues to seek a mechanistic explanation for the ability of glycogen synthase to not only catalyze the incorporation of glucose into glycogen but also elucidate the role of glycogen synthase in covalent phosphate incorporation. The mechanistic enigma surrounding glycosyl transfer and the widespread significance of the reactions catalyzed by GTs make investigation of their catalytic mechanism important because it is central both to a fundamental understanding of their chemistry and toward therapeutic applications in diseases such as Lafora disease.In this paper, we report the crystal structure of the allosterically activated form of yeast glycogen synthase (Gsy2p) in complexes with either UDP⋅Glc or UMP⋅GCP. In each structure, one of the four subunits of Gsy2p has captured a catalytically relevant form of the nucleotide and donor sugar molecule, whereas the other subunits retain only the nucleotide. NMR studies of UDP-Glc hydrolysis by Gsy2p were used to verify the stereochemistry of the reaction products and unexpectedly revealed the production of GCP. The structural similarities observed in the Gsy2p complexes with either Glc or GCP to the Escherichia coli glycogen synthase and the maltodextrin phosphorylase structure bound to 1-deoxyglucose and maltodextran are consistent with the use of a common mechanism of glucosyl transfer in GT-B enzymes.     

Learning relational policies from electronic health record access logs

Modern healthcare organizations (HCOs) are composed of complex dynamic teams to ensure clinical operations are executed in a quick and competent manner. At the same time, the fluid nature of such environments hinders administrators' efforts to define access control policies that appropriately balance patient privacy and healthcare functions. Manual efforts to define these policies are labor-intensive and error-prone, often resulting in systems that endow certain care providers with overly broad access to patients' medical records while restricting other providers from legitimate and timely use. In this work, we propose an alternative method to generate these policies by automatically mining usage patterns from electronic health record (EHR) systems. EHR systems are increasingly being integrated into clinical environments and our approach is designed to be generalizable across HCOs, thus assisting in the design and evaluation of local access control policies. Our technique, which is grounded in data mining and social network analysis theory, extracts a statistical model of the organization from the access logs of its EHRs. In doing so, our approach enables the review of predefined policies, as well as the discovery of unknown behaviors. We evaluate our approach with 5 months of access logs from the Vanderbilt University Medical Center and confirm the existence of stable social structures and intuitive business operations. Additionally, we demonstrate that there is significant turnover in the interactions between users in the HCO and that policies learned at the department-level afford greater stability over time.     

Enabling realistic health data re-identification risk assessment through adversarial modeling

ObjectiveRe-identification risk methods for biomedical data often assume a worst case, in which attackers know all identifiable features (eg, age and race) about a subject. Yet, worst-case adversarial modeling can overestimate risk and induce heavy editing of shared data. The objective of this study is to introduce a framework for assessing the risk considering the attacker’s resources and capabilities.Materials and MethodsWe integrate 3 established risk measures (ie, prosecutor, journalist, and marketer risks) and compute re-identification probabilities for data subjects. This probability is dependent on an attacker’s capabilities (eg, ability to obtain external identified resources) and the subject’s decision on whether to reveal their participation in a dataset. We illustrate the framework through case studies using data from over 1 000 000 patients from Vanderbilt University Medical Center and show how re-identification risk changes when attackers are pragmatic and use 2 known resources for attack: (1) voter registration lists and (2) social media posts.ResultsOur framework illustrates that the risk is substantially smaller in the pragmatic scenarios than in the worst case. Our experiments yield a median worst-case risk of 0.987 (where 0 is least risky and 1 is most risky); however, the median reduction in risk was 90.1% in the voter registration scenario and 100% in the social media posts scenario. Notably, these observations hold true for a wide range of adversarial capabilities.ConclusionsThis research illustrates that re-identification risk is situationally dependent and that appropriate adversarial modeling may permit biomedical data sharing on a wider scale than is currently the case.     

The inhibition of human glutathione S-transferases activity by plant polyphenolic compounds ellagic acid and curcumin.

Glutathione S-transferases (GSTs) are multifunctional detoxification proteins that protect the cell from electrophilic compounds. Overexpression of GSTs in cancer results in resistance to chemotherapeutic agents and inhibition of the over expressed GST has been suggested as an approach to combat GST-induced resistance. The inhibition of human recombinant GSTs by natural plant products was investigated in this study. Using 1-chloro-2,4 dinitrobenzene (CDNB) as a substrate, ellagic acid and curcumin were shown to inhibit GSTs A1-1, A2-2, M1-1, M2-2 and P1-1 with IC(50) values ranging from 0.04 to 5 microM whilst genistein, kaempferol and quercetin inhibited GSTs M1-1 and M2-2 only. The predominant mode of inhibition with respect to the G and H-sites were mixed inhibition and uncompetitive to a lesser extent. The K(i) (K(i)(')) values for ellagic acid and curcumin with respect to GSH and CDNB were in the range 0.04-6 microM showing the inhibitory potency of these polyphenolic compounds. Ellagic acid and curcumin also showed time- and concentration-dependent inactivation of GSTs M1-1, M2-2 and P1-1 with curcumin being a more potent inactivator than ellagic acid. These results facilitate the understanding of the interaction of human GSTs with plant polyphenolic compounds with regards to their role as chemomodulators in cases of GST-overexpression in malignancies.