Effects of osmolytes under crowding conditions on the properties of muscle glycogen phosphorylase b.

The study of the relationship between the activity and stability of enzymes under crowding conditions in the presence of osmolytes is important for understanding the functioning of a living cell. The effect of osmolytes (trehalose and betaine) on the secondary and tertiary structure and activity of muscle glycogen phosphorylase b (Phb) under crowding conditions created by PEG 2000 and PEG 20000 was investigated using dynamic light scattering, differential scanning calorimetry, circular dichroism spectroscopy, fluorimetry and enzymatic activity assay. At 25 °C PEGs increased Phb activity, but PEG 20000 to a greater extent. Wherein, PEG 20000 significantly destabilized its tertiary and secondary structure, in contrast to PEG 2000. Trehalose removed the effects of PEGs on Phb, while betaine significantly reduced the activating effect of PEG 20000 without affecting the action of PEG 2000. Under heat stress at 48 °C, the protective effect of osmolytes under crowding conditions was more pronounced than at room temperature, and the Phb activity in the presence of osmolytes was higher in these conditions than in diluted solutions. These results provide important insights into the complex mechanism, by which osmolytes affect the structure and activity of Phb under crowding conditions.

Combined action of chemical chaperones on stability, aggregation and oligomeric state of muscle glycogen phosphorylase b.

Chemical chaperones are a class of small molecules, which enhance protein stability, folding, inhibit protein aggregation, and are used for long-term storage of therapeutic proteins. The combined action of chemical chaperones trehalose, betaine and lysine on stability, aggregation and oligomeric state of muscle glycogen phosphorylase b (Phb) has been studied. Dynamic light scattering data indicate that the affinity of trehalose to Phb increased in the presence of betaine or lysine at both stages (stage of nucleation and aggregate growth) of enzyme aggregation at 48 °C, in contrast, the affinity of betaine to the enzyme in the presence of lysine remained practically unchanged. According to differential scanning calorimetry and analytical ultracentrifugation data, the mixture of trehalose and betaine stabilized Phb stronger than either of them in total. Moreover, the destabilizing effect of lysine on the enzyme was almost completely compensated by trehalose and only partially by betaine. The main protective effect of the mixtures of osmolytes and lysine is associated with their influence on the dissociation/denaturation stage, which is the rate-limiting one of Phb aggregation. Thus, a pair of chaperones affects the stability, oligomeric state, and aggregation of Phb differently than individual chaperones.

Synthesis, Kinetic and Conformational Studies of 2-Substituted-5-(ß-d-glucopyranosyl)-pyrimidin-4-ones as Potential Inhibitors of Glycogen Phosphorylase.

Dysregulation of glycogen phosphorylase, an enzyme involved in glucose homeostasis, may lead to a number of pathological states such as type 2 diabetes and cancer, making it an important molecular target for the development of new forms of pharmaceutical intervention. Based on our previous work on the design and synthesis of 4-arylamino-1-(ß-d-glucopyranosyl)pyrimidin-2-ones, which inhibit the activity of glycogen phosphorylase by binding at its catalytic site, we report herein a general synthesis of 2-substituted-5-(ß-d-glucopyranosyl)pyrimidin-4-ones, a related class of metabolically stable, C-glucosyl-based, analogues. The synthetic development consists of a metallated heterocycle, produced from 5-bromo-2-methylthiouracil, in addition to protected d-gluconolactone, followed by organosilane reduction. The methylthio handle allowed derivatization through hydrolysis, ammonolysis and arylamine substitution, and the new compounds were found to be potent (µM) inhibitors of rabbit muscle glycogen phosphorylase. The results were interpreted with the help of density functional theory calculations and conformational analysis and were compared with previous findings.

Effect of arginine on stability and aggregation of muscle glycogen phosphorylase b.

Arginine (Arg) is frequently used in biotechnology and pharmaceutics to stabilize protein preparations. When using charged ions like Arg, it is necessary to take into account their contribution to the increase in ionic strength, in addition to the effect of Arg on particular processes occurring under the conditions of constancy of ionic strength. Here, we examined contribution of ionic strength (0.15 and 0.5 M) to the effects of Arg on denaturation, thermal inactivation and aggregation of skeletal muscle glycogen phosphorylase b (Phb). Dynamic light scattering, analytical ultracentrifugation, differential scanning calorimetry, circular dichroism and enzymatic activity assay were used to assess the effects of Arg at constant ionic strength compared with the effects of ionic strength alone. We found that high ionic strength did not affect the secondary structure of Phb, but changed conformation of the protein. Such a destabilization of the enzyme causes an increase in the initial rate of aggregation and inactivation of Phb thereby affecting its denaturation. Binding of Arg causes additional changes in the protein conformation, weakening the bonds between monomers in the dimer. This causes the dimer to dissociate into monomers, which rapidly aggregate. Thus, Arg acts on these processes much stronger than just ionic strength.

Chaperone-Like Activity of HSPB5: The Effects of Quaternary Structure Dynamics and Crowding.

Small heat-shock proteins (sHSPs) are ATP-independent molecular chaperones that interact with partially unfolded proteins, preventing their aberrant aggregation, thereby exhibiting a chaperone-like activity. Dynamics of the quaternary structure plays an important role in the chaperone-like activity of sHSPs. However, relationship between the dynamic structure of sHSPs and their chaperone-like activity remains insufficiently characterized. Many factors (temperature, ions, a target protein, crowding etc.) affect the structure and activity of sHSPs. The least studied is an effect of crowding on sHSPs activity. In this work the chaperone-like activity of HSPB5 was quantitatively characterized by dynamic light scattering using two test systems, namely test systems based on heat-induced aggregation of muscle glycogen phosphorylase b (Phb) at 48 °C and dithiothreitol-induced aggregation of α-lactalbumin at 37 °C. Analytical ultracentrifugation was used to control the oligomeric state of HSPB5 and target proteins. The possible anti-aggregation functioning of suboligomeric forms of HSPB5 is discussed. The effect of crowding on HSPB5 anti-aggregation activity was characterized using Phb as a target protein. The duration of the nucleation stage was shown to decrease with simultaneous increase in the relative rate of aggregation of Phb in the presence of HSPB5 under crowded conditions. Crowding may subtly modulate sHSPs activity.

Comparative effects of trehalose and 2-hydroxypropyl-ß-cyclodextrin on aggregation of UV-irradiated muscle glycogen phosphorylase b.

Chemical chaperones are a class of small molecules which enhance folding and prevent aggregation of proteins. Investigation of their effects on the processes of protein aggregation is of importance for further understanding of implication of protein aggregation in neurodegenerative diseases, as well as for solving biotechnological tasks. The effects of chemical chaperones trehalose and 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) on the kinetics of aggregation of UV-irradiated muscle glycogen phosphorylase b (UV-Phb) at 37 °C have been studied. The process of thermal aggregation of UV-Phb includes a slow stage of structural reorganization of the UV-Phb molecule, nucleation stage and fast attachment of structurally reorganized UV-Phb molecules to nuclei formed during the nucleation stage. It was shown that both trehalose and HP-ß-CD increased the duration of the nucleation phase and slowed down the rate of structural reorganization of the UV-Phb molecule. This conclusion has been confirmed by the circular dichroism data. In the absence of chaperones, 82% UV-Phb aggregates, whereas in the presence of HP-ß-CD or trehalose the portion of aggregated protein decreases to 70 and 66%, respectively. The data on analytical ultracentrifugation demonstrated that in the presence of these additives the size of protein aggregates decreased. Analysis of the combined effect of trehalose and HP-ß-CD on UV-Phb aggregation showed that protein aggregation was independently affected by trehalose and HP-ß-CD.

Design, Synthesis, and Use of Novel Photoaffinity Probes in Measuring the Serum Concentration of Glycogen Phosphorylase.

A procedure to measure the serum concentration of glycogen phosphorylase during acute myocardial infarction is presented. This method was based on the synthesis of photoaffinity probes, and used the semiquantitative protein electrophoretic mobility shift technique. Three novel photoaffinity probes bearing different secondary tags were synthesized. Their potency was evaluated in an enzyme inhibition assay against rabbit muscle glycogen phosphorylase a (RMGPa). The inhibitory activity of probe 1 was only 100-fold less potent than the mother compound CP-320626. The photoaffinity labeling experiments were also performed, and a protein with molecular weight (MW) of about 90⁻100 kDa, which was consistent with the MW of GP, was clearly labeled by probe 1. A semiquantitative evaluation of the GP level in serum with probe 1 was also performed. The results showed that the protein band with a MW of about 90⁻100 kDa was tagged, and the concentration of the protein in serum was found to be between 25 and 50 ng/mL. Mass spectrometric analysis revealed that alpha-1,4 glucan phosphorylase (GPMM) was well-preserved in the bands.

Effect of ionic strength and arginine on aggregation of UV-irradiated muscle glycogen phosphorylase b.

In this work the effect of ionic strength and arginine on the kinetics of aggregation of UV-irradiated muscle glycogen phosphorylase b (UV-Phb) was studied using dynamic light scattering at 37 °C at various ionic strengths (0.02-0.7 M). Under these conditions the rate-limiting stage of the overall aggregation process is the structural reorganization of UV-Phb, which can be characterized by the first order rate constant kI. It was shown that an increase in NaCl concentration caused a decrease in the kI value, suggesting a slowdown of the UV-Phb structural reorganization. Circular dichroism data confirmed this conclusion. Arginine is widely used in biotechnology as an agent suppressing protein aggregation. However, arginine is a charged molecule, and, when studying the action of arginine on protein aggregation, the effects of ionic strength should be taken into account. To evaluate the effect of arginine, experiments were conducted at fixed values of ionic strength (0.15 M and 0.5 M). It was shown that at a low ionic strength arginine (0-0.13 M) accelerated the process of protein aggregation, whereas at higher ionic strength arginine (0-0.48 M) acted as an aggregation suppressor.

A new interpretation of sulfate activation of rabbit muscle glycogen phosphorylase.

It is widely known that sulfate ion at high concentration serves like an allosteric activator of glycogen phosphorylase (GP). Based on the crystallographic studies on GP, it has been assumed that the sulfate ion is bound close to the phosphorylatable Ser14 site of nonactivated GP, causing a conformational change to catalytically-active GP. However, there are also reports that sulfate ion inhibits allosterically-activated GP by preventing the phosphate substrate from attaching to the catalytic site. In the present study, using a high concentration of sulfate ion, significant enhancement of GP activity was observed when macromolecular glycogen was used as substrate but not when smaller maltohexaose was used. In glycogen solution, nonreducing-end glucose residues are localized on the surface of glycogen and are not distributed homogenously in the solution. Using cyclodextrin-immobilized column chromatography, we found that sulfate at high concentration promoted GP-dextrin binding through the dextrin-binding site (DBS) located away from the catalytic site. This result is consistent with the properties of the DBSs found in glycogen-debranching enzyme and ß-amylase. Therefore, we propose a new interpretation of the sulfate activation of GP, wherein sulfate ions at high concentration promote glycogen-binding to the DBS directly, and glycogen-binding to the catalytic site indirectly. Our findings were successfully applied to the affinity purification of porcine brain GP.

Kinetic regime of thermal aggregation of holo- and apoglycogen phosphorylases b.

To characterize the role of pyridoxal 5'-phosphate in stabilization of the conformation of muscle glycogen phosphorylase b (Phb), the mechanism of thermal aggregation for holo- and apoforms of Phb has been studied using dynamic light scattering. The order of aggregation with respect to the protein (n) for aggregation of holoPhb at 48°C is equal to 0.5 suggesting that the dissociative mechanism of denaturation is operative and denaturation is followed by rapid aggregation stage. In the case of aggregation of apoPhb at 37°C n=2 and the rate-limiting stage is aggregation of unfolded protein molecules.

Checking for reversibility of aggregation of UV-irradiated glycogen phosphorylase b under crowding conditions.

It is believed that the initial stages of protein aggregation are reversible and can be reversed by simple dilution, whereas prolonged exposure to factors responsible for denaturing proteins (for example, to elevated temperatures) results in the formation of irreversible aggregates. A new approach has been developed to discriminate the stage of the formation of reversible aggregates. Aggregation of UV-irradiated glycogen phosphorylase b (UV-Phb) was studied at 10, 25 and 37 °C in the presence of crowders (polyethylene glycol and Ficoll-70) using dynamic light scattering and analytical ultracentrifugation (pH 6.8; 0.1M NaCl). The dilution of the protein solution in the course of aggregation at 10 °C results in the breakdown of protein aggregates suggesting that the aggregation process is reversible. When aggregation of UV-Phb is studied at 37 °C, reversibility is lacking. Chemical chaperones (arginine, proline) induce the breakdown of protein aggregates of UV-Phb formed at 10 °C. In the experiments carried out at 37 °C in the presence of crowder the addition of arginine results in disintegration of protein aggregates only at early stages of the aggregation process. It is assumed that general pathway of protein aggregation includes the formation of reversible, completely dissociable, partly dissociable and irreversible aggregates.

Effect of crowding on several stages of protein aggregation in test systems in the presence of α-crystallin.

Macromolecular crowding can facilitate protein-protein interactions in the cell, in particular aggregation processes. To characterize the anti-aggregation activity of chaperones under conditions mimicking the crowded environment in the cell, two basic test systems are used. Test systems of the first type are based on aggregation of target proteins undergoing unfolding under different factors. Dithithreitol-induced aggregation of α-lactalbumin is used as such a system. The increase in the duration of lag phase after the addition of the crowder (polyethylene glycol; PEG) to the system containing α-crystallin has been interpreted as a retardation of the stages that are the rate-limiting stages of the general process of aggregation (the nucleation stage and the stages of clusterization of nuclei). Test systems of the second type are based on aggregation of UV-irradiated proteins. Such test systems permit investigating the effects of different agents directly on the stages of aggregation of unfolded protein. UV-irradiated glycogen phosphorylase b (Phb) is used as a target protein. Analysis of the initial rate of aggregation after the addition of PEG at different points in time to the mixture of UV-irradiated Phb and α-crystallin allowed estimating the time of half-conversion for the structural rearrangement of the primary UV-irradiated Phb-α-crystallin complex.

McArdle Disease: Update of Reported Mutations and Polymorphisms in the PYGM Gene.

McArdle disease is an autosomal-recessive disorder caused by inherited deficiency of the muscle isoform of glycogen phosphorylase (or "myophosphorylase"), which catalyzes the first step of glycogen catabolism, releasing glucose-1-phosphate from glycogen deposits. As a result, muscle metabolism is impaired, leading to different degrees of exercise intolerance. Patients range from asymptomatic to severely affected, including in some cases, limitations in activities of daily living. The PYGM gene codifies myophosphoylase and to date 147 pathogenic mutations and 39 polymorphisms have been reported. Exon 1 and 17 are mutational hot-spots in PYGM and 50% of the described mutations are missense. However, c.148C>T (commonly known as p.R50X) is the most frequent mutation in the majority of the studied populations. No genotype-phenotype correlation has been reported and no mutations have been described in the myophosphorylase domains affecting the phosphorylated Ser-15, the 280's loop, the pyridoxal 5'-phosphate, and the nucleoside inhibitor binding sites. A newly generated knock-in mouse model is now available, which renders the main clinical and molecular features of the disease. Well-established methods for diagnosing patients in laboratories around the world will shorten the frequent ∼20-year period stretching from first symptoms appearance to the genetic diagnosis.

Clinical and molecular characterization of McArdle's disease in Brazilian patients.

McArdle's disease, a glycogen storage disease type V, is caused by a deficiency of the enzyme myophosphorylase, encoded by the PYGM gene. Worldwide distribution of mutations has revealed interesting data about the prevalence of mutations and population migrations. Currently, more than 100 mutations in the PYGM gene have been described, with some recurrent mutations in the different populations. However, no molecular studies of McArdle's disease were reported in Brazilian patients. Here, we describe the clinical phenotype and genotype of 10 patients from 8 unrelated Brazilian families. Among the 10 patients (3 females, 7 males), eight presented with the typical phenotype, with exercise intolerance, cramps, and myalgia; one patient showed permanent muscle weakness; and one patient showed a mild phenotype. Molecular analysis identified 5 different mutations in the 8 families, both in homozygosis or compound heterozygosis state. Four of them had already been described (p.R50X, p.T692kfs30, p.K609K, and p.G455R), and one, pI513V, is a novel heterozygous mutation. The common nonsense p.R50X mutation was found in 6 of the 8 families, being therefore the commonest mutation in the Brazilian population as well. Other mutations previously reported in European patients were also found in the patients in this study, which was expected considering the European ancestry of the Brazilian population.

Cellular regulation by protein phosphorylation.

A historical account of the discovery of reversible protein phosphorylation is presented. This process was uncovered in the mid 1950s in a study undertaken with Edwin G. Krebs to elucidate the complex hormonal regulation of skeletal muscle glycogen phosphorylase. Contrary to the known activation of this enzyme by AMP which serves as an allosteric effector, its hormonal regulation results from a phosphorylation of the protein by phosphorylase kinase following the activation of the latter by Ca(2+) and ATP. The study led to the establishment of the first hormonal cascade of successive enzymatic reactions, kinases acting on kinases, initiated by cAMP discovered by Earl Sutherland. It also showed how two different physiological processes, carbohydrate metabolism and muscle contraction, could be regulated in concert.

Rac1 protein regulates glycogen phosphorylase activation and controls interleukin (IL)-2-dependent T cell proliferation.

Small GTPases of the Rho family have been implicated in important cellular processes such as cell migration and adhesion, protein secretion, and/or gene transcription. In the lymphoid system, these GTPases participate in the signaling cascades that are activated after engagement of antigen receptors. However, little is known about the role that Rho GTPases play in IL-2-mediated responses. Here, we show that IL-2 induces Rac1 activation in Kit 225 T cells. We identified by mass spectrometry the muscle isoform of glycogen phosphorylase (PYGM) as a novel Rac1 effector molecule in IL-2-stimulated cells. The interaction between the active form of Rac1 (Rac1-GTP) and PYGM was established directly through a domain comprising amino acids 191-270 of PYGM that exhibits significant homology with the Rac binding domain of PAK1. The integrity of this region was crucial for PYGM activation. Importantly, IL-2-dependent cellular proliferation was inhibited upon blocking both the activation of Rac1 and the activity of PYGM. These results reveal a new role for Rac1 in cell signaling, showing that this GTPase triggers T cell proliferation upon IL-2 stimulation by associating with PYGM and modulating its enzymatic activity.

N-(4-Substituted-benzoyl)-N'-(ß-d-glucopyranosyl)ureas as inhibitors of glycogen phosphorylase: Synthesis and evaluation by kinetic, crystallographic, and molecular modelling methods.

N-(4-Substituted-benzoyl)-N'-(ß-d-glucopyranosyl) ureas (substituents: Me, Ph, Cl, OH, OMe, NO(2), NH(2), COOH, and COOMe) were synthesised by ZnCl(2) catalysed acylation of O-peracetylated ß-d-glucopyranosyl urea as well as in reactions of O-peracetylated or O-unprotected glucopyranosylamines and acyl-isocyanates. O-deprotections were carried out by base or acid catalysed transesterifications where necessary. Kinetic studies revealed that most of these compounds were low micromolar inhibitors of rabbit muscle glycogen phosphorylase b (RMGPb). The best inhibitor was the 4-methylbenzoyl compound (K(i)=2.3µM). Crystallographic analyses of complexes of several of the compounds with RMGPb showed that the analogues exploited, together with water molecules, the available space at the ß-pocket subsite and induced a more extended shift of the 280s loop compared to RMGPb in complex with the unsubstituted benzoyl urea. The results suggest the key role of the water molecules in ligand binding and structure-based ligand design. Molecular docking study of selected inhibitors was done to show the ability of the binding affinity prediction. The binding affinity of the highest scored docked poses was calculated and correlated with experimentally measured K(i) values. Results show that correlation is high with the R-squared (R(2)) coefficient over 0.9.

Effect of 2-hydroxypropyl-ß-cyclodextrin on thermal stability and aggregation of glycogen phosphorylase b from rabbit skeletal muscle.

The study of the kinetics of thermal aggregation of glycogen phosphorylase b (Phb) from rabbit skeletal muscles by dynamic light scattering at 48°C showed that 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) accelerated the aggregation process and induced the formation of the larger protein aggregates. The reason of the accelerating effect of HP-ß-CD is destabilization of the protein molecule under action of HP-ß-CD. This conclusion was supported by the data on differential scanning calorimetry and the kinetic data on thermal inactivation of Phb. It is assumed that destabilization of the Phb molecule is due to preferential binding of HP-ß-CD to intermediates of protein unfolding in comparison with the original native state. The conclusion regarding the ability of the native Phb for binding of HP-ß-CD was substantiated by the data on the enzyme inhibition by HP-ß-CD. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 986-993, 2010.

Effect of GroEL on thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscle.

The suppression of the thermal aggregation of glycogen phosphorylase b (Phb) from rabbit skeletal muscle by the chaperonin GroEL is studied using dynamic light scattering. It is shown that the decrease in the rate of Phb aggregation under the action of GroEL is due to the transition of the aggregation process from the kinetic regime, wherein the rate of aggregation is limited by diffusion of the interacting particles, to a regime where the sticking probability for the colliding particles becomes lower than one (reaction-limited cluster-cluster aggregation). The analytical-ultracentrifugation data show that elevated temperatures induce dissociation of the dimeric Phb. The formation of a complex between the denatured monomeric form of Phb and the dissociated forms of GroEL is detected during heating at 46 degrees C.

Glycogen phosphorylase a inhibitors with a phenethylphenylphthalimide skeleton derived from thalidomide-related alpha-glucosidase inhibitors and liver X receptor antagonists.

Novel glycogen phosphorylase a (GPa) inhibitors with a phenethylphenylphthalimide skeleton were prepared based on alpha-glucosidase inhibitors and liver X receptor (LXR) antagonists derived from thalidomide. Their structure-activity relationships were analyzed. Some of the compounds thus prepared showed potent inhibitory activity against rabbit muscle GPa with more than 10-fold greater efficacy than a typical GPa inhibitor, 1,4-dideoxy-1,4-imino-D-arabinitol.