A century of exercise physiology: key concepts in regulation of glycogen metabolism in skeletal muscle.

Glycogen is a branched, glucose polymer and the storage form of glucose in cells. Glycogen has traditionally been viewed as a key substrate for muscle ATP production during conditions of high energy demand and considered to be limiting for work capacity and force generation under defined conditions. Glycogenolysis is catalyzed by phosphorylase, while glycogenesis is catalyzed by glycogen synthase. For many years, it was believed that a primer was required for de novo glycogen synthesis and the protein considered responsible for this process was ultimately discovered and named glycogenin. However, the subsequent observation of glycogen storage in the absence of functional glycogenin raises questions about the true role of the protein. In resting muscle, phosphorylase is generally considered to be present in two forms: non-phosphorylated and inactive (phosphorylase b) and phosphorylated and constitutively active (phosphorylase a). Initially, it was believed that activation of phosphorylase during intense muscle contraction was primarily accounted for by phosphorylation of phosphorylase b (activated by increases in AMP) to a, and that glycogen synthesis during recovery from exercise occurred solely through mechanisms controlled by glucose transport and glycogen synthase. However, it now appears that these views require modifications. Moreover, the traditional roles of glycogen in muscle function have been extended in recent years and in some instances, the original concepts have undergone revision. Thus, despite the extensive amount of knowledge accrued during the past 100 years, several critical questions remain regarding the regulation of glycogen metabolism and its role in living muscle.

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.

Improving Peptide Fragmentation for Hydrogen-Deuterium Exchange Mass Spectrometry Using a Time-Dependent Collision Energy Calculator.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is becoming a popular technique for interrogating biological systems. In recent years, advancements have been made to increase peptide coverage for proteins that resist digestion such as antibodies and membrane proteins. These methods commonly include using alternative digestion enzymes or longer chromatographic gradients, which may be expensive or time-consuming to implement. Here, we recommend an efficient proteomics-based approach to increase peptide confidence and coverage. A major filtering parameter for peptides in HDX is the number of product ions detected; this is a result of the collision energy (CE) applied within the MS. A traditional linear ramp achieves optimal CE for only short periods of time. More product ions will be created and detected if optimal CE can be achieved for a longer period of time. As a result, the coverage, redundancy, and data confidence are all increased. We achieved this by implementing a mobility-dependent CE look up table (LUT) which increases the CE as a function of mobility. We developed a program to calculate the optimal CE for a set of peptides and MS settings based on initial reference samples. We demonstrated the utility of the CE LUT on three protein samples including the soluble phosphorylase B, IgG2, and the membrane-stabilized AcrB. We showed that applying a CE LUT provided 8.5-50% more peptides compared to a linear CE ramp. The results demonstrate that a time-dependent CE LUT is a quick and inexpensive method to increase data confidence and peptide abundance for HDX-MS experiments.

Breast cancers utilize hypoxic glycogen stores via PYGB, the brain isoform of glycogen phosphorylase, to promote metastatic phenotypes.

In breast cancer, tumor hypoxia has been linked to poor prognosis and increased metastasis. Hypoxia activates transcriptional programs in cancer cells that lead to increased motility and invasion, as well as various metabolic changes. One of these metabolic changes, an increase in glycogen metabolism, has been further associated with protection from reactive oxygen species damage that may lead to premature senescence. Here we report that breast cancer cells significantly increase glycogen stores in response to hypoxia. We found that knockdown of the brain isoform of an enzyme that catalyzes glycogen breakdown, glycogen phosphorylase B (PYGB), but not the liver isoform, PYGL, inhibited glycogen utilization in estrogen receptor negative and positive breast cancer cells; whereas both independently inhibited glycogen utilization in the normal-like breast epithelial cell line MCF-10A. Functionally, PYGB knockdown and the resulting inhibition of glycogen utilization resulted in significantly decreased wound-healing capability in MCF-7 cells and a decrease in invasive potential of MDA-MB-231 cells. Thus, we identify PYGB as a novel metabolic target with potential applications in the management and/or prevention of metastasis in breast cancer.

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry.

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 µg of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Protective manifestation of bacoside A and bromelain in terms of cholinesterases, gamma-amino butyric acid, serotonin level and stress proteins in the brain of dichlorvos-intoxicated mice.

The objective of the study was to evaluate the neuroprotective effects of bacoside A and bromelain against dichlorvos-incited toxicity. Healthy 6-8-week old, male Swiss mice were administered subacute doses of dichlorvos (40 mg/kg bw), bacoside A (5 mg/kg bw) and bromelain (70 mg/kg bw). AChE, BChE, GABA, serotonin and total protein content and their expressions were used for determination of toxic action of dichlorvos. Protective effects of bacoside A and bromelain were evaluated on the same parameters. Exposure to dichlorvos leads to significant decline in activities of AChE (p < 0.01, p < 0.001), BChE (p < 0.05) and GABA (p < 0.01) and total protein levels (p < 0.01). Antioxidant treatment significantly increased the activities of AChE (p < 0.01, p < 0.001), BChE (p < 0.05), GABA (p < 0.01) and total protein level (p < 0.05) compared to those in dichlorvos-treated mice. Overexpression of Hsp 70 protein and underexpression of phosphorylase a and b, catalase SOD and GPx were observed after dichlorvos exposure which suggests the oxidative stress. The results indicate that dichlorvos-induced neuronal damage which results in the generation of molecular expression of proteins is in agreement with the biochemical data ameliorated by bacoside A and bromelain.

Berberine attenuates axonal transport impairment and axonopathy induced by Calyculin A in N2a cells.

Berberine is a primary component of the most functional extracts of Coptidis rhizome used in traditional Chinese medicine for centuries. Recent reports indicate that Berberine has the potential to prevent and treat Alzheimer's disease (AD). The previous studies reported that Calyculin A (CA) impaired the axonal transport in neuroblastoma-2a (N2a) cells. Berberine attenuated tau hyperphosphorylation and cytotoxicity induced by CA. Our study aimed at investigating the effects of Berberine on the axonal transport impairment induced by CA in N2a cells. The results showed that Berberine could protect the cell from CA -induced toxicity in metabolism and viability, as well as hyperphosphorylation of tau and neurofilaments (NFs). Furthermore, Berberine could reverse CA-induced axonal transport impairment significantly. Berberine also partially reversed the phosphorylation of the catalytic subunit of PP-2A at Tyrosine 307, a crucial site negatively regulating the activity of PP-2A, and reduced the levels of malondialdehyde and the activity of superoxide dismutase, markers of oxidative stress, induced by CA. The present work for the first time demonstrates that Berberine may play a role in protecting against CA-induced axonal transport impairment by modulating the activity of PP-2A and oxidative stress. Our findings also suggest that Berberine may be a potential therapeutic drug for AD.

Surface induced dissociation yields quaternary substructure of refractory noncovalent phosphorylase B and glutamate dehydrogenase complexes.

Ion mobility (IM) and tandem mass spectrometry (MS/MS) coupled with native MS are useful for studying noncovalent protein complexes. Collision induced dissociation (CID) is the most common MS/MS dissociation method. However, some protein complexes, including glycogen phosphorylase B kinase (PHB) and L-glutamate dehydrogenase (GDH) examined in this study, are resistant to dissociation by CID at the maximum collision energy available in the instrument. Surface induced dissociation (SID) was applied to dissociate the two refractory protein complexes. Different charge state precursor ions of the two complexes were examined by CID and SID. The PHB dimer was successfully dissociated to monomers and the GDH hexamer formed trimeric subcomplexes that are informative of its quaternary structure. The unfolding of the precursor and the percentages of the distinct products suggest that the dissociation pathways vary for different charge states. The precursors at lower charge states (+21 for PHB dimer and +27 for GDH hexamer) produce a higher percentage of folded fragments and dissociate more symmetrically than the precusors at higher charge states (+29 for PHB dimer and +39 for GDH hexamer). The precursors at lower charge state may be more native-like than the higher charge state because a higher percentage of folded fragments and a lower percentage of highly charged unfolded fragments are detected. The combination of SID and charge reduction is shown to be a powerful tool for quaternary structure analysis of refractory noncovalent protein complexes, as illustrated by the data for PHB dimer and GDH hexamer.

Thermal denaturation and aggregation of apoform of glycogen phosphorylase b. Effect of crowding agents and chaperones.

The effect of protein and chemical chaperones and crowders on thermal stability and aggregation of apoform of rabbit muscle glycogen phosphorylase b (apoPhb) has been studied at 37°C. Proline suppressed heat-induced loss in ability of apoPhb to reconstitution at 37°C, whereas α-crystallin did not reveal a protective action. To compare the antiaggregation activity of intact and crosslinked α-crystallins, an adsorption capacity (AC) of a protein chaperone with respect to a target protein was estimated. This parameter is a measure of the antiaggregation activity. Crosslinking of α-crystallin results in 11-fold decrease in the initial AC. The nonlinear character of the relative initial rate of apoPhb aggregation versus the [intact α-crystallin]/[apoPhb] ratio plot is indicative of the decrease in the AC of α-crystallin with increasing the [α-crystallin]/[apoPhb] ratio and can be interpreted as an evidence for dynamic chaperone structure and polydispersity of α-crystallin-target protein complexes. As for chemical chaperones, a semisaturation concentration of the latter was used as a characteristic of the antiaggregation activity. A decrease in the semisaturation concentration for proline was observed in the presence of the crowders (polyethylene glycol and Ficoll-70).

From protein complexes to subunit backbone fragments: a multi-stage approach to native mass spectrometry.

Native mass spectrometry (MS) is becoming an important integral part of structural proteomics and system biology research. The approach holds great promise for elucidating higher levels of protein structure: from primary to quaternary. This requires the most efficient use of tandem MS, which is the cornerstone of MS-based approaches. In this work, we advance a two-step fragmentation approach, or (pseudo)-MS(3), from native protein complexes to a set of constituent fragment ions. Using an efficient desolvation approach and quadrupole selection in the extended mass-to-charge (m/z) range, we have accomplished sequential dissociation of large protein complexes, such as phosporylase B (194 kDa), pyruvate kinase (232 kDa), and GroEL (801 kDa), to highly charged monomers which were then dissociated to a set of multiply charged fragmentation products. Fragment ion signals were acquired with a high resolution, high mass accuracy Orbitrap instrument that enabled highly confident identifications of the precursor monomer subunits. The developed approach is expected to enable characterization of stoichiometry and composition of endogenous native protein complexes at an unprecedented level of detail.

Alkylated trihydroxyacetophenone as a MALDI matrix for hydrophobic peptides.

Hydrophobic peptides are difficult to detect in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), because of the hydrophilic properties of conventional matrices and the low affinity for hydrophobic peptides. Recently, we reported on alkylated dihydroxybenzoic acid (ADHB) as a matrix additive for hydrophobic peptides; however, the peptides were detected in the rim of the matrix-analyte dried spot. Here, we report on a novel matrix, alkylated trihydroxyacetophenone (ATHAP), which is a 2,4,6-trihydroxyacetophenone derivative incorporating a hydrophobic alkyl chain on the acetyl group and thus is expected to have an affinity for hydrophobic peptides. ATHAP increased the sensitivity of hydrophobic peptides 10-fold compared with α-cyano-4-hydroxycinnamic acid (CHCA), in which the detection of hydrophilic peptides was suppressed. The peptides were detected throughout the entire matrix-analyte dried spot using ATHAP, overcoming the difficulty of finding a "sweet spot" when using ADHB. In addition, ATHAP functioned alone as a matrix, unlike ADHB as an additive. In phosphorylase b digests analysis, hydrophobic peptides, which were not detected with CHCA for 1 pmol, were detected with this matrix, confirming that ATHAP led to increased sequence coverage and may extend the range of target analytes in MALDI-MS.

Effect of crowding and chaperones on self-association, aggregation and reconstitution of apophosphorylase b.

It was shown that the rate of reconstruction of muscle glycogen phosphorylase b (Phb) from apoenzyme and pyridoxal 5'-phosphate decreased under crowding conditions. The effect of crowding was counteracted by chaperones (α-crystallin and proline). Sedimentation analysis shows that crowding stimulates the formation of high-molecular-weight associates at 25 °C, whereas chaperones stabilize small oligomers. The study of the kinetics of apoPhb aggregation at 37 °C showed that the anti-aggregation activity of chaperones decreased under crowding conditions. When studying the sedimentation behavior of the mixture of apoPhb and α-crystallin, the complexes between unfolded apoPhb and dissociated forms of α-crystallin were observed. It is assumed that these complexes are responsible for realization of the chaperone-like activity of α-crystallin under crowding conditions.

Synthesis of branched polysaccharides with tunable degree of branching.

An in vitro enzyme-catalyzed tandem reaction using the enzymes phosphorylase b from rabbit muscle and Deinococcus geothermalis glycogen branching enzyme (Dg GBE) to obtain branched polyglucans with tunable degree of branching (2% ÷ 13%) is presented. The tunable degree of branching is obtained by varying the reaction conditions such as pH value, the choice of reducing agent and its concentration and reaction time. Linear amylose is formed by the phosphorylase-catalyzed propagation of glucose-1-phosphate while Dg GBE introduces branching points on the α-(1→6) position by relocating short oligosaccharide chains. Our results show that the best way to obtain different degrees of branching with this set of enzymes is by regulation of the reaction time.

Size exclusion chromatography with multi detection in combination with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry as a tool for unraveling the mechanism of the enzymatic polymerization of polysaccharides.

Determination of the size distributions of natural polysaccharides is a challenging task. More advantageous for characterization are well-defined synthetic (hyper)-branched polymers. In this study we concentrated on synthetic amylopectin analogues in order to obtain and compare all available data for different distributions and size dependence of molecular weights. Two groups of well-defined synthetic branched polysaccharides were synthesized via an in vitro enzyme-catalyzed reaction using the enzyme phosphorylase b from rabbit muscle and Deinococcus geothermalis glycogen branching enzyme. Synthetic polymers had a tunable degree of branching (2%-13% determined via (1)H NMR) and a tunable degree of polymerization (30-350 determined indirectly via UV spectrometry). The systems used for separation and characterization of branched polysaccharides were SEC-DMSO/LiBr and multi detection (refractive index detector, viscosity detector, and multi angle light scattering detector) and SEC-water/0.02% NaN(3); and SEC-50 mM NaNO(3)/0.02% NaN(3) and multi detection. Additionally the side chain length distribution of enzymatically debranched polysaccharides was investigated by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. With this combination of characterization techniques, we were able not only to characterize the amylopectin analogues but also to solve parts of the molecular mechanism of their enzymatic polymerization. Moreover our materials showed potential to be standards in the field of natural polysaccharides characterization.

Determination of ¹5N-incorporation into plant proteins and their absolute quantitation: a new tool to study nitrogen flux dynamics and protein pool sizes elicited by plant-herbivore interactions.

Herbivory leads to changes in the allocation of nitrogen among different pools and tissues; however, a detailed quantitative analysis of these changes has been lacking. Here, we demonstrate that a mass spectrometric data-independent acquisition approach known as LC-MS(E), combined with a novel algorithm to quantify heavy atom enrichment in peptides, is able to quantify elicited changes in protein amounts and (15)N flux in a high throughput manner. The reliable identification/quantitation of rabbit phosphorylase b protein spiked into leaf protein extract was achieved. The linear dynamic range, reproducibility of technical and biological replicates, and differences between measured and expected (15)N-incorporation into the small (SSU) and large (LSU) subunits of ribulose-1,5-bisphosphate-carboxylase/oxygenase (RuBisCO) and RuBisCO activase 2 (RCA2) of Nicotiana attenuata plants grown in hydroponic culture at different known concentrations of (15)N-labeled nitrate were used to further evaluate the procedure. The utility of the method for whole-plant studies in ecologically realistic contexts was demonstrated by using (15)N-pulse protocols on plants growing in soil under unknown (15)N-incorporation levels. Additionally, we quantified the amounts of lipoxygenase 2 (LOX2) protein, an enzyme important in antiherbivore defense responses, demonstrating that the approach allows for in-depth quantitative proteomics and (15)N flux analyses of the metabolic dynamics elicited during plant-herbivore interactions.

Alkylated dihydroxybenzoic acid as a MALDI matrix additive for hydrophobic peptide analysis.

Hydrophobic peptides are generally difficult to detect using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) because the majority of MALDI matrixes are hydrophilic and therefore have a low affinity for hydrophobic peptides. Here, we report on a novel matrix additive, o-alkylated dihydroxybenzoic acid (ADHB), which is a 2,5-dihydroxybenzoic acid (DHB) derivative incorporating a hydrophobic alkyl chain on a hydroxyl group to improve its affinity for hydrophobic peptides, thereby improving MALDI-MS sensitivity. The addition of ADHB to the conventional matrix α-cyano-4-hydroxycinnamic acid (CHCA) improved the sensitivity of hydrophobic peptides 10- to 100-fold. The sequence coverage of phosphorylase b digest was increased using ADHB. MS imaging indicated that hydrophobic peptides were enriched in the rim of a matrix/analyte dried spot when ADHB was used. In conclusion, the addition of ADHB to the standard matrix led to improved sensitivity of hydrophobic peptides by MALDI-MS.

Concentration dependence of chaperone-like activities of α-crystallin, αB-crystallin and proline.

Chaperone-like activities of α-crystallin, αB-crystallin and proline were studied using a test system based on aggregation of UV-irradiated glycogen phosphorylase b (Phb) from rabbit skeletal muscle. The biphasic character of the dependence of the initial rate of aggregation (v(agg)) of UV-irradiated Phb on the concentration of α-crystallin or αB-crystallin is indicative of the existence of two types of chaperone-protein substrate complexes differing significantly in affinity between the components of the complex. The dependence of v(agg) on the proline concentration is sigmoid (Hill coefficient is equal to 1.6) suggesting that the positive cooperative interactions between the proline molecules bound on the surface of the protein particles occur. When studying the combined suppressive action of α-crystallin and proline on aggregation of UV-irradiated Phb, a slight antagonism between proline used at a fixed concentration (0.15M) and α-crystallin was observed. At higher concentration of proline (0.5M) each chaperone acts independent of one another.

The σ-hole phenomenon of halogen atoms forms the structural basis of the strong inhibitory potency of C5 halogen substituted glucopyranosyl nucleosides towards glycogen phosphorylase b.

C5 halogen substituted glucopyranosyl nucleosides (1-(ß-D-glucopyranosyl)-5-X-uracil; X=Cl, Br, I) have been discovered as some of the most potent active site inhibitors of glycogen phosphorylase (GP), with respective K(i) values of 1.02, 3.27, and 1.94 µM. The ability of the halogen atom to form intermolecular electrostatic interactions through the σ-hole phenomenon rather than through steric effects alone forms the structural basis of their improved inhibitory potential relative to the unsubstituted 1-(ß-D-glucopyranosyl)uracil (K(i) =12.39 µM), as revealed by X-ray crystallography and modeling calculations exploiting quantum mechanics methods. Good agreement was obtained between kinetics results and relative binding affinities calculated by QM/MM-PBSA methodology for various substitutions at C5. Ex vivo experiments demonstrated that the most potent derivative (X=Cl) toward purified GP has no cytotoxicity and moderate inhibitory potency at the cellular level. In accordance, ADMET property predictions were performed, and suggest decreased polar surface areas as a potential means of improving activity in the cell.

Does the crowded cell-like environment reduce the chaperone-like activity of α-crystallin?

The effect of crowding on the chaperone-like activity of α-crystallin has been studied using aggregation of UV-irradiated glycogen phosphorylase b (Phb) from rabbit skeletal muscle as an aggregation test system. The merit of this test system is the possibility of testing agents that directly affect the stage of aggregation of the protein molecules. It was shown that the solution of Phb denatured by UV contained aggregates with a hydrodynamic radius of 10.4 nm. These aggregates are relatively stable at 20 °C; however, they reveal a tendency to stick further in the presence of crowding agents. The study of the effect of α-crystallin on the aggregation of UV-irradiated Phb in the presence of the crowding agents by dynamic light scattering at 37 °C showed that under crowding conditions the antiaggregation ability of α-crystallin was weakened. On the basis of the analytical ultracentrifugation, size-exclusion chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis data, the scheme of interaction of UV-irradiated Phb and α-crystallin has been proposed. It is assumed that chaperone-target protein complexes of two types are formed, namely, the complexes of dissociated forms of α-crystallin with a protein substrate and high-mass α-crystallin-denatured protein complexes. The complexes of the first type reveal a weak propensity to aggregate even under crowding conditions. The complexes of the second type are characterized by the lower rate of aggregation in comparison with that of original UV-irradiated Phb. However, crowding stimulates the rate of aggregation of these complexes, resulting in the above-mentioned decrease in the chaperone-like activity of α-crystallin.