Major Advances in Brain Glycogen Research: Understanding of the Roles of Glycogen Have Evolved from Emergency Fuel Reserve to Dynamic, Regulated Participant in Diverse Brain Functions.

Brain glycogen is extremely difficult to study because it is very labile to physiological status and postmortem autolysis, and glycogen degradative enzymes are rapidly activated by metabolites and signaling molecules. Glycogen is predominantly located within astrocytes in adult brain, and abnormal glycogen metabolism in neurons has lethal consequences. Diverse distribution of glycogen among subcellular compartments suggests local regulation and different functional roles, and recent studies have revealed critically important roles for glycogen in normal brain function and Lafora disease.

Introduction to the Thematic Minireview Series: Brain glycogen metabolism.

The synthesis of glycogen allows for efficient intracellular storage of glucose molecules in a soluble form that can be rapidly released to enter glycolysis in response to energy demand. Intensive studies of glucose and glycogen metabolism, predominantly in skeletal muscle and liver, have produced innumerable insights into the mechanisms of hormone action, resulting in the award of several Nobel Prizes over the last one hundred years. Glycogen is actually present in all cells and tissues, albeit at much lower levels than found in muscle or liver.

The regulation of glycogenolysis in the brain.

The key regulatory enzymes of glycogenolysis are phosphorylase kinase, a hetero-oligomer with four different types of subunits, and glycogen phosphorylase, a homodimer. Both enzymes are activated by phosphorylation and small ligands, and both enzymes have distinct isoforms that are predominantly expressed in muscle, liver, or brain; however, whole-transcriptome high-throughput sequencing analyses show that in brain both of these enzymes are likely composed of subunit isoforms representing all three tissues. This Minireview examines the regulatory properties of the isoforms of these two enzymes expressed in the three tissues, focusing on their potential regulatory similarities and differences.

Structural characterization of the catalytic γ and regulatory β subunits of phosphorylase kinase in the context of the hexadecameric enzyme complex.

In the tightly regulated glycogenolysis cascade, the breakdown of glycogen to glucose-1-phosphate, phosphorylase kinase (PhK) plays a key role in regulating the activity of glycogen phosphorylase. PhK is a 1.3 MDa hexadecamer, with four copies each of four different subunits (α, β, γ and δ), making the study of its structure challenging.

The structure of the large regulatory α subunit of phosphorylase kinase examined by modeling and hydrogen-deuterium exchange.

Phosphorylase kinase (PhK), a 1.3 MDa regulatory enzyme complex in the glycogenolysis cascade, has four copies each of four subunits, (αβγδ) , and 325 kDa of unique sequence (the mass of an αβγδ protomer). The α, β and δ subunits are regulatory, and contain allosteric activation sites that stimulate the activity of the catalytic γ subunit in response to diverse signaling molecules.

Protein Structural Analysis via Mass Spectrometry-Based Proteomics.

Modern mass spectrometry (MS) technologies have provided a versatile platform that can be combined with a large number of techniques to analyze protein structure and dynamics. These techniques include the three detailed in this chapter: (1) hydrogen/deuterium exchange (HDX), (2) limited proteolysis, and (3) chemical crosslinking (CX). HDX relies on the change in mass of a protein upon its dilution into deuterated buffer, which results in varied deuterium content within its backbone amides.

The regulatory α and β subunits of phosphorylase kinase directly interact with its substrate, glycogen phosphorylase.

The selective phosphorylation of glycogen phosphorylase (GP) by its only known kinase, phosphorylase kinase (PhK), keeps glycogen catabolism tightly regulated. In addition to the obligatory interaction between the catalytic γ subunit of PhK and the phosphorylatable region of GP, previous studies have suggested additional sites of interaction between this kinase and its protein substrate. Using short chemical crosslinkers, we have identified direct interactions of GP with the large regulatory α and β subunits of PhK.

What Mutagenesis Can and Cannot Reveal About Allostery.

Allosteric regulation of protein function is recognized to be widespread throughout biology; however, knowledge of allosteric mechanisms, the molecular changes within a protein that couple one binding site to another, is limited. Although mutagenesis is often used to probe allosteric mechanisms, we consider herein what the outcome of a mutagenesis study truly reveals about an allosteric mechanism. Arguably, the best way to evaluate the effects of a mutation on allostery is to monitor the allosteric coupling constant (Qax), a ratio of the substrate binding constants in the absence versus presence of an allosteric effector.

Activation of Phosphorylase Kinase by Physiological Temperature.

In the six decades since its discovery, phosphorylase kinase (PhK) from rabbit skeletal muscle has usually been studied at 30 °C; in fact, not a single study has examined functions of PhK at a rabbit's body temperature, which is nearly 10 °C greater. Thus, we have examined aspects of the activity, regulation, and structure of PhK at temperatures between 0 and 40 °C. Between 0 and 30 °C, the activity at pH 6.

Mass Spectrometric Analysis of Surface-Exposed Regions in the Hexadecameric Phosphorylase Kinase Complex.

Phosphorylase kinase (PhK) is a 1.3 MDa (αβγδ)4 enzyme complex, in which αβγδ protomers associate in D2 symmetry to form two large octameric lobes that are interconnected by four bridges. The approximate locations of the subunits have been mapped in low-resolution cryo-electron microscopy structures of the complex; however, the disposition of the subunits within the complex remains largely unknown.

A model for activation of the hexadecameric phosphorylase kinase complex deduced from zero-length oxidative crosslinking.

Phosphorylase kinase (PhK) is a hexadecameric (αβγδ)(4) enzyme complex that upon activation by phosphorylation stimulates glycogenolysis. Due to its large size (1.3 MDa), elucidating the structural changes associated with the activation of PhK has been challenging, although phosphoactivation has been linked with an increased tendency of the enzyme's regulatory β-subunits to self-associate.

Physicochemical changes in phosphorylase kinase induced by its cationic activator Mg(2+).

For over four decades free Mg(2+) ions, that is, those in excess of MgATP, have been reported to affect a wide variety of properties of phosphorylase kinase (PhK), including its affinity for other molecules, proteolysis, chemical crosslinking, phosphorylation, binding to certain monoclonal antibodies, and activity, which is stimulated. Additionally, for over three decades Mg(2+) has been known to act synergistically with Ca(2+) , another divalent activator of PhK, to affect even more properties of the enzyme. During all of this time, however, no study has been performed to determine the overall effects of free Mg(2+) ions on the physical properties of PhK, even though the effects of Ca(2+) ions on PhK's properties are well documented.

Structure and location of the regulatory β subunits in the (αβγδ)4 phosphorylase kinase complex.

Phosphorylase kinase (PhK) is a hexadecameric (αβγδ)(4) complex that regulates glycogenolysis in skeletal muscle. Activity of the catalytic γ subunit is regulated by allosteric activators targeting the regulatory α, β, and δ subunits. Three-dimensional EM reconstructions of PhK show it to be two large (αβγδ)(2) lobes joined with D(2) symmetry through interconnecting bridges.

Mass spectrometry reveals differences in stability and subunit interactions between activated and nonactivated conformers of the (αβγδ)4 phosphorylase kinase complex.

Phosphorylase kinase (PhK), a 1.3 MDa enzyme complex that regulates glycogenolysis, is composed of four copies each of four distinct subunits (α, β, γ, and δ). The catalytic protein kinase subunit within this complex is γ, and its activity is regulated by the three remaining subunits, which are targeted by allosteric activators from neuronal, metabolic, and hormonal signaling pathways.

A review of methods used for identifying structural changes in a large protein complex.

This chapter explores the structural responses of a massive, hetero-oligomeric protein complex to a single allosteric activator as probed by a wide range of chemical, biochemical, and biophysical approaches. Some of the approaches used are amenable only to large protein targets, whereas others push the limits of their utility. Some of the techniques focus on individual subunits, or portions thereof, while others examine the complex as a whole.

The glucoamylase inhibitor acarbose is a direct activator of phosphorylase kinase.

Phosphorylase kinase (PhK), an (alphabetagammadelta)(4) complex, stimulates energy production from glycogen in the cascade activation of glycogenolysis. Its large homologous alpha and beta subunits regulate the activity of the catalytic gamma subunit and account for 81% of PhK's mass. Both subunits are thought to be multidomain structures, and recent predictions based on their sequences suggest the presence of potentially functional glucoamylase (GH15)-like domains near their amino termini.

Expressed phosphorylase b kinase and its alphagammadelta subcomplex as regulatory models for the rabbit skeletal muscle holoenzyme.

Understanding the regulatory interactions among the 16 subunits of the (alphabetagammadelta)(4) phosphorylase b kinase (PhK) complex can only be achieved through reconstructing the holoenzyme or its subcomplexes from the individual subunits. In this study, recombinant baculovirus carrying a vector containing a multigene cassette was created to coexpress in insect cells alpha, beta, gamma, and delta subunits corresponding to rabbit skeletal muscle PhK. The hexadecameric recombinant PhK (rPhK) and its corresponding alphagammadelta trimeric subcomplex were purified to homogeneity with proper subunit stoichiometries.

Physicochemical changes in phosphorylase kinase associated with its activation.

Phosphorylase kinase (PhK) regulates glycogenolysis through its Ca(2+)-dependent phosphorylation and activation of glycogen phosphorylase. The activity of PhK increases dramatically as the pH is raised from 6.8 to 8.

The regulatory beta subunit of phosphorylase kinase interacts with glyceraldehyde-3-phosphate dehydrogenase.

Skeletal muscle phosphorylase kinase (PhK) is an (alphabetagammadelta) 4 hetero-oligomeric enzyme complex that phosphorylates and activates glycogen phosphorylase b (GP b) in a Ca (2+)-dependent reaction that couples muscle contraction with glycogen breakdown. GP b is PhK's only known in vivo substrate; however, given the great size and multiple subunits of the PhK complex, we screened muscle extracts for other potential targets. Extracts of P/J (control) and I/lnJ (PhK deficient) mice were incubated with [gamma- (32)P]ATP with or without Ca (2+) and compared to identify potential substrates.

CrossSearch, a user-friendly search engine for detecting chemically cross-linked peptides in conjugated proteins.

Chemical cross-linking and high resolution MS have been integrated successfully to capture protein interactions and provide low resolution structural data for proteins that are refractive to analyses by NMR or crystallography. Despite the versatility of these combined techniques, the array of products that is generated from the cross-linking and proteolytic digestion of proteins is immense and generally requires the use of labeling strategies and/or data base search algorithms to distinguish actual cross-linked peptides from the many side products of cross-linking. Most strategies reported to date have focused on the analysis of small cross-linked protein complexes (<60 kDa) because the number of potential forms of covalently modified peptides increases dramatically with the number of peptides generated from the digestion of such complexes.

Structural evidence for co-evolution of the regulation of contraction and energy production in skeletal muscle.

Skeletal muscle phosphorylase kinase (PhK) is a Ca(2+)-dependent enzyme complex, (alpha beta gamma delta)(4), with the delta subunit being tightly bound endogenous calmodulin (CaM). The Ca(2+)-dependent activation of glycogen phosphorylase by PhK couples muscle contraction with glycogen breakdown in the "excitation-contraction-energy production triad." Although the Ca(2+)-dependent protein-protein interactions among the relevant contractile components of muscle are well characterized, such interactions have not been previously examined in the intact PhK complex.

Differential inhibition of cytosolic PEPCK by substrate analogues. Kinetic and structural characterization of inhibitor recognition.

The mechanisms of molecular recognition of phosphoenolpyruvate (PEP) and oxaloacetate (OAA) by cytosolic phosphoenolpyruvate carboxykinase (cPEPCK) were investigated by the systematic evaluation of a variety of PEP and OAA analogues as potential reversible inhibitors of the enzyme against PEP. The molecules that inhibit the enzyme in a competitive fashion were found to fall into two general classes. Those molecules that mimic the binding geometry of PEP, namely phosphoglycolate and 3-phosphonopropionate, are found to bind weakly (millimolar Ki values).