Architecture and activation of human muscle phosphorylase kinase.

The study of phosphorylase kinase (PhK)-regulated glycogen metabolism has contributed to the fundamental understanding of protein phosphorylation; however, the molecular mechanism of PhK remains poorly understood. Here we present the high-resolution cryo-electron microscopy structures of human muscle PhK. The 1.3-megadalton PhK α4ß4γ4δ4 hexadecamer consists of a tetramer of tetramer, wherein four αßγδ modules are connected by the central ß4 scaffold. The α- and ß-subunits possess glucoamylase-like domains, but exhibit no detectable enzyme activities. The α-subunit serves as a bridge between the ß-subunit and the γδ subcomplex, and facilitates the γ-subunit to adopt an autoinhibited state. Ca2+-free calmodulin (δ-subunit) binds to the γ-subunit in a compact conformation. Upon binding of Ca2+, a conformational change occurs, allowing for the de-inhibition of the γ-subunit through a spring-loaded mechanism. We also reveal an ADP-binding pocket in the ß-subunit, which plays a role in allosterically enhancing PhK activity. These results provide molecular insights of this important kinase complex.

Report of an Iranian child with chronic abdominal pain and constipation diagnosed as glycogen storage disease type IX: a case report.

BACKGROUND: Glycogen storage disease type IX is a rare disorder that can cause a wide variety of symptoms depending on the specific deficiency of the phosphorylase kinase enzyme and the organs it affects. CASE PRESENTATION: A 4-and-a-half-year-old Caucasian girl was referred to our clinic with a liver biopsy report indicating a diagnosis of glycogen storage disease. Prior to being referred to our clinic, the patient had been under the care of pediatric gastroenterologists. The patient's initial symptoms included chronic abdominal pain, constipation, and elevated liver transaminase. With the help of the pediatric gastroenterologists, cholestasis, Wilson disease, and autoimmune hepatitis were ruled out. Given that glycogen storage diseases type I and type III are the most common, we initially managed the patient with frequent feedings and a diet that included complex carbohydrates such as a corn starch supplement and a lactose restriction. Following an unfavorable growth velocity and hepatomegaly during the follow-up period, genetic analysis was conducted, which revealed a novel mutation of the phosphorylase kinase regulatory subunit beta gene- a c.C412T (P.Q138x) mutation. As the diagnosis of glycogen storage disease type IX was confirmed, the treatment regimen was altered to a high protein diet (more than 2 g/kg/day) and a low fat diet. CONCLUSION: Given the mild and varied clinical manifestations of glycogen storage disease type IX, it is possible for the diagnosis to be overlooked. It is important to consider glycogen storage disease type IX in children who present with unexplained hepatomegaly and elevated transaminase levels. Furthermore, due to the distinct management of glycogen storage disease type IX compared with glycogen storage disease type I and glycogen storage disease type III, genetic analysis is essential for an accurate diagnosis.

A Mouse Model of Glycogen Storage Disease Type IX-Beta: A Role for Phkb in Glycogenolysis.

Glycogen storage disease type IX (GSD-IX) constitutes nearly a quarter of all GSDs. This ketotic form of GSD is caused by mutations in phosphorylase kinase (PhK), which is composed of four subunits (α, ß, γ, δ). PhK is required for the activation of the liver isoform of glycogen phosphorylase (PYGL), which generates free glucose-1-phosphate monomers to be used as energy via cleavage of the α -(1,4) glycosidic linkages in glycogen chains. Mutations in any of the PhK subunits can negatively affect the regulatory and catalytic activity of PhK during glycogenolysis. To understand the pathogenesis of GSD-IX-beta, we characterized a newly created PHKB knockout (Phkb−/−) mouse model. In this study, we assessed fasting blood glucose and ketone levels, serum metabolite concentrations, glycogen phosphorylase activity, and gene expression of gluconeogenic genes and fibrotic genes. Phkb−/− mice displayed hepatomegaly with lower fasting blood glucose concentrations. Phkb−/− mice showed partial liver glycogen phosphorylase activity and increased sensitivity to pyruvate, indicative of partial glycogenolytic activity and upregulation of gluconeogenesis. Additionally, gene expression analysis demonstrated increased lipid metabolism in Phkb−/− mice. Gene expression analysis and liver histology in the livers of old Phkb−/− mice (>40 weeks) showed minimal profibrogenic features when analyzed with age-matched wild-type (WT) mice. Collectively, the Phkb−/− mouse recapitulates mild clinical features in patients with GSD-IX-beta. Metabolic and molecular analysis confirmed that Phkb−/− mice were capable of sustaining energy homeostasis during prolonged fasting by using partial glycogenolysis, increased gluconeogenesis, and potentially fatty acid oxidation in the liver.

Lung adenocarcinoma-derived vWF promotes tumor metastasis by regulating PHKG1-mediated glycogen metabolism.

Tumor metastasis is a series of complicated biological events. Hematogenous metastasis mediated by von Willebrand factor (vWF) is critical in tumor metastasis. However, the source of vWF and its role in tumor metastasis are controversial, and the further mechanism involved in mediating tumor metastasis is still unclear. In this study, we first demonstrated that lung adenocarcinoma cells could express vWF de novo and promotes tumor metastasis. Through the analysis of transcriptome sequencing, the metastasis promotion effect of vWF may be related to phosphorylase kinase subunit G1 (PHKG1), a catalytic subtype of phosphorylase kinase (PhK). PHKG1 was highly expressed in lung adenocarcinoma patients and led to poor prognosis. Further experiments found that lung adenocarcinoma-derived vWF induced the upregulation of PHKG1 through the PI3K/AKT pathway to promote glycogenolysis. Glycogen was funneled into glycolysis, leading to increased metastasis. Tumor metastasis assayed in vitro and in vivo showed that knockdown of PHKG1 or synergistic injection of phosphorylase inhibition based on the overexpression of vWF could inhibit metastasis. In summary, our research proved that lung adenocarcinoma-derived vWF may mediate tumor metastasis by regulating PHKG1 to promote glycogen metabolism and suggested potential targets for inhibition of lung adenocarcinoma metastasis.

Maximal Multistage Shuttle Run Test-induced Myalgia in a Patient with Muscle Phosphorylase B Kinase Deficiency.

Muscle phosphorylase b kinase (PHK) deficiency is a rare mild metabolic disorder caused by mutations of the PHKA1 gene encoding the αM subunit of PHK. A 16-year-old boy experienced myalgia during the maximal multistage 20-m shuttle run test targeting the maximal oxygen consumption. Although an ischemic forearm exercise test was normal, a muscle biopsy revealed subsarcolemmal glycogen accumulation. He harbored a novel insertion mutation in the PHKA1 gene that resulted in premature termination of the αM subunit close to the C-terminus. Compared with previously reported cases, his reduction in PHK activity was relatively mild.

Genome-wide analysis of expression QTL (eQTL) and allele-specific expression (ASE) in pig muscle identifies candidate genes for meat quality traits.

BACKGROUND: Genetic analysis of gene expression level is a promising approach for characterizing candidate genes that are involved in complex economic traits such as meat quality. In the present study, we conducted expression quantitative trait loci (eQTL) and allele-specific expression (ASE) analyses based on RNA-sequencing (RNAseq) data from the longissimus muscle of 189 Duroc × Luchuan crossed pigs in order to identify some candidate genes for meat quality traits. RESULTS: Using a genome-wide association study based on a mixed linear model, we identified 7192 cis-eQTL corresponding to 2098 cis-genes (p ≤ 1.33e-3, FDR ≤ 0.05) and 6400 trans-eQTL corresponding to 863 trans-genes (p ≤ 1.13e-6, FDR ≤ 0.05). ASE analysis using RNAseq SNPs identified 9815 significant ASE-SNPs in 2253 unique genes. Integrative analysis between the cis-eQTL and ASE target genes identified 540 common genes, including 33 genes with expression levels that were correlated with at least one meat quality trait. Among these 540 common genes, 63 have been reported previously as candidate genes for meat quality traits, such as PHKG1 (q-value = 1.67e-6 for the leading SNP in the cis-eQTL analysis), NUDT7 (q-value = 5.67e-13), FADS2 (q-value = 8.44e-5), and DGAT2 (q-value = 1.24e-3). CONCLUSIONS: The present study confirmed several previously published candidate genes and identified some novel candidate genes for meat quality traits via eQTL and ASE analyses, which will be useful to prioritize candidate genes in further studies.

Intracellular calcium leak lowers glucose storage in human muscle, promoting hyperglycemia and diabetes.

Most glucose is processed in muscle, for energy or glycogen stores. Malignant Hyperthermia Susceptibility (MHS) exemplifies muscle conditions that increase [Ca2+]cytosol. 42% of MHS patients have hyperglycemia. We show that phosphorylated glycogen phosphorylase (GPa), glycogen synthase (GSa) - respectively activated and inactivated by phosphorylation - and their Ca2+-dependent kinase (PhK), are elevated in microsomal extracts from MHS patients' muscle. Glycogen and glucose transporter GLUT4 are decreased. [Ca2+]cytosol, increased to MHS levels, promoted GP phosphorylation. Imaging at ~100 nm resolution located GPa at sarcoplasmic reticulum (SR) junctional cisternae, and apo-GP at Z disk. MHS muscle therefore has a wide-ranging alteration in glucose metabolism: high [Ca2+]cytosol activates PhK, which inhibits GS, activates GP and moves it toward the SR, favoring glycogenolysis. The alterations probably cause these patients' hyperglycemia. For basic studies, MHS emerges as a variable stressor, which forces glucose pathways from the normal to the diseased range, thereby exposing novel metabolic links.

Animals and humans move by contracting the skeletal muscles attached to their bones. These muscles take up a type of sugar called glucose from food and use it to fuel contractions or store it for later in the form of glycogen. If muscles fail to use glucose it can lead to excessive sugar levels in the blood and a condition called diabetes. Within muscle cells are stores of calcium that signal the muscle to contract. Changes in calcium levels enhance the uptake of glucose that fuel these contractions. However, variations in calcium have also been linked to diabetes, and it remained unclear when and how these 'signals' become harmful. People with a condition called malignant hyperthermia susceptibility (MHS for short) have genetic mutations that allow calcium to leak out from these stores. This condition may result in excessive contractions causing the muscle to over-heat, become rigid and break down, which can lead to death if left untreated. A clinical study in 2019 found that out of hundreds of patients who had MHS, nearly half had high blood sugar and were likely to develop diabetes. Now, Tammineni et al. ­ including some of the researchers involved in the 2019 study ­ have set out to find why calcium leaks lead to elevated blood sugar levels. The experiments showed that enzymes that help convert glycogen to glucose are more active in patients with MHS, and found in different locations inside muscle cells. Whereas the enzymes that change glucose into glycogen are less active. This slows down the conversion of glucose into glycogen for storage and speeds up the breakdown of glycogen into glucose. Patients with MHS also had fewer molecules that transport glucose into muscle cells and stored less glycogen. These changes imply that less glucose is being removed from the blood. Next, Tammineni et al. used a microscopy technique that is able to distinguish finely separated objects with a precision not reached before in living muscle. This revealed that when the activity of the enzyme that breaks down glycogen increased, it moved next to the calcium store. This effect was also observed in the muscle cells of MHS patients that leaked calcium from their stores. Taken together, these observations may explain why patients with MHS have high levels of sugar in their blood. These findings suggest that MHS may start decades before developing diabetes and blood sugar levels in these patients should be regularly monitored. Future studies should investigate whether drugs that block calcium from leaking may help prevent high blood sugar in patients with MHS or other conditions that cause a similar calcium leak.

Proteomic analysis of infected primary human leucocytes revealed PSTK as potential treatment-monitoring marker for active and latent tuberculosis.

Markers for monitoring clearance of Mycobacterium tuberculosis (Mtb) infection during anti-TB drug treatment could facilitate management of tuberculosis (TB) treatment, but are lacking. We aimed to screen for Mtb clearance markers from in-vitro-infected leucocytes and to evaluate these markers in followed-up active TB (ATB) patients and latent TB (LTBI) cases after anti-TB drug treatment. Extracellular proteins from primary leucocytes infected with each of the Mtb lineages (East-Asian, Indo-Oceanic, Euro-American and the laboratory strain H37Rv) were screened as possible clearance markers. Leucocytes infected with Staphylococcus aureus acted as controls. The proteomic analysis was performed using GeLC-MS/MS. Several quantitative and qualitative candidate clearance markers were found. These proteins were suppressed during the infection stage of all Mtb lineages and re-expressed after bacillary clearance. PSTK, FKBP8 and MGMT were common clearance markers among the four Mtb lineages in our model. Only PSTK was a potential clearance marker based on western blot validation analysis from culture supernatants. The PSTK marker was further validated with western blot analysis using serum samples (n = 6) from ATB patients and LTBI cases during anti-TB drug treatment, and from healthy controls (n = 3). Time-dependent increase of PSTK was found both in ATB and LTBI patients during the course of anti-TB drug treatment, but not in healthy controls. We have demonstrated that PSTK is a potential treatment-monitoring marker for active and latent TB.

Phosphorylase Kinase ß Represents a Novel Prognostic Biomarker and Inhibits Malignant Phenotypes of Liver Cancer Cell.

Glycogen phosphorylase kinase ß-subunit (PHKB) is a regulatory subunit of phosphorylase kinase (PHK), involving in the activation of glycogen phosphorylase (GP) and the regulation of glycogen breakdown. Emerging evidence suggests that PHKB plays a role in tumor progression. However, the function of PHKB in HCC progression remains elusive. Here, our study revealed that the expression of PHKB significantly decreased in HCC tissues, and the low expression of PHKB could serve as an independent indicator for predicting poor prognosis in HCC. Functional experiments showed that PHKB knockdown significantly promoted cell proliferation both in vitro and in vivo, whereas PHKB overexpression resulted in opposing effects. Additionally, in vitro assays revealed that the over (or high) expression of PHKB greatly hindered HCC cell invasion and increased apoptosis rates. Also, we found that the over (or high) expression of PHKB effectively suppressed the epithelial-mesenchymal transition, which was further confirmed by our clinical data. Intriguingly, the biological function of PHKB in HCC was independent of glycogen metabolism. Mechanically, PHKB could inhibit AKT and STAT3 signaling pathway activation in HCC. Collectively, our data demonstrate that PHKB acts as a novel prognostic indicator for HCC, which exerts its suppression function via inactivating AKT and STAT3. Our data might provide novel insights into progression and facilitate the development of a new therapeutic strategy for HCC.

Differential expression profile analysis of PSTK-regulated mRNAs in podocytes.

This study aimed to elucidate the precise mechanisms underlying the protective effects of phosphoseryl-tRNA kinase (PSTK) against cisplatin-induced podocyte injury. PSTK overexpression and knockdown vectors were generated and transfected into murine podocyte cells-5. PSTK levels were measured, and transcriptome sequencing was conducted. Differential expression analysis was performed to identify messenger RNAs (mRNAs) that were positively and negatively correlated with PSTK. We selected 10 candidate genes identified via real-time quantitative polymerase chain reaction and Western blot analysis for further analysis. As expected, PSTK levels were significantly higher in PSTK-overexpressing podocytes and significantly lower in PSTK-knockdown podocytes. PSTK overexpression resulted in the upregulation of 122 genes and downregulation of 372 genes in podocytes. On the other hand, PSTK knockdown resulted in the upregulation of 231 genes and downregulation of 445 genes. Furthermore, the analysis revealed that 11 genes were positively correlated with PSTK, whereas 20 genes were negatively correlated with PSTK. The obtained PSTK-regulated genes were primarily involved in molecular function, biological process, and cellular component, as well as the angiogenesis pathway. The Wnt family member 10A levels were significantly higher after PSTK overexpression, but were significantly lower after PSTK knockdown. In addition, Na+/K+ ATPase subunit α-2 and matrix metalloproteinase 9 levels were significantly downregulated after PSTK overexpression, but significantly upregulated upon PSTK knockdown. Cell proliferation was significantly increased upon PSTK overexpression, but significantly decreased upon PSTK suppression. The results of this study not only identified several significant PSTK-regulated genes for further validation, but also provided insights into the mechanisms underlying the protective effects of PSTK on podocytes.

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. Additionally, the activity, structure, and regulation of the remaining enzyme necessary for glycogenolysis, glycogen-debranching enzyme, are also reviewed.

Circulating Glucagon 1-61 Regulates Blood Glucose by Increasing Insulin Secretion and Hepatic Glucose Production.

Glucagon is secreted from pancreatic α cells, and hypersecretion (hyperglucagonemia) contributes to diabetic hyperglycemia. Molecular heterogeneity in hyperglucagonemia is poorly investigated. By screening human plasma using high-resolution-proteomics, we identified several glucagon variants, among which proglucagon 1-61 (PG 1-61) appears to be the most abundant form. PG 1-61 is secreted in subjects with obesity, both before and after gastric bypass surgery, with protein and fat as the main drivers for secretion before surgery, but glucose after. Studies in hepatocytes and in ß cells demonstrated that PG 1-61 dose-dependently increases levels of cAMP, through the glucagon receptor, and increases insulin secretion and protein levels of enzymes regulating glycogenolysis and gluconeogenesis. In rats, PG 1-61 increases blood glucose and plasma insulin and decreases plasma levels of amino acids in vivo. We conclude that glucagon variants, such as PG 1-61, may contribute to glucose regulation by stimulating hepatic glucose production and insulin secretion.

Glycogen metabolism in brain and neurons - astrocytes metabolic cooperation can be altered by pre- and neonatal lead (Pb) exposure.

Lead (Pb) is an environmental neurotoxin which particularly affects the developing brain but the molecular mechanism of its neurotoxicity still needs clarification. The aim of this paper was to examine whether pre- and neonatal exposure to Pb (concentration of Pb in rat offspring blood below the "threshold level") may affect the brain's energy metabolism in neurons and astrocytes via the amount of available glycogen. We investigated the glycogen concentration in the brain, as well as the expression of the key enzymes involved in glycogen metabolism in brain: glycogen synthase 1 (Gys1), glycogen phosphorylase (PYGM, an isoform active in astrocytes; and PYGB, an isoform active in neurons) and phosphorylase kinase ß (PHKB). Moreover, the expression of connexin 43 (Cx43) was evaluated to analyze whether Pb poisoning during the early phase of life may affect the neuron-astrocytes' metabolic cooperation. This work shows for the first time that exposure to Pb in early life can impair brain energy metabolism by reducing the amount of glycogen and decreasing the rate of its metabolism. This reduction in brain glycogen level was accompanied by a decrease in Gys1 expression. We noted a reduction in the immunoreactivity and the gene expression of both PYGB and PYGM isoform, as well as an increase in the expression of PHKB in Pb-treated rats. Moreover, exposure to Pb induced decrease in connexin 43 immunoexpression in all the brain structures analyzed, both in astrocytes as well as in neurons. Our data suggests that exposure to Pb in the pre- and neonatal periods results in a decrease in the level of brain glycogen and a reduction in the rate of its metabolism, thereby reducing glucose availability, which as a further consequence may lead to the impairment of brain energy metabolism and the metabolic cooperation between neurons and astrocytes.

Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis.

Ferroptosis is form of regulated nonapoptotic cell death that is involved in diverse disease contexts. Small molecules that inhibit glutathione peroxidase 4 (GPX4), a phospholipid peroxidase, cause lethal accumulation of lipid peroxides and induce ferroptotic cell death. Although ferroptosis has been suggested to involve accumulation of reactive oxygen species (ROS) in lipid environments, the mediators and substrates of ROS generation and the pharmacological mechanism of GPX4 inhibition that generates ROS in lipid environments are unknown. We report here the mechanism of lipid peroxidation during ferroptosis, which involves phosphorylase kinase G2 (PHKG2) regulation of iron availability to lipoxygenase enzymes, which in turn drive ferroptosis through peroxidation of polyunsaturated fatty acids (PUFAs) at the bis-allylic position; indeed, pretreating cells with PUFAs containing the heavy hydrogen isotope deuterium at the site of peroxidation (D-PUFA) prevented PUFA oxidation and blocked ferroptosis. We further found that ferroptosis inducers inhibit GPX4 by covalently targeting the active site selenocysteine, leading to accumulation of PUFA hydroperoxides. In summary, we found that PUFA oxidation by lipoxygenases via a PHKG2-dependent iron pool is necessary for ferroptosis and that the covalent inhibition of the catalytic selenocysteine in Gpx4 prevents elimination of PUFA hydroperoxides; these findings suggest new strategies for controlling ferroptosis in diverse contexts.

Discovery of a Genetic Metabolic Cause for Mauriac Syndrome in Type 1 Diabetes.

A mechanistic cause for Mauriac syndrome, a syndrome of growth failure and delayed puberty associated with massive liver enlargement from glycogen deposition in children with poorly controlled type 1 diabetes, is unknown. We discovered a mutation in the catalytic subunit of liver glycogen phosphorylase kinase in a patient with Mauriac syndrome whose liver extended into his pelvis. Glycogen phosphorylase kinase activates glycogen phosphorylase, the enzyme that catalyzes the first step in glycogen breakdown. We show that the mutant subunit acts in a dominant manner to completely inhibit glycogen phosphorylase kinase enzyme activity and that this interferes with glycogenolysis causing increased levels of glycogen in human liver cells. It is known that even normal blood glucose levels physiologically inhibit glycogen phosphorylase to diminish glucose release from the liver when glycogenolysis is not needed. The patient's mother possessed the same mutant glycogen phosphorylase kinase subunit, but did not have diabetes or hepatomegaly. His father had childhood type 1 diabetes in poor glycemic control, but lacked the mutation and had neither hepatomegaly nor growth failure. This case proves that the effect of a mutant enzyme of glycogen metabolism can combine with hyperglycemia to directly hyperinhibit glycogen phosphorylase, in turn blocking glycogenolysis causing the massive liver in Mauriac disease.

Sensitive, nonradioactive assay of phosphorylase kinase through measurement of enhanced phosphorylase activity towards fluorogenic dextrin.

Glycogen phosphorylase (GP) exists in two interconvertible forms, GPa (phosphorylated form, high activity) and GPb (nonphosphorylated form, low activity). Phosphorylase kinase (PhK) catalyses the phosphorylation of GPb and plays a key role in the cascade system for regulating glycogen metabolism. In this study, we developed a highly sensitive and nonradioactive assay for PhK activity by measuring the enhanced GP activity towards a pyridylaminated maltohexaose. The enhanced GP activity (ΔA) was calculated by the following formula: ΔA = A(+) - A(0), where A(+) and A(0) represent the GP activities of the PhK-treated and PhK-nontreated samples, respectively. Using a high-performance liquid chromatograph equipped with a fluorescence spectrophotometer, the product of GP activity could be isolated and quantified at 10 fmol. This method does not require the use of any radioactive compounds and only 1 µg of GPb per sample was needed to obtain A(+) and A(0) values. The remarkable reduction in GPb concentration enabled us to discuss an interesting new role for glycogen in PhK activity.

Interplay between the chalcone cardamonin and selenium in the biosynthesis of Nrf2-regulated antioxidant enzymes in intestinal Caco-2 cells.

Selenoenzymes and nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated phase II enzymes comprise key components of the cellular redox and antioxidant systems, which show multiple interrelations. Deficiency of the micronutrient selenium (Se) and impaired biosynthesis of selenoproteins have been reported to result in induction of Nrf2 target genes. Conversely, transcription of the selenoenzymes glutathione peroxidase 2 (GPx2) and thioredoxin reductase 1 (TrxR1) is up-regulated upon Nrf2 activation. Here, we have studied the interplay between Se and the secondary plant metabolite cardamonin, an Nrf2-activating chalcone, in the regulation of Nrf2-controlled antioxidant enzymes. Se-deficient and Se-repleted (sodium selenite-supplemented) human intestinal Caco-2 cells were exposed to cardamonin. Uptake of cardamonin by the Caco-2 cells was independent of their Se status. Cardamonin strongly induced gene expression of GPx2 and TrxR1. However, cardamonin treatment did not result in elevated GPx or TrxR activity and protein levels, possibly relating to a concomitant down-regulation of O-phosphoseryl-tRNA(Sec) kinase (PSTK), an enzyme involved in translation of selenoprotein mRNAs. On the other hand, induction of the Nrf2-regulated enzyme heme oxygenase 1 (HO-1) by cardamonin was diminished in Se-replete compared to Se-deficient cells. Our findings suggest that cardamonin interferes with the biosynthesis of Nrf2-regulated selenoenzymes, in contrast to the Nrf2-activating isothiocyanate compound sulforaphane, which has been shown earlier to synergize with Se-mediated cytoprotection. Conversely, the cellular Se status apparently affects the cardamonin-mediated induction of non-selenoprotein antioxidant enzymes such as HO-1.

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 6.8 of nonphosphorylated PhK predictably increased; however, between 30 and 40 °C, there was a dramatic jump in its activity, resulting in the nonactivated enzyme having a far greater activity at body temperature than was previously realized. This anomalous change in properties between 30 and 40 °C was observed for multiple functions, and both stimulation (by ADP and phosphorylation) and inhibition (by orthophosphate) were considerably less pronounced at 40 °C than at 30 °C. In general, the allosteric control of PhK's activity is definitely more subtle at body temperature. Changes in behavior related to activity at 40 °C and its control can be explained by the near disappearance of hysteresis at physiological temperature. In important ways, the picture of PhK that has emerged from six decades of study at temperatures of ≤30 °C does not coincide with that of the enzyme studied at physiological temperature. The probable underlying mechanism for the dramatic increase in PhK's activity between 30 and 40 °C is an abrupt change in the conformations of the regulatory ß and catalytic γ subunits between these two temperatures.

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

Phosphorylase kinase (PhK) is a 1.3 MDa (αßγδ)4 enzyme complex, in which αßγδ 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. We have used partial proteolysis and chemical footprinting in combination with high-resolution mass spectrometry to identify surface-exposed regions of the intact nonactivated and phospho-activated conformers. In addition to the known interaction of the γ subunit's C-terminal regulatory domain with the δ subunit (calmodulin), our exposure results indicate that the catalytic core of γ may also anchor to the PhK complex at the bottom backside of its C-terminal lobe facing away from the active site cleft. Exposed loops on the α and ß regulatory subunits within the complex occur at regions overlapping with tissue-specific alternative RNA splice sites and regulatory phosphorylatable domains. Their phosphorylation alters the surface exposure of α and ß, corroborating previous biophysical and biochemical studies that detected phosphorylation-dependent conformational changes in these subunits; however, for the first time, specific affected regions have been identified.

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

Phosphorylase kinase (PhK) is a hexadecameric (αßγδ)(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 ß-subunits to self-associate. Here we report the effect of a peptide mimetic of the phosphoryltable N-termini of ß on the selective, zero-length, oxidative crosslinking of these regulatory subunits to form ß-ß dimers in the nonactivated PhK complex. This peptide stimulated ß-ß dimer formation when not phosphorylated, but was considerably less effective in its phosphorylated form. Because this peptide mimetic of ß competes with its counterpart region in the nonactivated enzyme complex in binding to the catalytic γ-subunit, we were able to formulate a structural model for the phosphoactivation of PhK. In this model, the nonactivated state of PhK is maintained by the interaction between the nonphosphorylated N-termini of ß and the regulatory C-terminal domains of the γ-subunits; phosphorylation of ß weakens this interaction, leading to activation of the γ-subunits.