The Experts below are selected from a list of 3435 Experts worldwide ranked by ideXlab platform
Manfred W Kilimann - One of the best experts on this subject based on the ideXlab platform.
-
fatal congenital heart glycogenosis caused by a recurrent activating r531q mutation in the γ2 subunit of amp activated protein Kinase prkag2 not by Phosphorylase Kinase deficiency
American Journal of Human Genetics, 2005Co-Authors: Barbara Burwinkel, John W Scott, Christoph Buhrer, Frank K H Van Landeghem, Gerald F Cox, Callum Wilson, Grahame D Hardie, Manfred W KilimannAbstract:Fatal congenital nonlysosomal cardiac glycogenosis has been attributed to a subtype of Phosphorylase Kinase deficiency, but the underlying genes and mutations have not been identified. Analyzing four sporadic, unrelated patients, we found no mutations either in the eight genes encoding Phosphorylase Kinase subunits or in the two genes encoding the muscle and brain isoforms of glycogen Phosphorylase. However, in three of five patients, we identified identical heterozygous R531Q missense mutations of the PRKAG2 gene, which encodes the γ2-subunit of AMP-activated protein Kinase, a key regulator of energy balance. Biochemical characterization of the recombinant R531Q mutant protein showed >100-fold reduction of binding affinities for the regulatory nucleotides AMP and ATP but an enhanced basal activity and increased phosphorylation of the α-subunit. Other PRKAG2 missense mutations were previously identified in patients with autosomal dominant hypertrophic cardiomyopathy with Wolff-Parkinson-White syndrome, characterized by juvenile-to-adult clinical onset, moderate cardiac glycogenosis, disturbed excitation conduction, risk of sudden cardiac death in midlife, and molecular perturbations that are similar to—but less severe than—those observed for the R531Q mutation. Thus, recurrent heterozygous R531Q missense mutations in PRKAG2 give rise to a massive nonlysosomal cardiac glycogenosis of fetal symptomatic onset and rapidly fatal course, constituting a genotypically and clinically distinct variant of hypertrophic cardiomyopathy with Wolff-Parkinson-White syndrome. R531Q and other PRKAG2 mutations enhance the basal activity and α-subunit phosphorylation of AMP-activated protein Kinase, explaining the dominant nature of PRKAG2 disease mutations. Since not all cases displayed PRKAG2 mutations, fatal congenital nonlysosomal cardiac glycogenosis seems to be genetically heterogeneous. However, the existence of a heart-specific primary Phosphorylase Kinase deficiency is questionable, because no Phosphorylase Kinase mutations were found.
-
severe phenotype of Phosphorylase Kinase deficient liver glycogenosis with mutations in the phkg2 gene
Pediatric Research, 2003Co-Authors: Barbara Burwinkel, Manfred W Kilimann, Terje Rootwelt, E A Kvittingen, Pranesh ChakrabortyAbstract:Phosphorylase Kinase-deficient liver glycogenosis manifests in infancy with hepatomegaly, growth retardation, and elevated plasma aminotransferases and lipids. It can be caused by mutations in three different genes of Phosphorylase Kinase subunits: PHKA2, PHKB, and PHKG2. It is usually a benign condition, often with complete resolution of symptoms during puberty. A minority of patients displays a more severe phenotype with symptomatic fasting hypoglycemia and abnormal liver histology that may progress to cirrhosis. Three patients with liver cirrhosis in childhood analyzed previously all had PHKG2 mutations. This suggested that this genotype may generally cause a more severe clinical manifestation, but to date PHKG2 mutations have been identified in only seven patients. Here, we report mutation analysis in three new patients with liver Phosphorylase Kinase deficiency and recurrent hypoglycemia, liver fibrosis, and lack of glucagon response but no overt cirrhosis. In all three patients, PHKG2 mutations were found (H89fs[insC], E157K, D215N, W300X). Three of these mutations are novel, bringing the total number of distinct human PHKG2 mutations to 11, found in 10 patients. We conclude that liver Phosphorylase Kinase deficiency with a severe phenotype, with or without cirrhosis, is indeed often caused by PHKG2 mutations. These patients require active measures to maintain normoglycemia (raw cornstarch, nocturnal tube feeding), which may also alleviate growth retardation and the development of abnormal liver histology.
-
Muscle glycogenosis with low Phosphorylase Kinase activity: mutations in PHKA1, PHKG1 or six other candidate genes explain only a minority of cases
European Journal of Human Genetics, 2003Co-Authors: Barbara Burwinkel, Paula R Clemens, Bin Hu, Anja Schroers, Shimon W Moses, Yoon S Shin, Dieter Pongratz, Matthias Vorgerd, Manfred W KilimannAbstract:Muscle-specific deficiency of Phosphorylase Kinase (Phk) causes glycogen storage disease, clinically manifesting in exercise intolerance with early fatiguability, pain, cramps and occasionally myoglobinuria. In two patients and in a mouse mutant with muscle Phk deficiency, mutations were previously found in the muscle isoform of the Phk α subunit, encoded by the X-chromosomal PHKA1 gene (MIM # 311870). No mutations have been identified in the muscle isoform of the Phk γ subunit ( PHKG1 ). In the present study, we determined Q1the structure of the PHKG1 gene and characterized its relationship to several pseudogenes. In six patients with adult- or juvenile-onset muscle glycogenosis and low Phk activity, we then searched for mutations in eight candidate genes. The coding sequences of all six genes that contribute to Phk in muscle were analysed: PHKA1, PHKB, PHKG1 , CALM1, CALM2 and CALM3 . We also analysed the genes of the muscle isoform of glycogen Phosphorylase ( PYGM ), of a muscle-specific regulatory subunit of the AMP-dependent protein Kinase ( PRKAG3 ), and the promoter regions of PHKA1 , PHKB and PHKG1 . Only in one male patient did we find a PHKA1 missense mutation (D299V) that explains the enzyme deficiency. Two patients were heterozygous for single amino-acid replacements in PHKB that are of unclear significance (Q657K and Y770C). No sequence abnormalities were found in the other three patients. If these results can be generalized, only a fraction of cases with muscle glycogenosis and a biochemical diagnosis of low Phk activity are caused by coding, splice-site or promoter mutations in PHKA1, PHKG1 or other Phk subunit genes. Most patients with this diagnosis probably are affected either by elusive mutations of Phk subunit genes or by defects in other, unidentified genes.
-
Phosphorylase Kinase deficient liver glycogenosis progression to cirrhosis in infancy associated with phkg2 mutations h144y and l225r
Journal of Medical Genetics, 2000Co-Authors: Barbara Burwinkel, M S Tanner, Manfred W KilimannAbstract:Editor—Deficiency of Phosphorylase Kinase (Phk), a regulatory protein Kinase in glycogen metabolism, is the most frequent cause of hepatic glycogen storage disease (GSD). Patients typically present as infants with hepatomegaly, growth retardation, and raised triglycerides, cholesterol, and transaminases. Compared to other types of liver GSD, the condition is usually mild and its course is benign such that patients may even become asymptomatic as they grow up. Hypoglycaemia and lactic acidosis, for example, are uncommon in Phk deficiency in contrast to glucose-6-phosphatase deficiency (GSD type I). Hepatic architecture typically remains normal, unlike GSD III (debranching enzyme deficiency) in which hepatic fibrosis is common, and unlike GSD IV (branching enzyme deficiency) which usually progresses to cirrhosis in infancy. Only two infants with Phk deficiency and cirrhosis have been reported. Development of fibrosis and even cirrhosis was found in five older Japanese patients, but it remains to be clarified whether this observation can be generalised and also applies to other ethnic groups.1-5 Phk is a complex enzyme consisting of four different subunits, (αβγδ)4, and isoforms or splice variants exist for each subunit. This gives rise to genetic and phenotypic heterogeneity of Phk deficiency.6 A muscle specific form of Phk deficiency is caused by mutations in the gene for the muscle isoform of the α subunit, PHKA1 , which resides on the long arm of the X chromosome, whereas liver Phk deficiency can be caused by mutations in three genes: PHKA2 …
-
a mutation in glut2 not in Phosphorylase Kinase subunits in hepato renal glycogenosis with fanconi syndrome and low Phosphorylase Kinase activity
Human Genetics, 1999Co-Authors: Barbara Burwinkel, Sami A Sanjad, Essam Alsabban, Abbas Alabbad, Manfred W KilimannAbstract:Fanconi-Bickel syndrome is characterized by hepato-renal glycogenosis with severe renal tubular dysfunction and rickets. It has recently been found to be associated with GLUT2 mutations in three families. In another family, low activities of liver Phosphorylase Kinase (Phk) have been observed, suggesting that Fanconi-Bickel syndrome might be genetically heterogeneous. We have analyzed this family for mutations in the GLUT2 gene and in the three Phk subunit genes that can cause liver glycogenosis (PHKA2, PHKB, and PHKG2). The coding sequences of all three Phk genes are normal but we have identified a homozygous missense mutation (Pro417Leu) in GLUT2. The affected proline residue is completely conserved in all mammalian glucose permease isoforms and even in bacterial sugar transporters and is believed to be critical for the passage of glucose through the permease. Seven affected individuals from different branches of the same large consanguineous sibship all are homozygous for this mutation. These findings indicate that there is no specific subtype of genetic Phk deficiency giving rise to hepato-renal glycogenosis. Rather, they provide further evidence that Fanconi-Bickel syndrome is caused by GLUT2 mutations. The low Phk activity is probably a secondary phenomenon that contributes to the deposition of glycogen in response to the intracellular glucose retention caused by GLUT2 deficiency.
Barbara Burwinkel - One of the best experts on this subject based on the ideXlab platform.
-
fatal congenital heart glycogenosis caused by a recurrent activating r531q mutation in the γ2 subunit of amp activated protein Kinase prkag2 not by Phosphorylase Kinase deficiency
American Journal of Human Genetics, 2005Co-Authors: Barbara Burwinkel, John W Scott, Christoph Buhrer, Frank K H Van Landeghem, Gerald F Cox, Callum Wilson, Grahame D Hardie, Manfred W KilimannAbstract:Fatal congenital nonlysosomal cardiac glycogenosis has been attributed to a subtype of Phosphorylase Kinase deficiency, but the underlying genes and mutations have not been identified. Analyzing four sporadic, unrelated patients, we found no mutations either in the eight genes encoding Phosphorylase Kinase subunits or in the two genes encoding the muscle and brain isoforms of glycogen Phosphorylase. However, in three of five patients, we identified identical heterozygous R531Q missense mutations of the PRKAG2 gene, which encodes the γ2-subunit of AMP-activated protein Kinase, a key regulator of energy balance. Biochemical characterization of the recombinant R531Q mutant protein showed >100-fold reduction of binding affinities for the regulatory nucleotides AMP and ATP but an enhanced basal activity and increased phosphorylation of the α-subunit. Other PRKAG2 missense mutations were previously identified in patients with autosomal dominant hypertrophic cardiomyopathy with Wolff-Parkinson-White syndrome, characterized by juvenile-to-adult clinical onset, moderate cardiac glycogenosis, disturbed excitation conduction, risk of sudden cardiac death in midlife, and molecular perturbations that are similar to—but less severe than—those observed for the R531Q mutation. Thus, recurrent heterozygous R531Q missense mutations in PRKAG2 give rise to a massive nonlysosomal cardiac glycogenosis of fetal symptomatic onset and rapidly fatal course, constituting a genotypically and clinically distinct variant of hypertrophic cardiomyopathy with Wolff-Parkinson-White syndrome. R531Q and other PRKAG2 mutations enhance the basal activity and α-subunit phosphorylation of AMP-activated protein Kinase, explaining the dominant nature of PRKAG2 disease mutations. Since not all cases displayed PRKAG2 mutations, fatal congenital nonlysosomal cardiac glycogenosis seems to be genetically heterogeneous. However, the existence of a heart-specific primary Phosphorylase Kinase deficiency is questionable, because no Phosphorylase Kinase mutations were found.
-
severe phenotype of Phosphorylase Kinase deficient liver glycogenosis with mutations in the phkg2 gene
Pediatric Research, 2003Co-Authors: Barbara Burwinkel, Manfred W Kilimann, Terje Rootwelt, E A Kvittingen, Pranesh ChakrabortyAbstract:Phosphorylase Kinase-deficient liver glycogenosis manifests in infancy with hepatomegaly, growth retardation, and elevated plasma aminotransferases and lipids. It can be caused by mutations in three different genes of Phosphorylase Kinase subunits: PHKA2, PHKB, and PHKG2. It is usually a benign condition, often with complete resolution of symptoms during puberty. A minority of patients displays a more severe phenotype with symptomatic fasting hypoglycemia and abnormal liver histology that may progress to cirrhosis. Three patients with liver cirrhosis in childhood analyzed previously all had PHKG2 mutations. This suggested that this genotype may generally cause a more severe clinical manifestation, but to date PHKG2 mutations have been identified in only seven patients. Here, we report mutation analysis in three new patients with liver Phosphorylase Kinase deficiency and recurrent hypoglycemia, liver fibrosis, and lack of glucagon response but no overt cirrhosis. In all three patients, PHKG2 mutations were found (H89fs[insC], E157K, D215N, W300X). Three of these mutations are novel, bringing the total number of distinct human PHKG2 mutations to 11, found in 10 patients. We conclude that liver Phosphorylase Kinase deficiency with a severe phenotype, with or without cirrhosis, is indeed often caused by PHKG2 mutations. These patients require active measures to maintain normoglycemia (raw cornstarch, nocturnal tube feeding), which may also alleviate growth retardation and the development of abnormal liver histology.
-
Muscle glycogenosis with low Phosphorylase Kinase activity: mutations in PHKA1, PHKG1 or six other candidate genes explain only a minority of cases
European Journal of Human Genetics, 2003Co-Authors: Barbara Burwinkel, Paula R Clemens, Bin Hu, Anja Schroers, Shimon W Moses, Yoon S Shin, Dieter Pongratz, Matthias Vorgerd, Manfred W KilimannAbstract:Muscle-specific deficiency of Phosphorylase Kinase (Phk) causes glycogen storage disease, clinically manifesting in exercise intolerance with early fatiguability, pain, cramps and occasionally myoglobinuria. In two patients and in a mouse mutant with muscle Phk deficiency, mutations were previously found in the muscle isoform of the Phk α subunit, encoded by the X-chromosomal PHKA1 gene (MIM # 311870). No mutations have been identified in the muscle isoform of the Phk γ subunit ( PHKG1 ). In the present study, we determined Q1the structure of the PHKG1 gene and characterized its relationship to several pseudogenes. In six patients with adult- or juvenile-onset muscle glycogenosis and low Phk activity, we then searched for mutations in eight candidate genes. The coding sequences of all six genes that contribute to Phk in muscle were analysed: PHKA1, PHKB, PHKG1 , CALM1, CALM2 and CALM3 . We also analysed the genes of the muscle isoform of glycogen Phosphorylase ( PYGM ), of a muscle-specific regulatory subunit of the AMP-dependent protein Kinase ( PRKAG3 ), and the promoter regions of PHKA1 , PHKB and PHKG1 . Only in one male patient did we find a PHKA1 missense mutation (D299V) that explains the enzyme deficiency. Two patients were heterozygous for single amino-acid replacements in PHKB that are of unclear significance (Q657K and Y770C). No sequence abnormalities were found in the other three patients. If these results can be generalized, only a fraction of cases with muscle glycogenosis and a biochemical diagnosis of low Phk activity are caused by coding, splice-site or promoter mutations in PHKA1, PHKG1 or other Phk subunit genes. Most patients with this diagnosis probably are affected either by elusive mutations of Phk subunit genes or by defects in other, unidentified genes.
-
Phosphorylase Kinase deficient liver glycogenosis progression to cirrhosis in infancy associated with phkg2 mutations h144y and l225r
Journal of Medical Genetics, 2000Co-Authors: Barbara Burwinkel, M S Tanner, Manfred W KilimannAbstract:Editor—Deficiency of Phosphorylase Kinase (Phk), a regulatory protein Kinase in glycogen metabolism, is the most frequent cause of hepatic glycogen storage disease (GSD). Patients typically present as infants with hepatomegaly, growth retardation, and raised triglycerides, cholesterol, and transaminases. Compared to other types of liver GSD, the condition is usually mild and its course is benign such that patients may even become asymptomatic as they grow up. Hypoglycaemia and lactic acidosis, for example, are uncommon in Phk deficiency in contrast to glucose-6-phosphatase deficiency (GSD type I). Hepatic architecture typically remains normal, unlike GSD III (debranching enzyme deficiency) in which hepatic fibrosis is common, and unlike GSD IV (branching enzyme deficiency) which usually progresses to cirrhosis in infancy. Only two infants with Phk deficiency and cirrhosis have been reported. Development of fibrosis and even cirrhosis was found in five older Japanese patients, but it remains to be clarified whether this observation can be generalised and also applies to other ethnic groups.1-5 Phk is a complex enzyme consisting of four different subunits, (αβγδ)4, and isoforms or splice variants exist for each subunit. This gives rise to genetic and phenotypic heterogeneity of Phk deficiency.6 A muscle specific form of Phk deficiency is caused by mutations in the gene for the muscle isoform of the α subunit, PHKA1 , which resides on the long arm of the X chromosome, whereas liver Phk deficiency can be caused by mutations in three genes: PHKA2 …
-
a mutation in glut2 not in Phosphorylase Kinase subunits in hepato renal glycogenosis with fanconi syndrome and low Phosphorylase Kinase activity
Human Genetics, 1999Co-Authors: Barbara Burwinkel, Sami A Sanjad, Essam Alsabban, Abbas Alabbad, Manfred W KilimannAbstract:Fanconi-Bickel syndrome is characterized by hepato-renal glycogenosis with severe renal tubular dysfunction and rickets. It has recently been found to be associated with GLUT2 mutations in three families. In another family, low activities of liver Phosphorylase Kinase (Phk) have been observed, suggesting that Fanconi-Bickel syndrome might be genetically heterogeneous. We have analyzed this family for mutations in the GLUT2 gene and in the three Phk subunit genes that can cause liver glycogenosis (PHKA2, PHKB, and PHKG2). The coding sequences of all three Phk genes are normal but we have identified a homozygous missense mutation (Pro417Leu) in GLUT2. The affected proline residue is completely conserved in all mammalian glucose permease isoforms and even in bacterial sugar transporters and is believed to be critical for the passage of glucose through the permease. Seven affected individuals from different branches of the same large consanguineous sibship all are homozygous for this mutation. These findings indicate that there is no specific subtype of genetic Phk deficiency giving rise to hepato-renal glycogenosis. Rather, they provide further evidence that Fanconi-Bickel syndrome is caused by GLUT2 mutations. The low Phk activity is probably a secondary phenomenon that contributes to the deposition of glycogen in response to the intracellular glucose retention caused by GLUT2 deficiency.
Gerald M. Carlson - One of the best experts on this subject based on the ideXlab platform.
-
structural characterization of the catalytic γ and regulatory β subunits of Phosphorylase Kinase in the context of the hexadecameric enzyme complex
Protein Science, 2018Co-Authors: Mary Ashley Rimmer, Antonio Artigues, Owen W Nadeau, Gerald M. CarlsonAbstract: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. Using hydrogen-deuterium exchange, we have analyzed the regulatory β subunit and the catalytic γ subunit in the context of the intact non-activated PhK complex to study the structure of these subunits and identify regions of surface exposure. Our data suggest that within the non-activated complex the γ subunit assumes an activated conformation and are consistent with a previous docking model of the β subunit within the cryoelectron microscopy envelope of PhK.
-
the structure of the large regulatory α subunit of Phosphorylase Kinase examined by modeling and hydrogen deuterium exchange
Protein Science, 2018Co-Authors: Mary Ashley Rimmer, Antonio Artigues, Owen W Nadeau, Yang Zhang, Jianyi Yang, Gerald M. CarlsonAbstract:Phosphorylase Kinase (PhK), a 1.3 MDa regulatory enzyme complex in the glycogenolysis cascade, has four copies each of four subunits, (αβγδ)4 , 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. Due to its size and complexity, no high resolution structures have been solved for the intact complex or its regulatory α and β subunits. Of PhK's four subunits, the least is known about the structure and function of its largest subunit, α. Here, we have modeled the full-length α subunit, compared that structure against previously predicted domains within this subunit, and performed hydrogen-deuterium exchange on the intact subunit within the PhK complex. Our modeling results show α to comprise two major domains: an N-terminal glycoside hydrolase domain and a large C-terminal importin α/β-like domain. This structure is similar to our previously published model for the homologous β subunit, although clear structural differences are present. The overall highly helical structure with several intervening hinge regions is consistent with our hydrogen-deuterium exchange results obtained for this subunit as part of the (αβγδ)4 PhK complex. Several low exchanging regions predicted to lack ordered secondary structure are consistent with inter-subunit contact sites for α in the quaternary structure of PhK; of particular interest is a low-exchanging region in the C-terminus of α that is known to bind the regulatory domain of the catalytic γ subunit.
-
the regulatory α and β subunits of Phosphorylase Kinase directly interact with its substrate glycogen Phosphorylase
Biochemical and Biophysical Research Communications, 2017Co-Authors: Jackie A Thompson, Gerald M. CarlsonAbstract: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. These newfound interactions were found to be sensitive to ligands that bind PhK.
-
mass spectrometric analysis of surface exposed regions in the hexadecameric Phosphorylase Kinase complex
Biochemistry, 2015Co-Authors: Mary Ashley Rimmer, Antonio Artigues, Owen W Nadeau, Maria T Villar, Victor Vasquezmontes, Gerald M. CarlsonAbstract: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. 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 β 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 β, 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
Protein Science, 2015Co-Authors: Jackie A Thompson, Owen W Nadeau, Gerald M. CarlsonAbstract: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. Here we report the effect of a peptide mimetic of the phosphoryltable N-termini of β on the selective, zero-length, oxidative crosslinking of these regulatory subunits to form β-β dimers in the nonactivated PhK complex. This peptide stimulated β-β dimer formation when not phosphorylated, but was considerably less effective in its phosphorylated form. Because this peptide mimetic of β 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 β and the regulatory C-terminal domains of the γ-subunits; phosphorylation of β weakens this interaction, leading to activation of the γ-subunits.
Owen W Nadeau - One of the best experts on this subject based on the ideXlab platform.
-
structural characterization of the catalytic γ and regulatory β subunits of Phosphorylase Kinase in the context of the hexadecameric enzyme complex
Protein Science, 2018Co-Authors: Mary Ashley Rimmer, Antonio Artigues, Owen W Nadeau, Gerald M. CarlsonAbstract: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. Using hydrogen-deuterium exchange, we have analyzed the regulatory β subunit and the catalytic γ subunit in the context of the intact non-activated PhK complex to study the structure of these subunits and identify regions of surface exposure. Our data suggest that within the non-activated complex the γ subunit assumes an activated conformation and are consistent with a previous docking model of the β subunit within the cryoelectron microscopy envelope of PhK.
-
the structure of the large regulatory α subunit of Phosphorylase Kinase examined by modeling and hydrogen deuterium exchange
Protein Science, 2018Co-Authors: Mary Ashley Rimmer, Antonio Artigues, Owen W Nadeau, Yang Zhang, Jianyi Yang, Gerald M. CarlsonAbstract:Phosphorylase Kinase (PhK), a 1.3 MDa regulatory enzyme complex in the glycogenolysis cascade, has four copies each of four subunits, (αβγδ)4 , 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. Due to its size and complexity, no high resolution structures have been solved for the intact complex or its regulatory α and β subunits. Of PhK's four subunits, the least is known about the structure and function of its largest subunit, α. Here, we have modeled the full-length α subunit, compared that structure against previously predicted domains within this subunit, and performed hydrogen-deuterium exchange on the intact subunit within the PhK complex. Our modeling results show α to comprise two major domains: an N-terminal glycoside hydrolase domain and a large C-terminal importin α/β-like domain. This structure is similar to our previously published model for the homologous β subunit, although clear structural differences are present. The overall highly helical structure with several intervening hinge regions is consistent with our hydrogen-deuterium exchange results obtained for this subunit as part of the (αβγδ)4 PhK complex. Several low exchanging regions predicted to lack ordered secondary structure are consistent with inter-subunit contact sites for α in the quaternary structure of PhK; of particular interest is a low-exchanging region in the C-terminus of α that is known to bind the regulatory domain of the catalytic γ subunit.
-
mass spectrometric analysis of surface exposed regions in the hexadecameric Phosphorylase Kinase complex
Biochemistry, 2015Co-Authors: Mary Ashley Rimmer, Antonio Artigues, Owen W Nadeau, Maria T Villar, Victor Vasquezmontes, Gerald M. CarlsonAbstract: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. 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 β 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 β, 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
Protein Science, 2015Co-Authors: Jackie A Thompson, Owen W Nadeau, Gerald M. CarlsonAbstract: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. Here we report the effect of a peptide mimetic of the phosphoryltable N-termini of β on the selective, zero-length, oxidative crosslinking of these regulatory subunits to form β-β dimers in the nonactivated PhK complex. This peptide stimulated β-β dimer formation when not phosphorylated, but was considerably less effective in its phosphorylated form. Because this peptide mimetic of β 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 β and the regulatory C-terminal domains of the γ-subunits; phosphorylation of β weakens this interaction, leading to activation of the γ-subunits.
-
mass spectrometry reveals differences in stability and subunit interactions between activated and nonactivated conformers of the αβγδ 4 Phosphorylase Kinase complex
Molecular & Cellular Proteomics, 2012Co-Authors: Laura A Lane, Gerald M. Carlson, Owen W Nadeau, Carol V RobinsonAbstract: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. The regulation of activity of the PhK complex from skeletal muscle has been studied extensively; however, considerably less is known about the interactions among its subunits, particularly within the non-activated versus activated forms of the complex. Here, nanoelectrospray mass spectrometry and partial denaturation were used to disrupt PhK, and subunit dissociation patterns of non-activated and phospho-activated (autophosphorylation) conformers were compared. In so doing, we have established a network of subunit contacts that complements and extends prior evidence of subunit interactions obtained from chemical crosslinking, and these subunit interactions have been modeled for both conformers within the context of a known three-dimensional structure of PhK solved by cryoelectron microscopy. Our analyses show that the network of contacts among subunits differs significantly between the nonactivated and phospho-activated conformers of PhK, with the latter revealing new interprotomeric contact patterns for the β subunit, the predominant subunit responsible for PhK's activation by phosphorylation. Partial disruption of the phosphorylated conformer yields several novel subcomplexes containing multiple β subunits, arguing for their self-association within the activated complex. Evidence for the theoretical αβγδ protomeric subcomplex, which has been sought but not previously observed, was also derived from the phospho-activated complex. In addition to changes in subunit interaction patterns upon phospho-activation, mass spectrometry revealed a large change in the overall stability of the complex, with the phospho-activated conformer being more labile, in concordance with previous hypotheses on the mechanism of allosteric activation of PhK through perturbation of its inhibitory quaternary structure.
L N Johnson - One of the best experts on this subject based on the ideXlab platform.
-
the structure of Phosphorylase Kinase holoenzyme at 9 9 a resolution and location of the catalytic subunit and the substrate glycogen Phosphorylase
Structure, 2009Co-Authors: Catherine Venienbryan, Nikos G Oikonomakos, Nicolas Boisset, Vasiliki Skamnaki, Nicolas Bischler, Slavica Jonic, Nick Brown, L N JohnsonAbstract:Phosphorylase Kinase (PhK) coordinates hormonal and neuronal signals to initiate the breakdown of glycogen. The enzyme catalyzes the phosphorylation of inactive glycogen Phosphorylase b (GPb), resulting in the formation of active glycogen Phosphorylase a. We present a 9.9 A resolution structure of PhK heterotetramer (αβγδ)4 determined by cryo-electron microscopy single-particle reconstruction. The enzyme has a butterfly-like shape comprising two lobes with 222 symmetry. This three-dimensional structure has allowed us to dock the catalytic γ subunit to the PhK holoenzyme at a location that is toward the ends of the lobes. We have also determined the structure of PhK decorated with GPb at 18 A resolution, which shows the location of the substrate near the Kinase subunit. The PhK preparation contained a number of smaller particles whose structure at 9.8 A resolution was consistent with a proteolysed activated form of PhK that had lost the α subunits and possibly the γ subunits.
-
structural and functional studies of rabbit skeletal muscle Phosphorylase Kinase
2008Co-Authors: S Jonic, L N Johnson, C Carriere, E Larquet, Vasiliki Skamnaki, Catherine Venienbryan, J P Mornon, I Callebaut, N BoissetAbstract:During a physical exercice, the activity of the Phosphorylase Kinase (PhK) is very high, promoting an optimal level of glucose in skeletal muscle. Its major role is to catalyze the conversion of the inactive Glycogene Phosphorylase (GPb) to an active form (GPa) by phosphorylation. The PhK is composed of 4 copies of the α, β, γ and δ subunits forming a hexadecameric structure. This structure is organised into 2 lobes with D2 symmetry. Each lobe comprises 2 protomers of α β γ δ, with bridges connecting the 2 lobes. The α and β subunits account for 2/3 of the protein mass (1.3×106 Da); the similarity of α and β sequences is probably due to gene duplication. The δ subunit is a typical calcium binding protein, a calmodulin, which is bound tighly to the complex. The γ subunit is the catalytic subunit, which functions to phosphorylate serine and threonine residues on PhK α and β, and GPb.
-
structural characterization of ca2 cam in complex with the Phosphorylase Kinase phk5 peptide
FEBS Journal, 2005Co-Authors: Atlanta G Cook, L N Johnson, James M McdonnellAbstract:Phosphorylase Kinase (PhK) is a large hexadecameric enzyme consisting of four copies of four subunits: (αβγδ)4. An intrinsic calmodulin (CaM, the δ subunit) binds directly to the γ protein Kinase chain. The interaction site of CaM on γ has been localized to a C-terminal extension of the Kinase domain. Two 25-mer peptides derived from this region, PhK5 and PhK13, were identified previously as potential CaM-binding sites. Complex formation between Ca2+/CaM with these two peptides was characterized using analytical gel filtration and NMR methods. NMR chemical shift perturbation studies showed that while PhK5 forms a robust complex with Ca2+/CaM, no interactions with PhK13 were observed. 15N relaxation characteristics of Ca2+/CaM and Ca2+/CaM/PhK5 complexes were compared with the experimentally determined structures of several Ca2+/CaM/peptide complexes. Good fits were observed between Ca2+/CaM/PhK5 and three structures: Ca2+/CaM complexes with peptides from endothelial nitric oxide synthase, with smooth muscle myosin light chain Kinase and CaM Kinase I. We conclude that the PhK5 site is likely to have a direct role in Ca2+-regulated control of PhK activity through the formation of a classical ‘compact’ CaM complex.
-
catalytic mechanism of Phosphorylase Kinase probed by mutational studies
Biochemistry, 1999Co-Authors: Vicky T Skamnaki, Martin E M Noble, Nikos G Oikonomakos, David J Owen, E D Lowe, G Lowe, L N JohnsonAbstract:The contributions to catalysis of the conserved catalytic aspartate (Asp149) in the Phosphorylase Kinase catalytic subunit (PhK; residues 1−298) have been studied by kinetic and crystallographic methods. Kinetic studies in solvents of different viscosity show that PhK, like cyclic AMP dependent protein Kinase, exhibits a mechanism in which the chemical step of phosphoryl transfer is fast and the rate-limiting step is release of the products, ADP and phosphoprotein, and possibly viscosity-dependent conformational changes. Site-directed mutagenesis of Asp149 to Ala and Asn resulted in enzymes with a small increase in Km for glycogen Phosphorylase b (GPb) and ATP substrates and dramatic decreases in kcat (1.3 × 104 for Asp149Ala and 4.7 × 103 for Asp149Asn mutants, respectively). Viscosometric kinetic measurements with the Asp149Asn mutant showed a reduction in the rate-limiting step for release of products by 4.5 × 103 and a significant decrease (possibly as great as 2.2 × 103) in the rate constant characte...
-
the crystal structure of a Phosphorylase Kinase peptide substrate complex Kinase substrate recognition
The EMBO Journal, 1997Co-Authors: Edward D Lowe, Martin E M Noble, Vicky T Skamnaki, Nikos G Oikonomakos, David J Owen, L N JohnsonAbstract:The structure of a truncated form of the gamma-subunit of Phosphorylase Kinase (PHKgammat) has been solved in a ternary complex with a non-hydrolysable ATP analogue (adenylyl imidodiphosphate, AMPPNP) and a heptapeptide substrate related in sequence to both the natural substrate and to the optimal peptide substrate. Kinetic characterization of the phosphotransfer reaction confirms the peptide to be a good substrate, and the structure allows identification of key features responsible for its high affinity. Unexpectedly, the substrate peptide forms a short anti-parallel beta-sheet with the Kinase activation segment, the region which in other Kinases plays an important role in regulation of enzyme activity. This anchoring of the main chain of the substrate peptide at a fixed distance from the gamma-phosphate of ATP explains the selectivity of PHK for serine/threonine over tyrosine as a substrate. The catalytic core of PHK exists as a dimer in crystals of the ternary complex, and the relevance of this phenomenon to its in vivo recognition of dimeric glycogen Phosphorylase b is considered.