Peroxisomal disorders disorders Peroxisomal Guy Besley Guy Besley Willink Biochemical Genetics Unit, Willink Biochemical Genetics Unit, Manchester Children’s Hospital, Manchester Children’s Hospital, Manchester M27 4HA Manchester M27 4HA BIMDG-York 3 July 2003
Peroxisomes Peroxisomes • Found in all nucleated mammalian cells • Found in all nucleated mammalian cells • Initially identified • Initially identified histologically histologically as as microbodies in 1950s in 1950s microbodies • First evidence of biological function in 1960s • First evidence of biological function in 1960s • Catalase • Catalase ‘marker’ enzyme to remove H ‘marker’ enzyme to remove H 2 O 2 2 O 2 generated by several oxidases oxidases generated by several • Many other important functions now known • Many other important functions now known
Peroxisome morphology morphology Peroxisome • Generally appear as spherical single • Generally appear as spherical single membrane- -bound, bodies of 0.1 bound, bodies of 0.1 – – 1.0 1.0 µ m in membrane µ m in diameter diameter • The number per cell varies depending on • The number per cell varies depending on demand. Most numerous in liver and kidney, demand. Most numerous in liver and kidney, few in fibroblasts few in fibroblasts • Localised by • Localised by histochemical histochemical staining for staining for catalase (DAB) or using (DAB) or using immunocytochemical immunocytochemical catalase methods methods • Peroxisomes • Peroxisomes are formed from pre are formed from pre- -existing existing organelles organelles • In rodents but not man induction can be • In rodents but not man induction can be stimulated by clofibrate clofibrate, , plasticisors plasticisors or or stimulated by hypolipidaemic drugs drugs hypolipidaemic
Liver EM – – DAB stain DAB stain Liver EM
Peroxisomal functions functions Peroxisomal • The importance of these functions is • The importance of these functions is illustrated by the severe consequences of illustrated by the severe consequences of peroxisomal dysfunction, especially in dysfunction, especially in peroxisomal peroxisome biogenesis defects biogenesis defects peroxisome • • Although these defects were first reported Although these defects were first reported in 1973 by Goldfischer Goldfischer, it was not until a , it was not until a in 1973 by decade later that the biochemical decade later that the biochemical abnormalities were really recognised abnormalities were really recognised
Peroxisomal functions in man functions in man Peroxisomal • Distinct fatty acid • Distinct fatty acid β -oxidation system, oxidation system, β - especially of very long straight- -chain chain especially of very long straight fatty acids (C12- -27) 27) fatty acids (C12 • Biosynthesis of ether phospholipids • Biosynthesis of ether phospholipids (plasmalogens plasmalogens) ) ( • The • The α -oxidation of 3 oxidation of 3- -methyl branched methyl branched α - chain fatty acids (phytanate phytanate) ) chain fatty acids ( • Biosynthesis of • Biosynthesis of isoprenoids isoprenoids
Other peroxisomal peroxisomal functions in functions in Other man man • Removal of • Removal of glyoxylate glyoxylate via via alanine alanine: :glyoxylate glyoxylate amino transferase amino transferase • Oxidation of • Oxidation of pipecolic pipecolic acid derived from L acid derived from L- - lysine lysine • Oxidation of • Oxidation of glutaryl glutaryl- -CoA CoA • Elongation of fatty acids • Elongation of fatty acids • Unlike mitochondria, the energy generated by • Unlike mitochondria, the energy generated by various oxidation steps generates heat and various oxidation steps generates heat and H 2 O 2 , not ATP. H 2 O 2 , not ATP.
Very long chain fatty acid Very long chain fatty acid metabolism metabolism • Very long chain fatty acids (C22 • Very long chain fatty acids (C22- -26) are 26) are preferentially oxidised by the peroxisome peroxisome. . preferentially oxidised by the • They are taken up into • They are taken up into peroxisomes peroxisomes following following activation to their CoA CoA ester ester activation to their • Uptake is via a specific ABC • Uptake is via a specific ABC- -transporter in transporter in the peroxisomal membrane, which is ATP- - the peroxisomal membrane, which is ATP dependent dependent • Referred to as ALDP which exists as a homo • Referred to as ALDP which exists as a homo- - or heterodimer heterodimer or
Peroxisome β -oxidation Phytanic acid THCA VLCFA (C26) Pristanic acid branched chain Straight chain acyl-CoA oxidase acyl-CoA oxidase bifunctional enzyme (hydratase + dehydrogenase) Thiolase 2 Thiolase 1 (Sterol carrier protein X) VLCFA (C24) Trimethyl tridecanoyl-CoA choloyl-CoA
Phytanic acid α -oxidation Phytanic acid Phytanoyl-CoA phytanoyl-CoA hydroxylase 2-hydroxy-phytanoyl-CoA lyase and dehydrogenase Pristanic acid
Plasmalogens – – ether ether- -linked linked Plasmalogens phospholipids phospholipids H 2 COCH=CHR R’COOCH O H 2 C-O-P-CH 2 CH 2 NH 3 O Plasmalogens represent 5 – 20% membrane phospholipids (30% in myelin) and protect against photosensitivity
Plasmalogen biosynthesis biosynthesis Plasmalogen Acyl- -CoA CoA Acyl Gly- -3 3- -P P DHAP Gly DHAP DHAP- -AT AT DHAP CoASH CoASH Acyl- -DHAP DHAP Acyl Long- -chain alcohol chain alcohol Long Alkyl- -DHAP DHAP Alkyl synthase synthase Fatty acid Fatty acid Alkyl- -DHAP DHAP Alkyl Alkyl/acyl acyl- -DHAP: DHAP: Alkyl/ plasmalogens plasmalogens NAD(P)H oxidoreductase oxidoreductase NAD(P)H Alkyl- -Gly Gly- -3 3- -P P Alkyl
Peroxisome biogenesis biogenesis Peroxisome • Peroxisomal • Peroxisomal proteins are synthesised on free proteins are synthesised on free polyribosomes polyribosomes • They must carry a specific peptide signal to • They must carry a specific peptide signal to direct them to pre- -existing existing peroxisomes peroxisomes direct them to pre • Most matrix proteins carry the • Most matrix proteins carry the Peroxisome Peroxisome Targetting Signal (PTS1) serine Signal (PTS1) serine- -lysine lysine- -leucine leucine Targetting (SKL) (SKL) • A second signal (PTS2) is required for some • A second signal (PTS2) is required for some enzyme proteins, including thiolase thiolase, , phytanoyl phytanoyl- - enzyme proteins, including CoA hydroxylase hydroxylase and DHAP and DHAP- -AT, alkyl AT, alkyl- -DHAP DHAP CoA synthase synthase • Peroxisome • Peroxisome membrane proteins do not use PTS1 membrane proteins do not use PTS1 or PTS2 for targetting targetting or PTS2 for
PEX genes PEX genes • Through studies on • Through studies on peroxisome peroxisome function and function and defects in biogenesis a number of genes have defects in biogenesis a number of genes have been identified been identified • These genes encode mostly • These genes encode mostly peroxisome peroxisome integral membrane proteins essential for the integral membrane proteins essential for the assembly and biosynthesis of peroxisomes peroxisomes assembly and biosynthesis of • The proteins are referred to as • The proteins are referred to as peroxins peroxins and and the genes PEX genes the genes PEX genes • Mutations in PEX genes are responsible for • Mutations in PEX genes are responsible for peroxisome biogenesis disorders ( biogenesis disorders (Zellweger Zellweger) ) peroxisome and RCDP and RCDP
Peroxisomal disorders and PEX disorders and PEX Peroxisomal gene disorders: two major gene disorders: two major categories (www. categories (www.peroxisome peroxisome.org) .org) • PEX gene defects • PEX gene defects – Peroxisome Peroxisome biogenesis disorders including: biogenesis disorders including: – Zellweger syndrome, neonatal syndrome, neonatal Zellweger adrenoleukodytrophy and infantile and infantile Refsum Refsum adrenoleukodytrophy disease and Rhizomelic Rhizomelic chondrodysplasia chondrodysplasia disease and punctata (PTS2 defect (PTS2 defect - - PEX7 mutations) PEX7 mutations) punctata • Single enzyme defects (X • Single enzyme defects (X- -ALD, ALD, β - β - oxidation defects, Refsum Refsum etc) etc) oxidation defects,
Peroxisome biogenesis disorders biogenesis disorders Peroxisome • Zellweger • Zellweger syndrome syndrome – – most severe phenotype most severe phenotype – Typical Typical dysmorphology dysmorphology, profound , profound hypotonia hypotonia, , – hepatic dysfunction, neonatal presentation hepatic dysfunction, neonatal presentation • Neonatal • Neonatal adrenoleukodystrophy adrenoleukodystrophy – Intermediate phenotype Intermediate phenotype – • Infantile • Infantile Refsum Refsum disease disease – – milder phenotype milder phenotype – Retinopathy, deafness, mild Retinopathy, deafness, mild dysmorphism dysmorphism – • 11 complementation groups, group 1 (PEX1 • 11 complementation groups, group 1 (PEX1 mutations account for 65% patients) includes mutations account for 65% patients) includes all phenotypes all phenotypes
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