Molecular organization of peroxisomal enzymes: protein-protein interactions in the membrane and in the matrix.
The beta-oxidation of fatty acids in peroxisomes produces hydrogen peroxide (H2O2), a toxic metabolite, as a bi-product. Fatty acids beta-oxidation activity is deficient in X-linked adrenoleukodystrophy (X-ALD) because of mutation in ALD-gene resulting in loss of very long chain acyl-CoA synthetase (VLCS) activity. It is also affected in disease with catalase negative peroxisomes as a result of inactivation by H2O2. Therefore, the following studies were undertaken to delineate the molecular interactions between both the ALD-gene product (adrenoleukodystrophy protein, ALDP) and VLCS as well as H2O2 degrading enzyme catalase and proteins of peroxisomal beta-oxidation. Studies using a yeast two hybrid system and surface plasmon resonance techniques indicate that ALDP, a peroxisomal membrane protein, physically interacts with VLCS. Loss of these interactions in X-ALD cells may result in a deficiency in VLCS activity. The yeast two-hybrid system studies also indicated that catalase physically interacts with L-bifunctional enzyme (L-BFE). Interactions between catalase and L-BFE were further supported by affinity purification, using a catalase-linked resin. The affinity bound 74-kDa protein, was identified as L-BFE by Western blot with specific antibodies and by proteomic analysis. Additional support for their interaction comes from immunoprecipitation of L-BFE with antibodies against catalase as a catalase- L-BFE complex. siRNA for L-BFE decreased the specific activity and protein levels of catalase without changing its subcellular distribution. These observations indicate that L-BFE might help in oligomerization and possibly in the localization of catalase at the site of H2O2 production in the peroxisomal beta-oxidation pathway.
Makkar
Randhir S
RS
The Charles Darby Children's Research Institute, Department of Pediatrics, Medical University of South Carolina, Charleston, SC 29425, USA.
Contreras
Miguel A
MA
Paintlia
Ajaib S
AS
Smith
Brian T
BT
Haq
Ehtishamul
E
Singh
Inderjit
I
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D015415:Biomarkers / Q000032:analysis; D002374:Catalase / Q000032:analysis / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D003066:Coenzyme A Ligases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004746:Enoyl-CoA Hydratase / Q000378:metabolism*; D005109:Extracellular Matrix / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D007535:Isomerases / Q000378:metabolism*; D008099:Liver / Q000166:cytology; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D063994:Peroxisomal Bifunctional Enzyme; D020675:Peroxisomes / Q000201:enzymology* / Q000648:ultrastructure; D011485:Protein Binding; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D013347:Subcellular Fractions / Q000378:metabolism; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
D015415:Biomarkers; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D020543:Proteome; D012333:RNA, Messenger; D015094:3-Hydroxyacyl CoA Dehydrogenases; D002374:Catalase; C576253:EHHADH protein, human; D004746:Enoyl-CoA Hydratase; D063994:Peroxisomal Bifunctional Enzyme; D007535:Isomerases; D003066:Coenzyme A Ligases; D000098859:Long-Chain-Fatty-Acid-CoA Ligase