Molecular organization of peroxisomal enzymes: Protein–protein interactions in the membrane and in the matrix

Abstract

The β-oxidation of fatty acids in peroxisomes produces hydrogen peroxide (H 2O 2), a toxic metabolite, as a bi-product. Fatty acids β-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 H 2O 2. 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 H 2O 2 degrading enzyme catalase and proteins of peroxisomal β-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 H 2O 2 production in the peroxisomal β-oxidation pathway.