title: Perturbation of neuronal cobalamin transport by lysosomal enzyme inhibition.
abstract: Cbl (cobalamin) utilization as an enzyme cofactor is dependent on its efficient transit through lysosomes to the cytosol and mitochondria. We have previously proposed that pathophysiological perturbations in lysosomal function may inhibit intracellular Cbl transport with consequences for down-stream metabolic pathways. In the current study, we used both HT1080 fibroblasts and SH-SY5Y neurons to assess the impact that protease inhibitors, chloroquine and leupeptin (N-acetyl-L-leucyl-L-leucyl-L-argininal), have on the distribution of [57Co]Cbl in lysosomes, mitochondria and cytosol. Under standard cell culture conditions the distribution of [57Co]Cbl in both neurons and fibroblasts was ~5% in lysosomes, 14% in mitochondria and 81% in cytosol. Treatment of cells with either 25 μM chloroquine or 40 μM leupeptin for 48 h significantly increased the lysosomal [57Co]Cbl levels, by 4-fold in fibroblasts and 10-fold in neurons, and this was associated with reduced cytosolic and mitochondrial [57Co]Cbl concentrations. Based on Western blotting of LAMP2 in fractions recovered from an OptiPrep density gradient, lysosomal Cbl trapping was associated with an expansion of the lysosomal compartment and an increase in a subpopulation of lysosomes with increased size and density. Moreover, the decreased mitochondrial Cbl that was associated with lysosomal Cbl trapping was correlated with decreased incorporation of [14C] propionate into cellular proteins/macromolecules, indicating an inhibition of Cbl-dependent Mm-CoA (methylmalonyl-coenzyme A) mutase activity. These results add support to the idea that lysosomal dysfunction may significantly impact upon Cbl transport and utilization.
authors.affiliation: Illawarra Health and Medical Research Institute, University of Wollongong, NSW 2522, Australia.|School of Biological Sciences, University of Wollongong, NSW 2522, Australia.
authors.forename: Hua
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Illawarra Health and Medical Research Institute, University of Wollongong, NSW 2522, Australia.|School of Biological Sciences, University of Wollongong, NSW 2522, Australia.
authors.forename: Kalani
authors.initials: K
authors.lastname: Ruberu
authors.affiliation: Illawarra Health and Medical Research Institute, University of Wollongong, NSW 2522, Australia.|School of Biological Sciences, University of Wollongong, NSW 2522, Australia.
authors.forename: Hongyun
authors.initials: H
authors.lastname: Li
authors.affiliation: Illawarra Health and Medical Research Institute, University of Wollongong, NSW 2522, Australia brettg@uow.edu.au.|School of Biological Sciences, University of Wollongong, NSW 2522, Australia.
authors.forename: Brett
authors.initials: B
authors.lastname: Garner
mesh_terms: D001693:Biological Transport, Active / Q000187:drug effects; D045744:Cell Line, Tumor; D002738:Chloroquine / Q000494:pharmacology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology*; D052119:Lysosomal-Associated Membrane Protein 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D008247:Lysosomes / Q000276:immunology*; D009474:Neurons / Q000201:enzymology*; D014805:Vitamin B 12 / Q000378:metabolism*
chemical_list: D015853:Cysteine Proteinase Inhibitors; C497811:LAMP2 protein, human; D007976:Leupeptins; D052119:Lysosomal-Associated Membrane Protein 2; D002738:Chloroquine; C032854:leupeptin; D014805:Vitamin B 12
keywords: lysosome; mitochondria; neurodegeneration; subcellular-fractionation; vitamin B12