title: Proteomic analysis of cathepsin B- and L-deficient mouse brain lysosomes.
abstract: Cathepsins B and L are lysosomal cysteine proteases which have been implicated in a variety of pathological processes such as cancer, tumor angiogenesis, and neurodegeneration. However, only a few protein substrates have thus far been described and the mechanisms by which cathepsins B and L regulate cell proliferation, invasion, and apoptosis are poorly understood. Combined deficiency of both cathepsins results in early-onset neurodegeneration in mice reminiscent of neuronal ceroid lipofuscinoses in humans. Therefore, we intended to quantify accumulated proteins in brain lysosomes of double deficient mice. A combination of subcellular fractionation and LC-MS/MS using isobaric tagging for relative and absolute quantitation (iTRAQ) allowed us to simultaneously assess wildtype and cathepsin B(-/-)L(-/-) cerebral lysosomes. Altogether, 19 different proteins were significantly increased in cathepsin B(-/-)L(-/-) lysosomes. Most elevated proteins had previously been localized to neuronal biosynthetic, recycling/endocytic or lysosomal compartments. A more than 10-fold increase was observed for Rab14, the Delta/Notch-like epidermal growth factor-related receptor (DNER), calcyon, and carboxypeptidase E. Intriguingly, immunohistochemistry demonstrated that Rab14 and DNER specifically stain swollen axons in double deficient brains. Since dense accumulations of expanded axons are the earliest phenotypic and pathognomonic feature of cathepsin B(-/-)L(-/-) brains, our data suggest a role for cathepsins B and L in recycling processes during axon outgrowth and synapse formation in the developing postnatal central nervous system.
authors.affiliation: Department of Human Genetics, University of Würzburg, Biozentrum, Am Hubland, D-97074 Würzburg, Germany.
authors.forename: Sonja
authors.initials: S
authors.lastname: Stahl
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Reinders
authors.forename: Esther
authors.initials: E
authors.lastname: Asan
authors.forename: Walther
authors.initials: W
authors.lastname: Mothes
authors.forename: Ernst
authors.initials: E
authors.lastname: Conzelmann
authors.forename: Albert
authors.initials: A
authors.lastname: Sickmann
authors.forename: Ute
authors.initials: U
authors.lastname: Felbor
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001369:Axons / Q000201:enzymology; D001923:Brain Chemistry / Q000235:genetics* / Q000502:physiology; D002401:Cathepsin B / Q000172:deficiency* / Q000235:genetics* / Q000502:physiology; D056668:Cathepsin L; D002403:Cathepsins / Q000172:deficiency* / Q000235:genetics* / Q000502:physiology; D003546:Cysteine Endopeptidases / Q000172:deficiency* / Q000235:genetics* / Q000502:physiology; D018507:Gene Expression Regulation, Developmental; D008247:Lysosomes / Q000201:enzymology* / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D020543:Proteome / Q000235:genetics; D013569:Synapses / Q000201:enzymology
chemical_list: D020543:Proteome; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L; C534278:Ctsl protein, mouse