title: Loss-of-function mutations in Rab escort protein 1 (REP-1) affect intracellular transport in fibroblasts and monocytes of choroideremia patients.
abstract: BACKGROUND
  Choroideremia (CHM) is a progressive X-linked retinopathy caused by mutations in the CHM gene, which encodes Rab escort protein-1 (REP-1), an escort protein involved in the prenylation of Rabs. Under-prenylation of certain Rabs, as a result of loss of function mutations in REP-1, could affect vesicular trafficking, exocytosis and secretion in peripheral cells of CHM patients.
  
  
  RESULTS
  To evaluate this hypothesis, intracellular vesicle transport, lysosomal acidification and rates of proteolytic degradation were studied in monocytes (CD14+ fraction) and primary skin fibroblasts from the nine age-matched controls and thirteen CHM patients carrying 10 different loss-of-function mutations. With the use of pHrodo BioParticles conjugated with E. coli, collagen I coated FluoSpheres beads and fluorescent DQ ovalbumin with BODYPY FL dye, we demonstrated for the first time that lysosomal pH was increased in monocytes of CHM patients and, as a consequence, the rates of proteolytic degradation were slowed. Microarray analysis of gene expression revealed that some genes involved in the immune response, small GTPase regulation, transcription, cell adhesion and the regulation of exocytosis were significantly up and down regulated in cells from CHM patients compared to controls. Finally, CHM fibroblasts secreted significantly lower levels of cytokine/growth factors such as macrophage chemoattractant protein-1 (MCP-1), pigment epithelial derived factor (PEDF), tumor necrosis factor (TNF) alpha, fibroblast growth factor (FGF) beta and interleukin (lL)-8.
  
  
  CONCLUSIONS
  We demonstrated for the first time that peripheral cells of CHM patients had increased pH levels in lysosomes, reduced rates of proteolytic degradation and altered secretion of cytokines. Peripheral cells from CHM patients expose characteristics that were not previously recognized and could used as an alternative models to study the effects of different mutations in the REP-1 gene on mechanism of CHM development in human population.
authors.affiliation: Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Bethesda, Maryland, USA. Strunnikovan@nei.nih.gov
authors.forename: Natalia V
authors.initials: NV
authors.lastname: Strunnikova
authors.forename: Jennifer
authors.initials: J
authors.lastname: Barb
authors.forename: Yuri V
authors.initials: YV
authors.lastname: Sergeev
authors.forename: Ashwin
authors.initials: A
authors.lastname: Thiagarajasubramanian
authors.forename: Christopher
authors.initials: C
authors.lastname: Silvin
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Munson
authors.forename: Ian M
authors.initials: IM
authors.lastname: Macdonald
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000378:metabolism*; D000293:Adolescent; D000328:Adult; D000368:Aged; D016022:Case-Control Studies; D015794:Choroideremia / Q000378:metabolism* / Q000473:pathology; D016207:Cytokines / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D005838:Genotype; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism*; D018942:Macrolides / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D009154:Mutation / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D010047:Ovalbumin / Q000378:metabolism; D010587:Phagocytosis / Q000187:drug effects; D010641:Phenotype; D021381:Protein Transport / Q000187:drug effects; D055815:Young Adult
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C081787:CHM protein, human; C085460:CHML protein, human; D016207:Cytokines; D036341:Intercellular Signaling Peptides and Proteins; D018942:Macrolides; C057620:bafilomycin A; D010047:Ovalbumin