Investigating copper-regulated protein expression in Menkes fibroblasts using antibody microarrays.
Neurodegenerative illnesses are characterized by aberrant metabolism of biometals such as copper (Cu), zinc (Zn) and iron (Fe). However, little is known about the metabolic effects associated with altered metal homeostasis. In this study, we used an in vitro model of altered Cu homeostasis to investigate how Cu regulates cellular protein expression. Human fibroblasts containing a natural deletion mutation of the Menkes (MNK) ATP7A Cu transporter (MNK deleted) were compared to fibroblasts overexpressing ATP7A (MNK transfected). Cultures of MNK-transfected (Low-Cu) cells exhibited 95% less intracellular Cu than MNK-deleted (High-Cu) cells. Comparative proteomic analysis of the two cell-lines was performed using antibody microarrays, and significant differential protein expression was observed between Low-Cu and High-Cu cell-lines. Western blot analysis confirmed the altered protein expression of Ku80, nexilin, L-caldesmon, MAP4, Inhibitor 2 and DNA topoisomerase I. The top 50 altered proteins were analysed using the software program Pathway Studio (Ariadne Genomics) and revealed a significant over-representation of proteins involved in DNA repair and maintenance. Further analysis confirmed that expression of the DNA repair protein Ku80 was dependent on cellular Cu homeostasis and that Low-Cu levels in fibroblasts resulted in elevated susceptibility to DNA oxidation.
Du
Tai
T
Centre for Neuroscience, The University of Melbourne, Victoria, Australia.
La Fontaine
Sharon L
SL
Abdo
Michael
M
Bellingham
Shayne A
SA
Greenough
Mark
M
Volitakis
Irene
I
Cherny
Robert A
RA
Bush
Ashley I
AI
Hudson
Peter J
PJ
Camakaris
James
J
Mercer
Julian F
JF
Crouch
Peter J
PJ
Masters
Colin L
CL
Perreau
Victoria M
VM
White
Anthony R
AR
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D034961:Antigens, Nuclear / Q000096:biosynthesis; D001692:Biological Transport; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019295:Computational Biology / Q000379:methods; D003300:Copper / Q000737:chemistry*; D000073840:Copper-Transporting ATPases; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000096:biosynthesis; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D000072200:Ku Autoantigen; D019636:Neurodegenerative Diseases / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010100:Oxygen / Q000737:chemistry; D040081:Protein Array Analysis; D040901:Proteomics / Q000379:methods; D012984:Software
D034961:Antigens, Nuclear; D027682:Cation Transport Proteins; D004268:DNA-Binding Proteins; D003300:Copper; D004247:DNA; D000251:Adenosine Triphosphatases; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; C081184:ATP7A protein, human; D000073840:Copper-Transporting ATPases; D010100:Oxygen