TY - JOUR
T1 - Mapping wild-type and R345W fibulin-3 intracellular interactomes
AU - Hulleman, John D.
AU - Genereux, Joseph C.
AU - Nguyen, Annie
N1 - Funding Information:
This work was funded in part by an endowment from the Roger and Dorothy Hirl Research Fund (JDH) , a National Eye Institute Visual Science Core Grant ( EY020799 ), an unrestricted grant from Research to Prevent Blindness , and a Career Development Award from Research to Prevent Blindness (JDH).
Publisher Copyright:
© 2016 Elsevier Ltd
PY - 2016/12/1
Y1 - 2016/12/1
N2 - Fibulin-3 (F3) is an important, disulfide-rich, extracellular matrix glycoprotein that has been associated with a number of diseases ranging from cancer to retinal degeneration. An Arg345Trp (R345W) mutation in F3 causes the rare, autosomal dominant macular dystrophy, Malattia Leventinese. The purpose of this study was to identify and validate novel intracellular interacting partners of wild-type (WT) and R345W F3 in retinal pigment epithelium cells. We used stable isotope labeling by amino acids in cell culture (SILAC) to generate ‘heavy’ and ‘light’ isotopically labeled ARPE-19 cell populations which were subsequently infected with adenovirus encoding for FLAG-tagged WT or R345W F3. After immunoprecipitation, interacting proteins were identified by multidimensional protein identification technology (MudPIT). We identified sixteen new intracellular F3 interacting partners, the vast majority of which are involved in protein folding and/or degradation in the endoplasmic reticulum (ER). Eight of these interactions (ANXA5, ERdj5, PDIA4, P4HB, PDIA6, RCN1, SDF2L1, and TXNDC5) were verified at the western blotting level. These F3 interactome results can serve as the basis for pursuing targeted genetic or pharmacologic approaches in an effort to alter the fate of either WT or mutant F3.
AB - Fibulin-3 (F3) is an important, disulfide-rich, extracellular matrix glycoprotein that has been associated with a number of diseases ranging from cancer to retinal degeneration. An Arg345Trp (R345W) mutation in F3 causes the rare, autosomal dominant macular dystrophy, Malattia Leventinese. The purpose of this study was to identify and validate novel intracellular interacting partners of wild-type (WT) and R345W F3 in retinal pigment epithelium cells. We used stable isotope labeling by amino acids in cell culture (SILAC) to generate ‘heavy’ and ‘light’ isotopically labeled ARPE-19 cell populations which were subsequently infected with adenovirus encoding for FLAG-tagged WT or R345W F3. After immunoprecipitation, interacting proteins were identified by multidimensional protein identification technology (MudPIT). We identified sixteen new intracellular F3 interacting partners, the vast majority of which are involved in protein folding and/or degradation in the endoplasmic reticulum (ER). Eight of these interactions (ANXA5, ERdj5, PDIA4, P4HB, PDIA6, RCN1, SDF2L1, and TXNDC5) were verified at the western blotting level. These F3 interactome results can serve as the basis for pursuing targeted genetic or pharmacologic approaches in an effort to alter the fate of either WT or mutant F3.
KW - Endoplasmic reticulum
KW - Fibulin-3
KW - Malattia Leventinese
KW - Mass spectrometry
KW - MudPIT
KW - SILAC
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U2 - 10.1016/j.exer.2016.10.017
DO - 10.1016/j.exer.2016.10.017
M3 - Article
C2 - 27777122
AN - SCOPUS:84993978561
SN - 0014-4835
VL - 153
SP - 165
EP - 169
JO - Experimental Eye Research
JF - Experimental Eye Research
ER -