[Using GST-tag to capture protein interaction of an interferon-inducible systemic lupus associated gene, IFIT1].
OBJECTIVE
To investigate the protein-to-protein interaction of interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), a newly discovered systemic lupus erythematosus (SLE) related up-regulated gene, and its possible function.


METHODS
Peripheral blood of 40 SLE patients was obtained to extract total RNA and synthesized cDNA. Real-time PCR was used to determine the IFIT1 expression at transcript level. Peripheral blood of another 10 SLE patients was extracted to obtain specimens of white blood cell lysate. Molecular cloning and a modified gluthion S-transferase (GST)-pull down assay were used to capture the protein interacting with IFIT1 in the specimens of white blood cell lysate. MALDI-TOF mass spectrometry (MS) was preformed to identify the captured protein that could interact with IFIT1. Twenty-nine sex and age-matched healthy persons were used as controls.


RESULTS
By real-time PCR showed that the IFIT1Delta Ct value (x +/- s) was 2.344 +/- 1.200 in the SLE patients and was 3.734 +/- 1.274 in the controls (P < 0.001), showing a significant up-regulation in SLE patients. IFIT1 was cloned and GST-IFIT1 fusion protein was expressed in Escherichia coli. GST-IFIT1 fusion protein was further purified using Glutathione Sepharose 4B column, and was treated as bait to capture prey from peripheral white blood cell lysate of SLE patients. MALDI-TOF MS detected protein interaction between Rho/Rac guanine nucleotide exchange factor and IFIT1.


CONCLUSIONS
IFIT1 may interact with Rho/Rac guanine nucleotide exchange factor, and regulate the activation of Rho/Rac proteins, thus being involved in the pathogenesis of SLE.
Ye
Shuang
S
Department of Rheumatology, Renji Hospital, Clinical Center of Rheumatic Diseases and Institute of Rheumatology, Shanghai 2nd Medical University, Shanghai 200001, China.
Pang
Hai
H
Gu
Yue-ying
YY
Shen
Nan
N
Chen
Shun-le
SL
Chen
Xing-guo
XG
Hua
Jing
J
Qian
Jie
J
Rao
Zi-he
ZH
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D020869:Gene Expression Profiling / Q000379:methods; D005982:Glutathione Transferase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C484290:IFIT1 protein, human; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D020741:rho GTP-Binding Proteins