Screening of LRRK2 interactants by yeast 2-hybrid analysis.
OBJECTIVE
To isolate and identify the potential binding partners of LRRK2, a gene linked to both dominant familial form and sporadic form of Parkinson's disease, thus to further our knowledge of its function.


METHODS
We used a sequence containing full-length of COR domain and part of ROC and MAPKKK domain as bait. The bait amplified by polymerase chain reaction (PCR) was then cloned into a yeast expression plasmid pGBKT7. After being sequenced and analyzed, pGBKT7-bait was transformed into the yeast strain AH109. Western blot was performed to confirm the expression of pGBKT7-bait in AH109 yeast strain. Then human fetal brain cDNA library was transformed into that yeast strain, which could express pGBKT7-bait fusion protein. The yeast strain which contained pGBKT7-bait and human fetal brain cDNA library was plated on quadruple dropout medium (SD/-Trp/-Leu/-His/-Ade) containing X-alpha-gal. We retested these positive colonies using 2 independent yeast strains AH109 contained pGBKT7-bait or pGBKT7, respectively. At last, these plasmids isolated from these true positive colonies were analyzed by bioinformatics.


RESULTS
We obtained 9 true positive colonies, these colonies were sequenced, and we performed sequence Blast in GenBank. Three colonies of the 9 positive colonies were not in open reading-frames. Among other 6 colonies, there were known proteins including spermatid perinuclear RNA-binding protein (STRBP) and BCL2-associated athanogene 5 isoform b (BAG5), as well as unknown proteins including tyrosine phosphatase non-receptor type (PTPN23), l(3)mbt-like 3 isoform b (L3MBTL3), RALY RNA binding protein-like isoform 1 (RALYL), and Homo sapiens mRNA for KIAA1783 protein, partial cds (KIAA1783).


CONCLUSIONS
True positive colonies of LRRK2 are successfully obtained by the yeast 2-hybrid. Our screened proteins may provide a new research clue for revealing biological functions of LRRK2, pathogenesis of Parkinson's disease, and other neurodegerations.
Zheng
Xiao-ye
XY
National Laboratory of Medical Genetics, Central South University, Changsha 410078, China.
Yang
Mei
M
Tan
Jie-qiong
JQ
Pan
Qian
Q
Long
Zhi-gao
ZG
Dai
He-ping
HP
Xia
Kun
K
Xia
Jia-hui
JH
Zhang
Zhuo-hua
ZH
D048868:Adaptor Proteins, Signal Transducing; D026481:Blotting, Far-Western; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D015723:Gene Library; D006801:Humans; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D010300:Parkinson Disease / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D054558:Protein Tyrosine Phosphatases, Non-Receptor / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; C496515:BAG5 protein, human; D002352:Carrier Proteins; D008869:Microtubule-Associated Proteins; D016601:RNA-Binding Proteins; C490551:STRBP protein, human; C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D017346:Protein Serine-Threonine Kinases; D054558:Protein Tyrosine Phosphatases, Non-Receptor