TY - JOUR
T1 - Isolation of cDNAs encoding cellular drug-binding proteins using a novel expression cloning procedure
T2 - Drug-western
AU - Tanaka, Hideki
AU - Ohshima, Nobuko
AU - Hidaka, Hiroyoshi
PY - 1999
Y1 - 1999
N2 - A rapid and convenient new method for isolating the genes encoding cellular drug-binding proteins is described. This method, drug-western, is based on the use of the drug conjugated with a marker molecule as a probe for the screening of a cDNA library. Unlike the other methods, this method allows us to identify the genes for trace amounts of cellular drug-binding proteins without purification. We have used this approach to isolate human cDNA clones encoding binding proteins of HMN-154 ((E)-4-[2-[2-(p-methoxy-benzene- sulfonamide) phenyl]ethenyl] pyridine), a novel benzenesulfonamide anticancer compound (Katoh and Hidaka, 1997). The proteins encoded by two of the isolated clones are identical to NF-YB, B subunit of nuclear transcription factor NF-Y, and thymosin β-10, respectively. Recombinants of both proteins bind specifically to HMN-154 in vitro. Comparison of amino acid sequences between these proteins shows the sequence similarity in a short amino acid stretch [K(X)AKXXK]. Deletion or mutation of this region causes the significant loss of binding of both proteins to HMN-154. Furthermore, HMN- 154 inhibits DNA binding of NF-Y to the human major histocompatibility complex class II human leukocyte antigen DRA Y-box sequence in a dose- dependent manner. Interestingly, other binding proteins identified by this method also possess the same or a similar motif. These results clearly demonstrate that NF-YB and thymosin β-10 are specific cellular binding proteins of HMN-154 and that this shared region is necessary for the binding to HMN-154. Hence, this new method is thought to be useful for the identification of drug-binding proteins.
AB - A rapid and convenient new method for isolating the genes encoding cellular drug-binding proteins is described. This method, drug-western, is based on the use of the drug conjugated with a marker molecule as a probe for the screening of a cDNA library. Unlike the other methods, this method allows us to identify the genes for trace amounts of cellular drug-binding proteins without purification. We have used this approach to isolate human cDNA clones encoding binding proteins of HMN-154 ((E)-4-[2-[2-(p-methoxy-benzene- sulfonamide) phenyl]ethenyl] pyridine), a novel benzenesulfonamide anticancer compound (Katoh and Hidaka, 1997). The proteins encoded by two of the isolated clones are identical to NF-YB, B subunit of nuclear transcription factor NF-Y, and thymosin β-10, respectively. Recombinants of both proteins bind specifically to HMN-154 in vitro. Comparison of amino acid sequences between these proteins shows the sequence similarity in a short amino acid stretch [K(X)AKXXK]. Deletion or mutation of this region causes the significant loss of binding of both proteins to HMN-154. Furthermore, HMN- 154 inhibits DNA binding of NF-Y to the human major histocompatibility complex class II human leukocyte antigen DRA Y-box sequence in a dose- dependent manner. Interestingly, other binding proteins identified by this method also possess the same or a similar motif. These results clearly demonstrate that NF-YB and thymosin β-10 are specific cellular binding proteins of HMN-154 and that this shared region is necessary for the binding to HMN-154. Hence, this new method is thought to be useful for the identification of drug-binding proteins.
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U2 - 10.1124/mol.55.2.356
DO - 10.1124/mol.55.2.356
M3 - Article
C2 - 9927629
AN - SCOPUS:0033052886
SN - 0026-895X
VL - 55
SP - 356
EP - 363
JO - Molecular Pharmacology
JF - Molecular Pharmacology
IS - 2
ER -