Patent Application: US-44263099-A

Abstract:
aminoacyl - trna synthetases are the enzymes catalyzing ligation of their cognate amino acids and trnas . human glutaminyl - trna synthetase consists of the unique n - terminal extension and the c - terminal catalytic domain . here , we found that the n - and c - domains of qrs interacted with pro - apoptotic mediator , daxx , and its downstream kinase , ask1 , respectively . the experimental results suggest that qrs may inhibit the ask1 activity via two different ways . first , its c - terminal domain made direct inhibitory interaction with ask1 . second , it inhibited the pro - apoptotic interaction between daxx and ask1 . qrs also blocked the daxx - ask1 mediated apoptosis . thus , qrs is not only an enzyme for protein synthesis but also plays a regulatory role in apoptosis

Description:
in the present work , we found that human qrs also modulates apoptotic process . human qrs contains 775 amino acids ( fig1 ), consisting of the n - terminal extension and c - terminal catalytic domains . we previously reported that its n - terminal 236 amino acid extension is involved in the interactions with other arss within the multi - ars complex ( 8 ). although the n - terminal extension of rrs is associated with other arss , it also interacts with pro - emapii ( 6 ). we thus searched for the cellular molecules interacting with the n - terminal extension of qrs using yeast two hybrid system ( 9 ). from the screening of about 500 , 000 clones of human fetal brain cdna library ( invitrogen ), four positive clones encoding different c - terminal parts of human daxx were isolated ( data not shown ). the smallest clone among them was the c - terminal 137 aa polypeptide . ( fig1 daxx - c ), indicating that this part would interact with the n - terminal extension of qrs . human daxx was originally identified to be associated to fas to mediate apoptotic signal ( 10 ) to its downstream kinase , ask1 ( 11 ). the activation of ask1 then results in activation of jun n - terminal kinase ( jnk ) and p38 for cellular apoptosis ( 12 ). the interaction between qrs and daxx was further refined by yeast two hybrid analysis . the full - length , n - and c - domains of qrs were tested for the interaction with daxx - f and - c . daxx - c interacted with qrs - f and - n but very weakly with qrs - c ( fig2 a . upper panel ). interestingly , the interaction of daxx - f with qrs was weaker than that of daxx - c , suggesting that the c - terminal region of daxx may be masked somehow by its n - domain . we thus tested whether the n - and c - domains of daxx interact with each other . as expected , daxx - n interacted with its own c - domain but not with qrs , supporting this possibility ( fig2 a , lower panel ). daxx - c also showed the interaction with ask1 , suggesting that this region contains residues interacting with ask1 . the interaction between the daxx - c and qrs - n was confirmed by in vitro pull down experiment . daxx - c and qrs - n were expressed as his and gst fusion proteins , respectively . the purified two fusion proteins were then mixed and precipitated with the gst resin . daxx - c was co - precipitated with qrs - n , confirming the direct interaction between the two molecules ( fig2 b ). since ask1 is the immediate downstream kinase of daxx , we tested whether qrs also interacts with ask1 . the interaction of the two proteins was determined by co - immunoprecipitation . each of qrs - f , - n and - c was co - expressed with ha - tagged ask1 in 293 cells . after confirming expression of these proteins ( fig3 a , wcl ), ha - ask1 was precipitated with anti - ha antibody . qrs - f and - c , but not - n , were co - precipitated with ha - ask1 ( fig3 a ). the interaction of qrs - c and ask1 was also confirmed by in vitro pull down assay ( data not shown ). since the n - terminal 649 aa peptide was previously determined to be a regulatory domain interacting with daxx ,( fig1 ), we tested whether qrs interacts with ask1 - n or - c . qrs was precipitated with ask1 - f and - n although the interaction with ask1 - n was weaker than that with ask1 - f ( fig3 b ). however , we could not test whether qrs also interacted with ask1 - c for its instability ( data not shown ). although the interaction of qrs with daxx was shown by genetic and in vitro pull down assays ( fig2 a and b ), daxx was not co - immunoprecipitated with qrs while it was with ask1 ( fig4 a ). these results suggest that daxx and qrs may be differently compartmentalized or do not associate in the cell . we thus tested whether the cellular interaction between the two molecules depends on the expression of ask 1 . when the three proteins were co - expressed , qrs was co - precipitated with daxx ( fig4 a ). however , the interaction of ask1 with daxx was decreased by qrs , indicating the competition of qrs with ask1 for the binding to daxx . expression of the three proteins was confirmed in whole cell lysate ( fig4 b ). the direct interaction of qrs with ask1 ( fig3 ), and its inhibition of the interaction between ask1 and daxx ( fig4 ) suggest that qrs may inhibit the ask1 activity . to test this possibility , qrs - f , - n and - c were expressed in 293 cells with ha - ask1 . the kinase activity of ask1 was measured using myelin basic protein ( mbp ) as an exogenous substrate ( 13 ). the ask1 activity was inhibited by the expression of qrs - f and - c but not of qrs - n ( fig4 a ), indicating the direct inhibitory effect of qrs - c on the ask1 activity . we then tested whether qrs can block the daxx - induced ask1 activity . for the experiment , we used the c - terminal 242 aa peptide of daxx ( daxx - ca ) that constitutively activates ask1 ( 11 ) and gst - sek1 ( 12 ) as a reaction substrate . the active mutant of daxx ( daxx - ca ) activated ask1 but qrs inhibited the ask1 activity enhanced by daxx ( fig5 b ). the activation of ask1 triggers its downstream apoptotic cascades ( 12 ). since qrs inhibited the activity of ask1 , it is expected to block apoptosis mediated by ask1 . we tested this possibility by expressing these molecules in hela cells ( fig6 ). each of qrs , daxx - ca or ask1 alone did not induce a significant apoptosis . co - expression of ask1 and daxx - ca then enhanced apoptotic cells to almost 11 %. additional expression of qrs then decreased the apopotic cells to below than 2 %, suggesting that qrs blocks the daxx - ask1 induced apoptosis . the cellular interaction of daxx and qrs was dependent on ask1 although qrs and ask1 competed for the daxx binding ( fig4 ). these results can be explained by a couple of different models . first , ask1 may change the cellular distribution of daxx . although daxx was first reported to be associated with fas embedded in cellular membrane , the following reports suggested that it is localized in nucleus interacting with different nuclear factors ( 14 , 15 ). if this is the case , qrs in cytoplasm would not meet daxx in nucleus unless one protein is translocated to other compartment . daxx may be localized in cytosol for the pro - apoptotic interaction with ask1 , and qrs may inhibit the formation of this complex . second , the conformation of daxx may be changed by the interaction with ask1 to open its c - terminal domain that has a higher affinity to qrs . then , qrs may attack this complex to block the activity of ask 1 . in this sense , it is worth noting that ask1 bound to the full - length daxx better than qrs whereas the opposite was the case for the interaction with daxx - c ( fig2 a ). understanding regulatory and molecular mechanisms for the interactions between these three molecules await further investigation . the relationship of arss to apoptosis has been previously reported in the cases of human tyrosyl - and arginyl - trna synthetases . the former contained the pro - apoptotic cytokine domains within its structure while the latter harbors the precursor of cytokine . here , we report that human qrs could repress apoptosis via bipartite interactions with two consecutive pro - apoptotic molecules , daxx and ask1 ( fig1 ). these reports suggest that arss play a pivotal role between cell proliferation and death , not only as a catalysis but also as a regulator . the dna fragment encoding the n - terminal 236 aa of human qrs was isolated from plexa vector [ rho , 1999 # 6878 ] and subcloned into pet28a ( novagen ). the his - qrs - n was expressed in e . coli bl21 ( de3 ) and purified using nickel affinity chromatography following the manufacturer &# 39 ; s instruction . the purified qrs - n was then used to prepare polyclonal rabbit antibody as described previously [ park , 1999 # 7366 ]. 293 and hela cells were grown in dmem supplemented 10 % fetal bovine serum ( fbs ), penicillin , and streptomycin . the cells were transfected with plasmid dna by using geneporter ( gts , san diego , calif .) according to manufacture &# 39 ; s protocol . twenty four hours after transfection , cells ( 100 - mm dish ) were washed twice in ice - cold phosphate - buffered saline ( pbs ) and lysed in 300 μl of ip - lysis buffer [ 50 mm hepes ( ph 7 . 4 ), 1 % np40 , 150 mm nacl , 10 % glycerol , 1 mm edta ] supplemented with 1 mm phenylmethylsulfonyl fluoride and 5 μg / ml aprotinin . extracts ( 1 mg of protein ) were diluted in ip buffer ( 1 ml ), and immunoprecipitated with 5 μg of antibody and 50 μl of a slurry of protein a - agarose beads ( sigma ) for 3 hour at 4 ° c . the immunoprecipitates were washed three times with ip - lysis buffer , and boiled in laemmli &# 39 ; s loading buffer . aliquots of cell lysates and immunoprecipitates resolved on sds - page were transferred to nitrocellulose membranes and probed with antibodies as specified , followed by secondary antibody conjugated with horseradish peroxidase ( pierce ). after washing , proteins were detected by enhanced chemiluminescence ( amersham ). cells ( 100 - mm dish ) were transfected with plasmid dna . twenty four hours after transfection , cells were lysed with a buffer solution containing 20 mm tris - hcl ( ph 7 . 5 ), 12 β - glycerophosphate , 150 mm nacl , 5 mm egta , 10 mm naf , 1 % triton x - 100 , 0 . 5 % deoxycholate , 3 mm dtt , 1 mm sodium orthovanadate , 1 mm pmsf , and 5 μg / ml aprotinin ). the lysates ( 1 mg ) were immunoprecipitated with antibody ( 5 μg ) and 50 μl of protein a - agarose for 4 hours . the beads were washed twice with a solution containing 150 mm nacl , 20 mm tris - hcl ( ph7 . 5 ), 5 mm egta , 2 mm dtt and 1 mm pmsf and once with a reaction buffer containing 20 mm tris - hci ( ph 7 . 5 ) and 20 mm mgcl 2 . mbp ( 40 μg / ml ) or gst - sek1 ( 20 μg / ml ) was incubated with the immunoprecipitates in a reaction buffer containing 20 mm tris - hcl ( ph 7 . 5 ), 20 mm mgcl 2 and 0 . 5 μci of [ 2 - 32p ] atp for 10 min at 30 ° c . samples were analyzed by sds - page with an autoradiography . cells were plated onto 6 - well dishes the day before transfection for hela cells . the cells were by geneporter ( gts ) according to manufacturer &# 39 ; s protocol . twenty four hours after transfection , x - gal staining was done for 4 hours to overnight according to manufacturer &# 39 ; s protocol ( invitrogen ). the percentage of apoptotic cells was determined by the number of blue cells with apoptotic morphology divided by the total number of blue cells . specific apoptosis was calculated as the percentage of blue cells with apoptotic morphology in each experimental condition minus the percentage of blue cells with apoptotic morphology in empty vector - transfected cells . at least 500 cells from three random fields were counted in each experiment , and the data shown are the average and sd of at least three independent experiment . to make b42 - daxx full clone , pcr was carried out with a set of primers [ forward : 5 ′- ccggaattccggatggccaccgctaacagc ( seq id no . 1 ) and backward : 5 ′- ccgctcgagcggctaatcagagtctgagagc ( seq id no . 2 )] using prk5 - flag - daxx ( a kind gift from dr . x . yang ) as a template and the product was cloned into pgex4t - 1 . b42 - daxx was constructed by insertion of the pcr product which was digested out by ecori and xhoi from pgex - daxx . b42 - daxx - c was a clone ( encoding daxx g604 - d740 ) isolated from screening of yeast two hybrid using lexa - qrs - n as a bait . to make lexa - qrs full clone , the qrs full gene was cut out from pgex4t - 1 - qrs by ecori and noti , and ligated into peg202 , a lexa vector . the plasmid , pgex4t - 1 - qrs was made by the insertion of the qrs full gene from pm191 ( a kind gift from dr . k . shiba ) into pgex4t - 1 using xhoi and noti sites . to make qrs - c clone , pcr was carried out with a set of primers [ forward : 5 ′- ccgctcgagatgaactacaagaccccaggct ( seq id no . 3 ) and backward : 5 ′- atttgcggccgctctagaactagt ( seq id no . 4 )] using pm191 as a template and the product encoding from n237 to the c - terminal end , v775 of qrs was cloned into pgex4t - 1 . lexa - qrs - c was constructed by insertion of the pcr product digested with ecori and noti from pgex - qrs - c . to make qrs - n clone , pcr was carried out with a set of primers [ forward : 5 ′- aatgaattcatggcggctctagactcc ( seq id no . 5 ) and backward : 5 ′- ccggtcgactcactcaccaggcttgtggaa ( seq id no . 6 )] using pm191 as a template and the product encoding from the n - terminus to e236 of qrs was cloned into pgex4t - 1 . lexa - qrs - c was constructed by insertion of the pcr digested with ecori and noti from pgex - qrs - c into peg202 . 1 . k . shiba , h . motegi , p . schimmel , trends biochem . sci . 22 , 453 ( 1997 ). 2 . m . mirande , prog . nucleic acid res . mol . biol . 40 , 95 ( 1991 ). 3 . l . l . kisselev and a . d . wolfson , prog . nucleic acid res . mol . biol , 48 , 83 ( 1994 ). 4 . d . c . h . yang , in current topics in cellular regulation , 34 , pp . 101 - 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