Patent Application: US-68512207-A

Abstract:
members of the ipah superfamily constitute a novel class of e3 ubiquitin ligases which are useful for engineering products which modulate trafficking and destruction of target proteins inside a cell and useful targets for identifying new antimicrobial molecules which modulate , especially inhibit , e3 ligases .

Description:
ipah9 . 8 and ssph1 have been discovered to exhibit e3 ubiquitin ligase activity . in addition to ubiquitin , a yeast substrate for ipah9 . 8 ( ste7 ) and a mammalian substrate for ssph1 ( pkn1 ) were identified . ubiquitin was removed from ubiquitinated ubch5b upon incubation with ipah9 . 8 or ssph1 ; this latter activity corresponding to the hydrolysis of the thioester bond linking ubiquitin to the e2 is not equivalent to the activity of de - ubiquitinating enzymes hydrolyzing the amide bond linking ubiquitin to target proteins ( amerik and hochstrasser , 2004 ). it might correspond either to the total consumption of the ubiquitinated e2 by the polyubiquitination activity of ipah9 . 8 and ssph1 towards ubiquitin or to the transfer of ubiquitin onto ipah9 . 8 and ssph1 prior to its transfer onto the substrate , as described for hect - domain e3s ( ardley and robinson , 2005 ; scheffner et al ., 1995 ). although the latter hypothesis is consistent with the observation that the cys residue conserved in all ipah family members is required for ipah9 . 8 activities both in yeast and in vitro , ubiquitin - ipah9 . 8 and ubiquitin - ssph1 intermediates could not be detected . the hect domain of e3s and the c - terminal domain of ipah proteins do not share sequence similarities ; furthermore , residues surrounding the catalytic cys residue in hect - domain e3s and the conserved cys residue in ipah proteins are different . accordingly , ipah superfamily members constitute a novel class of e3 ubiquitin ligases . co - opting the ubiquitination pathway , either to promote or prevent ubiquitination of host proteins , is emerging as a common strategy employed by pathogens using t3s systems to down regulate host responses . the effector avrptob from the tomato pathogen p . syringae is structurally similar to u - box and ring - finger e3s , possesses autoubiquitination and presumably ubiquitin ligase activities towards host proteins , and blocks signalling cascades that limit infection by activating the cell death program ( abramovitch et al ., 2006 ; janjusevic et al ., 2006 ). through an unknown mechanism , the p . syringae effector hopm1 promotes the proteasome - dependent degradation of the arabidopsis protein atmin7 and inhibits vesicle trafficking required to mount a cell wall - based defense to infection ( nomura et al ., 2006 ). the salmonella effector sopa has recently been shown to be a hect - like e3 endowed with an autoubiquitination activity ( zhang et al ., 2006 ). the shigella effector ospg , encoded in the same operon as ipah9 . 8 , is a kinase that binds ubiquitinated e2s , prevents ubiquitination of phospho - i □ b □ and dampens inflammation in the host ( kim et al ., 2005 ). as shown here , the salmonella effector ssph1 is an e3 ubiquitin ligase for pkn1 , a protein kinase involved in the nf - κb pathway and activated upon cell infection ( haraga and miller , 2006 ). the s . flexneri chromosomally - encoded ipah proteins have been reported to play a role in dampening inflammation ( ashida et al ., 2007 ). the lrr - containing n - terminal domain of ipahs is likely involved in protein - protein interactions and substrate recognition ; the yersinia effector yopm containing only lrrs related to those of ipahs ( fig2 c ) can act as a scaffolding protein that brings host kinases together ( mcdonald et al ., 2003 ) and the lrr domain of ssph1 interacts with pkn1 ( haraga and miller , 2006 ). in hela cells infected by s . flexneri for 90 min , a significant decrease in the amount of the mapkk mek1 , mek3 , and ikkα was not observed , suggesting that these proteins are not degraded upon invasion of epithelial cells . the substrates of ipah proteins in human cells remain to be identified . the demonstration that ipah9 . 8 and ssph1 are e3 ubiquitin ligases permits determination of the function of these proteins and their homologues during infection by identifying their target ( s ), possibly protein kinases , in host cells . shigella produces multiple ipahs that differ in their lrr domain and , likewise , some other pathogens contain several genes encoding ipah homologues . this diversity suggests that each of these pathogens uses a repertoire of e3 ubiquitin ligases to promote degradation of several host proteins . many bacteria pathogenic for plants or animals , including shigella spp . responsible for shigellosis in humans , use a type iii secretion apparatus to inject effector proteins into host cells . effectors alter cell signalling and host responses induced upon infection , however , their activities have been elucidated in very few cases . utilizing saccharomyces cerevisiae as a surrogate host , the examples below show that the shigella effector protein ipah9 . 8 interrupts pheromone response signalling by promoting the proteasome - dependent destruction of the mapkk ste7 . in vitro , ipah9 . 8 displayed ubiquitin ligase activity towards ubiquitin and ste7 . replacement of a cys residue invariant among ipah homologues of plant and animal pathogens abolished ipah9 . 8 activities . the examples also show that the ipah homologue ssph1 from salmonella enterica can ubiquitinate ubiquitin and pkn1 , a previously identified partner of interaction of ssph1 . these results demonstrate that ipah superfamily members constitute a novel class of e3 ubiquitin ligases . to gain insight to ipah9 . 8 activity , saccharomyces cerevisiae was employed as a surrogate model . yeast producing flag - tagged ipah9 . 8 under the control of the gal promoter were not impaired in their ability to grow at elevated temperatures , in the presence of a variety of ions , or under high and low osmotic stresses . detection of mating pheromone by a g protein - coupled receptor activates an archetypal mapk signalling cascade , inducing both arrest of the cell cycle and transcription of mating genes . the pheromone α - factor diffusing from a disk causes cell cycle arrest in matα cells , resulting in a halo of inhibited growth ( hoffman et al ., 2002 ). upon exposure to α - factor , wild - type yeast producing ipah9 . 8 failed to form a halo and to induce expression of a pheromone - responsive fus1 - lacz reporter gene , indicating that ipah9 . 8 interferes with the pheromone response pathway and acts on or upstream of the mapk fus3 . to identify the target of ipah9 . 8 , yeast strains were used that were altered in the signalling cascade . overproduction of the g protein α subunit ste4 activates the signalling pathway and promotes growth arrest ( cole et al ., 1990 ); production of ipah9 . 8 rescued this phenotype , i . e . allowed growth , indicating that ipah9 . 8 acts downstream of ste4 . the constitutively active variant of the mapkkk ste11 encoded by the allele ste11 - 4 promotes elevated transcription of pheromone responsive genes , even in the absence of pheromone ( stevenson et al ., 1992 ). growth of the strain sy2625 harbouring a fus1 - his3 pheromone - inducible reporter is dependent on signalling through the pheromone response pathway on a medium lacking histidine and containing 3 - amino triazole ( evangelista et al ., 1997 ). sy2625 containing a plasmid encoding ste11 - 4 , but not those containing the vector , were his + , consistent with activation of the pathway by ste11 - 4 and transcription of fus1 - his3 . in contrast , yeast containing plasmids encoding ste11 - 4 and ipah9 . 8 were his − , indicating that ipah9 . 8 interrupts signalling at or downstream of ste11 , on either the mapk fus3 or the mapkk ste7 . immunoblot analysis indicated that the amount of ste7 , but not of ste11 and fus3 , was drastically reduced in wild - type yeast producing ipah9 . 8 , regardless of stimulation by α - factor . upon phosphorylation by ste11 , ste7 is ubiquitinated and , following removal of ubiquitin chains by the specific deubiquitinase ubp3 , is degraded by the proteasome ( wang et al ., 2003 ). in both ubp3 , and ste11δ cells , production of ipah9 . 8 still resulted in the disappearance of ste7 , indicating that ipah9 . 8 - mediated disappearance of ste7 is independent of the known ste7 degradation pathway . blockage of signalling downstream of ste11 suggested that ipah9 . 8 should rescue sst2δ cells defective for the gtpase activating protein encoded by sst2 ; these cells are unable to dampen signalling and can not grow in the presence of pheromone ( dohlman et al ., 1996 ). indeed , production of ipah9 . 8 allowed sst2δ cells to grow in the presence of pheromone ( fig1 a and 1b ). ipah7 . 8 , another ipah family member from shigella , also rescued the pheromone - induced growth arrest of sst2δ cells ( fig1 a ), indicating that ipah9 . 8 and ipah7 . 8 have similar activities in yeast . the strong phenotype of sst2δ cells producing ipah proteins was used to perform a functional analysis of ipah domains . the nine different ipahs encoded by the virulence plasmid and the chromosome ( yang et al ., 2005 ) consist of a ≈ 250 - residue variable n - terminal domain containing six to eight 20 - residue leucine - rich repeats ( lrr ) and a ≈ 300 - residue conserved c - terminal domain . production of neither ipah9 . 8 - nter nor ipah9 . 8 - cter ( fig1 c ) rescued growth of sst2δ cells exposed to pheromone ( fig1 a ), indicating that both domains of ipah9 . 8 are required for the function in yeast . sequence comparisons revealed that the ipah c - terminal domain shares 25 - 40 % identity with two groups of proteins of bacteria that contain a t3s system and are pathogens of plants , fish , and mammals ( fig4 ). one group includes eighteen ≈ 600 - residue proteins from shigella spp ., yersinia pestis ( and y . pseudotuberculosis ), salmonella enterica , edwardsiella ictaluri , bradyrhizobium japonica , and rhizobium sp . strain ngr234 and the other includes fifteen ≈ 1500 - residue proteins from pseudomonas putida , p . entomophila , p . fluorescens , and p . syringae . in both groups , the conserved domain is c - terminal and preceded by lrrs ( fig2 c ). the presence of one cys residue among the nine residues that are identical in all members of the ipah family suggested that its thiol group might be involved in catalysis . to test this hypothesis , cys - 337 of ipah9 . 8 was replaced by ala in ipah9 . 8 - c337a . although ipah9 . 8 - c337a was produced in similar amounts to ipah9 . 8 ( fig1 b ), it did not allow sst2δ cells to grow in the presence of pheromone ( fig1 a ). circular dichroism measurements in the far - uv and near - uv regions on purified gst - ipah9 . 8 and gst - ipah9 . 8 - c337a showed that these two proteins have similar secondary and tertiary structure contents , suggesting that the cys residue conserved in all ipah homologues is involved in function rather than in structure . to test if the ipah - mediated disappearance of ste7 required proteasome function , a yeast strain carrying the cim5 - 1 allele encoding a component of the 26 - s proteasome that is functional at 25 ° c . but not at 37 ° c . ( ghislain et al ., 1993 ) was used . ste7 was present in cim5 - 1 yeast producing ipah9 . 8 - c337a at both temperatures and ipah9 . 8 at 37 ° c . but was not present in yeast producing ipah9 . 8 at 25 ° c . ( fig3 a ). moreover , the proteasome inhibitor mg132 prevented the disappearance of ste7 provoked by ipah9 . 8 in a mg132 - permeable erg6 mutant ( lee and goldberg , 1998 ). thus , the disappearance of ste7 promoted by ipah9 . 8 is proteasome - dependent . the observation that the degradation of ste7 promoted by ipah9 . 8 is proteasome - dependent led us to test in vitro if ipah9 . 8 might be involved in an ubiquitination pathway . it was discovered that the e2 enzyme ubch5b was apparently not ubiquitinated by e1 in the presence of gst - ipah9 . 8 ( fig2 b ). furthermore , after ubiquitination of ubch5b , addition of gst - ipah9 . 8 to the reaction mixture promoted the removal of ubiquitin from ubch5b ( fig3 b ). this activity was not observed when gst - ipah9 . 8 - c337a was added to the reaction mixture ( fig2 b ), indicating that it required the cys residue of ipah9 . 8 . the amide linkage , but not the thioesther linkage , of ubiquitin to ubiquitinated proteins is resistant to dithiothreitol ( dtt ). in reactions containing ipah9 . 8 , but not in those containing ipah9 . 8 - c337a or lacking ubch5b , a dtt - resistant ubiquitinated protein of the size of gst - ipah9 . 8 ( fig3 ) was detected . since some e3 ubiquitin ligases possess an autoubiquitination activity ( beaudenon et al ., 2005 ), these results indicate that ipah9 . 8 is an e3 ubiquitin ligase . ubiquitin biotinylated on lys residues can not support polyubiquitination reactions . to test if ipah9 . 8 could polyubiquitinate proteins , reactions were performed using ha - tagged ubiquitin , instead of biotinylated ubiquitin . anti - ha antibodies detected a ladder of ubiquitinated proteins , from 24 to & gt ; 200 kda , in reactions performed in the presence of gst - ipah9 . 8 , but not in the presence of gst - ipah9 . 8 - c337a ( fig3 c ). anti - ubch5b antibodies detected a single species corresponding to ubch5b ( 18 kda ), indicating that ubch5b was not polyubiquitinated ( fig2 c ). the sizes of species detected by anti - ha antibodies were multiples of the size of ha - ubiquitin ( 9 kda ), indicating that the molecule that was polyubiquitinated is ubiquitin . using k48r and k63r ubiquitin variants , it was found that ipah9 . 8 catalyzed the formation of polyubiquitin chains on lys - 48 , but not lys - 63 . polyubiquitinated proteins using the e2 ubch7 were not detected . these results demonstrate that ipah9 . 8 is endowed with ubiquitin ligase activity towards ubiquitin and uses ubch5b , but not ubch7 , as an e2 . to test whether ipah9 . 8 might ubiquitinate ste7 , purified active mapk complexes containing ste7 , as well as ste11 - 4 and the mapk kss1 , were incubated with native ubiquitin , e1 , ubch5b , and gst - ipah9 . 8 or gst - ipah9 . 8 - c337a . both the non - and mono - ubiquitinated forms of ipah9 . 8 were detected using anti - ipah antibodies ( fig2 d ), confirming the autoubiquitination activity of ipah9 . 8 . in addition to ste7 , larger species that formed only in the presence of ipah9 . 8 were detected by anti - ste7 antibodies ( fig2 d ). thus , ipah9 . 8 is an e3 ubiquitin ligase for ste7 ; the proteasome - dependent disappearance of ste7 in yeast was likely due to the degradation of ste7 following its polyubiquitination by ipah9 . 8 and ipah7 . 8 . the ipah homologue ssph1 is an e3 ubiquitin ligase for pkn1 ssph1 , one of the salmonella enterica typhimurium homologues of ipah , has been shown to interact with the mammalian protein kinase pkn1 ( haraga and miller , 2006 ). to test whether ssph1 shares activities with ipah9 . 8 , a gst - ssph1 recombinant protein was purified . as described above for ipah9 . 8 , ssph1 was endowed with the activities ( i ) to remove ubiquitin from ubiquitinated ubch5b , ( ii ) to autoubiquitinate , ( iii ) and to polyubiquitinate ha - tagged ubiquitin ( fig3 ). ubiquitination of pkn1 by ssph1 ; ssph1 , ipah9 . 8 , or ipah9 . 8 - c337a was incubated with e1 , native ubiquitin , ubch5b , and gst - pkn1 was tested . when the reaction was performed in the presence of ssph1 , anti - pkn1 antibodies detected an additional species migrating at a size & gt ; 250 kda ( fig3 c ). these results demonstrated that ssph1 is an e3 ubiquitin ligase that can use both ubiquitin and pkn1 as substrates . plasmids encoding flag - tagged ipah9 . 8 , ipah9 . 8 - c337a , ipah9 . 8 - cter , ipah9 . 8 - nter , and ipah7 . 8 were derivatives of the vector pfl38cii / pgal1 containing the gal promoter ( badis et al ., 2004 ). ycp50 - ste11 - 4 carrying ste11 - 4 under the control of its own promoter and prs316 - gal - ste4 carrying ste4 under the control of the gal promoter have been described ( dohlman et al ., 1995 ; stevenson et al ., 1992 ). yeast strains are described in table s1 . ubch7 , e1 , ubiquitin , ha - ubiquitin , biotinylated ubiquitin , ubiquitin - k48r and - k63r , horseradish peroxidase - coupled avidin , mg132 , and anti - ubch5 antibodies were purchased from boston biochem . anti - iκbα , - ubiquitin , - ste7 , - fus3 , - mek3 , - i_kα , - kss1 , and - pkn1 antibodies were purchased from santa cruz biotechnology . purified gst - pkn1 was purchased from invitrogen . anti - mek1 antibodies and purified active mek1 were purchased from upstate cell signaling solutions . the mating pheromone α - factor was purchased from sigma . his - tagged ubch5b was prepared as described ( kim et al ., 2005 ). gst - ipah9 . 8 , gst - ipah9 . 8 - c337a , and gst - ssph1 were prepared as described ( mavris et al ., 2002 ). complexes containing ste11 - 4 , ste7 , and kss1 were prepared as described ( breitkreutz et al ., 2001 ) and eluted from flag m2 - agarose affinity gel ( sigma ) using a flag peptide . ubiquitination of ubch5b - his by e1 was performed in a 40 - μl reaction mixture containing buffer a ( 25 mm tris . hcl ( ph 7 . 5 ), 50 mm nacl , 5 mm atp , 10 mm mgcl 2 , 0 . 1 mm dtt ), 2 μg of biotinylated ubiquitin , 0 . 5 μg of e1 , and 2 μg of e2 in the presence , or not , of 1 μg of gst - ipah9 . 8 , gst - ipah9 . 8 - c337a , or gst - ssph1 . reactions were incubated at 37 ° c . for 1 h and stopped by the addition of an equal volume of laemmli sample buffer ( 62 . 5 mm tris - hcl , ph 6 . 8 , 10 % glycerol , 2 % sds , 0 . 0005 % bromophenol blue ) containing , or not , 100 mm dtt . ubiquitination reactions were carried out in the same manner except that 2 μg of ha - tagged ubiquitin , ubiquitin , ubiquitin - 48r or - 3r were used instead of biotinylated ubiquitin . approximately 1 μg of ste11 - 4 : ste7 : kss1 complexes , or 0 . 4 μg of gst - pkn1 , was incubated in buffer a with 5 μg of ubiquitin , 0 . 5 μg of e1 , and 2 μg of e2 in the presence , or not , of 0 . 6 μg of gst - ipah9 . 8 , gst - ipah9 . 8 - c337a , or gst - ssph1 . reaction mixtures were separated by sds / page , transferred onto a nitrocellulose membrane and probed with specific antibodies or peroxidase - coupled streptavidin when biotinylated ubiquitin was used . dna fragments encoding ipah9 . 8 , ipah9 . 8 - nter , ipah9 . 8 - cter , and ipah7 . 8 were amplified by pcr and cloned as xbai - noti fragments into the vector pfl38cii / pgal1 containing the gal promoter ( badis et al ., 2004 ) to create plasmids pjr001 , pjr002 , pjr003 , and pjr004 , respectively . the 3 ′ oligonucleotides encoded the flag epitope followed by a stop codon and a xbai site . ycp50 - ste11 - 4 carrying ste11 - 4 under the control of its own promoter and prs316 - gal - ste4 carrying ste4 under the control of the gal promoter have been described ( dohlman et al ., 1995 ; stevenson et al ., 1992 ). an ecori - spei fragment from pjr001 encompassing the gal promoter and the sequence encoding ipah9 . 8 and the flag tag was cloned into plasmid prs425 ( sikorski and hieter , 1989 ) to create pjr005 . site directed mutagenesis of ipah9 . 8 codon 337 ( tgt encoding cys ) carried by plasmid prt7 ( mavris et al ., 2002 ) encoding gst - ipah9 . 8 was performed using a stratagene quick change ii kit to create plasmid pjr006 encoding the ipah9 . 8 - c337a variant ( gct encoding ala ). to construct pjr007 encoding ipah9 . 8 - flag , a ndei - pvuii fragment from pjr006 was transformed into yeast by4741 along with bsabi - bsiwi digested pjr001 and transformants were plated on ura - medium ; plasmids were rescued from ura + prototrophs . to construct pjr008 encoding gst - ssph1 , a the ssph1 gene was amplified by pcr from salmonella typhimurium atcc 14028 and cloned as a bamhi - xhoi fragment into the vector pgex - 6p1 . all plasmid insertions were confirmed by dna sequencing . invasive wild - type shigella flexneri 5 m90t - sm ( allaoui et al ., 1992 ) and its mxie derivative sf1060 ( mavris et al ., 2002 ) were used for infection . plasmids were propagated in escherichia coli dh5α ( end a1 hsdr17 sup e44 thi1 reca1 gyra rela1 laczya - argf ). to create jry101 , the integrating plasmid pfc23 ( o &# 39 ; 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