Patent Abstract:
the present invention relates to the use of yops as caspase inhibitors . more specifically , it relates to the use of yope and yopt as inhibitors of caspase - 1 activity . the inhibitor can be used to treat caspase - 1 - related pathologies , such as inflammatory diseases and to inhibit caspase - 1 - related and / or - mediated cell death .

Detailed Description:
plasmids and antibodies . wild - type ( wt ) yope , yopt and yoph were amplified by polymerase chain reaction from the e40 ( pyv40 ) plasmid ( sory et al ., 1995 ) and cloned in frame with an n - terminal e - tag into pcaggs - etag ( heyninck et al ., 1999 ) cut with noti and xmai restriction enzymes . the inactive mutants ( m ) yope r144a and yopt c139s were generated by overlapping polymerase chain reaction using mutated primers . pyv40 plasmids for specific yop effector knockouts have been described previously ( iriarte and cornelis , 1998 ; mills et al ., 1997 ). expression plasmids for yope wt and yope m , which were used for complementation of yope knockout strains , were a gift from dr . l . j . mota . the cdna of human rhoa , rac1 , cdc42 and their corresponding dominant - negative ( t 17 → n 17 in rac1 and cdc42 , t 19 → n 19 in rhoa ) and constitutive - active ( q 61 → l 61 in cdc42 , q 63 → l 63 in rhoa , g 12 → v 12 in rac1 ) mutants have been described previously ( sander et al ., 1999 ), and were a kind gift of dr . j . piette . the cdna of rho family members and caspase - 1 were amplified by polymerase chain reaction and cloned in frame with an n - terminal e - tag or flag - tag into pcaggs ( niwa et al ., 1991 ) cut with noti and xmai restriction enzymes . overlapping polymerase chain reaction using constitutive - active rac1 as a template and mutated primers generated the constitutive - active mutants rac1 ca - f37l and rac1 ca - y40h , respectively . the mouse proil - 1β cdna was cloned into pcaggs - etag vector with an additional ha - tag at the 3 ′- end of the proil - 1β cdna . all constructs were confirmed by dna sequence analysis . the expression plasmid for n - terminal flag - tagged human asc ( pcr3 . v66 - met - flag - asc ) and myc - tagged limk - 1 constructs were kind gifts of dr . jurg tschopp and dr . pico caroni ( basel , switzerland ), respectively . the β - galactosidase - encoding plasmid put651 was purchased from cayla ( toulouse , france ). a rabbit polyclonal antibody against recombinant murine caspase - 1 was prepared by the centre d &# 39 ; economie rurale ( laboratoire d &# 39 ; hormonologie animale , marloie , belgium ). bacterial strains and growth conditions . escherichia coli top10 or mc1061 were used for standard manipulations ; e . coli sm10 lambda pir + was used to deliver mobile plasmids into y . enterocolitica ( cornelis et al ., 1998 ). e . coli strains were routinely grown at 37 ° c . in tryptic soy broth or on tryptic soy agar plates containing the appropriate antibiotics . y enterocolitica bacteria were grown at 25 ° c . in brain - heart infusion ( bhi ; difco ) or on tryptic soy agar plates containing the appropriate antibiotics . y . enterocolitica e40 strains and derivatives have been described before ( iriarte and cornelis , 1998 ; mills et al ., 1997 ). for infections , bacteria were diluted to an od 0 . 1 in fresh bhi medium and incubated at 25 ° c . for 120 minutes . subsequently , yop secretion was induced by incubation for 30 minutes in a shaking water bath ( 110 rpm ) at 37 ° c . prior to infection bacteria were washed with rpmi1640 . culture , infection and transfection of cells . the murine macrophage cell line mf4 . 4 ( desmedt et al ., 1998 ), and the human embryonic kidney cell line hek293t were cultured at 37 ° c . in rpmi1640 or dmem , respectively , supplemented with 10 % fbs , 2 mm l - glutamine , penicillin ( 100 u / ml ), streptomycin sulphate ( 100 μg / ml ), sodiumpyruvate ( 1 mm ) and β - mercaptoethanol ( 2 × 10 − 5 m ). prior to infection , mf4 / 4 cells were seeded in medium without antibiotics . after 15 hours , cells were infected at a multiplicity of infection ( m . o . i .) of 50 with the relevant y . enterocolitica strains that were grown at 37 ° c . under conditions for moderate yop induction ( see above ). extracellular bacteria were killed two hours after infection by adding gentamicin ( 50 μg / ml ). hek293t cells were plated in 6 - well plates at 2 × 10 5 cells per well and transiently transfected by calcium phosphate - dna coprecipitation . twenty - four hours after transfection , medium was removed and cells were lysed in 300 μl lysis buffer ( 50 mm hepes , ph 7 . 6 , 200 mm nacl , 0 . 1 % np40 , 5 mm edta ). proteins were separated by sds - page and analyzed by western blotting with rabbit polyclonal anti - caspase - 1 and anti - il - 1 β antibodies ( r & amp ; d systems ), respectively , with mouse monoclonal anti - flaghrp ( sigma - aldrich ) or anti - e - taghrp antibodies ( pharmacia biotech ). immunoreactivity was revealed with the enhanced chemiluminescence method ( nen ™ renaissance , nen life sciences products ). ldh release was assayed using cytotox - one homogeneous membrane integrity assay as described by the manufacturers protocol ( promega ). β - galactosidase release was assayed using the galactostar reporter gene assay system ( tropix , applera belgium n . v .) yope inhibits the release of il - 1β in y . enterocolitica - infected macrophages yersinia has previously been shown to prevent nf - κb activation in infected cells in a yopp - dependent manner ( ruckdeschel et al ., 2001 ). therefore , it could be expected that the expression of nf - κb - dependent genes would be strongly increased in cells infected with a yopp - deficient strain ( yopp − ) compared to cells infected with wild - type ( wt ) yersinia . to verify this , we compared the amount of il - 1β and il - 6 in the supernatant of mf4 / 4 macrophages that were infected with either yopp − or wt yersinia enterocolitica . to our surprise , only the levels of il - 6 but not those of il - 1β were increased in the supernatant of yopp − - infected cells ( fig1 , panel a ). nevertheless , intracellular il - 1β levels in the corresponding cell lysates were considerably higher in cells infected with the yopp − strain ( fig1 , panel a , inset ). it should be mentioned that only the precursor form of il - 1β could be detected in these cell extracts . the above observations indicated a role for another yop effector protein in the regulation of il - 1β maturation and its release in the cell supernatant . therefore , we infected macrophages with a y . enterocolitica strain that is deficient for all six yop effectors ( δhopemt , fig1 , panel b ), which indeed resulted in a large increase of il - 1β levels in the supernatant ( fig1 , panel b ). all together , the above results demonstrate that , besides the yopp - mediated down - regulation of il - 1β production at the transcriptional level , other yop effectors could specifically inhibit the maturation and release of il - 1β . to analyze which yop effector controls the release of il - 1β , we constructed five double knockout y . enterocolitica strains , which are negative for yopp and for any one of the five other yops : yoppe − , yoppt − , yoppo − , yopph − and yoppm − strains . after infection of macrophages with each of these double - deficient bacteria , we analyzed again the secretion of il - 1β in the supernatant . il - 1β release was still strongly inhibited in macrophages infected with the double mutant bacteria yoppt − , yoppo − , yopph − and yoppm − , although a small but reproducible increase in il - 1β could be detected in yoppt − - infected cells ( fig1 , panel b ). however , cells infected with the yoppe − train released significantly higher levels of il - 1β , suggesting that yope is at least partially responsible for the inhibition of il - 1β release as seen upon infection with wt or yopp - deficient yersinia . since il - 1 levels released by yoppe − and yoppt − - infected cells were still lower than those released from cells infected with the δhopemt mutant , one could expect that cells infected with a triple yoppet mutant would release comparable amounts of il - 1β as those infected with the δhopemt mutant . however , we were unable to see a reproducible difference between cells infected with yoppet − or yoppe − mutants . as shown in fig1 , panel b , infection of macrophages with a yersinia strain ( pyv − ), which forms no functional secretion apparatus , does not induce caspase - 1 activation . this suggests that the initial signal for caspase - 1 activation may be initiated from one or more components of the secretion apparatus . a possible hypothesis could be that the secretion apparatus activates rho gtpases which may result in cytoskeletal rearangements leading to intracellular k + loss and activation of the inflammasome . yope is a gap for rho gtpases , in particular rac1 ( andor et al ., 2001 ), switching them off by accelerating gtp hydrolysis ( von pawel - rammingen et al ., 2000 ). to analyze if the gap activity of yope is required for inhibition of il - 1β release , we complemented the yoppe − strain with wild - type yope ( yope wt ) or with a mutant of yope ( yope m ) that lacks the gap activity ( fig1 , panel c ). complementation of the yoppe − strain with yope wt , but not with yope m , restored the potential of yersinia to prevent the release of il - 1β in the medium of infected macrophages . in contrast , intracellular expression levels of proil - 1β were independent of yope ( fig1 , panel c ). a previous report showed that installation of the yersinia secretion apparatus into the cell membrane of the macrophage results in pore formation and the release of cytosolic proteins such as lactate dehydrogenase ( ldh ), which could be prevented by yope . moreover , a role for rho gtpases and rearrangements of the cytoskeleton in the regulation of this pore formation was illustrated ( viboud and bliska , 2001 ). in agreement with the latter observation , yope deficiency also resulted in a significant increase of ldh and proil - 1β release in our experiments ( fig1 , panels c and d , respectively ), which coincided with an apoptotic morphology of the cells . however , also a clear band corresponding to mature active il - 1β could be seen in the supernatant of yoppe - infected cells , implicating that the increased il - 1β levels measured in the elisa experiments are not just due to an increase in proil - 1β release . the latter was further confirmed by the fact that identical results could be obtained when the il - 1 levels in the supernatant were measured with a ctll cell proliferation assay that detects specifically mature bio - active il - 1β ( vandenabeele et al ., 1990 ). these results indicate that the gap activity of yope is responsible for the inhibition of the maturation and secretion of il - 1β in yersinia - infected macrophages . moreover , this also implies a role for rho gtpases in the process of il - 1β maturation and il - 1β release . because il - 1β maturation is mediated by caspase - 1 , we next analyzed the effect of yope on caspase - 1 - mediated il - 1β maturation in hek293t cells that were transiently transfected with procaspase - 1 and proil - 1β . in these conditions , overexpression of procaspase - 1 induces its autocatalytic processing to an active p20 / p10 form , resulting in the maturation of the 33 kda proil - 1β to the bio - active 17 kda form ( fig2 , panel a ). moreover , caspase - 1 auto - activation also results in the induction of cell death , which can be assayed by the release of cotransfected β - galactosidase into the medium ( fig2 , panels a , c and e ). additional co - expression of yope wt , but not of the catalytically inactive mutant yope m , inhibited the autocatalytic processing of procaspase - 1 as well as the proteolytic maturation of proil - 1β . furthermore , also β - galactosidase release into the medium was partially inhibited . this demonstrates that yope can interfere with the autocatalytic processing of caspase - 1 through its gap activity , leading to a decrease in proil - 1β maturation and caspase - 1 - mediated cell death . interestingly , co - expression of yopt had a similar inhibitory effect on the proteolytic activation of procaspase - 1 and proil - 1β ( fig2 , panel a ). yopt functions as a cysteine protease that cleaves off the prenylated c - terminus of rho , rac and cdc42 , leading to their release from the plasma membrane and their irreversible inactivation ( shao et al ., 2002 ). a mutant of yopt ( yopt m ) that has lost its proteolytic activity on rho gtpases , was no longer able to prevent the processing of procaspase - 1 and proil - 1β . in contrast to yope and yopt , co - expression of the effector protein yoph , which is a tyrosine phosphatase that has been shown to dephosphorylate proteins from focal adhesions and other signaling complexes ( black and bliska , 1997 ; black et al ., 2000 ; persson et al ., 1997 ), had no effect on caspase - 1 activity . although yoph can lead to a rearrangement of the actin cytoskeleton ( grosdent et al ., 2002 ), an effect on rho gtpases has not been reported . these experiments show that yope and yopt can interfere with the autocatalytic processing and the activation of caspase - 1 by interfering with the function of rho gtpases . the inhibitory effect of yopt expression on il - 1β maturation is somewhat contradictory to the lack of a significant increase in il - 1β release in cells infected with a yoppt − double knockout as described in our previous experiments ( fig1 , panel b ). most likely , this can be explained by a difference in the yop concentration in cells upon overexpression or infection , respectively . in this context , it should be mentioned that whereas yope and yopt inactivate rhoa , rac 1 , and cdc42 in vitro ( andor et al ., 2001 ; shao et al ., 2002 ), when they are injected into cells by yersinia , they are remarkably specific , inactivating selectively rac1 and rhoa , respectively ( aepfelbacher et al ., 2003 ; andor et al ., 2001 ). these results suggest a major role for rac1 in caspase - 1 activation . in order to confirm the role of rho gtpases , and in particular rac1 , in the proteolytic activation of caspase - 1 , hek293t cells were cotransfected with procaspase - 1 , proil - 1β , β - galactosidase , and constitutive - active ( ca ) mutants of rhoa , rac1 and cdc42 . we hypothesized from our previous experiments that the constitutive - active rho gtpases should promote the autocatalytic processing of procaspase - 1 and the corresponding maturation and secretion of il - 1β . indeed , il - 1β levels were significantly increased in the supernatant of cells overexpressing rhoa ca , cdc42 ca or rac1 ca . titration of the transfected plasmid dna concentration clearly showed that rac1 ca is much more efficient then rhoa or cdc42 ( fig2 , panel b ), which is in agreement with the more pronounced release of il - 1β in yoppe − versus yoppt − - infected cells ( fig1 , panel b ) and the described preferential effect of yope on rac1 ( andor et al ., 2001 ). as expected , rac1 ca also enhanced the autoproteolytic activation of procaspase - 1 and the release of β - galactosidase ( fig2 , panel c ), whereas rhoa ca and cdc42 ca did not . reversely , transfection of a dominant - negative mutant of rac1 ( rac1 dn ) resulted in a decrease of caspase - 1 auto - activation and , consequently , in a decrease of il - 1β and β - galactosidase release ( fig2 , panels d and e ). it should be mentioned that the ectopic expression levels of rac1 that are needed to affect caspase - 1 activation are extremely low , being under the detection limit in the case of rac1 dn ( fig2 , panel e ). all together , the above observations imply an important role for rho gtpases , especially rac1 , in the regulation of caspase - 1 activity . pharmaceutical modulation of rho gtpase activity affects caspase - 1 activation and il - 1β maturation to further confirm the role of rho gtpases in caspase - 1 activation and il - 1β production , we analyzed the effect of clostridium difficile toxin b . the latter is a glucosyltransferase that covalently links a glucose moiety on a critical threonine residue of rho , rac and cdc42 ( prepens et al ., 1996 ; wilkins and lyerly , 1996 ), thus impairing the docking of the gtpases on their effectors . similarly , we also tested the effect of the geranylgeranyl transferase inhibitor ggti - 2147 , which prevents the prenylation and membrane localization of rho gtpases ( vasudevan et al ., 1999 ). western blot analysis revealed that treatment of procaspase - 1 and proil - 1β - expressing hek293t cells with toxin b or ggti - 2147 significantly prevents the proteolytic auto - activation of caspase - 1 and maturation of proil - 1β ( fig3 ). these results further demonstrate that rho gtpases play an important role in the regulation of caspase - 1 activation , and the corresponding caspase - 1 - mediated maturation and release of il - 1 β . the effect of rac1 on caspase - 1 activation is independent of its effect on the jnk pathway , but is mediated by lim kinase - 1 the functions of rho gtpases , first assigned to the regulation of the organization of the actin cytoskeleton , have been extended to many other cellular processes , including activation of the c - jun n - terminal kinase ( jnk ) ( bishop and hall , 2000 ). moreover , rac - induced cytoskeleton reorganization and jnk activation are the result of independent rac - induced signaling pathways ( lamarche et al ., 1996 ; westwick et al ., 1997 ). to dissect which signaling pathway is important in the rac1 - induced - activation of caspase - 1 , we used specific point mutants of rac1 ca that are defective in either jnk activation ( rac1 ca - y40h ) or actin reorganization ( rac1 ca - f37l ) ( lamarche et al ., 1996 ; westwick et al ., 1997 ). transfection of cells with rac1 ca or rac1 ca - y40h promoted the proteolytic activation of cotransfected procaspase - 1 , as well as the corresponding release of mature il - 1 into the medium , to a similar extent ( fig4 , panel a ). in contrast , cotransfection with rac1 ca - f37l was unable to promote the activation of caspase - 1 and the release of il - 1 . therefore , we can conclude that rac1 - mediated signaling to jnk activation is not involved in caspase - 1 activation . in contrast , caspase - 1 activation seems to be limited to the rac1 - mediated control of the actin cytoskeleton . limk - 1 participates in rac1 - mediated actin cytoskeletal reorganization by phosphorylating cofilin ( arber et al ., 1998 ; yang et al ., 1998 ). overexpression of constitutive active limk - 1 could promote the activation of caspase - 1 , confirming the importance of the cytoskeleton in the activation process . moreover , a dominant negative form of limk - 1 could abrogate the rac1 signaling pathway towards caspase - 1 activation ( fig4 , panel b ). the latter suggests that the signaling pathway from rac1 towards caspase - 1 activation could involve limk - 1 . the molecular mechanism of caspase - 1 activation is still largely unknown . the caspase recruitment domain - ( card -) containing protein asc has been shown to function as a caspase - 1 activating adaptor protein by mediating the assembly of a caspase - 1 signaling complex that promotes the activation of caspase - 1 and the proteolytic maturation of proil - 1β ( srinivasula et al ., 2002 ; wang et al ., 2002 ). to analyze if rac1 and limk - 1 can modulate the asc - mediated activation of caspase - 1 , we cotransfected hek293t cells with expression vectors for procaspase - 1 , asc , proil - 1β and either yope , yopt , limk - 1 dn or rac1 dn . western blot analysis of caspase - 1 , as well as analysis of the production of bio - active il - 1β shows that asc - mediated caspase - 1 activation can be affected by rac1 dn and limk - 1 dn , as well as by the yop effector proteins yope and yopt ( fig5 , panel a ). to analyze if rac1 ca could promote the asc - mediated caspase - 1 activation , we co - expressed asc or rac1 ca with procaspase - 1 to a level that does not lead to a significant activation of caspase - 1 . however , co - expression of constitutive - active rac1 ca and asc with procaspase - 1 resulted in a strong activation of caspase - 1 ( fig5 , panel b ). these data suggest that rac1 and limk - 1 can regulate the asc - mediated activation of caspase - 1 . we were unable to study the effect of rac on asc - induced caspase - 1 oligomerization because overexpression of asc led to the redistribution of caspase - 1 oligomers to the insoluble cell fraction , which is consistent with the previously described formation of filament - like aggregates upon asc overexpression ( masumoto et al ., 2001 ). however , caspase - 1 oligomerization can be forced by overexpression in hek293t cells . to analyze further whether rac1 modulates caspase - 1 activation by affecting its oligomerization , we analyzed in a co - immunoprecipitation experiment whether rac1 , yope and yopt could affect caspase - 1 oligomerization by making use of two enzymatically inactive procaspase - 1 molecules that were tagged with a different epitope - tag . as illustrated in fig6 , a dominant - negative rac mutant ( rac1 dn ), as well as yope and yopt , could prevent the oligomerization of procaspase - 1 , with yops being much more efficient then rac1 dn . in contrast , coexpression of constitutive active rac1 ( rac1 ca ) could promote the formation of procaspase - 1 oligomers . these experiments demonstrate that rac1 modulates caspase - 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