h ~ 7
~ i
~ METHOD FOR REGIJLA~ION OF NF~,cB
i
.~ This application is a continuation-in-part application of U.S. Patent Appli~ion
Serial No. 08/117,863, filed September 7, 1993.
BACKGROUND OF THE INVEN~iON
,~,
~;
5 1. Fieldof ~7e Inven~on
-,i This invention relates generaily to the field of regulation of 3en~ expression anc
~ specifically to the regulation of transcnption factor NF-,cB through re~u~n of
' degradation of the NF-KB inhibitor, I~
2. Descnption of ~elated Art
-, 10 The nuciear factor-kappa B (NF-I~B) is an inducible ~anscrip~ion factor which
, participates in the regulation of muitiple cellular genes a~er trea~nent of cells
with factors such as phorbol ester, lipopolysaccharWe (US), interieuhir~1 (IL-1)and tumor necrosis factor O. These gene~ are involved in th~ immedlate ea~y
~1
,-~ processes of immune, acute phase, and inflammatoly responses. NF-~cB has
.~
15 also been implic~ted in the transcriptional activa~on of se~eral viruses, most
notably ~e type 1 human immunodeficiency vin-s (HIV-1) (Nabel, et ~1., N~tur~,
;~:711, 1987; Kaufrnan, et~l., MoL Cell. Biol., 7:3759. g8n-
NF-.cB is a dimeric transcription factor ~at binds and reS~ulates ~ene
expre~ion thruugh decameric cis a~ins3 ~B DNA IT~f~. Al~ough a p50/pB5
20 heterodimer has ~raditionally been referred ~o æ NF-~cB and remains ~e
prototypica and most abundant fonn, it has been reco~nked reeen~y ~at
; several distinct but ~osely related homo- ard heterodimeric ~actors are
?1 responsible for ~tB site~pendent DNA bTnding ac~vity and regula0on. The
v various dimerio factors are composed of members of the family of Rel-re ated
'! 25 polypeptides. One subclas~ of this family, d~nguished by its proteolyac
. A :
.'Jf, :.. '.,i,............... " ..
-2-
processing from precursor forms and lack of recognized acavation domains,
inciudes p50 (NFKB1) and p50B (NFKE~2, p52), whsreas the second subciass
contains recognked ac~vation domain~s and ~udes p65 (RelA), RelB, ~Rei,
v-Rel, and ~e Drosophila pr~tein Dorsal. AJI Rel rebteci member~ share a 300
5 amino acid region of homology, r~sponsible far DNA binding and dimerization,
cailed the Rel homology domain. In the cyloplasm, NF-,cB and Rel proteirls
form a "Rel compleX'.
Ac~vation of the NF-ICB transcrip~on factor and various related forms can be
initiated by a variety of agents, including TNF~, phorboi 12-rnyris~te 13 acetste
10 (PMA), interleukin-1 (IL-1) and interleukin-2 (IL-2). Activation proceeds through
a post-translational event in which preformod cytoplasmic NF~,cB in the Rel
complex is released from a cytoplasmic inhl~itory protein, kB~ cBa inhibits
transac~vation of the p50/p65 heterodimer, by binding to ~e p65 component,
blocking the dimer's translocation to the nucleus. kB~ also inhibits complexes
15 containing c-R~I or RelB. I,cB~ blocks binding of various NF-leB dimers to ~eB
binding sites in DNA in vitro. ~;
ihorbol e~ter and TNF~ induction of nucbar i-ocale~on of NF-,cB is assooiated
wffll both the degrada~on of preformed l,ci3~ and the ac~vation of l~ gene
expression. Transfection 3tud~ inciicate ~ ~ kB~ gene i~ ~pecificaiy
20 induced by the 65 kilodaiton transactiva~n~ subunit of NF-ICB (Sun, et ~1.,
ScJence, ~:1912, 1993). As~iatTon of the nswly syn~ed l~ with p~5
r~stores Trl~racellular TnhibTbon of NF-ICB Di~lA b~ ac~ and probr~s the
sunhvaJ of thTs labile inhibTtor. lhese shJdbS show that NF-,~cB controls the
expression ~f l~ by means of an inducibb au~oregulatory pa~way.
25 NF-leB gene regulatTon is involved in many pathologicai even~ inaiuding
progression of acquired immune deficiency disease (AIDS), the acute phase
response and the acffva~on of immune and ~ndo~elial cells during to~c shock,
~3i~ ~
allosraft rejeotion, and radia~on resporses. Recently, cell-free exp~nents
alluded to the possibili~ ~t kB~ phosph~a~on might play a role in the
inac~vation and release af the inhib tor (Ghosh, et ~1., N~ure, 344:~78, 1~go)
and transient l~eBa phosphorylation hæ bff~n observed in several in vho
5 activation studies (Brown, et al., Proc. Nstl A~d. Sci., U.S~ 2~ 1993;
Beg,etal., l Mol. Cell.8101., 13:3301,1993). Surprisingly,forthefirsttimeffle
present invention unequivocally establishes ~at i~ is not phosphorylation of I~cB~
that causes dissociation of kB~ from the ReJ complex, but rather the phosphor-
ylation renders ~t suscepb~le to proteolysis.
10 Therefore, identification of the nature of l~cBa p~otease is critical for identific~on
of specific inhibnors of the protease which would be effective as anti-inflarnma-
tory and immunosuppressn~e agents. The presern invention provides
methodologies for iden~ such inhibnors and pro~des a group of inhibitors
as well.
" ~ "" ~ ",, ~ " , ~,,
h ~
SUMMARY OF TH"""" INVENTiON
.
The present invention is~, based on ~I~,e s~ minai di,~scove,~ th~t ~,e enzyTe
responsible i.*,r degradation of l,c~ is a chymot,ly~"~"in-like serine protease
. which reco~j4nizes p','n,osphorylated L?ci3~. i3ecause of this finding, the invention
`J 6 "~rovides a ~thod of identif~ing a comi~,ition which affe~,~, degradat"on of~
, ",c"3~ comprlising incubating components ,nc,uding the eompo,~e,~,ition, J,ci3~, and
~, a chym~,~rypsin-like serine proteæe under eonditions sufficient to allow the
componerns to interact and measuring t,l~ effect on the protease caused by
the eom,~,n. The invention ,~,so indudes a method of identifying a
~; 10 compositionwhich affacts degradation of ki3~ eomprising incubating compone-
.,
;~ nt~s comprising the composition, an induce,r of NF-I~B, and an indicator cell, and
.~
~i de~,~,t3~ing NF-~B a~Aty~.
,,
7 rne inve,n,tion a"so includes a method of treating an immunopathologi~i disor-
der assooi~t"3d ~w,~ith NF-,cB gene activation in a subject, ~mprising admink,~er-
3 15 ing to the subject a tnerape,uth~ ai,ly ef,t'~ve arnou"nt of a,n inhibitor of a ch~
~, t,ry~psin-like serine protease whieh des,rade~e, I,tc"3a.
, .,
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; ,
~,
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~3i~ 7
BRIEF DESCRIPTION Of THE DRAWINGS
Fl&URE 1a shows a Western blot ana~sis of ~e effect of phorbol 12-rr~te
1~ac~tate (PMA)-trea~ner~ on lleBa in 70V3 pr~8 cells.
.,
FIGURE 1b shows an autoradiogram of DNA from PMA treated 7~Z13 pre-B
.:i, 5 cells probed with æP-labeled DNA encompassing ~e NF-ICB bincling motif of
tha mouse ~ light chain enharlcer.
FIGURE 1 c shows ~e effe~ of IL-1, US and TNF treabments on the ac~vatbn
;J of NF-,cB and stability of l~ in 70Z/3 cells and HeLa oells. Sections of
,~, Western blots and EMSAs (fluorograms *om native gels) are shown.
~,
FIGURE 2a shows EMSA (upper panel) and a Western blot ~ower pan~l) of the
effec~ of cyclohexamide on the stability of l,c~ and the acffvation of NF-KB in
-~ 70V3 cells ~fter PMA stimulation.
~1
.
FIGURE 2b shows a densitometric quantitation of l,cB~ after Westem blot
analysis of cells treated vnth cyciohexarnide aione (le~ panel) or treated with
PMA for the indicated ~mes after 1 hour pr~trea~n~r~ cycloh~xamide
(right pane~.
.,
FIGURE 3a snow~ the ~t of ~tosyl L-phenyblanine chlorome~ylketone
tTPCK) on ~e induction by PMA on the a~ion of NF-,cB and ~tabili~ o~ LcB~
in 70Y3 cslls.
FIGURES 3b and 3c show the effect ~f TPCK on ~e activation of NF-ICB by IL-
-~ 1, US, and PMA in 70Z/3 cslls.
:~,
~!
.
FIGURE 3d shows the ~ect of pyrrolidinedithiocarbamate (PDTC) on ~e
activation of NF-ICB and s abilny of l/cBc. in 70V3 cells.
., ,
5, FIGURE 4a shows NF-~cB ac~vity as determined by HlV-LTR-Luc a~vity. Jurkat
il cells were stimulated wi~ the follo~nng reagents: (P), PMA (phorbol 12
myrislated 1~acetate), 10 ng/ml; (1), A231~7 Ca~ ionophore, 1~M; ,(3), anti-
CD3 (OKT3), ~g/ml; (28~ CD28 (monoclonal antibody 9.3), 3 ~,~/ml; (P/~,
PMA ~ Ca~+ ionophore 1 I,c~ transfected Jurkat cells; (NSj non-stimulated
(0.1%, DMSO only).
. I ,
i~ FIGURE 4b shows an ebctromobilny shin assay (EMSA) of the effec~ of
"~' 10 various stimuli on NF-ICB acavity.
:
i .
i~ FIGURE 4c is a Western blot showing phosphorylation of I~cB¢s after sbmulation
of cells with various stim~i.
FIGURE 5a shows kineacs of modffication of l,~B~ alter PMA and ~ +
~ ionophore (P/l) stimulation.
il
FIGURE 5b shows I~ degradation after trea~nent of cells with NF-~cB
blockers, T~CK, PDTC and CsA.
.~ :
FIGURE 6a shows an EMSA of cells ~eat~d wi~ N-acetyl L0u-Le~NorleucJnal
,, (AcLLnL) and the effect an I,cB~ phosphorylaffon and degrada~on.
FIGURE 6b shows an EMSA of the AcLLnL~treated later ~nne poirns and ~e
. 20 effecton NF-ICB acffvi~.
FIGURE 6c shows West~n blot analysis of immunoprecipitation of Rel A and
after AcLlnL treatrnent of cells.
~,
:
,,, . . . - . :
"~
'~13 ~,5 ~ ~1
. FIGURE 7 shows the effects of various p~p~de aldehyd~ inhibitors on I~
degradation foilowing lNF~., IL-1 or PllftA stimula~on of cells.
,i FIGURE 8 shows the effect of various peptide aldehyde inhibitors on NF-hB
'~ promoter.
5 FIGURE 9 shows an EMSA and Western blot of dose response of NF-ICB DNA
binding and I~ degradation of benzyloxycarbony~Leu-Leu-phenylalaninal (Z-
LLF) and AcLLnL
.~1
,
"I
~, .
,
..
DETAILED DESCRIPTION OF ThiE INVENTiON
`' The present invention pro~ncies a method of identifying a compo ition which
affects degradation of I~cB~ This method can be accomplished by incubating
componerlts comprising the composition, I,cB~l, and a chymotrypsin-like serine
-s 5 protease under condibons sufficient ts allow the components to interact and
measuring the effect on the protease caused by the compssition.
The NF-,cB transcription factor complex is secjuestered in the cytoplasm by the
inhibitory protein, I,ci~. Various cellular s~imuli reieve thls inhibition by
mechanisms which are mostiy unknown, leading to NF-.~B nuclear localization
and transactiva~on of it~ tar~et genes. Thus, NF-~cB anci l~eB~ are invoived in
a tightly cor~rolled mechanism of regulation.
..
The present invention describes the protease which degrades I~cB as a
chymotrypsin-like serine protease and describes inhibitors of ~e protease.
~'According to the invention, the surprising discovery that the l~ protease is
15a chymotrypsin~ike serine protease provides a useful screening tool for
-compositions which affect the protease.
:!,
Thus, the invention provides a m~i for ider~ing composi~ which aff~t
deçjrada~on of I~cB~, such as by inhibiting or stimula~ ~e prot~ase, and thus
;may be effec~ve as an~flammatory and immunosuppre~ive dn~e. The
20method comprises incubathg component~, which include the composition to
-ibe tested, phosphorylated l~tB and the protease, under conditions sufficient
to allow the components to hteract, then subsequer~y measurir~ the effect the
composition on the protease. The observed effect on ~e protease may be
e-ther inhibitory or sbmulatory. For exarnple, a composition which inhibits the
25protease will prever~ the protease from degrading I~ , thereby prohibiting NF-I-B from being translocat~d to nucl~us and inhibiting transac~on of genes
,
" .
. '.' ' ,,, ~
by NF-,cB. Tha effect the composibon has on the stability of kB~. can be
determined by various m~odologies including immunobgical, nucbic acid
and protein analyses. The l,cB~. can be label~d 50 that its fate can ~e
d2termined. Examples of labels ir clude a radioisotope, a fluorescent
compound, a bioluminescent compound, a chemiluminescent compound, a
metal chelator or an enzylT e. Those of ordinary skill in the art will be able to
ascertain such, using routine experimentation.
The method of the inver~ion for idenfffying a compostion which aflects
~I degradation of LcB~. Tay further include the addi~on of NF-~cB, prefe~bty
t$ 10 bound to or complexed with IICB¢.. The chymotrypsin-like serine pro~ease whch
degrades I~ may be chymotrypsin. A candidate c~ytoplasmic protease which
- can degrade l,~B~ following stimulation i9 a ubiquitous 700kD mul~subunit
proteosome which has chyrno~rypsin~ace ac~vity ~initsky, et ~1., Biochem~sby,
~:9421, 1992). A proteosome as described herain is a multicatalytic enzylT e
1 15 complex.
rl The invention also includes a method of iden~fying a composition which allec~s
degradation of kB~ comprising incubating cornponents including the
composition to be tested, an induoer of NF-,cB and an indicator cell, and
detec~ng NF-~cB ac~. The inducer of NF-ICB can be added prbr ~ o,r
20 followina the addition ot ~e composi~on to be tested. Preferably, it is addedalter the composition is adcled. Inducers of NF-ICB include IL-1, IL-2, TNF~,
phorl~ol ester and US. Other induc~ will be l<nown to ~c~e of shïl in a.t
~ ~ .
, .~
The method of the imention is perfonneci in an indicator cell. An Sndcator
cellU i~ one in which ac~v#tion of NF-,cB can be detected. E)~ampl~ c~
25 mammalian host indicator cells include the pr~B cell lin~, 70Z13, Jurla~ T,
COS, BHK, 293, CHO, HepG2, and HeLa cells. Other cell lines can be u~ed
as indicator cells, as long æ the level of NF-leB can be detected. The cel'~ can
.'
, .
.
' X. ' ' ' .
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.
:
be recombinantly modffied to contain an expression vactor which encodes one
or more additional copies of the ~B binding rno~f, preferably operaffvely linkedto a raporter gene. The cells can also be nK~d to express l~ and NF-ICB.
Preferably, the expression vector which encodes NF-~cB, contains the coding
5 region for the p65 subunit of NF-,~B, to which l~ binds.
. .,
The host cell may be a yeast modffled by recombinant DNA to express NF-,cB,
a chymotrypsin-like gene derived ~rom a cDNA expression library and ,cB motif
~nked to a reporter gene. The expression of the chymotrypsin-like protease
would result in I~cB dsgradation and ac~on of NF-~cB, hence inducaon of
10 reporter activity. In Ule presence of the composition, the chymotrypsin-like
protease would be inhibited resulting in no reporter ac~ . Exampbs of
markers typioally used in yeast reporter s~ include ,~ galactosidase ~s~al),
HIS3 and LEU2 nutrient selection markers.
~, lhe report~r gen0 is a phenotypically ~ntifiable marker for detecbon of
15 stimulation or inhibibon of NF-~cB ac~vatio L Markers preferiably usied in the
present invsr~on include the LUC gene whose expression is detectable by a
luciferase i~y. Examples of markers typically used in prokiaryotic expression
vectors include antibiotic resistance genes for iarnpicillin (s-lactamases),
~, tetracycline iand chloramphenicol (chbramphenicol acetyltransferase~.
20 Exiarnples of such ~kers typically used in marnmalian expression vectors,
-~ which are preferable for the present inver~on, include ~e ~sne hr adeno~ine
deiaminase (ADA), aminog~coside phos~nsFeriase (n~o, G418), dihydro-
folate recluctase (DHFR), hygromyan-B-pilos~otriansferase (HPH), ~ymidine
Wnase tTI~, xar~ine guanin~ phosphoribosyltransferse QtGPRT, gpt) and ~-
25 gaiactosidase ~s~ai).
Transformation of a host call wi~ recon~ DNA may bs oarried out by
~' conventionai tschniqu~s whidl are w~ll known to those skillsd in the art.
!
'
C~
Where the host is prokaryotic, such as E. coli, compet~t cells which are
capable of ONA uptake can be prepared from cells harvested after exponential
! grow~ phase and subsequen~y treat~d by the CaC12 rn~od by proc~dures
well known in ~he art. Altematively, MgCI2 or RbCI can be used. Transforma-
5 ffon can also be performed after forming a protoplast of the host cell or by
electroporabon.
When the host is a eukaryote, which is preferable in the method of the
invention, such methods of transfection of DNA as caldum phosphate co-
precipitates, conventional mectlan cal procedures such as microinjaction,
.~,
10 electroporation, insertion of a plasmid encased Tn liposomes, or virus vectors
.3, may be used. Eukaryotic cells can also be cotransformed w;th DNA sequenc-
es encoding ~e polypeptides of the inven~on, and a second foreign DNA
mobcule ercoding a selsctable phenotype, sudl ~ the herpe~ simplex
thymidine kinase gene. Another me~od is to use a eukaryotic viral vector,
15 such as simian virus 40 (SV40) or bovine papilloma virus, to transien~y infect
d~ or transform eukaryobc cells and express the protein. (Eu~yo~c V7r~1 V~ctors,
l~ Cold Spring Harbor Laboratory, Gluzman ed., 1982).
~, In the present invention, ICB bindin~ motif polynucleotide sequences, preferably
opera~vely linked to a reporter g0ne, I~ and NF-,cB polyruJcleotide sequenc-
20 es may be ir~rted irlto r~ombinant expression vectors. The term "recombi~
n~nt ~xpression vector" refers to a plasmici, virus or other vehicie known in the
art ~at has be~n manipulatec by insertion or incorporation of the ~enetic
saquencqs. Such expression vectors contain a promoter sequence which
facilitates the 0fficient transcription of the inserted genetic sequence of the host
25 The expression vector typic~lly contains an origin of replication and a promoter,
as well as speciflc genes which allow phenotypic selection of the transformed
cells. Vectors su-table for use in the present invention inciude, but are not
3 limit~d to ~e 17-bas~d expr~ssion vectorfor expression in bacteria (Rosenberg
.j ~
., ,
~3..~
-12-
et ~I., Gene ~:125, 1987), the pMSXND expression vector for expression in
marnmalian oells (L~e ~d Nathans, J. Biol. Chem. ~:~1, 1988) and
baculovirus-derived vector~ for expression in ir~sect cells. The DNA ~egment
can be present in the ~ctor opsrably linked to regulatory elements, for
5 example, a promoter (e.~, 17, metallothionein 1, or polyhedron promoters).
i
Detection of NF-,cB ac~ in the method of ~e invention can be detected by
measuring the level ~f ~e gene product of the reporter gene. Methods of
detection may be immunological, by nucleic acid analysis, by protein analysis,
by nutrient selection, or by enzyrnatic assay, for exarnple. Other common
10 methods will be known to those of skill in the art.
In another embodiment, the invention provides a method of treating an
immunopathological disorder associated wWl NF-ICB gene ac~ion in a
subject. Preferably, the immunopathological disorder is associated wffl TNF,
IL-1 or IL~ produ tion. The method eompris~s administering to the ~ubject a
15 therapsutically effective amount of an inhibitor of a chymo~ypsin like eerineprotease which dagrades kB~. The term "immunopathological disorder~ refers
to any disease which irNolves the immune response or immunity in generai.
~herapeutically effec~ve' as used herein, refers to that arnount of inhibitor ~at
is of sufFicient quarffity to ameliorate the cause of the NF-~B disorder.
20 ~Amelioratea refers to a bssenin~ of the detrimentai effect of ~e disorder in the
pa~ent receiving the th~rapy. ~he subject of ~e inve~n is preferably a
~; human, however, it can be envisioned ~at any animal wi~ a NF-.~B ~rder
can be treated by ~e msthod of ~e inver~on, for exampb, a SCID mouse
grafbd with human bone marr~w (hum~eed SCID). E1camples of immuno-
25 pathological disorders which can be b-eated by ~e method of the invcn~on
include acquired immunodeficiency disorder (AIDS), toxic shock syndrome,
allograft rejection, ultraviolet and radiation rsspons~s, and disorders associated
the ac~vation of T cells, B cell~ and macrophag~s during ths immune
'I ~
f~f~3f~f ~
resfponse and the acute phase respQfnse anci disorders associatfKi with
af~vanced cancer suoh as tumor necrosis factor-mediated cachexia
The invention indudes a me~od of moffduiatins the activation of a virus
asscociated with NF-~iB transactivaftion comfprising administering to the subject
S a modulating effective amount of an inhilf~r of a chymotrypsin-like serine
protease which degrades l,cii~. The tsrm modulates referis to either inhibWng
or stimulating the activation of a ~rus. Any virus which is trani acffvatfs~f I by NF-
~eB is included, for examph3f, human immunodffeficienfcy virus (HJV~. Essenff'aily,
any disorder which is etiologioaifly linked to NF-ffeB/II/efBcfff would 'l~e considerfcdf
10 susceptible to treatment.
The present inventiion provides inhibitors of the protease whieh are peptide
aldehydes. The peptide alldehyde prfsfferably has~ the formula:
R1i~2fLI~rCH~S~
wherein R~ i9 an optionall amine prc~ective group; L i~ leudne; R2 ie
5
optiona-i and has the formub -(CH2)n~CO~ wherein
I~H3+
n is 1-5 and wherein (CH2)n is a branched ar straight chain aikyi; and R3 is an
optional hydrophobic amino aad, except for me~iionine. The amine protec~ve
20 ~roup is prsferabiy N-ace~l, benzyl carbonyF or butyl. Exa~Tple~ of such
~tide aidehyde inhibitors inciude proteosome irfhibitors such as N-ace~Leu-
iLeu-Norleucinai (AcLLnL) and benzyl~carbonyl Leu-Leu-Phenyaiarfinai
(ZUJ:). Other pep~ide aldehydes are usehJ as inhibi~ors in the method~ of ~
hventon inciude N-acetyl-Aia-Leu-L~u-Norbucinal (AcALLnL). Most preferred
25 are thiose peptide aldehyd6s where ~ie arr~no acid position R3 is subsfflutec-i
wWl a hydrophobic amino acid. Examples of i~3 include alanine valine, leucine,
isoleucine, proline and phenylalarine. Least preferred is a i~iptide aldehyde
,~
wherein the R3 is a charged arnino acid. Most pref0rably, the inhibitor is
benzyloxycarbonyl-Leu-Leu-Pher~lalaninal (ZLLF).
The inhibitor of the invention can be administered parenteraliy by inJection or
by gradual infusion over time. The inhibitor can be administered intravenous~,
intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, or
extracorporeally. Methods for delivery of the inhibitor also include orally, by
encapsulabon in microspheres or pro~einoids, by aerosol delivery to the lun~s,
or transdermally by iontophoresis or transdermal electroporation. O~her
methods of administration will ~e known to those skilled in the art
Preparations for parenteral administration of an inhibitor of the invention include
sterile aqueous or non-aqueous solu~ions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene glycol,
~`
vegetable oils such as olive oil, and injectable organic esters such as ethyl
oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsione
15 or suspensions, including saline and buffered media. Parenteral v~hicles
include sodium chloride solution, Ringe~s dextrose, dextrose and sodium
chloride, lactated Ringer's, or foced oils. Intravenous vehides include fluid and
nutrient replenishers, electrolyte replenishers (such as those based on Rin~er'sdex~ose), and the like. Preserv~ves and o~er addi~ves may al~o be presen~
20 8uch as, for example, antimicrobials, anti~xidan~, chela~ng agents, and inert~3 ~ases and the like.
.~
'~; In another embodiment, the invention provides a phannacelnicaJ composition
for inhibi~ng a protease which degrades kB~, comprising a ~erapeu~
effec~ve arnount of an inhibitor of a chymotrypsin-like serine prcteas0 which
25 degrades IJcB. The inhibitor is preferably a pepbde aldehyde and has the
formula:
,.~;. . :
. r ~ , , ', : '
' '~..' '
:, : .:. :
:
R~ R2UR3-CHO
wherein R~ is an optional ~nine prote~ve group; L is leudne; R2 is
.~ H
optional and has the formula -(CH2)n~0~ wherein
n is 1-5 and wherein (CH~n is a branched or straight chain alkyl; and R3 i~ an
, optional hydrophobic ar~uno acid, except for me~ionine.
;'-,; .
Preferably R3 is selected from the group consisting of alanine, valine, bucine,
isoleucine, proline and ph~ylalanine. The amine protective group i~ preferably
10 selected from N-acetyl, benzyloxycarbonyl and butyl. Exarnples of pepffde
;~ aldehyde inhibitors usef~d in the pharmaceutical composition of ~e inven~on
includeN-acetyl-Leu-Le~Norleucinal(AcLLnL) ,N-acetyl-Ala-Leu-Leu-Norleucinal
(AcALLnL), and benzyb~carbonyl-Leu-Leu-phenylalaninai (ZLLf). A
therapeutically effective amount of inhibitor is that amount that inhibit~ l,ci~degradation by greatar than 5096 and preferably grsater than 80%.
~, The following ~xarnples are intended to illustrate but not limit the imention.
While ~ey are typTcai of tllose that might be used, other procedures known to
those skilbd in Ule art may aitematively be used.
'7 EXAMPLES
20 ~he following examples ~how ff~at T-cell acffv~ion result~ in th6 ph~
tion of ~ and that ~is is a rapid physiolo~ evern that is dependent on
appropriate Iymphocyte costimulation. The fact~a~ ths inducible phosphoryla-
tion is abolished by several distinct NF-,cB blo~dng re~gents, indic~
- essential role in the ac~v~ion process. A key discove~y was that in vi~/o I~
25 phosphorylation does not cause its dissociation from the ~el eomplsx, but
¦ ra~er renders l~ susc~ptible to proteolysis by the chymotrypsin-lik6 er~ne.
"r . ~ ,
~3~ 3~
i, E~MPl.E 1
Y FAT~ OF l/cB~ AFIER NF-~cB AC:TIVAIION
~ .
The fate of kB~ after traatment of cells wWl various NF-,c~ac~vating stimuli wasinvestigated. 70Z/3 cells were culbured in RPM~1640 medium (Gibco BRL)
supplemented with 1096 fetal calf serum and 50~M 2-mercaptoethanol. Two ml
aliquots with approximately æ3 x 106 suspended cells were treated WWl 50
ng/ml2 PMA (Sigma), 50 U/ml IL~ hringer-Mannheim) or 15 ~.0/ml US
:'! (Sigma) for various times. HeLa cells were cultured in DMEM supplemented
3 with 1096 fetal caff serum and 1% L~lutamine, and treated with 200 Ulml
recombinant human TNF~ (Genzyme). Stimulation was stopped by cooling on
ice and immediate centrifugation of cells for 5 sec in an Eppendorf microfu~e.
Cell pelle~ (108 cells) were !ysed with 60 ~1 ~f a hi~h-salt extraction buffer
t (Baeuerle, P~, et ~1., Science. ~,:540 646. 1988). Supematant~ of ~ysate~
(from a 1~min. centrifu~ation at 13,000 rpm in an Eppendorf Microfuye~ were
used for analysis by b~th Westem bb~ng and electrophoretic mobili~y shffl
assay (EMSA), a~ described (Zabel, et ~1., EMBO J.. ~ 201, 1983). For SDS-
3 PAGE and Westem blot~ng, 30 ~-l aliqw~ of extrac~ were mKed with 1 5 ~ 1 of
t SDS sample buffer (Schreck, R., et ~1., J. Exp. M~d., ~:1181, 199~), boiled
and subjeeted to SDS-PAGE on 1æ5% polyacrybrnide mini gels (Biorad).
Ii .
Proteins were transfened from gels onto Immobilon TM fiKers (0.45 ~m;
Millipore) usin0 a sem~dry blotang d~Ace (Biora~ hr 1 hour at 15U/æ5
m~Vall2. Biotting effiaency wa~ mu~tored by prot~in stainin~ of flKers wi~
Ponc~au S (SeNa). FiHers were blocked over~ight in Tris-buffered saline
containing 0.1% (vh) Tween-20 (TBST), 5% skim milk powder (Nes~e) and 1%
BSA. Tha filter was ~en incubated for 1 hour at r~m temperature wffl an'd-
IgG in a dialyzed eluat~ from ffie k~ affinty column diluted 1:100 in
blockin~ buffer. After 30 minutes of washing in T7BST, ~e filter wa~ incubated
in a 1:4000 dilution of goat anti-rabbit bG/horse radish peroxidass conjugate
,,
; "' ' . ` ', ' . ', ~ . ~ " , '''
.
'~ ~ 3 ~
.~ ~
-17-
(Biorad) in blocking bufFer. After 30 minutes of washing in TBST, the fli~r was
treated with ECL detec~ion reagent (Arnersham) and exposed to Kodak )tF~ film
for less than 1 min. For EMSA, 2 ~11 aliquots of extract~ were added to a
binding mix containing 100 mM KCI, 20 mM HEPES, pH 7.9, æ5 mM
s d thiothreitol, 0.5 mM phenylmethyl sulfonylfluoride (PMSF), 0.2% Nonidet P 40,
5% Ficoll, 20 ~9 BSA, 3 ~.9 poly(dl~C) and 10,000 Gp.m. (Cerenkov counting)
of a 32P-labeled doubl~s~anded ,cB oligonucieotide probe (Gibco BRL).
Specificity of the protein-DNA complex was verified by immunoreacb~ity with
g a polyclonal antibody specific for Rel ~ After a 20 min-incubation on ice,samples were subjected to electrophoresis on native 4% polyacrylamide gels
run in 0.5x TBE. Dried gels were exposed to Kodak XR film at -70-C
ovemight Human IIcB~ was produced in E. coli and purified as described.
Two milligrams of 6 x Hi~tagged, purified human kB produced in Escherichla
coli (Zabel, et ~1., EMBO J., supra; Henkel, et al., Cell, Ç~:1121, 19~2) were
1s coupled to 0.5 ml cyanogen bromid~ac~vated sepharose 4CL~B (Pharmacia),
~, according to the instruction of the manufacturer. Specifio I~G in 2 ml rabbit
antiserum raised against kB (Zabel, et ~1., supr~) was ~ffini~y-purified (Henkel,
et ~ supr~) on l,e~sepharose. After e~densive washes wi~ phosphate-
buffered saline and 2 column volumes of 0.1M glydn~HCI, pH æ7, speclfic
antibodies were eluted ~nth 2 volumes of 4M guanidinium hydrochloride. The
eluate was extensively dialyzed against Tris buffered saline and u~ed for
Westem blot~ng at a dilution of 1:100 in blocking buffer (Henkel, et~l., supr~).
i
Cells were treated wi~ PMA for indic~ted periods of ~me (FIGURE 1a, lanes
2-9). Proteins in total cell extracts were separated by SDS-PAGE and
transferred onto a membrane fil~er followed by Westem b~ analysis using anti-
A~ IgG and decoration with peroxidas~labelled an~d~abbi~ IgG. The arrow
indicates the position of I~ . FIGURE 1b shows ~e effect of PMA on the
I i DNA-binding activity of NF-~eB. Total cell extracts from con~ol (lane 1) and
3 I PMA-tr~ated cell~ (lan~s 2-6) were incubat~ a æP~abell~d DNA prob~
,
c
-18
encompassing the NF-ICB binding motif from the mouse ~c light chain enhance~
(Sen, et ~1., Cell 47:921-928, 1986) followed by analysis of DNAbindin~
activities using electrophoreffc mobili~ shift assay (EMSA). A fluorogram of a
s nativa gel is shown. FIGURE 1c shows the eff~t of IL-1g, US and TNF on ~e
ac~vation of NF-/~B and stability of l,~B~. 70Z/3 cells (upper and middle
panels) or HeLa cells ~wer panel) were treated with IL-1g (lanes 24), US
anes ~8) or TNF~ (lanes 10-13) for the indicated periods of ffme folbwed by
analysis of total cell extracts for NF-,c~DNA complex is shown by a filled
arrowhead, the posiffon of uncomplexed DNA probe by an open arrowhead
and the position of the kB~ band by an arrow.
In extracts from unstimulated 70Z/3 pre-B colls, I,cB-speciflc IgG detected a
: single 38K band on Western blots (FIGURE 1a, lane 1) which was not seen
wi~ the s~cond antibody alone. Be~Neen 2 and 5 minutes after the addition
of phorbol 12-myristate 1~acetate (PMA) to cell cultures, I~ almost
1s completely disappeared from cells (FIGURE 1a, compare lanes 3 and 4). The
3 speciflc immunoreactivity reappeared after 40 minutes (FIGURE 1a, lane 7).
'~, The disappearance of kB~ coincided wi~ ~e appearance of NF-,~B DNA-
binding ac~vity in total cells e~*racts (FIGURE 1b, compare lanes 2 and 3),
suggesting a causal relationship betNeen ~e h~.ro events. Interleukh (IL-lp),
lipopoiy~accharide (US) and tumor necrosis factor~ tTNF~) all indu~ a
decay of l~cB in 70Z/3 or HsLa cells (FIGURE 1c), showin~ ~at distinct stimuli
induc~d the same reac~on. AJthough most of the l~ had already decayed
5 minute~ a~er s~mulatbn with PMA, TNF-Q and IL 1~, it took more ~ 15
minutes before there wa~ a rapid decay of ~ inhibitor in LPS-stimulated 70iV3
cells. ûespite ~ese kinetic differences, ~e depletion of I~ was in each case
coincident with ~he appearance of NF-~B ac~ . In contrast to ano~r ~tudy
(Brown, et ~1., Proc. N~. Acad. Sci., U.SA, 90:2532, 1993), no transient
change in ~e electrop~wrebc mobili~y of I~ was apparent after induction wi~
~e various stimuli, even when phosphatase inhibitors were included in ~e ~sis
,~J',' ' ~ ,
;~ ~
buffsr. Mor30ver, no breakdown products of ~B~ c~uld be detected. ~se
a po yclonal ar~ibody was used, it is unak~y ~at only one epitope of 1,cB was
lost or rnod~ied upon sbmulation.
EXAMPLE 2
EFFECT OF A PROTEIN SYNTHESIS INHIBITOR
ON THE STABIUTY QF l~cBa
70Z13 cells were treated with 25 ~.g/ml cycloheximide (Sigma). The protein
syn~esis inhibHor cydoheximide (CHX~ ac~vates NF-,cB in 70Z/3 cell~. At 10
~, ~,g/ml, prot~in syr thesis is inhibHed to 90% in 70Z/3 cells. Cell culture, extract
preparaaon, EMSA, SD~PAGE and West~m blofflng were as described above
in Example 1. Densitometric scanning was done w;~ a Howtek Scanmaster
3 and data analyzed by ~e sofhNare Quanffly One Version z.æ
In FIGURE 2a, 70Z/3 cells were treated wi~ cycloheximide (CH~ for 1 hour
followed by stimulation with PMA for ~e indicated periods of ffme. Total cell
extracts were assayed for NF-ICB acffvity by EMSA (upper panel). A section of
an autoradiogram from a naffve gel is shom with a filbd arrowhead indic~ting
~e position of ~e NF-,c~DNA complex. AJiquots of ~e cell extrac~s were
assaycd for l~ by Westem blo~n~ (lower panel). llle position of the k8~
band is indicat~d by an arrow. FIGURE 2b shows ~e half-life of l,tB~ in protein
21) synthesis-arre~d cslls. Cells wera treated wi~ C~K alone (le~ paneq or, ~er
a 1 hour traa~n6nt wi~ Cl K, induced with PMA for the indicated ffmes (righ~
panel). Total c~ll extracts of equal protein content were analyzed by westem
blottng followed by densitometric quar~tabon o~ tha 38K kB~ band in
fluorograms. The alTounts of I~B~ d~tected wi~ C~DC trea~nant alone ~eft
2~ panel; open trianglss) and after PMA stimulation (rlght pan~l, filled trian~les) are
shown on a haK-logarithmic plot. Dot~ed lines indi~ta the portion of the slopes
., ~
used for estimation of t 1/2 values. The dashed line in the right panel indicates
.~, the decay of I~ in the absence of PMA, as determinec in ~e left panei.
.~
Following a 1 hour treatment of 70Z/3 cells wffl C~D(, the DNA binding of NF-
,cB was inciuc~d only weakly (FIGURE 2a, lane 1, upper panel), anc significant
5 bvels of IKB~ were still prcserlt (iower panel). This shows ~at interference w~th
the normai tumover rate of kB~ is not sufficient for a rapid ac~vation of NF-I-B.
When C~ eated cells were stimulated with PMA, a rapid decay of l,c8~ and
further induction of NF-i~B binding ac~ were observed w~th kinetice
indistinguishable frsm those observed in the absence of C~Dt (compare
~ 10 FIGURE 2a with FIGURE 1a). But CH)t prevented the reappearance of l~cB~
seen after a ~40 minutes treatment w th P.MA alone (compare FIGURE ~. v~th
FIGURE 1a), consisterrt with the finding that l,c.~ is newly synthesized under
transc~iptional control by NF-.eB. A haff-lifa of .approximately 138 minutes wa~~i deter.rnined for l.e,B<~, in C.~ treated 70Z/3 cells (FIGURE .2b). If protein
.' 15 sy~thesis-arrested cells were s~mulated ~th P.MA, the half-l~e of~ l,c.~. was
reduc~d to only 1.5 minutes during th.e ph.~ of its fastest decay, showing ~.at
P.MA induced a t HO orders of magnitude decre.ase in the ha!f-life of l.eB~..
The f.unc~on~. sign.iflca.nce o~ the induable deca.y of l.~B~. for ac~.ation ~f NF-.~cB
was tested by e~.re of cell ~.ltu.res to various protease inhibitor~. 8k
.20 dis'dnst .serine prctease inhibitors with c~notrypsin-like specificity efficien~y
prevent ~e induc~on of NF-,~B DNA-bindir~ ac~vity in response to PMA and
;~ TNF~ (Table 1). Cons~ve DNA-bindir~ activities, includin~ Oct-1, were not
affecteci and no significant cell death was observed by a dye exclusion assay
and phase~ontrast microscopy at inhibitor concentra~ons preventing NF~B
g5 ac~vation. Leupeptin, antipain and the ~ypsin inhibitor tosyl-Lys~e~ylaster
were not effecffve, even at high concer~ions.
TAE~LE t
THE EFFECT OF VARIOUS PROTEASE IN111~ITO~
ON THE ACTIVATION QF NF~,cB
Protease inhibitor ,Jurkat mZ~
~-Z Tosyl-Phe-chloromethyiketone (TPCK~ 20* 20
i Benzyloxycarbonyl-Leu-Tyr-
chloromethylketone (21 YCK) 10 10
'i Tosyl-Lys-chloromethylk~tone (lLC19 1D0 100
3,4-Dichloroisocoumarh 20 ND
N-Benzoyl-L-Tyr~thylester (BTEE) 1,000 ND
N-Acetyl-DL-Ph~Z~-Naph~ylester (APNE) 200 200
Jurkat and 70/3 cell cultures were incubatcd with the listed compounds at
15 various conc~Zn~rations for 30 miswtes and ~en stimulated by ei~r TNF~
(Jurkat cells) or PMA pOV3). Total cell or nuclear extracts were prepared and
analyzed for NF-,cB ac~ using EMSA.
.~ ,
~IC90 (I~M). 90% inhibitory concentration; ND, not determined. Jurlcat T cells
were cultured as desibecl (Schreclc~ R, e~ ~1., EMBO J., ~Q:2247, 19g2). ~l
20 compounds (Sigma) were di~ d in DMSO and cells pretr~ated at various
concentrations for 30 min. EMSA was don~ a~ described (H~nkel, et 81., Cell,
~:1121, 1992).
The effect of tosyl-Phe chloromethylketone (TPCK~ inves~ated in more
detail. Cell culture, extract preparation, EMSA, SD~PAGE and Westem
2~ blotUng w~re as de~bed above. 70Z/3 cells were prebeated wi~ M
TPCK or TLCK (Sigma) dissolved in dime~ylsulphoxide (DMSO~. Control
,0
-æ-
cultures received an equivalent arr~t of DMSO. Cell cultures were treated
wi~ the arnmonium salt of PDTC (Sigma).
,
~ FIGlJRE 3a shows ~e effect of TPCK on ac~vation of NF-I~B and stabirty of
S kB~. 70Z13 cells w~re stimula~ed with PMA for the indicated times in the
absence (left panel) or presence of 25 ~M TPCK (1 hour pretreatment). Cell
extracts were analyzed for NF-.~B ac~vity by EMSA (upper panels) and for I~
~' levels by Western blotting (lower panel). Ar~ arrow indicates the position of the
38K l,tB~ band in Westem blots. The faint lower band is nonspecific because
,J its abundance was strongly reduced when affini~y-purified antibody as u~sd.
The pos-tion of the NF-I~DNA complex in the seotions of fluorograms i~
indicated by filled arrowheads. (b) The effect of TPCK on th~ activation of NF-
,cB by IL~ , US and PMA in 70V3 cells; Co. Control cells. Cells were traated
with TPCK 10 minutes before stimu~on ~ ~8) or, hr 10 min, follov~in~
treatments of cells for 30 minutes wffl IL-1 (L) and PMA (P), and for 60 minuteswnh US lL) (lanes 1~12). Total o~ll extracts from cortrol (lanes 1~) and
TPCK-treated cells (lanes ~12) were analyzed for NF-~B activity by EMSA (c)
The effect of TLCK on the activation of NF-ICB by PMA 70Z/3 cells wer~ t
untreated (lane 1) or treated for 10 minu~es wWl e~her 25 pM TPCK (lane 2) or
25 ~.M TLCK (lane 3) followed by addition of PMA Cell extracts were analyzed
by EMSA for NF-,cB activity. (d) Tl~ effect of ~e antio~ddant PDTC. Cell~
were treated for the indicated periods of time wi~ PMA In the absence or
presence of 100 ~M PDTC added 1 hour before stimulation. Cell extract~ were
analyzed for NF-ICB ac~ y by EMSA (upper panel~) and for II-B levels by
Westem blotting (lower panel).
,~
Treabnent of cells with 25 ~.M TPCK hl~ inhibited the induction of NF-~B
ac~ (FIGIJRE 3a, upper panels) as well as ~e d~cay of I~cB~ in response
to PMA (lower panels). ~h~ haK~aximal inhibitory concerltration (IC~) of
TPCK was about ~ ~M. The protease inhibitor prevented the acbvation of NF-
~ I
J
- ~131J~ j
,cB by IL~ and US in 70Z/3 cells ~FIGURE 3b, lanes 6 and n and bq TNF~
in various other oells lin0s. TPCK did not strongly int~rfer~ with NF-ICB acb'~hty
when added after stimulation wi~ PMA, IL-1 or US but seemed to arrest a
furthar activation of the factor (FIGURE 3b, lanes 1~12). It is unlike~y that
5 TPCK acted on pro~ein kinase C (PKC) because IL-1 and TNF~ activate NF-ICB
independer~y of PMA-inducible PKC isoerzymes (Bomsztyk, etal., C~ll. Regul.
3~ 329,1991; Schreck, R., etal., J. Biol. Chem., ~:8339,1990). Furthermora,
TPCK did not interfere with early signalling events of the TNF receptor. TLCK,
an inhibitor of trypsin-like serine proteases, which is highly related in structure
10 and chemical ac~vity to l~CK, could not prevent ac~ on of NF-~cB at a
concentration of 2i5 ~IM (FIGURE 3c, lane 3), but only at 100 ~M (Table 1). The
` pharmacological data strongly support the requirement of proteolytic
- degradation of l,~B~ for the activation of NF-,cB. The inhibitor profile suggests
the involvement of a serine prot~ase with chymo~ypsin like speci~city, which
15 we will refer to as l,cB~ protease. i~ough it cannot be ruled out that the
protaaise inhibitors interfered with another prot001yac step located upstream
from the l,~ protease, ~is appears unlikely in view of ~e diverisity of NF-~-B
activators, which might have very few up~tream pa~ways in common.
Reac~ve oxygen intennediates (ROls) seem to play a role as messenger~ in
20 the ac~vation of NF-,.B by many inducing conditions. A potent arftioxidant
inhibitor of NF-,tB ac~vation is pyrrolidimedithiocarbanate (PDTC). No
'St
ign ficant decay of kB~ or a~tion of NF-ICB was seen after PMA stimulation
in 70V3 c~lls pr~eated wi~ 100 ~.M PDTC, (FIGURE 3d), ~uggesting ~e
. proteolysis of l~ is co~trolled by ROls. PDTC doas not int~ re with ~e
25 PMA-induc~d membrane association and kinase ac~vity of PKC, which argues
against a direct phosphorylation of l,cB~ by PKC in intact :ells.
... .
.
,
,A
, ' ~
: - ~s j,
-2
`:
~M~
NF-IcE~ ACTIV~TION IN JURKAI C!ELL~ REQlllRE$~0STlMULdT10
.` ~12
~NYOLVF~THE PHO~lO~YLAT19N ANI:) DEGRADATION~E~
5 Physiological ac~vation of T Iyrnphocytes recluires cos~mulaffon ~rough ~e
TCR/CD3 receptor and CD28 (Fraser, J.D., et~l., /mmunol. Today, 14:357-362,
1993; Unsby, P.S., et ~1., Annu. Rev. Immunol., 11:191-212, 1993; Umlauf,
S.W., et aJ., Immunol. Rev., ~3:177-197, 1993), which can be mimicked by
suboptimai PMA concentra~on~ in combir~n wi~ the en~a~ement of one
0 receptor or Ca+~ ionophora (Fraser, J.D., ~ ~1., supr~; Unsley, P.S., et ~1.,
supr~; Umbuf, S.W., et al., supra; Crabtree, G.R., SGlence, ~:355-361, 1989;
Su, B., et ~1., Cell, 77:7Z7-736, 1994). Previou3 studies indicated that NF-I~B
ac~ on is enhanced by cosbmulabon (Sen, R., et ~1., Cell, ~:921-92B, 1986;
Lenardo, M..l., et ~1., Cell, 5B:2Z7-229, 1989; Mattila, P.S., et ~1., EM80 J..
3 15 9:4425 4433, 1990; Fran~, B., et ~J., EMB0 J., ~:8~1~70, 1994).
I
The steps involved in ~e a~bon of NF~cB, include kB~ elimination through
7 the appearance of the nuclear DNA binding ac~ y, and the transcriptional
acavity. NF-~B acbvation was measured ~hrough ffie reporter ac~vi~ of ~e
HIV-LTR promoter, wnich is lar~ely controlled by NF--cB in conjunction wi~ ~e
HIV Tat protdn (Nabel, G., e~ ~1., N~ture, ~:711-713, 198n. Jurkat cslls were
1 ~timulat~d wi~ ~e fo~wing reagents: (P), PMA (phorbol-12 myrsla~ed 13-
ace~te), 10 n~/ml; (1), A23187 Ca+' ionophore, 1~M; (3), anti-CD3 (OK~3),
5~/ml; (28) an~-CD28 (monocbnal antibody 9.3), 3 ~,~lml; (P/~, PMA + Ca~+
ionophore in I~ trar~fected Jurkat cells; ~NS) non-s~mulabd (0.1%, DMSO
only). Jurkat cells were ~an~ted by electroporation (Su, B., ~t ~1., supr~)
with 1 ~g HIV-LTR-WC (Israel, S., e~ ~1., Gene, ~ 146, 1991) and 4
~,, PB~TAT. Control cultures were ~transf~d with 8 ~9 Rc-CMV-l~B~ (~abel,
. ¦ U., et 81., EMBO J., ~:201-211, 1~93). FoUowinçl 30 hours bansfec~on, ~e
:`
~ :
:
cell~ were stimulated for 8 hours and luciferase ac~ y wa~ determined using
the Promega lucif~rase assay system.
. .
~' Suboellular protein extrac~s were prepared essentialiy according to Beg, AA
et al., supra. Following 20 minutes stimubtion of Jurkat cells, cytosolic extracts
s (50 ~-g/point) were analyzed by Westem blot, and nuclear extracts (5 ~ g/point)
'~ by EMSA, as described (Henkel, T., et al., Nature, 365~ 185, 1993).
.. , .
Rabbit anti-l,cB~ was prepared against the GST~I/cB~ fusion protein by standard
methods. The specif~y of ~e antibodies was confirmed in competition assay~
using recombinant IIcB~ protein.
10 FIGURE 4 shows that neither Ca++ ionophore alone, nor ligatlon of either CD3
or CD28, induced reporter ac~ y, while PMA alone at 1 û n~/ml enhanced ~e
~i~ ` basai activity by only h~o-fold. On ~e o~er hand, a combination of PMA wW~
soluble anti-CD3 antibodies, soluble ar~CD28 a~tibodies or Ca++ ionophore,
;' enhanced reporter ac~vity by 4 to 9 ~old (FIGURE 4a). The costimulated
1s activity of the HlV-promo~er was dearly NF-ICB depender~t as attested to by the
findin~ that it was completely blocked by overexpression of ~e IIcB~ co-
~, transfected with the rqporter plasmid (FIGURE 4a, $/P/i).
, .. .
The costimula'don requirements o~ NF-,cB were also evident in an electromobilityshm assay (EMSA) (FIGURE 4b). The upper sec~on of the EMSA shows ~e
20 NF-,cB complex composed of RelA (p65) and NF-I-B (p50) (arrow). Whereas
single s~muli were barely s~ien~ to induce NF-I-B ac~vity in EMSA,
combinations of PMA wi~ ~ither Ca++ ionophore, anti~CD3 or ar~-CD28 and
costimulation with the h~o antibodies enhanceci ~e NF-ICB DNA binding ac~
10 to 20 fold over ~e level induc~d by a single stimulus (FIGURE 4b).
, ~ .
,~ ~
;,
.
~ia~ ~
-26
Parallel to NF-.eB ac~vitiy, ~e fate of l~c~ following cell treatmerlt with ~e
various stimuli wae inv~stigated. Single stimuJi which failed to activate NF-~-Bdid not affect ~e migrabon or intensity of the I~ ignal in a Westem blot
Jurkat cells were treated fer 8 minutes with the indicated stimuli a~ in FIGURE
5 4a.
!
In contrast, T cell costirnulation resulted in the reduced intensity of the l~
signal and ~e appearance of a slow migrating form of l,~ (FIGURE 4c).
Alkaline phosphatase treatment of cytosolic extracts from stimulated cells
reversed the slow migration of ~e novel I~ band, indicating it was a
10 phosphorylated form of l~ . The arrow indicates the l,cB~ non-modffied band.
A non-specific band of lower mol~ular weight appears in the bottom of the
blot and is not affected by cell stimulation.
J
? EXAMPLE 4
`~ INHlBlTlON OF l,cB~ PHOSPHORYLbTlON BLOCKSlTS
1~ 15 I~JDUCI~LE DEGRADATION
On the basis of the results in Example 3, phosphorylation of l~ , in parallel
to NF-ICB act vation, is induced according to ~he physiob~ical requirements for
T cell ac~vation. Cell stimulation and extract prepar;~tion was as describsd
i above. Jurlcat cells were treated prior to stimulation wW~ .M TPCK
(Boehringer) or 400 ng/ml CsA (Sandoz) for 30 min, or with 100 ~.M PDTC
(Sigma) for 1 hour. Duration of Jurl<at cell sbmulabon ~Ath PMA (10 ng/ml) and
Ca+~ ionophore (1~.M) was determined by Western blot analysis. I~ arrow
; indicates the position of the non rnodified kB~ and ~e * arrow, ~e pos tion of
the modified I~ fonn. Duration of Jurkat sbmulabon with PMA and Ca++
ionophore was as indicated.
?
. ' .
t~"':~'' ' , ' ' " ' , ~'''''''','`'' ~' . ~' ' '
"~
l~netic studies of the kB~ modification procsss ravealed that concotr7itant ~nththe appearance of phoeph4~ted l~cBa, the non-modified kB~ band dimir~
ished (FIGURE 5a). Upon densitometry an~, the two l~ nal~ at 7 min.
post stimulation equaled the single l~ signal from non-stimulated cells.
5 Likewise7 be~ween 7 and 9 min., the intensity of the phosphorylated l~ signal
equaled the consecu~ve signal loss of the non-modified band (FIGURE 5a).
These features show ~at the decay of bhe non-phosphorylated kB~ signal
occurs subsequent to its modifiication, and that the decay of the pho~phorylat-
ed band is due to degradation.
-~ 10 To test the hypothesis that l~sBQ degradab'on requires its prior phosphorylation,
three different reagents were used which ha~re been shown to bbck NF-/cB
ac~ivity: Tosyl-Phe cloromethylketone (TPCK) (Henk01, T., et al., supra), I
pyrrolidinedithiocarbamate (PDTC) (Henkel, T., et al., supra; Scherck, R., et al.,
J. Exp. Med., 175:1181-1194, 1992; Sun, S.C., etaJ., Science, ~:1912-1915,
1993j and cyclosporine A (CsA) (Su, B., et ~1., supra).
~1 ,
-~ TPCK, a serine protease inhibitor and PDTC a powerful antioxidant blocked
t, both the indudble modification and degrad~ition of l,cB~ completely (FIGURE
5b). Cyclosporine A, a powerful immunosuppressive drug and inhibitor of ~e
3, phosphatase calcineurin (Scluiber, S.L, CeJI, ~Q:365-3~, 1~92), abolished
-~ 20 most of ~e inducible kB phosphorylation ancl degradation (FIGURE 5b). The
phosphorylation of kB~ in cdls treated wWl CsA prior to stimub~on wa~
initiated at a lower efliciency and barely developed during the 20 min. assay.
The ~act that all three reagents, each with it3 individual mode of action, blocked
,. ~ both the induable I~cB~ modification and its degradation, indicates ~at
~ .
~ 25 phosphorylation is a prerequisite for l~ degrada~on.
~3i~7
` ,~
.
EXAII~pLE 5
l~ PHOSPHORYU~TIQN IS NC21' ~;UFFI~IENT FOR NF-ICB ACTlVATlOt~
,,
., Since the stability of I~ wae shown to be enhanced by complexing to Rsl A
(p65), it was suggested that mod~ication of kB~ could lead to it~ dissociation
~-; 5 and destabilization (Sun, S.C., et ~1., supta~ 1993; Scott, M.L, et ~1., Genes ~
~!, Dev., 7:12661Z76, 1993; Rioe, N.R., et ~I., EMBO J., ~:4685~5, 19~3;
; Chiao, P.J., et ~1., Proc. NaV. Ac~d Sci. USA, 91:2~ 1994). Once
-, di~sociation is induced through I~ phosphoryla:~on, the released Rel proteins
would be free to translocate to the nucieus and bind to the kB site. Sub~
1Q quent degradabon of the released ki-3~ wouW complement the dissociation
stsp ensuring its irreversibility. The previous Examples (1 and 2) lec to the
preliminary characteriz~tion of the l,ci3~ protease as a serine protease likely
related to chymotrypsin (Henkei, T., et ~1., supr~). A candidate cytopiasmic
.~i
-~ probase which could eliminate LcB~ following stimulation ie Ule ubiquitous 700
~5 kD multisubunH proteosome with its distinc~ive chmotrypsin-like ac~
~Vinitsky, A., et ~I., 8iochemistry, ~:9421-942~ (1992)).
:-q,
~ ~ N-acetyl-Leu-Leu-Norieucinai (AcLLnL) is a reversible non-allcylating proteosorne
"~ inhibitor not likeiy to disturb post-translation mociffica~on events. ~herefore, ~e
effect of AcLLnL on l~ proc~ssing was ~iieci. Immunoprscipitation (-iP)
j~ 20 was performed by incubabng 500 ~ cytosoî~ ~xbact in 200 ~ i Hepe~ buffer
(20 ml Jl), pH 7.6, ~ining 10 mM Ka~ prot~ and phosphatase Tnhibitors
(Su, et ~1., supra), wi~ 3 ~1 antiserum. Following 18 hours incuba~on at 0-C,
IPs were recovered from protein A Sepharose b~ads and subjected to Westsrn
r ~ blot analysis with anti-l,cB~. Anti-Rel A serum was a rabbit polyclonaJ antisenan
prepared against the C-terrninus ponion of Rel 1~ In Fl~;URE 6a, c~lls w~re
pretreated for 4 hours with 50 ~g/mî AcLLnL, stimulated wi~ PMA (10 ng/ml)
- and Ca++ ionophore (1 ~.M) for the time intervals indicated and analyzed by
Westem blot. llle arrow indioates the non-rnodi~ . AcLLnL and o~efr
,,
.
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peptide aidehydes utilized in the E~OEImples were synthesked by standard
- me~ods known in the aff't
Pretreatment of Jurkat eells with the proteosome inhibitor, Acilni_ did not
interfere with l~ phosphorylation, but complsteiy blockeci l,ti~ degradation
'f 5 (FIGURE 6a). Some variations were observeci in the ratio of the two l~cBa
formsf, with accumulation of the phffosphorylated form peaking 20 minutes after
cell stimulation. Nevertheless, the sum of the two forms remained constant, as
detennined by densitometry analysis.
f Nuciear ex~acts corresponding to the two late time points were tested for the
effsct of AcLLnL on NF-~B activity (FIGURE 6b). In FIGURE 3b, the duration
of s~mulation in ~e presence (+) or absence (-) of AcLLnL i8 indicated. In ~e
upper sec~on of the EMSA autoradiogram, the arrow indicates the NF-/~B
complex. In the presence of the pro~ase inhibitor, neariy all of the inducible
NF-~eB activity was abolished irrespective of the phosphorylation status cff I~
Hence, I,cB~ phospholylation per se is not sufficient to enable the nuclear
, ~anslocation of NF-,cB, but must be accompanied by its proteoly~c elimination.
ough not wanting to be bound by a particular tlleory, these resul~s could
3 be explained by two different models. ~he first ~ntail5 phosphsrylation
~f dependent kB~Rel dissociation and subsequent d~ilization of IIcB~, wi~
i' 20 proteolysisprevenbng l~ rea~socbtlon. If so, inacffvated cell, cytopla~mic
complexes would not be exp~ted to con~ain phoephorylated I~ and Rel A
Altematively, phosphoryla~on could tag l,cB~ hr proteolysi~. In the even~ it
should be possible to detect a transbnt complex containing Rel A and
phosphorylatsd, but intact l,cB~.
3 ~ 25 The composition of ~e Rel A complex was assesssd by immunoprecip~tation
wffll anti-Rel A serum (FIGURE 6c, lane 4) or con~ol serum (C, lane 3) followed
b
i
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:
by Westem blot analysis wi~ ar~ . FIGURE 6c shows that kB~ phosphor-
ylation doe~ not lead to its dissociation from the Rel complex. l~ 2 and 4
show immunoprecip tates (IP) with anti-P~el A serum; lane 3, IP wi~ control an~
i, cAbl serum; lanes 1 and 5 are reference points (prior to IP) corr0sponding to
the 7 minute point *om FIGURE 4a and the 30 minute point from FIGURE 6a,
` respec~rely. In the presence o~ phosphalase inhibitors, the 18 hour3
~!~ immunoprecipitation of cytoplasmic extracts from an ear~y time point post
stimulation with anti-Rel A, resulted in the recovery of equal amounts of
phosphorylated and non-phosphorylated l,~B~ in a complex with Rel A. Similar
results were obtained with another anti-Rel A serum ~/initsky, A. et ~ upra).
In the presence of AcllnL Rel A immunocomplexes from late (FIGURE ~c,
lane 2) or early post-s~mulation time-points, contained intact phosphorylated
It is therefore clear that the affinity of LcB~ to Rel A does not dlminish
~, following its induced phosphorylation. me complex behveen Rel A anci ~e
modffied l,cB~ perssts as long a~ it is not subject to the cell'~ proteoly~c
machinery (FIGURE 6, a+c).
i -
E3(AMPLE ô
INHlBlTiON OF l,c13~ DEGRADAIION BY PEPIIDE Al~PEillYDES
~i
he r~sults in Example 5 s;howed ~at the pep~e aldehyde, AcLlnL ef~
. 20 inhibited llcB degradation, therefore otner psptide aldehyd~ were exarnined
;~ for inhibition of IICB degraWon.
:.
Human HeLa S3 or Hep G2 cells were pretre~d for 4 hours wffll the indicat~d
peptide aldehyde prior to simulation with eithsr TN~ (20s~g/ml), IL-1(20 ng/ml)
or PMA (100 ~,g/ml). The cells were harvested 20 mlnute~ after hduc~on and
the exterlt of llcB degradation was sxamined via Westem blot a~si5 usin9 I~-B
~ specific antisera FK;URE 7 summarize~ the ability of related pep~de
, aldehydes to block l,tB degrada~on induced by a varie~ of ager~s which
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ac~vate NF-,cB. (+++) indicates greater than 85% inhibition, (++) indlcates
greater ~an 6096 inhibition and (-) indicates r o inhibiffon of l,cB de~rada~on
was observed.
, ,
~ Benzyloxycarbonyl-Leu-Leu-phenylalaninal (ZLLF) was the most potent inhibitor
P S of proteolysis of 1~ , followed by AcUnL and AcALLnL Substih~on of the
~ third amino acid with methionine or ar0inine fa~ed to inhibit l,cB~ de~radation.
~,
.
EICAMPL~
PEI?TIC~E ALDEHYDES INHI~IT TRANS~RIPllON F~OM NF-,cB
DEPEN~ENl~OMQTORS IN Vl~/O
,~ ,
10 The pepffde aldehyde, AcLLnLwas examined for inhibibon of transcription from
NF-,c~dependent promoters in vivo. Human Hep G2 cells were electroporat0d
(220V, 960uF) wi~i either 2X kB-LUC (J1~LUC) or RSV-WC (10~9/0.5 x 107
cell~). 22 hours after electroporated, the cells were treated withi the indicated
peptide aldehyde (200 ~.M) for 3 hours, and subsequer~ mulated wWl lNF~
15 (20ng/ml) where indicated. The cells were ~en harvested 6 hours post-
induc~on and the level of lucifera~ act~ was determined. FIGURE 8 ~hows
~e re~ults.
~, ,
This ~xperiment demons~rates ~at AcLLnL salectiYaly inhibi~s ~anscription of
~3 /~B clepsnder~ promoters in vivo. Fu~iermoir~, the reilated pe,~de aldehyde,
20 Ac~LR, had no effect on ,cB~ependerit transaiption~i activaffon.
.1 .
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~2-
25~pll~8
, DOSE RESPONSE OF NF-dS DNA 13INDING AND I~B~
DEGRADAnQN T~ E PEP~I~LDEll~m~PROTEOSC2Me
INHIBITORS Z-l.,U~:HO AND Ac-llnLCHQ
.~
5 Two pep~dde aldehydes, one with phenylaline subsff~tion (ZILF) and one wi~
~! norleudnal (~cLLnL) were ~xamined for NF,cB DNA binding in a dose response
,~ assay.
Hela cells were pretreated for 5 hours with ~e indcated concen~ation of
pep~de aldehyde inhibitor or left untreated and then stimulated with TNF~
10 (1 5ng/ml) for 20 minutes The cells ware then harvested for whob cell extractpreparation Extrac~s were analyzed for NF-~B DNA bindin~ a~y by
electrophoretic mobility shift assay (EMSA) and relative l,ci~ protein levels wsre
measured by Westem bl~t anaiysis as shown in FIGURE 9.
The resuits show that at concen~ations of 1~.M to 200 ~M, AcilnL and aLF
15 inhibited TNF~ induced NF-~.B ac~vity in a dose dependent manner. Similarly,
IKB~ degradation was mhimai in ~e presence of the psptide aldehydes.
The i~ at ~e bottom d FIGURE 9 shows ~e IC30 for aLF and AcLLnL a~
detQnnined by densitsmetric scanning of the EMSA and Westem blots. IC~
for ZLLF was 3.4 ~M and 1æ1 ~M for AcLLnL
20 Th~refore, the ultimate rob of inducible I~ phospl~ylation is, most likely, to
tag l~t~ for degradabon. A cytopbsmic protease, sudl as ~e proteosome, .-
would recognée ~e tagged I~ wi~in ~e Rel complex and degrade ~
,
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r ~
,~ The foregoin~ is meant to illustrate, but not to ~nit, the scope of the invenaon.
Indeed,~os~ of wdinary sldll in the art can readay en~sion and produce fw~er
~3 emboduner ts, based on ~e teaching~ herein, without undue experimentabon.
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