The generation of cytotoxic T cell epitopes and their generation for cancer immunotherapy Kessler, J.

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(1)The generation of cytotoxic T cell epitopes and their generation for cancer immunotherapy Kessler, J.. Citation Kessler, J. (2009, October 27). The generation of cytotoxic T cell epitopes and their generation for cancer immunotherapy. Retrieved from https://hdl.handle.net/1887/14260 Version:. Corrected Publisher’s Version. License:. Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden. Downloaded from:. https://hdl.handle.net/1887/14260. Note: To cite this publication please use the final published version (if applicable)..

(2) CHAPTER 6.

(3) 6 RETPAHC. Submitted for publication.

(4) Novel antigen-processing pathways for cytotoxic T cell recognition 1! " 1! #$ "%2! " &'! ( 4! ") *+, .1, Arnoud de Ru1, Nadine van Montfoort1! 3. 5 *$huijsen1! ( * $6! 7 8) 6! :4!

(5) ; <)1! => ? "7! . " B$%7! $ =C, Birgitta Tomkinson9! F>

(6) G10! = ( I2! ;) 3 : )4! > * 4! 3 ;JK >1, = ,1#! ? B) 1#! ( (% 1#. 1 ; % ..> . ) * ) 7%> ! ) # () ! )! 7.

(7) ) 2 > %O * .+P! () ?> % >.8 ) # *! *! &. ' = ;" : ! (> & ! ) () " ! * ! #" 4 ; % ( > ) > * .! ) % ">> * ! # % >$! < ! #" 6 : # > % ">)! # % *..! *..! #) ) . 6 ; % B ! ) # () ! )! 7

(8) ) 7 ) > % .! ) #! )! 7

(9) ) C Laboratory % * .

(10) > ) ! : > % ( S:(U! ( ! ) 9 ; % () * . ) ( 8 ! # #! #! ") 10 Division of Cell * ! 7

(11) ) >! .).! 7

(12) ) # 8>) F> >). ABSTRACT < 7 . S7U $ > % . 8 + ) ) ) 8 . > >% ? 8) molecules these peptides need to have an appropriate C-terminal anchor residue, which is classi ) 8 . .< )% ) ) .. .

(13) ) 8> +. 8>

(14) +. % ) ) 7. ) S7B=U +. .. ) ) 3 )X .. 8 ) )> +.> % >. +X 7 7 % ) ) 7B= 8 ) ) 8 + functional protein degradation products into class I ligands, thereby strengthening the immune )% > ) .

(15) .

(16) INTRODUCTION " ) SC U ) 8 class I molecules, called CTL epitopes when I) 8 ;C+ cytotoxic T lymphocytes S7U! )>) )> > > turnover in the cytosol of full length and misfolded proteins into peptides and eventu % . ) [+'\ 7 )) process is accomplished by the multicatalytic proteasome complex and by cytosolic pepti) [!'\ 7) ) S7==U []\! 7B= [6! \ ) . ) [!\ 8 implicated in degradation, but the contribu % S) U) .) > % .) )) )> S C U %> cytosolic destruction through translocation 7= ) . >>. S5:U! 8 ..)

(17) +. 8 5:=^5:= [!\ ) .8 ). 8% 8 >) >% The absence of C-terminal excision of CTL epi .+8) [_!C\! gether with a failure to detect C-terminal trim. 5:! ) the current notion that solely the proteasome 8 < +.> [!\ endopeptidases, however, may also produce ) +.> X % 8)ing from protein degradation products lack > ))! ) . 8 < X % )+ )) . > [`+'\ > . >) $ to not inhibit completely, this suggests the existence of a partially proteasome-independent % ) " % 7== ) )> +.> % )X) 7 S% . ,

(18) %U []\! 8> 8 ) % 7== % ) )) >$ [6\. Other peptidases liberating the C-terminus of ) $ 7 )X such peptidases, we began an in-depth investigation of the generation of a CTL epitope with an unambiguously proteasome-independent +.>. Cleavage C-terminally to a lysine is not readily .) 8 . [ \ ! < % +'! . > that binds peptides with a C-terminal lysine, is particularly insensitive to proteasome in8 [`!\ 7% ! ) % CTL epitope with a proteasome-independent +.> ) +' 7 . 5?"z5 S=:`{+`C) from tumor ) =:(5 [_\ ) by binding prediction and bound with high } +' S"> ? U ; % _+. =:C+{C >X) {" somes showed no cleavage after the epitope’s +. +`C ~$ +.> !

(19) +.> 8) 8 } . 8% &>+`{ S? U 7). . . S("^("U )X) 5?"z5 ) ) >) % . +' >% % 6 +' S 6 ! =:(5+! %) +'U S"> ? U ! 7 ) =:`{+`C epitope exogenously )) +'! } I) 6 +' ) >. < =:(5 ) +' S? *U X. > % 7 7 recognized the exogenously loaded 11-, 12- and '+. +. <)) % S5?"z5 , ^:^ U! 8> ) +' 8) } S"> ? U > ) S"> ? U 3 >) %> % 7.

(20) .

(21) 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208. A. FLKEGACDELFSYLIEKVKRKKNVLRL C K562-A3 MZ1257 94.15 K562-A2. 75. IFNγ prod. (pg/ml). % Specific lysis. B. 50. 25. 30000. 9-m er c-prot. 20000. i-prot. m ock. 10000. 0. 0 0. 25. ET ratio. 50. 1 μM. 200 nM. Digest concentration loaded. Figure 1. PRA!"!#

(22) $%%&'

(23) ( ' ' ) *' +

(24) '

(25)

(26) ( terminus. = . ) % _+. =:C+{C S5?"z5 ! 8 )U ; S >8 U 8 ..> + > >. {" . .8 > * ) ) %X 8% &>+`{ ?. +C` +.> .. %. ))) > _ % )) . S )U ).) 8 . . ; ) . . > * : 8 7 +5?"z5 % >$. 6 +' ) . S:U (6_ ) `]6 S =:(5+ ) +'+U 6 + S++U I) 8 % % 6+. =:190-214 S5?"z5 , :

(27) ,: !

(28) + .>U )) % ..> + > . S"5( % U ; )! )) 5 : S+'+U ) +>8) 7 +5?"z5 *$ >) 6+. >8 >8) '_ > I. S. $ )U! F> ) 5?"z5 ) S`+.U. to monitor the generation of the epitope and its C-terminally extended variants in digests % =:(5 ) 7 . ) % 6+. =:190-214 S5?"z5

(29) +.>U )) =:(5+ +'+ cells were not recognized by the CTL S? U! X. .+)) % +.>.

(30)

(31)

(32)

(33) 7% ! ) % )SU +.> % 5?"z5 ?! 7 8) 6 +' ) with the metallopeptidase inhibitor o-phenanthroline, whereas other inhibitors had no S? U " 7== 8 8>-. 8)) 8 ) 8 >. []\! further excluded the potential involvement of 7== []!C\ 8 ) ) :

(34) % S:

(35) U S"> ? 'U " )! ) )% )! < ) F>) ~> 6+. >8 , :S+?U

(36) S=:190-204, epitope in bold) to digestion % S) 8 < . U % 6 +' ? '_ )! + % ! 8 F> )8 +`6 ) ~> S?U > {! 8 ~> S? *U 7 % ) ) ) . X )X) 8 ("^(" S? *! "> ? ]U

(37) )-.

(38) '.

(39) B fluorescence (OD). 100. % CTL recognition. Fr. 37 1200. 75 50 25. 900. 2500. FL-pep. + Phen. % KCl. 2000 1500. 600 1000 500 0 25 27 29 31 33 35 37 39 41 43 45 47. u + ntre Br a + efe ted Bu ld ta in bi A n + Ph did e + ena Le n t u h + pep . C a tin + pto C a pri + lpep l Ap ti ro n t + inin In su lin. C. 5 4 3 2 1. 300 0. 0. mM KCl. A. Fraction number. 1 2 3. D. Western NRD. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25. ELFSYLIEKVKRKKNVLRLCCKKLK IFNγ prod. (pg/ml). 6000 4000 2000 0 200 nM. 40 nM. 8 nM. Digest concentration loaded. siRNA siRNA control NRD. IFNγ prod. (pg/ml). m ock dig. c-proteas. dig. i-proteas. dig. nardilysin dig.. 8000. 3000 2500 2000 1500 1000 500 0 si-Control si-NRD. K562-A3. 900 600 300 0 si-Control si-NRD. HeLa-A3. Figure 2. Nardilysin produces C-terminal extended precursors of the PRA!"!# epitope. 6 +' ) ) >) S{{ U ) )) 8 S ) ! . ) U ) +>8) 7 S"5( % <.U * % >) % . < >. S +) )) ( ) % )U % 6 +' +>8) % '{ . ~> >8 5?"zS+)8U 5 , :S+?U

(40) 8 SF> .8 U SU % S .(U 7 . >8 X 8 ;+ 8 $

(41) +. .. ) )>) ~> S) U ? '_ ) 8 ";"+=&5 ) 8) ) = )X) 8 ("^(" S8) U . 8 ! SU $! S'U + +>8>! S]U . ) S6U ) S "> ? ]U ; % 6+. =:190-214 S ! 8 )U >X) )! ..> + . > . % '{ .

(42) ) } 8) +. ''` % ) +%. S U! 8) +. ] % %. S U ) 8) '+. ` % %. S 'U ; S)! .! . $U )! )) +'+ cells and co-incubated with the CTL to assess gen % +> ; 7 % 6 +' ) +' 8 %) < :

(43) > ) S

(44) :;U + :

(45) S"5( % % <.U ..> 8 ) 8 ) % 6 +' % . ]

(46) .

(47) lysin, hitherto not implicated in class I antigen processing, is a cytosolic endopeptidase of the pitrilysin family of zinc-metalloproteases and >8F> > <) S % ` ) #& )8 ^^8. ^#&^5"7= X,> 6C]_CU 7)! ) % 6+. =:190-214 S , :

(48) ,: U >X) ) ) } )> % +. <)) +! + ) '+. % 8 } % +{{! +{ ) +{ S? U! ) X of nardilysin before or between two basic )> [{!\ >8 > '{ ))! 7 } I) the nardilysin-digest, due to the presence of 5?"z5 , ^:^ %.! the proteasomal digest was not recognized S? U ? >! ) F>) cells for epitope generation because small in8 :

(49) S:

(50) U+.)) > % ) 6 +' ) +' S! =:(5+! +'U )>) 8 7 S? ;U! ) < % ) +' ) 7 S"> ? 6U 7 ! X) . ) indispensable for the endogenous C-terminal + % =:`{+`C epitope by producing cleavages after Lys-200, Arg-201 and Lys-202, but it does not excise the exact C.> S+`CU.

(51)

(52) *+ ,' '

(53) < >>) )X % dase that generates the correct C-terminus of *> % ! >8F> > < ) X! . )) 7B=! > ) >+)) I. B :

(54) +. .)) > % 7B= 6 +' strongly reduced CTL recognition, whereas suppression of the other peptidases had no S? 'U! ) 7B= . ) > % 5?z"5 ( ! >X) 7B= ) } directly behind the epitope’s C-terminal Lys`C )+)) + ) '+. > S=:190-201/202 5?"z5 , :^ U 7>! 7B= ) % > +. residues, in accordance with its cleavage pref [!'\! 8 } )> < . S? '*U ))! 7B=+) % +. =:190-201 loaded on I) . } .. S? 'U its preference for peptides 6 to 17 aa in length []\! ) ) 8 7B= S"> ? *! ) ) U! )ing that pre-processing by nardilysin into the +. ) '+. F>) .$ + > >8 7B= 7 X.) +) % 6+. =:190-214 ) ) 7B=! >) )> % 5?"z5 S)X) 8 . .! ) U )! ) >8+) I) . }ciently by the CTL – due to the presence of the epitope – than the digest with nardilysin alone ) > S"> ? *U 7>! +.> % 5?"z5 ) > F> 8 ) ) 7B= Generation of the N-terminus of the PRA!"!#-epitope The N-terminus of the epitope is generated by } +$ . 8% &>+`{ S? U 7 . 8 $) 8 X 8 =

(55) >,"B [6\ % +$ proteasome activity as shown in a digestion of.

(56) 6.

(57) % CTL recognition. A 100. 75 50. 75 50 25 0 si-Control si-TOP K562-A3 (si-stable). 0. Western TOP. -C on tro l + si + -T si -N OP eu ro ly s. + si -ID E K5 62 -A 2. 25. +. si. % CTL recognition. 100. B 1. 2. 3. 4. 5. 6. 7 8. 9 10 11 12 13. ELFSYLIEKVKRK ELFSYLIEKVKR ELFSYLIEKVK IFNγ prod. (pg/ml). C 5000 4000. siRNA Control. siRNA TOP. 9-mer 25% 11.1% 0.8%. 12-m er, m ock dig. 12-m er, 10 m in dig. 12-m er, 1 h dig. 9-m er. 3000 2000 1000 0. 16 nM 3.2 nM 640 pM Digest concentration loaded. _+. =:_+`C S"> ? _!*U ))! CTL recognition of cells treated with this in8 )>) S"> ? _U The general proteasome inhibitor epoxomycin only partially inhibited cleavage before the

(58) +.> S"> ? _!*U ! when cells were treated with epoxomycin the % 5?"z5 .$) ) S"> ? _U! $ )> )>) . < .+ + ' ) TAP-dependence of PRA!"!# Thus, together, the proteasome, nardilysin and 7B= )> .. S. "> ? CU <)! presentation of the epitope was completely. Figure 3. TOP produces the C-terminus !# * ' ' / % 7 r % 6 +' siently transfected with pools of siRNA )>< > 7B=! > ) > )) I. S;5U + :

(59) S{{ % U : 7 % 6 +' 8 %) < :

(60) > 7B= + :

(61) "5( % % > <. ..> 8 7B= 8 ) % 8 %) 6 +' * ; % '+! + ) +. >8 =:190-202/201/200 S5?"z5 , ^:^ ! 8 )U >X) 7B= >8 * ) ) %X 8) +`C ? . >8 % %. ) S>) +)) >8U . 5?"z5 + % '{ . ) ) 7 % +. =:190-201 S5?"z5 , :U )) 8 7B= % { . 7 ) ) ) )) +'+ 5 : 7 +)) +. S. $ )U I) : % F> ) 5?"z5 ) S`+.U %. )) 5: 8 7= S"> ? Ù 7 5?"z5 ) } ) 8 7= 5:! +! ) '+. +> } ) S"> ? `*U 7% ! )+ )>) cursors are available in the cytosol for process 8 7B=! .. % its production – can rapidly escape from cyto )> 8 5: General roles of nardilysin and TOP in class I antigen processing 7 X ) . ) ) 7B= 7 )> ; these enzymes, either apart or together, have a . .

(62) .

(63) Nardilysin in principle can support the generation of both the C-terminus and the N-termi> % S"> ? C*U 8 % matics analysis of all 1620 published ligands of the most prevalent class I molecules with their ~$ ) )8 . % % ) [{!\ )

(64) +.> +.> 6 % ) S78 U 7 . % %F> ) S8U +.> % ) ) 8 +' > S+'! +! + CU )

(65) +.> % +*_ ) S78 ) U 3 <.) >X) ) > <) + ' ) +*_ ) . % ))! +. % +'+) overlapping immunodominant epitopes from ,+ & _ [ \ } )>) S? ]U . %) ) S) U . ! '6 % +*_ ) 8 )> S=U < ) . 8) + =! > ) + S78 ) U

(66) ) } ) ) 8%

(67) +.> % > % +*_ ligands tested and in three cases produced the epitope’s C-terminus; among these are immu ) . % . ,+ ] ) 5*

(68) ' [_\ S? ]*!U

(69) ! % > % ) )X) > be proteasome-independent as studied with . 8 S )U ['\ To demonstrate nardilysin-dependence of these epitopes during processing in cells, ) >) 8 :

(70) 5*

(71) '+ > )>) 8 7 + *_{6+) 5*

(72) '6C+. S::z;5U [_\ S? ];U 5) > of the minimal epitope with its N-terminal. ~$ X.) )+)pendence to reside in the N-terminal epitope S? ];U 7>! > . ) < ) 5:! ) F>) %

(73) +. 8 % 5*

(74) '6C+. B> sults help to explain the strong over-represen % ) = +*_ )! residues that contribute at that position only modestly to binding capacity but enforce pep) 8 [C\3 )sin cleaves before a basic doublet, it may also liberate ligands with a non-basic C-terminus ) 8 +! +! +*C ) + *'6 S"> ? C*U )) ! )8 motif is often present within 4 aa of the N- or +.> S78 U! > .>+ .. % + )> 7 . % is present directly at or within 4 aa of the N- or +.> % { ) I) S78 U *> ) .. 8% . +8 [\ S ? ] "?; + U! ~> . 8 ) )8 . %

(75) ) . ) 8) ) vaccine design, the deliberate insertion of the nardilysin cleavage motif between CTL epit + F> . 8 >%> . } 7B= 8 )) ) [ \! 8 . [6\ ) F> < % 5?"z5 8 7B= < 7 %) % 7B= % X +. )> [!'\ 8 ) F> X []!`\ % . >8 > _ []\! ) 7B= with potential C-terminus generating capac ?>. ! >)X) + . % 7B= 8 ) ['{\.

(76) _.

(77) A. LSGGELDRWEKIRLRPGG KKK YKLKHIVWA KIRLRPGG K (aa. 18-26) RLRPGG KKK (aa. 20-28) MADKPDMGEI ASFDKAKL K K TETQEKNTLP. B GRA KRR MQYN RR FVNVVPTFG KKK GPNANS SIMKWNRE RRR LQIEDFEAR IALLPLLQAE GPPPPHYEG RR MGPPVGGH R R GPSRYGPQY GYHWSLMERD RR ISGVDRYY VSKGLENID RL RR SRTPL RRR FSRSPI RRKR SRSSERGR FASLRMARAN ARLFGIRA K R AKEAAEQDVE GYRIMY KKR T KR LVVFDAR (-Ct) NPPIPVGEIY KR WIILGLNK. C. epitope prod. 10 min 30 min. RFLRGKWQ RR Y RR IYDLIEL. 35.8%. 62.5%. IFNγ prod. (pg/ml). D 4500 3000 1500 0 si-Control si-NRD. B-LCL. si-Control si-NRD. K562-B27-mini. 7% ! ) ~<8 % 7B= )> ) 8 . >8> S= ) =U > >) % 7B= +.> % 5?"z5 =+>8> ) }! 8> < % = = vented digestion, production of the epitope S5?"z5 U 6 % { . % ) S"> ? {U = )> 8 >8F> .)) % occurred, but did not prevent epitope-produc S"> ? {U 8 } 7 % ) S"> ? {*U ; % =+>8>) !. ,0 4/*

(78)

(79)

(80) $% antigen processing. Digestions b >X) ) % +'! + ) +*_ ) ) * ) )> > )+ . % ; . '{ . * ) ! 6 % ) +%. ! 6 % %. ; 8 >X) ) % S>U '{+. ,+ _ C+'_ . +'+) 7 S ))! >))U ) S U '{+. . + +'{ . ++) ) "?; S>))U 7 <. $ % . "z?=57 )8 S%)U * ; 8 >X) ) S'{ .U % long peptides encompassing eight published +*_{6 ) ) S>))U % . "z?=57 )8 > ~$ ) S 8 .U ]{" 8 . "'{ ]{+6 )+> &:(` 6_+ _ :8 > '+'

(81) ;+>8F> <) )> 6+ " ' ;

(82) +8) *"" { + {'] {" 8 . ' CC+` ;

(83) + directed RNA polymerases 7 kDa polypeptide 6{+6C ) ,+ ] 6+_]! is an immunodominant CTL epitope presented +*_{6 S :3&

(84) U )J +*'6 ) 7

(85) ===,&5z S % )U ; 8 >X) ) % {+. :?:& 3::z::z;5 S5*

(86) ' ] + . ! +*_{6 ) >))U = )> % % { ) '{ . >8 )) ; % 8 7 +::z;5 % > > *+ S+*_{6+) express 5*

(87) '! 8 < :

(88) > ) :

(89) : 7 % 6 +*_+. ! 6 < +*_{6 ) . (?:& 3::z::z;5 S5*

(90) ' ]_+. ! epitope underlined), with or without suppressed ) 8 8 :

(91) < . C

(92) .

(93) on the other hand, resulted in generation of S5?"z5XU = S) F>U! &> S"> ? {U! 8> % )> 7 >) 8 } % 5?"z5X % . =+)> S ,! ! U )^ 8 ) } .)) % S% =! 7! ! 7U 7 % ! &> =+ ! ! 8 > 3 ) +F> .! 7B= )>) +. > S"z"?=5=5 ) ,;&;, [_\U S"> ? {U 7 ) ) 7B= )> from a wide array of C-terminally extended > 7% ! 7B= cessing is likely balanced between its anti-epit ) + ) limiting presentation – by partial destruction – of epitopes whose correct C-terminus has ) 8 .) 8 SU . S$ "

(94) ?5 [6!_\U! )! )! )> +. X % binding by trimming C-terminally extended > $ > Importantly, this new model is in accordance with a recent analysis of cytosolic substrates ) )> % 7B=! ). 7B= both destroys and generates peptides of the % ) ['\ 7 >) showed, in line with our results, that many 7B=+ )> +.>. % ) ) 7B=! '{ ) 17 aa respectively, prior to their action, protein hydrolysis by the proteasome or other . $ F>) . 7 % ) )) ) ) 7B= )X ).)! 8> > ) ) that both enzymes are capable of shaping and broadening the antigenic peptide repertoire, thereby expanding the options for successful immune responses to intracellular pathogens ) . The general implication of our study is that )) )> X for antigenic presentation due to lack of an appropriate C-terminal anchor residue can 8 ) 8 ) )^ 7B= > 8) F>. % . > *> % >8

(95) `.

(96) References . . ' . ] . 6 . . _ . C . ` . { . . : $ ! z $ ! & )8 = + . antigen processing for major histocompatibility .<

(97) ..> 6 _{+. __! {{] "

(98) ! )>) "! "8 ":! " ) 7! > ) X 8! .))! ) ) % ( + ..> : {_'+ ]! {{6 z) 3 =>.8 > % ) > ( ) ) > B ..> `_`+C ! {{_ : 5!

(99) J ! 8 ! *$>J 3! ! ;JK > 3!

(100) G .J % 7== .. . )) )> % ( ..> {]`6+6{ ! {{] z $ ! ( ! . ! 3! " z! 8. :! " 7! & )8 ! : $ 7 cytosolic endopeptidase, thimet oligopeptidase, destroys antigenic peptides and limits the extent % ( ..> C]`+]]{! {{' " 7! &% ! & )8 = % )) % ) ) 8 . a key role for thimet oligopeptidase and other . ) * . _`] _'+] _'! {{] > ! $ 7! & )8 ! : $ 7 distinct proteolytic processes in the generation of a major histocompatibility complex class I-pre) ) =

(101) ) " # " `]{C6{+ {C66! ``_ " I ! ;$ 7=! ; (! = .. *! :.. &! ") & % > stomatitis virus nucleoprotein cytotoxic T lympho F> .+)) ) +)) 5> ..> C]{`+]{' ! ``C *. (! & .. (!

(102) G )%ferences in the relationship between proteasome ) ( ) ) ..> C'+C`! ``C >$ ! &(! ( ! ,$ "! ( *?! I ,! 7! "8 I ! > ;?! 5) , = . ) ( ) for nonproteasomal epitope generation in the ..> +! ``C "I ! ) &> :! ".)$ &! $ "! ; *(! . *! (! % :! & > ( 7 . 8 and epoxomicin can be used to either up- or down> < ) ..> ] ]_+ 6_! {{{. >$ ! ( ! =) (! 5! 3 ?(! ;! "8 I ! > ;?! 5) , ; < % >. ( ) ) ) % . 8 ..> _+! {{ ' ( (! ! I ) = .+)) +*_ ) . % . . 8 ( = . `'! {{_ ] "% #! ( ! ".> ! ; > ?! 3 $ ! $ ! $ =! ; "! )> (! ) "! 3$ 7! ") ! ) ;! & ! &! I =(! : z! . % ) ) % (

(103) ..> ]'_6+'_`! {{' 6 5) = : % ) ) ( ) % 5> ..> 'C {`+ '! {{C 7I "! = *! *>$ "! " B! .. ! "I ((! I =(! :.. &! ") ! I> & ( ) ( pathway by combining predictions of proteasomal ! 7= ) ( 8) ( % " {6+{'_! {{6 _ ! *$.

(104) ! *+, . "! ,)J$ =! , =! .+ (! , ;! * &! (&! "J ! ;JK > 3! B) ?! B :! (% ( 5} )X % +SU{{+ presented cytotoxic T lymphocyte epitopes in ) <) >. =:(5 8 .+.)) ) 5< () `'_'+CC! {{ C & 5! =%% &! 3. (! >+I (! *>. 3! 5. !

(105) ). & ant protease with potential to substitute for some %> % . " C'`_C+`C! ``` ` ?>. =! (! 5 ! "II =! :II &! =II> ! ,! ". ! &! >) B! B "! 7. : >.

(106) :; ) . ) ) <) X )> ) . ) )> > & . ]_'C+]6! ``C { (! > 5! > (! " =$ ! > ! * ">) >8 X % ) S

(107) + )8 U * . _6`6]6+`66! {{{ (! B$ B! & ). ! ( ! > (! > ! *

(108) ) ) . 8 * . ]'`+ ]]! {{'. '{

(109) .

(110) . ' . ] . 6 . . _ . C . ` . '{ . ' . &! ;) =(! * 7. ) X ) % % cleavage near the C-terminus and product inhibi % . $ % ) ) * . '{C S = U]6+6{! ``6 B ,! & :! : ,! ? 5"! " ! . ! > (! > 7.> ) ) % combinant metallooligopeptidases neurolysin and . ) 5> * . `]' + ]'']! {{ B ,! . (! ( :! ? 5"! . ! > (! > ">8 X characterization of recombinant metallo oligo) . ) ) > * . ]{]]_+]]6! {{ " =?! ".> 5! )I :B! den Nieuwendijk AM, Leeuwenburgh MA, van der ( &! *(! B$% "! ? +.8 8 ) +8) probe for the caspase-like activity of the protea . * () . _']{+']{6! {{_ %) (! % 57! z! "$ ! %) (! (

(111) ! *>

(112) ! "I (5! z ! &! z> &! ( ! : $ ! 7(! ! : 8 5"! (! 3$ *; ) ;) = ;" 8> " ;CSU 7 : ,+ = " () ']{'! {{ * $ (! (> :! 7 . 3! > (&! :$ * ; +*_ >8 . ..> ) . 5+* > ) 5< () _CC_`+CC_! ``' 8 !

(113) J ! ! ! ;J% > 3! : 5!

(114) G > 5) + *_ F> (

(115) +7. ;8 =) > =) "8 = ..> _ `_+_{! {{ ". ! = 7! 78 ,! ;! &>$. (! 3 % ?<8 >8 recognition by thimet oligopeptidase as revealed 8 )> >) * . 'CC66+ ! {{6 " ! = ?! * ,! ) . ! ? 5" 7. ) S5 ']]6U! > % ( * . * : ..> 666`+6`6! ``` * ;! ( ! :> ! (! > ?(! ! " ! I$ ?! ? 5"! ?$ ; % > >8 ) )> % . ) S5 ']]6U >. .8 $) `' * . C]]{6+] ! {{`.

(116) '.

(117) %5 TABLE 1. Proportion of HLA class I ligands potentially excised by nardilysin. HLA class I. Ligands. a. b. C-dir. (%). c. C-ind. (%). d. N-dir. (%). e. N-ind. (%). f. Total (%). HLA-A1. 86. 5.8. 7.0. 0.0. 3.5. 16.3. HLA-A2. 515. 1.7. 5.0. 2.5. 4.5. 13.7. HLA-A3. 121. 10.7. 5.8. 4.1. 5.0. 25.6. HLA-A11. 71. 11.3. 2.8. 1.4. 7.0. 22.5. 46 (238). 21.7 13.0. 17.4 7.1. 2.2 2.9. 0.0 4.6. 41.3 27.6. 51. 2.0. 3.9. 5.9. 9.8. 21.6 16.2. HLA-A68 g Cumulative A3-type HLA-A24 HLA-B7. 68. 2.9. 7.4. 1.5. 4.4. HLA-B8. 52. 0.0. 5.8. 0.0. 3.8. 9.6. HLA-B2701. 8. 0.0. 12.5. 37.5. 25.0. 75.0. HLA-B2702. 18. 0.0. 5.6. 61.1. 5.6. 72.3. HLA-B2703. 29. 3.4. 3.4. 62.1. 0.0. 68.9. HLA-B2704. 52. 5.8. 3.8. 38.5. 3.8. 51.9. HLA-B2705. 185. 7.6. 5.9. 31.4. 4.3. 49.2. HLA-B2706. 38. 5.3. 7.9. 26.3. 10.5. 50.0. HLA-B2707. 3. 0.0. 0.0. 66.7. 0.0. 66.7. 56 (389). 1.8 5.4. 7.1 5.9. 26.8 35.2. 3.6 4.9. 39.3 51.4. HLA-B2709 h Cumulative B27 HLA-B35. 11. 9.1. 9.1. 9.1. 9.1. 36.4. HLA-B3501. 20. 0.0. 5.0. 0.0. 5.0. 10.0. HLA-B44. 53. 3.8. 1.9. 1.9. 15.1. 22.7. HLA-B60 (B4001). 34. 2.9. 5.9. 0.0. 8.8. 17.6. HLA-B61 (B4002). 7. 0.0. 14.3. 0.0. 0.0. 14.3. HLA-B62 (B1501). 96. 4.2. 5.2. 5.2. 5.2. 19.8. 1620. 4.8. 5.7. 10.4. 5.2. 26.1. Cumulative a. Number of ligands per class I molecule. For each allele, all ligands in the SYFPEITHI database were analysed for presence of NRD-motifs in the ligand and its N-term. and C-term. ~anking regions.. b c d c f g h. C-dir.; NRD-motif present leading to direct production of the C-terminus. C-ind.; indirect production of C-terminus: NRD-motif present within 4 aa of the C-terminus. N-dir.; NRD-motif present leading to direct production of the N-terminus. N-ind.; indirect production of N-terminus: NRD-motif present within 4 aa of the N-terminus. Percentage of ligands per allele that may be either N-term. or C-term. dependent on NRD cleavage. Cumulative percentages of NRD-motif containing ligands for HLA-A3, HLA-A11 and HLA-A68. Cumulative percentages of NRD-motif containing ligands for HLA-B27 subtypes.. '

(118) .

(119) TABLE 2. HLA-B27 ligands and nardilysin-cleavage motifs, exempliXed for HLA-B2703. Ligands in flanking regions and dibasic-motifs. a. Source protein and position of ligand. KLQEEERERRDNYVPEVSALDQEIIE. 40S ribosomal protein S17 aa.79-87. VAEVDKVTGRFNGQFKTYAICGAIRR. 40S ribosomal protein S21 aa.44-53. GLIKLVSKHRAQVIYTRNTKGGDAPA. 40S ribosomal protein S25 aa.103-111. KNYDPQKDKRFSGTVRLKSTPRPKFS. 60S ribosomal protein L10A (Csa-19) aa.46-54. NNLKARNSFRYNGLIHRKTVGVEPAA. 60S ribosomal protein L28 aa.37-45. LFESWCTDKRNGVIIAGYCVEGTLAK. Cleavage and polyadenylation specif. Fact. 73kDa aa.348-357. AIRPQIDLKRYAVPSAGLRLFALHAS. DNA dep. protein kinase catal. subunit Q13 aa.263-272. DIPHALREKRTTVVAQLKQLQAETEP. EIF-3 protein 6 aa.73-81. IPDVITYLSRLRNQSVFNFCAIPQVM. Farnesyl-diphosphate farneysltransferase 1 aa.278-286. VDLVRFTEKRYKSIVKYKTAFYSFYL. Farnesyl pyrophosphate synthetase aa.191-199. LEKIIQVGNRIKFVIKRPELLTHSTT. General transcription factor II aa.532-540. GTVALREIRRYQKSTELLIRKLPFQR. Histone H3.3 aa.52-60. EVECATQLRRFGDKLNFRQKLLNLIS. Immediate early response protein APR aa.30-38. VGEKIALSRRVEKHWRLIGWGQIRRG. Initiation factor eIF-2 gamma aa.444-452. VHFKDSQNKRIDIIHNLKLDRTYTGL. KIAA1197 aa.399-407. IWQLSSSLKRFDDKYTLKLTFISGRT. Microsomal sign. peptidase 25kDa Su aa.164-172. HWSLMERDRRISGVDRYYVSKGLENI. NADH-ubiquinone oxidoreductase aa.52-60. SPDDKYSRHRITIKKRFKVLMTQQPR. Nop10p aa.44-52. DFDWNLKHGRVFIIKSYSEDDIHRSI. Ny-Ren antigen aa.410-418. PLLLTEEEKRTLIAEGYPIPTKLPLT. OASIS protein aa.267-275. MLSTILYSRRFFPYYVYNIIGGLDEE. Proteasome Su C5 aa.99-107. VKGPRGTLRRDFNHINVELSLLGKKK. Ribosomal protein L9 aa.35-43. SLVKGGLCRRVLVQVSYAIGVSHPLS. S-adenosylmethionine synthetase gamma aa.312-320. RGDSVIVVLRNPLIAGK ( C-term. protein). Small nuclear ribonucleoprotein SM D2 aa.110-118. KDSKTDRLKRFGPNVPALLEAIDDAY. SMC6 protein aa.478-487. PRLAILYAKRASVFVKLQKPNAAIRD. ST13-like tumor suppressor aa.149-157. KLEAINELIRFDHIYTKPLVLEIPSE. XBP1 aa.281-289. a. All HLA-B2703 ligands in the SYFPEITHI database (at: www.syfpeithi.de) are listed and were analysed for the presence of dibasic nardilysin cleavage motifs. Ligand is underlined and nardilysin cleavage motifs are printed bold..

(120) ''.

(121) 6678% 9 A % Competition. 75. ELFSYLIEK. (9-mer). ELFSYLIEKV (10-mer) ELFSYLIEKVK (11-mer) ELFSYLIEKVKR (12-mer) ELFSYLIEKVKRK (13-mer). 50. 25. 0 0 2.5. 5 7.5 10 12.5 15 17.5 20 22.5 25. Peptide concentration (μM). B ELFSYLIEK ELASYLIEK ELFAYLIEK ELFRYLIEK ELFSALIEK ELFSYAIEK ELFSYLAEK ELFSYLIAK. C. CTL recognition ++ +/++ ++ ++ 9-m er 11-m er 12-m er 13-m er. IFNJ prod. (pg/ml). 25000 20000 15000 10000 5000 0 200 nM. 40 nM. 8 nM. 1.6 nM. Peptide concentration loaded. 0''/,0 /$%%&

(122)

(123) ,

(124) ( ,

(125)

(126) * ' '

(127) C-terminally extended precursors. A *) } % +' % . S=:`{+`CU 5?"z5 ) +. ) {+! +! + ) '+. ).) . +8) 8) ! {+. 8) )> $ % +. +' 8) B + )% 7 S7:U+ )> 5?"z5 ) ">8> + % 5?"z5 )! . ) ) 8 ! )) *+ 5 : S+'+) and co-incubated with the CTL clone that was raised against the exogenously )) 5?"z5 ) 7 % )) SU! S^+U S+U S )) 8) +' ) U 7 > . 192=+"+7194 F> S)) 8 <U F>) % 7: ! % +. <)) % 5?"z5 8 7 C 7 % +. <)) % 5?"z5 ) 7 +.! +. ) '+. =:190-200/201/202 S5?"z5 , ^:^ U ) ) )) *+ 5 : S+'+U 7 +>8) 7 % ] ) ?

(128) ¤+ )> .>). ']

(129) .

(130) B. 1.00. 100. 0.75. 75. 0.50. 50. 0.25. 25. 0.00. 0. Percent B. OD 214 nm. A. IFNJ prod. (pg/ml). 1500. 1000. 500. 0. C. 1.00. 100. 0.75. 75. 0.50. 50. 11-m er 12/13-m er. 0.25. 25. 3 6 8 11 14 16 19 22 24 27 30 32 35 38 40 43 46 48 51 54 56 59. 0.00. Percent B. OD 214 nm. 9-m er. 0. Fraction number (1-60). D. 130 243.1 390.2 477.2 640.3 753.4 866.5 955.5. -. L. F. S. Y. L. I. E. -. K. bn. 1012.6 899.5 752.4 665.4 502.3 389.2 276.1 147.1 yn *. Relative intenstity. E. y4. y5 y6. s. 0''/,0 :/

(131)

(132) PRA!"!#$%%&'

(133) ' ' / A =+ X % +' ) ) >X) % . 6 +' S¥{10U +' . > ..> } >X) % . ! ) )+>) peptides were separated in 60 fractions by + =! ) S))U % ' ]{ 8> * ' . S% <! B;] <! *U B : 8 7 +5?"z5 %. { =+% )) *+ 5 : S+'+) to detect the fraction containing >) )+ ? I) 7 C =+ X % % .<> % `+. 5?"z5 peptide and its C-terminal extended 11-, +! ) '+. 5?"z5 , ^:^ S=:190-200/201/202U 7 ducted identical to the separation of cell >% <) ) S U 7 `+.! +.! +. ) '+. ) >) % ! `! ) '! S X.) 8 . . U D 7 ("^(" >. % .Î 6_' SU present in fraction 21 of the eluted peptides )X) 5?"z5 . 8+ + >)) 7 ("^(" >. of the eluted peptide was identical to the ("^(" >. % 5?"z5 ) S U =$ )) " >) 8 <) 8 > peptide fragment-ions, however, were also ("^(" >. % ) 7 $ )) $ >) 8$ >) $. s. y3 ss s. s s. y2. s. y7 *. *. s. 0. 200. 300. 400. 500. 600. 700. 800. 900. 1000. M/Z.

(134) '6.

(135) A. B. FLKEGACDELFSYLIEKVKRKKNVLRL. substrate (83.7%). EGACDELFSYLIEKVKRKKNVLRL. fragment 1.9%. CDELFSYLIEKVKRKKNVLRL. fragment 1.2%. LFSYLIEKVKRKKNVLRL. fragment 5.3%. YLIEKVKRKKNVLRL. fragment 7.9%. F naftLKEGA C D E L F S Y L I E K V K R K K N V L R L. C FIP VEVLVD L F L K E G A C D E L F S Y L I E K V KR K K N V L R L % CTL recognition. D 100. 50. 0. si-control si-TPPII K562-A3. 0''/,0 &/66

(136) ,

(137) * ' ' / A ) >X) 7== % _+. =:C+{C 5?"z5 ) S8 )U .)) 7 %. 8) % ) ) F>X) % >..) .>) 8 . . ) + >8 ) >8F> ) %. B . ) >X) 7== % _+. =:C+{C! . )X) >8 the N-terminal phenylalanine a 2-naphylsulfonyl group is attached to protect the substrate from N. < +)

(138) )> )) S ) U C ) >X) 7== % '_+. =:_+{C 7 ) ) $ 8 $ 7==+.))

(139) +. < +)

(140) )> )) S ) U 7 ! > F> . % ' % .

(141) +.> % +. )X) =:C+{C substrate and its subseF> ) %.! ) $ < +) ..+ % 7== D 7 % 6 +' 8 %) < :

(142) > 7== + :

(143) S"5( % % <.U. '

(144) .

(145) A 72.0 186.0 299.1 398.2 511.3 624.4 711.4 768.4 839.5 953.5 1082.5 1139.6 -. A. N. L. V. L. L. S. G. A. N. E. G. K. bn. - 1214.6 1100.6 987.5 888.4 775.3 662.3 575.2 518.2 447.2 333.1 204.1 147.1 yn. Relative Intensity. 100. b3. b2. y9 y8 y7. y6. 50. y10. b4 b5. y11. 0 100. 200. 300. 400. 500. 600. 700. 800. 900. 1000. 1100 1200. m/z. B Band 1. 2. 3. a. MS/MS. b. Sequence identified. c. Source protein. 507.6 (3+). IKGEHPGLSIGDVAK. High mobility group protein. 531.6 (3+). KHPDASVNFSEFSK. High mobility group protein. 796.9 (2+). KHPDASVNFSEFSK. High mobility group protein. 536.3 (2+). LAGLEEALQK. Keratin. d. 739.8 (2+). DSLENTLTETEAR. Keratin. 426.2 (2+). GLTGGFGSR. Keratin. 813.4 (2+). LNVEVDAAPTVDLNR. Keratin. 519.7 (2+). AQYDDIASR. Keratin. 514.6 (2+). TAVCDIPPR. -tubulin. 508.3 (2+). DVNAAIATIK. -tubulin. 912.9 (2+). VGINYQPPTVVPGGDLAK. -tubulin. 470.9 (3+). QLFHPEQLITGK. -tubulin. 4. no precursors found. 5. 643.3 (2+). ANLVLLSGANEGK. e. Nardilysin. 0''/,0 4/

(146)

(147)

(148)

(149)

(150)

(151)

(152) "/&;<=:%& / Anion-ex % '_ % 6 +' ! ) ) % ~> >8 5?"zS+)8U5 , :S+?U

(153) ! ) 8 ";"+=&5 ) 8) ) S ? *U 7 )X) 8 ). . . S("^("U A 7 ("^(" >. % .Î ]'' SU % >) 8) 6 S *U )) )X) )

(154) ,"&

(155) 5& % . ) . 8+ ) + >)) B )X 8 ("^(" % 8) ' . 8 S8) U! $ S8) U! ) + +>8> S8) 'U

(156) SU = 8) ) % . ";"+=&5 S8U ( SU % ("^(" SU F> )X) 8 ("^(" S)U $ . % % >) $) % . SU % . 8) 6! ) %> X.) 8 . % %. 6' _ SU! 8 F z77;?7 ) _`' SU F 55?""?55 S) U.

(157) '_.

(158) 0''/,0 </( ,

(159)

(160)

(161)

(162) > ?'

(163) ,$ %& / A : 8 7 +5?"z5 % +' +< ) S+'+

(164) :;+) as .>) 8 ?

(165) ¤ 5"

(166) ) +< +' ) 8 5&?= < 7 > 5&?= < > ) < >) % > ( ¦ "5( % % > <. B B . % +' %) :5"+

(167) :;+5&?= ) ) % % % 5&?=+ 5&?=+ < ! > +< ). A % CTL recognition. 150 125 100 75 50 25 0. Vector NRD+ HeLa-A3. B 150 120 90 60 30 0 0 10. 1. 10. 10. 2. 10. 3. 10. 4. F L1-H. 214. 213. 212. 211. 210. 209. 208. 207. 206. 205. 203 204. 202. 201. 199 200. 198. 197. 196. 195. 194. 193. 192. TOP Nardilysin 191. 190. A. ELFSYLIEKVKRKKNVLRLCCKKLK 1 2 3 4 5 6 7 8 9 10 11 12 13. IFNJ prod. (pg/ml). B 4000. TOP + NRD dig. NRD only dig.. 3000. TOP only dig. m ock digest. 2000 1000 0 200 nM. 40 nM. 8 nM. Digest concentration loaded. 0''/,0 =/( ,

(168)

(169) 6%!":4 co-digested with nardilysin and TOP. A ">8 6+. =:190-214 S5?"z5 , :

(170) ,: ! 8 )%)U % ) ) 7B= )) +) ) ) 7B= S U! <. ' % ) %. .. . 5?"z5 ! ) 8 . . B 7 % 6+. =:190-214 )) % 8 ) S

(171) :;U! 7B=! 8 ) "5( % % <. )) . ! 6+. >8) 7B= )) > .. S ) %.U! % . 8 % 7 . ) % _+. =:190-206 7B= ) 8 % S+.U S) U. 'C

(172) .

(173) A FIPVEVLVDLFLKEGACDELFSYLIEK ELFSYLIEK B. Relative generation + Epoxom. + Casp. Inh. 40% 0%. IFNJ prod. (pg/ml). mock digest Proteasome dig. Prot. + Epoxom. Prot. + Casp. Inh.. 4000 3000 2000 1000 0 1 μM. 200 nM. Digest concentration loaded. % CTL recognition. C 150. 100. 50. VS. I. LAA. +. PS +. in. +. Ep. ox. as C +. om. p.. di el ef. br. yc. In. A n. ed at tre +. un. h.. 0. 0''/,0 ;/* 8 +

(174) 0 ' 0 * ' @ >, > of the proteasome. A = . _+. =:_+`C S +.>U ; % .) ..> + ) > >. {" . % 8 % 8 % < . S{ §(U =

(175) >,"B S ! >) 6{ §(U! X 8 % +$ . ) 8>) 8% &>+`{ S8 U +%. ) .. %. S +C` +.>U ))) > 6 % )) . % :> % ..> + ) > . .8 7 } % % 8 )) % ) > 8 7 <. ) . . > B 7 % ) % _+. =:_+`C 7 >8 )) > . S U 8 % 8 % < . =

(176) >,"B S U ; )! )) +'+ cells and co-incubated with the CTL +5?"z5

(177) _+. >8) '_ > . S. $ )U C : 7 % 6 +' ) . 8 < . S §(U! =" S{ §(U ) )<''," S+," 6{ §(U 8 % =

(178) >,"B S ! 6{ §(U .>) > ?

(179) ¤+ :% {{ 6 +' ) S . )U! 8> >) 8$ >) 6 +' ) ) ..) ) 8%) 8 $ +< "5( % <. )).

(180) '`.

(181) 207. 208. 206. 205. 204. 203. 202. 201. 199. 198. 197. 196. 195. 194. Nardilysin 193. 192. 191. 190. 189. 188. 187. 186. 185. 184. 183. 182. Proteasome. 200. A. FLKEGACDELFSYLIEKVKRKKNVLRL 1 2 3 4 5 6 7 8 9 10 11 12 13. ELFSYLIEKVK TOP. ELFSYLIEKVKR ELFSYLIEKVKRK ELFSYLIEK B A3-supertype Epitope. B B X X X X. Epitope. X B B X X X. Epitope. X X X X B B. X X X X B B X X X X X B B B B X X X X. Epitope HLA-B27 Epitope Epitope. A. B. 60. % Inhibition. 1600. IFNJ prod. (pg/ml). Suppl. Figure 8. Mechanism of epitope generation and role of nardilysin in class I processing. A: The proteasome, nardilysin ) 7B= + % =:`{+`C 7 ) } % SU . 8% &>+`{! SU % ) behind Lys-200, Arg-201 and Lys{ ) S'U % 7B= 8) +`C )+ )) + ) '+. cursors together generating the +.> B: " ) ) % presented peptides depending on the position of the dibasic motif S*! 8 U. 1200 800. 50 40 30 9-m er GRIDKPILK 13-m er 12-mer. 20. 400 10 0. 0. K562-A3-control. K562-A3-ICP47. 0. 10. 20. 30. 40. 50. Concentration test peptide. 0''/,0 !/%6 '

(182)

(183)

(184)

(185)

(186) 6%!"!# epitope and its precursors. A 7=+)) % 5?"z5 6 +' )>) 7=+8 =]_ S 6 +'+=]_U 8 $ 7=+.)) ) ! ) >) % 7 +5?"z5 :% 6 +' )>) . > S 6 +'+ U "5( % % <. B: 5} % 7= % `+. 5?"z5 ) +. ) '+. +. <)) S5?"z5 , :^ U 7 ) ) . 7=+ ~> 8) % ) $ 8 ) } =) &:; = >) ) +*_ ) ) $ 8 } ) 8 7= ]{

(187) .

(188) A 10 min 30 min. % epitope. 30 20 10 0. PKQRCDNWESHVLYIATFMG VIDPFWCLYNHAEGMTSQKR. % destroyed epitpe. ELFSYLIEK-XKRK (P1’). 10 min 30 min. 40 30 20 10 0. PKQRCDNWESHVLYIATFMG VIDPFWCLYNHAEGMTSQKR. ELFSYLIEK-XKRK (P1’). 10000. IFNJ prod. (pg/ml). B. ELFSYLIEX-VKRK (P1). 8000. 10 min 30 min. ELFSYLIEX-VKRK (P1). C C. Epitope production 10 min. 30 min. 1h. SLYSFPEPEAVKRK. 5.7%. 17.5%. 24.6%. VLDGLDVLLVKRK. 5.0%. 6.3%. 4.5%. SLYSFPEPEARRFV. 6.6%. 16.4%. 25.8%. VLDGLDVLLRRFV. 14.0%. 19.2%. 17.6%. ELFSYLIEKRRFV. 2.7%. 10.5%. 21.5%. 6000 4000 2000 0. PKQRCDNWESHVLYIATFMG. ELFSYLIEK-XKRK (P1’). Suppl. Figure 10. The epitope-generating capacity of TOP. A ; 8 7B= % % '+. 5?"z5 , : S=:190-202), where the residues at the =+ ) =+ > >) 7B= S5?"z5=+= : U >8>) % 8 )> 7 ) '+. F> >)) . S 8 =+ ) =+ >8> U ) )) 8 ) 8 ) ) )> # + )> S5?"z5 5?"z5 % =+ ) =+>8> ! U % { . ) '{ . )) ) F>X) % >..) .>) 8 . . ">8> )) ) } % + )> { . + )> S ) ) > %. % U % { . ) '{ . )) % >..) B 7 % 7B=+)) '+. % =:190-202 >8> = S5?"z5 + = : U 7 ) )! )) +'+ 5 : ! +>8) 7 +5?"z5 ) ?

(189) ¤+ .>) : % ) '+. F> )) 8 ) 8 ) ) )> C: % >X) 7B= +.> % ) 7 % . > X +. <)) 5 ) "z"?=5=5 S=:]+6U! ,;&;, S=:{{+{CU ~$ F> , : ::?, ) 5?"z5 S=:`{+`CU ~$ F> ::?, 5 )> % {! '{ ) { . >8 % >8 7B= )) ) <) % >..) .>) 8 . ..

(190) ].

(191) METHODS Cell lines Cell lines used were erythroleukemia cell line 6 S> < =:(5U! . S=:(5+), renal cell . S:U (6_! +`]6 S8 ! =:(5+ ) +'+U ) (C6 S+'+! =:(5+U S: )) 8 ; 5 ,)! #(! 7

(192) )U ) *+ 5 : S+'+ ) =:(5+ U =:(5+< ) 8 =: 6 ! > ! ) )>) < +'! + + *_{6 S)) 6 +'! 6 +! 6 + *_ ) +'U > + ) +' ) :" .) ['\ S )) 8 ) ( .$$! ) 5<. . ! ) # () U +*_{6 ) .) >% < % +'! + ) +*_{6 .>) 8 ~ . > . 8 ) &=+'! **_ ) 3 ' S) U +' overexpressing nardilysin were made by trans% :5"+5&?=

(193) :; ;

(194) S:5"+

(195) :;+5&?=U S

(196) :; . X >.

(197) :;U % % ) 5&?=+ ) 5&?=+ > ! > +< ) 6 +' < 7= 8 =]_ ) S 6 +'+=]_ ) 6 +'+ U .) 8 )> > :"+:5"+&?= .) > =]_ 6 +*_{6 was stably transfected with a plasmid encoding . (?:& 3::z V S5*

(198) ' ]_+. ! <

(199) +. methionine; CTL epitope in bold), transfected ) . . lines were cultured in complete culture me)>. % (;( S* $! ,! *>.U >.) C % % >. S?" =! I! >U! {{ #^ . ) .( +>.

(200) =) I) 8 )+ strategies on an automated multiple peptide I S" ! (>"! 3! &.U > ?. +. ? ~> )! ~> lently coupled to the cysteine residue using 6+S ) .) U~> S?>$ . &! *>! "I)U 7 F> )>! ?. ++S;8U+B 8) % .

(201) . S?U ? 8 $ %

(202) + terminal trimming in digestion experiments, the N-terminus of the peptide was blocked +>% > ) =+>X) ) )) 8 . .

(203)

(204)

(205) . 6 +' S6¥{9) were homogenized in 8> S{ .( 7+! .( ;77! .( NaN'! 6 .(

(206) ! { .( (2! { .( 5;7! { ! .( 7=! 6{ .(

(207) ?! { .(

(208) C,B4! _6U > a Dounce glass . I 7 . fuged % { . {!{{{ ¥ g, and the super %>) % {{!{{{ ¥ g 8 . . ) 7== 7 X) X) { §. X 7 ) S { % % {6 .U 8 < . > . >. S: 6^6! ..U ) S{ .U % . { 6{{ .( 8> ~ % .^. "X ) activity in the fractions was assessed with in-. ]

(209) .

(210) F>) ~> 6+. >8 5?"zS+)8U5 , :S+?U

(211) S=:190-204U ? ~> S?U+ group was attached to a cysteine in stead of the > 7 % .) >8 X % {6 ¨( >8 8> S'{ .( 7! { .( ! 6 .( (2! .( ;77! _6U 8 % .( % ~> ! )> % F> % . ~> ! .>) > ' { . < ) ] { . . ? S{ §U $ S% ' ]{U > ";" .) ) ) massie Brilliant Blue according to standard )> ") 8) ) % . % '_! >8J) ) )8) [''\ ) ) > . .

(212)

(213) } % ) % +' .>) with a competition-based cellular peptide 8) )8) > [']\ In this assay the capacity of test peptides to . >% <) 8) % ~> S?U+8) reference peptide that is known to bind with } . > % ) 7 8 % ~> 8) % ) 8) by the competitor test peptide was calculated > % % .> S+S(?reference and competitor peptide (?no reference peptideU ^ S(?reference peptide (?no reference peptideUU ¥ {{ 7 8) } % ) <) )) 8 6{ 8) % ?+8) % ) S6{U 6{ 6 .( )) } 8) ) 6 .( © 6{ 6 .( . )) .) } 8) 7 ?+8) % ) >) % +' ,?=S?U

(214) [']\ RNA interference mediated suppression of peptidases RNA interference mediated suppression of ) < 6 +'! 6 +*_ ) +' 8) 8 transfection with pools of four interfering oligonucleotide RNA duplexes for each ) S&5

(215) B(5 "(:7 % . Dharmacon) and/or by stable transfection % "#=5:+> S % % . ) : *)!

(216) ) >U ) :

(217) F> 7% % "#=5:+> .) 6 +' ) 6 + B27 was performed by electroporation, and +' ?>&5

(218) 5 S: Molecular Biochemicals, Indianapolis) accord > % .>%> Cells stably expressing siRNA were obtained by culturing in the presence of puromycine S §^. % 6 +' ) *_ ) { §^ . % +'U 7 % % :

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