besides other targets like kinases and membrane receptors , proteases are one of the most effective targets for drug discovery [ 1 , 2 ] . to date , protease - inhibitors have been widely developed whereas protease - activators , which have great potential in medical and scientific uses [ 3 , 4 ] , have been far less developed due to technical difficulties . these include the fact that the activation mechanism , which is usually regulated by the binding of ligands , that is , allosteric effectors , rather than by substrates , is very difficult to predict in detail from theory . in contrast , the inhibition mechanism can be principally assigned to competitive binding of inhibitors to the active site or steric hindrance of the normal action of enzyme . the latter can be inferred from the molecular structure at atomic resolution [ 7 , 8 ] .    to address the above difficulty , we have recently developed a systemic in vitro evolution method ( called erapansy ( evolutionary rapid panning analysis system ) )   for screening of protease - regulating peptide aptamers based on mrna ( and cdna ) display [ 1012 ] and a substrate - function link ( sf - link ) method . this method was described in the successful identification of cathepsin e- ( cate- ) inhibitory peptide aptamers . as a next step , we attempted to obtain cate - activity - enhancing peptide aptamers at neutral ph ( ph 7.4 ) using this method since cate was reported to induce apoptosis of cancer cells under physiological conditions ( ph 7.4 ) . in this process , cate catalyzes the release of trail ( tumor necrosis factor - related apoptosis - inducing ligand ) without affecting normal cells . thus , cate - activity - enhancing peptide aptamers that function at neutral ph may be promising reagents for cancer therapeutics . intriguingly , a similar approach that utilizes a microenvironmentally activated protease has been developed , and clinical trials using protease - activated prodrugs for prostate cancer are path - finding approaches in this field .   in order to identify cate - activity - enhancing peptide aptamers at neutral ph , we first developed an assay system for measuring the unusual activity of cathepsin e at ph 7.4 instead of ph 4.5 and then performed the general scheme of in vitro evolution ( erapansy ) for obtaining protease - activating peptides based on inverse sf - link selection . we describe here the successful development of one promising peptide aptamer ( s3 ) that enhanced cate - activity by up to 260% in vitro and which induced apoptosis in cancer cells ( hela ) . through these experiments , we have demonstrated and confirmed the effectiveness of this method for in vitro evolution and its wide applicability . a fluorogenic substrate susceptible at neutral ph to the protease cathepsin e , which usually functions at acidic ph , was designed as per reference : [ nma - gly - gly - arg - arg - ser - gly - thy - cys - gly(dnp)-d - arg - nh2 ] . the peptide was custom - synthesized at peptide institute ( osaka , japan ) and confirmed to be an effective substrate at ph 7.4 . the peptide was dissolved in dimethyl sulfoxide ( dmso ) at 10  mm and stored in a deep freezer until use . the enzyme cate was prepared from   rat spleen according to the method previously described   and immobilized on n - hydroxysuccinimide - activated sepharose beads ( ge healthcare , usa ) using the amine - coupling method and stored in , 100  mm phosphate buffer ( ph 7.4 ) at 4c . cate - activation by peptide aptamers was assayed by the method described previously   but with a few modifications . briefly , the cate ( 20  nm ) solution preincubated with a peptide aptamer in a 1  :  1 molar ratio in the selection buffer ( 50  mm tris - hcl , 100  mm nacl , ph 7.4 ) at 25c for 10  min . was combined with the fluorogenic substrate ( 5  m ) and the enzyme reaction performed at 37c for 1  h. the reaction product was measured by fluorescence intensity at 440  nm with an excitation wavelength of 340  nm using a fluorescence microplate reader , infinite 200 ( tecan , japan ) . the percentage of activation ( a ) was calculated by fitting the data to the following equation : 
 ( 1)a=100sfbfcfbf  [ %  activation ] , 
							 where sf   represents the fluorescence intensity of the positive reaction consisting of the enzyme ( cate ) , the fluorogenic substrate , and a possible peptide activator ; cf is that of the control reaction mixture consisting of cate and fluorogenic substrate ( without peptide activator ) ; bf is the background fluorescence of the negative reaction mixture consisting of only the fluorogenic substrate . the primary and the secondary libraries were constructed according to the protocols described in . in brief , the primary library was constructed by y - ligation - based block shuffling ( ylbs ) . the peptide sequences obtained from the primary library selection were cluster - analyzed and used to design the subsequently constructed blocks ( figure 2 ) . using these blocks , the resultant library contained all of the arbitrarily shuffled blocks with a different number of blocks ( 14 blocks ; figure 2(b ) ) , thus termed an all - steps - all - combinations ( asac ) library . prior to the in vitro selection , the variable sequences were integrated into the cdna - tagged - sf - link peptide construct . the preparation of the construct was performed following the method previously reported   and is partially appearing in figure 1 . the cdna displayed - sf - link construct having an intervening cate - substrate sequence ( figure 1 ) was subjected to the inverse sf - link selection method . an inverse function - based selection strategy was adopted to obtain cate - activating peptides at neutral ph ( ph 7.4 ) and is described below . first , in order to remove the nonspecific binding peptides , the cdna - tagged sf - link - peptide library ( see figure 1 ) was treated with the selection buffer ( 50  mm tris - hcl , 100  mm nacl , 5  mm mgcl2 , ph 7.4 ) containing 5  l of nhs - activated sepharose beads ( 90  m mean particle diameter , resuspended in 100% isopropanol ) at 25c for 10  min . the supernatant containing unbound cdna - tagged - sf - link peptide molecules was collected after removing the beads by spinning down and used in the succeeding experiment . the cdna - tagged - sf - link peptide library thus obtained was incubated with cate - immobilized beads ( about 5  pmol cate immobilized ) at 25c for 10  min . those peptides which bind to beads ( in other words , peptides with affinity for cate ) , were collected by spinning and immediately resuspended in reaction buffer ( 50  mm tris - hcl , 1  m nacl , ph 7.4 ) and incubated at 37c for 1  h. ( 1st round ) , 30  min . the incubation time decreases as the selection round progresses leading to enrichment of rapidly cleaved species . the supernatant expected to contain cate - activators was collected , pcr - treated , and used as the library for the next selection round . the molecules thus selected were subjected to conventional cloning , sequencing analyses , synthesis , and activity assay . for the rapid synthesis of peptides , in vitro translation using the coding sequences of selected peptides was used . the dna construct consisting of the sequences for the relevant peptide , the streptavidin - binding peptide , and the protease factor xa recognition site was prepared by way of pcr and then transcribed and finally translated in vitro according to the method previously reported in . those peptides which were cathepsin e - activating in these experiments were subjected to commercial chemical synthesis ( bex corporation ltd . , an spr instrument , biacore 2000 ( ge healthcare , uk ) , was used for measuring kd for the binding of cathepsin e and selected peptides . cathepsin e was immobilized onto biacore sensor chip cm5 ( ge healthcare , uk ) by the amine - coupling method using an amine coupling kit ( ge healthcare , uk ) . immobilization of cathepsin e was performed using running buffer ( 50  mm sodium acetate , 100  mm nacl , ph 4.5 ) . to determine dissociation constants , four different concentrations of selected peptides the peptide interactions were performed against cate using the running buffer ( 50  mm tris - hcl , 100  mm nacl , ph 7.4 ) . the binding data were analyzed with the 1  :  1 binding with mass transfer model in the biaevaluation software version 4  :  1 ( biacore ) . hela cells ( 1  10 cells / well in 96-well plate ) were treated with cathepsin e ( 7.7  m ) alone and also in addition with peptide aptamer ( p3 , selected from the primary library ) using three different molar ratios of cathepsin e : peptide aptamer = 1  :  10 ; 1  :  100 ; 1  :  1000 in 100  l of serum - free opti - mem at 37c for 20  h.   the cells ' viability was measured with the use of a cell counting kit-8 ( dojindo molecular technologies , japan ; figure 4(a ) ) . the same method was also used for measuring the effect of cathepsin e and peptide aptamer ( s3 , selected from the secondary library ) on hela cells . the cell viability was measured with cell counting kit - f ( dojindo molecular technologies , japan ; figure 4(b ) ) . two - way analysis of variance ( student 's t - test ) with repeated measures was used . to identify cate - activity - enhancing peptides at neutral ph , we adopted an in vitro evolution method ( erapansy )   based on the inverse sf - link selection approach which is shown schematically in figure 1 . in brief , the overall strategy consists of the primary library construction and selection ( mass selection and clonal selection ) followed by construction and enrichment through selection of a secondary library ( a peptide library generated by block shuffling of all - step - all - combinations using the primary library selection products ) .   in the mass selection ( i.e. , dealing with a population of molecules as a whole ) , the variable sequences ( possible  functional  sequences ) were integrated into the initial dna construct flanked by the substrate peptide sequence ( step 1 in figure 1 ) . namely , each peptide was attached to the dna molecule which consisted of coding regions for t7 promoter , kozak , his - tag , factor xa recognition sequence , variable sequence , cate substrate sequence , antigenic sequence for the flag antibody , and puromycin linker binding region ( plbr ; figure 1 ) . the cdna display method , a derivative of mrna display [ 10 , 11 ] , was applied to this dna library to obtain the peptide library with the cdna - tagged sf - link . to prepare the cdna display construct , a puromycin linker dna was bound to an mrna molecule through the hybridization with the plbr sequence ( step 2 in figure 1 ) . the puromycin - linked mrna thus formed can covalently bind to the nascent peptide via the puromycin , a mimic of an aminoacyl trna , generating the mrna display construct . to increase the stability , the mrna display construct was converted to the cdna display one by the action of reverse - transcriptase . the peptide library with the cdna - tagged sf - link construct thus obtained contains a random peptide ( functional part ) and a cate - substrate sequence   ( step 3 in figure 1 ) and was subjected to the inverse sf - link selection ( step 4a4d , for details see materials and methods ) devised for screening of the cate - activity - enhancing peptide aptamers . the essential difference between the sf - link selection method ( to identify inhibitors ) and the inverse sf - link selection method ( to identify activators ) is that the noncleaved peptides were discarded while the cleaved peptides were retained as shown in step 4c .   in the clonal selection ( i.e. , screening of the molecules individually on a clone - by - clone basis ) , the peptide aptamers thus obtained from mass selection were cloned , sequenced , and cluster analyzed ( step 4e ) . some of clones were subjected to in vitro peptide translation   and the cate - enhancing activity confirmed in an activity assay as described in the materials and methods . after the primary library generation and selection , the secondary library generation and selection results in a proven elevation in the activity of selected cate - peptide activators ( step 4f ) . the peptide aptamers thus obtained from the primary library ( p1 , p2 , and p3 ) enhanced the cate - activity by around 10 to 30% ( measured using peptides obtained by in vitro translation synthesis ) and such activities were also confirmed by using chemical synthesis of peptides ( table 2(a ) ) . notably , the peptide with the highest activity in the primary library selection was an insertion - carrying peptide ( p3 ) of 13 amino acids while others are of normal size ( octapeptide ) . the resulting cate - activity - enhancing peptide aptamers thus obtained from the primary library were cluster - analyzed to provide sequence information on possible functional blocks which could be used for generating the secondary library ( asac library ; table 1 ; and figure 2(a ) ) . the secondary cdna - tagged sf - link peptide library thus constructed was subjected to the inverse sf - link selection as described above ( figure 1 ) . the selection products from this library demonstrated higher activity than those selected from the primary library ( figure 3(a ) and table 2 ) . the average increase in the cate - enhancing activity of the top three hits selected out of ~50 clones sequence - analyzed was significant with peptides demonstrating more than 160% activity and one peptide ( s3 ) showing an activity increase of up to 260% cate activity ( p < .0003 ; table 2 ) , which is two - fold higher activity than that ( 130% ) of the best peptide ( p3 ; p < .05 ) obtained from the primary library selection when measured using in vitro translation synthesized peptide ( figure 3(a ) ) . however , such values are known to have a relatively large fluctuation   since the amount of the final product ( peptide ) generated by in vitro translation is an estimation value based on the initial amount of the reactant due to their very small n amount ( ~pmole ) . therefore , peptide ( s3 ) was chemically synthesized , and its cate - enhancing activity was measured ( table 2(b ) ) . the value thus obtained was lower than that obtained from in vitro translation , which is a trend generally observed with this approach . as a result , a higher activity ( 168% ) than the best peptide in the primary screen ( 130% ) was confirmed , proving the steady rise in the activity of peptides in this approach . the dissociation constant of the peptide ( s3 ) was found to be 3.89  10 m by the spr method , thus showing a relatively strong binding affinity ( table 2(b ) and figure 3(b ) ) . the selection thus performed using the inverse sf - link method was able to identify cate - activity - enhancing peptide aptamers at neutral ph ( figure 1 and table 2 ) , indicating the effectiveness of the inverse sf - link selection method . the effectiveness of the secondary library selection is clearly shown in figure 3 . in order to evaluate the activities of selected peptides ( p3 and s3 obtained from the primary and the secondary libraries , resp . ; table 2 ) , we next tested the combined effect of cate and selected peptide aptamers on hela cells . the cells were treated with cate alone or with cate and peptide aptamers ( p3 and s3 ) in a dose - dependent manner . any decrease in cellular viability was analyzed ( for details see material and methods ) . cells treated with cate and a peptide aptamer , p3 or s3 , at a molar ratio of 1  :  100 showed around a 1.5- and 3-fold increase in the level of apoptosis in comparison to cells treated with cate alone ( p < .05 ; figure 4 ) , respectively . these positive results corroborate our current approach of using an in vitro evolution method modified with the inverse sf - link selection for identifying protease activity - enhancing peptide aptamers . cate is reported to have the ability to induce apoptosis in cancer cells , a process which must occur under physiological conditions ( i.e. , at neutral ph ) . in this study , the activity of cate , which is known to usually function at acidic ph ( ph 4.5 ) , was measured at neutral ph ( ph 7.4 ) using a novel substrate as described in materials and methods . the peptides that can enhance cate activity under the assay conditions were selected as shown in table 2 . in this paper , we address two subjects : ( i ) selection of activity - enhancing , rather than inhibiting peptides and ( ii ) performance of evolution , not only selection , of peptides ( proteins ) . both points are discussed here . it is well known that finding activating reagents is more difficult than finding inhibitory ones since the latter has the clear target of the substrate - binding site . therefore , a lot of inhibitory reagents have been developed but far fewer activating ones [ 4 , 5 , 20 , 21 ] . the sf- ( substrate - function- ) link method developed recently   has the potential to select protease - activating peptides if it is operated in the inverse mode owing to its construction design ( see figure 1 ) , and this technique has been successfully exploited for the first time in this paper . this has resulted in the finding that the majority of selected peptides are cate - activating species ( partly shown in table 2 ) . this is noteworthy since starting with the same library ( the affinity selection product ) , we could obtain the opposite type of functional peptides ( activators ) by adopting the inverse mode of function - based selection using the sf - link method in which swiftly cleaved off cdna - tagged sf - link peptide molecules ( which are a conjugate of information and functional molecules , see figure 1 step 4c , were collected ) . the cathepsin e - activity - enhancing peptides ( p1 , p3 , s2 , and s3 ) identified herein contain common features being composed of a charged head group and a large hydrophobic tail ( detergent - like peptides ) . the activation may be due to some combination of enzyme stabilization in solution , loosening of the protein secondary structure , or alteration of adsorption of the enzyme to the surface of containers during the enzyme reaction . interestingly , in the spr measurements , the secondary library selection resulted in obtaining peptides with increased activity without much change in affinities ( similar kd values for p3 , from the primary library , and s3 , from the secondary library and see table 2 and figure 3(b ) ) . those peptides ( p1 and s2 ) which contain a single charged amino acid embedded in hydrophobic ones showed abnormal behaviors in the spr measurement ( data not shown ) , probably indicating strong nonspecific ( hydrophobic ) binding to the supporting plate even though a surface - blocking agent ( 1  m ethanolamine - hcl , ph 8.5 ) was used . therefore , these peptides may need to be modified into a more soluble structure for advanced applications such as a drug seed . in addition , the evaluation of selected cate - activity - enhancing peptides on cells provides definitive proof that our current in vitro evolution method based on the inverse sf - link selection is effective . in addition , this study also confirms the effectiveness of the erapansy method that allows the molecular library to progress towards higher activity . although it may be early to draw the conclusion that this is a proven method for obtaining much improved molecules , the following can be stated safely : the primary library selection is equivalent to a search for module - like peptides out of a huge number of meaningless peptides while the construction of the secondary library is analogous to module - shuffling ( or exon shuffling ) , which is assumed to have worked during the evolution of proteins . an interesting issue to be addressed in the future concerns the size of blocks ( or module ) which holds meaning ( or function ) , though , in this study , it was rather arbitrarily determined to be a tetrapeptide , which was ultimately effective . in other words , the way , by which natural proteins evolved , holds promise for the artificial development of proteins . according to recent genome analysis , during the evolution of life , the recombination mechanism strongly governed and contributed to the generation of novel proteins [ 24 , 25 ] . this fact is also supportive of our approach where drastic recombination of sequences ( block shuffling ) has been employed together with point mutation in the construction of the secondary library .          in conclusion , this study has succeeded in introducing an assay system for cate activity at neutral ph ( ph 7.4 ) and in obtaining cate - activating peptides by employing the successive in vitro evolution method ( erapansy ) based on inverse sf - link selection . these results endorse the notion that the method employed here is applicable for obtaining activity - enhancing peptides for proteases at large . such a notion , of course , needs to be strengthened by succeeding studies adopting this method .
the aspartic protease cathepsin e has been shown to induce apoptosis in cancer cells under physiological conditions . therefore , cathepsin e - activity - enhancing peptides functioning in the physiological ph range are valuable potential cancer therapeutic candidates . here 
 , we have used a general in vitro selection method ( evolutionary rapid panning analysis system ( erapansy ) ) , based on inverse substrate - function link ( sf - link ) selection to successfully identify cathepsin e - activity - enhancing peptide aptamers at neutral ph . 
 a successive enrichment of peptide activators was attained in the course of selection . 
 one such peptide activated cathepsin e up to 260% , had a high affinity ( kd ; 300  nm ) , and had physiological activity as demonstrated by its apoptosis - inducing reaction in cancerous cells . 
 this method is expected to be widely applicable for the identification of protease - activity - enhancing peptide aptamers .