The Experts below are selected from a list of 17241 Experts worldwide ranked by ideXlab platform
Ingemar Björk - One of the best experts on this subject based on the ideXlab platform.
-
grAfting of feAtures of CystAtins c or b into the n terminAl region or second binding loop of CystAtin A stefin A substAntiAlly enhAnces inhibition of cysteine proteinAses
Biochemistry, 2003Co-Authors: Alona Pavlova, Ingemar BjörkAbstract:ReplAcement of the three N-terminAl residues preceding the conserved Gly of CystAtin A by the corresponding 10-residue long segment of CystAtin C increAsed the Affinity of the inhibitor for the mAjor lysosomAl cysteine proteinAse, cAthepsin B, by ApproximAtely 15-fold. This tighter binding wAs predominAntly due to A higher overAll AssociAtion rAte constAnt. ChArActerizAtion of the interAction with An inActive Cys29 to AlA vAriAnt of cAthepsin B indicAted thAt the higher rAte constAnt wAs A result of An increAsed Ability of the N-terminAl region of the chimeric inhibitor to promote displAcement of the cAthepsin B occluding loop in the second binding step. The low dissociAtion rAte constAnt for the binding of CystAtin A to cAthepsin B wAs retAined by the chimeric inhibitor, which therefore hAd A higher Affinity for this enzyme thAn Any nAturAl CystAtin identified so fAr. In contrAst, the N-terminAl substitution negligibly Affected the Ability of CystAtin A to inhibit pApAin. However, substitutions of Gly75 in the second binding loop of CystAtin A by Trp or His, mAking the loop similAr to those of CystAtins C or B, respectively, increAsed the Affinity for pApAin by ApproximAtely 10-fold. This enhAnced Affinity wAs due to both A higher AssociAtion rAte constAnt And A lower dissociAtion rAte constAnt. Modeling of complexes between the two vAriAnts And pApAin indicAted the possibility of fAvorAble interActions being estAblished between the substituting residues And the enzyme. The second-loop substitutions negligibly Affected or moderAtely reduced the Affinity for cAthepsin B. Together, these results show thAt the inhibitory Ability of CystAtins cAn be substAntiAlly improved by protein engineering.
-
the role of the second binding loop of the cysteine proteAse inhibitor CystAtin A stefin A in stAbilizing complexes with tArget proteAses is exerted predominAntly by leu73
FEBS Journal, 2002Co-Authors: Alona Pavlova, Sergio Estrada, Ingemar BjörkAbstract:The Aim of this work wAs to elucidAte the roles of individuAl residues within the flexible second binding loop of humAn CystAtin A in the inhibition of cysteine proteAses. Four recombinAnt vAriAnts of the inhibitor, eAch with A single mutAtion, L73G, P74G, Q76G or N77G, in the most exposed pArt of this loop were generAted by PCR-bAsed site-directed mutAgenesis. The binding of these vAriAnts to pApAin, cAthepsin L, And cAthepsin B wAs chArActerized by equilibrium And kinetic methods. MutAtion of Leu73 decreAsed the Affinity for pApAin, cAthepsin L And cAthepsin B by ≈ 300-fold, >10-fold And ≈ 4000-fold, respectively. MutAtion of Pro74 decreAsed the Affinity for cAthepsin B by ≈ 10-fold but minimAlly Affected the Affinity for the other two enzymes. MutAtion of Gln76 And Asn77 did not Alter the Affinity of CystAtin A for Any of the proteAses studied. The decreAsed Affinities were cAused exclusively by increAsed dissociAtion rAte constAnts. These results show thAt the second binding loop of CystAtin A plAys A mAjor role in stAbilizing the complexes with proteAses by retArding their dissociAtion. In contrAst with CystAtin B, only one Amino-Acid residue of the loop, Leu73, is of principAl importAnce for this effect, Pro74 Assisting to A minor extent only in the cAse of cAthepsin B binding. The contribution of the second binding loop of CystAtin A to proteAse binding vAries with the proteAse, being lArgest, ≈ 45% of the totAl binding energy, for inhibition of cAthepsin B.
-
CystAtin inhibition of cAthepsin b requires dislocAtion of the proteinAse occluding loop demonstrAtion by releAse of loop Anchoring through mutAtion of his110
FEBS Letters, 2000Co-Authors: Alla Pavlova, Magnus Abrahamson, Joanne C Krupa, John S Mort, Ingemar BjörkAbstract:CystAtins A And C were both shown to inhibit cAthepsin B by A two-step mechAnism, involving An initiAl weAk interAction followed by A conformAtionAl chAnge. Disruption of the mAjor sAlt bridge Anchoring the occluding loop of cAthepsin B to the mAin body of the enzyme by mutAtion of His110 to AlA converted the binding to An AppArent one-step reAction. The second step of CystAtin binding to cAthepsin B must therefore be due to the inhibitor hAving to Alter the conformAtion of the enzyme by displAcing the occluding loop to Allow A tight complex to be formed. CystAtin A wAs AppreciAbly less effective in displAcing the loop thAn CystAtin C, resulting in A considerAbly lower overAll inhibition rAte constAnt.
-
the contribution of n terminAl region residues of CystAtin A stefin A to the Affinity And kinetics of inhibition of pApAin cAthepsin b And cAthepsin l
Biochemistry, 1999Co-Authors: Sergio Estrada, And Alla Pavlova, Ingemar BjörkAbstract:The Affinity And kinetics of binding of three N-terminAlly truncAted vAriAnts of the cysteine proteinAse inhibitor CystAtin A to cysteine proteinAses were chArActerized. Deletion of Met-1 only minimAlly Altered the inhibitory properties of the protein. However, deletion Also of Ile-2 resulted in reduced Affinities of 900-, ≥3-, And 200-fold for pApAin And cAthepsins L And B, respectively. Further truncAtion of Pro-3 substAntiAlly increAsed the inhibition constAnts to ∼0.5 μM for pApAin And cAthepsin L And to 60 μM for cAthepsin B, reflecting AdditionAlly 2 × 103-, 2 × 104-, And 400-fold decreAsed Affinities, respectively. The reductions in Affinity shown by the lAtter mutAnt indicAte thAt the N-terminAl region contributes About 40% of the totAl free energy of binding of CystAtin A to cysteine proteinAses. Moreover, Pro-3 And to A lesser extent Ile-2 Are the residues responsible for this binding energy. The reduced Affinities for pApAin And cAthepsin L were due only to higher dissociAtion rAte constAnts, w...
-
hydrophobic sequences cAn substitute for the wild type n terminAl sequence of CystAtin A stefin A in tight binding to cysteine proteinAses selection of high Affinity n terminAl region vAriAnts by phAge displAy
FEBS Journal, 1999Co-Authors: Karin Ylinenjarvi, Mikael Widersten, Ingemar BjörkAbstract:A phAge-displAy librAry of the cysteine-proteinAse inhibitor, CystAtin A, wAs constructed in which vAriAnts with the four N-terminAl Amino Acids rAndomly mutAted were expressed on the surfAce of filAmenteous phAge. Screening of this librAry for binding to pApAin gAve predominAntly vAriAnts with A glycine residue in position 4. This finding is in Agreement with previous conclusions thAt glycine in this position is essentiAl for tight binding of CystAtin A to cysteine proteinAses by Allowing optimAl interAction of the N-terminAl region of the inhibitor with the enzyme. In contrAst, the first three residues of the vAriAnts obtAined by the screening were more vAriAble. Two vAriAnts were identified with similAr Affinities for pApAin As the wild-type inhibitor, but with these residues, VAl-Phe-Thr- or Ile-Leu-Leu, differing AppreciAbly from those of the wild-type, Met-Ile-Pro. Other sequences of the N-terminAl region, presumAbly mAinly hydrophobic, cAn thus substitute for the wild-type sequence And contribute similAr energy to the inhibitor–proteinAse interAction. The two vAriAnts binding tightly to pApAin differed in their Affinity for cAthepsin B, demonstrAting thAt CystAtin vAriAnts with increAsed selectivity for A pArticulAr tArget cysteine proteinAse cAn be obtAined by phAge-displAy technology.
Marja Jaattela - One of the best experts on this subject based on the ideXlab platform.
-
cAthepsin b Acts As A dominAnt execution proteAse in tumor cell Apoptosis induced by tumor necrosis fActor
Journal of Cell Biology, 2001Co-Authors: Lasse Foghsgaard, Dorte Wissing, Daniel Mauch, Ulrik Lademann, Lone Bastholm, Marianne Boes, Folmer Elling, Marcel Leist, Marja JaattelaAbstract:DeAth receptors cAn trigger cell demise dependent or independent of cAspAses. In WEHI-S fibrosArcomA cells, tumor necrosis fActor (TNF) induced An increAse in cytosolic cAthepsin B Activity followed by deAth with Apoptotic feAtures. Surprisingly, this process wAs enhAnced by low, but effectively inhibiting, concentrAtions of pAn-cAspAse inhibitors. ContrAry to cAspAse inhibitors, A pAnel of phArmAcologicAl cAthepsin B inhibitors, the endogenous cAthepsin inhibitor CystAtin A As well As Antisense-mediAted depletion of cAthepsin B rescued WEHI-S cells from Apoptosis triggered by TNF or TNF-relAted Apoptosis-inducing ligAnd. Thus, cAthepsin B cAn tAke over the role of the dominAnt execution proteAse in deAth receptor-induced Apoptosis. The conservAtion of this AlternAtive execution pAthwAy wAs further exAmined in other tumor cell lines. Here, cAthepsin B Acted As An essentiAl downstreAm mediAtor of TNF-triggered And cAspAse-initiAted Apoptosis cAscAde, whereAs Apoptosis of primAry cells wAs only minimAlly dependent on cAthepsin B. These dAtA imply thAt cAthepsin B, which is commonly overexpressed in humAn primAry tumors, mAy hAve two opposing roles in mAlignAncy, reducing it by its proApoptotic feAtures And enhAncing it by its known fAcilitAtion of invAsion.
-
cAthepsin b Acts As A dominAnt execution proteAse in tumor cell Apoptosis induced by tumor necrosis fActor
Journal of Cell Biology, 2001Co-Authors: Lasse Foghsgaard, Dorte Wissing, Daniel Mauch, Ulrik Lademann, Lone Bastholm, Marianne Boes, Folmer Elling, Marcel Leist, Marja JaattelaAbstract:DeAth receptors cAn trigger cell demise dependent or independent of cAspAses. In WEHI-S fibrosArcomA cells, tumor necrosis fActor (TNF) induced An increAse in cytosolic cAthepsin B Activity followed by deAth with Apoptotic feAtures. Surprisingly, this process wAs enhAnced by low, but effectively inhibiting, concentrAtions of pAn-cAspAse inhibitors. ContrAry to cAspAse inhibitors, A pAnel of phArmAcologicAl cAthepsin B inhibitors, the endogenous cAthepsin inhibitor CystAtin A As well As Antisense-mediAted depletion of cAthepsin B rescued WEHI-S cells from Apoptosis triggered by TNF or TNF-relAted Apoptosis-inducing ligAnd. Thus, cAthepsin B cAn tAke over the role of the dominAnt execution proteAse in deAth receptor-induced Apoptosis. The conservAtion of this AlternAtive execution pAthwAy wAs further exAmined in other tumor cell lines. Here, cAthepsin B Acted As An essentiAl downstreAm mediAtor of TNF-triggered And cAspAse-initiAted Apoptosis cAscAde, whereAs Apoptosis of primAry cells wAs only minimAlly dependent on cAthepsin B. These dAtA imply thAt cAthepsin B, which is commonly overexpressed in humAn primAry tumors, mAy hAve two opposing roles in mAlignAncy, reducing it by its proApoptotic feAtures And enhAncing it by its known fAcilitAtion of invAsion.
Marcel Leist - One of the best experts on this subject based on the ideXlab platform.
-
cAthepsin b Acts As A dominAnt execution proteAse in tumor cell Apoptosis induced by tumor necrosis fActor
Journal of Cell Biology, 2001Co-Authors: Lasse Foghsgaard, Dorte Wissing, Daniel Mauch, Ulrik Lademann, Lone Bastholm, Marianne Boes, Folmer Elling, Marcel Leist, Marja JaattelaAbstract:DeAth receptors cAn trigger cell demise dependent or independent of cAspAses. In WEHI-S fibrosArcomA cells, tumor necrosis fActor (TNF) induced An increAse in cytosolic cAthepsin B Activity followed by deAth with Apoptotic feAtures. Surprisingly, this process wAs enhAnced by low, but effectively inhibiting, concentrAtions of pAn-cAspAse inhibitors. ContrAry to cAspAse inhibitors, A pAnel of phArmAcologicAl cAthepsin B inhibitors, the endogenous cAthepsin inhibitor CystAtin A As well As Antisense-mediAted depletion of cAthepsin B rescued WEHI-S cells from Apoptosis triggered by TNF or TNF-relAted Apoptosis-inducing ligAnd. Thus, cAthepsin B cAn tAke over the role of the dominAnt execution proteAse in deAth receptor-induced Apoptosis. The conservAtion of this AlternAtive execution pAthwAy wAs further exAmined in other tumor cell lines. Here, cAthepsin B Acted As An essentiAl downstreAm mediAtor of TNF-triggered And cAspAse-initiAted Apoptosis cAscAde, whereAs Apoptosis of primAry cells wAs only minimAlly dependent on cAthepsin B. These dAtA imply thAt cAthepsin B, which is commonly overexpressed in humAn primAry tumors, mAy hAve two opposing roles in mAlignAncy, reducing it by its proApoptotic feAtures And enhAncing it by its known fAcilitAtion of invAsion.
-
cAthepsin b Acts As A dominAnt execution proteAse in tumor cell Apoptosis induced by tumor necrosis fActor
Journal of Cell Biology, 2001Co-Authors: Lasse Foghsgaard, Dorte Wissing, Daniel Mauch, Ulrik Lademann, Lone Bastholm, Marianne Boes, Folmer Elling, Marcel Leist, Marja JaattelaAbstract:DeAth receptors cAn trigger cell demise dependent or independent of cAspAses. In WEHI-S fibrosArcomA cells, tumor necrosis fActor (TNF) induced An increAse in cytosolic cAthepsin B Activity followed by deAth with Apoptotic feAtures. Surprisingly, this process wAs enhAnced by low, but effectively inhibiting, concentrAtions of pAn-cAspAse inhibitors. ContrAry to cAspAse inhibitors, A pAnel of phArmAcologicAl cAthepsin B inhibitors, the endogenous cAthepsin inhibitor CystAtin A As well As Antisense-mediAted depletion of cAthepsin B rescued WEHI-S cells from Apoptosis triggered by TNF or TNF-relAted Apoptosis-inducing ligAnd. Thus, cAthepsin B cAn tAke over the role of the dominAnt execution proteAse in deAth receptor-induced Apoptosis. The conservAtion of this AlternAtive execution pAthwAy wAs further exAmined in other tumor cell lines. Here, cAthepsin B Acted As An essentiAl downstreAm mediAtor of TNF-triggered And cAspAse-initiAted Apoptosis cAscAde, whereAs Apoptosis of primAry cells wAs only minimAlly dependent on cAthepsin B. These dAtA imply thAt cAthepsin B, which is commonly overexpressed in humAn primAry tumors, mAy hAve two opposing roles in mAlignAncy, reducing it by its proApoptotic feAtures And enhAncing it by its known fAcilitAtion of invAsion.
Hynda K Kleinman - One of the best experts on this subject based on the ideXlab platform.
-
role of lAminin 1 And tgf betA 3 in AcinAr differentiAtion of A humAn submAndibulAr glAnd cell line hsg
Journal of Cell Science, 1996Co-Authors: Matthew P Hoffman, Maura C Kibbey, John J Letterio, Hynda K KleinmanAbstract:Previous studies show thAt culturing An immortAlized humAn submAndibulAr glAnd cell line (HSG) on MAtrigel, A bAsement membrAne extrAct, induces cytodifferentiAtion. We hAve further defined this model system And identified fActors involved in HSG cell AcinAr development And cyto-differentiAtion. AcinAr development is mArked by cell migrAtion into multi-cellulAr sphericAl structures, cell proliferAtion And Apoptosis of the centrAlly locAlized cells. In Addition, functionAl differentiAtion wAs determined by indirect immunofluorescence And immunoblot AnAlysis for CystAtin, A sAlivAry glAnd AcinAr cell-specific protein found to be produced by differentiAted HSG cells. MAtrigel contAins multiple extrAcellulAr mAtrix proteins, however, lAminin-1 wAs identified As the mAjor mAtrix component thAt induced HSG cell AcinAr development And cytodifferentiAtion. Antibodies AgAinst specific components of MAtrigel And AgAinst cell surfAce Adhesion molecules were Added to cells in culture to identify components importAnt for HSG cell AcinAr differentiAtion. ImmunostAining of HSG cell Acini identified TGF-betA 2 And betA 3 As the predominAnt isoforms within the cells. NeutrAlizing Antibodies directed AgAinst TGF-betA 3 significAntly decreAsed (P
-
role of lAminin 1 And tgf betA 3 in AcinAr differentiAtion of A humAn submAndibulAr glAnd cell line hsg
Journal of Cell Science, 1996Co-Authors: Matthew P Hoffman, Maura C Kibbey, John J Letterio, Hynda K KleinmanAbstract:Previous studies show thAt culturing An immortAlized humAn submAndibulAr glAnd cell line (HSG) on MAtrigel, A bAsement membrAne extrAct, induces cytodifferentiAtion. We hAve further defined this model system And identified fActors involved in HSG cell AcinAr development And cyto-differentiAtion. AcinAr development is mArked by cell migrAtion into multi-cellulAr sphericAl structures, cell proliferAtion And Apoptosis of the centrAlly locAlized cells. In Addition, functionAl differentiAtion wAs determined by indirect immunofluorescence And immunoblot AnAlysis for CystAtin, A sAlivAry glAnd AcinAr cell-specific protein found to be produced by differentiAted HSG cells. MAtrigel contAins multiple extrAcellulAr mAtrix proteins, however, lAminin-1 wAs identified As the mAjor mAtrix component thAt induced HSG cell AcinAr development And cytodifferentiAtion. Antibodies AgAinst specific components of MAtrigel And AgAinst cell surfAce Adhesion molecules were Added to cells in culture to identify components importAnt for HSG cell AcinAr differentiAtion. ImmunostAining of HSG cell Acini identified TGF-betA 2 And betA 3 As the predominAnt isoforms within the cells. NeutrAlizing Antibodies directed AgAinst TGF-betA 3 significAntly decreAsed (P < or = 0.0002) the size of Acini formed. These results indicAte thAt multiple components, including lAminin-1 And TGF-betA 3, contribute to HSG cell AcinAr development. This model system will be useful to study AcinAr differentiAtion And sAlivAry glAnd-specific protein expression in vitro.
Sergio Estrada - One of the best experts on this subject based on the ideXlab platform.
-
the role of the second binding loop of the cysteine proteAse inhibitor CystAtin A stefin A in stAbilizing complexes with tArget proteAses is exerted predominAntly by leu73
FEBS Journal, 2002Co-Authors: Alona Pavlova, Sergio Estrada, Ingemar BjörkAbstract:The Aim of this work wAs to elucidAte the roles of individuAl residues within the flexible second binding loop of humAn CystAtin A in the inhibition of cysteine proteAses. Four recombinAnt vAriAnts of the inhibitor, eAch with A single mutAtion, L73G, P74G, Q76G or N77G, in the most exposed pArt of this loop were generAted by PCR-bAsed site-directed mutAgenesis. The binding of these vAriAnts to pApAin, cAthepsin L, And cAthepsin B wAs chArActerized by equilibrium And kinetic methods. MutAtion of Leu73 decreAsed the Affinity for pApAin, cAthepsin L And cAthepsin B by ≈ 300-fold, >10-fold And ≈ 4000-fold, respectively. MutAtion of Pro74 decreAsed the Affinity for cAthepsin B by ≈ 10-fold but minimAlly Affected the Affinity for the other two enzymes. MutAtion of Gln76 And Asn77 did not Alter the Affinity of CystAtin A for Any of the proteAses studied. The decreAsed Affinities were cAused exclusively by increAsed dissociAtion rAte constAnts. These results show thAt the second binding loop of CystAtin A plAys A mAjor role in stAbilizing the complexes with proteAses by retArding their dissociAtion. In contrAst with CystAtin B, only one Amino-Acid residue of the loop, Leu73, is of principAl importAnce for this effect, Pro74 Assisting to A minor extent only in the cAse of cAthepsin B binding. The contribution of the second binding loop of CystAtin A to proteAse binding vAries with the proteAse, being lArgest, ≈ 45% of the totAl binding energy, for inhibition of cAthepsin B.
-
the n terminAl region of CystAtin A stefin A binds to pApAin subsequent to the two hAirpin loops of the inhibitor demonstrAtion of two step binding by rApid kinetic studies of CystAtin A lAbeled At the n terminus with A fluorescent reporter group
Protein Science, 2000Co-Authors: Sergio Estrada, Steven T Olson, Elke RaubsegallAbstract:The three-dimensionAl structures of CystAtins, And other evidence, suggest thAt the flexible N-terminAl region of these inhibitors mAy bind to tArget proteinAses independent of the two rigid hAirpin loops forming the remAinder of the inhibitory surfAce. In An Attempt to demonstrAte such two-step binding, which could not be identified in previous kinetics studies, we introduced A cysteine residue before the N-terminus of CystAtin A And lAbeled this residue with fluorescent probes. Binding of AANS- And AEDANS-lAbeled CystAtin A to pApAin resulted in ApproximAtely 4-fold And 1.2-fold increAses of probe fluorescence, respectively, reflecting the interAction of the N-terminAl region with the enzyme. Observed pseudo-first-order rAte constAnts, meAsured by the loss of pApAin Activity in the presence of A fluorogenic substrAte, for the reAction of the enzyme with excess AANS-CystAtin A increAsed lineArly with the concentrAtion of the lAtter. In contrAst, pseudo-first-order rAte constAnts, obtAined from meAsurements of the chAnge of probe fluorescence with either excess enzyme or lAbeled inhibitor, showed An identicAl hyperbolic dependence on the concentrAtion of the reActAnt in excess. This dependence demonstrAtes thAt the binding occurs in two steps, And implies thAt the lAbeled N-terminAl region of CystAtin A interActs with the proteinAse in the second step, subsequent to the hAirpin loops. The compArAble Affinities And dissociAtion rAte constAnts for the binding of lAbeled And unlAbeled CystAtin A to pApAin indicAte thAt the lAbel did not AppreciAbly perturb the interAction, And thAt unlAbeled CystAtin therefore Also binds in A similAr two-step mAnner. Such independent binding of the N-terminAl regions of CystAtins to tArget proteinAses After the hAirpin loops mAy be chArActeristic of most CystAtin-proteinAse reActions.
-
the contribution of n terminAl region residues of CystAtin A stefin A to the Affinity And kinetics of inhibition of pApAin cAthepsin b And cAthepsin l
Biochemistry, 1999Co-Authors: Sergio Estrada, And Alla Pavlova, Ingemar BjörkAbstract:The Affinity And kinetics of binding of three N-terminAlly truncAted vAriAnts of the cysteine proteinAse inhibitor CystAtin A to cysteine proteinAses were chArActerized. Deletion of Met-1 only minimAlly Altered the inhibitory properties of the protein. However, deletion Also of Ile-2 resulted in reduced Affinities of 900-, ≥3-, And 200-fold for pApAin And cAthepsins L And B, respectively. Further truncAtion of Pro-3 substAntiAlly increAsed the inhibition constAnts to ∼0.5 μM for pApAin And cAthepsin L And to 60 μM for cAthepsin B, reflecting AdditionAlly 2 × 103-, 2 × 104-, And 400-fold decreAsed Affinities, respectively. The reductions in Affinity shown by the lAtter mutAnt indicAte thAt the N-terminAl region contributes About 40% of the totAl free energy of binding of CystAtin A to cysteine proteinAses. Moreover, Pro-3 And to A lesser extent Ile-2 Are the residues responsible for this binding energy. The reduced Affinities for pApAin And cAthepsin L were due only to higher dissociAtion rAte constAnts, w...
-
the role of gly 4 of humAn CystAtin A stefin A in the binding of tArget proteinAses chArActerizAtion by kinetic And equilibrium methods of the interActions of CystAtin A gly 4 mutAnts with pApAin cAthepsin b And cAthepsin l
Biochemistry, 1998Co-Authors: Sergio Estrada, Jonathan P Waltho, M Nycander, N J Hill, C J Craven, Ingemar BjörkAbstract:The importAnce of the evolutionArily conserved Gly-4 residue for the Affinity And kinetics of interAction of CystAtin A with severAl cysteine proteinAses wAs Assessed by site-directed mutAgenesis. Even the smAllest replAcement, by AlA, resulted in ∼1000-, ∼10- And ∼6000-fold decreAsed Affinities for pApAin, cAthepsin L, And cAthepsin B, respectively. Substitution by Ser gAve further 3−8-fold reductions in Affinity, whereAs the lArgest decreAses, >105-fold, were observed for mutAtions to Arg And Glu. The kinetics of inhibition of pApAin by the mutAnts with smAll side chAins, AlA And Ser, were compAtible with A one-step bimoleculAr reAction similAr to thAt with wild-type CystAtin A. The decreAsed Affinities of these mutAnts for pApAin And cAthepsin L were due exclusively to increAsed dissociAtion rAte contAnts, but the reduced Affinities for cAthepsin B were due Also to decreAsed AssociAtion rAte constAnts. The lAtter finding indicAtes thAt the intAct N-terminAl region serves As A guide directing CystAtin A...
-
chArActerizAtion by spectroscopic kinetic And equilibrium methods of the interAction between recombinAnt humAn CystAtin A stefin A And cysteine proteinAses
Biochemical Journal, 1995Co-Authors: Ewa Pol, Sergio Estrada, S L Olsson, T W Prasthofer, Ingemar BjörkAbstract:AbstrAct The neAr-UV spectroscopic chAnges induced by the binding of recombinAnt humAn CystAtin A to pApAin were AppreciAbly different from those induced by CystAtin C, reflecting mAinly interActions involving the single tryptophAn of CystAtin C, Trp-106. CystAtin A bound tightly And rApidly to pApAin And cAthepsin L, with dissociAtion equilibrium constAnts of ApproximAtely 10(-11)-10(-13) M And AssociAtion rAte constAnts of 3 x 10(6)-5 x 10(6) M-1.s-1. These Affinities Are At leAst 50-100-fold higher thAn previously reported vAlues. The kinetics of binding to pApAin were consistent with A simple reversible bimoleculAr reAction mechAnism, indicAting thAt CystAtin A, like chicken CystAtin And CystAtin C, binds to pApAin with no AppreciAble conformAtionAl AdAptAtion of either reActing protein. CystAtin A bound more weAkly to Actinidin And cAthepsins B, C And H, with dissociAtion equilibrium constAnts of 10(-8)-10(-9) M. The weAker binding to cAthepsin B wAs lArgely due to A considerAbly reduced AssociAtion rAte constAnt (ApproximAtely 4 x 10(4) M-1.s-1), consistent with the 'occluding loop' of cAthepsin B mArkedly restricting the Access of CystAtin A to the Active site. The lower Affinities for Actinidin And cAthepsins C And H were due pArtly to lower AssociAtion rAte constAnts (2 x 10(5)-6 x 10(5) M-1.s-1) but primArily to higher dissociAtion rAte constAnts. The mode of binding of CystAtin A to inActivAted pApAins indicAted thAt there is AppreciAbly less spAce Around the Active-site cysteine of pApAin in the complex with CystAtin A thAn in the complexes with chicken CystAtin And CystAtin C. An N-terminAlly truncAted form of CystAtin A, lAcking the first six residues, hAd considerAbly lower Affinity for pApAin thAn the full-length inhibitor, consistent with An intAct N-terminAl region being of importAnce for proteinAse binding.
