메뉴 건너뛰기




Volumn 56, Issue 4, 2013, Pages 1443-1454

Effect of helical conformation and side chain structure on γ-secretase inhibition by β-peptide foldamers: Insight into substrate recognition

Author keywords

[No Author keywords available]

Indexed keywords

AMYLOID BETA PROTEIN; FUNCTIONAL GROUP; GAMMA SECRETASE; PRESENILIN 1;

EID: 84874590103     PISSN: 00222623     EISSN: 15204804     Source Type: Journal    
DOI: 10.1021/jm301306c     Document Type: Article
Times cited : (24)

References (59)
  • 1
    • 79953854897 scopus 로고    scopus 로고
    • Alzheimer's disease: The challenge of the second century
    • Holtzman, D. M.; Morris, J. C.; Goate, A. M. Alzheimer's disease: the challenge of the second century Sci. Transl. Med. 2011, 3, 77sr1
    • (2011) Sci. Transl. Med. , vol.3
    • Holtzman, D.M.1    Morris, J.C.2    Goate, A.M.3
  • 2
    • 0028169925 scopus 로고
    • Visualization of Aβ42(43) and Aβ40 in senile plaques with end-specific Aβ monoclonals: Evidence that an initially deposited species is Aβ42(43)
    • Iwatsubo, T.; Odaka, A.; Suzuki, N.; Mizusawa, H.; Nukina, N.; Ihara, Y. Visualization of Aβ42(43) and Aβ40 in senile plaques with end-specific Aβ monoclonals: evidence that an initially deposited species is Aβ42(43) Neuron 1994, 13, 45-53
    • (1994) Neuron , vol.13 , pp. 45-53
    • Iwatsubo, T.1    Odaka, A.2    Suzuki, N.3    Mizusawa, H.4    Nukina, N.5    Ihara, Y.6
  • 3
    • 66349133387 scopus 로고    scopus 로고
    • Secretase inhibitors and modulators for Alzheimer's disease treatment
    • Tomita, T. Secretase inhibitors and modulators for Alzheimer's disease treatment Expert Rev. Neurother. 2009, 9, 661-679
    • (2009) Expert Rev. Neurother. , vol.9 , pp. 661-679
    • Tomita, T.1
  • 4
    • 76849086405 scopus 로고    scopus 로고
    • The secretases: Enzymes with therapeutic potential in Alzheimer disease
    • De Strooper, B.; Vassar, R.; Golde, T. The secretases: enzymes with therapeutic potential in Alzheimer disease Nat. Rev. Neurol. 2010, 6, 99-107
    • (2010) Nat. Rev. Neurol. , vol.6 , pp. 99-107
    • De Strooper, B.1    Vassar, R.2    Golde, T.3
  • 5
    • 84655164279 scopus 로고    scopus 로고
    • γ-Secretase inhibitors and modulators for Alzheimer's disease
    • Wolfe, M. S. γ-Secretase inhibitors and modulators for Alzheimer's disease J. Neurochem. 2012, 120 (Suppl. 1) 89-98
    • (2012) J. Neurochem. , vol.120 , Issue.SUPPL. 1 , pp. 89-98
    • Wolfe, M.S.1
  • 6
    • 70350059834 scopus 로고    scopus 로고
    • Recent advances in the identification of γ-secretase inhibitors to clinically test the Aβ oligomer hypothesis of Alzheimer's disease
    • Kreft, A. F.; Martone, R.; Porte, A. Recent advances in the identification of γ-secretase inhibitors to clinically test the Aβ oligomer hypothesis of Alzheimer's disease J. Med. Chem. 2009, 52, 6169-6188
    • (2009) J. Med. Chem. , vol.52 , pp. 6169-6188
    • Kreft, A.F.1    Martone, R.2    Porte, A.3
  • 7
    • 79851473226 scopus 로고    scopus 로고
    • γ-Secretase modulators as potential disease modifying anti-Alzheimer's drugs
    • Oehlrich, D.; Berthelot, D. J.-C.; Gijsen, H. J. M. γ-Secretase modulators as potential disease modifying anti-Alzheimer's drugs J. Med. Chem. 2011, 54, 669-698
    • (2011) J. Med. Chem. , vol.54 , pp. 669-698
    • Oehlrich, D.1    Berthelot, D.J.-C.2    Gijsen, H.J.M.3
  • 14
    • 33744949267 scopus 로고    scopus 로고
    • C-terminal fragment of presenilin is the molecular target of a dipeptidic γ-secretase-specific inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-L- alanyl]-S-phenylglycine t-butyl ester)
    • DOI 10.1074/jbc.M513012200
    • Morohashi, Y.; Kan, T.; Tominari, Y.; Fuwa, H.; Okamura, Y.; Watanabe, N.; Sato, C.; Natsugari, H.; Fukuyama, T.; Iwatsubo, T.; Tomita, T. C-terminal fragment of presenilin is the molecular target of a dipeptidic γ-secretase-specific inhibitor DAPT (N -[ N -(3,5-difluorophenacetyl)- l -alanyl]- S -phenylglycine t -butyl ester J. Biol. Chem. 2006, 281, 14670-14676 (Pubitemid 43855171)
    • (2006) Journal of Biological Chemistry , vol.281 , Issue.21 , pp. 14670-14676
    • Morohashi, Y.1    Kan, T.2    Tominari, Y.3    Fuwa, H.4    Okamura, Y.5    Watanabe, N.6    Sato, C.7    Natsugari, H.8    Fukuyama, T.9    Iwatsubo, T.10    Tomita, T.11
  • 15
    • 79959636033 scopus 로고    scopus 로고
    • The many substrates of presenilin/γ-secretase
    • Haapasalo, A.; Kovacs, D. M. The many substrates of presenilin/γ- secretase J. Alzheimer's Dis. 2011, 25, 3-28
    • (2011) J. Alzheimer's Dis. , vol.25 , pp. 3-28
    • Haapasalo, A.1    Kovacs, D.M.2
  • 16
    • 43049163813 scopus 로고    scopus 로고
    • Substrate specificity of γ-secretase and other intramembrane proteases
    • Beel, A. J.; Sanders, C. R. Substrate specificity of γ-secretase and other intramembrane proteases Cell. Mol. Life Sci. 2008, 65, 1311-1334
    • (2008) Cell. Mol. Life Sci. , vol.65 , pp. 1311-1334
    • Beel, A.J.1    Sanders, C.R.2
  • 20
    • 9444226868 scopus 로고    scopus 로고
    • The world of β- And γ-peptides comprised of homologated proteinogenic amino acids and other components
    • Seebach, D.; Beck, A. K.; Bierbaum, D. J. The world of β- and γ-peptides comprised of homologated proteinogenic amino acids and other components Chem. Biodiversity 2004, 1, 1111-1239
    • (2004) Chem. Biodiversity , vol.1 , pp. 1111-1239
    • Seebach, D.1    Beck, A.K.2    Bierbaum, D.J.3
  • 21
    • 0035471135 scopus 로고    scopus 로고
    • β-Peptides: From structure to function
    • Cheng, R. P.; Gellman, S. H.; DeGrado, W. F. β-Peptides: from structure to function Chem. Rev. 2001, 101, 3219-3232
    • (2001) Chem. Rev. , vol.101 , pp. 3219-3232
    • Cheng, R.P.1    Gellman, S.H.2    Degrado, W.F.3
  • 23
    • 3242759017 scopus 로고    scopus 로고
    • Discovery of a subnanomolar helical D-tridecapeptide inhibitor of γ-secretase
    • DOI 10.1021/jm049788c
    • Bihel, F.; Das, C.; Bowman, M. J.; Wolfe, M. S. Discovery of a subnanomolar helical d -tridecapeptide inhibitor of γ-secretase J. Med. Chem. 2004, 47, 3931-3933 (Pubitemid 38970954)
    • (2004) Journal of Medicinal Chemistry , vol.47 , Issue.16 , pp. 3931-3933
    • Bihel, F.1    Das, C.2    Bowman, M.J.3    Wolfe, M.S.4
  • 24
    • 0025345233 scopus 로고
    • Structural characteristics of α-helical peptide molecules containing Aib residues
    • DOI 10.1021/bi00481a001
    • Karle, I. L.; Balaram, P. Structural characteristics of α-helical peptide molecules containing Aib residues Biochemistry 1990, 29, 6747-6756 (Pubitemid 20225472)
    • (1990) Biochemistry , vol.29 , Issue.29 , pp. 6747-6756
    • Karle, I.L.1    Balaram, P.2
  • 25
    • 0001210551 scopus 로고
    • Non-standard amino acids in peptide design and protein engineering
    • Balaram, P. Non-standard amino acids in peptide design and protein engineering Curr. Opin. Struct. Biol. 1992, 2, 845-851
    • (1992) Curr. Opin. Struct. Biol. , vol.2 , pp. 845-851
    • Balaram, P.1
  • 26
    • 0037178109 scopus 로고    scopus 로고
    • Mimicry of host-defense peptides by unnatural oligomers: Antimicrobial β-peptides
    • DOI 10.1021/ja0260871
    • Porter, E. A.; Weisblum, B.; Gellman, S. H. Mimicry of host-defense peptides by unnatural oligomers: antimicrobial β-peptides J. Am. Chem. Soc. 2002, 124, 7324-7330 (Pubitemid 34670441)
    • (2002) Journal of the American Chemical Society , vol.124 , Issue.25 , pp. 7324-7330
    • Porter, E.A.1    Weisblum, B.2    Gellman, S.H.3
  • 28
    • 0024278714 scopus 로고
    • Helix geometry in proteins
    • Barlow, D. J.; Thornton, J. M. Helix geometry in proteins J. Mol. Biol. 1988, 201, 601-619
    • (1988) J. Mol. Biol. , vol.201 , pp. 601-619
    • Barlow, D.J.1    Thornton, J.M.2
  • 29
    • 77957302949 scopus 로고    scopus 로고
    • Crystallographic characterization of 12-helical secondary structure in β-peptides containing side chain groups
    • Choi, S. H.; Guzei, I. a; Spencer, L. C.; Gellman, S. H. Crystallographic characterization of 12-helical secondary structure in β-peptides containing side chain groups J. Am. Chem. Soc. 2010, 132, 13879-13885
    • (2010) J. Am. Chem. Soc. , vol.132 , pp. 13879-13885
    • Choi, S.H.1    Guzei, I.A.2    Spencer, L.C.3    Gellman, S.H.4
  • 30
    • 0027624925 scopus 로고
    • CD of proline-rich polypeptides: Application to the study of the repetitive domain of maize glutelin-2
    • Rabanal, F.; Ludevid, M. D.; Pons, M.; Giralt, E. CD of proline-rich polypeptides: application to the study of the repetitive domain of maize glutelin-2 Biopolymers 1993, 33, 1019-1028 (Pubitemid 23184437)
    • (1993) Biopolymers , vol.33 , Issue.7 , pp. 1019-1028
    • Rabanal, F.1    Ludevid, M.D.2    Pons, M.3    Giralt, E.4
  • 32
    • 0035838894 scopus 로고    scopus 로고
    • An efficient route to either enantiomer of trans-2- aminocyclopentanecarboxylic acid
    • DOI 10.1021/jo010279h
    • LePlae, P. R.; Umezawa, N.; Lee, H. S.; Gellman, S. H. An efficient route to either enantiomer of trans -2-aminocyclopentanecarboxylic acid J. Org. Chem. 2001, 66, 5629-5632 (Pubitemid 32878926)
    • (2001) Journal of Organic Chemistry , vol.66 , Issue.16 , pp. 5629-5632
    • LePlae, P.R.1    Umezawa, N.2    Lee, H.-S.3    Gellman, S.H.4
  • 34
    • 0001529568 scopus 로고    scopus 로고
    • Theoretical and experimental circular dichroic spectra of the novel helical foldamer poly[(1 R,2 R)- trans -2-aminocyclopentanecarboxylic acid]
    • Applequist, J.; Bode, K. A.; Appella, D. H.; Christianson, L. A.; Gellman, S. H. Theoretical and experimental circular dichroic spectra of the novel helical foldamer poly[(1 R,2 R)- trans -2-aminocyclopentanecarboxylic acid] J. Am. Chem. Soc. 1998, 120, 4891-4892
    • (1998) J. Am. Chem. Soc. , vol.120 , pp. 4891-4892
    • Applequist, J.1    Bode, K.A.2    Appella, D.H.3    Christianson, L.A.4    Gellman, S.H.5
  • 35
    • 0033603830 scopus 로고    scopus 로고
    • Synthesis and structural characterization of helix-forming β-peptides: Trans -2-aminocyclopentanecarboxylic acid oligomers
    • Appella, D. H.; Christianson, L. A.; Klein, D. A.; Richards, M. R.; Powell, D. R.; Gellman, S. H. Synthesis and structural characterization of helix-forming β-peptides: trans -2-aminocyclopentanecarboxylic acid oligomers J. Am. Chem. Soc. 1999, 121, 7574-7581
    • (1999) J. Am. Chem. Soc. , vol.121 , pp. 7574-7581
    • Appella, D.H.1    Christianson, L.A.2    Klein, D.A.3    Richards, M.R.4    Powell, D.R.5    Gellman, S.H.6
  • 36
    • 0028235205 scopus 로고
    • Benzophenone photophores in biochemistry
    • DOI 10.1021/bi00185a001
    • Dorman, G.; Prestwich, G. D. Benzophenone photophores in biochemistry Biochemistry 1994, 33, 5661-5673 (Pubitemid 24184566)
    • (1994) Biochemistry , vol.33 , Issue.19 , pp. 5661-5673
    • Dorman, G.1    Prestwich, G.D.2
  • 37
    • 34347251573 scopus 로고    scopus 로고
    • Signal peptide peptidases and gamma-secretase: Cousins of the same protease family?
    • DOI 10.1159/000101835
    • Fluhrer, R.; Haass, C. Signal peptide peptidases and γ-secretase: cousins of the same protease family? Neurodegener. Dis. 2007, 4, 112-116 (Pubitemid 47000404)
    • (2007) Neurodegenerative Diseases , vol.4 , Issue.2-3 , pp. 112-116
    • Fluhrer, R.1    Haass, C.2
  • 38
    • 5644240819 scopus 로고    scopus 로고
    • A signal peptide peptidase (SPP) reporter activity assay based on the cleavage of type II membrane protein substrates provides further evidence for an inverted orientation of the SPP active site relative to presenilin
    • DOI 10.1074/jbc.M405879200
    • Nyborg, A. C.; Jansen, K.; Ladd, T. B.; Fauq, A.; Golde, T. E. A signal peptide peptidase (SPP) reporter activity assay based on the cleavage of type II membrane protein substrates provides further evidence for an inverted orientation of the SPP active site relative to presenilin J. Biol. Chem. 2004, 279, 43148-43156 (Pubitemid 39372210)
    • (2004) Journal of Biological Chemistry , vol.279 , Issue.41 , pp. 43148-43156
    • Nyborg, A.C.1    Jansen, K.2    Ladd, T.B.3    Fauq, A.4    Golde, T.E.5
  • 40
    • 33745586176 scopus 로고    scopus 로고
    • Signal peptide peptidase: Biochemical properties and modulation by nonsteroidal antiinflanimatory drugs
    • DOI 10.1021/bi060597g
    • Sato, T.; Nyborg, A. C.; Iwata, N.; Diehl, T. S.; Saido, T. C.; Golde, T. E.; Wolfe, M. S. Signal peptide peptidase: biochemical properties and modulation by nonsteroidal antiinflammatory drugs Biochemistry 2006, 45, 8649-8656 (Pubitemid 44078885)
    • (2006) Biochemistry , vol.45 , Issue.28 , pp. 8649-8656
    • Sato, T.1    Nyborg, A.C.2    Iwata, N.3    Diehl, T.S.4    Saido, T.C.5    Golde, T.E.6    Wolfe, M.S.7
  • 41
    • 57749120456 scopus 로고    scopus 로고
    • Distinct pharmacological effects of inhibitors of signal peptide peptidase and γ-secretase
    • Sato, T.; Ananda, K.; Cheng, C. I.; Suh, E. J.; Narayanan, S.; Wolfe, M. S. Distinct pharmacological effects of inhibitors of signal peptide peptidase and γ-secretase J. Biol. Chem. 2008, 283, 33287-33295
    • (2008) J. Biol. Chem. , vol.283 , pp. 33287-33295
    • Sato, T.1    Ananda, K.2    Cheng, C.I.3    Suh, E.J.4    Narayanan, S.5    Wolfe, M.S.6
  • 42
    • 0035382627 scopus 로고    scopus 로고
    • The outstanding biological stability of β- and γ-peptides toward proteolytic enzymes: An in vitro investigation with fifteen peptidases
    • Frackenpohl, J.; Arvidsson, P. I.; Schreiber, J. V.; Seebach, D. The outstanding biological stability of β- and γ-peptides toward proteolytic enzymes: an in vitro investigation with fifteen peptidases ChemBioChem 2001, 2, 445-455 (Pubitemid 33723576)
    • (2001) ChemBioChem , vol.2 , Issue.6 , pp. 445-455
    • Frackenpohl, J.1    Arvidsson, P.I.2    Schreiber, J.V.3    Seebach, D.4
  • 43
    • 0037134881 scopus 로고    scopus 로고
    • Tolerance of acyclic residues in the β-peptide 12-helix: Access to diverse side-chain arrays for biological applications
    • DOI 10.1021/ja017869h
    • LePlae, P. R.; Fisk, J. D.; Porter, E. A.; Weisblum, B.; Gellman, S. H. Tolerance of acyclic residues in the β-peptide 12-helix: access to diverse side-chain arrays for biological applications J. Am. Chem. Soc. 2002, 124, 6820-6821 (Pubitemid 34633571)
    • (2002) Journal of the American Chemical Society , vol.124 , Issue.24 , pp. 6820-6821
    • LePlae, P.R.1    Fisk, J.D.2    Porter, E.A.3    Weisblum, B.4    Gellman, S.H.5
  • 44
    • 0000333938 scopus 로고    scopus 로고
    • 3-Amino Acids and Their Use as Building Blocks for the Solid-Phase Synthesis of β-Peptides
    • 3-amino acids and their use as building blocks for the solid-phase synthesis of β-peptides Helv. Chim. Acta 1998, 81, 187-206 (Pubitemid 128498257)
    • (1998) Helvetica Chimica Acta , vol.81 , Issue.2 , pp. 187-206
    • Guichard, G.1    Abele, S.2    Seebach, D.3
  • 45
    • 0031778381 scopus 로고    scopus 로고
    • Synthesis of Fmoc-β-homoamino acids by ultrasound-promoted wolff rearrangement
    • Müller, A.; Vogt, C.; Sewald, N. Synthesis of Fmoc-β-homoamino acids by ultrasound-promoted Wolff rearrangement Synthesis 1998, 837-841 (Pubitemid 28282806)
    • (1998) Synthesis , Issue.6 , pp. 837-841
    • Muller, A.1    Vogt, C.2    Sewald, N.3
  • 46
    • 0035913057 scopus 로고    scopus 로고
    • Peptide folding induces high and selective affinity of a linear and small β-peptide to the human somatostatin receptor 4
    • DOI 10.1021/jm010816q
    • Gademann, K.; Kimmerlin, T.; Hoyer, D.; Seebach, D. Peptide folding induces high and selective affinity of a linear and small β-peptide to the human somatostatin receptor 4 J. Med. Chem. 2001, 44, 2460-2468 (Pubitemid 32852166)
    • (2001) Journal of Medicinal Chemistry , vol.44 , Issue.15 , pp. 2460-2468
    • Gademann, K.1    Kimmerlin, T.2    Hoyer, D.3    Seebach, D.4
  • 49
    • 6344221515 scopus 로고    scopus 로고
    • Asymmetric synthesis of the cis - And trans -stereoisomers of 4-aminopyrrolidine-3-carboxylic acid and 4-aminotetrahydrofuran-3-carboxylic acid
    • Bunnage, M. E.; Davies, S. G.; Roberts, P. M.; Smith, A. D.; Withey, J. M. Asymmetric synthesis of the cis-and trans -stereoisomers of 4-aminopyrrolidine-3-carboxylic acid and 4-aminotetrahydrofuran-3-carboxylic acid Org. Biomol. Chem. 2004, 2, 2763-2776
    • (2004) Org. Biomol. Chem. , vol.2 , pp. 2763-2776
    • Bunnage, M.E.1    Davies, S.G.2    Roberts, P.M.3    Smith, A.D.4    Withey, J.M.5
  • 50
    • 14644390240 scopus 로고    scopus 로고
    • Structural role of glycine in amyloid fibrils formed from transmembrane α-helices
    • DOI 10.1021/bi047827g
    • Liu, W.; Crocker, E.; Zhang, W.; Elliott, J. I.; Luy, B.; Li, H.; Aimoto, S.; Smith, S. O. Structural role of glycine in amyloid fibrils formed from transmembrane α-helices Biochemistry 2005, 44, 3591-3597 (Pubitemid 40322028)
    • (2005) Biochemistry , vol.44 , Issue.9 , pp. 3591-3597
    • Liu, W.1    Crocker, E.2    Zhang, W.3    Elliott, J.I.4    Luy, B.5    Li, H.6    Aimoto, S.7    Smith, S.O.8
  • 53
    • 46749127360 scopus 로고    scopus 로고
    • The C-terminal PAL motif and transmembrane domain 9 of presenilin 1 are involved in the formation of the catalytic pore of the γ-secretase
    • Sato, C.; Takagi, S.; Tomita, T.; Iwatsubo, T. The C-terminal PAL motif and transmembrane domain 9 of presenilin 1 are involved in the formation of the catalytic pore of the γ-secretase J. Neurosci. 2008, 28, 6264-6271
    • (2008) J. Neurosci. , vol.28 , pp. 6264-6271
    • Sato, C.1    Takagi, S.2    Tomita, T.3    Iwatsubo, T.4
  • 54
    • 77953782609 scopus 로고    scopus 로고
    • Functional analysis of the transmembrane domains of presenilin 1: Participation of transmembrane domains 2 and 6 in the formation of initial substrate-binding site of γ-secretase
    • Watanabe, N.; Image Image, I. I.; Takagi, S.; Tominaga, A.; Image Image, I.; Tomita, T.; Iwatsubo, T. Functional analysis of the transmembrane domains of presenilin 1: participation of transmembrane domains 2 and 6 in the formation of initial substrate-binding site of γ-secretase J. Biol. Chem. 2010, 285, 19738-19746
    • (2010) J. Biol. Chem. , vol.285 , pp. 19738-19746
    • Watanabe, N.1    Image Image, I.I.2    Takagi, S.3    Tominaga, A.4    Image Image, I.5    Tomita, T.6    Iwatsubo, T.7
  • 55
    • 78649393418 scopus 로고    scopus 로고
    • Participation of transmembrane domain 1 of presenilin 1 in the catalytic pore structure of the γ-secretase
    • Takagi, S.; Tominaga, A.; Sato, C.; Tomita, T.; Iwatsubo, T. Participation of transmembrane domain 1 of presenilin 1 in the catalytic pore structure of the γ-secretase J. Neurosci. 2010, 30, 15943-15950
    • (2010) J. Neurosci. , vol.30 , pp. 15943-15950
    • Takagi, S.1    Tominaga, A.2    Sato, C.3    Tomita, T.4    Iwatsubo, T.5
  • 58
    • 0029839894 scopus 로고    scopus 로고
    • Efficient synthesis of (S)-methyl hexafluorovalinate
    • Keese, R.; Hinderling, C. Efficient synthesis of (S)-methyl hexafluorovalinate Synthesis 1996, 695-696 (Pubitemid 26295424)
    • (1996) Synthesis , Issue.6 , pp. 695-696
    • Keese, R.1    Hinderling, C.2
  • 59
    • 34250336128 scopus 로고    scopus 로고
    • Aβ42 overproduction associated with structural changes in the catalytic pore of γ-secretase: Common effects of Pen-2 N-terminal elongation and fenofibrate
    • DOI 10.1074/jbc.M611549200
    • Isoo, N.; Sato, C.; Miyashita, H.; Shinohara, M.; Takasugi, N.; Morohashi, Y.; Tsuji, S.; Tomita, T.; Iwatsubo, T. Aβ42 overproduction associated with structural changes in the catalytic pore of γ-secretase: common effects of Pen-2 N-terminal elongation and fenofibrate J. Biol. Chem. 2007, 282, 12388-12396 (Pubitemid 47100594)
    • (2007) Journal of Biological Chemistry , vol.282 , Issue.17 , pp. 12388-12396
    • Isoo, N.1    Sato, C.2    Miyashita, H.3    Shinohara, M.4    Takasugi, N.5    Morohashi, Y.6    Tsuji, S.7    Tomita, T.8    Iwatsubo, T.9


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.