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生物機能中分子の細胞導入の分子基盤:...
Transcript of 生物機能中分子の細胞導入の分子基盤:...
生物機能中分子の細胞導入の分子基盤:
細胞内送達ペプチドHAadの設計と評価
Introduction
京都大学化学研究所 二木史朗
Development of systems that effectively bring macromolecules into cytosolshould be beneficial for the understanding of biological significance of cytosoliccomponents by modulating their functions using macromolecules, and fordeveloping therapeutic systems based on these understandings. Our laboratorydeveloped a peptide “L17E” that delivers macromolecules into cytosol, by modifyingthe sequence of a hemolytic spider venom peptide M-lycotoxin (Akishiba, M., et
al., Nat. Chem. (2017)). L17E is capable of efficiently delivering various bioactiveproteins, including IgG, into cytosol. Specific recognition of cytosolic proteins andsuppression of glucocorticoid receptor-mediated transcription were thus achievedusing the targeting IgGs in the presence of L17E.
Results & Discussion
[Cells; HeLa, L17E; 40 μM,
Alexa488-hIgG; 1mg/mL,
treatment; 1hr in α-MEM(-
)]
A. Substitution of histidine to alanine residues
*cytotoxic
peptide name sequence
IWLTALKFLGK AAK AKQQLSKL-amide
IWLTALKFLGK AAK AKQQLSKL-amide
IWLTALKFLGK AAK AKQ LSKL-amide
IWLTALKFLGK AAK AKQ LSKL-amide
no peptide
DIC
Ale
xa
48
8
cell death
cell death
52.3%
B. Introduction of acidic amino acid with longer side chain
peptide name sequence
IWLTALKFLGKHAAKH AKQQLSKL-amide
IWLTALKFLGKHAAKH AKQQLSKL-amide
IWLTALKFLGKHAAKH AKQQLSKL-amide
peptide name sequence
IWLTALKFLGK AAK AKQQLSKL-amide
IWLTALKFLGK AAK AKQ LSKL-amide
ce
lls y
ield
ing
cyto
so
lic
de
xtr
an
sig
na
ls (
%)
0
25
50
75
100
40μM 15μM 20μM 30μM 40μM
L17E HAad
X; L-2-aminoadipic acid (Aad)
C. Combination of A and B
13.3% 69.9%0.90%
52.3%
DIC
Ale
xa
48
8 24.5% 62.6%
D. Leakage assay
F. HAad-mediated intracellular delivery of functional protein
DsRed EGFP
DsRed
EGFP
DsRed
EGFP
Cre + peptide
Cre-mediated
recombination
loxP STOP
40 mM 40 mM
52.3% 74.4%
[Cells; HeLa, peptides; 40 μM, Alexa488-dextran (10 kDa); 200 mg/mL,
treatment; 1 hr in serum-free a-MEM]
[Cells; HeLa, peptides; 40 μM, Alexa488-dextran (10 kDa); 200 mg/mL,
treatment; 1 hr in serum-free a-MEM]
[Cells; HeLa, peptides; 40 μM, Alexa488-dextran (10 kDa); 200 mg/mL, treatment; 1 hr in serum-free a-MEM. Results are presented as mean ± SE
(n=3).]
✓ His-to-Ala substitution yielded
an improved intracellular macromolecule-delivering activity.
✓ has no pH selectivity.
✓ has negatively-charged lipid-selective
and low pH-selective membrane destabilizing
activity.
no peptide
DIC
EG
FP
DsR
ed
me
rge
no peptide
Cre 5 mM Cre 10 mM
EG
FP
-po
sitiv
e r
ep
ort
er-
tra
nsfe
cte
d c
ells
(%
)0
20
40
60
80
100
no peptide L17E HAad no peptide L17E HAad
+ Cre 5 μM + Cre 10 μM
[Cells; HeLa expressing loxP-DsRed-STOP-
losP-EGFP reporter, peptides; 40 μM, Cre
recombinase; 5 or 10 μM, treatment; 1 hr in
serum-free a-MEM. Results are presented as mean ± SE (n=3).]
A. Substitution of
His to Ala
B. Substitution of
Glu to L-2-aminoadipic acid
CH3CH3 CH3CH3
✓ Substitution of Glu to Aad also yielded
an improved intracellular macromolecule-delivering activity.
CH3CH3
✓ By combination of both substitutions
HAad was obtained to display the highest efficacy.
-20
0
20
40
60
80
100
120
0.0001 0.001 0.01 0.1 1
L17E
PC 100% LUV, pH 7.4PC 75%/PG 25 % LUV, pH 7.4PC 100% LUV, pH 5.0PC 75%/PG 25 % LUV, pH 5.0
P/L
-20
0
20
40
60
80
100
120
0.0001 0.001 0.01 0.1 1
HAad
PC 100% LUV, pH 7.4PC 75%/PG 25 % LUV, pH 7.4PC 100% LUV, pH 5.0PC 75%/PG 25 % LUV, pH 5.0
P/L
PC, pH7.4
PC, pH5.0
PC, pH7.4
PC, pH5.0
PC/PG,
pH5.0
PC/PG,
pH7.4
PC/PG,
pH5.0
PC/PG,
pH7.4
L-2-aminoadipic
acid (Aad)GluAsp
[lipid concentration; 200μM, lipid composition; 100% POPC or 75% POPC and 25% POPG (mol:mol) encapsulating 12.5mM ANTS/45.0mM DPX, pH; 7.4 or 5.0,
incubation; 1hr at 37℃, leakage (%)=(Fobs-Fblank)/(Fmax-Fblank)*100]
[peptide concentration; 20μM, lipid concentration; 2mM, lipid composition; 100% POPC or 75% POPC and 25% POPG (mol:mol), pH; 7.4 or 5.0, temperature;
25°C,]
E. CD spectroscopy
✓ does not have a marked helical
structure even in the presence of LUVs.
✓ showed almost complete helical
structure in the presence of POPG at pH 5.0.-4x10
4
-3x104
-2x104
-1x104
0
1x104
200 210 220 230 240 250 260
L17E
POPC only LUV (pH 7.4)
POPC only LUV (pH 5.0)
POPC/POPG mix LUV (pH 7.4)
POPC/POPG mix LUV (pH 5.0)
WL (nm)
-4x104
-3x104
-2x104
-1x104
0
1x104
200 210 220 230 240 250 260
HAad
POPC only LUV (pH 7.4)
POPC only LUV (pH 5.0)
POPC/POPG mix LUV (pH 7.4)
POPC/POPG mix LUV (pH 5.0)
WL (nm)
PC, pH7.4PC, pH5.0
PC/PG,
pH5.0PC/PG,
pH7.4
PC, pH7.4PC, pH5.0
PC/PG,
pH5.0
PC/PG,
pH7.4
L17E HAadno peptidebright
field
EG
FP
DsR
ed
co
rona
l se
ctio
n
cut
Cre (5 mM) +
peptide (40 mM)
7 days
G. Intracerebroventrical administration of Cre and HAad
EGFP
DsRed
DsRed
Cre-mediated
recombination
EGFP DsRed
EGFP
loxP
H. Mode of action of HAad
L17E HAadno peptide
Ga
lectin
3
LLOMe
(positive control)
0
25
50
75
100
NH4Cl (-) NH4Cl (+) NH4Cl (-) NH4Cl (+)
L17E HAad
Cel
ls w
ith
cyt
oso
lic
dex
tran
sig
nal
s (%
)
0
25
50
75
100
CQ(-) CQ(+) CQ(-) CQ(+)
L17E HAad
Cel
ls w
ith
cyt
oso
lic
dex
tran
sig
nal
s (%
)*** ***
n.s.
******
n.s.
[mouse; adult female R26GGR mouse expressing loxP-
EGFP-STOP-loxP-DsRed reporter, peptide; 40 μM, Cre
recombinase; 5 μM, treatment; i.c.v. administration for 7
days]
STOP
➢ Immunocytochemistry of Galectin-3
➢ Inhibition of endosomal acidification
✓ In HAad-treated cells Galectin-3 was strongly localized
to disrupted endosomes (red puncta signal).
◆ We are deeply grateful to Dr. Sugiyama and his lab members (Laboratory Animal Science, Tsukuba Univ.) to perform this in vivo experiment.
intact endosome disrupted endosome
b-galactosides
Galectin-3
endosomolysis
Galectin-3 recognizing
b-galactosides
[Cells; HeLa, L17E; 40 μM, HAad; 40 μM, LLOMe; 1 mM, treatment; 1 hr in serum-free a-MEM. 1st antibody; mouse
anti-Galectin 3 antibody [A3A12], 2nd antibody; Alexa Fluor 488 goat anti-mouse IgG (H+L).]
[Cells; HeLa, L17E or HAad; 40 μM, Alexa488-dextran; 200 mg/mL,
NH4Cl: 25 mM, chloroquine (CQ); 100 mM, pretreatment; 30 min w/ or
w/ o NH4Cl or CQ, treatment; 1 hr in serum-free a-MEM w/ or w/o BafA1 or CQ. Results are presented as mean ± SE (n = 3).]
✓ In the presence of endosomal acidification inhibitors, the cytosolic
macromolecule-delivering activity of HAad was attenuated
to the same level of L17E.
✓ The increment of the activity from L17E to HAad would be due to the
obtained/enhanced endosomolytic activity.
To improve the cytosolic delivery activity of L17E, we focused on the helical structure formation of the peptidesdependent on endosomal pH decrease to more interact with endosomal membranes to improve the endosomolytic activity.We have succeeded in obtaining a peptide named “HAad”, by stepwise remodeling of L17E. HAad attained a significantlyimproved cytosolic delivery of various macromolecules (polydextran and Cre recombinase).
In detailed analysis, we have found that L17E has an unique mechanismof action: L17E stimulates polymerization of actin followed by induction ofmembrane ruffling. At that site L17E transiently permeabilize the ruffledplasma membrane and macromolecules enters into cytosol within 5 min.This mode of action is thought to be the crucial mechanism for cytosolicdelivery of macromolecules rather than endosomal destabilization.
Akishiba, M. and Futaki, S., Mol., Pharmaceutics (2019).
Injection to right
lateral ventricle
DA
PI_
DsR
ed
cro
ss s
ection
✓ HAad delivered Cre
even in vivo.