structural determinants of lipoprotein(a) pathogenicity · research council of canada, and pfizer;...
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Structural determinants of lipoprotein(a) pathogenicity
Marlys L. Koschinsky, PhD FAHA FNLAScientific & Executive DirectorRobarts Research InstituteProfessor, Dept. of Physiology & PharmacologySchulich School of Medicine & DentistryThe University of Western Ontario @RobartsDirector
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Disclosures
Marlys L. Koschinsky holds/has held research grants from Canadian Institutes of Health Research, Heart and Stroke Foundation of Canada, Natural Sciences and Engineering Research Council of Canada, and Pfizer; is a member of advisory boards for Sanofi and Amgen; has received speaker’s honoraria/consulting fees from Amgen, Regeneron, and Eli Lilly; and holds/has held research contracts with Sanofi, Ionis, Eli Lilly, and Abcentra (Cardiovax).
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Acknowledgements
Team KoschinskyDr. Amer YoussefJulia St. JohnMatthew BorrelliJustin ClarkBella XingAhmed Habib
Team BoffaTasnim RezaAbdullah MasoodiKevin ZhangJohn Ackersviller
AlumniDr. Corey ScipioneDr. Rocco RomagnuoloJackson McAineyMatthew Gemin
IRCMDr. Nabil SeidahDr. Annik Prat
UWODr. Robert HegeleDr. Murray JunopRobert Szabla
University of AmsterdamDr. Erik StroesRenate Hoogeveen
University of WashingtonDr. Santica Marcovina
University of HelsinkiDr. Kati ÖörniDr. Martina Lorey
University of California, San DiegoDr. Sam TsimikasDr. Joe Witztum
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Lp(a) assembly – the science is not settled
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Models from in vivo kineticsModel ILp(a) formed in or on hepatocytes
Model IIbLp(a)-apoBrecycled
Model IIaSome contribution from circulating LDL
Model IIIApo(a) recycled
Reyes-Soffer G, et al. J Lipid Res 2017;58:1756
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Evidence for coupling of apo(a) and Lp(a)-apoBbiosynthesis
• Oleate stimulates apo(a) secretion from HepG2 cells• Nassir F, et al. J Biol Chem 1998;273:17793
• Lp(a) levels are reduced by an MTP inhibitor (lomitapide) and by apoBantisense oligonucleotide (mipomersen)
• Samaha FF, et al. Nat Clin Pract Cardiovasc Med 2008;5:497• Santos RD, et al. ATVB 2015;35:689
• PCSK9 inhibitor (evolocumab) monotherapy reduces apo(a) PR• Watts GF et al. Eur Heart J 2018;39:2577
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PCSK9 enhances apoB secretion
HepG2 - 17KJ. Clark
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PCSK9 increases apo(a) secretionHepG2 - 17K
J. Clark
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HepG2 - 17K∆LBS7,8HepG2 - 17K
J. Clark
PCSK9 effect dependent on apo(a):apoB interaction
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Lomitapide decreases apoB secretionHepG2 - 17K
J. Clark
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Lomitapide decreases apo(a) secretionHepG2 - 17K
J. Clark
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HepG2 - 17K∆LBS7,8
Lomitapide effect dependent on apo(a):apoB interactionJ. Clark
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Effect of sortilin overexpression and knockdown on apo(a) secretion
HepG2 - 17KM. Gemin
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Sortilin effect dependent on apo(a):apoB interactionHepG2 - 17K∆LBS7,8HepG2 - 17K∆LBS10
M. Gemin
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Effect of SORT1 variants on apo(a) secretionapo(a)
J. Clark
Dr. R. Hegele
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HepG2 - 17K
HepG2
Effect of sortilin overexpression on apoB secretionJ. Clark
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Co-IP of apo(a) and apoB from lysates –REDUCING CONDITIONS
Dr. A. Youssef
Inp
-ve
⍺1-4
-ant
i-apo
(a)
Poly
ant
i-Apo
B
Inp
-ve
⍺1-4
-ant
i-apo
(a)
Poly
ant
i-Apo
B
Lysa
tes
Glycine ε-ACA
IP:
Med
iaIB: A
5 An
ti-ap
o(a)
[aa]100 mM
[aa]200 mM
[aa]200 mM
Apo(a)
Apo(a)
Apo(a)
HepG2 - 17K
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Co-IP of apo(a) and apoB from lysates –NON-REDUCING CONDITIONS
HepG2 - 17K
Dr. A. Youssef
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Colocalization of apo(a) and apoB intracellularlyDr. A. Youssef
CalnexinApo(a) ApoBDAPI Merged
TGN46Apo(a) ApoBDAPI Merged
LAMP1Apo(a) ApoBDAPI Merged
EEA1Apo(a) ApoBDAPI Merged
Triple Colocalization
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Mechanism?modified from Fisher E, et al. J Biomed Res 2014;28:178
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OxPL on apo(a) as a unifying hypothesis for the pathogenic effects of Lp(a)
Boffa MB, Koschinsky ML Nat Rev Cardiol 2019, in press
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OxPL on apo(a) and the NLRP3 inflammasome
Dr. M. Lorey dissertation:
Secretome analysis of human macrophages activated by microbial stimuli, http://urn.fi/URN:ISBN:978-951-51-3522-3
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Inflammasome induction in THP-1 macrophages
0 . 0
2 . 5
5 . 0
2 0 0
4 0 0
6 0 0
8 0 0
mR
NA
ex
pr
es
sio
n (
fold
)
no
rm
ali
ze
d t
o G
AP
DH
I L - 1 β
I L - 8
I L - 1 8
M. BorrelliDr. A. Youssef
Dr. K. Öörni
Dr. M. Lorey
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Structure-function relationships in KIV10
ε-ACA
Asp56 (mutated in non-human primates)
Trp72 (mutated in non-human primates)
His3
His31
His33
Thr64(Met)
Arg10(Gln)
pdb: 3kiv
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Mutation of His33 prevents apo(a) secretion
medium lysate
6K (mature)6K (immature)
250 kDa
17K 17K H33A
17K (mature)17K (immature)250 kDa
M. Borrelli
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Thr64: enhanced oxPL modification?KIV10-KV di-kringle constructs
DTT - + - + M64T D56A
DTT - + - + M64T D56A
IB: E06α-oxPL
IB: poly-clonal α-apo(a)re-probe
Elution progression
D56A M64TWt
ε-ACA
M. Borrelli
proteinstain
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Modeling of M64T substitution in KIV10
M64 model
T64 model (pdb: 3kiv)
• Energy minimization: Rosetta Relax
• Structure visualization: PyMOL
T64
M64
H31H33
R71
ε-ACA
R. SzablaDr. M. Junop
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High-level challenges
• Establish plausibility/mechanisms of phenomena arising from in vivo kinetic studies of Lp(a) production and clearance
(e.g. recycling of apo(a) and/or apoB; role of individual receptors)
• Tease apart lysine-binding and oxPL-binding functions of KIV10
• Establish tractable animal model for Lp(a) pathogenicity
• Corroborate pathogenic mechanisms in vivo