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

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).

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

Lp(a) assembly – the science is not settled

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

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

PCSK9 enhances apoB secretion

HepG2 - 17KJ. Clark

PCSK9 increases apo(a) secretionHepG2 - 17K

J. Clark

HepG2 - 17K∆LBS7,8HepG2 - 17K

J. Clark

PCSK9 effect dependent on apo(a):apoB interaction

Lomitapide decreases apoB secretionHepG2 - 17K

J. Clark

Lomitapide decreases apo(a) secretionHepG2 - 17K

J. Clark

HepG2 - 17K∆LBS7,8

Lomitapide effect dependent on apo(a):apoB interactionJ. Clark

Effect of sortilin overexpression and knockdown on apo(a) secretion

HepG2 - 17KM. Gemin

Sortilin effect dependent on apo(a):apoB interactionHepG2 - 17K∆LBS7,8HepG2 - 17K∆LBS10

M. Gemin

Effect of SORT1 variants on apo(a) secretionapo(a)

J. Clark

Dr. R. Hegele

HepG2 - 17K

HepG2

Effect of sortilin overexpression on apoB secretionJ. Clark

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

Co-IP of apo(a) and apoB from lysates –NON-REDUCING CONDITIONS

HepG2 - 17K

Dr. A. Youssef

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

Mechanism?modified from Fisher E, et al. J Biomed Res 2014;28:178

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

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

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

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

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

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

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

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