Structural determinants of lipoprotein(a) pathogenicity Marlys L. - - PowerPoint PPT Presentation

▶

Jan 28, 2024 25 likes •318 views

Structural determinants of lipoprotein(a) pathogenicity Marlys L. Koschinsky, PhD FAHA FNLA Scientific & Executive Director Robarts Research Institute Professor, Dept. of Physiology & Pharmacology Schulich School of Medicine &

SLIDE 1

Structural determinants of lipoprotein(a) pathogenicity

Marlys L. Koschinsky, PhD FAHA FNLA Scientific & Executive Director Robarts Research Institute Professor, Dept. of Physiology & Pharmacology Schulich School of Medicine & Dentistry The University of Western Ontario @RobartsDirector

SLIDE 2

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).

SLIDE 3

Acknowledgements

Team Koschinsky

Dr. Amer Youssef

Julia St. John Matthew Borrelli Justin Clark Bella Xing Ahmed Habib

Team Boffa

Tasnim Reza Abdullah Masoodi Kevin Zhang John Ackersviller

Alumni

Dr. Corey Scipione
Dr. Rocco Romagnuolo

Jackson McAiney Matthew Gemin

IRCM

Dr. Nabil Seidah
Dr. Annik Prat

UWO

Dr. Robert Hegele
Dr. Murray Junop

Robert Szabla

University of Amsterdam

Dr. Erik Stroes

Renate Hoogeveen

University of Washington

Dr. Santica Marcovina

University of Helsinki

Dr. Kati Öörni
Dr. Martina Lorey

University of California, San Diego

Dr. Sam Tsimikas
Dr. Joe Witztum

SLIDE 4

SLIDE 5

Lp(a) assembly – the science is not settled

SLIDE 6

Models from in vivo kinetics

Model I Lp(a) formed in or on hepatocytes Model IIb Lp(a)-apoB recycled Model IIa Some contribution from circulating LDL Model III Apo(a) recycled

Reyes-Soffer G, et al. J Lipid Res 2017;58:1756

SLIDE 7

Evidence for coupling of apo(a) and Lp(a)-apoB biosynthesis

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 apoB

antisense 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

SLIDE 8

PCSK9 enhances apoB secretion

HepG2 - 17K

J. Clark

SLIDE 9

PCSK9 increases apo(a) secretion

HepG2 - 17K

J. Clark

SLIDE 10

HepG2 - 17K∆LBS7,8 HepG2 - 17K

J. Clark

PCSK9 effect dependent on apo(a):apoB interaction

SLIDE 11

Lomitapide decreases apoB secretion

HepG2 - 17K

J. Clark

SLIDE 12

Lomitapide decreases apo(a) secretion

HepG2 - 17K

J. Clark

SLIDE 13

HepG2 - 17K∆LBS7,8

Lomitapide effect dependent on apo(a):apoB interaction

J. Clark

SLIDE 14

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

HepG2 - 17K

M. Gemin

SLIDE 15

Sortilin effect dependent on apo(a):apoB interaction

HepG2 - 17K∆LBS7,8 HepG2 - 17K∆LBS10

M. Gemin

SLIDE 16

Effect of SORT1 variants on apo(a) secretion

apo(a)

J. Clark
Dr. R. Hegele

SLIDE 17

HepG2 - 17K HepG2

Effect of sortilin overexpression on apoB secretion

J. Clark

SLIDE 18

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

REDUCING CONDITIONS

Dr. A. Youssef

Inp

⍺1-4- anti-apo(a) Poly anti-ApoB Inp

⍺1-4- anti-apo(a) Poly anti-ApoB Lysates Glycine ε-ACA IP: Media IB: A5 Anti-apo(a) [aa] 100 mM [aa] 200 mM [aa] 200 mM Apo(a) Apo(a) Apo(a)

HepG2 - 17K

SLIDE 19

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

NON-REDUCING CONDITIONS

HepG2 - 17K

Dr. A. Youssef

SLIDE 20

Colocalization of apo(a) and apoB intracellularly

Dr. A. Youssef

Calnexin Apo(a) ApoB DAPI Merged TGN46 Apo(a) ApoB DAPI Merged LAMP1 Apo(a) ApoB DAPI Merged EEA1 Apo(a) ApoB DAPI Merged

Triple Colocalization

SLIDE 21

Mechanism?

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

SLIDE 22

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

SLIDE 23

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:9 78-951-51-3522-3

SLIDE 24

Inflammasome induction in THP-1 macrophages

0 . 0 2 . 5 5 . 0 2 0 0 4 0 0 6 0 0 8 0 0

m R N A e x p r e s s i o n ( f o l d ) n o r m a l i z e d t o G A P D H I L - 1 β I L - 8 I L - 1 8

M. Borrelli
Dr. A. Youssef
Dr. K. Öörni
Dr. M. Lorey

SLIDE 25

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

SLIDE 26

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

SLIDE 27

Thr64: enhanced oxPL modification? KIV10-KV di-kringle constructs

DTT - + - + M64T D56A DTT - + - + M64T D56A

IB: E06 α-oxPL IB: poly- clonal α-apo(a) re-probe

Elution progression

D56A M64T Wt

ε-ACA

M. Borrelli

protein stain

SLIDE 28

Modeling of M64T substitution in KIV10

M64 model T64 model (pdb: 3kiv)

Energy minimization: Rosetta Relax
Structure visualization: PyMOL

T64 M64 H31 H33 R71 ε-ACA

R. Szabla
Dr. M. Junop

SLIDE 29

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