The Presentation by Dr. Mahra Nourbakhsh for Journal Club Possible role for DGAT in Hepatitis C Virus Assembly

1. The Involvement of
Diacylglycerol Acyltransferase
(DGAT) in HCV Assembly

2. Outline of presentation:
1. Initial phase of virion assembly and the role of
lipid droplets.
1. Structure of lipid droplets.
1. Formation of lipid droplets and role of
Diacylglycerol Acyltransferases (DGATs).
1. Interaction between HCV core protein and
DGATs. Selena

3. Release of the core from polyprotein by
SP and SPP from polyprotein
Mature core protein contains two
domains; D1, D2.
D2 contains two amphipathic alpha
helices separated by a hydrophobic loop
and interacts with phospholipid (PL)
layer in ER and later PL surrounding
LDs. Hope et. Al. J. Biol. Chem. 277, 4261–4270
Hope et. Al. J. Biol. Chem. 277, 4261–4270

4. JonesDM,McLauchlanJJ.Biol.Chem.2010;285:22733-22739
Association of core with LDs:
essential for infectious HCV production.
Mutations in D2 that disrupt the core-LD interaction also prevent
virus release. Boulant S et. al. J. Gen. Virol. 88,2204–2213
Miyanari et. al. Nat. Cell Biol. 9,1089–1097
Replication complex is recruited to the core-LD site in an NS5A
dependent manner. Miyanari et. al. Nat. Cell Biol. 9,1089–1097

5. Core Protein On LDs and NS5A on
ER Membrane Adjacent to LDs
Immunoelectron micrographs of LDs labelled with antibodies against
Core, NS5A or both using 15nm (core) and 10nm (NS5A) gold particles.
Core NS5A Core (arrow head)
NS5A (arrow)
Miyanari et. al. Nat. Cell Biol. 9,1089–1097

6. The later stage of HCV assembly:
1-Gain access to E1, E2 reside on luminal leaflet of ER membrane.
2-Obtaining lipid envelop.
3-Possible interaction with very low density lipoprotein pathway and secretion as LVP.
JonesDM,McLauchlanJJ.Biol.Chem.2010;285:22733-22739

7. Structure of Lipid Droplets
Is that all they have??
Only an inert cytoplasmic inclusion??

8. Structure of Lipid Droplets:
• Very dynamic organelles and heterogeneous in size, location and protein
contents.
• 27 proteins have been detected on lipid droplets obtained from HepG2
(hepatoma cells) and 17 in Huh7.
Fuji Fujimoto et. al , Biochim. Biophys. Acta Mol. C Res. 1644 : 47 – 59, Sato et. al. Biochem . 139 : 921 – 930
• Alteration of protein that coat lipid droplets in response to multiple factors,
including tissue-specific expression and metabolic state (basal vs. lipogenic vs.
lipolytic).
• Protein Groups:
1. The PAT protein family: Prelipin, ADRF, TIP 47
2. Membrane trafficking: Rab group of protein
3. Lipid metabolism 4. Chaperons: HSP70,…
5. Cytoskeleton: Actin, Vimentin 6. Endoplasmic Reticulum: Calnexin, PDI,…
7. Signaling proteins: Caveolin-1 8. Mitochondrial protein
9. Miscellaneous.

9. Esterification
Acyltransferases
Hydrolysis
Esterases(lipases)
Central Dogma in TG Synthesis

10. TG Biosynthesis occurs at the
ER.
Two major pathways:
1. Glycerol Phosphate (Kennedy)
Pathway: present in all tissue
1. Monoacylglycerol Pathway:
mostly in small intestine,
important for re-esterification of
partially hydrolyzed TGs

11. DGATs which mediate the final reaction of both pathways of TG biosynthesis are ER
residents.
Newly synthesized TG are thought to be deposited in the membrane, segregate between
the two membrane leaflets and bulge toward cytosol or ER lumen to form lipid droplets
or secreted by lipoprotein.

12. Possible mechanisms of LD
birth
1. Budding process: The PL
layer of LDs is derived from
cytosolic side of the ER
membrane
1. Hatching process: The PL
layer of LDs is derived from
both sides of ER membrane,
explains the presence of ER
proteins on LDs.

13. DGAT-1 and DGAT-2: Distinct Gene Families
DGAT-1: Little is known about its structure and topology
1. Gene: Chromosome 8, 17 exons, 10.62 kb
2. Protein: 500 aa, 50 Kda, 6 hydrophobic regions (transmembrane
domain), forms homotetramers
3. Fatty acyl binding domain at N-terminus
4. Diacylglycerol binding domain at C-terminal
5. Membrane bound protein with O-acetylation activity (MBOAT)

14. DGAT-1 and DGAT-2: Distinct Gene Families
DGAT-2: it is better known
1. Gene: Chromosome 11, 8 exons, 42.03 kb
2. Protein: 350-400 aa, 40-44 KDa, 2 hydrophobic regions (transmembrane
domain), both N and C-terminals toward the cytosol
3. Possible domain at N-terminal with DAG binding capacity
4. Catalytic domain at C-terminal (aa200-360), active site (aa 161-164)
5. Member of DAGAT gene family

15. Biochemistry of DGAT Enzymes
1. Both have DGAT activity in vivo and in vitro
2. DGAT-1 prefers unsaturated or mono saturated FA. DGAT-
2 prefers short to medium chain FA.
1. In general DGAT-2 is more potent than DGAT-1.
1. DGAT-2 more active in lower concentration of substrate
(low Km), DGAT-1 more active in higher concentration of
substrate (high Km).
1. DGAT-1 but not DGAT-2 has additional acyltransferase
activities beyond esterification of DG, such as acyl-
CoA:retinol acyltransferase (ARAT) and monoacylglycerol
acyltrasnferase (MGAT) activities.

16. Cellular Biology of DGAT Enzymes
DGAT-2 is responsible for
incorporating endogenously
synthesized monounsaturated
FA into TG and is involved in
bulk of TG synthesis.
DGAT-1is believed to be
involved in esterification of
exogenous FA taken up by cells
or in recycling of partially
hydrolyzed TG.

17. Physiologic Function of DGAT Enzymes
DGAT-2 deficiency is not compatible
with life.
90% less TG content of Dgat 2 -/- mice,
absence of TG in the liver.
Diminished barrier function of skin,
leading to rapid dehydration
DGAT-1 Can not compensate for the
loss of DGAT-2
Dgat-2 +/- (heterozygotes), normal

18. Physiologic Function of DGAT Enzymes
DGAT-1 deficiency is compatible with life.
50% reduction in adiposity. Maintain a lean
phenotype even on high fat diet.
The same amount of food but, delayed TG
absorption and more energy expenditure.
Dgat-1 +/- (heterozygotes), has less anti obesity
effect compare to (homozygotes).

19. DGAT Enzymes and Lipoprotein Metabolism
1. In hepatocytes and enterocytes may mediate lipoprotein
metabolism and regulate the flux of TG throughout the body.
2. Delayed intestinal absorption but not malabsorption in DGAT-1
deficient mice fed with high fat diet.
1. Theoretically two separate DGAT activities in hepatic
microsomes: cytosolic (overt) and ER luminal (latent) activities.
1. The latent fraction of DGAT activity within ER lumen where
TG bound for secretion is synthesized. DGAT-1 is considered to
be responsible (inconclusive data).
2. The overt DGAT activity (cytosolic); responsible for formation
of cytosolic LDs.

20. Summary
1. Lipid droplets are essential for initial phase of HCV assembly.
2. Lipid droplets are dynamic organelles (not just an inert store),
originally forms by deposition of TG between leaflets of ER
phospholipids bilayer.
3. DGATs (both 1 and 2) mediate the final step of TG biosynthesis
and are the link for formation of lipid droplets from TG.
4. DGAT-2 mostly mediates the formation of TG from newly
synthesized FA during normal daily life, in contrast DGAT-1 is
considered to be responsible for formation of TG from
exogenously supplied FA during fed state.
Does modulation of DGATs alter the HCV virion
assembly/secretion?