The Involvement of Diacylglycerol Acyltransferase

(DGAT) in HCV Assembly

Outline of presentation:

1. Initial phase of virion assembly and the role of lipid droplets.

2. Structure of lipid droplets.

3. Formation of lipid droplets and role of Diacylglycerol Acyltransferases (DGATs).

4. Interaction between HCV core protein and DGATs. Selena

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

Jones D M

, McL

auchlan J J. Biol. C

hem. 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

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

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.

Jones D M

, McL

auchlan J J. Biol. C

hem. 2010;285:22733-22739

Structure of Lipid Droplets

Is that all they have?? Only an inert cytoplasmic inclusion??

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 472. Membrane trafficking: Rab group of protein3. Lipid metabolism 4. Chaperons: HSP70,…5. Cytoskeleton: Actin, Vimentin 6. Endoplasmic Reticulum: Calnexin, PDI,

…7. Signaling proteins: Caveolin-1 8. Mitochondrial protein9. Miscellaneous.

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Esterases (lipases)

Central Dogma in TG Synthesis

TG Biosynthesis occurs at the ER.

Two major pathways:

1. Glycerol Phosphate (Kennedy) Pathway: present in all tissue

2. Monoacylglycerol Pathway: mostly in small intestine, important for re-esterification of partially hydrolyzed TGs

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.

Possible mechanisms of LD birth

1. Budding process: The PL layer of LDs is derived from cytosolic side of the ER membrane

2. Hatching process: The PL layer of LDs is derived from both sides of ER membrane, explains the presence of ER proteins on LDs.

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 kb2. Protein: 500 aa, 50 Kda, 6 hydrophobic regions (transmembrane

domain), forms homotetramers3. Fatty acyl binding domain at N-terminus4. Diacylglycerol binding domain at C-terminal5. Membrane bound protein with O-acetylation activity (MBOAT)

DGAT-1 and DGAT-2: Distinct Gene Families

DGAT-2: it is better known

1. Gene: Chromosome 11, 8 exons, 42.03 kb2. Protein: 350-400 aa, 40-44 KDa, 2 hydrophobic regions (transmembrane

domain), both N and C-terminals toward the cytosol3. Possible domain at N-terminal with DAG binding capacity4. Catalytic domain at C-terminal (aa200-360), active site (aa 161-164)5. Member of DAGAT gene family

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.

3. In general DGAT-2 is more potent than DGAT-1.

4. DGAT-2 more active in lower concentration of substrate (low Km), DGAT-1 more active in higher concentration of substrate (high Km).

5. 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.

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.

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

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).

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.

3. Theoretically two separate DGAT activities in hepatic microsomes: cytosolic (overt) and ER luminal (latent) activities.

4. 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).

5. The overt DGAT activity (cytosolic); responsible for formation of cytosolic LDs.

Summary1. 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?