James McManaman Division of Basic Reproductive Sciences

University of Colorado School of Medicine

• Neonatal Calories • Essential Fatty Acids • Fat Soluble Vitamins

• Ice Cream • Butter • Cheese

fat

what-when-how.com

• What determines the chemical and structural properties of MFG? • How is the interface between the lipid core and the MFG membrane established, and what are the molecular determinants of this interface?

CLD SV

J. Monks, In Preparation

CLD CLD

Milk Space

SV

Mito

• How are CLD plasma membrane docking interactions initiated ? • What determines envelopment? • How does envelopment affect PM molecular and structural organization and how do these changes contribute to secretion?

• What determines CLD production in the lactating mammary gland? • How are the steps of CLD production integrated to meet milk lipid secretion demands? • How are the cellular functions of CLD determined?

Martin and Parton, NatRev 2006

Robust Process Rodents synthesize and secrete their body weights in

lipid during a 20 day period of lactation Developmentally regulated

Timing is precise and understood Biochemical pathways are known and similar to other

lipogenic organs The mammary gland is experimentally accessible

Mouse genetics Exogenous gene expression using adenovirus

• Perilipins are among the most abundant CLD proteins • Structural proteins functioning in CLD assembly and regulation of TG storage and metabolism • ADPH is the principal perilipin on MG CLD

Transcript Protein

Russell et al J Lipid Res, 2007

ADPH/WGA/Nuc

Russell et al J Cell Science, 2011

WT ADPHdef a ba baa bb

pa ba baa bb

pp

Lactation Failure (1st Pregnancy)

C57B6 0/50

ADPHdef 50/50

CLD/TAG Lumen Size

WT ADPHdef

P16

P18

β-Casein/WGA

Russell et al J Cell Sci, 2011

Casein Levels

Casein IF

Adv GFP-ADPH

ADPHdef P16/P18

ADPHdef

a b c

d e f

g h i

a b c

d e f

g h i

a b c

d e f

g h i

a b c

d e f

g h i

a b c

d e f

g h i

GFP ATGL Merge

GFP

-AD

PH

Tran

sduc

ed

Non

-Tr

ansd

uced

G

FP -

Tran

sduc

ed

Adv GFP-ADPH

GFP ATGL Nuclei

P15 / P16

Russell et al J Cell Science,

2011

ADPHdef

Phenotype should be associated with the members of gene family of similar function Lipid droplets are regulated by perilipin

family members Gene should be common to the

phenotype in all species ADPH is enriched on CLD of all

mammalian species studied to date The gene must associated with a

specific phenotype ADPH is highly expressed in

differentiating milk secreting cells ADPH expression is coordinated with

CLD growth and accumulation Specific inactivation of the gene

should lead to a measurable loss of function ADPH depletion interferes with CLD

accumulation Replacement of the gene restores

function GFP-ADPH expression in milk secreting

cells rescues defects in CLD accumulation

ADPH is required for neutral lipid accumulation in the differentiating mammary gland

ADPH regulates neutral lipid accumulation by inhibiting ATGL from binding to the CLD surface

Balanced neutral lipid metabolism is required for normal alveolar maturation during mammary gland differentiation

Fulfillment of “Koch’s Postulate” Neutral Lipid Regulation in the

Differentiating Mammary Gland

Lipid homeostasis is critical for normal mammary gland differentiation/maturation - Impaired triglycerol synthesis and/or stabilization impair alveolar formation and are associated with lactation failure in mice.

Lipid metabolism genes associated with alveolar and lactation defects • GPAT4 • DGAT1 • ADPH • PKB1

Mather and Keenan Model

• Impaired milk lipid secretion in BTN and XOR deficient mice • ADPH, BTN and XOR co-localize on the APM during lactation • XOR binds to the C-terminal B 30.2 domain of BTN • Complex of ADPH, BTN and XOR can be isolated from bovine MFGM

PAT CLD TARGETING 4-Helix Bundle

75 200 220 425 1 115

• Proteolysis • TAG stability

• 11-mer helical repeats • CLD localization

• Predicted from structural studies of TIP47 McManaman et al, JLR, 2003; Orlicky et al JCS 2008

BTN KO mouse - Ogg et al, PNAS, 2004

Russell et al, J Lipid Res 2007

CLD size dramatically decreases between P18 and lactation day 2

Interference with milk lipid secretion results in retention of large CLD in mammary epithelial cells

Secretion Assay

AdGFP-ADPH1-220

WT Mice P17/L2

Chong et al JBC 2011

GFP-ADPH(1-220) GFP

NT Region of ADPH

53 62% helix -TFE

MW SS WB

25kD

ADPH (172-425)

Chong et al, JBC 2011

CD = 52% Helix

ADPH-CT(170-425) Ezrin Merge

Liposome Binding Functions

PC liposomes S S P P

25

37

50 75

ADPH Cells

TIP

47/B

TN C

ells

BTN TIP47

AD

PH

/BTN

Cel

ls BTN ADPH

ADPH is a multidomain protein that functions as an adaptor for milk lipid secretion

The ADPH CT 4-helix bundle is an independently folding membrane binding structure.

4-helix bundle directly interacts with membrane phospholipids by a process that is stabilized by dimerization.

In the presence of BTN, ADPH coated CLD become enveloped by the plasma membrane.

Membrane envelopment appears to be specific to ADPH coated CLD.

•ADPH acts as an adaptor to mediate contacts between CLD and phospholipids of the inner leaflet of the plasma membrane

ADPH dimers arrayed on the CLD surface alter PM properties inducing curvature that is stabilized by BTN recruitment.

XOR binding to the contact sites promotes completion of the envelopment process facilitating milk lipid secretion.

Chong et al, TEM, 2011

Elise Bales Brandi Chong Jenifer Monks Tanya Russell

David Orlicky Jerry Schaack Phil Reigan Kathryn Howell

Mark Ladinsky (CalTech)

Larry Chan (Baylor College of Medicine)

Bennie Chang (Baylor College of Medicine)

Ian Mather (U. Maryland)

• NIH 2R01HD045965 & 5P01HD038129 • UNCF/MERCK