becky wright: history 2002-3 postdoc (15months) cardiovascular genetics, rayne bldg, ucl...
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Becky Wright: historyBecky Wright: history
2002-3 Postdoc (15months) Cardiovascular Genetics, 2002-3 Postdoc (15months) Cardiovascular Genetics, Rayne Bldg, UCLRayne Bldg, UCLAssociation between variation in the Alpha-1-Antitrypsin (AAT) gene and risk of cardiovascular disease (CVD).
1998-20031998-2003 Ph.D. Ph.D. St. George's Hosp, Uni of LondonSt. George's Hosp, Uni of London
Insulin-like Growth Factor Binding Proteins (IGFBPs) as Co-coordinators of Human Ovarian Function.
1996-98 RA -Molecular Immunology, NIMR, LondonT cell selection and the invariant chain
1995-96 RA -Interface Analysis Centre, Uni. of BristolXPS and EM analysis of top 10-30Å materials surface
Cardiovascular genetics Cardiovascular genetics Aims of project:Aims of project:to determine the association of AAT genotypes with the risk of CVD
(particularly atherosclerosis)
Hypothesis:Hypothesis:Polymorphisms are related to changes in the level or activity of AAT Polymorphisms are related to changes in the level or activity of AAT
protein. Low levels/activity of AAT will promote atherosclerosis.protein. Low levels/activity of AAT will promote atherosclerosis.
Background:Background: Produced by liver (and monocytes) as part of the acute phase Produced by liver (and monocytes) as part of the acute phase
response (APR)response (APR) Serine protease inhibitor (SERPIN)Serine protease inhibitor (SERPIN) In particular, AAT prevents the degradation of elastin by serine In particular, AAT prevents the degradation of elastin by serine
proteases (ELASTASES) esp in lungs and vasculature.proteases (ELASTASES) esp in lungs and vasculature.Cf metalloproteases and TIMPCf metalloproteases and TIMP
90% of anti-elastase activity in lung is due to AAT90% of anti-elastase activity in lung is due to AAT
Possible mechanisms by which Possible mechanisms by which AAT could affect atherogenesisAAT could affect atherogenesis
AAT
C-36
Activation of monocytesFoam cell formation
Oxidised AAT+LDL
?
Clearance of LDL/oxAAT from plaque
Lipid accumulation/foam cell formation
Elastin
Elastin peptide
ELASTASEActivation of MMPs
Plaque ruptureVessel wall remodellingMonocyte transmigrationSMC migration
TIMP1
Hardening of vessel wallsIncreased blood pressure
Reactive oxygen species (ROS)
Suicide cleavage
AAT
C-36
Activation of monocytesFoam cell formation
Oxidised AAT+LDL
?
Clearance of LDL/oxAAT from plaque
Lipid accumulation/foam cell formation
Elastin
Elastin peptide
ELASTASEActivation of MMPs
Plaque ruptureVessel wall remodellingMonocyte transmigrationSMC migration
TIMP1
Hardening of vessel wallsIncreased blood pressure
Reactive oxygen species (ROS)
Suicide cleavage
Identification of novel polymorphisms in Identification of novel polymorphisms in the promoter of the promoter of AATAAT gene gene
Chromosome 14, within gene cluster of SERPINSChromosome 14, within gene cluster of SERPINS 2 mutually exclusive promoters in liver (endoderm) and 2 mutually exclusive promoters in liver (endoderm) and
macrophages (mesoderm)macrophages (mesoderm) Alternative splicing of macrophage mRNAAlternative splicing of macrophage mRNA 3’ UTR in liver3’ UTR in liver
1AIA
Identification of novel polymorphisms in Identification of novel polymorphisms in the promoter of the promoter of AATAAT gene gene
1AIA
• Sequence databases (Ensembl, NCBI UC)-blast-predicted polys/ conflicts
• Sequencing (PCR, sequencing)
• Validation of polys-RFLP MADGE (PCR, RE, gel)-TaqMan (labeled probes)
Novel polymorphism
Genotyping of novel polys in Genotyping of novel polys in association studiesassociation studies
In linkage disequilibrium with each other and In linkage disequilibrium with each other and some other polyssome other polys
As such, association with increased progression As such, association with increased progression of athersclerosis in one study. No significant of athersclerosis in one study. No significant association seen in other studies (with different association seen in other studies (with different endpoints).endpoints). VV
213a213aGluGlu264 S264 S
LysLys342 Z342 Z
Glu Glu 376as376as
3 new 3 new snpssnps
ArgArg101101
****** ******
VV213a213a
****** ****** ****
GluGlu264 S264 S
**
LysLys342 Z342 Z
**
Glu3Glu37676
*** <0.0005** <0.005* <0.05
Functional analysis of novel polysFunctional analysis of novel polys Transplorer software: predicts Transplorer software: predicts
transcription factor binding sites on transcription factor binding sites on DNA sequencesDNA sequences
YY1 Oct 1CDP CR1 CDP CR3+HD
A allele G allele
In vitroIn vitro functional studies for new polymorphisms: functional studies for new polymorphisms:Splicing profile of AAT mRNA transcripts in stimulated Splicing profile of AAT mRNA transcripts in stimulated
macrophages of different genotypesmacrophages of different genotypes
RT-PCR spanning intronsRT-PCR spanning introns
1A 1B 1C II III IV V1A 1B 1C II III IV V
5’5’ 3’3’
1A1B1C Both1A1B1C Both1A1C1A1C
• mRNA transcript mRNA transcript profile visualised on profile visualised on agarose gelagarose gel
IGFBPs: role in the ovaryIGFBPs: role in the ovaryAims:Aims:To investigate the role of IGFBP-2 and -4 in folliculogenesisBackground:Background: The insulin-like growth factors (IGFs) are potent stimulators of ovarian
follicle function. They are modulated in their actions by a range of IGF binding proteins
(IGFBPs). Amongst the IGFBPS, IGFBP-2 and -4 are uniquely correlated with the
health of a follicle. They are preferentially proteolysed in healthy follicles but remain intact in atretic follicles.
IGFBP-4 caused potent inhibition of gonadotrophin-mediated steroidogenesis and its proteolysis is therefore implicated as a requirement for selection of a dominant follicle.
Hypothesis:Hypothesis:IGFBP-2 and -4 are acting as brakes on folliculogenesis. Local regulation of
production and proteolysis occurs within the follicular and ovarian micro-environment
Some pretty picturesSome pretty pictures
Effects of IGFBP-2 and -4 on Effects of IGFBP-2 and -4 on FSH-mediated steroidogenesisFSH-mediated steroidogenesis
0
20
40
60
E2
fm
ol/1
000c
ells
/48h
t t+BP-4 t+FSH t+FSH+ BP-4
0
0.5
1.5
2.5
3.5
4.5
t t+FSH 0.5 5.0 50 BP-4 (ng/ml)
+ t + FSH
d
cbcb
a
a
t t+ BP-2 BP-3 BP-4 FSH t+ FSH+
0
0.5
1
1.5
2
2.5
3
a
b
c
E2
nm
ol/1
000
cell
s/48
h
Cells cultured +/- gonadotrophin+/- BP-2,-4 for 48h[Steroid] determined in the medium by RIASimilar results for lactate production (as measured by an ELISA kit)
Status of IGFBP-4 within Status of IGFBP-4 within follicular cellsfollicular cells
granulosa theca
IGFBP-4 WIB of GCCM (visualized by ECL)
lane 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
NHS
30
28
24
kDa
Mechanism of IGFBP-4 effectsMechanism of IGFBP-4 effects
0
20
40
60
0
20
40
60
6h
3h
C LH LH+IGFBP-4
Pnmol/1000 cells/48h
(i)
a
b
a
b
c
c
t FSH FSH+ FSH+ FSH+ 8-br 8-br+ 8-br+ 8-br+ BP-4 BP-4 BP-4 BP-4 BP-4 BP-4 (50) (5) (0.5ng/ml) (50) (5) (0.5ng/ml)
Pn
mol
/100
0 ce
lls/
48h
0
15
30
45
a
d
c
b
c c
(i) GC (3-7mm follicles)
Timecourse
Effect on cAMP-mediated steroidogenesis
Localisation of IGFBP-4 in Localisation of IGFBP-4 in follicular cellsfollicular cells
28 kDa(glycosylated,Intact)
16.5 kDa(fragment)
48 kDa(dimers)
CM AW N M C
LegendCM=conditioned mediumAW=cell surface associated fraction
N=nuclear fraction M=membrane fractionC=cytosolic fraction
Fig. 8a
(i) (ii)
(iii) (iv)
(v) (vi)
5 m 5 m
5 m 5 m
10 m
20 m
(iv)(iii)
(ii)(i)
5 m
5 m 5 m
5 m
(v) (vi)
10 m
20 m
Possible mechanisms by which Possible mechanisms by which IGFBP-4 inhibits gonadotrophin-IGFBP-4 inhibits gonadotrophin-
mediated steroidogenesismediated steroidogenesis
PutativeBP-4receptor
PKA
Gn
cAMP
ATP
GDPGTP
AC
steroidogenesismitogenesis
Extracellular
Intracellular
GTP
GsGs
PKA
Gn
cAMP
ATP
GDPGTP
AC
steroidogenesismitogenesis
Extracellular
Intracellular
GTP
GsGs
IGFBP-4
IGFBP-4
(i) Schematic model of the Gn signal transduction pathway in follicular cells leading toincreased steroidogenesis.
(ii) Possible sites of action of IGFBP-4 in the Gn signal transduction pathway leadingto inhibition of steroidogenesis.
Gn receptor
ActivatedGs protein
-subunitlinked toGTP
Figure 14. Postulated schematic model of the mechanism bywhich IGFBP-4 inhibits Gn-stimulated steroidogenesis.
See legend overleaf.
Possible mechanisms by which Possible mechanisms by which IGFBP-4 inhibits gonadotrophin-IGFBP-4 inhibits gonadotrophin-
mediated steroidogenesismediated steroidogenesis
PutativeBP-4receptor
PKA
Gn
cAMP
ATP
GDPGTP
AC
steroidogenesismitogenesis
Extracellular
Intracellular
GTP
GsGs
PKA
Gn
cAMP
ATP
GDPGTP
AC
steroidogenesismitogenesis
Extracellular
Intracellular
GTP
GsGs
IGFBP-4
IGFBP-4
(i) Schematic model of the Gn signal transduction pathway in follicular cells leading toincreased steroidogenesis.
(ii) Possible sites of action of IGFBP-4 in the Gn signal transduction pathway leadingto inhibition of steroidogenesis.
Gn receptor
ActivatedGs protein
-subunitlinked toGTP
Figure 14. Postulated schematic model of the mechanism bywhich IGFBP-4 inhibits Gn-stimulated steroidogenesis.
See legend overleaf.
Regulation of IGFBP-4 proteinRegulation of IGFBP-4 protein
0
5
10
15
20
25
30
Arb
itra
ry O
D u
nit
s
of 2
8KD
a p
rote
in
c t 10 1ng/ml
t+FSH
25%
48%
(ii) Mean optical density of 28kDa isoform of IGFBP-4
t+FSH t+FSH
28 kDa
NHS
NHS H A A A H H A A A H
NHS H A A HNHS H A A H
Follicular fluid
Granulosa cells
Regulation of IGFBP-4 proteaseRegulation of IGFBP-4 protease
NAS PS H A H H A A
c+tt+LH
Large Follicle
BP-4 PS +++
Intact IGFBP-4
IGFBP-4fragments
Fig. 13b(i) Protease assay of GCCM (19mm)
Follicular fluidGranulosa cells
The effects of cell culture The effects of cell culture on IGFBP profileon IGFBP profile
1 3 4 7 11
30
28
24
kDa
3 4 7 11 1 3 4 7 11day ofincubation
Large follicles Small follicles Large follicles
0
1 00
2 00
1 3 4 7 110
100
200
300
1 3 4 7 11 0
100
200
300
3 4 7 11
OD
Arb
itra
ry u
nit
s
30 kDa
28 kDa
24 kDa
day ofincubation
Role of IGFBP-4 in the ovaryRole of IGFBP-4 in the ovary
FSH LHdue to steroids bydominant follicle
Atresia of remainder of cohort
BP-4 protease inhibitor
Figure 18. Schematic model for the role of IGFBP-4 in the ovary.
The concentration of IGFBP-4 protease in a follicle at the selection stageis dependent on GC number and GC responsiveness to proteaseregulatory factors, i.e. FSH. At selection stage, the follicle with thehighest concentration of IGFBP-4 protease and lowest levels of inhibitor,has the greatest ability to produce steroids in response to Gn and IGF-II.This follicle is able to become dominant over the remaining cohort and bythe increased production of E2, decrease the circulating levels of FSH andacquire receptors to LH on the GC. This results in further proteolysis ofIGFBP-4 and a reduction in the production of IGFBP-4 and proteaseinhibitor.The reduction in FSH levels due to the selected follicle, causes theproduction of IGFBP-4 to increase and proteolysis to decrease. This leadsto increased inhibition of Gn-stimulated steroidogenesis and eventuallyatresia.
proteolysed BP-4 Gn-mediated steroidogenesis
intact BP-4 inhibition ofGn-mediated steroidogenesis
Recruitment Selection
Growth and development ofdominant follicle
TechniquesTechniques Protein: - activity (functional assays, eg protease assays)Protein: - activity (functional assays, eg protease assays)
- concentration (ELISA, RIA)- concentration (ELISA, RIA) -status (WIB)-status (WIB) -localisation (confocal, sub-cell fractionation+WIB)-localisation (confocal, sub-cell fractionation+WIB)
Cells: -micro-dissectionCells: -micro-dissection -primary cells-primary cells -cell lines (immortalised or finite lifespan)-cell lines (immortalised or finite lifespan) -viability (MTT, neutral red)-viability (MTT, neutral red) -cell proliferation (3H-thymidine incorporation, cell lysis, count)-cell proliferation (3H-thymidine incorporation, cell lysis, count)
Signaling: -cAMP analogues Signaling: -cAMP analogues -specific enzyme blockers-specific enzyme blockers
DNA: - determination of genotype (RFLP, TaqMan)DNA: - determination of genotype (RFLP, TaqMan) - detection of genetic polymorphisms (sequencing, databases, SSCP)- detection of genetic polymorphisms (sequencing, databases, SSCP) - measurement of gene expression (RT-PCR)- measurement of gene expression (RT-PCR) - functional analysis of polymorphisms (consensus sequence prediction - functional analysis of polymorphisms (consensus sequence prediction
software, luciferase assayssoftware, luciferase assays
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