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Studies on Intracellular Trafficking of Metals and Huntingtin Associated Cargos
林詠峯 Yung-Feng Lin, Ph.DDepartment of Human GeneticsEmory University, Atlanta, GA, USA
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Metallochaperones 金屬陪伴子
Rosenzweig, Science, 2002
Metal Metallo-chaperone
Target Protein
Cu CCS SOD1
Cox17 Sco1
Atx1 Ccc2
Hah1 (Atox1) ATP7A
ATP7B
CopZ CopY
CopA
Ni UreE urease
Fe Frataxin iron-sulfur clusters
heme
5
Kd of SOD1 for Cu: 10-15 MCytoplasmic free Cu: 10-18 M
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Arsenic is the most prevalent environmental toxic substance
6
http://www.atsdr.cdc.gov/cercla/07list.html
Total points: Toxicity + Frequency of occurrence + Potential for human exposure
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Arsenic chaperone
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ars operons in bacteria
R B CR B C
Many bacteria
R D A B C
R B C D A
DA R C
Klebsiella, Acidiphilium, Salmonella and Listeria sp.
Bacillus and Sinorhizobium sp.
Halobacterium sp.
E. Coli R773
What advantage of ArsD to provide for cells?
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ArsD confers a competitive advantage to cells growing in environmental concentrations of arsenic.
Day
0 2 4 6 8 10
% c
on
ten
t
0
20
40
60
80
100
arsDAB (X)
arsAB (Y)
9
AA
BB
AA
BB
DD
BamHI
BamHI
XbaI
arsAB arsDAB
XY
Z
Day 0 Day 9
arsAB Mixed
ZYX
Day 0 Day 9
0.5 kb
arsDABDay 0 Day 9
E. coli (∆ars) / pSE380 / 10 μM As(III)
Z
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ArsD increases the efficiency of the pump to lower intracellular As(III) through working with ArsA
• pACYC184 (PBAD; arsD) + pSE380 (PTRC; arsB or arsAB)
• E. coli AW3110 (∆ars)• 10 μM As(III)• [As] by ICP-MS
Time (min)
0 2 4 6 8 10 12
As(
III)
acc
um
ula
tio
n (
pm
ol/
109 c
ells
)
0
5
10
15
20
25
30
arsAB
arsB
arsDAB
10 M As(III)
arsarsDB
10
GlpFAS(III)
ArsD
Prote
in lad
der
∆ars
ArsB
ArsAB
ArsDAB
ArsD C12
/13A
AB
Coomassie Blue
ArsA a
nd A
rsD
60
10
ArsA
ArsD
ArsD 1-
118 C11
2/11
3AAB
ArsBADP
ATP
ArsAAS(III)AS(III)
(or arsDor arsA)
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AD Vector
ArsD and ArsA interactArsD and ArsA interact
AD-AAD-CAD-DAD-R
Controls
BD-RBD-C BD-DBD Vector BD-A
1:10
1:10
01:
1000
1:10
000
1 1:10
1:10
01:
1000
1:10
000
1 1:10
1:10
01:
1000
1:10
000
1 1:10
1:10
01:
1000
1:10
000
1 1:10
1:10
01:
1000
1:10
000
1Anti-ArsA
Anti-ArsD Anti-CadC
1 2 3 4 5 6 7 8 9 10
11
12
ArsA + + + + + + + + + +
ArsD + + + + + +
CadC + + + + +
Sb(III) + + +
MgCl2 + + + +
ATP + + + +
bBBr + + + + + + + + + +
A1 A2NBD1 NBD2
SS
S
ArsDSS
ArsD
S
• PhysicallyPhysically
• Through metal Through metal binding sitesbinding sites
• ArsA in ArsA in nucleotide-bound nucleotide-bound formform
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ArsD transfers metalloid to ArsA
0 1 2 3 4 5 6 7 8 9 10 11 12 130
5
10
15
20
25
ProteinSb(III)
Elution volume (ml)0 1 2 3 4 5 6 7 8 9 10 11 12 13
Co
nc
en
tra
tio
n ( M
)0
5
10
15
20
25
ProteinSb(III)
BSAMgATP
ArsAMgATP
Maltose
Maltose
ArsD
ArsD
12
MBP-ArsD + Sb(III) + amylose column + ArsA (or BSA)
P 1 2 3 4 6 7 9 11 12 13
ArsA-6xH
MBP-ArsD
A1 A2NBD1NBD2
SHSH
SH
ArsD
SS
ArsDS
A1 A2ATP ATP
SS
S
ArsDSHSH
3ArsDSH
ATP
Kd (µM)
As (III)
Sb(III)
ArsA ~1200 ~20
ArsD ~20 ~1.5
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ArsD increases the affinity of the ATPase for metalloids
[As(III)] M
0 20 40 60 80 100
AT
Pas
e A
ctiv
ity
(nm
ol/m
g/m
in)
0
50
100
150
200
250
+ ArsD (Vmax=294; K1/2=21 M)
- ArsD (Vmax=273; K1/2=1211 M)[Sb(III)] M
0 5 10 15 20 25
Ars
A A
TP
ase
Ac
tivi
ty (
mm
ol/m
g/m
in)
0
200
400
600
800
1000
+ArsD (Vmax=882 K1/2=1.5 M)
-ArsD (Vmax=844; K1/2=19.1 M)
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GlpF
As(III)
1
4As
ArsD
SS
ArsD
SHSH
ArsD
SSH
As
2 3
PiSHSH
ArsD
H+
As(III)
ArsB
H+
As(III)
ArsBAs
A1 A2ADP
SS
S
ArsD ArsD ArsD ArsD
3
A1 A2ATP ATP
SHSH
SH
A1 A2ATP ATP
SSH
S As
A1 A2ATPATP
SS
S
SH S SH SH
ADP
Intracellular trafficking of molecules is well regulated and important to health.
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Detroit, MI
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Neurodegenerative diseases and dysfunction of trafficking
Disease Protein Dysfunction Prevalence
Huntington’s disease Htt Dynein/dynactin Adaptor
1/10,000~ 300,000
Alzheimer’s disease Tau
APP
Microtubule associated protein Kinesin-1 adaptor
1/10~100 (old>young)
Parkinson’s disease α-synuclein
Parkin PINK1 DJ-1
Microtubule-associated protein Maintenance of mitochondria
1/300~3000
Amyotrophic lateral sclerosis (ALS)
p150Glued
SOD1
Motor associated protein Mitochondrial enzyme
1/10,000~ 50,000
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Trafficking pathology in Huntington disease (HD)
• HD has a single genetic single genetic cause, a well-defined neuropathology, and informative pre-manifest predictive genetic testing.
• Mutant Huntingtin (mHtt) retards HAP1HAP1 and inhibits HAP1 trafficking.
• It fails to transport BDNFBDNF efficiently.
• It interferes microtubule-based transport of mitochondriamitochondria and reduces ATPATP level in synaptosome.
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Hap1 and Htt in trafficking
Kinesin light chain (KLC)
Microtubule-dependent transport
14-3-3 Protein trafficking complex assembly
TBP Transcription factor
AHI1 Intracellular trafficking
Androgen receptor (AR)
Membrane receptor
References
1995
1997, 19981997
2002
2004
2003
2003
20062006
2007
2007200820092009
Hap1-interacting
proteins
proBDNF
KIF5 Microtubule-dependent transport
Neurotrophin precusor
19Salinas S et al, Curr Opin Cell Biol 2008
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HAP1 expression in hypothalamus
P3 P10 P15
50
75
100
% S
urv
ival
0P1 P2 P9
25
P3 P4 P6 P15
Li S et al., J. Neurosci, 2003
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Orexin neurons are located in the lateral hypothalamic area (LHA) and project to most parts of the brain
My study focuses on orexin neurons because of their importance and availability of orexin-Cre mice
Orexin expression HAP1 expression
Orexin neuronal function:
Coronal sections
LHDMH
VMH
ARC
FeedingLocomotor ActivitySleep/wakefulness
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TKTK NeoNeoE1E1
39513951 69576957
Cre Cre recombinaserecombinase
2 kb2 kb
TKTK
TKTK E1E1
2 kb2 kb
Hap1Hap1
Hap1-loxPHap1-loxP
Hap1-CKOHap1-CKO
3 Kb
1 Kb
5 Kb
WT
KO
M C T T Hy
HAP1-loxP
+Cre
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HAP1 Orexin A Merged
Het
Hom
Homozygous orexin-Hap1 knockout selectively depletes HAP1 in orexin neurons
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Reduced body weight and food intake in Orexin-Hap1 KO mice
Age (day)
0 10 20 30 40 60 80 100
Bo
dy
wei
gh
t (g
)
0
5
10
15
20
25
30
Orexin-Hap1 +/-Orexin-Hap1 -/-Orexin-Hap1+/+
Male Female
Fo
od
inta
ke (
g /
day
/ g
bo
dy
wei
gh
t)
0.0
0.1
0.2
0.3
0.4
+/- -/-
* *
Male Female
Wat
er i
nta
ke (
g /
day
/ g
bo
dy
wei
gh
t)
0.0
0.1
0.2
0.3
+/- -/-
wt ko
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activ
ity
Decreased locomotor activities in Orexin-HAP1 KO mice
Fed, n=32
Time
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7
Mo
vem
ent
(bea
m b
reak
s /
ho
ur)
0
200
400
600
800
1000
Orexin-Hap1 +/-
Orexin-Hap1 -/-
25
Fasted, n=32
Time
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7
Mo
vem
ent
(bea
m b
reak
s /
ho
ur)
0
200
400
600
800
1000
Orexin-Hap1 +/-
Orexin-Hap1 -/-
Male Female AllNig
htt
ime
acti
vity
(b
eam
bre
aks
/ h
ou
r)
0
200
400
600
800
WTFasted WT
Orexin-Hap1+/-
Fasted Orexin-Hap1+/-
Orexin-Hap1-/-
Fasted Orexin-Hap1-/-
****
****
*****
*
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Impaired orexin neuronal processes in Hap1 KO mouse brain
(+/–)
(–/–)
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Reduced orexin neuron population in Orexin-Hap1KO mouse brain
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Distance from V3 (m)
0 500 1000 1500 2000
Ore
xin
ne
uro
n n
um
be
r
0
20
40
60
80
1002m WT 2m KO 13m WT 13m KO 2m wt2m ko13m wt13m ko
2 m 13 m0
1000
2000
3000
4000 WT Hap1-KO
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Mouse brain fractionation in sucrose gradient
Fraction volume (ml)
0 2 4 6 8 10 12
Rel
ativ
e A
28
0 v
alu
e
0
20
40
60
80
100 Wild TypeHAP1-KO
5 ~ 45% Sucrose
Nucleotides
Synaptosomes & organelles
Cyt
opla
smic
pr
otei
ns
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Loss of Hap1 alters the distribution of trafficking proteins and cargos
Hap1WT
KO
5 ~ 45% Sucrose
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HAP1 deficiency
Intracellular trafficking impairment
Neuronal development defect or degeneration
30
Neuropathology
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Atlanta, GA
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Summary• Intracellular trafficking of certain metals/metalloids
is well regulated by metallochaperones.▫No arsenic chaperon has been identified in eukaryotes.▫Other small molecules may also require chaperones
intracellularly.
• Impairment of intracellular trafficking by HAP1 deficiency leads to neuropathology.▫Exact function of HAP1 is still not clear.▫Regulation of HAP1-partner interactions would be a
key to the regulation of intracellular trafficking. ▫There could be unknown HAP1 partners.
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Human Genome Project http://www.ornl.gov/sci/techresources/Huma
n_Genome/project/journals/insights.shtml•The total number of genes is estimated at
25,000, much lower than previous estimates of 80,000 to 140,000.
•Functions are unknown for more than 50% of discovered genes. ---- Last modified: Friday, October 09, 2009
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AcknowledgementEmory University,
Atlanta, GAXiao-Jiang LiShi-Hua LiGuoqing Sheng Jason SchroederChuan-En WangXingshun XuStephen Warren
中山醫學大學蔡淦仁 (Kan-Jen Tsai) 院長傅學樑楊宏基
Wayne State University, Detroit, MIBarry RosenMarco WongHiranmoy BhattacharjeeRussell Finley
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Conclusive thoughts
•I was like a boy playing on the sea-shore, and diverting myself now and then finding a smoother pebble or a prettier shell than ordinary, whilst the great ocean of truthtruth lay all undiscovered before me. Isaac Newton
• Science is Science is an imaginative adventure of the mind seeking truth seeking truth in a world of mystery. Sir Cyril Herman Hinshelwood (1897-1967) English chemist. Nobel prize 1956.
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ArsA binds more metalloid in the presence of ArsD
• As(III) + MBP-ArsD + ArsA-6xHis + MgATPγS
• Amylose or Ni resin + Gel filtration column
These data are consistent with transfer of metalloid from ArsD to ArsA.
A1 A2NBD1NBD2
SHSH
SH
ArsD
SS
ArsDS
As
A1 A2ATPγSATPγS
SS
S
ArsDSHSH
3ArsDSH
As
ATPγS
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P-Type ATPases
• They are a large group of of ion pumpsion pumps.
• They catalyze auto- phosphorylation of a key conserved aspartate residue within the pump.
Type IB: • Cu+, Ag+, Cu2+, Zn2+,
Cd2+, Pb2+ and Co2+.
• They are key elements for metal resistance and metal homeostasis in a wide range of organisms.
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A Checklist for Future Research from the Human Genome Project
• Exact gene number, exact locations, and functions • Gene regulation • DNA sequence organization • Chromosomal structure and organization • Noncoding DNA types, amount, distribution, information content, and
functions • Coordination of gene expression, protein synthesis, and post-translational
events • Interaction of proteins in complex molecular machines • Predicted vs experimentally determined gene function • Evolutionary conservation among organisms • Protein conservation (structure and function) • Proteomes (total protein content and function) in organisms • Correlation of SNPs (single-base DNA variations among individuals) with
health and disease • Disease-susceptibility prediction based on gene sequence variation • Genes involved in complex traits and multigene diseases • Complex systems biology, including microbial consortia useful for
environmental restoration • Developmental genetics, genomics
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Neurodegenerative diseases (examples)Huntington’s
ALS
Parkinson’s
Alzheimer’s
41