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

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Kd of SOD1 for Cu: 10-15 MCytoplasmic free Cu: 10-18 M

Arsenic is the most prevalent environmental toxic substance

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http://www.atsdr.cdc.gov/cercla/07list.html

Total points: Toxicity + Frequency of occurrence + Potential for human exposure

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?

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

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)

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

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

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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

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.

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

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

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 -/-

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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-/-

****

****

*****

*

Impaired orexin neuronal processes in Hap1 KO mouse brain

(+/–)

(–/–)

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

Mouse brain fractionation in sucrose gradient

Fraction volume (ml)

0 2 4 6 8 10 12

Rel

ativ

e A

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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

Loss of Hap1 alters the distribution of trafficking proteins and cargos

Hap1WT

KO

5 ~ 45% Sucrose

HAP1 deficiency

Intracellular trafficking impairment

Neuronal development defect or degeneration

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Neuropathology

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

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

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