The Challenge of Finding Where SOD Gets
Zinc: Identifying Novel Techniques to
Recombinantly Express Metallothionein
U n iv ers ity H o n ors Co lle g e a n d Bio r e sourc e Re s e a r ch T h e s is
U n d ergraduate Re s e a r che r: N ic k B. We s t
P r in ciple I n v estigator: D r. Jo seph Beck man
S e c o n dary Men to r: D r. D a n ie l S u d a kin
Amyotrophic Lateral Sclerosis (ALS)
ALS
5,600 Americans
Diagnosed Every
Year With ALS
20% of patients live past
5 years and 10% live past
10 years
[Doctor Tipster, 2011]
Introduction
•Amyotrophic Lateral Sclerosis
• Mutations in Cu,Zn-Superoxide Dismutase
•Where does SOD get Zinc?
• Metallothionein
Superoxide Dismutase (SOD) and Familial ALS (fALS)
Daniel Rosen
• Correlation
between
mutant SOD
and fALS
Mutated SOD
• 130
mutations
discovered
fALS vs. sALS
• Clinically
similar
• Mutant SOD
Accounts for
2% of Total
Cases
Mutated SOD
Controversial
Mechanism
of Disease
Familial ALS
Onset
Superoxide Dismutase (SOD) Structure
Homodimer
• Cys57-Cys146 disulfide bond
Unusual Stability
• Copper and zinc are coordinated
with specific amino acids
• Copper and zinc must be bound
for proper SOD functioning
[Roberts. Journal of
Molecular Biology. 2007]
Superoxide Dismutase Function
O2- + Cu(II)ZnSOD  O2 + Cu(I)ZnSOD
(1)
O2- + Cu(I)ZnSOD + 2H+  H2O2 + Cu(II)ZnSOD (2)
[Hart. Biochemistry. 1999]
[Roberts. Journal of
Molecular Biology. 2007]
Zinc Deficient SOD and fALS
[Roberts. Journal of
Molecular Biology. 2007]
Zinc Deficient SOD Toxic Gain of Function
Nitric Oxide (NO)
• Created by Nitric Oxide
Synthase (NOS)
• O2- reacts 3 times faster with
NO than with WT,SOD
Peroxynitrite (ONOO-)
• Formed from NO and O2-
SOD-Cu2+ + ONOO-  SOD-CuO….NO2+
SOD-CuO---NO2+ + H---Tyr  SOD-Cu2+ + HO-
+ NO2-Tyr
[Beckman, Nature. 1993]
Proposed Mechanism for ALS
Mutant SOD
Zinc Deficient
SOD
SOD Toxic Gain of
Function
Nitration of HSP90
Motor Neuron Cell
Programmed Death
ALS Onset
The Mystery of Where SOD Receives Zinc
Metallothionein (MT)
Margoshes and Vallee discovered a
cadmium binding protein in horse liver
(1957).
Vallee and Kägi called the cadmium
binding protein Metallothionein and
note its ability to bind zinc (1961).
Maret and Vallee proposed a novel
cell functioning of MT not as an
anti-oxidant protein, but as a zinc
concentration regulator (1998).
atg
M
gat
D
ccg
P
aac
N
tgc
C
agc
S
tgt
C
gca
A
gct
A
ggt
G
gac
D
tcc
S
tgc
C
acc
T
tgt
C
gcc
A
ggc
G
tct
S
tgc
C
aaa
K
tgt
C
aaa
K
gaa
E
tgc
C
aaa
K
tgt
C
act
T
agt
S
tgc
C
aag
K
aaa
K
agc
S
tgc
C
tgt
C
tcc
S
tgc
C
tgt
C
ccg
P
gtg
V
ggc
G
tgc
C
gca
A
aaa
K
tgt
C
gca
A
cga
Q
ggc
G
tgc
C
atc
I
tgt
C
aaa
K
ggc
G
gct
A
agc
S
gac
D
aag
K
tgc
C
agc
S
tgt
C
tgc
C
gct
A
tga
Stop
[Kojima, 1976]
Metallothionein Structure
Thionein
• Apo (non metal) form of MT
• No known tertiary structure
• Molecular Weight is 6-7 kDa
2 Domains
• α Domain binds 3 zinc atoms
• β Domain binds 4 zinc atoms
University of Zurich
Metallothionein Cellular Function
[Maret, Vallee. Proceedings of the
National Academy of Sciences.
1998]
[Maret. Journal of Biological
Inorganic Chemistry. 2011]
Theoretical Connection of MT and SOD
Reduced Form of
Thionein Binds
Zinc
Zinc Bound
Metallothionein
Interacts With an
Oxidizing Agent
Oxidizing Agent
Causes Release
of Zinc to
Apoenzyme
SOD Could Be A
Potential
Apoenzyme
Target
Research Objectives
Create Recombinant
MT Gene
• Insert MT gene into
pTYB11 plasmid
Express MT with
BL21(DE3) pLysS
System
• Insert MTpTYB11
plasmid into
competent
BL21(DE3) pLysS
cells
• Induce with IPTG
Express MT with
Shuffle T7 System
• Insert MTpTYB11
plasmid into
competent Shuffle
T7 cells
• Induce with IPTG
Methods – Create Recombinant MT Gene
SapI
MT Gene
Intein Protein Coding
Region (Purple)
pTYB11
Plasmid
EcoRI
Results – Create Recombinant MT Gene
Indirect Verification of Correct Insertion
1% Agarose Gel
MTpTYB11
pTYB11
(Control)
Results – Create Recombinant MT Gene
pTYB11 DNA Sequencing
ggcttagtag tctcggttaa cgcagaacct gctaaggttg acatgaatgt caccaaacat aaaattagtt
ggcttagtag tctcggttaa cgcagaacct gctaaggttg acatgaatgt caccaaacat aaaattagtt
atgctattta tatgtctggt ggagatgttt tgcttaacgt tctttcgaag tgtgccggct ctaaaaaatt
atgctattta tatgtctggt ggagatgttt tgcttaacgt tctttcgaag tgtgccggct ctaaaaaatt
caggcctgct cccgccgctg cttttgcacg tgagtgccgc ggattttatt tcgagttaca agaattgaag
caggcctgct cccgccgctg cttttgcacg tgagtgccgc ggattttatt tcgagttaca agaattgaag
gaagacgatt attatgggat tactttatct gatgattctg atcatcagtt tttgcttgga tcccaggttg
gaagacgatt attatgggat tactttatct gatgattctg atcatcagtt tttgcttgga tcccaggttg
ttgtacagaa c [MT sequence begins here]
ttgtacagaa c [MT sequence begins here]
- pTYB11 Sequence
- CGRB Sequence
Results – Create Recombinant MT Gene
MT DNA Sequencing
atggatccga actgcagctg tgcagctggt gactcctgca cctgtgccgg ctcttgcaaa
atggatccga actgcagctg tgcagctggt gactcctgca cctgtgccgg ctcttgcaaa
tgtaaagaat gcaaatgtac tagttgcaag aaaagctgct gttcctgctg tccggtgggc
tgtaaagaat gcaaatgtac tagttgcaag aaaagctgct gttcctgctg tccggtgggc
tgcgcaaaat gtgcacgagg ctgcatctgt aaaggcgcta gcgacaagtg cagctgttgc
tgcgcaaaat gtgcacgagg ctgcatctgt aaaggcgcta gcgacaagtg cagctgttgc
gcttga
gcttga
- MT Sequence
- CGRB Sequence
Methods -Express MT with BL21(DE3) pLysS System
St. Edwards
Univ.
University of
Zurich
Results -Express MT with BL21(DE3) pLysS System
SDS Gel – BL21 Expression
~63 kDa
Overcoming Challenges
Express MT with BL21(DE3) pLysS
System
• T7 RNA Polymerase
• T7 Lysozyme
• OmpT protease
• Thioredoxin
• Glutaredoxin
• Lon protease
Express MT with Shuffle T7
System
• T7 RNA Polymerase
• T7 Lysozyme deficient
• OmpT protease deficient
• Lon protease deficient
• Glutaredoxin and thioredoxin
deficient
• DsbC chaperone protein
Methods – Express MT with Shuffle T7
St. Edwards
Univ.
University of
Zurich
Results – Express MT with Shuffle T7
BL21 Expression
~63 kDa
Shuffle T7 Expression
~63 kDa
Results – Express MT with Shuffle T7
Li-Cor Western Blot
Shuffle T7
Samples
Liver
Positive
Control
Shuffle T7 Samples
Liver
Positive
Control
Discussion
Successful creation of
recombinant plasmid!! Verified
by DNA sequencing.
Unsuccessful expression in BL21
System. Verified by SDS gel.
Successful expression of
intein protein with Shuffle T7.
Verified by SDS gel.
Inconclusive expression of MT
using Shuffle T7. Analyzed using
Western Blot.
Future Plans – In A Perfect World
Zinc
Bound
MT
Zinc
Bound
SOD?
Cu,-SOD
Zinc
Bound
MT
Cu,Zn
SOD?
Cu,Zn
SOD
Apo
Thionein
Zinc
Bound
MT
Apo SOD
Implications of MT Research
Process to Purify
Metallothionein
Characterization
of SOD
Postranslational
Metal Binding
Sequence of
Events
Increased
Understanding
of ALS
Acknowledgements
•Beckman Lab
•
•
•
•
•
•
Principle Investigator – Dr. Joseph Beckman
Nathan Lopez
Pamela Beilby
Ed Labut
Jared Williams
Nick Rosson
•Environmental and Molecular Toxicology
Department
• Secondary Mentor – Dr. Daniel Sudakin
•Howard Hughes Medical Institute
• Kevin Ahern
•Undergraduate, Research, Innovation,
Scholarship, Creativity Program (URISC)
•Environmental Health Sciences Center
•Bioresource Research
• Department Head – Dr. Kate Field
• Advisor – Wanda Crannell
•Friends and Family
• Russ and Mary West
• Reva West, J.D. and Audrey Cant, Jamie and Tony
Haddock
• Alex Sutherland
• Fraternity of Phi Gamma Delta
Thank You
Are there any questions?