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People Sulfur Because of A Disulfide Bond
The Role of Thioredoxin in Tuberculosis
Messmer SMART Team: Giovanni Rodriguez, Kevonna Nathaniel, Anwuri Osademe, David Gonzalez
Advisor: Ms. Carol Johnson
Mentor: Daniel Sem, Ph.D., Department of Chemistry, Marquette University
Abstract
Role of Thioredoxin In Bacterium Cell Survival
Tuberculosis, a disease caused by the bacterium, Mycobacterium
tuberculosis (M.tb) affects about one-third of the world’s
population, killing 2 million people each year. The bacteria reside
in macrophages of the respiratory tract of infected individuals.
Macrophages are a type of immune cell whose function is to
engulf and kill foreign substances such as bacteria that invade the
body. Macrophages do this by bleaching, or oxidizing, bacterial
cell proteins, rendering the bacterial cell susceptible to cell death.
To protect against these lethal oxidative attacks by macrophages,
two bacterial cell proteins, Thioredoxin C (TrxC) and Thioredoxin
Reductase (TrxR), function to reduce the oxidized proteins, thus
stabilizing them and enabling the survival of the bacteria. To
accomplish this protective reduction and maintain redox
homeostasis in the bacterial cell, TrxC donates electrons to the
oxidized bacterial cell proteins, becoming oxidized in the process.
In order to continue to donate electrons to protect the cell, TrxC
itself must now gain electrons (be reduced). TrxR is the protein
that donates electrons to oxidized TrxC converting it back to the
reduced form, continuing the redox cycle. NADPH then reduces
the oxidized TrxR with its electrons stored in a tightly bound FAD.
To accomplish this redox cycle, TrxC binds to TrxR through a
disulfide bond, and stabilized by a hydrophobic pocket on TrxC
that fits into a crevice on TrxR. If this reaction can be prevented,
the protective redox cycle of TrxC/TrxR could be stopped thus
leading to cell death of the Mycobacterium tuberculosis,
preventing many deaths.
Introduction
• Tuberculosis is one of the deadliest diseases known, infecting over
one third of the world’s population
•There are almost 2 million TB-related deaths worldwide each year,
mostly in third world countries
• The incidence of TB in developed countries is significantly lower
than in developing countries
Figure 1a:
Estimated new
cases of TB
worldwide
Phagosome
Bacterium
Leads to cell survival
Fig. 2a- Phagocytosis of M.tb bacterium by alveolar
macrophage. Fusion of M.tb with lysosome in
macrophage forms a phagolysosome leading to
destruction and exocytosis of bacterium, preventing
disease.
Phagolysosomes
Reduced MTB cell
protein
Receptors
Thioredoxin
Phagocytosis
e-
e-
Fig. 2b- Reactive oxidative species in macrophage
phagolysosome oxidizes the bacterial cell proteins
leading to bacterial cell death. Reduction of bacterial
cell proteins by a redox cycle (Fig. 3) involving
Thioredoxin (TrxC) allows the bacteria to survive
causing progression of the disease.
Lysosome
Reactive oxidative species
Oxidized MTB
cell protein
•H202
•NO
•O2-1
Soluble debris
Leads to death of bacterium
Exocytosis
Fig. 2a- Alveolar Macrophage
Engulfing and Destroying M.tb
Fig. 2b –Phagolysosome In Macrophage with
Thioredoxin reducing oxidized M.tb cellular proteins
A Physical model of TrxC and TrxR Complex
Redox Cycle and Formation of Critical Disulfide Bond between TrxC and TrxR
NADP
To protect itself from oxidative attack by host macrophages and avoid
cell death, Thioredoxin (TrxC ) reduces the oxidized cellular proteins
and, in turn, is itself oxidized. To continue the redox process, the
reducing power of TrxC must be restored via a redox pathway involving
Thiroredoxin Reductase ( TrxR) and the cofactors FADH2 and NADPH.
The figure to the right shows the steps in this redox pathway and how
TrxC goes from the oxidized state, to the reduced state becoming HSTrxC-SH. The formation of a disulfide bond between TrxC and TrxR is
crucial to this redox pathway and to the survivial of the bacterium:
NADPH
1.
e-
Cellular Proteins
FAD
Cellular Proteins
FADH2
2.
5.
e-
e-
TrxR
1. NADPH reduces (passes an electron to) FAD to FADH2
2. FADH2 reduces TrxR to HS-TrxR- SH
3. A disulfide bridge forms between Cys 139 of TrxR and Cys 350 of
TrxC and electrons are passed from TrxR to TrxC.
4. TrxC is reduced to HS-TrxC-SH
5. Oxidized bacterial cell proteins are now reduced by TrxC leading to
survival of the TB bacterium.
Fig. 5
Oxidized State
Reduced State
HS-TrxC-SH
3.
HS-TrxR-SH
4.
Fig. 5- Shows TrxC (orchid) bonded to TrxR (cyan).
The blue portions of the figure are the amino acids of
the hydrophobic pocket that form a crevice on TrxC
that TrxR fits into. In order for TrxR to bind to TrxC,
as described in Fig 3, a disulfide bond (yellow) must
form between Cys 139 of TrxR and Cys 350 of TrxC.
Preventing this bond from forming could save
millions from Tuberculosis.
TrxC
e-
Cys 350
TrxR
Fig. 3
TrxC
e-
TrxCTrxR_Cofact.pdb
Cys 139
Identification of Amino Acids of Trx C and TrxC/TrxR Complex by NMR
http://thefastertimes.com/globalpandemics/2009/12/17/tb-or-not-tb-meeting-millenium-development-goals/
Tuberculosis is caused by the bacterium Mycobacterium
tuberculosis (M.tb). This bacterium attacks the lungs, but can also
attack the kidneys, spine and brain. Tuberculosis can be fatal if not
treated but not everyone becomes sick when infected with TB.
Fig.1b
http://www2.bakersfieldcollege.edu/bio16/22_Resppictures.htm
Figure 1b shows how the disease enters the body of the host. In this
picture, the bacteria are in the air, and can be breathed in by a
person. The bacteria travel through the respiratory system, into the
alveoli. Once the macrophage detects that there is a pathogen in the
body, they try to kill it before any harm is done. Normally the
macrophage can kill the bacteria, however in TB the bacteria use
redox strategies (Fig.2-3) to survive the attacks of the macrophage.
Fig. 4a: Each cross peak corresponds to a backbone amide
(NH) for an amino acid in TrxC and has been overlaid for
TrxC alone (blue), or complexed with TrxR NADPH cofactor
bound (green).
•Only two amino acids, Cys 139 of TrxR and Cys 350 of
TrxC, have cross peaks that disappear showing that these two
cysteines form the critical disulfide bond in the TrxC-TrxR
complex (data not shown).
•A group of amino acids disappear, due to a process called
“exchange broadening,” suggesting that these amino acids are
undergoing a conformational change and are no longer in the
same position. The disappearance of the cross peaks shows
which amino acids form the hydrophobic interface between
TrxC and TrxR. These data allow a physical model of the
complex to be constructed (Fig. 5 )
•Fig. 4b: Model of TrxC showing amino acids (in blue) that
form the hydrophobic interface between TrxC and TrxR.
•Fig 4c is an image of the complex between TrxR and TrxC
showing bonding at the hydrophobic interface and the critical
disulfide bond
Conclusion
Fig. 4a
T67
T35
W36
V78
C37
F32
Fig. 4b
Fig. 4c
Two million people die each year from tuberculosis,
mostly in third world countries. TB infects mainly
respiratory tissues. Alveolar macrophages try to kill the
bacterium by oxidizing bacterial cell proteins but the
bacterium is able to fight back and prevent cell death
by reducing these cell proteins back to their original
state via the oxidation of the protein Thioredoxin. To
continue to provide protection for the bacterium, TrxC
must be reduced (gain electrons) in a redox cycle
involving the formation of a complex between
Thioredoxin and Thioredoxin Reductase and NADPH
cofactors involving a disulfide bond between the two
proteins. This bond allows TrxR to donate electrons to
TrxC which then donates electrons to the oxidized TB
cell proteins. This bond is crucial to the survival of the
bacteria. If this bond can be prevented, the oxidized
TB cell proteins can be stopped from being reduced
thus leading to the death of the TB bacterium. In
conclusion, if we can prevent this critical disulfide
bond from forming, then we can save the lives of
millions who are infected by the bacterium.
A SMART Team project supported by the National Institutes of Health (NIH)-National Center for Research Resources Science Education Partnership Award (NCCR-SEPA) and an NIH CTSA Award to the Medical College of Wisconsin..