Download Exposure to the metalloid arsenic is a daily occurrence as a result of

A. Abdul Ajees, M. Sc., M. Phil., Ph.D.
[email protected]
Research Proposal
Introduction:
Arsenic, a metalloid, is found in water, soil, and air from natural and anthropogenic sources. It is present in both
inorganic and organic forms, as well as in different oxidation states (-3, +3 and +5). Widely distributed in Earth’s crust,
inorganic arsenicals dissolve from the bedrock into water, thereby causing contamination of food and drinking water.
Prolonged arsenic exposure is associated with a wide variety of human diseases, including skin, vascular and neurological
disorders and cancer (1). Several districts of West Bengal, Bihar, Uttar Pradesh, Tamil Nadu in India, as well as other
countries such as Bangladesh, Taiwan, Japan and Mexico have arsenic levels above the WHO maximum permissible limit
(50 μg/ml) (2). As a result of its ubiquity, health effects, and potential for human exposure, arsenic ranks first on the
Superfund List of Hazardous Chemicals in USA (http://www.atsdr.Cdc.gov/cercla/07list.html).
The reduction of pentavalent arsenical to trivalency plays a decisive role in determining its toxicity and
elimination of inorganic arsenic and will be the focus of our research. Arsenate reductases that catalyze the two-electron
reduction of inorganic arsenate (As(V)) to arsenite (As(III)) have been reported in both prokaryotes and eukaryotes. In
prokaryotes, the arsC gene product of Escherichia coli plasmid R773 as well as Staphylococcus aureus plasmid pI258
exhibit arsenate reductase activity. In eukaryotes, the Cdc25 family of protein tyrosine phosphatases that use glutaredoxin
and glutathione as reductant, catalyze As(V) reduction.
Background and Specific Aim:
Three isoforms of Cdc25 have been identified in humans, Cdc25A, Cdc25B, and Cdc25C, each having several
splice variants (3), and belong to the rhodanese/Cdc25 superfamily (4). These isoforms uniquely function to
dephosphorylate phosphotyrosine and phosphothreonine residues on specific cyclin-dependent kinases, thus playing
critical roles in G1-S and G2-M transitions, and in mitosis, thereby regulating cell division. The N-termini of Cdc25
enzymes are 20-25% identical in sequence with each other and contain several phosphorylation sites, as well as nuclear
import and export signals (5). The C-termini of the Cdc25 PTPases show over 60% sequence similarity with each other
and contain the catalytic center. We investigated the possibility whether the catalytic domains of Cdc25A, Cdc25B, and
Cdc25C might adventitiously catalyze the reduction of As(V) to As(III) (6). The results of our experiments demonstrate
that the catalytic domain of either human Cdc25B or Cdc25C reduce As(V) in the presence of human glutaredoxin (Grx1)
and glutathione reductase, suggesting that this may be the coupled system for the reduction of As(V) in humans. Based
on these results, we plan to test our hypothesis and accomplish the overall objective of this proposal by pursuing the
following two specific aims:
1. Crystallization and structural studies of human Cdc25B and Cdc25C reductase.
(a) Crystallization trials of full length Cdc25B and Cdc25C.
Human Cdc25B is a 65-kDa protein containing 580
amino acid residues. The catalytic domain of Cdc25B (Cdc25B-cd) forms between 370-580 residues. To date, more than
15 structures of Cdc25B-cd are available in the Protein Data Bank. Despite extensive studies on human Cdc25, the
molecular basis for its function is still elusive, in part because of lack of a structure of the full-length protein. We plan to
clone and purify the full-length human Cdc25B. This will be followed with crystallization trials and structure-function
relationship will be examined. Similar approaches will be taken to determine the structure of full-length human Cdc25C.
(b) Crystallization trials of Cdc25B-cd with arsenate. We have shown that As(V) is a competitive inhibitor of Cdc25Bcd phosphatase activity, with an apparent Ki of 0.4 mM (6), suggesting that the site can recognize either arsenate or
phosphate. Crystallization trials of Cdc25B-cd with arsenate as a ligand will be carried out to better understand the
mechanism of Cdc25B arsenate reductase activity.
(c) Crystallization trials of Cdc25B-cd mutations. The catalytic domains of Cdc25 enzymes contain a C-X5-R signature
motif, where C is the catalytic cysteine, the five X residue form a loop, and R is a highly conserved arginine. The cysteine
in the C-X5-R signature motif at the active site acts as a nucleophile in the phosphatase reaction mechanism (7).
Alteration of either the conserved cysteine or arginine to alanine resulted in loss of both phosphatase and arsenate
reductase activity, suggesting that these residues are critical for catalysis (6). The five X residues interact with their
backbone amides toward the functionality of the active site. We plan to perform a scanning alanine mutagenesis of the
five X residues (Glu474, Phe475, Ser476, Ser477, and Glu478) as well as the conserved histidine (His472) that is present
1
A. Abdul Ajees, M. Sc., M. Phil., Ph.D.
His472
Arg479
Glu474
Cys473
Phe475
Ser476
Glu478
Ser477
[email protected]
in the signature motif, and study the effects of the mutation on both phosphatase and
arsenate reductase activity (Figure 1). Each altered protein will be examined by
crystallographic studies.
Figure 1. Catalytic domain of Cdc25B. The C-X5-R- active-site loop region of Cdc25B
(PDB 1QB0) is shown as green. The residues are shown as ball-and-stick model with the
nitrogen, sulfur, and oxygen atoms colored as blue, yellow, and red respectively. Hydrogen
bonds are depicted as dotted lines.
2. Crystallization and structural studies of human Cdc25B-cd with glutaredoxin.
We have shown that Cdc25-cd requires human glutaredoxin (Grx1) for arsenate reduction (6). The human genome
encodes for two dicysteinic glutaredoxin. Grx1 is a cytosolic protein (8), whereas Grx2 is found in mitochondria (9),
although Grx2 variants have been found in the cytosol (10). Human Grx1 contains the active site sequence Cys-Pro-TyrCys, which is also observed in E. coli glutaredoxins (Grx1, Grx2, and Grx3) and S. cerevisiae glutaredoxins (Grx1 and
Grx2). On the other hand, human Grx2 contains the Cys-Ser-Tyr-Cys motif, and Grx2 has been shown to catalyze the
reduction of glutathionylated substrates with almost identical catalytic efficiencies as Grx1 (11). Furthermore, it has been
shown that Grx2 is a substrate for NADPH and thioredoxin reductase, which efficiently reduced both the active site
disulfide and the GSH-glutaredoxin intermediate formed in the reduction of glutathionylated substrates (11). It would be
therefore interesting to examine the efficiency of both human Grx1 and Grx2 in Cdc25B-cd catalyzed As(V) reduction.
To gain insight into the interaction between Cdc25B and human glutaredoxins, we employed ClusPro 2.0
(http://cluspro.bu.edu) for docking analysis. Docking studies were performed considering Cdc25B-cd (PDB ID: 1QB0) as
the receptor and either human Grx1 (PDB ID: 1B4Q) or Grx2 (PDB ID: 2FLS) as the ligand. The model predicts that 8
Grx1 residues are within 4 Å of 8 residues of Cdc25B. Grx1 residues Lys19, Tyr24, Arg27, Arg67, Val69, Ser83, Asp84,
and Ser87 are predicted to be within 3.2 Å of Cdc25B residues Thr428, Tyr430, Leu445, Arg447, Glu478, and Arg482.
Similarly, docking studies between human Grx2 and Cdc25B produced several top scoring solutions. The final model
which reflects the closest proximity of the two active sites shows that 17 Grx2 residues are within 4 Å of 14 residues of
Cdc25B. Grx2 residues such as Tyr39, Asn66, Gln69, Asp70, Arg79, Thr80, Val81, and Thr95 are predicted to be within
3.2 Å of Cdc25B residues Thr428, Tyr430, Glu431, Arg447, Met531, and His533. Co-crystallization trials of human
Cdc25B-cd with Grx1 and Cdc25B-cd with Grx2 will be carried out to test these predictions (Figure 2).
Results obtained from these investigations will provide a fundamental understanding about arsenic biotransformation
in humans. Understanding arsenic metabolism is of public health concern, as millions of people in India as well as
elsewhere in the world chronically consumes drinking water that contains high
concentrations of inorganic arsenic. This study will be conducted in collaboration with
Dr. Hiranmoy Bhattacharjee (Associate Profesoor, Florida International University,
USA). Dr. Bhattacharjee will provide the necessary clones and mentor the biochemical
experiments (letter of collaboration enclosed).
A
B
Figure 2. Modeling of Cdc25B-Grx complex. Docking of the catalytic domain of Cdc25B (green cartoon) is
shown with A. human Grx1, and B. human Grx2 (both Grxs are shown as orange surface). Residues that
form strong hydrogen bonds between Cdc25B and Grx1 or Grx2 are shown in yellow. The C-X5-R active site
motif of CdC25B is shown in deep blue.
References
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Bhattacharjee H, Sheng J, Ajees AA, Mukhopadhyay R, & Rosen BP (2010) Biochemistry 49, 802-809.
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Zhang ZY (2003) Acc Chem Res 36, 385-392.
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A. Abdul Ajees, M. Sc., M. Phil., Ph.D.
[email protected]
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