2009 Beckman Scholars at Carnegie Mellon
Kathleen McCann, Department of Biological Sciences, Carnegie Mellon University
Genetic Polymophisms and Splicing of RNA for the Human Dopamine Reuptake Transporter
The transporter protein DAT (encoded by gene SLC6A3) plays a critical role in re-uptake of the neurotransmitter dopamine from synapses in the brain. In this way, it helps control the intensity and duration of dopaminergic signaling. Accordingly, DAT has been implicated in schizophrenia, bipolar disorder, and Parkinson’s disease. Previous studies identified statistical associations between common DNA sequence variations (single-nucleotide polymorphisms: SNPs) within the third intron of SLC6A3 and risk for schizophrenia in two large Caucasian samples (Talkowski et al 2008). Using a primate-specific computational model, we predicted a novel 108 nt cassette exon (“E3b”) within intron 3. This exon was flanked by four schizophrenia-associated SNPs, three of which fell within predicted splicing regulatory signals. E3b introduces multiple in-frame premature stop codons into the SLC6A3 mRNA, truncating the DAT protein. The premature stop codons in E3b are predicted to trigger nonsense-mediated decay of the mRNA, which would thus only accumulate to low levels (as observed), and this can explain why the exon had not been discovered previously. This suggests that E3b serves a negative regulatory function to control the level of DAT expression. Analysis of minigene constructs bearing either risk-associated or non-risk haplotypes revealed that the risk-associated alleles at the flanking SNP positions are associated with increased inclusion of E3b in cell transfection assays. This data supports our hypothesis that risk-associated SNPs contribute to schizophrenia by increasing expression of E3b, thereby reducing the expression of functional DAT, which in turn should lead to increased dopaminergic activity. To determine if the risk alleles at E3b are both necessary and sufficient for increased E3b inclusion, I have swapped the region surrounding E3b such that the non-risk minigene is risk at E3b. Transient transfections of the swap construct and RT-PCR analysis show that the SNPs at E3b are primarily responsible for increased E3b inclusion. This supports a functional connection between the SNPs and splicing alterations at E3b.
• Talkowski et al (2007) Human Molecular Genetics 17: 747-758
Natalie Weir, Department of Biological Sciences, Carnegie Mellon University
Auto-Oxidation of Human Hemoglobin and the Roles of Distal Heme Pocket Substitutions
Background: Hemoglobin (Hb) is continually undergoing redox reactions. The superoxide generated during auto-oxidation initiates a cascade of reactions which are a source of red-cell induced oxidative stress. In the cell-free solutions of these experiments, auto-oxidation prevents Hb from reversibly binding O2, rendering Hb physiologically inactive. This study investigates the auto-oxidation reaction of human normal adult hemoglobin (Hb A), recombinant Hbs (rHbs) with mutations in the distal heme pocket, recombinant cross-linked Hbs, and recombinant octameric Hbs. This research also explores the effects of oxygen affinity and tetramer-dimer dissociation on the rate and mechanism of auto-oxidation.
Methods: rHbs with single amino acid substitutions at helical positions E11 and B10 have been expressed in Escherichia coli and purified, as well as di-α linked and octameric hemoglobins. These rHbs include: rHb (αV62L), rHb (αV62I), rHb (βV67L), rHb (βV67I), rHb (αL29W), rHb (αL29F), di-α rHb [Arg(141α1)GlyVal(1α2)], rHb (diαL29F), rHb (diαL29W), and rHb(βG83C), an octameric Hb. Auto-oxidation measurements were conducted over a range of Hb concentrations in MES buffer (pH 6.5) for 66 hours at 35 °C.
Hypothesis: Heme environment can play a role in regulating the auto-oxidation and oxygen affinity of Hb. To test this hypothesis, amino-acid substitutions at E11 and B10 in the heme pockets have been created. Tetramer-dimer dissociation can accelerate the rate of auto-oxidation. Recombinant cross-linked Hbs and octameric Hbs, which do not dissociate into dimers, have been created to test this effect.
Results: A monophasic nature of auto-oxidation has been observed for Hb A and a biphasic nature for all other rHbs. Oxidation was accelerated at low concentrations as a result of tetramer-dimer dissociation. The amplitudes of the fast and slow phases of the biphasic auto-oxidation curves decrease and increase, respectively, as concentration increases.
Conclusion: Results of rHb auto-oxidation studies indicate that the distal heme pocket strongly influences the rate of Hb oxidation. Additionally, each of the studied rHbs exhibits distinct structural and functional effects, suggesting that each mutation uniquely affects the heme pocket environment.