Browsing by Author "Perez-Miller, Samantha"

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    Catalytic Contribution of Threonine 244 in Human ALDH2
    (Elsevier, 2013) González-Segura, Lilian; Ho, K.-K.; Perez-Miller, Samantha; Weiner, Henry; Hurley, Thomas D.; Biochemistry and Molecular Biology, School of Medicine
    Amongst the numerous conserved residues in the aldehyde dehydrogenase superfamily, the precise role of Thr-244 remains enigmatic. Crystal structures show that this residue lies at the interface between the coenzyme-binding and substrate-binding sites with the side chain methyl substituent oriented toward the B-face of the nicotinamide ring of the NAD(P)(+) coenzyme, when in position for hydride transfer. Site-directed mutagenesis in ALDH1A1 and GAPN has suggested a role for Thr-244 in stabilizing the nicotinamide ring for efficient hydride transfer. Additionally, these studies also revealed a negative effect on cofactor binding which is not fully explained by the interaction with the nicotinamide ring. However, it is suggestive that Thr-244 immediately precedes helix αG, which forms one-half of the primary binding interface for the coenzyme. Hence, in order to more fully investigate the role of this highly conserved residue, we generated valine, alanine, glycine and serine substitutions for Thr-244 in human ALDH2. All four substituted enzymes exhibited reduced catalytic efficiency toward substrate and coenzyme. We also determined the crystal structure of the T244A enzyme in the absence and presence of coenzyme. In the apo-enzyme, the alpha G helix, which is key to NAD binding, exhibits increased temperature factors accompanied by a small displacement toward the active site cysteine. This structural perturbation was reversed in the coenzyme-bound complex. Our studies confirm a role for the Thr-244 beta methyl in the accurate positioning of the nicotinamide ring for efficient catalysis. We also identify a new role for Thr-244 in the stabilization of the N-terminal end of helix αG. This suggests that Thr-244, although less critical than Glu-487, is also an important contributor toward coenzyme binding.
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    Expression and purification of functional human glycogen synthase-1 (hGYS1) in insect cells
    (Elsevier, 2013) Khanna, May; Imasaki, Tsuyoshi; Chikwana, Vimbai M.; Perez-Miller, Samantha; Hunter, Gerald O.; Mosley, Amber; Takagi, Yuichiro; Hurley, Thomas D.; Biochemistry and Molecular Biology, School of Medicine
    We have successfully expressed and purified active human glycogen synthase-1 (hGYS1). Successful production of the recombinant hGYS1 protein was achieved by co-expression of hGYS1 and rabbit glycogenin (rGYG1) using the MultiBac baculovirus expression system (BEVS). Functional measurements of activity ratios of hGYS1 in the absence and presence of glucose-6-phosphate and treatment with phosphatase indicate that the expressed protein is heavily phosphorylated. We used mass spectrometry to further characterize the sites of phosphorylation, which include most of the known regulatory phosphorylation sites, as well as several sites unique to the insect cell over-expression. Obtaining large quantities of functional hGYS1 will be invaluable for future structural studies as well as detailed studies on the effects on specific sites of phosphorylation.
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    Inhibition of the Ubc9 E2 SUMO-conjugating enzyme-CRMP2 interaction decreases NaV1.7 currents and reverses experimental neuropathic pain
    (Lippincott, Williams & Wilkins, 2018-10) François-Moutal, Liberty; Dustrude, Erik T.; Wang, Yue; Brustovetsky, Tatiana; Dorame, Angie; Ju, Weina; Moutal, Aubin; Perez-Miller, Samantha; Brustovetsky, Nickolay; Gokhale, Vijay; Khanna, May; Khanna, Rajesh; Pharmacology and Toxicology, School of Medicine
    We previously reported that destruction of the small ubiquitin-like modifier (SUMO) modification site in the axonal collapsin response mediator protein 2 (CRMP2) was sufficient to selectively decrease trafficking of the voltage-gated sodium channel NaV1.7 and reverse neuropathic pain. Here, we further interrogate the biophysical nature of the interaction between CRMP2 and the SUMOylation machinery, and test the hypothesis that a rationally designed CRMP2 SUMOylation motif (CSM) peptide can interrupt E2 SUMO-conjugating enzyme Ubc9-dependent modification of CRMP2 leading to a similar suppression of NaV1.7 currents. Microscale thermophoresis and amplified luminescent proximity homogeneous alpha assay revealed a low micromolar binding affinity between CRMP2 and Ubc9. A heptamer peptide harboring CRMP2's SUMO motif, also bound with similar affinity to Ubc9, disrupted the CRMP2-Ubc9 interaction in a concentration-dependent manner. Importantly, incubation of a tat-conjugated cell-penetrating peptide (t-CSM) decreased sodium currents, predominantly NaV1.7, in a model neuronal cell line. Dialysis of t-CSM peptide reduced CRMP2 SUMOylation and blocked surface trafficking of NaV1.7 in rat sensory neurons. Fluorescence dye-based imaging in rat sensory neurons demonstrated inhibition of sodium influx in the presence of t-CSM peptide; by contrast, calcium influx was unaffected. Finally, t-CSM effectively reversed persistent mechanical and thermal hypersensitivity induced by a spinal nerve injury, a model of neuropathic pain. Structural modeling has now identified a pocket-harboring CRMP2's SUMOylation motif that, when targeted through computational screening of ligands/molecules, is expected to identify small molecules that will biochemically and functionally target CRMP2's SUMOylation to reduce NaV1.7 currents and reverse neuropathic pain.