التفاصيل البيبلوغرافية
| العنوان: |
Biochemical and hydrogen-deuterium exchange studies of the single nucleotide polymorphism Y649C in human platelet 12-lipoxygenase linked to a bleeding disorder. |
| المؤلفون: |
Tran, Michelle1 (AUTHOR), Signorelli, Rachel L.2 (AUTHOR), Yamaguchi, Adriana3 (AUTHOR), Chen, Eefie1 (AUTHOR), Holinstat, Michael3 (AUTHOR), Iavarone, Anthony T.4 (AUTHOR), Offenbacher, Adam R.1,2 (AUTHOR) offenbachera17@ecu.edu, Holman, Theodore1 (AUTHOR) holman@ucsc.edu |
| المصدر: |
Archives of Biochemistry & Biophysics. Jan2023, Vol. 733, pN.PAG-N.PAG. 1p. |
| مصطلحات موضوعية: |
*BLOOD platelet aggregation, *SINGLE nucleotide polymorphisms, *HYDROGEN-deuterium exchange, *BLOOD platelets, *BINDING sites, *DIFFERENTIAL scanning calorimetry, *LIPOSOMES, *THROMBIN receptors |
| مستخلص: |
Human platelet 12-lipoxygenase (h12-LOX) is responsible for the formation of oxylipin products that play an important role in platelet aggregation. Single nucleotide polymorphisms (SNPs) of h12-LOX have been implicated in several diseases. In this study, we investigate the structural, dynamical, and functional impact of a h12-LOX SNP that generates a tyrosine-to-cysteine mutation at a buried site (Y649C h12-LOX) and was previously ascribed with reduced levels of 12(S)-hydroxyeicosatetraenoic acid (12S-HETE) production in isolated platelets. Herein, in vitro Michaelis-Menten kinetics show reduced catalytic rates for Y649C compared to WT h12-LOX at physiological or lower temperatures. Both proteins exhibited similar melting temperatures, metal content, and oligomerization state. Liposome binding for both proteins was also dependent upon the presence of calcium, temperature, and liposome composition; however, the Y649C variant was found to have lowered binding capacity to liposomes compared to WT at physiological temperatures. Further, hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments revealed a regional defined enhancement in the peptide mobility caused by the mutation. This increased instability for the mutation stemmed from a change in an interaction with an arched helix that lines the substrate binding site, located ≥15 Å from the mutation site. Finally, differential scanning calorimetry demonstrated a reduced protein (un)folding enthalpy, consistent with the HDX results. Taken together, these results demonstrate remarkable similarity between the mutant and WT h12-LOX, and yet, subtle changes in activity, membrane affinity and protein stability may be responsible for the significant physiological changes that the Y649C SNP manifests in platelet biology. [Display omitted] • A human 12-LOX SNP forming a Y649C mutation is linked to a bleeding disorder. • In vitro kinetics show that Y649C h12-LOX exhibits reduced rates of 12S-HETE production at 37 °C. • Y649C h12-LOX exhibits lowered liposome binding properties at 37 °C. • HDX-MS reveals a long-range network of altered protein dynamics stemming from the mutation site. • Our results identify new structural and functional insights into this human 12-LOX SNP. [ABSTRACT FROM AUTHOR] |
| قاعدة البيانات: |
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