SEMINAR: Research in Progress

 Nidhi Gera, PhD

Research Assistant Professor

Parise Lab

Exploiting platelet lipid metabolism to prevent thrombosis

Human platelets are essential mediators of both hemostasis and thrombosis. A comprehensive knowledge of the platelet proteome is necessary for understanding thrombotic mechanisms and discovering new antiplatelet therapies. Using a chemoproteomics approach, we identified a novel lipid deacetylase in platelets, arylacetamidedeacetylase-like 1 (AADACL1/NCEH1).  We found that inhibition of this enzyme impairs agonist-induced platelet aggregation. Human platelets treated with the AADACL1 specific inhibitor JW480 or the AADACL1 lipid substrate 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG) exhibited decreased Ca+2 flux, PKC activation, granule secretion and  platelet aggregation. Experiments with P2Y12-/- and CalDAG GEFI1-/- mice revealed that the P2Y12 pathway is the predominate target of HAG-mediated inhibition of platelet aggregation. HAG likely mediates its inhibitory effects via conversion to a phosphorylated metabolite, 1-O-hexadecyl-2-acetyl-sn-glycerol-3-phosphate (HAGP). We found that HAGP interacts directly with the C1a domains of two distinct PKC isoforms and blocks PKC kinase activity in vitro.  Finally, AADACL1 inhibition in vivo reduces platelet aggregation and protects rats against FeCl3-induced arterial thrombosis.  In summary, our data support a model whereby AADACL1 inhibition shifts the platelet ether lipidome to an inhibitory axis of HAG and HAGP that impairs PKC activation, granule secretion and recruitment of platelets to sites of vascular damage. 

Zhijie Li, PhD 

Postdoctoral Fellow
Carter Lab

Aminoacyl-tRNA synthetases may have evolved from molten globular precursors

To represent an early stage in the evolution of contemporary aminoacyl-tRNA synthetases (aaRSs), the structurally invariant 120~130-residue Urzymes (Ur-: suffix for primitive, original) have been designed and consists of a minimal catalytic domain that retains a considerable fraction of the catalytic activity of the native aaRSs. We previously described experimental evidence that the Urzymes acylate tRNA far faster than the uncatalyzed rate of nonribosomal peptide bond formation from activated amino acids, and Urzymes exhibit ~60% of the contemporary catalytic proficiencies. Previous NMR study of the TrpRS Urzyme renatured from inclusion bodies suggested that it functions as a catalytically active molten globule (1). This study focuses on LeuRS urzyme. Like the TrpRS Urzyme, LeuRS is also purified from inclusion bodies and its HSQC spectra showed that LeuRS Urzyme also lacks extensive tertiary structure. Together with the PPi exchange assays, it indicates that LeuRS Urzyme is also catalytically active molten globular. A transition-state analog, AQP, may trigger the conformational ordering expected for a catalytic molten globule in transition state.

References

1. Sapienza, P.J., Li, L., Williams, T., Lee, A.L. and Carter, C.W., Jr. (2016) An Ancestral Tryptophanyl-tRNA Synthetase Precursor Achieves High Catalytic Rate Enhancement without Ordered Ground-State Tertiary Structures. ACS Chemical Biology, 11, 1661−1668