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Austin, TX, United States

Li W.,1 University Station C0800 | Crutchfield A.,1 University Station C0800 | Hodge K.,1 University Station C0800 | Matthews W.,1 University Station C0800 | And 4 more authors.
Biochemistry | Year: 2016

The human asparaginase-like protein 1 (hASRGL1) is a member of the N-terminal nucleophile (Ntn) family that hydrolyzes l-asparagine and isoaspartyl-dipeptides. The nascent protein folds into an αβ-βα sandwich fold homodimer that cleaves its own peptide backbone at the G167-T168 bond, resulting in the active form of the enzyme. However, biophysical studies of hASRGL1 are difficult because of the curious fact that intramolecular cleavage of the G167-T168 peptide bond reaches only ≤50% completion. We capitalized upon our previous observation that intramolecular processing increases thermostability and developed a differential scanning fluorimetry assay that allowed direct detection of distinct processing intermediates for the first time. A kinetic analysis of these intermediates revealed that cleavage of one subunit of the hASRGL1 subunit drastically reduces the processing rate of the adjacent monomer, and a mutagenesis study showed that stabilization of the dimer interface plays a critical role in this process. We also report a comprehensive analysis of conserved active site residues and delineate their relative roles in autoprocessing and substrate hydrolysis. In addition to glycine, which was previously reported to selectively accelerate hASRGL1 cleavage, we identified several novel small molecule activators that also promote intramolecular processing. The structure-activity analysis supports the hypothesis that multiple negatively charged small molecules interact within the active site of hASRGL1 to act as a base in promoting cleavage. Overall, our investigation provides a mechanistic understanding of the maturation process of this Ntn hydrolase family member. © 2016 American Chemical Society.

Wang T.,1 University Station C0800 | McElroy A.,1 University Station C0800 | Halaney D.,University of Texas Health Science Center at San Antonio | Vela D.,Texas Heart Institute | And 7 more authors.
Lasers in Surgery and Medicine | Year: 2015

Background and Objectives Atherosclerosis and plaque rupture leads to myocardial infarction and stroke. A novel hybrid optical coherence tomography (OCT) and two-photon luminescence (TPL) fiber-based imaging system was developed to characterize tissue constituents in the context of plaque morphology. Study Design/Materials and Methods Ex vivo coronary arteries (34 regions of interest) from three human hearts with atherosclerotic plaques were examined by OCT-TPL imaging. Histological sections (4μm in thickness) were stained with Oil Red O for lipid, Von Kossa for calcium, and Verhoeff-Masson Tri-Elastic for collagen/elastin fibers and compared with imaging results. Results Biochemical components in plaques including lipid, oxidized-LDL, and calcium, as well as a non-tissue component (metal) are distinguished by multi-channel TPL images with statistical significance (P<0.001). TPL imaging provides complementary optical contrast to OCT (two-photon absorption/emission vs scattering). Merged OCT-TPL images demonstrate the distribution of lipid deposits in registration with detailed plaque surface profile. Conclusions Results suggest that multi-channel TPL imaging can effectively identify lipid sub-types and different plaque components. Furthermore, fiber-based hybrid OCT-TPL imaging simultaneously detects plaque structure and composition, improving the efficacy of vulnerable plaque detection and characterization. Lasers Surg. Med. 47:485-494, 2015. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

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