TAILS Identifies Candidate Substrates and Biomarkers of ADAMTS7, a Therapeutic Protease Target in Coronary Artery Disease.

Loss-of-function mutations in the secreted enzyme ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) are associated with protection for coronary artery disease. ADAMTS7 catalytic inhibition has been proposed as a therapeutic strategy for treating coronary artery disease; however, the lack of an endogenous substrate has hindered the development of activity-based biomarkers. To identify ADAMTS7 extracellular substrates and their cleavage sites relevant to vascular disease, we used TAILS (terminal amine isotopic labeling of substrates), a method for identifying protease-generated neo-N termini. We compared the secreted proteome of vascular smooth muscle and endothelial cells expressing either full-length mouse ADAMTS7 WT, catalytic mutant ADAMTS7 E373Q, or a control luciferase adenovirus. Significantly enriched N-terminal cleavage sites in ADAMTS7 WT samples were compared to the negative control conditions and filtered for stringency, resulting in catalogs of high confidence candidate ADAMTS7 cleavage sites from our three independent TAILS experiments. Within the overlap of these discovery sets, we identified 24 unique cleavage sites from 16 protein substrates, including cleavage sites in EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1/Fibulin-3). The ADAMTS7 TAILS preference for EFEMP1 cleavage at the amino acids 123.124 over the adjacent 124.125 site was validated using both endogenous EFEMP1 and purified EFEMP1 in a binary in vitro cleavage assay. Collectively, our TAILS discovery experiments have uncovered hundreds of potential substrates and cleavage sites to explore disease-related biological substrates and facilitate activity-based ADAMTS7 biomarker development.

Coronary Disease Association With ADAMTS7 Is Due to Protease Activity.

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Assessing optimal: inequalities in codon optimization algorithms.

BACKGROUND: Custom genes have become a common resource in recombinant biology over the last 20 years due to the plummeting cost of DNA synthesis. These genes are often "optimized" to non-native sequences for overexpression in a non-native host by substituting synonymous codons within the coding DNA sequence (CDS). A handful of studies have compared native and optimized CDSs, reporting different levels of soluble product due to the accumulation of misfolded aggregates, variable activity of enzymes, and (at least one report of) a change in substrate specificity. No study, to the best of our knowledge, has performed a practical comparison of CDSs generated from different codon optimization algorithms or reported the corresponding protein yields. RESULTS: In our efforts to understand what factors constitute an optimized CDS, we identified that there is little consensus among codon-optimization algorithms, a roughly equivalent chance that an algorithm-optimized CDS will increase or diminish recombinant yields as compared to the native DNA, a near ubiquitous use of a codon database that was last updated in 2007, and a high variability of output CDSs by some algorithms. We present a case study, using KRas4B, to demonstrate that a median codon frequency may be a better predictor of soluble yields than the more commonly utilized CAI metric. CONCLUSIONS: We present a method for visualizing, analyzing, and comparing algorithm-optimized DNA sequences for recombinant protein expression. We encourage researchers to consider if DNA optimization is right for their experiments, and work towards improving the reproducibility of published recombinant work by publishing non-native CDSs.

Cationic zinc is required for factor XII recruitment and activation by stimulated platelets and for thrombus formation in vivo.

BACKGROUND: Although divalent zinc (Zn2+ ) is known to bind factor (F)XII and affect its sensitivity to autoactivation, little is known about the role of Zn2+ in the binding of FXII to platelets, where FXII activation is thought to occur in vivo, and the function of Zn2+ during thrombus formation following vascular injury remains poorly understood. OBJECTIVES: To evaluate the role of Zn2+ in platelet-dependent FXIIa generation. METHODS: FXII binding to platelets and FXII activation by stimulated platelets were assessed using flow cytometry and a platelet-dependent thrombin generation assay. The mouse cremaster laser injury model was used to evaluate the impact of Zn2+ chelation on thrombus formation in vivo. RESULTS: Our data demonstrate that stimulated platelets support FXII-dependent thrombin generation and that FXII activation by platelets requires the presence of Zn2+ . By contrast, thrombin generation by stimulated endothelial cells occurred independently of FXII and Zn2+ . Using flow cytometry, we found that FXII-fluorescein-5-isothiocyanate binds to the surfaces of stimulated platelets in a specific and Zn2+ -dependent manner, whereas resting platelets demonstrated minimal binding. Other physiologically-relevant divalent cations are unable to support this interaction. Consistent with these findings, the Zn2+ -specific chelator ethylenediaminetetraacetic acid calcium disodium salt confers thromboprotection in the mouse cremaster laser injury model without causing increased bleeding. We observed an identical phenotype in FXII null mice tested in the same system. CONCLUSIONS: Our results suggest a novel role for Zn2+ in the binding and activation of FXII at the platelet surface, an interaction that appears crucial to FXII-dependent thrombin generation but dispensable for hemostasis.