Jonathan Lam

ADAMTS13 Substrate Recognition of von Willebrand Factor A2 Domain

ADAMTS13 controls the multimeric size of circulating von Willebrand factor (VWF) by cleaving the Tyr1605–Met1606 bond in theA2 domain. To examine substrate recognition, we expressed in bacteria and purified three A2 (VWF76-(1593–1668), VWF115-(1554–1668), VWFA2-(1473–1668)) and one A2-A3 (VWF115-A3-(1554–1874)) domain fragments. Using high pressure liquid chromatography analysis, the initial rates of VWF115 cleavage by ADAMTS13 at different substrate concentrations were determined, and from this the kinetic constants were derived (Km 1.61 μm; kcat 0.14 s–1), from which the specificity constant kcat/Km was calculated, 8.70 × 104 m–1 s–1. Similar values of the specificity constant were obtained for VWF76 and VWF115-A3. To identify residues important for recognition and proteolysis of VWF115, we introduced certain type 2A von Willebrand disease mutations by site-directed mutagenesis. Although most were cleaved normally, one (D1614G) was cleaved ∼8-fold slower. Mutagenesis of additional charged residues predicted to be in close proximity to Asp1614on the surface of the A2 domain (R1583A, D1587A, D1614A, E1615A, K1617A, E1638A, E1640A) revealed up to 13-fold reduction in kcat/Km for D1587A, D1614A, E1615A, and K1617A mutants. When introduced into the intact VWFA2 domain, proteolysis of the D1587A, D1614A, and E1615A mutants was also slowed, particularly in the presence of urea. Surface plasmon resonance demonstrated appreciable reduction in binding affinity between ADAMTS13 and VWF115 mutants (KD up to ∼1.3 μm), compared with VWF115 (KD 20 nm). These results demonstrate an important role for Asp1614 and surrounding charged residues in the binding and cleavage of the VWFA2 domain by ADAMTS13.

Further characterization of ADAMTS-13 inactivation by thrombin

Summary.  Background: The multimeric size and platelet‐tethering function of von Willebrand factor (VWF) are modulated by the plasma metalloprotease, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS‐13). In vitro ADAMTS‐13 is susceptible to proteolytic inactivation by thrombin. Objectives: In this study, we aimed to characterize the inactivation of ADAMTS‐13 by thrombin and to assess its physiological significance. Methods and results: By N‐terminal sequencing of cleavage products, and by mutagenesis, we identified the principal thrombin cleavage sites in ADAMTS‐13 as R257 and R1176. Using a library of 76 thrombin mutants, we highlighted the functional importance of exosite I on thrombin in the proteolysis of ADAMTS‐13. Proteolysis of ADAMTS‐13 by thrombin caused an 8‐fold reduction in its affinity for VWF that contributed to its loss of VWF‐cleaving function. Intriguingly, thrombin‐cleaved ADAMTS‐13 both bound and proteolyzed a short recombinant VWF A2 domain substrate (VWF115) normally. Following activation of coagulation in normal plasma, endogenous ADAMTS‐13, but not added ADAMTS‐13, appeared resistant to coagulation‐induced fragmentation. An estimation of the Km for ADAMTS‐13 proteolysis by thrombin was appreciably higher than the physiological concentration of ADAMTS‐13. This was corroborated by the comparatively low affinity of ADAMTS‐13 for thrombin (KD 95 nm). Conclusions: Together, our data suggest that ADAMTS‐13 is protected from rapid proteolytic inactivation by thrombin in normal plasma. Whether this remains the case under pathological situations involving elevated/sustained generation of thrombin remains unclear.

cAMP-dependent Protein Kinase A (PKA) Signaling Induces TNFR1 Exosome-like Vesicle Release via Anchoring of PKA Regulatory Subunit RIIβ to BIG2

The 55-kDa TNFR1 (type I tumor necrosis factor receptor) can be released to the extracellular space by two mechanisms, the proteolytic cleavage and shedding of soluble receptor ectodomains and the release of full-length receptors within exosome-like vesicles. We have shown that the brefeldin A-inhibited guanine nucleotide exchange protein BIG2 associates with TNFR1 and selectively modulates the release of TNFR1 exosome-like vesicles via an ARF1- and ARF3-dependent mechanism. Here, we assessed the role of BIG2 A kinase-anchoring protein (AKAP) domains in the regulation of TNFR1 exosome-like vesicle release from human vascular endothelial cells. We show that 8-bromo-cyclic AMP induced the release of full-length, 55-kDa TNFR1 within exosome-like vesicles via a protein kinase A (PKA)-dependent mechanism. Using RNA interference to decrease specifically the levels of individual PKA regulatory subunits, we demonstrate that RIIβ modulates both the constitutive and cAMP-induced release of TNFR1 exosome-like vesicles. Consistent with its AKAP function, BIG2 was required for the cAMP-induced PKA-dependent release of TNFR1 exosome-like vesicles via a mechanism that involved the binding of RIIβ to BIG2 AKAP domains B and C. We conclude that both the constitutive and cAMP-induced release of TNFR1 exosome-like vesicles occur via PKA-dependent pathways that are regulated by the anchoring of RIIβ to BIG2 via AKAP domains B and C. Thus, BIG2 regulates TNFR1 exosome-like vesicle release by two distinct mechanisms, as a guanine nucleotide exchange protein that activates class I ADP-ribosylation factors and as an AKAP for RIIβ that localizes PKA signaling within cellular TNFR1 trafficking pathways.

The Role of Tyrosine Kinases in the Pathogenesis and Treatment of Lung Disease

Tyrosine kinases (TKs) are key regulators of signal transduction pathways that modulate essential biological processes, such as cellular differentiation, metabolism and proliferation, as well as, protein synthesis, cell cycle progression and apoptosis (Schlessinger, 2000). These enzymes modify protein function by transferring phosphate groups from adenosine triphosphate (ATP) or guanosine triphosphate (GTP) to free hydroxyl groups on tyrosine residues. Under physiological conditions, kinase activity is regulated by protein phosphatases that dephosphorylate and inactivate signaling pathways. However, these signaling pathways can become dysregulated in lung diseases, such as non-small cell lung cancer (NSCLC), asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). This chapter will provide an overview of the pathways mediated by receptor and non-receptor tyrosine kinases, as well as their role in the pathogenesis of lung disease. Furthermore, the emerging role of TK inhibitors (TKIs) for the treatment of NSCLC, asthma, COPD and IPF will be discussed.