Organisms respond to circumstances threatening the cellular protein homeostasis by activation of heat-shock transcription factors (HSFs), which play important roles in stress resistance, development, and longevity. Of the four HSFs in vertebrates (HSF1-4), HSF1 is activated by stress, whereas HSF2 lacks intrinsic stress responsiveness. The mechanism by which HSF2 is recruited to stress-inducible promoters and how HSF2 is activated is not known. However, changes in the HSF2 expression occur, coinciding with the functions of HSF2 in development. Here, we demonstrate that HSF1 and HSF2 form heterotrimers when bound to satellite III DNA in nuclear stress bodies, subnuclear structures in which HSF1 induces transcription. By depleting HSF2, we show that HSF1-HSF2 heterotrimerization is a mechanism regulating transcription. Upon stress, HSF2 DNA binding is HSF1 dependent. Intriguingly, when the elevated expression of HSF2 during development is mimicked, HSF2 binds to DNA and becomes transcriptionally competent. HSF2 activation leads to activation of also HSF1, revealing a functional interdependency that is mediated through the conserved trimerization domains of these factors. We propose that heterotrimerization of HSF1 and HSF2 integrates transcriptional activation in response to distinct stress and developmental stimuli.

Leukocyte migration to sites of inflammation is regulated by several endothelial adhesion molecules. Vascular adhesion protein-1 (VAP-1) is unique among the homing-associated molecules as it is both an enzyme that oxidizes primary amines and an adhesin. Although granulocytes can bind to endothelium via a VAP-1-dependent manner, the counter-receptor(s) on this leukocyte population is(are) not known. Here we used a phage display approach and identified Siglec-9 as a candidate ligand on granulocytes. The binding between Siglec-9 and VAP-1 was confirmed by in vitro and ex vivo adhesion assays. The interaction sites between VAP-1 and Siglec-9 were identified by molecular modeling and confirmed by further binding assays with mutated proteins. Although the binding takes place in the enzymatic groove of VAP-1, it is only partially dependent on the enzymatic activity of VAP-1. In positron emission tomography, the 68 Gallium-labeled peptide of Siglec-9 specifically detected VAP-1 in vasculature at sites of inflammation and cancer. Thus, the peptide binding to the enzymatic groove of VAP-1 can be used for imaging conditions, such as inflammation and cancer. (Blood. 2011;118(13):3725-3733) IntroductionLeukocyte migration from the blood into the nonlymphoid tissues is a hallmark of inflammation. Several molecules on the endothelial cell surface and their counter-receptors on leukocytes mediate a multistep adhesion cascade featuring tethering, rolling, activation, adhesion, crawling, and transmigration phases. 1,2 Vascular adhesion protein-1 (VAP-1/AOC3) is an endothelial cell molecule that is rapidly translocated from the intracellular storage granules to the endothelial cell surface on inflammation. It contributes to several steps in the extravasation cascade and controls trafficking of lymphocytes, granulocytes, and monocytes to sites of inflammation. VAP-1 has unique features distinct from other conventional adhesion molecules because, besides being an adhesin, it is also an enzyme. It catalyzes oxidative deamination of primary amines and produces hydrogen peroxide, aldehyde, and ammonium. 3 The end products of the enzymatic activity are highly potent inflammatory mediators and can up-regulate other adhesion molecules, such as E-and P-selectin, ICAM-1, and VCAM-1. 4,5 We recently found the first lymphocyte ligand for VAP-1, Siglec-10. 6 It is expressed on B cells, monocytes, and eosinophils but is absent from granulocytes. 7 However, VAP-1 is also involved in granulocyte migration to sites of inflammation. This has been demonstrated in studies with acute inflammation models (peritonitis, lung, and air pouch inflammation) in mouse. In these studies, significant reduction in granulocyte migration to sites of inflammation was obtained with a function blocking anti-VAP-1 antibody and a small molecular inhibitor against VAP-1. [8][9][10] Contribution of VAP-1 both at the rolling and transmigration steps during leukocyte extravasation has been demonstrated, and the enzymatic activity of VAP-1 seems to be important in these proc...

The expression of human vascular adhesion protein-1 (hVAP-1) is induced at sites of inflammation where extravasation of lymphocytes from blood to the peripheral tissue occurs. We have solved the X-ray structure of hVAP-1, a human copper amine oxidase (CAO), which is distinguished from other CAOs in being membrane-bound. The dimer structure reveals some intriguing features that may have fundamental roles in the adhesive and enzymatic functions of hVAP-1, especially regarding the role of hVAP-1 in inflammation, lymphocyte attachment, and signaling. Firstly, Leu469 at the substrate channel may play a key role in controlling the substrate entry; depending on its conformation, it either blocks or gives access to the active site. Secondly, sugar units are clearly observed at two of the six predicted N-glycosylation sites. Moreover, mutagenesis analysis showed that all of the predicted sites were glycosylated in the protein used for crystallization. Thirdly, the existence of a solvent-exposed RGD motif at the entrance to each active site in hVAP-1 suggests that it may have a functional role.

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Leukocytes migrate from the blood into areas of inflammation by interacting with various adhesion molecules on endothelial cells. Vascular adhesion protein-1 (VAP-1) is a glycoprotein expressed on inflamed endothelium where it plays a dual role: it is both an enzyme that oxidizes primary amines and an adhesin that is involved in leukocyte trafficking to sites of inflammation. Although VAP-1 was identified more than 15 years ago, the counterreceptor(s) for VAP-1 on leukocytes has remained unknown. Here we have identified Siglec-10 as a leukocyte ligand for VAP-1 using phage display screenings. The binding between Siglec-10 and VAP-1 was verified by different adhesion assays, and this interaction was also consistent with molecular modeling. Moreover, the interaction between Siglec-10 and VAP-1 led to increased hydrogen peroxide production, indicating that Siglec-10 serves as a substrate for VAP-1. Thus, the Siglec-10-VAP-1 interaction seems to mediate lymphocyte adhesion to endothelium and has the potential to modify the inflammatory microenvironment via the enzymatic end products. IntroductionMaintenance of an adequate immune defense is largely dependent on migration of circulating leukocytes from the vasculature into the surrounding tissue. Extravasation of leukocytes from the blood into the tissues is controlled by a multistep cascade, which involves numerous adhesion and signaling molecules. First, leukocytes tether and roll on the vascular endothelium, after which they adhere more strongly, arrest, and finally diapedese through the vessel wall. 1,2 One of the endothelial molecules involved in this cascade is a 180-kDa homodimeric glycoprotein, vascular adhesion protein-1 (VAP-1). 3 VAP-1 is mainly expressed on vascular endothelium, on smooth muscle cells, and on adipocytes. 4 It is rapidly translocated to the endothelial cell surface on inflammation, where it supports recruitment of leukocytes. 5,6 VAP-1 is involved in the rolling, firm adhesion, and transmigration phases of the extravasation cascade. Besides being an adhesin, VAP-1 is also an enzyme (semicarbazide sensitive amine oxidase, SSAO) and catalyzes oxidative deamination of a primary amine to an aldehyde with concomitant release of hydrogen peroxide and ammonium. 4 We and others have shown that VAP-1 supports leukocyte recruitment to sites of inflammation via both enzyme-activity-dependent and enzyme-activity-independent ways. Monoclonal anti-VAP-1 antibodies, which do not block the enzymatic activity of VAP-1, still block the binding of leukocytes to the endothelium in vitro and in vivo, and inhibitors of the enzymatic function of VAP-1 are able to effectively prevent the interaction between leukocytes and endothelium in vitro and in vivo. 7,8 Moreover, it has been recently shown that the end products generated by the catalytic activity of VAP-1, especially hydrogen peroxide, play a role in the regulation of other homing-associated molecules and thus enhance the inflammatory response. 9,10 Although VAP-1 has been known already for a long time and ...