Structural model of a2-subunit N-terminus and its binding interface for Arf-GEF CTH2: Implication for regulation of V-ATPase, CTH2 function and rational drug design

We have previously identified the interaction between mammalian V-ATPase a2-subunit isoform and cytohesin-2 (CTH2) and studied molecular details of binding between these proteins. In particular, we found that six peptides derived from the N-terminal cytosolic domain of a2 subunit (a2N1–402) are involved in interaction with CTH2 (Merkulova, Bakulina, Thaker, Grüber, & Marshansky, 2010). However, the actual 3D binding interface was not determined in that study due to the lack of high-resolution structural information about a-subunits of V-ATPase. Here, using a combination of homology modeling and NMR analysis, we generated the structural model of complete a2N1–402 and uncovered the CTH2-binding interface. First, using the crystal-structure of the bacterial M. rubber Icyt-subunit of A-ATPase as a template (Srinivasan, Vyas, Baker, & Quiocho, 2011), we built a homology model of mammalian a2N1–352 fragment. Next, we combined it with the determined NMR structures of peptides a2N368–395 and a2N386–402 of the C-terminal section of a2N1–402. The complete molecular model of a2N1–402 revealed that six CTH2 interacting peptides are clustered in the distal and proximal lobe sub-domains of a2N1–402. Our data indicate that the proximal lobe sub-domain is the major interacting site with the Sec7 domain of first CTH2 protein, while the distal lobe sub-domain of a2N1–402 interacts with the PH-domain of second CTH2. Indeed, using Sec7/Arf-GEF activity assay we experimentally confirmed our model. The interface formed by peptides a2N1–17 and a2N35–49 is involved in specific interaction with Sec7 domain and regulation of GEF activity. These data are critical for understanding of the cross-talk between V-ATPase and CTH2 as well as for the rational drug design to regulate their function

r84, a Novel Therapeutic Antibody against Mouse and Human VEGF with Potent Anti-Tumor Activity and Limited Toxicity Induction

Vascular endothelial growth factor (VEGF) is critical for physiological and pathological angiogenesis. Within the tumor microenvironment, VEGF functions as an endothelial cell survival factor, permeability factor, mitogen, and chemotactic agent. The majority of these functions are mediated by VEGF-induced activation of VEGF receptor 2 (VEGFR2), a high affinity receptor tyrosine kinase expressed by endothelial cells and other cell types in the tumor microenvironment. VEGF can also ligate other cell surface receptors including VEGFR1 and neuropilin-1 and -2. However, the importance of VEGF-induced activation of these receptors in tumorigenesis is still unclear. We report the development and characterization of r84, a fully human monoclonal antibody that binds human and mouse VEGF and selectively blocks VEGF from interacting with VEGFR2 but does not interfere with VEGF∶VEGFR1 interaction. Selective blockade of VEGF binding to VEGFR2 by r84 is shown through ELISA, receptor binding assays, receptor activation assays, and cell-based functional assays. Furthermore, we show that r84 has potent anti-tumor activity and does not alter tissue histology or blood and urine chemistry after chronic high dose therapy in mice. In addition, chronic r84 therapy does not induce elevated blood pressure levels in some models. The ability of r84 to specifically block VEGF∶VEGFR2 binding provides a valuable tool for the characterization of VEGF receptor pathway activation during tumor progression and highlights the utility and safety of selective blockade of VEGF-induced VEGFR2 signaling in tumors

Prealternate molt-migration in Rusty Blackbirds and its implications for stopover biology

To achieve greater understanding of the full annual cycles of birds, it is critical to describe the spatial nature of little-understood phases. One of the least understood aspects of avian annual cycles is the ecology of molt: the periodic replacement of feathers. While work on the spatial nature of molt in migratory passerines has increasingly found incidences of species and populations completing molt during migration, this work has been limited entirely to prebasic flight feather molt. We examined the prevalence and progression of contour feather molt in a migratory songbird, the Rusty Blackbird (Euphagus carolinus), during spring stopover. We found that 98% of birds exhibited a partial prealternate molt during stopover, primarily in the head region. Furthermore, molt intensity peaked in the middle of the migration period and was negatively associated with fat score. This is the first evidence in the passerine literature of an obligate prealternate molt completed during migration, which is in many ways similar to the molt strategy of a variety of shorebirds (Families Charadriidae and Scolopacidae). These findings could prove crucial to understanding the constraints on spring migration in this declining species. Furthermore, we argue that molt schedules such as those of the Rusty Blackbird and shorebirds should be referred to as “prealternate molt-migration,” broadening the traditional definition of molt-migration beyond prebasic flight feather molt

MMP14 is a novel target of PTH signaling in osteocytes that controls resorption by regulating soluble RANKL production

Parathyroid hormone (PTH) affects the skeleton by acting on osteocytes (Ots) in bone through yet unclear mechanisms. We report that matrix metalloproteinase 14 (MMP14) expression/activity are increased in bones from mice with genetic constitutive activation (ca) of the PTH receptor 1 (PTH1R) in Ots (caPTH1ROt) and in bones from mice exposed to elevated PTH levels but not in mice lacking [conditional knockout (cKO)] the PTH1R in Ots (cKOPTH1ROt). Furthermore, PTH upregulates MMP14 in human bone cultures and in Ot-enriched bones from floxed control mice but not from cKOPTH1ROt mice. MMP14 activity increases soluble receptor activator of NF-κΒ ligand production, which in turn, stimulates osteoclast differentiation and resorption. Pharmacologic inhibition of MMP14 activity reduced the high bone remodeling exhibited by caPTH1ROt mice or induced by chronic PTH elevation and decreased bone resorption but allowed full stimulation of bone formation induced by PTH injections, thereby potentiating bone gain. Thus, MMP14 is a new member of the intricate gene network activated in Ots by PTH1R signaling that can be targeted to adjust the skeletal responses to PTH in favor of bone preservation.-Delgado-Calle, J., Hancock, B., Likine, E. F., Sato, A. Y., McAndrews, K., Sanudo, C., Bruzzaniti, A., Riancho, J. A., Tonra, J. R., Bellido, T. MMP14 is a novel target of PTH signaling in osteocytes that controls resorption by regulating soluble RANKL production

Pleiotropic Stromal Effects of Vascular Endothelial Growth Factor Receptor 2 Antibody Therapy in Renal Cell Carcinoma Models1

The benefits of inhibiting vascular endothelial growth factor (VEGF) signaling in cancer patients are predominantly attributed to effects on tumor endothelial cells. Targeting non-endothelial stromal cells to further impact tumor cell growth and survival is being pursued through the inhibition of additional growth factor pathways important for the survival and/or proliferation of these cells. However, recent data suggest that VEGF receptor (VEGFR)-specific inhibitors may target lymphatic vessels and pericytes in addition to blood vessels. Here, in fact, we demonstrate that DC101 (40 mg/kg, thrice a week), an antibody specific to murine VEGFR2, significantly reduces all three of these stromal components in subcutaneous (SKRC-29) and orthotopic (786-O-LP) models of renal cell carcinoma (RCC) established in nu/nu athymic mice. Sunitinib (40 mg/kg, once daily), a receptor tyrosine kinase inhibitor of VEGFR2 and other growth factor receptors, also caused significant loss of tumor blood vessels in RCC models but had weaker effects than DC101 on pericytes and lymphatic vessels. In combination, sunitinib did not significantly add to the effects of DC101 on tumor blood vessels, lymphatic vessels, or pericytes. Nevertheless, sunitinib increased the effect of DC101 on tumor burden in the SKRC-29 model, perhaps related to its broader specificity. Our data have important implications for combination therapy design, supporting the conclusion that targeting VEGFR2 alone in RCC has the potential to have pleiotropic effects on tumor stroma