Structure of the proteolytic enzyme PAPP-A with the endogenous inhibitor stanniocalcin-2 reveals its inhibitory mechanism.

The metzincin metalloproteinase PAPP-A plays a key role in the regulation of insulin-like growth factor (IGF) signaling by specific cleavage of inhibitory IGF binding proteins (IGFBPs). Using single-particle cryo-electron microscopy (cryo-EM), we here report the structure of PAPP-A in complex with its endogenous inhibitor, stanniocalcin-2 (STC2), neither of which have been reported before. The highest resolution (3.1 Å) was obtained for the STC2 subunit and the N-terminal approximately 1000 residues of the PAPP-A subunit. The 500 kDa 2:2 PAPP-A·STC2 complex is a flexible multidomain ensemble with numerous interdomain contacts. In particular, a specific disulfide bond between the subunits of STC2 and PAPP-A prevents dissociation, and interactions between STC2 and a module located in the very C-terminal end of the PAPP-A subunit prevent binding of its main substrate, IGFBP-4. While devoid of activity towards IGFBP-4, the active site cleft of the catalytic domain is accessible in the inhibited PAPP-A·STC2 complex, as shown by its ability to hydrolyze a synthetic peptide derived from IGFBP-4. Relevant to multiple human pathologies, this unusual mechanism of proteolytic inhibition may support the development of specific pharmaceutical agents, by which IGF signaling can be indirectly modulated.

Growth of Endothelial Cells in Space and in Simulated Microgravity - a Comparison on the Secretory Level.

BACKGROUND/AIMS: Endothelial cells exposed to the Random Positioning Machine (RPM) reveal three different phenotypes. They grow as a two-dimensional monolayer and form three-dimensional (3D) structures such as spheroids and tubular constructs. As part of the ESA-SPHEROIDS project we want to understand how endothelial cells (ECs) react and adapt to long-term microgravity. METHODS: During a spaceflight to the International Space Station (ISS) and a subsequent stay onboard, human ECs (EA.hy926 cell line) were cultured for 12 days in real microgravity inside an automatic flight hardware, specially designed for use in space. ECs were cultivated in the absence or presence of vascular endothelial growth factor, which had demonstrated a cell-protective effect on ECs exposed to an RPM simulating microgravity. After cell fixation in space and return of the samples, we examined cell morphology and analyzed supernatants by Multianalyte Profiling technology. RESULTS: The fixed samples comprised 3D multicellular spheroids and tube-like structures in addition to monolayer cells, which are exclusively observed during growth under Earth gravity (1g). Within the 3D aggregates we detected enhanced collagen and laminin. The supernatant analysis unveiled alterations in secretion of several growth factors, cytokines, and extracellular matrix components as compared to cells cultivated at 1g or on the RPM. This confirmed an influence of gravity on interacting key proteins and genes and demonstrated a flight hardware impact on the endothelial secretome. CONCLUSION: Since formation of tube-like aggregates was observed only on the RPM and during spaceflight, we conclude that microgravity may be the major cause for ECs' 3D aggregation.