Vitronectin and Plasminogen Activator Inhibitor-1 Form Higher-Order Complexes that Localize to the Extracellular Matrix and Adopt Adhesive Properties

Vitronectin is a human circulatory protein that is recruited to the extracellular matrix (ECM) during tissue remodeling pathways linked to injury, inflammation, and tumor metastasis. Whereas circulating vitronectin is monomeric, the tissue-associated form is multimeric and contains multivalent binding sites for both cell-surface receptors and components of the extracellular matrix. In addition, matrix-associated vitronectin directly regulates plasmin-regulated matrix proteolysis through the localization and stabilization of the serine protease inhibitor, plasminogen activator inhibitor type-1 (PAI-1). As a component of the ECM, vitronectin adopts a pro-adhesive role, providing a unique regulatory link between cell adhesion and pericellular proteolysis. The mechanism by which vitronectin interconverts between a monomeric, circulating protein and a multimeric ECM component remains to be established. Vitronectin shows a high degree of conformational flexibility, and in vitro studies demonstrate that a number of chaotropic agents and macromolecular ligands like PAI-1 are able to induce the multimerization of vitronectin into higher-order forms. The induced multimeric form of vitronectin shows similar ligand-binding properties to that of the matrix-associated form, with a potential for multivalent interactions with biological partners including cell- surface receptors and ECM components. The following dissertation serves to summarize a graduate research project designed to address the working hypothesis that PAI-1 represents a physiological cofactor for the conversion of vitronectin from a circulating, monomeric form to a matrix-associated, activated form. PAI-1-binding to vitronectin induces the formation of higher-order complexes that display altered adhesive properties distinct from the circulating (monomeric) form of vitronectin. These altered adhesive properties arise from the formation of multivalent binding sites for both cell surfaces and components of the ECM. Formation of vitronectin/PAI-1 complexes follows a step-wise assembly process that is dependent on protein concentration and time, ultimately leading to an increased accumulation of vitronectin at the cell-matrix interface. To address this hypothesis, biophysical studies were designed to evaluate the mechanism of assembly of PAI-1/vitronectin complexes and the pathway leading to multimerization of vitronectin. These studies were performed in parallel with cell- and matrix-binding experiments to establish a correlation between PAI-1-induced multimerization with enhanced adhesive properties and and conversion of vitronectin into a matrix-associated form

A mechanism for assembly of complexes of vitronectin and plasminogen activator inhibitor-1 from sedimentation velocity analysis

Plasminogen activator inhibitor-1 (PAI-1) and vitronectin are cofactors involved in pathological conditions such as injury, inflammation, and cancer, during which local levels of PAI-1 are increased and the active serpin forms complexes with vitronectin. These complexes become deposited into surrounding tissue matrices, where they regulate cell adhesion and pericellular proteolysis. The mechanism for their co-localization has not been elucidated. We hypothesize that PAI-1-vitronectin complexes form in a stepwise and concentration-dependent fashion via 1:1 and 2:1 intermediates, with the 2:1 complex serving a key role in assembly of higher order complexes. To test this hypothesis, sedimentation velocity experiments in the analytical ultracentrifuge were performed to identify different PAI-1-vitronectin complexes. Analysis of sedimentation data invoked a novel multisignal method to discern the stoichiometry of the two proteins in the higher-order complexes formed (Balbo, A., Minor, K. H., Velikovsky, C. A., Mariuzza, R. A., Peterson, C. B., and Schuck, P. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 81-86). Our results demonstrate that PAI-1 and vitronectin assemble into higher order forms via a pathway that is triggered upon saturation of the two PAI-1-binding sites of vitronectin to form the 2:1 complex. This 2:1 PAI-1-vitronectin complex, with a sedimentation coefficient of 6.5 S, is the key intermediate for the assembly of higher order complexes

Connectivity and systemic resilience of the Great Barrier Reef

Australia’s iconic Great Barrier Reef (GBR) continues to suffer from repeated impacts of cyclones, coral bleaching, and outbreaks of the coral-eating crown-of-thorns starfish (COTS), losing much of its coral cover in the process. This raises the question of the ecosystem’s systemic resilience and its ability to rebound after large-scale population loss. Here, we reveal that around 100 reefs of the GBR, or around 3%, have the ideal properties to facilitate recovery of disturbed areas, thereby imparting a level of systemic resilience and aiding its continued recovery. These reefs (1) are highly connected by ocean currents to the wider reef network, (2) have a relatively low risk of exposure to disturbances so that they are likely to provide replenishment when other reefs are depleted, and (3) have an ability to promote recovery of desirable species but are unlikely to either experience or spread COTS outbreaks. The great replenishment potential of these ‘robust source reefs’, which may supply 47% of the ecosystem in a single dispersal event, emerges from the interaction between oceanographic conditions and geographic location, a process that is likely to be repeated in other reef systems. Such natural resilience of reef systems will become increasingly important as the frequency of disturbances accelerates under climate change

Global urban environmental change drives adaptation in white clover

Urbanization transforms environments in ways that alter biological evolution. We examined whether urban environmental change drives parallel evolution by sampling 110,019 white clover plants from 6169 populations in 160 cities globally. Plants were assayed for a Mendelian antiherbivore defense that also affects tolerance to abiotic stressors. Urban-rural gradients were associated with the evolution of clines in defense in 47% of cities throughout the world. Variation in the strength of clines was explained by environmental changes in drought stress and vegetation cover that varied among cities. Sequencing 2074 genomes from 26 cities revealed that the evolution of urban-rural clines was best explained by adaptive evolution, but the degree of parallel adaptation varied among cities. Our results demonstrate that urbanization leads to adaptation at a global scale

Spinal cord injury-induced plasticity in the mouse-the crossed phrenic phenomenon

The crossed phrenic phenomenon (CPP) describes respiratory functional plasticity that arises following spinal cord injury. Cervical spinal cord hemisection rostral to the phrenic nucleus paralyzes the ipsilateral hemidiaphragm by interrupting the descending flow of respiratory impulses from the medulla to phrenic motoneurons in the spinal cord. This loss of activity converts some synapses on phrenic motoneurons from a "functionally ineffective" state pre-hemisection to a "functionally latent" state post-hemisection. If the animal is subjected to respiratory stress by transecting the contralateral phrenic nerve, this latent respiratory pathway is activated and function is restored to the paralyzed hemidiaphragm. The mechanisms underlying this plasticity are not well-defined, particularly at the molecular level. Therefore, we explored whether it was possible to demonstrate the CPP in mice, a species amenable to a molecular genetic approach. We show the CPP qualitatively in mice using electromyographic (EMG) recordings from the diaphragm. Interestingly, our data also suggest that in the mouse latent fibers in the ventral funiculus ipsilateral to an anatomically incomplete hemisection may also play a role in the CPP. In particular, we examined the inter-operative delay time between the spinal cord injury and contralateral phrenicotomy required for a response. As the inter-operative delay was reduced, the proportion of mice displaying the CPP decreased from 95% for overnight animals, 86% in 4-8\ua0h, to 77% for 1-2\ua0h mice, and less than 28% for animals receiving a phrenicotomy under 0.5\ua0h post-spinal cord lesion. This is the first study to demonstrate the CPP in mice

Studying multiprotein complexes by multisignal sedimentation velocity analytical ultracentrifugation

Protein interactions can promote the reversible assembly of multiprotein complexes, which have been identified as critical elements in many regulatory processes in cells. The biophysical characterization of assembly products, their number and stoichiometry, and the dynamics of their interactions in solution can be very difficult. A classical first-principle approach for the study of purified proteins and their interactions is sedimentation velocity analytical ultracentrifugation. This approach allows one to distinguish different protein complexes based on their migration in the centrifugal field without isolating reversibly formed complexes from the individual components. An important existing limitation for systems with multiple components and assembly products is the identification of the species associated with the observed sedimentation rates. We developed a computational approach for integrating multiple optical signals into the sedimentation coefficient distribution analysis of components, which combines the size-dependent hydrodynamic separation with discrimination of the extinction properties of the sedimenting species. This approach allows one to deduce the stoichiometry and to assign the identity of the assembly products without prior assumptions of the number of species and the nature of their interaction. Although chromophoric labels may be used to enhance the spectral resolution, we demonstrate the ability to work label-free for three-component protein mixtures. We observed that the spectral discrimination can synergistically enhance the hydrodynamic resolution. This method can take advantage of differences in the absorbance spectra of interacting solution components, for example, for the study of protein–protein, protein–nucleic acid or protein–small molecule interactions, and can determine the size, hydrodynamic shape, and stoichiometry of multiple complexes in solution

Interactions of plasminogen activator inhibitor-1 with vitronectin involve an extensive binding surface and induce mutual conformational rearrangements

In order to explore early events during the association of plasminogen activator inhibitor-1 (PAI-1) with its cofactor vitronectin, we have applied a robust strategy that combines protein engineering, fluorescence spectroscopy, and rapid reaction kinetics. Fluorescence stopped-flow experiments designed to monitor the rapid association of PAI-1 with vitronectin indicate a fast, concentration-dependent, biphasic binding of PAI-1 to native vitronectin but only a monophasic association with the somatomedin B (SMB) domain, suggesting that multiple phases of the binding interaction occur only when full-length vitronectin is present. Nonetheless, in all cases, the initial fast interaction is followed by slower fluorescence changes attributed to a conformational change in PAI-1. Complementary experiments using an engineered, fluorescently silent PAI-1 with non-natural amino acids showed that concomitant structural changes occur as well in native vitronectin. Furthermore, we have measured the effect of vitronectin on the rate of insertion of the reactive center loop into β-sheet A of PAI-1 during reaction with target proteases. With a variety of PAI-1 variants, we observe that both full-length vitronectin and the SMB domain have protease-specific effects on the rate of loop insertion but that the two exhibit clearly different effects. These results support a model for PAI-1 binding to vitronectin in which the interaction surface extends beyond the region of PAI-1 occupied by the SMB domain. In support of this model are recent results that define a PAI-1-binding site on vitronectin that lies outside the somatomedin B domain (Schar, C. R., Blouse, G. E., Minor, K. H., and Peterson, C. B. (2008) J. Biol. Chem. 283, 10297-10309) and the complementary site on PAI-1 (Schar, C. R., Jensen, J. K., Christensen, A., Blouse, G. E., Andreasen, P. A., and Peterson, C. B. (2008) J. Biol. Chem. 283, 28487-28496). © Copyright 2009 by the American Chemical Society