Effect of temperature and nucleotide on the binding of BiP chaperone to a protein substrate.

BiP (immunoglobulin heavy-chain binding protein) is a Hsp70 monomeric ATPase motor that plays broad and crucial roles in maintaining proteostasis inside the cell. Structurally, BiP is formed by two domains, a nucleotide-binding domain (NBD) with ATPase activity connected by a flexible hydrophobic linker to the substrate-binding domain. While the ATPase and substrate binding activities of BiP are allosterically coupled, the latter is also dependent on nucleotide binding. Recent structural studies have provided new insights into BiP's allostery; however, the influence of temperature on the coupling between substrate and nucleotide binding to BiP remains unexplored. Here, we study BiP's binding to its substrate at the single molecule level using thermo-regulated optical tweezers which allows us to mechanically unfold the client protein and explore the effect of temperature and different nucleotides on BiP binding. Our results confirm that the affinity of BiP for its protein substrate relies on nucleotide binding, by mainly regulating the binding kinetics between BiP and its substrate. Interestingly, our findings also showed that the apparent affinity of BiP for its protein substrate in the presence of nucleotides remains invariable over a wide range of temperatures, suggesting that BiP may interact with its client proteins with similar affinities even when the temperature is not optimal. Thus, BiP could play a role as a "thermal buffer" in proteostasis.

Determination of protein-protein interactions at the single-molecule level using optical tweezers.

Biomolecular interactions are at the base of all physical processes within living organisms; the study of these interactions has led to the development of a plethora of different methods. Among these, single-molecule (in singulo) experiments have become relevant in recent years because these studies can give insight into mechanisms and interactions that are hidden for ensemble-based (in multiplo) methods. The focus of this review is on optical tweezer (OT) experiments, which can be used to apply and measure mechanical forces in molecular systems. OTs are based on optical trapping, where a laser is used to exert a force on a dielectric bead; and optically trap the bead at a controllable position in all three dimensions. Different experimental approaches have been developed to study protein­protein interactions using OTs, such as: (1) refolding and unfolding in trans interaction where one protein is tethered between the beads and the other protein is in the solution; (2) constant force in cis interaction where each protein is bound to a bead, and the tension is suddenly increased. The interaction may break after some time, giving information about the lifetime of the binding at that tension. And (3) force ramp in cis interaction where each protein is attached to a bead and a ramp force is applied until the interaction breaks. With these experiments, parameters such as kinetic constants (koff, kon), affinity values (KD), energy to the transition state ΔG≠, distance to the transition state Δx≠ can be obtained. These parameters characterize the energy landscape of the interaction. Some parameters such as distance to the transition state can only be obtained from force spectroscopy experiments such as those described here.

PKD2/polycystin-2 induces autophagy by forming a complex with BECN1.

Macroautophagy/autophagy is an intracellular process involved in the breakdown of macromolecules and organelles. Recent studies have shown that PKD2/PC2/TRPP2 (polycystin 2, transient receptor potential cation channel), a nonselective cation channel permeable to Ca2+ that belongs to the family of transient receptor potential channels, is required for autophagy in multiple cell types by a mechanism that remains unclear. Here, we report that PKD2 forms a protein complex with BECN1 (beclin 1), a key protein required for the formation of autophagic vacuoles, by acting as a scaffold that interacts with several co-modulators via its coiled-coil domain (CCD). Our data identified a physical and functional interaction between PKD2 and BECN1, which depends on one out of two CCD domains (CC1), located in the carboxy-terminal tail of PKD2. In addition, depletion of intracellular Ca2+ with BAPTA-AM not only blunted starvation-induced autophagy but also disrupted the PKD2-BECN1 complex. Consistently, PKD2 overexpression triggered autophagy by increasing its interaction with BECN1, while overexpression of PKD2D509V, a Ca2+ channel activity-deficient mutant, did not induce autophagy and manifested diminished interaction with BECN1. Our findings show that the PKD2-BECN1 complex is required for the induction of autophagy, and its formation depends on the presence of the CC1 domain of PKD2 and on intracellular Ca2+ mobilization by PKD2. These results provide new insights regarding the molecular mechanisms by which PKD2 controls autophagy.Abbreviations: ADPKD: autosomal dominant polycystic kidney disease; ATG: autophagy-related; ATG14/ATG14L: autophagy related 14; Baf A1: bafilomycin A1; BCL2/Bcl-2: BCL2 apoptosis regulator; BCL2L1/BCL-XL: BCL2 like 1; BECN1: beclin 1; CCD: coiled-coil domain; EBSS: Earle's balanced salt solution; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GOLGA2/GM130: golgin A2; GST: glutathione s-transferase; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; NBR1: NBR1 autophagy cargo receptor; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PKD2/PC2: polycystin 2, transient receptor potential cation channel; RTN4/NOGO: reticulon 4; RUBCN/RUBICON: rubicon autophagy regulator; SQSTM1/p62: sequestosome 1; UVRAG: UV radiation resistance associated; WIPI2: WD repeat domain, phosphoinositide interacting 2.

Application of Force to a Syndecan-4 Containing Complex With Thy-1-αVß3 Integrin Accelerates Neurite Retraction.

Inflammation contributes to the genesis and progression of chronic diseases, such as cancer and neurodegeneration. Upregulation of integrins in astrocytes during inflammation induces neurite retraction by binding to the neuronal protein Thy-1, also known as CD90. Additionally, Thy-1 alters astrocyte contractility and movement by binding to the mechano-sensors αVß3 integrin and Syndecan-4. However, the contribution of Syndecan-4 to neurite shortening following Thy-1-αVß3 integrin interaction remains unknown. To further characterize the contribution of Syndecan-4 in Thy-1-dependent neurite outgrowth inhibition and neurite retraction, cell-based assays under pro-inflammatory conditions were performed. In addition, using Optical Tweezers, we studied single-molecule binding properties between these proteins, and their mechanical responses. Syndecan-4 increased the lifetime of Thy-1-αVß3 integrin binding by interacting directly with Thy-1 and forming a ternary complex (Thy-1-αVß3 integrin + Syndecan-4). Under in vitro-generated pro-inflammatory conditions, Syndecan-4 accelerated the effect of integrin-engaged Thy-1 by forming this ternary complex, leading to faster neurite retraction and the inhibition of neurite outgrowth. Thus, Syndecan-4 controls neurite cytoskeleton contractility by modulating αVß3 integrin mechano-receptor function. These results suggest that mechano-transduction, cell-matrix and cell-cell interactions are likely critical events in inflammation-related disease development.

Connexins in Astrocyte Migration.

Astrocytes have long been considered the supportive cells of the central nervous system, but during the last decades, they have gained much more attention because of their active participation in the modulation of neuronal function. For example, after brain damage, astrocytes become reactive and undergo characteristic morphological and molecular changes, such as hypertrophy and increase in the expression of glial fibrillary acidic protein (GFAP), in a process known as astrogliosis. After severe damage, astrocytes migrate to the lesion site and proliferate, which leads to the formation of a glial scar. At this scar-forming stage, astrocytes secrete many factors, such as extracellular matrix proteins, cytokines, growth factors and chondroitin sulfate proteoglycans, stop migrating, and the process is irreversible. Although reactive gliosis is a normal physiological response that can protect brain cells from further damage, it also has detrimental effects on neuronal survival, by creating a hostile and non-permissive environment for axonal repair. The transformation of astrocytes from reactive to scar-forming astrocytes highlights migration as a relevant regulator of glial scar formation, and further emphasizes the importance of efficient communication between astrocytes in order to orchestrate cell migration. The coordination between astrocytes occurs mainly through Connexin (Cx) channels, in the form of direct cell-cell contact (gap junctions, GJs) or contact between the extracellular matrix and the astrocytes (hemichannels, HCs). Reactive astrocytes increase the expression levels of several proteins involved in astrocyte migration, such as αvß3 Integrin, Syndecan-4 proteoglycan, the purinergic receptor P2X7, Pannexin1, and Cx43 HCs. Evidence has indicated that Cx43 HCs play a role in regulating astrocyte migration through the release of small molecules to the extracellular space, which then activate receptors in the same or adjacent cells to continue the signaling cascades required for astrocyte migration. In this review, we describe the communication of astrocytes through Cxs, the role of Cxs in inflammation and astrocyte migration, and discuss the molecular mechanisms that regulate Cx43 HCs, which may provide a therapeutic window of opportunity to control astrogliosis and the progression of neurodegenerative diseases.

Single-molecule measurements of the effect of force on Thy-1/αvß3-integrin interaction using nonpurified proteins.

Thy-1 and αvß3 integrin mediate bidirectional cell-to-cell communication between neurons and astrocytes. Thy-1/αvß3 interactions stimulate astrocyte migration and the retraction of neuronal prolongations, both processes in which internal forces are generated affecting the bimolecular interactions that maintain cell-cell adhesion. Nonetheless, how the Thy-1/αvß3 interactions respond to mechanical cues is an unresolved issue. In this study, optical tweezers were used as a single-molecule force transducer, and the Dudko-Hummer-Szabo model was applied to calculate the kinetic parameters of Thy-1/αvß3 dissociation. A novel experimental strategy was implemented to analyze the interaction of Thy-1-Fc with nonpurified αvß3-Fc integrin, whereby nonspecific rupture events were corrected by using a new mathematical approach. This methodology permitted accurately estimating specific rupture forces for Thy-1-Fc/αvß3-Fc dissociation and calculating the kinetic and transition state parameters. Force exponentially accelerated Thy-1/αvß3 dissociation, indicating slip bond behavior. Importantly, nonspecific interactions were detected even for purified proteins, highlighting the importance of correcting for such interactions. In conclusion, we describe a new strategy to characterize the response of bimolecular interactions to forces even in the presence of nonspecific binding events. By defining how force regulates Thy-1/αvß3 integrin binding, we provide an initial step towards understanding how the neuron-astrocyte pair senses and responds to mechanical cues.

αVß3 Integrin regulates astrocyte reactivity.

BACKGROUND: Neuroinflammation involves cytokine release, astrocyte reactivity and migration. Neuronal Thy-1 promotes DITNC1 astrocyte migration by engaging αVß3 Integrin and Syndecan-4. Primary astrocytes express low levels of these receptors and are unresponsive to Thy-1; thus, inflammation and astrocyte reactivity might be necessary for Thy-1-induced responses. METHODS: Wild-type rat astrocytes (TNF-activated) or from human SOD1G93A transgenic mice (a neurodegenerative disease model) were used to evaluate cell migration, Thy-1 receptor levels, signaling molecules, and reactivity markers. RESULTS: Thy-1 induced astrocyte migration only after TNF priming. Increased expression of αVß3 Integrin, Syndecan-4, P2X7R, Pannexin-1, Connexin-43, GFAP, and iNOS were observed in TNF-treated astrocytes. Silencing of ß3 Integrin prior to TNF treatment prevented Thy-1-induced migration, while ß3 Integrin over-expression was sufficient to induce astrocyte reactivity and allow Thy-1-induced migration. Finally, hSOD1G93A astrocytes behave as TNF-treated astrocytes since they were reactive and responsive to Thy-1. CONCLUSIONS: Therefore, inflammation induces expression of αVß3 Integrin and other proteins, astrocyte reactivity, and Thy-1 responsiveness. Importantly, ectopic control of ß3 Integrin levels modulates these responses regardless of inflammation.

Integrin-mediated transactivation of P2X7R via hemichannel-dependent ATP release stimulates astrocyte migration.

Our previous reports indicate that ligand-induced αVß3 integrin and Syndecan-4 engagement increases focal adhesion formation and migration of astrocytes. Additionally, ligated integrins trigger ATP release through unknown mechanisms, activating P2X7 receptors (P2X7R), and the uptake of Ca(2+) to promote cell adhesion. However, whether the activation of P2X7R and ATP release are required for astrocyte migration and whether αVß3 integrin and Syndecan-4 receptors communicate with P2X7R via ATP remains unknown. Here, cells were stimulated with Thy-1, a reported αVß3 integrin and Syndecan-4 ligand. Results obtained indicate that ATP was released by Thy-1 upon integrin engagement and required the participation of phosphatidylinositol-3-kinase (PI3K), phospholipase-C gamma (PLCγ) and inositol trisphosphate (IP3) receptors (IP3R). IP3R activation leads to increased intracellular Ca(2+), hemichannel (Connexin-43 and Pannexin-1) opening, and ATP release. Moreover, silencing of the P2X7R or addition of hemichannel blockers precluded Thy-1-induced astrocyte migration. Finally, Thy-1 lacking the integrin-binding site did not stimulate ATP release, whereas Thy-1 mutated in the Syndecan-4-binding domain increased ATP release, albeit to a lesser extent and with delayed kinetics compared to wild-type Thy-1. Thus, hemichannels activated downstream of an αVß3 integrin-PI3K-PLCγ-IP3R pathway are responsible for Thy-1-induced, hemichannel-mediated and Syndecan-4-modulated ATP release that transactivates P2X7Rs to induce Ca(2+) entry. These findings uncover a hitherto unrecognized role for hemichannels in the regulation of astrocyte migration via P2X7R transactivation induced by integrin-mediated ATP release.

Generation of superoxide radicals by copper-glutathione complexes: redox-consequences associated with their interaction with reduced glutathione.

The interaction between Cu(2+) ions and GSH molecules leads to the swift formation of the physiologically occurring Cu(I)-[GSH](2) complex. Recently, we reported that this complex is able to reduce molecular oxygen into superoxide in a reversible reaction. In the present study, by means of fluorescence, luminescence, EPR and NMR techniques, we investigated the superoxide-generating capacity of the Cu(I)-[GSH](2) complex, demonstrated the occurrence and characterized the chemical nature of the oxidized complex which is formed upon removing of superoxide radicals from the former reaction, and addressed some of the redox consequences associated with the interaction between the Cu(I)-[GSH](2) complex, its oxidized complex form, and an in-excess of GSH molecules. The interaction between Cu(I)-[GSH](2) and added GSH molecules led to an substantial exacerbation of the ability of the former to generate superoxide anions. Removal of superoxide from a solution containing the Cu(I)-[GSH](2) complex, by addition of Tempol, led to the formation and accumulation of Cu(II)-GSSG. Interaction between the latter complex and GSH molecules permitted the re-generation of the Cu(I)-[GSH](2) complex and led to a concomitant recovery of its superoxide-generating capacity. Some of the potential redox and biological implications arising from these interactions are discussed.