Alejandra Sáenz

Self-assembly of spider silk proteins is controlled by a pH-sensitive relay.

Nature’s high-performance polymer, spider silk, consists of specific proteins, spidroins, with repetitive segments flanked by conserved non-repetitive domains. Spidroins are stored as a highly concentrated fluid dope. On silk formation, intermolecular interactions between repeat regions are established that provide strength and elasticity. How spiders manage to avoid premature spidroin aggregation before self-assembly is not yet established. A pH drop to 6.3 along the spider’s spinning apparatus, altered salt composition and shear forces are believed to trigger the conversion to solid silk, but no molecular details are known. Miniature spidroins consisting of a few repetitive spidroin segments capped by the carboxy-terminal domain form metre-long silk-like fibres irrespective of pH. We discovered that incorporation of the amino-terminal domain of major ampullate spidroin 1 from the dragline of the nursery web spider Euprosthenops australis (NT) into mini-spidroins enables immediate, charge-dependent self-assembly at pH values around 6.3, but delays aggregation above pH 7. The X-ray structure of NT, determined to 1.7 Å resolution, shows a homodimer of dipolar, antiparallel five-helix bundle subunits that lack homologues. The overall dimeric structure and observed charge distribution of NT is expected to be conserved through spider evolution and in all types of spidroins. Our results indicate a relay-like mechanism through which the N-terminal domain regulates spidroin assembly by inhibiting precocious aggregation during storage, and accelerating and directing self-assembly as the pH is lowered along the spider’s silk extrusion duct.

High-resolution structure of a BRICHOS domain and its implications for anti-amyloid chaperone activity on lung surfactant protein C

BRICHOS domains are encoded in > 30 human genes, which are associated with cancer, neurodegeneration, and interstitial lung disease (ILD). The BRICHOS domain from lung surfactant protein C proprotein (proSP-C) is required for membrane insertion of SP-C and has anti-amyloid activity in vitro. Here, we report the 2.1 Å crystal structure of the human proSP-C BRICHOS domain, which, together with molecular dynamics simulations and hydrogen-deuterium exchange mass spectrometry, reveals how BRICHOS domains may mediate chaperone activity. Observation of amyloid deposits composed of mature SP-C in lung tissue samples from ILD patients with mutations in the BRICHOS domain or in its peptide-binding linker region supports the in vivo relevance of the proposed mechanism. The results indicate that ILD mutations interfering with proSP-C BRICHOS activity cause amyloid disease secondary to intramolecular chaperone malfunction.

Physical properties and surface activity of surfactant-like membranes containing the cationic and hydrophobic peptide KL4

Surfactant‐like membranes containing the 21‐residue peptide KLLLLKLLLLKLLLLKLLLLK (KL4), have been clinically tested as a therapeutic agent for respiratory distress syndrome in premature infants. The aims of this study were to investigate the interactions between the KL4 peptide and lipid bilayers, and the role of both the lipid composition and KL4 structure on the surface adsorption activity of KL4‐containing membranes. We used bilayers of three‐component systems [1,2‐dipalmitoyl‐phosphatidylcholine/1‐palmitoyl‐2‐oleoyl‐phosphatidylglycerol/palmitic acid (DPPC/POPG/PA) and DPPC/1‐palmitoyl‐2‐oleoyl‐phosphatidylcholine (POPC)/PA] and binary lipid mixtures of DPPC/POPG and DPPC/PA to examine the specific interaction of KL4 with POPG and PA. We found that, at low peptide concentrations, KL4 adopted a predominantly α‐helical secondary structure in POPG‐ or POPC‐containing membranes, and a β‐sheet structure in DPPC/PA vesicles. As the concentration of the peptide increased, KL4 interconverted to a β‐sheet structure in DPPC/POPG/PA or DPPC/POPC/PA vesicles. Ca2+ favored α⇆β interconversion. This conformational flexibility of KL4 did not influence the surface adsorption activity of KL4‐containing vesicles. KL4 showed a concentration‐dependent ordering effect on POPG‐ and POPC‐containing membranes, which could be linked to its surface activity. In addition, we found that the physical state of the membrane had a critical role in the surface adsorption process. Our results indicate that the most rapid surface adsorption takes place with vesicles showing well‐defined solid/fluid phase co‐existence at temperatures below their gel to fluid phase transition temperature, such as those of DPPC/POPG/PA and DPPC/POPC/PA. In contrast, more fluid (DPPC/POPG) or excessively rigid (DPPC/PA) KL4‐containing membranes fail in their ability to adsorb rapidly onto and spread at the air–water interface.

C-terminal, endoplasmic reticulum-lumenal domain of prosurfactant protein C - structural features and membrane interactions

Surfactant protein C (SP‐C) constitutes the transmembrane part of prosurfactant protein C (proSP‐C) and is α‐helical in its native state. The C‐terminal part of proSP‐C (CTC) is localized in the endoplasmic reticulum lumen and binds to misfolded (β‐strand) SP‐C, thereby preventing its aggregation and amyloid fibril formation. In this study, we investigated the structure of recombinant human CTC and the effects of CTC–membrane interaction on protein structure. CTC forms noncovalent trimers and supratrimeric oligomers. It contains two intrachain disulfide bridges, and its secondary structure is significantly affected by urea or heat only after disulfide reduction. The postulated Brichos domain of CTC, with homologs found in proteins associated with amyloid and proliferative disease, is up to 1000‐fold more protected from limited proteolysis than the rest of CTC. The protein exposes hydrophobic surfaces, as determined by CTC binding to the environment‐sensitive fluorescent probe 1,1′‐bis(4‐anilino‐5,5′‐naphthalenesulfonate). Fluorescence energy transfer experiments further reveal close proximity between bound 1,1′‐bis(4‐anilino‐5,5′‐naphthalenesulfonate) and tyrosine residues in CTC, some of which are conserved in all Brichos domains. CTC binds to unilamellar phospholipid vesicles with low micromolar dissociation constants, and differential scanning calorimetry and CD analyses indicate that membrane‐bound CTC is less structurally ordered than the unbound protein. The exposed hydrophobic surfaces and the structural disordering that result from interactions with phospholipid membranes suggest a mechanism whereby CTC binds to misfolded SP‐C in the endoplasmic reticulum membrane.

Fluidizing effects of C-reactive protein on lung surfactant membranes: protective role of surfactant protein A.

The purpose of this study was to investigate how surfactant membranes can be perturbed by C‐reactive protein (CRP) and whether surfactant protein A (SP‐A) might overcome CRP‐induced surfactant membrane alterations. The effect of CRP on surfactant surface adsorption was evaluated in vivo after intratracheal instillation of CRP into rat lungs. Insertion of CRP into surfactant membranes was investigated through monolayer techniques. The effect of CRP on membrane structure was studied through differential scanning calorimetry and fluorescence spectroscopy and microscopy using large and giant unilamellar vesicles. Our results indicate that CRP inserts into surfactant membranes and drastically increases membrane fluidity, resulting in surfactant inactivation. At 10% CRP/phospholipid weight ratio, CRP causes disappearance of liquid‐ordered/liquid‐disordered phase coexistence distinctive of surfactant membranes. SP‐A, the most abundant surfactant lipoprotein structurally similar to C1q, binds to CRP (Kd=56±8 nM), as determined by solid‐phase binding assays and dynamic light scattering. This novel SP‐A/CRP interaction reduces CRP insertion and blocks CRP effects on surfactant membranes. In addition, intratracheal coinstillation of SP‐A+ CRP into rat lungs prevents surfactant inhibition induced by CRP, indicating that SP‐A/CRP interactions might be an important factor in vivo in controlling harmful CRP effects in the alveolus.—Sáenz, A., López‐Sánchez, A., Mojica‐Lázaro, J., Martínez‐Caro, L., Nin, N., Bagatolli, L. A., Casals, C. Fluidizing effects of C‐reactive protein on lung surfactant membranes: protective role of surfactant protein A. FASEB J. 24, 3662–3673 (2010). www.fasebj.org

Surfactant Protein A Prevents IFN-γ/IFN-γ Receptor Interaction and Attenuates Classical Activation of Human Alveolar Macrophages

Lung surfactant protein A (SP-A) plays an important function in modulating inflammation in the lung. However, the exact role of SP-A and the mechanism by which SP-A affects IFN-γ–induced activation of alveolar macrophages (aMϕs) remains unknown. To address these questions, we studied the effect of human SP-A on rat and human aMϕs stimulated with IFN-γ, LPS, and combinations thereof and measured the induction of proinflammatory mediators as well as SP-A’s ability to bind to IFN-γ or IFN-γR1. We found that SP-A inhibited (IFN-γ + LPS)–induced TNF-α, iNOS, and CXCL10 production by rat aMϕs. When rat macrophages were stimulated with LPS and IFN-γ separately, SP-A inhibited both LPS-induced signaling and IFN-γ–elicited STAT1 phosphorylation. SP-A also decreased TNF-α and CXCL10 secretion by ex vivo–cultured human aMϕs and M-CSF–derived macrophages stimulated by either LPS or IFN-γ or both. Hence, SP-A inhibited upregulation of IFN-γ–inducible genes (CXCL10, RARRES3, and ETV7) as well as STAT1 phosphorylation in human M-CSF–derived macrophages. In addition, we found that SP-A bound to human IFN-γ (KD = 11 ± 0.5 nM) in a Ca2+-dependent manner and prevented IFN-γ interaction with IFN-γR1 on human aMϕs. We conclude that SP-A inhibition of (IFN-γ + LPS) stimulation is due to SP-A attenuation of both inflammatory agents and that the binding of SP-A to IFN-γ abrogates IFN-γ effects on human macrophages, suppressing their classical activation and subsequent inflammatory response.

Natural Anti-Infective Pulmonary Proteins: In Vivo Cooperative Action of Surfactant Protein SP-A and the Lung Antimicrobial Peptide SP-BN

The anionic antimicrobial peptide SP-BN, derived from the N-terminal saposin-like domain of the surfactant protein (SP)-B proprotein, and SP-A are lung anti-infective proteins. SP-A–deficient mice are more susceptible than wild-type mice to lung infections, and bacterial killing is enhanced in transgenic mice overexpressing SP-BN. Despite their potential anti-infective action, in vitro studies indicate that several microorganisms are resistant to SP-A and SP-BN. In this study, we test the hypothesis that these proteins act synergistically or cooperatively to strengthen each other’s microbicidal activity. The results indicate that the proteins acted synergistically in vitro against SP-A– and SP-BN–resistant capsulated Klebsiella pneumoniae (serotype K2) at neutral pH. SP-A and SP-BN were able to interact in solution (Kd = 0.4 μM), which enabled their binding to bacteria with which SP-A or SP-BN alone could not interact. In vivo, we found that treatment of K. pneumoniae–infected mice with SP-A and SP-BN conferred more protection against K. pneumoniae infection than each protein individually. SP-A/SP-BN–treated infected mice showed significant reduction of bacterial burden, enhanced neutrophil recruitment, and ameliorated lung histopathology with respect to untreated infected mice. In addition, the concentrations of inflammatory mediators in lung homogenates increased early in infection in contrast with the weak inflammatory response of untreated K. pneumoniae–infected mice. Finally, we found that therapeutic treatment with SP-A and SP-BN 6 or 24 h after bacterial challenge conferred significant protection against K. pneumoniae infection. These studies show novel anti-infective pathways that could drive development of new strategies against pulmonary infections.

Beneficial effects of synthetic KL4­surfactant in experimental lung transplantation

The aim of this study was to investigate whether intratracheal administration of a new synthetic surfactant that includes the cationic, hydrophobic 21-residue peptide KLLLLKLLLLKLLLLKLLLLK (KL4), might be effective in reducing ischaemia–reperfusion injury after lung transplantation. Single left lung transplantation was performed in Landrace pigs 22 h post-harvest. KL4 surfactant at a dose of 25 mg total phospholipid·kg body weight−1 (2.5 mL·kg body weight−1) was instilled at 37°C to the donor left lung (n = 8) prior to explantation. Saline (2.5 mL·kg body weight−1; 37°C) was instilled into the donor left lung of the untreated group (n = 6). Lung function in recipients was measured during 2 h of reperfusion. Recipient left lung bronchoalveolar lavage (BAL) provided native cytometric, inflammatory marker and surfactant data. KL4 surfactant treatment recovered oxygen levels in the recipient blood (mean±sd arterial oxygen tension/inspiratory oxygen fraction 424±60 versus 263±101 mmHg in untreated group; p=0.01) and normalised alveolar–arterial oxygen tension difference. Surfactant biophysical function was also recovered in KL4 surfactant-treated lungs. This was associated with decreased C-reactive protein levels in BAL, and recovery of surfactant protein A content, normalised protein/phospholipid ratios, and lower levels of both lipid peroxides and protein carbonyls in large surfactant aggregates. These findings suggest an important protective role for KL4 surfactant treatment in lung transplantation.

Surfactant protein A (SP-A)-tacrolimus complexes have a greater anti-inflammatory effect than either SP-A or tacrolimus alone on human macrophage-like U937 cells.

Intratracheal administration of immunosuppressive agents to the lung is a novel treatment after lung transplantation. Nanoparticles of tacrolimus (FK506) might interact with human SP-A, which is the most abundant lipoprotein in the alveolar fluid. This study was undertaken to determine whether the formation of FK506/SP-A complexes interferes with FK506 immunosuppressive actions on stimulated human macrophage-like U937 cells. We found that SP-A was avidly bound to FK506 (K(d) = 35 ± 4nM), as determined by solid phase-binding assays and dynamic light scattering. Free FK506, at concentrations ≤ 1 μM, had no effect on the inflammatory response of LPS-stimulated U937 macrophages. However, coincubation of FK506 and SP-A, at concentrations where each component alone did not affect LPS-stimulated macrophage response, significantly inhibited LPS-induced NF-κB activation and TNF-alpha secretion. Free FK506, but not FK506/SP-A, functioned as substrate for the efflux transporter P-glycoprotein. FK506 bound to SP-A was delivered to macrophages by endocytosis, since several endocytosis inhibitors blocked FK506/SP-A anti-inflammatory effects. This process depended partly on SP-A binding to its receptor, SP-R210. These results indicate that FK506/SP-A complexes have a greater anti-inflammatory effect than either FK506 or SP-A alone and suggest that SP-A strengthened FK506 anti-inflammatory activity by facilitating FK506 entrance into the cell, overcoming P-glycoprotein.

Folding and Intramembraneous BRICHOS Binding of the Prosurfactant Protein C Transmembrane Segment

Background: Amyloidogenic human lung surfactant protein C (SP-C) is a transmembrane peptide generated from pro-SP-C, containing a luminal BRICHOS domain. Results: BRICHOS inserts partly in ER membranes, binds unfolded SP-C, and prevents misfolding. Conclusion: Disease-associated pro-SP-C mutations result in loss of membrane insertion and binding to SP-C. Significance: Co-translational folding of transmembrane pro-SP-C is inefficient and membrane insertion of BRICHOS promotes correct folding. Surfactant protein C (SP-C) is a novel amyloid protein found in the lung tissue of patients suffering from interstitial lung disease (ILD) due to mutations in the gene of the precursor protein pro-SP-C. SP-C is a small α-helical hydrophobic protein with an unusually high content of valine residues. SP-C is prone to convert into β-sheet aggregates, forming amyloid fibrils. Natures way of solving this folding problem is to include a BRICHOS domain in pro-SP-C, which functions as a chaperone for SP-C during biosynthesis. Mutations in the pro-SP-C BRICHOS domain or linker region lead to amyloid formation of the SP-C protein and ILD. In this study, we used an in vitro transcription/translation system to study translocon-mediated folding of the WT pro-SP-C poly-Val and a designed poly-Leu transmembrane (TM) segment in the endoplasmic reticulum (ER) membrane. Furthermore, to understand how the pro-SP-C BRICHOS domain present in the ER lumen can interact with the TM segment of pro-SP-C, we studied the membrane insertion properties of the recombinant form of the pro-SP-C BRICHOS domain and two ILD-associated mutants. The results show that the co-translational folding of the WT pro-SP-C TM segment is inefficient, that the BRICHOS domain inserts into superficial parts of fluid membranes, and that BRICHOS membrane insertion is promoted by poly-Val peptides present in the membrane. In contrast, one BRICHOS and one non-BRICHOS ILD-associated mutant could not insert into membranes. These findings support a chaperone function of the BRICHOS domain, possibly together with the linker region, during pro-SP-C biosynthesis in the ER.