Regulation of p38 MAP kinase by anastellin is independent of anastellin's effect on matrix fibronectin

Anastellin is an angiogenesis inhibitor derived from the first type III repeat of fibronectin (FN). Anastellin binds to fibronectin and promotes the polymerization of soluble fibronectin into a highly polymerized form termed superfibronectin. In addition, anastellin also causes remodeling of pre-existing fibronectin matrix and modulates cell signaling pathways in both endothelial cells and fibroblasts. In the present study, we address the relationship of anastellin's effects on fibronectin matrix to its effects on p38 MAP kinase (MAPK) activation. Using a mutant form of anastellin which binds to fibronectin matrix, but does not stimulate formation of superfibronectin, we demonstrate that the activation of p38 MAPK by anastellin is not dependent on the formation of superfibronectin. The mutant form of anastellin does stimulate matrix remodeling, but experiments using FN^?/? cells show that the effect of anastellin on p38-MAPK activation is completely independent of fibronectin. Anastellin was able to activate p38 MAPK on cells in suspension as well as on cells null for β1 integrins, suggesting that anastellin activity did not require ligation of integrins. These data suggest that the activation of p38 MAPK by anastellin is independent of anastellin's effects on fibronectin matrix organization.     

Domain unfolding plays a role in superfibronectin formation

Superfibronectin (sFN) is a fibronectin (FN) aggregate that is formed by mixing FN with anastellin, a fragment of the first type III domain of FN. However, the mechanism of this aggregation has not been clear. In this study, we found that anastellin co-precipitated with FN in a ratio of approximately 4:1, anastellin:FN monomer. The primary binding site for anastellin was in the segment (III)1-3, which bound three molecules of anastellin and was able to form a precipitate without the rest of the FN molecule. Anastellin binding to (III)3 caused a conformational change in that domain that exposed a cryptic thermolysin-sensitive site. An additional anastellin binds to (III)11, where it enhances thermolysin digestion of (III)11. An engineered disulfide bond in (III)3 inhibited both aggregation and protease digestion, suggesting that the stability of (III)3 is a key factor in sFN formation. We propose a three-step model for sFN formation: 1) FN-III domains spontaneously unfold and refold; 2) anastellin binds to an unfolded domain, preventing its refolding and leaving it with exposed hydrophobic surfaces and beta-sheet edges; and 3) these exposed elements bind to similar exposed elements on other molecules, leading to aggregation. The model is consistent with our observation that the kinetics of aggregation are first order, with a reaction time of 500-700 s. Similar mechanisms may contribute to the assembly of the native FN matrix.     

Identification of the region responsible for fibril formation in the CAD domain of caspase-activated DNase

Caspase-activated DNase (CAD) has a compact domain at its N-terminus (CAD domain, 87 amino acid residues), which comprises one alpha-helix and five beta-strands forming a single sheet. The CAD domain of CAD (CAD-CD) forms amyloid fibrils containing alpha-helix at low pH in the presence of salt. To obtain insights into the mechanism of amyloid fibril formation, we identified the peptide region essential for fibril formation of CAD-CD and the region responsible for the salt requirement. We searched for these regions by constructing a series of deletion and point mutants of CAD-CD. Fibril formation by these CAD-CD mutants was examined by fluorescence analysis of thioflavin T and transmission electron microscopy. C-Terminal deletion and point mutation studies revealed that an aromatic residue near the C-terminus (Trp81) is critical for fibril formation. In addition, the main chain conformation of the beta5 strand, which forms a hydrophobic core with Trp81, was found to be important for the fibril formation by CAD-CD. The N-terminal 30 amino acid region containing two beta-strands was not essential for fibril formation. Rather, the N-terminal region was found to be responsible for the requirement of salt for fibril formation.     

Fibronectin aggregation and assembly: the unfolding of the second fibronectin type III domain

The mechanism of fibronectin (FN) assembly and the self-association sites are still unclear and contradictory, although the N-terminal 70-kDa region ((I)1-9) is commonly accepted as one of the assembly sites. We previously found that (I)1-9 binds to superfibronectin, which is an artificial FN aggregate induced by anastellin. In the present study, we found that (I)1-9 bound to the aggregate formed by anastellin and a small FN fragment, (III)1-2. An engineered disulfide bond in (III)2, which stabilizes folding, inhibited aggregation, but a disulfide bond in (III)1 did not. A gelatin precipitation assay showed that (I)1-9 did not interact with anastellin, (III)1, (III)2, (III)1-2, or several (III)1-2 mutants including (III)1-2KADA. (In contrast to previous studies, we found that the (III)1-2KADA mutant was identical in conformation to wild-type (III)1-2.) Because (I)1-9 only bound to the aggregate and the unfolding of (III)2 played a role in aggregation, we generated a (III)2 domain that was destabilized by deletion of the G strand. This mutant bound (I)1-9 as shown by the gelatin precipitation assay and fluorescence resonance energy transfer analysis, and it inhibited FN matrix assembly when added to cell culture. Next, we introduced disulfide mutations into full-length FN. Three disulfide locks in (III)2, (III)3, and (III)11 were required to dramatically reduce anastellin-induced aggregation. When we tested the disulfide mutants in cell culture, only the disulfide bond in (III)2 reduced the FN matrix. These results suggest that the unfolding of (III)2 is one of the key factors for FN aggregation and assembly.     

The physical state of fibronectin matrix differentially regulates morphogenetic movements in vivo

This study demonstrates that proper spatiotemporal expression and the physical assembly state of fibronectin (FN) matrix play key roles in the regulation of morphogenetic cell movements in vivo. We examine the progressive assembly and 3D fibrillar organization of FN and its role in regulating cell and tissue movements in Xenopus embryos. Expression of the 70 kD N-terminal fragment of FN blocks FN fibril assembly at gastrulation but not initial FN binding to integrins at the cell surface. We find that fibrillar FN is necessary to maintain cell polarity through oriented cell division and to promote epiboly, possibly through maintenance of tissue-surface tension. In contrast, FN fibrils are dispensable for convergence and extension movements required for axis elongation. Closure of the migratory mesendodermal mantle was accelerated in the absence of a fibrillar matrix. Thus, the macromolecular assembly of FN matrices may constitute a general regulatory mechanism for coordination of distinct morphogenetic movements.     

Syndecan-1 regulates cell migration and fibronectin fibril assembly

Corneal scarring is a major cause of blindness worldwide and can result from the deposition of abnormal amounts of collagen fibers lacking the correct size and spacing required to produce a clear cornea. Collagen fiber formation requires a preformed fibronectin (FN) matrix. We demonstrate that the loss of syndecan1 (sdc1) in corneal stromal cells (CSC) impacts cell migration rates, the sizes and composition of focal and fibrillar adhesions, the activation of integrins, and the assembly of fibronectin into fibrils. Integrin and fibronectin expression are not altered on sdc1-null CSCs. Cell adhesion, spreading, and migration studies using low compared to high concentrations of FN and collagen I (CNI) or vitronectin (VN) with and without activation of integrins by manganese chloride show that the impact of sdc1 depletion on integrin activation varies depending on the integrin-mediated activity evaluated. Differences in FN fibrillogenesis and migration in sdc1-null CSCs are reversed by addition of manganese chloride but cell spreading differences remain. To determine if our findings on sdc1 were specific to the cornea, we compared the phenotypes of sdc1-null dermal fibroblasts with those of CSCs. We found that without sdc1, both cell types migrate faster; however, cell-type-specific differences in FN expression and its assembly into fibrils exist between these two cell types. Together, our data demonstrate that sdc1 functions to regulate integrin activity in multiple cell types. Loss of sdc1-mediated integrin function results in cell-type specific differences in matrix assembly. A better understanding of how different cell types regulate FN fibril formation via syndecans and integrins will lead to better treatments for scarring and fibrosis.     

Opposing effects of collagen I and vitronectin on fibronectin fibril structure and function

Extracellular matrix fibronectin fibrils serve as passive structural supports for the organization of cells into tissues, yet can also actively stimulate a variety of cell and tissue functions, including cell proliferation. Factors that control and coordinate the functional activities of fibronectin fibrils are not known. Here, we compared effects of cell adhesion to vitronectin versus type I collagen on the assembly of and response to, extracellular matrix fibronectin fibrils. The amount of insoluble fibronectin matrix fibrils assembled by fibronectin-null mouse embryonic fibroblasts adherent to collagen- or vitronectin-coated substrates was not significantly different 20 h after fibronectin addition. However, the fibronectin matrix produced by vitronectin-adherent cells was ~ 10-fold less effective at enhancing cell proliferation than that of collagen-adherent cells. Increasing insoluble fibronectin levels with the fibronectin fragment, anastellin did not increase cell proliferation. Rather, native fibronectin fibrils polymerized by collagen- and vitronectin-adherent cells exhibited conformational differences in the growth-promoting, III-1 region of fibronectin, with collagen-adherent cells producing fibronectin fibrils in a more extended conformation. Fibronectin matrix assembly on either substrate was mediated by α5β1 integrins. However, on vitronectin-adherent cells, α5β1 integrins functioned in a lower activation state, characterized by reduced 9EG7 binding and decreased talin association. The inhibitory effect of vitronectin on fibronectin-mediated cell proliferation was localized to the cell-binding domain, but was not a general property of αvβ3 integrin-binding substrates. These data suggest that adhesion to vitronectin allows for the uncoupling of fibronectin fibril formation from downstream signaling events by reducing α5β1 integrin activation and fibronectin fibril extension.     

Deacylated pulmonary surfactant protein SP-C transforms from alpha-helical to amyloid fibril structure via a pH-dependent mechanism: an infrared structural investigation

Bovine pulmonary surfactant protein C (SP-C) is a hydrophobic, alpha-helical membrane-associated lipoprotein in which cysteines C4 and C5 are acylated with palmitoyl chains. Recently, it has been found that the alpha-helix form of SP-C is metastable, and under certain circumstances may transform from an alpha-helix to a beta-strand conformation that resembles amyloid fibrils. This transformation is accelerated when the protein is in its deacylated form (dSP-C). We have used infrared spectroscopy to study the structure of dSP-C in solution and at membrane interfaces. Our results show that dSP-C transforms from an alpha-helical to a beta-type amyloid fibril structure via a pH-dependent mechanism. In solution at low pH, dSP-C is alpha-helical in nature, but converts to an amyloid fibril structure composed of short beta-strands or beta-hairpins at neutral pH. The alpha-helix structure of dSP-C is fully recoverable from the amyloid beta-structure when the pH is once again lowered. Attenuated total reflectance infrared spectroscopy of lipid-protein monomolecular films showed that the fibril beta-form of dSP-C is not surface-associated at the air-water interface. In addition, the lipid-associated alpha-helix form of dSP-C is only retained at the surface at low surface pressures and dissociates from the membrane at higher surface pressures. In situ polarization modulation infrared spectroscopy of protein and lipid-protein monolayers at the air-water interface confirmed that the residual dSP-C helix conformation observed in the attenuated total reflectance infrared spectra of transferred films is randomly or isotropically oriented before exclusion from the membrane interface. This work identifies pH as one of the mechanistic causes of amyloid fibril formation for dSP-C, and a possible contributor to the pathogenesis of pulmonary alveolar proteinosis.     

Cryo-electron microscopy structure of an SH3 amyloid fibril and model of the molecular packing

Amyloid fibrils are assemblies of misfolded proteins and are associated with pathological conditions such as Alzheimer's disease and the spongiform encephalopathies. In the amyloid diseases, a diverse group of normally soluble proteins self-assemble to form insoluble fibrils. X-ray fibre diffraction studies have shown that the protofilament cores of fibrils formed from the various proteins all contain a cross-beta-scaffold, with beta-strands perpendicular and beta-sheets parallel to the fibre axis. We have determined the threedimensional structure of an amyloid fibril, formed by the SH3 domain of phosphatidylinositol-3'-kinase, using cryo-electron microscopy and image processing at 25 A resolution. The structure is a double helix of two protofilament pairs wound around a hollow core, with a helical crossover repeat of approximately 600 A and an axial subunit repeat of approximately 27 A. The native SH3 domain is too compact to fit into the fibril density, and must unfold to adopt a longer, thinner shape in the amyloid form. The 20x40-A protofilaments can only accommodate one pair of flat beta-sheets stacked against each other, with very little inter-strand twist. We propose a model for the polypeptide packing as a basis for understanding the structure of amyloid fibrils in general.     

Understanding the elasticity of fibronectin fibrils: unfolding strengths of FN-III and GFP domains measured by single molecule force spectroscopy.

While it is well established that fibronectin (FN) matrix fibrils are elastic, the mechanism of fibril elasticity during extension is still debated. To investigate the molecular origin of FN fibril elasticity, we used single molecule force spectroscopy (SMFS) to determine the unfolding behavior of a recombinant FN-III protein construct that contained eight FN-III domains ((1-8)FN-III) and two green fluorescent protein (GFP) domains. FN-III domains were distinguished from GFP domains by their shorter unfolding lengths. The unfolding strengths of both domains were determined for a wide range of pulling rates (50 to 1,745 nm/s). We found that the mechanical stabilities of FN-III and GFP domains were very similar to each other over the entire range of pulling speeds. FN fibrils containing GFP remain brightly fluorescent, even when stretched, meaning that GFP domains remain largely folded. Since GFP and FN-III have equal unfolding strengths, this suggests that FN-III domains are not extensively unraveled in stretched FN fibrils. Our results thus favor an alternative model, which invokes a conformational change from a compact to an extended conformation, as the basis for FN fibril elasticity.     

Surface-bound basement membrane components accelerate amyloid-β peptide nucleation in air-free wells: An in vitro model of cerebral amyloid angiopathy

Cerebral amyloid angiopathy is caused by deposition of the amyloid β-peptide which consists of mainly 39–40 residues to the cortical and leptomeningeal vessel walls. There are no definite in vitro systems to support the hypothesis that the vascular basement membrane may act as a scaffold of amyloid β-peptide carried by perivascular drainage flow and accelerate its amyloid fibril formation in vivo. We previously reported the critical roles of interfaces and agitation on the nucleation of amyloid fibrils at low concentrations of amyloid β-peptide monomers. Here, we reproduced the perivascular drainage flow in vitro by using N-hydroxysuccinimide-Sepharose 4 Fast flow beads as an inert stirrer in air-free wells rotated at 1 rpm. We then reproduced the basement membranes in the media of cerebral arteries in vitro by conjugating Matrigel and other proteins on the surface of Sepharose beads. These beads were incubated with 5 μM amyloid β(1–40) at 37 °C without air, where amyloid β(1–40) alone does not form amyloid fibrils. Using the initiation time of fibril growth kinetics (i.e., the lag time of fibril growth during which nuclei, on-pathway oligomers and protofibrils are successively formed) as a parameter of the efficiency of biological molecules to induce amyloid fibril formation, we found that basement membrane components including Matrigel, laminin, fibronectin, collagen type IV and fibrinogen accelerate the initiation of amyloid β-peptide fibril growth in vitro. These data support the essential role of vascular basement membranes in the development of cerebral amyloid angiopathy.     

Normal transthyretin and synthetic transthyretin fragments form amyloid-like fibrils in vitro

In two general forms of amyloidosis, senile systemic amyloidosis and several familial amyloidoses the amyloid fibrils are built up by transthyretin and fragments of the molecule. It has previously been demonstrated that other amyloid fibril proteins e.g. atrial natriuretic factor and islet amyloid polypeptide form amyloid-like fibrils in vitro. In this study we used normal transthyretin and synthetic polypeptides corresponding to segments of the transthyretin molecule. We show that normal transthyretin and two of our tested polypeptides, which corresponded to the beta-strands A and G, easily form amyloid-like fibrils in vitro.     

A single mutation in an SH3 domain increases amyloid aggregation by accelerating nucleation, but not by destabilizing thermodynamically the native state

We investigated the relationship between thermodynamic stability and amyloid aggregation propensity for a set of single mutants of the alpha-spectrin SH3 domain (Spc-SH3). Whilst mutations destabilizing the domain at position 56 did not enhance fibrillation, the N47A mutation increased the rate of amyloid fibril formation by 10-fold. Even under conditions of identical thermodynamic stability, the aggregation rate was much higher for the N47A mutant than for the WT domain. We conclude that the N47A mutation does not change the apparent mechanism of fibrillation or the morphology of the amyloid fibrils, and that its amyloidogenic property is due to its effect upon the rate of the conformational events leading to nucleation and not to its overall destabilizing effect.     

Phenolsulfonphthalein, but not phenolphthalein, inhibits amyloid fibril formation: implications for the modulation of amyloid self-assembly

The study of the mechanism of amyloid fibril formation and its inhibition is of key medical importance due to the lack of amyloid assembly inhibitors that are approved for clinical use. We have previously demonstrated the potent inhibitory potential of phenolsulfonphthalein, a nontoxic compound that was approved for diagnostic use in human subjects, on aggregation of islet amyloid polypeptide (IAPP) that is associated with type 2 diabetes. Here, we extend our studies on the mechanism of action of phenolsulfonphthalein by comparing its antiamyloidogenic effect to a very similar compound that is also approved for human use, phenolphthalein. While these compounds have very similar primary chemical structures, they significantly differ in their three-dimensional conformation. Our results clearly demonstrated that these two compounds had completely different inhibitory potencies: While phenolsulfonphthalein was a very potent inhibitor of amyloid fibril formation by IAPP, phenolphthalein did not show significant antiamyloidogenic activity. This behavior was observed with a short amyloid fragment of IAPP and also with the full-length polypeptide. The NMR spectrum of IAPP 20-29 in the presence of phenolsulfonphthalein showed chemical shift deviations that were different from the unbound or phenolphthalein-bound peptide. Differential activity was also observed in the inhibition of insulin amyloid formation by these two compounds, and density-gradient experiments clearly demonstrated the different inhibitory effect of the two compounds on the formation of prefibrillar assemblies. Taken together, our studies suggest that the three-dimensional arrangement of the polyphenol phenolsulfonphthalein has a central role in its amyloid formation inhibition activity.     

Does the location of a mutation determine the ability to form amyloid fibrils?

We have previously reported studies of fibril formation by a set of protein G B1 domain (beta1) variants, with mutations located around the central parallel beta-strands. In this study, we designed multiple mutations in the edge strands of beta1 to create proteins with a stability range comparable to that of the set of central mutants. All the edge variants are able to form amyloid fibrils when they are incubated at their melting temperatures. This result suggests that overall protein stability is the key determinant for amyloid formation and not the specific location of destabilizing mutations. The edge strand and variants cross-seed with each other and with members of the central variant family. Interesting fibrillar morphology was observed in some cross-seeding cases and its implications for a better understanding of nucleation and elongation events are discussed.     

Structure and aggregation mechanism of beta(2)-microglobulin (83-99) peptides studied by molecular dynamics simulations

Many human neurodegenerative diseases are associated with amyloid fibril formation. The human 99-residue beta(2)-microglobulin (beta2m) is one of the most intensively studied amyloid-forming proteins. Recent studies show that the C-terminal fragments 72-99, 83-89, and 91-96 form by themselves amyloid fibrils in vitro and play a significant role in fibrillization of the full-length beta2m protein under acidic pH conditions. In this work, we have studied the equilibrium structures of the 17-residue fragment 83-99 in solution, and investigated its dimerization process by multiple molecular dynamics simulations. We find that an intertwined dimer, with the positions of the beta-strands consistent with the results for the monomer, is a possible structure for two beta2m(83-89) peptides. Based on our molecular-dynamics-generated dimeric structure, a protofibril model is proposed for the full-length beta2m protein.     

Long noncoding RNA lnc-ABCA12-3 promotes cell migration,invasion, and proliferation by regulating fibronectin 1 in esophageal squamous cell carcinoma

Long noncoding RNAs (lncRNAs) have been shown to play important roles in human cancers, including esophageal squamous cell carcinoma (ESCC). We previously demonstrated that a novel lncRNA, lnc-ABCA12-3, was overexpressed in ESCC tissues. However, the exact function of lnc-ABCA12-3 is unknown. In the current study, we aimed to evaluate the expression of lnc-ABCA12-3 in ESCC and to explore the potential mechanism of lnc-ABCA12-3 in cell migration, invasion, and proliferation. We showed that lnc-ABCA12-3 was upregulated in ESCC tumor tissues and cell lines. The increased expression of lnc-ABCA12-3 was positively associated with advanced tumor-node-metastasis stages and poor prognosis. The knockdown of lnc-ABCA12-3 inhibited the cell migration, invasion, and proliferation abilities of KYSE-510 and Eca-109 cells. We also found that fibronectin 1 (FN1) was upregulated in ESCC tumor tissues. The expression of FN1 messenger RNA was positively correlated with the expression of lnc-ABCA12-3 in ESCC tumor tissues. After lnc-ABCA12-3 knockdown, the expression of FN1 was downregulated. In addition, the overexpression of FN1 restored the abilities of cell migration, invasion and proliferation in Eca-109 cells. Further studies indicated that lnc-ABCA12-3 acted as a competing endogenous RNA for miR-200b-3p to regulate FN1 expression. In conclusion, these results suggest that lnc-ABCA12-3 is a novel oncogene in tumorigenesis and that its high expression is related to a poor prognosis for patients with ESCC. lnc-ABCA12-3 promotes cell migration, invasion, and proliferation via the regulation of FN1 in ESCC. Our data suggest that lnc-ABCA12-3 might serve as a potential prognostic biomarker and therapeutic target for ESCC.     

The GB1 amyloid fibril: recruitment of the peripheral beta-strands of the domain swapped dimer into the polymeric interface

Three-dimensional domain swapping has been evoked as a mechanism for oligomerization of proteins. Here, we show for the immunoglobulin-binding domain B1 of streptococcal protein G (GB1) that fibril formation is observed readily for variants that exist as domain-swapped dimers. No fibril was formed by a revertant that exhibits the stable wild-type GB1 fold or a mutant comprising a highly destabilized, fluctuating ensemble of conformers. Structural features of the GB1 amyloid fibril were characterized by cysteine disulfide cross-linking. Residues in the outer edge beta-strands of the domain-swapped dimer readily form intermolecular disulfide bonds prior to and during fibril formation. On the basis of these data, a structural model for the assembly of domain-swapped dimers into a polymeric structure of the GB1 fibril is proposed.     

3D domain-swapped human cystatin C with amyloidlike intermolecular beta-sheets

Oligomerization of human cystatin C (HCC) leads to amyloid deposits in brain arteries, and this process is greatly accelerated with a naturally occurring L68Q variant. The crystal structures of N-truncated and full-length HCC (cubic form) showed dimer formation via three-dimensional (3D) domain swapping, and this observation has led to the suggestion that an analogous domain-swapping mechanism, but propagated in an open-ended fashion, could be the basis of HCC fibril formation. Here we report that full-length HCC, when crystallized in a new, tetragonal form, dimerizes by swapping the same secondary structure elements but with a very different overall structure generated by the flexibility of the hinge linking the moveable elements. The beta-strands of the beta-cores of the two folding units of the present dimer are roughly parallel, while they formed an angle of about 100 degrees in the previous two structures. The dimers pack around a crystallographic dyad by extending their molecular beta-sheets in an intermolecular context. At the other edge of the molecular beta-sheet, side-chain-side-chain hydrogen bonds propagate the beta-structure in the same direction. In consequence, a supramolecular crystal structure is generated, with all the beta-strands of the domain-swapped dimers being perpendicular to one crystallographic direction. This observation is relevant to amyloid aggregation of HCC, as X-ray diffraction studies of amyloid fibrils show them to have ordered, repeating structure, consistent with the so-called cross-beta structure, in which extended polypeptide chains are perpendicular to the fiber axis and form infinite beta-sheets that are parallel to this axis.     

Reciprocal Interactions between Membrane Bilayers and S. aureus PSMα3 Cross-α Amyloid Fibrils Account for Species-Specific Cytotoxicity

Phenol-soluble modulin α3 (PSMα3) is a functional amyloid secreted by the pathogenic bacterium Staphylococcus aureus. This 22-residue peptide serves as a key virulence determinant, toxic to human cells via the formation of unique cross-α amyloid-like fibrils. We demonstrate that bilayer vesicles accelerated PSMα3 fibril formation, and the fibrils, in turn, inserted deeply into bilayers mimicking mammalian cell membranes, accounting for PSMα3 cellular toxicity. Importantly, a mere amphipathic helical conformation was not a sufficient determinant for membrane-activity of PSMα3, pointing to the functional role of cross-α fibrils. In contrast to deep insertion of PSMα3 into mammalian membrane bilayers, the peptide only interacted with the surface of bilayers mimicking bacterial membranes, which might be related to its lack of antibacterial activity. Together, our data provide mechanistic insight into species-specific toxicity of a key bacterial amyloid virulence factor via reciprocal interactions with membranes, and open new perspectives into amyloid-related cytotoxicity mediated by helical fibril structures.