Formation of amyloid-like fibrils by self-association of a partially unfolded fibronectin type III module

Litvinovich, Sergei V.; Brew, Shelesa A.; Aota, Shin-ichi; Akiyama, Steven K.; Haudenschild, Christian C.; Ingham, Kenneth C.

doi:10.1006/jmbi.1998.1863

Cited by 194 publications

(138 citation statements)

References 51 publications

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“…1B,C), in agreement with previously published images of such artificial Fn fibers (Ejim et al, 1993;Wojciak-Stothard et al, 1997). Indeed, cryo-scanning electron microscopic images suggest that Fn fibers exist as 'cables' comprised of individual fibrous strands of ~5-15 nm in diameter and larger (Chen et al, 1978;Dzamba and Peters, 1991;Peters et al, 1998;Singer, 1979) which are proposed to be held together by hydrogen bonds, intermolecular beta-strand swapping (Briknarova et al, 2003;Litvinovich et al, 1998), disulfide bonds which are potentially formed by cryptic disulfide isomerase activity (Langenbach and Sottile, 1999), and other weak electrostatic interactions (Chen and Mosher, 1996;Morla et al, 1994). Although the exact location and properties of these bonds are unknown, it has been observed that they are strong enough to render cell-derived Fn fibers irreversibly insoluble in 1% de-oxycholate (McKeown-Longo and Mosher, 1983), which is a phenomenon we observed with manually deposited Fn fibers as well (data not shown).…”

Section: Manually Deposited Fn Fibers Bundle Into Fiber Cables Similasupporting

confidence: 89%

Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage

et al. 2008

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In response to growing needs for quantitative biochemical and cellular assays that address whether the extracellular matrix (ECM) acts as a mechanochemical signal converter to co-regulate cellular mechanotransduction processes, a new assay is presented where plasma fibronectin fibers are manually deposited onto elastic sheets, while force-induced changes in protein conformation are monitored by fluorescence resonance energy transfer (FRET). Fully relaxed assay fibers can be stretched at least 5-6 fold, which involves Fn domain unfolding, before the fibers break. In native fibroblast ECM, this full range of stretch-regulated conformations coexists in every field of view confirming that the assay fibers are physiologically relevant model systems. Since alterations of protein function will directly correlate with their extension in response to force, the FRET vs. strain curves presented herein enable the mapping of fibronectin strain distributions in 2D and 3D cell cultures with high spatial resolution. Finally, cryptic sites for fibronectin's N-terminal 70-kD fragment were found to be exposed at relatively low strain, demonstrating the assay's potential to analyze stretch-regulated protein-rotein interactions.

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Section: Manually Deposited Fn Fibers Bundle Into Fiber Cables Similasupporting

confidence: 89%

Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage

et al. 2008

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“…Force-induced unfolding of FnIII modules provides a basis for one proposed mechanism of Fn self-assembly into fibrils. As neighboring Fn molecules refold upon release of tension, they may noncovalently bind each other by undergoing ␤-strand or ␤-sheet exchange between FnIII modules (29).…”

Section: Discussionmentioning

confidence: 99%

Fibronectin extension and unfolding within cell matrix fibrils controlled by cytoskeletal tension

Baneyx

Baugh

Vogel

2002

Proc. Natl. Acad. Sci. U.S.A.

333

306

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Evidence is emerging that mechanical stretching can alter the functional states of proteins. Fibronectin (Fn) is a large, extracellular matrix protein that is assembled by cells into elastic fibrils and subjected to contractile forces. Assembly into fibrils coincides with expression of biological recognition sites that are buried in Fn's soluble state. To investigate how supramolecular assembly of Fn into fibrillar matrix enables cells to mechanically regulate its structure, we used fluorescence resonance energy transfer (FRET) as an indicator of Fn conformation in the fibrillar matrix of NIH 3T3 fibroblasts. Fn was randomly labeled on amine residues with donor fluorophores and site-specifically labeled on cysteine residues in modules FnIII7 and FnIII15 with acceptor fluorophores. Intramolecular FRET was correlated with known structural changes of Fn in denaturing solution, then applied in cell culture as an indicator of Fn conformation within the matrix fibrils of NIH 3T3 fibroblasts. Based on the level of FRET, Fn in many fibrils was stretched by cells so that its dimer arms were extended and at least one FnIII module unfolded. When cytoskeletal tension was disrupted using cytochalasin D, FRET increased, indicating refolding of Fn within fibrils. These results suggest that cell-generated force is required to maintain Fn in partially unfolded conformations. The results support a model of Fn fibril elasticity based on unraveling and refolding of FnIII modules. We also observed variation of FRET between and along single fibrils, indicating variation in the degree of unfolding of Fn in fibrils. Molecular mechanisms by which mechanical force can alter the structure of Fn, converting tensile forces into biochemical cues, are discussed. E xtracellular matrices (ECM) are complex supramolecular assemblies that control cell signaling and behavior. While many ECM proteins have been characterized, little is known about how matrix assembly alters their structure and confers functions not present in individual proteins. Less is known about mechanisms by which cell contractile forces applied to protein assemblies regulate protein function. Many ECM proteins, such as fibronectin (Fn), laminin, and thrombospondin, are large (Ͼ100 kDa), multifunctional proteins composed of repeating, structurally defined modules often less than 100 aa each. The multimodular structure may serve as a convenient way to integrate multiple functions in one molecule. For example, some modules carry cell adhesion sites, whereas others carry sites for binding other proteins and for self-assembly. Exposure of functional sites may be controlled by the organization of such sites in extracellular matrices, and by the application of force by cells (1-3). However, the molecular mechanisms regulating ECM protein assembly and the exposure of binding sites remain unclear.Fn is an ECM protein that undergoes cell-mediated assembly into insoluble, elastic fibrils. In blood and when secreted by cells such as fibroblasts, Fn exists as a soluble dimer. The two strand...

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“…Its partial solvation was observed in all simulations only when the four FN-III modules were mechanically stretched from the twisted to the aligned state. Unique to FN-III 7 , an additional hydrogen bond between the side chain of Tyr 34 O and the backbone hydrogen of Ser 26 HN was shown to consistently break before the transition to the aligned state. This hydrogen bond is significant because it is part of the previously mentioned hydrophobic core, and it mechanically connects the upper and lower ␤-sheets.…”

Section: Correlating Plateaus In Extension Time Curves With Conformatmentioning

confidence: 99%

Comparison of the early stages of forced unfolding for fibronectin type III modules

Craig

Krammer

Schulten

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

123

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The structural changes accompanying stretch-induced early unfolding events were investigated for the four type III fibronectin (FN-III) modules, FN-III 7, FN-III8, FN-III9, and FN-III10 by using steered molecular dynamics. Simulations revealed that two main energy barriers, I and II, have to be overcome to initiate unraveling of FN-III's tertiary structure. In crossing the first barrier, the two opposing ␤-sheets of FN-III are rotated against each other such that the ␤-strands of both ␤-sheets align parallel to the force vector (aligned state). All further events in the unfolding pathway proceed from this intermediate state. A second energy barrier has to be overcome to break the first major cluster of hydrogen bonds between adjacent ␤-strands. Simulations revealed that the height of barrier I varied significantly among the four modules studied, being largest for FN-III 7 and lowest for FN-III10, whereas the height of barrier II showed little variation. Key residues affecting the mechanical stability of FN-III modules were identified. These results suggest that FN-III modules can be prestretched into an intermediate state with only minor changes to their tertiary structures. T he mechanical properties of fibronectin (FN) and its extracellular matrix fibers have drawn considerable attention recently. FN is a 450-to 500-kDa dimeric protein composed of more than 20 modules per monomer. FN primarily consists of three structurally homologous modules, FN-I, FN-II, and FN-III, presented schematically in Fig. 1A. Cells assemble FN into fibrillar networks that provide mechanical stability to the extracellular matrix and connective tissue. Furthermore, cells have been found to mechanically stretch FN fibers by up to four times their normal length (1). This wide extension range is attributed to the unfolding of individual FN modules under tension, which are presumed to refold when tension is released (2, 3). The FN-III modules are of particular interest because they contain several cell recognition sites, including the synergy site on FN-III 9 and an RGD loop on FN-III 10 (4). Beyond its role in FN, the FN-III motif is structurally ubiquitous and found in 2% of all animal proteins (5). The FN-III motif is a Greek key ␤-sandwich (Fig. 1B) with four ␤-strands in the upper sheet and three in the lower sheet (6).Mechanical forces, in addition to chemical cues, have been implicated in playing a critical role in regulating the functional states of FN. Binding sites buried within the protein in its native state, also referred to as cryptic sites, can be exposed by denaturation, or potentially through mechanical stretching (7,8). Forced unfolding of the FN-III 10 module also has been suggested to modify exposure of the RGD loop, thus influencing FNЈs accessibility to integrins (9). Mechanical forces, furthermore, are essential in initiating FN fibril assembly (10), potentially through exposure of cryptic sites, and͞or by swapping complementary ␤-strands of partially unfolded FN-III modules during refolding (11,12). The functional ...

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Formation of amyloid-like fibrils by self-association of a partially unfolded fibronectin type III module

Cited by 194 publications

References 51 publications

Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage

Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage

Fibronectin extension and unfolding within cell matrix fibrils controlled by cytoskeletal tension

Comparison of the early stages of forced unfolding for fibronectin type III modules

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