BACKGROUND
after glycation  <cit> , sonication  <cit> , or incubation at high temperature  <cit> , numerous proteins can be converted to amyloid-like fibrils with common properties such as more notable β-structure, fibrillar morphology, protofilament substructure, cross-β diffraction pattern, and binding to congo red or amyloid-specific dye thioflavin t   <cit> .
the formation of amyloid fibrils can be promoted by the presence of ethanol  <cit> , sodium dodecyl sulfate   <cit> , or other anionic detergents  <cit> .
however, these methods generally require a high concentration of protein, vigorous shaking, or the addition of fibril seed and are not easy to isolate fibrillar protein from others such as oligomers, protofibrils or non-fibrillar proteins that may also exist in the solution  <cit> .
amyloid fibrils are cytocytotoxic  <cit> , however, it is still debatable as to whether oligomers, mature fibrils, protofibrils or target cellular types are the most critical factors in dictating cellular damage  <cit> .
study of disease-associated proteins such as the amyloid-β peptide , α-synuclein, and transthyretin shows that common features of specific types of aggregates rather than specific amino acid sequences are positively related to cytotoxicity  <cit> .
amyloid oligomers from several different proteins have been shown to share the ability to permeabilize cellular membranes and lipid bilayers, which is thus proposed as the primary toxic mechanism of amyloid pathogenesis  <cit> .
lysozyme amyloid oligomers and fibrils, however, exert cytotoxicity by acting within different time-scales and via apoptosis-like and necrosis-like cell death, respectively  <cit> .
study with human islet amyloid polypeptide , the major constituent of islet amyloid, on the other hand, shows that growth of hiapp fibrils at the cellular membrane, rather than pre-formed oligomers or fibrils are responsible for the observed membrane damage  <cit> .
recently, it has been demonstrated with specific integrin-blocking antibodies that amyloid-β peptide  deposition and neurotoxicity in human cortical primary neurons are mediated through α2β <dig> and αvβ <dig> integrin receptors  <cit> .
whether binding of aβ to integrin results in change of down stream signaling molecules such as akt, bad, forkhead transcription factors, glycogen synthase kinase- <dig> , caspase- <dig> and eventually lead to apoptosis  <cit> , however, has not been clarified.
here we used a detergent-assisted refolding method  <cit>  developed previously for the refolding of recombinant capsid protein vp <dig> of foot-and-mouth disease virus  to promote the formation of fibrillar proteins in general.
we chose a model protein, bsa, for evaluating fibrillation of protein using the new method.
furthermore, the fibrillar bsa processed by this new method was used to investigate the cellular effect and signaling cascade of amyloid-like fibrils.
our findings suggest that amyloid-like fibrillar bsa induced cellular cytotoxicity and apoptosis via modulating the integrin/focal adhesion kinase /akt/gsk-3β/caspase- <dig> pathway.
RESULTS
conversion of globular protein into fibril by column chromatography
under certain extreme conditions such as high temperature, glycation, or sonication, proteins can be refolded and converted into fibrils  <cit> .
in this study, bsa was refolded by being dissolved in 1% sds solution, passed through the gel filtration column superdex- <dig> and eluted with buffer solution containing  <dig> mm tris-hcl, ph  <dig> ,  <dig> mm edta,  <dig>  m nacl, and  <dig> % sds .
the refolded bsa protein obtained from the superdex- <dig> column , like amyloid fibrils, exhibited enhanced fluorescence level of amyloid-specific dye tht in a dose-dependent manner , as compared with bsa not processed by the superdex- <dig> column.
these results were substantiated by transmission electron microscopy  analysis showing bsa with a globular structure  and bsa-s <dig> with a fibril structure .
to investigate the effect of gel-filtration column pore size on the formation of fibrils, bsa was eluted with same buffer through a gel filtration column with a smaller pore size  than that of the superdex- <dig> column.
tem analysis revealed that bsa eluted from superdex- <dig> , like bsa, showed a globular structure  and no enhanced tht fluorescence .
the effect of column matrix on fibrillar protein formation was further examined with a hw55s gel filtration column with pore size similar to that of superdex- <dig> but a different matrix composite.
bsa eluted from hw55s chromatography , like bsa-s <dig>  displayed a fibrillar structure .
these data suggest that a molecular sizing column such as superdex- <dig>  or hw55s with a pore size more than  <dig> kda promotes the formation of amyloid-like fibrillar proteins in the presence of a low concentration of sds detergent.
we also investigated whether other detergents have the same effect as sds.
bsa solution in the presence of 1% zwittergent 3– <dig>  which retains its zwitterionic character over a wide range of ph, was eluted from a superdex- <dig> column with a buffer solution containing  <dig> mm tris-hcl, ph  <dig> ,  <dig> mm edta,  <dig>  m nacl, and  <dig> % zwittergent 3– <dig>  tem revealed the bsa protein obtained from a superdex- <dig> column with zwittergent 3– <dig> , like bsa-s <dig>  with a fibrillar structure .
these data suggest that not only anionic detergent but also zwitterionic detergent are effective in facilitating fibrillar protein formation.
fibrillar bsa induces apoptosis in bhk- <dig> cells
since amyloid-like fibrils are cytotoxic to neuronal cells  <cit> , we examined whether fibrillar bsas induced by our method are cytotoxic to cells, we treated bhk- <dig> cells with various concentrations of bsa-s <dig>  bsa-zwit, or bsa-hw55s in serum-free medium for  <dig> h. bsa-s <dig>  bsa-zwit, and bsa-hw55s were all cytotoxic to cells in a dose-dependent manner .
bsa-zwit exhibited the strongest cytotoxicity among all tested.
at  <dig>  μm concentration, it induced almost 100% cytotoxicity, whereas bsa-s <dig> induced 35% and bsa-hw55s 10% cytotoxicity.
the ic <dig> for bsa-zwit, bsa-s <dig> and bsa-hw55s was  <dig> ,  <dig>  and more than  <dig> μm, respectively.
as controls, two globular proteins, bsa and bsa-s <dig> induced little, if any, cytotoxicity to cells .
interestingly, the cytotoxicity induced by all fibril bsas  in bhk- <dig> cells was stronger than amyloid aβ25– <dig> which induced a mere 10% cytotoxicity at concentrations as high as  <dig> μm after  <dig> h incubation .
significant number of cell death induced by aβ25– <dig> could be observed after  <dig> h incubation .
pre-treating bhk- <dig> cells with increasing concentrations of globular bsa  did not reverse the cytotoxicity induced by bsa-s <dig> .
to examine whether f-bsa-induced cytotoxicity was related to cellular apoptosis, dapi staining and measurement of caspase- <dig> activity were performed.
our results revealed that f-bsa induced nuclei condensation  and increased caspase- <dig> activity  as compared with g-bsa and aβ25– <dig> .
taken together, these results suggest that treatment with f-bsa induces cytotoxicity and apoptosis of cells and this effect of f-bsa is not reversed by treatment with g-bsa.
fibrillar bsa induces apoptosis via integrin/fak/akt/gsk-3β pathway
being cytotoxic to bhk- <dig> cells, f-bsa was also cytotoxic to cancer cells such as t47d cells  .
to examine whether the apoptotic effects of f-bsa is via integrins known to modulate various processes such as cell proliferation, morphology, migration, and apoptosis  <cit> , t47d cells were pre-treated with anti-integrin α5β <dig> antibody for  <dig> min, then incubated with f-bsa  for  <dig> h in serum-free medium.
cell viability results revealed that pre-treating t47d cells with anti-integrin α5β <dig> antibody but not goat igg diminished the cytotoxic effect of f-bsa .
the interaction between f-bsa and integrin was further verified by immunoprecipitation.
incubation of control beads or integrin α5β <dig> protein-linked beads with g-bsa or f-bsa revealed that f-bsa but not g-bsa bound to integrin α5β <dig> .
we then investigated whether the molecules involved in the integrin signaling-pathway cascade, such as fak, akt and gsk-3β, are affected by f-bsa.
f-bsa dephosphorylated fak at tyr  <dig> but not tyr 576/ <dig> in a time-dependent manner .
western blot analysis revealed that f-bsa dephosphorylated both akt and gsk-3β .
the effect of f-bsa on akt and gsk-3β phosphorylation could be reversed by pre-treating cells with anti-integrin α5β <dig> antibody .
in comparison, g-bsa, as well as anti-integrin α5β <dig> antibody, had no effect on the phosphorylation of akt .
thus, f-bsa induces apoptosis via an integrin/fak/akt/gsk-3β/caspase- <dig> pathway.
discussion
although proteins usually fold to their native and functional states, under certain conditions, some proteins may fail to fold correctly and form fibrillar products with abnormal biological effects  <cit> .
one notable example of incorrectly folded proteins is amyloid aggregate, which is associated with alzheimer's disease and transmissible spongiform encephalopathy  <cit> .
in the present study, we used a simple column chromatography process to induce native proteins, in this case, globular bsa, to undergo fibrillization in the presence of detergent  .
our findings are thus consistent with a previous proposal that numerous proteins with diverse structures, including both disease and non-disease associated proteins, are capable of forming fibril amyloid  <cit> .
aging proteins at 37°c may convert them into amyloid fibrils, but, in most cases, aging requires days to weeks  <cit> .
fibril formation can also be accelerated by sds  <cit>  but still requires vigorous stirring overnight at 37°c with the addition of fibril seeds  <cit>  or  <dig> days of incubation at room temperature  <cit> .
in addition, unless aggregates form and precipitate out, these methods cannot be used to isolate fibrillar from non-fibrillar proteins.
our column chromatography process converts globular protein into a fibrillar form in an efficient manner and effectively separates the fibril from other forms of the protein.
it is thus a novel approach for producing fibrillar proteins.
we show that superdex- <dig> and hw55s columns are more efficient than superdex- <dig> columns in converting proteins into fibrillar forms.
even though superdex- <dig> and hw55s have different matrix composite , they have similar pore size , which is larger than that of superdex- <dig> .
although bsa-s <dig> was more potent than bsa-hw55s , these findings seem to suggest, nonetheless, that an appropriate bead pore size plays a crucial role in the column-induced fibril formation, because fibrillization was less with superdex- <dig>  a likely explanation is that the proteins are reshaped to fibrillar form after entering the pores and passing through the long channel inside the column beads.
alternatively, the reshaping may be a consequence of the greater overall stability resulting from factors such as an increase in van der waals interactions in the column bead.
the explanation remains to be verified by further studies.
pertinhez et al.
found that low concentrations of the anionic detergent sds promoted fibril formation  <cit>  and proposed that sds might stimulate aggregation, perhaps by associating with the peptide molecules; once such aggregates are formed, intermolecular β-sheet interactions are likely to develop readily.
several anionic detergents have subsequently been found to be effective for fibrillization  <cit> .
we found that the effect of zwittergent 3– <dig> on the formation of cytotoxic bsa was similar to or even better than that of sds .
tem showed that there was few, if any, bsa oligomer or protofibril in the eluate of the column chromatography, both bsa-s <dig> and bsa-zwit existed in the fibrillar form .
the length of bsa-zwit was in average two fold longer than bsa-s <dig> .
as similar concentration of fibrillar bsa-zwit and bsa-s <dig> was used in the cytotoxic assay, the difference in potency is likely due to factor other than concentration of amyloid fibrils.
we are thus the first to report that zwitterionic detergents are also effective for promoting fibril formation.
in addition, zwittergent 3– <dig>  unlike sds, contains both anionic and cationic properties, so fibril formation may be stimulated by not only anionic but also cationic properties of the detergents.
further elucidating whether the detergents in this process enhance fibril formation by increasing unfolding or providing stability of the incorrectly refolded proteins, or both, will be interesting.
although g-bsa is not a ligand for integrin , f-bsa caused cellular apoptosis by binding to integrin α5β <dig> .
recently, wright et al.
<cit>  reported that amyloid-β peptide  bound to α2β <dig> and αvβ <dig> integrin to activate pyk <dig>  as well as phosphorylated paxillin and induced neurotoxicity.
the authors did not, however, detect any effect in the phosphorylation of fak  <cit> .
here, we report that f-bsa mediated cell apoptosis by binding to integrin α5β <dig>  which leads to the dephosphorylation of fak , akt and gsk-3β.
the f-bsa produced in this study therefore seemed to deactivate an integrin signaling pathway via a mechanism different from that induced by aβ.
wright et al.
used human cortical neurons, whereas we used bhk- <dig> and t47d, so whether this difference in mechanism of action is due to the difference in target cells or integrin receptors remains to be clarified.
aβ and amyloid polypeptides usually undergo multistage assemblies such as oligomers, protofibrils to become fibrils  <cit> .
even though it is still debatable which state of the polypeptide is more toxic to the cells, the existing data seem to suggest that different assembled macromolecular forms may exert cytotoxicity via different mechanism of actions including membrane perturbation, apoptosis, necrosis etc.
<cit> .
although fibril proteins have been shown to cause caspase-related apoptosis  <cit> , the details have not been explored.
in this study, we found that fibrillar bsa causes apoptosis by modulating the integrin/fak/akt/gsk-3β/caspase- <dig> signaling pathway.
because bsa does not contain rgd, a unique binding motif for integrin, it will be interesting to analyze whether the mechanism of binding of fibrillar bsa to integrin is the same as molecules with rgd in their sequence such as vitronectin.
of note, even though some of the rgd-containing peptides are cytotoxic, others, such as fibronectin, are not  <cit> .
in comparison, almost all fibrillar proteins have been documented to cause cytotoxicity  <cit> .
thus, elucidating whether the mechanism of action of all fibrillar proteins in causing cytotoxicity is due to the involvement of a shared structural motif will be of interest.
detergents and column chromatography have routinely been used to obtain membrane proteins close to their native status  <cit> .
however, whether the isolated membrane proteins are cytotoxic and/or apoptotic has not been reported.
our process could be used to isolate a few membrane proteins to elucidate whether they are fibrillar and apoptotic.
if the membrane proteins isolated in this manner do cause apoptosis, then the structure analysis of membrane proteins isolated with the assistance of detergents particularly those with sds and zwittergents might require re-examination.
CONCLUSIONS
we report on a novel process to convert globular proteins such as bsa into fibrillar form.
unlike globular bsa, which is non-cytotoxic, fibrillar bsa caused cell death by modulating the integrin/fak/akt/gsk-3β/caspase- <dig> signaling pathway.
these findings may be useful for improving our understanding of the pathogenesis of amyloid-like fibrils and applicable for the development of better therapeutics that targets the underlying mechanism of the etiologic agents.

 after glycation, sonication, or incubation at high temperature, numerous proteins can be converted to amyloid-like fibrils with common properties such as more notable β-structure, fibrillar morphology, protofilament substructure, cross-β diffraction pattern, and binding to congo red or amyloid-specific dye thioflavin t. the formation of amyloid fibrils can be promoted by the presence of ethanol, sodium dodecyl sulfate, or other anionic detergents. however, these methods generally require a high concentration of protein, vigorous shaking, or the addition of fibril seed and are not easy to isolate fibrillar protein from others such as oligomers, protofibrils or non-fibrillar proteins that may also exist in the solution. amyloid fibrils are cytocytotoxic, however, it is still debatable as to whether oligomers, mature fibrils, protofibrils or target cellular types are the most critical factors in dictating cellular damage. study of disease-associated proteins such as the amyloid-β peptide, α-synuclein, and transthyretin shows that common features of specific types of aggregates rather than specific amino acid sequences are positively related to cytotoxicity. amyloid oligomers from several different proteins have been shown to share the ability to permeabilize cellular membranes and lipid bilayers, which is thus proposed as the primary toxic mechanism of amyloid pathogenesis. lysozyme amyloid oligomers and fibrils, however, exert cytotoxicity by acting within different time-scales and via apoptosis-like and necrosis-like cell death, respectively. study with human islet amyloid polypeptide, the major constituent of islet amyloid, on the other hand, shows that growth of hiapp fibrils at the cellular membrane, rather than pre-formed oligomers or fibrils are responsible for the observed membrane damage. recently, it has been demonstrated with specific integrin-blocking antibodies that amyloid-β peptide deposition and neurotoxicity in human cortical primary neurons are mediated through α2β and αvβ integrin receptors. whether binding of aβ to integrin results in change of down stream signaling molecules such as akt, bad, forkhead transcription factors, glycogen synthase kinase-, caspase- and eventually lead to apoptosis, however, has not been clarified. here we used a detergent-assisted refolding method developed previously for the refolding of recombinant capsid protein vp of foot-and-mouth disease virus to promote the formation of fibrillar proteins in general. we chose a model protein, bsa, for evaluating fibrillation of protein using the new method. furthermore, the fibrillar bsa processed by this new method was used to investigate the cellular effect and signaling cascade of amyloid-like fibrils. our findings suggest that amyloid-like fibrillar bsa induced cellular cytotoxicity and apoptosis via modulating the integrin/focal adhesion kinase /akt/gsk-3β/caspase- pathway.  conversion of globular protein into fibril by column chromatography under certain extreme conditions such as high temperature, glycation, or sonication, proteins can be refolded and converted into fibrils. in this study, bsa was refolded by being dissolved in 1% sds solution, passed through the gel filtration column superdex- and eluted with buffer solution containing mm tris-hcl, ph, mm edta, m nacl, and % sds. the refolded bsa protein obtained from the superdex- column, like amyloid fibrils, exhibited enhanced fluorescence level of amyloid-specific dye tht in a dose-dependent manner, as compared with bsa not processed by the superdex- column. these results were substantiated by transmission electron microscopy analysis showing bsa with a globular structure and bsa-s with a fibril structure. to investigate the effect of gel-filtration column pore size on the formation of fibrils, bsa was eluted with same buffer through a gel filtration column with a smaller pore size than that of the superdex- column. tem analysis revealed that bsa eluted from superdex-, like bsa, showed a globular structure and no enhanced tht fluorescence. the effect of column matrix on fibrillar protein formation was further examined with a hw55s gel filtration column with pore size similar to that of superdex- but a different matrix composite. bsa eluted from hw55s chromatography, like bsa-s displayed a fibrillar structure. these data suggest that a molecular sizing column such as superdex- or hw55s with a pore size more than kda promotes the formation of amyloid-like fibrillar proteins in the presence of a low concentration of sds detergent. we also investigated whether other detergents have the same effect as sds. bsa solution in the presence of 1% zwittergent 3– which retains its zwitterionic character over a wide range of ph, was eluted from a superdex- column with a buffer solution containing mm tris-hcl, ph, mm edta, m nacl, and % zwittergent 3– tem revealed the bsa protein obtained from a superdex- column with zwittergent 3–, like bsa-s with a fibrillar structure. these data suggest that not only anionic detergent but also zwitterionic detergent are effective in facilitating fibrillar protein formation. fibrillar bsa induces apoptosis in bhk- cells since amyloid-like fibrils are cytotoxic to neuronal cells, we examined whether fibrillar bsas induced by our method are cytotoxic to cells, we treated bhk- cells with various concentrations of bsa-s bsa-zwit, or bsa-hw55s in serum-free medium for h. bsa-s bsa-zwit, and bsa-hw55s were all cytotoxic to cells in a dose-dependent manner. bsa-zwit exhibited the strongest cytotoxicity among all tested. at μm concentration, it induced almost 100% cytotoxicity, whereas bsa-s induced 35% and bsa-hw55s 10% cytotoxicity. the ic for bsa-zwit, bsa-s and bsa-hw55s was, and more than μm, respectively. as controls, two globular proteins, bsa and bsa-s induced little, if any, cytotoxicity to cells. interestingly, the cytotoxicity induced by all fibril bsas in bhk- cells was stronger than amyloid aβ25– which induced a mere 10% cytotoxicity at concentrations as high as μm after h incubation. significant number of cell death induced by aβ25– could be observed after h incubation. pre-treating bhk- cells with increasing concentrations of globular bsa did not reverse the cytotoxicity induced by bsa-s. to examine whether f-bsa-induced cytotoxicity was related to cellular apoptosis, dapi staining and measurement of caspase- activity were performed. our results revealed that f-bsa induced nuclei condensation and increased caspase- activity as compared with g-bsa and aβ25–. taken together, these results suggest that treatment with f-bsa induces cytotoxicity and apoptosis of cells and this effect of f-bsa is not reversed by treatment with g-bsa. fibrillar bsa induces apoptosis via integrin/fak/akt/gsk-3β pathway being cytotoxic to bhk- cells, f-bsa was also cytotoxic to cancer cells such as t47d cells. to examine whether the apoptotic effects of f-bsa is via integrins known to modulate various processes such as cell proliferation, morphology, migration, and apoptosis, t47d cells were pre-treated with anti-integrin α5β antibody for min, then incubated with f-bsa for h in serum-free medium. cell viability results revealed that pre-treating t47d cells with anti-integrin α5β antibody but not goat igg diminished the cytotoxic effect of f-bsa. the interaction between f-bsa and integrin was further verified by immunoprecipitation. incubation of control beads or integrin α5β protein-linked beads with g-bsa or f-bsa revealed that f-bsa but not g-bsa bound to integrin α5β. we then investigated whether the molecules involved in the integrin signaling-pathway cascade, such as fak, akt and gsk-3β, are affected by f-bsa. f-bsa dephosphorylated fak at tyr but not tyr 576/ in a time-dependent manner. western blot analysis revealed that f-bsa dephosphorylated both akt and gsk-3β. the effect of f-bsa on akt and gsk-3β phosphorylation could be reversed by pre-treating cells with anti-integrin α5β antibody. in comparison, g-bsa, as well as anti-integrin α5β antibody, had no effect on the phosphorylation of akt. thus, f-bsa induces apoptosis via an integrin/fak/akt/gsk-3β/caspase- pathway. discussion although proteins usually fold to their native and functional states, under certain conditions, some proteins may fail to fold correctly and form fibrillar products with abnormal biological effects. one notable example of incorrectly folded proteins is amyloid aggregate, which is associated with alzheimer's disease and transmissible spongiform encephalopathy. in the present study, we used a simple column chromatography process to induce native proteins, in this case, globular bsa, to undergo fibrillization in the presence of detergent. our findings are thus consistent with a previous proposal that numerous proteins with diverse structures, including both disease and non-disease associated proteins, are capable of forming fibril amyloid. aging proteins at 37°c may convert them into amyloid fibrils, but, in most cases, aging requires days to weeks. fibril formation can also be accelerated by sds but still requires vigorous stirring overnight at 37°c with the addition of fibril seeds or days of incubation at room temperature. in addition, unless aggregates form and precipitate out, these methods cannot be used to isolate fibrillar from non-fibrillar proteins. our column chromatography process converts globular protein into a fibrillar form in an efficient manner and effectively separates the fibril from other forms of the protein. it is thus a novel approach for producing fibrillar proteins. we show that superdex- and hw55s columns are more efficient than superdex- columns in converting proteins into fibrillar forms. even though superdex- and hw55s have different matrix composite, they have similar pore size, which is larger than that of superdex-. although bsa-s was more potent than bsa-hw55s, these findings seem to suggest, nonetheless, that an appropriate bead pore size plays a crucial role in the column-induced fibril formation, because fibrillization was less with superdex- a likely explanation is that the proteins are reshaped to fibrillar form after entering the pores and passing through the long channel inside the column beads. alternatively, the reshaping may be a consequence of the greater overall stability resulting from factors such as an increase in van der waals interactions in the column bead. the explanation remains to be verified by further studies. pertinhez et al. found that low concentrations of the anionic detergent sds promoted fibril formation and proposed that sds might stimulate aggregation, perhaps by associating with the peptide molecules; once such aggregates are formed, intermolecular β-sheet interactions are likely to develop readily. several anionic detergents have subsequently been found to be effective for fibrillization. we found that the effect of zwittergent 3– on the formation of cytotoxic bsa was similar to or even better than that of sds. tem showed that there was few, if any, bsa oligomer or protofibril in the eluate of the column chromatography, both bsa-s and bsa-zwit existed in the fibrillar form. the length of bsa-zwit was in average two fold longer than bsa-s. as similar concentration of fibrillar bsa-zwit and bsa-s was used in the cytotoxic assay, the difference in potency is likely due to factor other than concentration of amyloid fibrils. we are thus the first to report that zwitterionic detergents are also effective for promoting fibril formation. in addition, zwittergent 3– unlike sds, contains both anionic and cationic properties, so fibril formation may be stimulated by not only anionic but also cationic properties of the detergents. further elucidating whether the detergents in this process enhance fibril formation by increasing unfolding or providing stability of the incorrectly refolded proteins, or both, will be interesting. although g-bsa is not a ligand for integrin, f-bsa caused cellular apoptosis by binding to integrin α5β. recently, wright et al. reported that amyloid-β peptide bound to α2β and αvβ integrin to activate pyk as well as phosphorylated paxillin and induced neurotoxicity. the authors did not, however, detect any effect in the phosphorylation of fak. here, we report that f-bsa mediated cell apoptosis by binding to integrin α5β which leads to the dephosphorylation of fak, akt and gsk-3β. the f-bsa produced in this study therefore seemed to deactivate an integrin signaling pathway via a mechanism different from that induced by aβ. wright et al. used human cortical neurons, whereas we used bhk- and t47d, so whether this difference in mechanism of action is due to the difference in target cells or integrin receptors remains to be clarified. aβ and amyloid polypeptides usually undergo multistage assemblies such as oligomers, protofibrils to become fibrils. even though it is still debatable which state of the polypeptide is more toxic to the cells, the existing data seem to suggest that different assembled macromolecular forms may exert cytotoxicity via different mechanism of actions including membrane perturbation, apoptosis, necrosis etc.. although fibril proteins have been shown to cause caspase-related apoptosis, the details have not been explored. in this study, we found that fibrillar bsa causes apoptosis by modulating the integrin/fak/akt/gsk-3β/caspase- signaling pathway. because bsa does not contain rgd, a unique binding motif for integrin, it will be interesting to analyze whether the mechanism of binding of fibrillar bsa to integrin is the same as molecules with rgd in their sequence such as vitronectin. of note, even though some of the rgd-containing peptides are cytotoxic, others, such as fibronectin, are not. in comparison, almost all fibrillar proteins have been documented to cause cytotoxicity. thus, elucidating whether the mechanism of action of all fibrillar proteins in causing cytotoxicity is due to the involvement of a shared structural motif will be of interest. detergents and column chromatography have routinely been used to obtain membrane proteins close to their native status. however, whether the isolated membrane proteins are cytotoxic and/or apoptotic has not been reported. our process could be used to isolate a few membrane proteins to elucidate whether they are fibrillar and apoptotic. if the membrane proteins isolated in this manner do cause apoptosis, then the structure analysis of membrane proteins isolated with the assistance of detergents particularly those with sds and zwittergents might require re-examination.  we report on a novel process to convert globular proteins such as bsa into fibrillar form. unlike globular bsa, which is non-cytotoxic, fibrillar bsa caused cell death by modulating the integrin/fak/akt/gsk-3β/caspase- signaling pathway. these findings may be useful for improving our understanding of the pathogenesis of amyloid-like fibrils and applicable for the development of better therapeutics that targets the underlying mechanism of the etiologic agents. 
numerous proteins can be converted to amyloid-like fibrils to increase cytotoxicity and induce apoptosis, but the methods generally require a high concentration of protein, vigorous shaking, or fibril seed. in this study, we have developed a novel process to convert native proteins into the fibrillar form. we induced bsa, a non-cytotoxic globular protein, to become fibril by a novel process involving superdex- column chromatography in the presence of anionic or zwittergenic detergent. we report on a novel process for converting globular proteins into fibrillar form to cause apoptosis by modulating the integrin/fak/akt/gsk-3β/caspase- signaling pathway. our findings may be useful for understanding the pathogenesis of amyloid-like fibrils and applicable for the development of better therapeutic agents that target the underlying mechanism of the etiologic agents. fibrillar bsa, but not globular bsa, bound to integrin, dephosphorylated focal adhesion kinase, akt and glycogen synthase kinase-3β. the column pore size was more important than column matrix composite in fibril formation. the fibrillar bsa induced apoptosis in bhk- cell as well as breast cancer cell line t47d. we used globular bovine serum albumin as a model protein to verify the properties of the fibrillar protein, investigated its cellular effects and studied the signaling cascade induced by the fibrillar protein.
 numerous proteins can be converted to amyloid-like fibrils to increase cytotoxicity and induce apoptosis, but the methods generally require a high concentration of protein, vigorous shaking, or fibril seed. as well, the detailed mechanism of the cytotoxic effects is not well characterized. in this study, we have developed a novel process to convert native proteins into the fibrillar form. we used globular bovine serum albumin as a model protein to verify the properties of the fibrillar protein, investigated its cellular effects and studied the signaling cascade induced by the fibrillar protein.  we induced bsa, a non-cytotoxic globular protein, to become fibril by a novel process involving superdex- column chromatography in the presence of anionic or zwittergenic detergent. the column pore size was more important than column matrix composite in fibril formation. the fibrillar bsa induced apoptosis in bhk- cell as well as breast cancer cell line t47d. pre-treating cells with anti-integrin antibodies blocked the apoptotic effect. fibrillar bsa, but not globular bsa, bound to integrin, dephosphorylated focal adhesion kinase, akt and glycogen synthase kinase-3β.  we report on a novel process for converting globular proteins into fibrillar form to cause apoptosis by modulating the integrin/fak/akt/gsk-3β/caspase- signaling pathway. our findings may be useful for understanding the pathogenesis of amyloid-like fibrils and applicable for the development of better therapeutic agents that target the underlying mechanism of the etiologic agents. 