BACKGROUND
prokaryotic and eukaryotic cells employ chaperones for guiding polypeptides during synthesis towards proper folding, for preventing misfolded proteins from aggregating, for re-establishing proper conformation or channeling misfolded polypeptides towards intracellular degradation.
under conditions of cell stress, such as heat stress or a high rate of protein synthesis, there is a higher amount of polypeptides misfolded, and in parallel there is a rapid increase of chaperone availability  <cit> .
as one of the dominant members of the chaperone family, heat shock protein   <dig> shows strong and preferential upregulation in various cell populations exposed to stress conditions  <cit> .
following the general principle of peptide chaperoning, hsp <dig> interacts with proteins by transiently binding to amino acid regions with distinct physicochemical properties.
detailed sequence analyses of hsp-chaperoned polypeptides identified stretches of at least seven amino acids with a core region of up to five hydrophobic amino acids as prominent binding motif for members of the hsp <dig> family  <cit> .
besides their function of peptide guidance, chaperones induced by stress serve as danger antigens to the innate immune system  <cit> , and those peptide regions of target proteins interacting with the peptide binding region of chaperones may be transferred onto mhc molecules.
this mechanism has been termed re-presentation  and may facilitate the induction of anti-tumor reactivity or the rise of autoimmunity  <cit> .
insulin is a primary product of protein synthesis of pancreatic β-islet cells.
the peptide hormone is generated from the precursor forms preproinsulin and proinsulin.
the mature, biologically active monomer of insulin is composed of an a- and b-chain; its structure is stabilized by intra- and inter-chain disulfide bonds.
interestingly, insulin represents a dominant antigen during the development of the immunological processes leading to pancreatic β-cell destruction and  type  <dig> diabetes.
although the hormone is a primary target of autoantibodies that emerge early in the prediabetic phase  <cit>  the stimulation of cell-mediated immune processes including the activation of insulin-reactive t-lymphocytes seems to be of major importance for the progression of β-cell-directed immune reactivity  <cit> .
in fact, insulin-specific t-cells can be isolated from human subjects both in the prediabetic phase and the onset of type  <dig> diabetes and are present in the diabetes-prone non-obese diabetic  mouse, an animal model of the human disease  <cit> .
in view of the chronic endoplasmatic reticulum stress conditions with enhanced chaperone activity observed for insulin producing pancreatic β-cells during islet inflammation in  type  <dig> diabetes as well as during metabolic stress in states of insulin resistance and obesity  <cit> , we hypothesized that  insulin interacts with chaperones.
in our experimental approach we therefore determined the ability of preproinsulin-derived 13-mer peptides, monomeric proinsulin or native insulin to bind to a chaperone.
in the current study we used bacterial hsp <dig> as the best characterized member of the large and evolutionary well-conserved hsp <dig> chaperone family  <cit> .
RESULTS
identification of dnak binding peptide regions in preproinsulin
the interaction of the  <dig> kda chaperone dnak with proinsulin was investigated by analyzing the binding of the chaperone to immobilized 13-mer peptides covering the entire length of the unprocessed precursor of the hormone.
soluble dnak showed differential affinities to the membrane-bound peptides.
as indicated by the staining intensities of the spots in figure 1a, four clusters of peptides covering four discrete regions of the preproinsulin molecule exhibited increased dnak retention capacities in a range similar to that of the control peptides c <dig>  c <dig> and c <dig> with well-documented high affinities to dnak.
one dnak binding region was located in the signal peptide  and a second in the c-peptide , connecting the proinsulin a- and b-chain.
two further dnak binding regions were located in the a-chain  and b-chain .
densitometric analysis of dnak eluted from the immobilized peptides indicated peak binding to peptides 1- <dig>  17- <dig>  36- <dig> and 46- <dig> .
the application of an algorithm developed to predict the dnak binding probability of peptides roughly confirmed the location of three of these binding regions in the preproinsulin molecule  .
in contrast to the experimental findings, the algorithm did not predict dnak binding for the a-chain region, but identified an additional dnak binding region at the position of the peptides  <dig> and  <dig> which are located in a region connecting the b-chain and the c-peptide.
as expected, the amino acid sequences of the control peptides yielded dnak binding with scores in a range of - <dig>   and - <dig>  .
the overlapping peptides with peak binding of dnak as determined in the solid phase assay were compared with the underlying peptide sequence of preproinsulin.
the corresponding dnak binding sequences in the preproinsulin molecule were amino acids s7- <dig> of the signal peptide, b9- <dig> of the b-chain, c15- <dig> of the c-peptide and a6- <dig> of the a-chain .
quantitative analysis of proinsulin peptide binding to dnak
electro-elution of dnak from immobilized peptides was performed in three consecutive cycles and the progress of peptide release was determined as an estimate of their dnak affinity.
the amounts of dnak eluted in the first cycle  correspond to the electroblots shown in figure 1a.
the amounts of dnak released during elution cycles  <dig> and  <dig> were compared with the release of dnak from the high affinity control ligand c <dig> .
most peptides retained dnak less than the control peptide during elution cycles  <dig> and  <dig> except for peptides  <dig> and  <dig>  of these two, peptide  <dig> exhibited better retention of dnak indicating highest affinity in this assay .
interestingly, the peptide  <dig>  corresponds to the insulin peptide b9- <dig>  which had been identified as an immunodominant epitope of autoimmune t-cell reactivity in type  <dig> diabetes.
therefore, we analyzed the interaction of dnak with peptide b11- <dig> also in a fluid phase assay.
we determined the capacity of a peptide to compete with the fluorescent high-affinity dnak ligand a-nr  for binding to dnak  <cit> .
increasing concentrations of non-labeled peptide b11- <dig> resulted in a decrease of the fluorescence signal reflecting effective displacement of a-nr from dnak by the peptide , corresponding to an apparent dissociation equilibrium constant  of  <dig>  ±  <dig>  μm of the competing peptide b11- <dig>  by contrast, peptide b18- <dig>  which is an adjacent peptide from the proinsulin b-chain region but without significant dnak-affinity, was much less effective in displacing a-nr .
binding of dnak to peptides versus intact insulin or monomeric proinsulin
next, we used the fluid phase assay to analyze whether dnak would interact with the intact insulin molecule.
native insulin, only poorly competed with a-nr   indicating that the steric configuration of insulin oligomers prevents effective interaction with dnak.
binding strength of insulin was in the range of the poorly binding peptide b18- <dig> .
for comparison, the sigma32-derived control peptide, a high-affinity dnak ligand, exhibited a dissociation constant of  <dig>  ±  <dig>  μm.
the latter was in the same range as found for insulin peptide b11- <dig> .
human insulin
peptide b18- <dig>
control peptide
peptide b11- <dig>
human proinsulin variant
the dnak affinity of insulin and insulin-derived peptides was determined in a competition assay as described in "methods".
lines indicate the disulfide bonds in the insulin and proinsulin-molecules.
the data show mean ± sd of three or four determinations.
finally, we investigated the possible binding of dnak to a proinsulin variant with decreased propensity to oligomer formation due to an inversion of the k-p sequence in the positions b <dig> and b <dig>  this proinsulin variant interacted with dnak at concentrations much lower than observed for insulin  .
discussion
our experiments demonstrate that the  <dig> kda heat shock protein dnak is able to bind to 13-mer peptide segments of the preproinsulin molecule and that the binding is limited to four distinct preproinsulin regions, amino acids s7- <dig> of the signal peptide, b9- <dig> of the b-chain, c15- <dig> of the c-peptide and a6- <dig> of the a-chain.
these peptide regions exhibit the leucine-rich motif found essential for binding of dnak  <cit> , usually a hydrophobic core of four to five residues enriched in l, but also in i, v, f and y, and two flanking regions enriched in basic residues.
of the proinsulin molecule, strongest binding occurred to peptide  <dig> , and this is probably mediated by a distinct hydrophobic motif formed by the three amino acid residues l-y-l in the central positions b <dig>  b <dig> and b <dig>  this conclusion is supported by the finding that the b-chain-derived peptide b18- <dig>  which comprises the six c-terminal residues of b11- <dig> but lacks the central l-y-l sequence or a corresponding hydrophobic motif exhibited no significant dnak affinity.
native insulin exhibited only low affinity to dnak.
this observation indicates that steric hindrance prevents effective interaction with the chaperone, possibly due to the conformation of the polypeptide heterodimer or the aggregation of insulin molecules into dimers, trimers or hexamers, rendering the regions b11- <dig> and a6- <dig> less accessible.
as deduced from the crystal structure of the insulin molecule http://swissmodel.expasy.org/repository, the b11- <dig> epitopes are buried at the interface between two mature insulin heterodimers, thus preventing access to the peptide binding site of dnak.
similarly, the a-chain region a6- <dig> comprises a number of amino acid residues localized at the interfaces of the insulin oligomers.
in particular, the surface-exposed e-residue in position a <dig> contributes to the stabilization of the dimer-dimer interfaces by its interaction with the f-residue in position  <dig> of the b-chain.
moreover, the c-residue in position a <dig> which is located at the surface of the insulin molecule, forms a stabilizing interchain disulfide bond with the c-residue in position  <dig> of the b-chain  <cit> .
to further elucidate a potential impact of steric conditions on dnak-insulin interactions we investigated the dnak binding of a proinsulin variant with an inversion of the k-p sequence in positions b <dig> and b <dig> causing a decreased tendency to oligomerization.
this variant indeed exhibited a much higher dnak affinity than mature insulin.
however, the kd obtained for the mature insulin molecule may only represent an estimate for the  chaperone affinity of the hormone, as the fluid phase system applied in our approach is optimised for studies on the interaction of chaperones with peptides rather than larger proteins.
nevertheless, previous studies which applied experimental systems designed to investigate the binding of dnak to polypeptides demonstrate weak, but significant interactions between dnak and full-length, native proteins  <cit> .
in those studies, dnak binding was found to depend on the accessible hydrophobic area of a protein and was characterized by dissociation constants of  <dig> -  <dig> μm, a range comparable to the kd of  <dig>  ±  <dig>  μm found for insulin in our current study.
taken together, these observation suggest that not only the insulin peptides but also mature proinsulin monomers can interact with dnak and that there is little interaction between dnak and native oligomeric forms of insulin.
in our current approach we selected insulin as the predominant β-cell specific  antigen.
dnak was selected as a representative member of the dominant and phylogenetically high conserved hsp <dig> family with the best characterized chaperone activity  <cit> .
although the members of the hsp <dig> family were found to exhibit some differences in peptide binding specificities  <cit>  extensive comparative analyses revealed a common binding specificity for peptide stretches of at least seven amino acid residues with a hydrophobic core  <cit>  thus largely resembling the hydrophobicity distribution pattern of the insulin-derived peptide b11- <dig>  therefore, our analyses on the interaction of potentially autoantigenic insulin peptides with the chaperone dnak was performed in a suitable model system which only partially reflects the situation in vivo, in human patients.
further detailed studies of the interaction of insulin and proinsulin with human hsp <dig> isoforms will be needed to fully understand the rules of insulin-chaperone interaction.
besides the chaperoning function, one other property of hsp <dig> family chaperones is the ability to induce t-cell immune responses to complexed peptides  <cit> , and a role of this pathway has been postulated for the induction of autoimmune disease  <cit> .
it therefore is noteworthy that the three dnak-binding regions of proinsulin, amino acids b9- <dig> of the b-chain, c15- <dig> of the c-peptide and a6- <dig> of the a-chain superimpose with the three major target regions for t-cell autoimmune reactivity in the nod mouse model or in humans with β-cell autoimmunity  <cit> .
in the mouse model, the b-chain region around b <dig> was identified as critical for disease development, i.e., mutating the native e in position b <dig> to q abolished t-cell reactivity to the peptide b9- <dig>  <cit> .
moreover, replacement of y to a in position b <dig> abrogated the response of b9- <dig> - reactive t-cell clones as well as the development of diabetes in vivo  <cit> .
i.e., the peptide b11- <dig> is closely associated with diabetes pathogenesis, and its diabetogenic potential depends on an intact core sequence, comprising the region which is important for the binding of this peptide to dnak.
as the insulin molecules with the mutated b-chains completely retain their metabolic activity  <cit>  it has to be assumed that a single amino acid-exchange does not impair the formation of the correct tertiary structure, a complex process that most likely involves proper binding and assistance by chaperones.
the findings from the mutated insulin molecules therefore further support the view, that the peptide binding motifs of chaperones are not determined by strictly defined amino acid sequences but by more roughly defined peptide properties, e.g.
by the hydrophobicity distribution pattern within a stretch of amino acids  <cit> .
in contrast, t-cell receptor-mediated peptide recognition is known to be a highly selective and largely amino acid sequence specific process.
based on these considerations it may be speculated that chaperones are able to bind defined sets of insulin peptides characterized by hydrophobic motifs.
chaperone-peptide complexes may be recognized by antigen presenting cells which perform the ultimate decision on the pathogenicity of a peptide by applying stringent, highly sequence-sensitive selection criteria during processing and re-presentation of the peptide in the context of mhc structures and co-stimulatory signals.
further studies are warranted to analyze whether enhanced binding of proinsulin monomers or misfolded insulin to hsp <dig> family chaperones during periods of β-cell stress contributes to the pathogenesis of autoimmune diabetes.
CONCLUSIONS
our observations indicate that the hsp <dig> chaperone dnak is able to bind to three discrete regions of the proinsulin molecule which are characterized by a central hydrophobic amino acid motif flanked by regions enriched for basic amino acids.
binding occurs to 13-mer peptides and to a proinsulin molecule modified to prevent oligomerization, but not to native insulin.
the three dnak-binding regions of proinsulin, aminoacids b9- <dig> of the b-chain, c15- <dig> of the c-peptide and a6- <dig> of the a-chain superimpose with the three major target regions for t-cell autoimmune reactivity in autoimmune diabetes.

 prokaryotic and eukaryotic cells employ chaperones for guiding polypeptides during synthesis towards proper folding, for preventing misfolded proteins from aggregating, for re-establishing proper conformation or channeling misfolded polypeptides towards intracellular degradation. under conditions of cell stress, such as heat stress or a high rate of protein synthesis, there is a higher amount of polypeptides misfolded, and in parallel there is a rapid increase of chaperone availability. as one of the dominant members of the chaperone family, heat shock protein shows strong and preferential upregulation in various cell populations exposed to stress conditions. following the general principle of peptide chaperoning, hsp interacts with proteins by transiently binding to amino acid regions with distinct physicochemical properties. detailed sequence analyses of hsp-chaperoned polypeptides identified stretches of at least seven amino acids with a core region of up to five hydrophobic amino acids as prominent binding motif for members of the hsp family. besides their function of peptide guidance, chaperones induced by stress serve as danger antigens to the innate immune system, and those peptide regions of target proteins interacting with the peptide binding region of chaperones may be transferred onto mhc molecules. this mechanism has been termed re-presentation and may facilitate the induction of anti-tumor reactivity or the rise of autoimmunity. insulin is a primary product of protein synthesis of pancreatic β-islet cells. the peptide hormone is generated from the precursor forms preproinsulin and proinsulin. the mature, biologically active monomer of insulin is composed of an a- and b-chain; its structure is stabilized by intra- and inter-chain disulfide bonds. interestingly, insulin represents a dominant antigen during the development of the immunological processes leading to pancreatic β-cell destruction and type diabetes. although the hormone is a primary target of autoantibodies that emerge early in the prediabetic phase the stimulation of cell-mediated immune processes including the activation of insulin-reactive t-lymphocytes seems to be of major importance for the progression of β-cell-directed immune reactivity. in fact, insulin-specific t-cells can be isolated from human subjects both in the prediabetic phase and the onset of type diabetes and are present in the diabetes-prone non-obese diabetic mouse, an animal model of the human disease. in view of the chronic endoplasmatic reticulum stress conditions with enhanced chaperone activity observed for insulin producing pancreatic β-cells during islet inflammation in type diabetes as well as during metabolic stress in states of insulin resistance and obesity, we hypothesized that insulin interacts with chaperones. in our experimental approach we therefore determined the ability of preproinsulin-derived 13-mer peptides, monomeric proinsulin or native insulin to bind to a chaperone. in the current study we used bacterial hsp as the best characterized member of the large and evolutionary well-conserved hsp chaperone family.  identification of dnak binding peptide regions in preproinsulin the interaction of the kda chaperone dnak with proinsulin was investigated by analyzing the binding of the chaperone to immobilized 13-mer peptides covering the entire length of the unprocessed precursor of the hormone. soluble dnak showed differential affinities to the membrane-bound peptides. as indicated by the staining intensities of the spots in figure 1a, four clusters of peptides covering four discrete regions of the preproinsulin molecule exhibited increased dnak retention capacities in a range similar to that of the control peptides c c and c with well-documented high affinities to dnak. one dnak binding region was located in the signal peptide and a second in the c-peptide, connecting the proinsulin a- and b-chain. two further dnak binding regions were located in the a-chain and b-chain. densitometric analysis of dnak eluted from the immobilized peptides indicated peak binding to peptides 1- 17- 36- and 46-. the application of an algorithm developed to predict the dnak binding probability of peptides roughly confirmed the location of three of these binding regions in the preproinsulin molecule. in contrast to the experimental findings, the algorithm did not predict dnak binding for the a-chain region, but identified an additional dnak binding region at the position of the peptides and which are located in a region connecting the b-chain and the c-peptide. as expected, the amino acid sequences of the control peptides yielded dnak binding with scores in a range of - and -. the overlapping peptides with peak binding of dnak as determined in the solid phase assay were compared with the underlying peptide sequence of preproinsulin. the corresponding dnak binding sequences in the preproinsulin molecule were amino acids s7- of the signal peptide, b9- of the b-chain, c15- of the c-peptide and a6- of the a-chain. quantitative analysis of proinsulin peptide binding to dnak electro-elution of dnak from immobilized peptides was performed in three consecutive cycles and the progress of peptide release was determined as an estimate of their dnak affinity. the amounts of dnak eluted in the first cycle correspond to the electroblots shown in figure 1a. the amounts of dnak released during elution cycles and were compared with the release of dnak from the high affinity control ligand c. most peptides retained dnak less than the control peptide during elution cycles and except for peptides and of these two, peptide exhibited better retention of dnak indicating highest affinity in this assay. interestingly, the peptide corresponds to the insulin peptide b9- which had been identified as an immunodominant epitope of autoimmune t-cell reactivity in type diabetes. therefore, we analyzed the interaction of dnak with peptide b11- also in a fluid phase assay. we determined the capacity of a peptide to compete with the fluorescent high-affinity dnak ligand a-nr for binding to dnak. increasing concentrations of non-labeled peptide b11- resulted in a decrease of the fluorescence signal reflecting effective displacement of a-nr from dnak by the peptide, corresponding to an apparent dissociation equilibrium constant of ± μm of the competing peptide b11- by contrast, peptide b18- which is an adjacent peptide from the proinsulin b-chain region but without significant dnak-affinity, was much less effective in displacing a-nr. binding of dnak to peptides versus intact insulin or monomeric proinsulin next, we used the fluid phase assay to analyze whether dnak would interact with the intact insulin molecule. native insulin, only poorly competed with a-nr indicating that the steric configuration of insulin oligomers prevents effective interaction with dnak. binding strength of insulin was in the range of the poorly binding peptide b18-. for comparison, the sigma32-derived control peptide, a high-affinity dnak ligand, exhibited a dissociation constant of ± μm. the latter was in the same range as found for insulin peptide b11-. human insulin peptide b18- control peptide peptide b11- human proinsulin variant the dnak affinity of insulin and insulin-derived peptides was determined in a competition assay as described in "methods". lines indicate the disulfide bonds in the insulin and proinsulin-molecules. the data show mean ± sd of three or four determinations. finally, we investigated the possible binding of dnak to a proinsulin variant with decreased propensity to oligomer formation due to an inversion of the k-p sequence in the positions b and b this proinsulin variant interacted with dnak at concentrations much lower than observed for insulin. discussion our experiments demonstrate that the kda heat shock protein dnak is able to bind to 13-mer peptide segments of the preproinsulin molecule and that the binding is limited to four distinct preproinsulin regions, amino acids s7- of the signal peptide, b9- of the b-chain, c15- of the c-peptide and a6- of the a-chain. these peptide regions exhibit the leucine-rich motif found essential for binding of dnak, usually a hydrophobic core of four to five residues enriched in l, but also in i, v, f and y, and two flanking regions enriched in basic residues. of the proinsulin molecule, strongest binding occurred to peptide, and this is probably mediated by a distinct hydrophobic motif formed by the three amino acid residues l-y-l in the central positions b b and b this conclusion is supported by the finding that the b-chain-derived peptide b18- which comprises the six c-terminal residues of b11- but lacks the central l-y-l sequence or a corresponding hydrophobic motif exhibited no significant dnak affinity. native insulin exhibited only low affinity to dnak. this observation indicates that steric hindrance prevents effective interaction with the chaperone, possibly due to the conformation of the polypeptide heterodimer or the aggregation of insulin molecules into dimers, trimers or hexamers, rendering the regions b11- and a6- less accessible. as deduced from the crystal structure of the insulin molecule http://swissmodel.expasy.org/repository, the b11- epitopes are buried at the interface between two mature insulin heterodimers, thus preventing access to the peptide binding site of dnak. similarly, the a-chain region a6- comprises a number of amino acid residues localized at the interfaces of the insulin oligomers. in particular, the surface-exposed e-residue in position a contributes to the stabilization of the dimer-dimer interfaces by its interaction with the f-residue in position of the b-chain. moreover, the c-residue in position a which is located at the surface of the insulin molecule, forms a stabilizing interchain disulfide bond with the c-residue in position of the b-chain. to further elucidate a potential impact of steric conditions on dnak-insulin interactions we investigated the dnak binding of a proinsulin variant with an inversion of the k-p sequence in positions b and b causing a decreased tendency to oligomerization. this variant indeed exhibited a much higher dnak affinity than mature insulin. however, the kd obtained for the mature insulin molecule may only represent an estimate for the chaperone affinity of the hormone, as the fluid phase system applied in our approach is optimised for studies on the interaction of chaperones with peptides rather than larger proteins. nevertheless, previous studies which applied experimental systems designed to investigate the binding of dnak to polypeptides demonstrate weak, but significant interactions between dnak and full-length, native proteins. in those studies, dnak binding was found to depend on the accessible hydrophobic area of a protein and was characterized by dissociation constants of - μm, a range comparable to the kd of ± μm found for insulin in our current study. taken together, these observation suggest that not only the insulin peptides but also mature proinsulin monomers can interact with dnak and that there is little interaction between dnak and native oligomeric forms of insulin. in our current approach we selected insulin as the predominant β-cell specific antigen. dnak was selected as a representative member of the dominant and phylogenetically high conserved hsp family with the best characterized chaperone activity. although the members of the hsp family were found to exhibit some differences in peptide binding specificities extensive comparative analyses revealed a common binding specificity for peptide stretches of at least seven amino acid residues with a hydrophobic core thus largely resembling the hydrophobicity distribution pattern of the insulin-derived peptide b11- therefore, our analyses on the interaction of potentially autoantigenic insulin peptides with the chaperone dnak was performed in a suitable model system which only partially reflects the situation in vivo, in human patients. further detailed studies of the interaction of insulin and proinsulin with human hsp isoforms will be needed to fully understand the rules of insulin-chaperone interaction. besides the chaperoning function, one other property of hsp family chaperones is the ability to induce t-cell immune responses to complexed peptides, and a role of this pathway has been postulated for the induction of autoimmune disease. it therefore is noteworthy that the three dnak-binding regions of proinsulin, amino acids b9- of the b-chain, c15- of the c-peptide and a6- of the a-chain superimpose with the three major target regions for t-cell autoimmune reactivity in the nod mouse model or in humans with β-cell autoimmunity. in the mouse model, the b-chain region around b was identified as critical for disease development, i.e., mutating the native e in position b to q abolished t-cell reactivity to the peptide b9-. moreover, replacement of y to a in position b abrogated the response of b9- - reactive t-cell clones as well as the development of diabetes in vivo. i.e., the peptide b11- is closely associated with diabetes pathogenesis, and its diabetogenic potential depends on an intact core sequence, comprising the region which is important for the binding of this peptide to dnak. as the insulin molecules with the mutated b-chains completely retain their metabolic activity it has to be assumed that a single amino acid-exchange does not impair the formation of the correct tertiary structure, a complex process that most likely involves proper binding and assistance by chaperones. the findings from the mutated insulin molecules therefore further support the view, that the peptide binding motifs of chaperones are not determined by strictly defined amino acid sequences but by more roughly defined peptide properties, e.g. by the hydrophobicity distribution pattern within a stretch of amino acids. in contrast, t-cell receptor-mediated peptide recognition is known to be a highly selective and largely amino acid sequence specific process. based on these considerations it may be speculated that chaperones are able to bind defined sets of insulin peptides characterized by hydrophobic motifs. chaperone-peptide complexes may be recognized by antigen presenting cells which perform the ultimate decision on the pathogenicity of a peptide by applying stringent, highly sequence-sensitive selection criteria during processing and re-presentation of the peptide in the context of mhc structures and co-stimulatory signals. further studies are warranted to analyze whether enhanced binding of proinsulin monomers or misfolded insulin to hsp family chaperones during periods of β-cell stress contributes to the pathogenesis of autoimmune diabetes.  our observations indicate that the hsp chaperone dnak is able to bind to three discrete regions of the proinsulin molecule which are characterized by a central hydrophobic amino acid motif flanked by regions enriched for basic amino acids. binding occurs to 13-mer peptides and to a proinsulin molecule modified to prevent oligomerization, but not to native insulin. the three dnak-binding regions of proinsulin, aminoacids b9- of the b-chain, c15- of the c-peptide and a6- of the a-chain superimpose with the three major target regions for t-cell autoimmune reactivity in autoimmune diabetes. 
the b-chain region b11- is located at the interface between two insulin molecules and not accessible in insulin oligomers. indeed, native insulin oligomers showed very low dnak affinity whereas a proinsulin molecule modified to prevent oligomerization showed good binding affinity. chaperones facilitate proper folding of peptides and bind to misfolded proteins as occurring during periods of cell stress. of a set of overlapping 13-mer peptides of human preproinsulin high affinity binding to dnak was found for the signal peptide and one further region in each proinsulin domain. intact insulin only weakly interacts with the hsp chaperone dnak whereas monomeric proinsulin and peptides from distinct proinsulin regions show substantial chaperone binding.
 chaperones facilitate proper folding of peptides and bind to misfolded proteins as occurring during periods of cell stress. complexes of peptides with chaperones induce peptide-directed immunity. here we analyzed the interaction of proinsulin with the best characterized chaperone of the hsp family, bacterial dnak.  of a set of overlapping 13-mer peptides of human preproinsulin high affinity binding to dnak was found for the signal peptide and one further region in each proinsulin domain. among the latter, peptides covering most of the b-chain region b11- exhibited strongest binding, which was in the range of known high-affinity dnak ligands, dissociation equilibrium constant of ± μm. the b-chain region b11- is located at the interface between two insulin molecules and not accessible in insulin oligomers. indeed, native insulin oligomers showed very low dnak affinity whereas a proinsulin molecule modified to prevent oligomerization showed good binding affinity.  intact insulin only weakly interacts with the hsp chaperone dnak whereas monomeric proinsulin and peptides from distinct proinsulin regions show substantial chaperone binding. strongest binding was seen for the b-chain peptide b 11- interestingly, peptide b11- represents a dominant autoantigen in type diabetes. 