BACKGROUND
pancreatic cancer is the seventh most common cause of cancer death in western society with a 5-year survival rate of approximately  <dig> % being the lowest 5-year relative survival rate of all cancers.
the incidence rate of this aggressive tumor is increasing and the treatment is ineffective with a poor prognosis  <cit> .
recently, gathering of data concerning genomics and proteomics studies of pancreatic cancer showed differential regulation of  <dig>  potential biomarkers  <cit> .
these include known oncogenes and cell cycle regulators, e.g., the ras oncogene  <cit> , p <dig>  <cit> , smad <dig>  <cit> , epidermal growth factor and epidermal growth factor receptor  <cit> , cyclin d <dig>  <cit> , cdk4i  <cit> , as well as insulin-like growth factor- <dig> receptor, stat- <dig> and the tyrosine kinase src  <cit> .
still, the molecular biology of the disease is not well understood and a more thorough investigation of the specific proteins is crucial
in an earlier study, we have revealed that endonuclein  <cit> , a cell cycle regulated wd-repeat protein, also contributes to the development of the protein phenotype of pancreatic cancer.
wd-repeat proteins usually consist of 4- <dig> wd-repeats forming an up to eight blade propeller structure creating a stable platform that may form complexes reversibly with several proteins  <cit> .
despite of the structural similarity between these members they are a functionally diversified group where all members seem to possess regulatory functions e.g.
proteins including cell division control proteins, coatomer b subunits, transcription initiation factors, microtube associate proteins, actin interacting proteins, as well as enzymes like protein phosphatase 2a and myosin heavy chain kinase a 3- <dig>  <cit> .
to date, the function of the various protein members have thus been ascribed to crucial regulatory roles in signal transduction, rna processing, gene expression, trafficking of vesicles, assembly of cytoskeletal components as well as participation in the regulation of the cell cycle  <cit> .
since several wd-repeat proteins are known to form multiprotein complexes by interacting with other proteins sometimes through their wd-repeat domains, we set forward studies to reveal which function that may be attached to endonuclein by identifying interacting proteins.
consequently, we have expressed recombinant endonuclein in escherichia coli and used the purified immobilized protein to affinity purify several endonuclein interacting proteins from a placental tissue extract.
a proteomic approach using two-dimensional gel electrophoresis  and tandem mass sepctrometry   <cit>  together with a number of proteins assays were used to further characterize the proteins.
the nature of the affinity purified proteins confirms that endonuclein may possess regulatory functions.
thus, among the interacting proteins, we have identified, are tip- <dig> , mt-ssb  and a number of stress related proteins belonging to the molecular chaperones.
thus, we have discovered and characterized a number of putative functions of endonuclein that will be discussed in light of the disclosure of the interacting partners.
RESULTS
in order to detect endonuclein interacting proteins, we immobilized recombinant endonuclein to a column and prepared a reference column without immobilized endonuclein.
similar volumes of placental tissue extract were added to both columns at neutral ph.
after a wash of both columns with  <dig> m nacl, proteins that interacted with the immobilized endonuclein were eluted with  <dig>  m glycine buffer  and subsequently neutralized with  <dig> m tris-hcl, ph  <dig>  both eluates from the endonuclein column and the reference column were concentrated and subjected to 2d-page as shown in fig  <dig>  several proteins were found to interact specifically with endonuclein as indicated with numbers in fig 1a.
a few proteins interacted non-specifically with the column matrix as given with arrowheads in fig 1b.
of several proteins that were subjected to partial internal sequencing by ms/ms, eight spots could be unambiguously identified as given in table  <dig>  the localization of the identified interacting proteins is in line with the previously established nuclear localization and cytoplasmic localization in the endoplasmic reticulum of endonuclein by immunoelectron and confocal laser scanning microscopy  <cit> .
each of the proteins will be described in detail below.
tfdltgi
ltpeeier
nelesyayslk
nucleus*
ililpsvtr
lsipqittk
*determined by laser scanning confocal microscopy in present work
identification of protein spots no.
<dig> and no.
16
protein spot no.
<dig> gave the peptide sequence eleeivqpiisk which was identified as a peptide located in the c-terminal of immunoglobulin heavy chain binding protein bip or grp <dig>  <cit> .
similarly, protein no.
<dig> gave  <dig> peptides  that also were found in bip.
peptide spots no.
<dig> and  <dig> with molecular masses around 25- <dig> kda thus represent c-terminal cleavage products of bip that has a molecular mass around  <dig> kda.
in order to verify the interaction with bip, we performed surface plasmon resonance experiments by flowing hamster bip that possesses more than 98% sequence identity to human bip over the immobilized endonuclein.
as seen from fig  <dig> these experiments verified that there was an interaction between the immobilized endonuclein and bip.
as a control recombinant gst revealed no detectable binding.
identification of protein spots no.
<dig> and no.
11
both spot no.
<dig> and no.
<dig> gave the sequence vseggpaeiaglqigdk that identified the protein as tax interaction protein  <dig>  tip- <dig>  <cit> .
the localization of protein spots no.
<dig> and no.
<dig> in the 2d gel is in agreement with the deduced molecular mass of  <dig>  kda and pi of  <dig>  for tip- <dig>  the surface plasmon resonance experiments shown in fig  <dig> were used to verify that recombinant tip- <dig> protein constructed as fusion protein with gst interacted with endonuclein.
separate control experiments revealed that pure gst protein did not interact with endonuclein.
to establish evidence that endonuclein and tip- <dig> are localized to the same subcellular compartment, we performed double immunocytochemistry on cultured hacat cells.
as shown in fig  <dig> the two proteins co-localize in the cytoplasm  and to some extend in the nucleus .
identification of protein spot no.
4
protein spot no.
<dig> revealed the peptide sequence tleveiepgvr that identified the protein as dnaj homolog subfamily b member  <dig>  dnajb <dig>  <cit> .
the molecular mass of the predicted protein is calculated to be  <dig>  kda and the pi to  <dig> , i.e.
very close to the experimental values observed, see fig  <dig>  by using the publicly available www server that is based on neuronal networks  <cit> , the n-terminal  <dig> amino acids were found to have the hydrophobic characteristics of a signal peptide for directing the protein into the er with a likely cleavage site between ala- <dig> and gly- <dig> which is consistent with the protein localization in strong association with the er membrane oriented towards the er lumen  <cit> .
the protein also exhibits a j domain after the signal sequence.
the j domain is a highly conserved domain of about  <dig> amino acids found in the hsp <dig> protein family that consist of over  <dig> members  <cit> .
further towards the c-terminal there is an rgd motif which may serve a role in cell adhesion, since this sequence can bind to the integrins  <cit> .
the different domains of dnajb <dig> are shown in fig 4a.
identification of protein spot no.
8
protein spot no.
<dig> gave the sequence tgaelvtcgsvlk that identified the protein as stromal cell-derived factor 2-like protein  <dig>  sdf2-like protein  <dig>  <cit> .
by analyzing the sequence using the www server  <cit> , we found a putative signal sequence with an indicated cleavage site between ala- <dig> and ala- <dig>  furthermore, the protein terminates with the hdel signal which has been identified as an er retrieval signal in some of the crec proteins  <cit>  suggesting that the protein is localized as a resident protein within the er.
the database search showed that the protein possesses a strong similarity to the stromal cell derived factor- <dig>  sdf- <dig>  <cit>  which has been reported to contain mir domains, since these are also found in protein o-mannosyl-transferases, inositol  <dig> ,5-trisphosphate receptors and ryanodine receptors  <cit> .
the three mir domains present in sdf2-like protein  <dig> are shown in fig 4b.
identification of protein spot no.
17
from spot no.
<dig>  we obtained two peptide sequences; sgdsevyqlgdvsqk and vgqdpvlr.
both peptides showed a perfect identity match to the mitochondrial single-stranded dna binding protein, mt-ssb  <cit> .
to further investigate this interaction, we performed double immunocytochemistry on cultured hacat cells .
in most cells, endonuclein and mt-ssb did not co-localize to the same cellular compartment .
however, few cells showed a clear presence of mt-ssb in the nucleus co-localizing with endonuclein .
this is consistent with the fact, that this protein has been reported to be mainly localized in the mitochondria, while small amounts have been detected within the nucleus and in the cytoplasm  <cit> .
in addition, it has been shown that mt-ssb is a transcription factor for the aα fibrinogen gene  <cit> .
the mt-ssb gene has been assigned to chromosome  <dig> at 7q <dig>  <cit> , spanning about  <dig> kb.
identification of protein spot no.
18
spot no.
<dig> revealed the peptides enkdvltftcepk, ililpsvtr, lsipqittk and lfipqittk.
by searching the databases, we found that these peptides corresponded to the pregnancy specific β- <dig> glycoprotein  <dig>  branch of the carcinoembryonic antigen family, where to date  <dig> proteins have been identified, psbg <dig> - psbg <dig>  <cit> .
the psgs are abundantly expressed in placenta during embryonic development and also in uterus, pancreas, testis and fetal liver.
the psgs contain signal sequences and are secreted proteins  <cit> .
many of them contain an rgd sequence which may serve a role in cell adhesion, since this sequence may bind to the integrins  <cit> .
the peptides identified  may correspond to several of the psbgs.
surface plasmon resonance experiments with a commercial preparation of psbg indeed verified that endonuclein may interact with members of this class of proteins .
ca2+-binding to endonuclein
binding of ca2+ to endonuclein was measured with the protein in solution using the rate dialysis technique  <cit> .
a scatchard plot of the data is shown in fig  <dig>  the data obtained may be interpreted by a model where endonuclein contains two ca2+-binding sites, each binding ca2+ with a dissociation constant, kd, of  <dig> μm at physiologic conditions.
discussion
endonuclein, previously known as ief ssp  <dig>  <cit>  and the yeast homologue pwp <dig>  are wd-repeat proteins that to date have been described in a very limited number of papers  <cit> .
in previous studies endonuclein has been reported to be up-regulated at the transcript level  <cit>  as well as at the protein level  <cit>  in adenocarcinoma of the pancreas.
up-regulation of this protein in cancer may be in line with the fact that endonuclein is identified as a cell cycle regulated protein synthesized at increasing levels and with nuclear focusing during interphase  <cit> .
up-regulation of the protein may promote the cell cycle in line with the observation that yeast cells with a null mutation of the homologous gene  grow slowly  <cit> .
pwp <dig> is involved in ribosome biosynthesis through its interaction with trans-acting ribosome biogenesis factors and with several ribosome protein subunits  <cit> .
in addition, it has recently been shown that pwp <dig> associates with the 25s ribosomal dna chromatin , and that pwp <dig> may play a role in regulation of rdna transcription  <cit> .
furthermore, the recent finding that endonuclein is downregulated during heat stress-induced apoptosis  <cit> , suggests that indeed this protein is involved in diverse important cellular functions.
since wd-repeat proteins as a rule possess regulatory functions rather than enzymatic  <cit> , we further explored the functional roles of endonuclein by biochemical analyses with emphasis on the identification and characterization of its interacting proteins.
endonuclein interacting proteins  may largely be classified into two groups, one that is involved in signal transduction from the cytosol to the nucleus and another that participates in chaperone activities in the er.
these localizations are in line with the previous observation by immunoelectron and laser scanning fluorescence microscopy  <cit>  that endonuclein localizes to the nucleus as well as to the cytoplasm in the endoplasmic reticulum indicating that the interactions observed may be of functional significance.
the results are summarized in fig  <dig>  in the cytosol endonuclein may interact with the tax interaction protein  <dig>  tip- <dig>  tip- <dig> was originally identified as a pdz domain protein interacting with the c-terminal of tax, a  <dig> kda nuclear phosphoprotein encoded by htlv- <dig>  <cit> , and recent studies have identified tip- <dig> as an atypical pdz protein that functions as a negative regulator of pdz-based scaffolding  <cit> .
tax is considered to be oncogenic by trans-activating a number of viral and cellular genes  <cit> , and it has been shown that overexpression of tip- <dig> reduced the proliferation and growth of colorectal cancer cells  <cit> .
since tax is not a dna binding protein per se, it executes its action through the binding of other cellular proteins.
however, which specific trans-activation pathway that is essential for transformation of cells still remains controversial  <cit> .
interestingly, tip- <dig> that contains a pdz domain has been reported to interact with the c-terminal of rhotekin  <cit> .
rhotekin was identified as a target protein of the rho family of small gtpases  <cit> .
a ternary complex of active rho with rhotekin and tip- <dig> produces a signal in the cytosol that triggers strong activation of the c-fos serum response element  <cit>  that also is activated by tax  <cit> .
how the signal from the ternary complex in the cytosol is transferred to the nucleus is unknown.
the interaction of endonuclein with tip- <dig> could thus have modulatory actions on the rho signal transduction pathway as well as on the pathway that leads to oncogenesis induced by tax.
such a modulatory effect on signal transduction pathways is further substantiated by the observation that endonuclein may interact with mt-ssb, a transcription factor for the aα fibrinogen gene  <cit> .
this gene is induced by interleukin- <dig>  which activates two signal transduction pathways; the janus kinases that phosphorylate a group of latent cytoplasmic transcription factors known as stats and the map kinase signaling cascade that activates the c/ebp transcription factors.
upon phosphorylation, stat- <dig> is translocated to the nucleus and generally binds to the il- <dig> response element  of the genes that it activates.
however, stat- <dig> does not associate with the il- <dig> re of the fibrinogen promoter.
instead mt-ssb is attached to this site.
upon il- <dig> stimulation mt-ssb transiently leaves the site and re-associates again later.
the further details of the molecular mechanism whereby il- <dig> trans-activates the aα fibrinogen gene remains to be investigated  <cit> .
endonuclein may thus putatively modulate the signal transduction pathway through its interaction with mt-ssb.
in the er, endonuclein may interact with proteins of the molecular chaperones that assist in the folding of proteins  <cit> .
the specific endonuclein interacting proteins identified was bip that is involved in protein translocation, folding, refolding and degradation of proteins in the er as well as reverse translocation of proteins from the er to the cytosol for ubiquitination and degradation in the proteasome  <cit> .
in presence of atp, bip interacts with the j domain of dnajb <dig> and may activate the atpase activity of bip during its action  <cit> .
both bip and dnajb <dig> have recently been implied as target genes in the mammalian unfolded protein response   <cit> .
endonuclein may thus through its interaction with dnajb <dig> and bip modulate the chaperone activity in the er.
finally, two other proteins of the er were observed to interact with endonuclein.
psbgs which are a group of secretory proteins that are abundantly expressed in the placenta during embryonic development and which may have immunomodulatory functions  <cit>  and sdf2-like protein  <dig> which is a novel protein that contains three mir domains.
the latter motifs are found in three classes of proteins in the er  <cit> , protein o-mannosyl-transferases, which catalyzes transfer of mannose from dolichyl activated mannose to seryl or threonyl residues of secretory proteins  <cit>  and two classes of intracellular ca2+-release channel proteins, the ryanodine receptors and the inositol  <dig> ,5-trisphosphate receptors  <cit> .
from these proteins, it is known that the mir domains in the protein o-mannosyl-transferases are essential for its enzymatic function  <cit>  whereas they are involved in binding of inositol  <dig> ,5-trisphosphate in the signal transduction pathway that releases ca2+ from intracellular stores.
in this respect, it may be of importance that endonuclein can bind two ca2+ ions, each with a kd of approximately  <dig> μm.
endonuclein thereby possesses the ability of binding ca2+ with the concentrations of free ca2+ that are present in the er ranging from about  <dig> μm in resting cells to below  <dig> μm after ca2+-mobilization  <cit> .
it is thus possible that the physiologic effect of endonuclein in the er is ca2+ dependent.
however, the specific functions of the mir domains in sdf2-like protein  <dig> must await further studies, although it may be anticipated that the domains presumably are involved in the binding to endonuclein since sdf2-like protein  <dig> almost exclusively consists of three mir domains .
CONCLUSIONS
in conclusion, we have found that the nature of the endonuclein interacting proteins confirms a regulatory role for endonuclein, especially with respect to modulation of signal transduction pathways to the nucleus and chaperone activities in the er.
the specific function of endonuclein in these processes will be the focus of future studies.

 pancreatic cancer is the seventh most common cause of cancer death in western society with a 5-year survival rate of approximately % being the lowest 5-year relative survival rate of all cancers. the incidence rate of this aggressive tumor is increasing and the treatment is ineffective with a poor prognosis. recently, gathering of data concerning genomics and proteomics studies of pancreatic cancer showed differential regulation of potential biomarkers. these include known oncogenes and cell cycle regulators, e.g., the ras oncogene, p, smad, epidermal growth factor and epidermal growth factor receptor, cyclin d, cdk4i, as well as insulin-like growth factor- receptor, stat- and the tyrosine kinase src. still, the molecular biology of the disease is not well understood and a more thorough investigation of the specific proteins is crucial in an earlier study, we have revealed that endonuclein, a cell cycle regulated wd-repeat protein, also contributes to the development of the protein phenotype of pancreatic cancer. wd-repeat proteins usually consist of 4- wd-repeats forming an up to eight blade propeller structure creating a stable platform that may form complexes reversibly with several proteins. despite of the structural similarity between these members they are a functionally diversified group where all members seem to possess regulatory functions e.g. proteins including cell division control proteins, coatomer b subunits, transcription initiation factors, microtube associate proteins, actin interacting proteins, as well as enzymes like protein phosphatase 2a and myosin heavy chain kinase a 3-. to date, the function of the various protein members have thus been ascribed to crucial regulatory roles in signal transduction, rna processing, gene expression, trafficking of vesicles, assembly of cytoskeletal components as well as participation in the regulation of the cell cycle. since several wd-repeat proteins are known to form multiprotein complexes by interacting with other proteins sometimes through their wd-repeat domains, we set forward studies to reveal which function that may be attached to endonuclein by identifying interacting proteins. consequently, we have expressed recombinant endonuclein in escherichia coli and used the purified immobilized protein to affinity purify several endonuclein interacting proteins from a placental tissue extract. a proteomic approach using two-dimensional gel electrophoresis and tandem mass sepctrometry together with a number of proteins assays were used to further characterize the proteins. the nature of the affinity purified proteins confirms that endonuclein may possess regulatory functions. thus, among the interacting proteins, we have identified, are tip-, mt-ssb and a number of stress related proteins belonging to the molecular chaperones. thus, we have discovered and characterized a number of putative functions of endonuclein that will be discussed in light of the disclosure of the interacting partners.  in order to detect endonuclein interacting proteins, we immobilized recombinant endonuclein to a column and prepared a reference column without immobilized endonuclein. similar volumes of placental tissue extract were added to both columns at neutral ph. after a wash of both columns with m nacl, proteins that interacted with the immobilized endonuclein were eluted with m glycine buffer and subsequently neutralized with m tris-hcl, ph both eluates from the endonuclein column and the reference column were concentrated and subjected to 2d-page as shown in fig several proteins were found to interact specifically with endonuclein as indicated with numbers in fig 1a. a few proteins interacted non-specifically with the column matrix as given with arrowheads in fig 1b. of several proteins that were subjected to partial internal sequencing by ms/ms, eight spots could be unambiguously identified as given in table the localization of the identified interacting proteins is in line with the previously established nuclear localization and cytoplasmic localization in the endoplasmic reticulum of endonuclein by immunoelectron and confocal laser scanning microscopy. each of the proteins will be described in detail below. tfdltgi ltpeeier nelesyayslk nucleus* ililpsvtr lsipqittk *determined by laser scanning confocal microscopy in present work identification of protein spots no. and no. 16 protein spot no. gave the peptide sequence eleeivqpiisk which was identified as a peptide located in the c-terminal of immunoglobulin heavy chain binding protein bip or grp. similarly, protein no. gave peptides that also were found in bip. peptide spots no. and with molecular masses around 25- kda thus represent c-terminal cleavage products of bip that has a molecular mass around kda. in order to verify the interaction with bip, we performed surface plasmon resonance experiments by flowing hamster bip that possesses more than 98% sequence identity to human bip over the immobilized endonuclein. as seen from fig these experiments verified that there was an interaction between the immobilized endonuclein and bip. as a control recombinant gst revealed no detectable binding. identification of protein spots no. and no. 11 both spot no. and no. gave the sequence vseggpaeiaglqigdk that identified the protein as tax interaction protein tip-. the localization of protein spots no. and no. in the 2d gel is in agreement with the deduced molecular mass of kda and pi of for tip- the surface plasmon resonance experiments shown in fig were used to verify that recombinant tip- protein constructed as fusion protein with gst interacted with endonuclein. separate control experiments revealed that pure gst protein did not interact with endonuclein. to establish evidence that endonuclein and tip- are localized to the same subcellular compartment, we performed double immunocytochemistry on cultured hacat cells. as shown in fig the two proteins co-localize in the cytoplasm and to some extend in the nucleus. identification of protein spot no. 4 protein spot no. revealed the peptide sequence tleveiepgvr that identified the protein as dnaj homolog subfamily b member dnajb. the molecular mass of the predicted protein is calculated to be kda and the pi to, i.e. very close to the experimental values observed, see fig by using the publicly available www server that is based on neuronal networks, the n-terminal amino acids were found to have the hydrophobic characteristics of a signal peptide for directing the protein into the er with a likely cleavage site between ala- and gly- which is consistent with the protein localization in strong association with the er membrane oriented towards the er lumen. the protein also exhibits a j domain after the signal sequence. the j domain is a highly conserved domain of about amino acids found in the hsp protein family that consist of over members. further towards the c-terminal there is an rgd motif which may serve a role in cell adhesion, since this sequence can bind to the integrins. the different domains of dnajb are shown in fig 4a. identification of protein spot no. 8 protein spot no. gave the sequence tgaelvtcgsvlk that identified the protein as stromal cell-derived factor 2-like protein sdf2-like protein. by analyzing the sequence using the www server, we found a putative signal sequence with an indicated cleavage site between ala- and ala- furthermore, the protein terminates with the hdel signal which has been identified as an er retrieval signal in some of the crec proteins suggesting that the protein is localized as a resident protein within the er. the database search showed that the protein possesses a strong similarity to the stromal cell derived factor- sdf- which has been reported to contain mir domains, since these are also found in protein o-mannosyl-transferases, inositol,5-trisphosphate receptors and ryanodine receptors. the three mir domains present in sdf2-like protein are shown in fig 4b. identification of protein spot no. 17 from spot no. we obtained two peptide sequences; sgdsevyqlgdvsqk and vgqdpvlr. both peptides showed a perfect identity match to the mitochondrial single-stranded dna binding protein, mt-ssb. to further investigate this interaction, we performed double immunocytochemistry on cultured hacat cells. in most cells, endonuclein and mt-ssb did not co-localize to the same cellular compartment. however, few cells showed a clear presence of mt-ssb in the nucleus co-localizing with endonuclein. this is consistent with the fact, that this protein has been reported to be mainly localized in the mitochondria, while small amounts have been detected within the nucleus and in the cytoplasm. in addition, it has been shown that mt-ssb is a transcription factor for the aα fibrinogen gene. the mt-ssb gene has been assigned to chromosome at 7q, spanning about kb. identification of protein spot no. 18 spot no. revealed the peptides enkdvltftcepk, ililpsvtr, lsipqittk and lfipqittk. by searching the databases, we found that these peptides corresponded to the pregnancy specific β- glycoprotein branch of the carcinoembryonic antigen family, where to date proteins have been identified, psbg - psbg. the psgs are abundantly expressed in placenta during embryonic development and also in uterus, pancreas, testis and fetal liver. the psgs contain signal sequences and are secreted proteins. many of them contain an rgd sequence which may serve a role in cell adhesion, since this sequence may bind to the integrins. the peptides identified may correspond to several of the psbgs. surface plasmon resonance experiments with a commercial preparation of psbg indeed verified that endonuclein may interact with members of this class of proteins. ca2+-binding to endonuclein binding of ca2+ to endonuclein was measured with the protein in solution using the rate dialysis technique. a scatchard plot of the data is shown in fig the data obtained may be interpreted by a model where endonuclein contains two ca2+-binding sites, each binding ca2+ with a dissociation constant, kd, of μm at physiologic conditions. discussion endonuclein, previously known as ief ssp and the yeast homologue pwp are wd-repeat proteins that to date have been described in a very limited number of papers. in previous studies endonuclein has been reported to be up-regulated at the transcript level as well as at the protein level in adenocarcinoma of the pancreas. up-regulation of this protein in cancer may be in line with the fact that endonuclein is identified as a cell cycle regulated protein synthesized at increasing levels and with nuclear focusing during interphase. up-regulation of the protein may promote the cell cycle in line with the observation that yeast cells with a null mutation of the homologous gene grow slowly. pwp is involved in ribosome biosynthesis through its interaction with trans-acting ribosome biogenesis factors and with several ribosome protein subunits. in addition, it has recently been shown that pwp associates with the 25s ribosomal dna chromatin, and that pwp may play a role in regulation of rdna transcription. furthermore, the recent finding that endonuclein is downregulated during heat stress-induced apoptosis, suggests that indeed this protein is involved in diverse important cellular functions. since wd-repeat proteins as a rule possess regulatory functions rather than enzymatic, we further explored the functional roles of endonuclein by biochemical analyses with emphasis on the identification and characterization of its interacting proteins. endonuclein interacting proteins may largely be classified into two groups, one that is involved in signal transduction from the cytosol to the nucleus and another that participates in chaperone activities in the er. these localizations are in line with the previous observation by immunoelectron and laser scanning fluorescence microscopy that endonuclein localizes to the nucleus as well as to the cytoplasm in the endoplasmic reticulum indicating that the interactions observed may be of functional significance. the results are summarized in fig in the cytosol endonuclein may interact with the tax interaction protein tip- tip- was originally identified as a pdz domain protein interacting with the c-terminal of tax, a kda nuclear phosphoprotein encoded by htlv-, and recent studies have identified tip- as an atypical pdz protein that functions as a negative regulator of pdz-based scaffolding. tax is considered to be oncogenic by trans-activating a number of viral and cellular genes, and it has been shown that overexpression of tip- reduced the proliferation and growth of colorectal cancer cells. since tax is not a dna binding protein per se, it executes its action through the binding of other cellular proteins. however, which specific trans-activation pathway that is essential for transformation of cells still remains controversial. interestingly, tip- that contains a pdz domain has been reported to interact with the c-terminal of rhotekin. rhotekin was identified as a target protein of the rho family of small gtpases. a ternary complex of active rho with rhotekin and tip- produces a signal in the cytosol that triggers strong activation of the c-fos serum response element that also is activated by tax. how the signal from the ternary complex in the cytosol is transferred to the nucleus is unknown. the interaction of endonuclein with tip- could thus have modulatory actions on the rho signal transduction pathway as well as on the pathway that leads to oncogenesis induced by tax. such a modulatory effect on signal transduction pathways is further substantiated by the observation that endonuclein may interact with mt-ssb, a transcription factor for the aα fibrinogen gene. this gene is induced by interleukin- which activates two signal transduction pathways; the janus kinases that phosphorylate a group of latent cytoplasmic transcription factors known as stats and the map kinase signaling cascade that activates the c/ebp transcription factors. upon phosphorylation, stat- is translocated to the nucleus and generally binds to the il- response element of the genes that it activates. however, stat- does not associate with the il- re of the fibrinogen promoter. instead mt-ssb is attached to this site. upon il- stimulation mt-ssb transiently leaves the site and re-associates again later. the further details of the molecular mechanism whereby il- trans-activates the aα fibrinogen gene remains to be investigated. endonuclein may thus putatively modulate the signal transduction pathway through its interaction with mt-ssb. in the er, endonuclein may interact with proteins of the molecular chaperones that assist in the folding of proteins. the specific endonuclein interacting proteins identified was bip that is involved in protein translocation, folding, refolding and degradation of proteins in the er as well as reverse translocation of proteins from the er to the cytosol for ubiquitination and degradation in the proteasome. in presence of atp, bip interacts with the j domain of dnajb and may activate the atpase activity of bip during its action. both bip and dnajb have recently been implied as target genes in the mammalian unfolded protein response. endonuclein may thus through its interaction with dnajb and bip modulate the chaperone activity in the er. finally, two other proteins of the er were observed to interact with endonuclein. psbgs which are a group of secretory proteins that are abundantly expressed in the placenta during embryonic development and which may have immunomodulatory functions and sdf2-like protein which is a novel protein that contains three mir domains. the latter motifs are found in three classes of proteins in the er, protein o-mannosyl-transferases, which catalyzes transfer of mannose from dolichyl activated mannose to seryl or threonyl residues of secretory proteins and two classes of intracellular ca2+-release channel proteins, the ryanodine receptors and the inositol,5-trisphosphate receptors. from these proteins, it is known that the mir domains in the protein o-mannosyl-transferases are essential for its enzymatic function whereas they are involved in binding of inositol,5-trisphosphate in the signal transduction pathway that releases ca2+ from intracellular stores. in this respect, it may be of importance that endonuclein can bind two ca2+ ions, each with a kd of approximately μm. endonuclein thereby possesses the ability of binding ca2+ with the concentrations of free ca2+ that are present in the er ranging from about μm in resting cells to below μm after ca2+-mobilization. it is thus possible that the physiologic effect of endonuclein in the er is ca2+ dependent. however, the specific functions of the mir domains in sdf2-like protein must await further studies, although it may be anticipated that the domains presumably are involved in the binding to endonuclein since sdf2-like protein almost exclusively consists of three mir domains.  in conclusion, we have found that the nature of the endonuclein interacting proteins confirms a regulatory role for endonuclein, especially with respect to modulation of signal transduction pathways to the nucleus and chaperone activities in the er. the specific function of endonuclein in these processes will be the focus of future studies. 
after chromatography the eluted proteins were further separated by two-dimensional gel electrophoresis and identified by tandem mass spectrometry. our results demonstrate that endonuclein interacts with several proteins indicating a broad function including signal transduction and chaperone activity. we have previously identified endonuclein as a cell cycle regulated wd-repeat protein that is up-regulated in adenocarcinoma of the pancreas. the interacting proteins were identified as; tax interaction protein, aα fibrinogen transcription factor, immunoglobulin heavy chain binding protein, human er-associated dnaj, endonuclein interaction protein, and pregnancy specific β- glycoproteins. using the cdna encoding human endonuclein, we have expressed and purified the recombinant protein from escherichia coli using metal affinity chromatography.
 we have previously identified endonuclein as a cell cycle regulated wd-repeat protein that is up-regulated in adenocarcinoma of the pancreas. now, we aim to investigate its biomedical functions.  using the cdna encoding human endonuclein, we have expressed and purified the recombinant protein from escherichia coli using metal affinity chromatography. the recombinant protein was immobilized to a column and by affinity chromatography several interacting proteins were purified from several litres of placenta tissue extract. after chromatography the eluted proteins were further separated by two-dimensional gel electrophoresis and identified by tandem mass spectrometry. the interacting proteins were identified as; tax interaction protein, aα fibrinogen transcription factor, immunoglobulin heavy chain binding protein, human er-associated dnaj, endonuclein interaction protein, and pregnancy specific β- glycoproteins. surface plasmon resonance analysis and confocal fluorescence microscopy were used to further characterize the interactions.  our results demonstrate that endonuclein interacts with several proteins indicating a broad function including signal transduction and chaperone activity. 