Functional Characterization of Two Secreted SEL1L Isoforms Capable of Exporting Unassembled Substrate

Abstract

SEL1L-A, a transmembrane glycoprotein residing in the endoplasmic reticulum (ER), is a component of the ER-associated degradation (ERAD) pathway. Alternative splicing generates two smaller SEL1L isoforms, -B and -C, that lack the SEL1L-A membrane-spanning region but retain some sel-1-like repeats, known to be involved in multi-protein interactions and signal transduction. In this study the functional characteristics of SEL1L-B and -C were investigated in human cell models. We show that these two isoforms are induced upon ER stress and activation of the unfolded protein response, together with SEL1L-A. Using transient transfection experiments (based on wild-type and mutant SEL1L constructs) combined with several biochemical tests we show that SEL1L-B and, more prominently, SEL1L-C are secreted glycoproteins. Although SEL1L-C is in monomeric form, SEL1L-B is engaged in intramolecular/intermolecular disulfide bonds. Both isoforms localize in secretory and degradative cellular compartments and in areas of cell-cell contact. However, whereas SEL1L-B is mainly associated with membranes, SEL1L-C shows the typical intralumenal localization of soluble proteins and is present in intercellular spaces. Furthermore, because of its peroxisomal domain, SEL1L-C localizes to peroxisomes. Both SEL1L-B and -C are involved in sorting and exporting unassembled Ig-μs but do not affect two other ERAD substrates, the null Hong Kong variant of α1-antitrypsin, and mutant α1-AT Z. Overall these findings suggest that SEL1L-B and -C participate to novel molecular pathways that, in parallel with ERAD, contribute to the disposure of misfolded/unfolded or orphan proteins through degradation or secretion. SEL1L-A, a transmembrane glycoprotein residing in the endoplasmic reticulum (ER), is a component of the ER-associated degradation (ERAD) pathway. Alternative splicing generates two smaller SEL1L isoforms, -B and -C, that lack the SEL1L-A membrane-spanning region but retain some sel-1-like repeats, known to be involved in multi-protein interactions and signal transduction. In this study the functional characteristics of SEL1L-B and -C were investigated in human cell models. We show that these two isoforms are induced upon ER stress and activation of the unfolded protein response, together with SEL1L-A. Using transient transfection experiments (based on wild-type and mutant SEL1L constructs) combined with several biochemical tests we show that SEL1L-B and, more prominently, SEL1L-C are secreted glycoproteins. Although SEL1L-C is in monomeric form, SEL1L-B is engaged in intramolecular/intermolecular disulfide bonds. Both isoforms localize in secretory and degradative cellular compartments and in areas of cell-cell contact. However, whereas SEL1L-B is mainly associated with membranes, SEL1L-C shows the typical intralumenal localization of soluble proteins and is present in intercellular spaces. Furthermore, because of its peroxisomal domain, SEL1L-C localizes to peroxisomes. Both SEL1L-B and -C are involved in sorting and exporting unassembled Ig-μs but do not affect two other ERAD substrates, the null Hong Kong variant of α1-antitrypsin, and mutant α1-AT Z. Overall these findings suggest that SEL1L-B and -C participate to novel molecular pathways that, in parallel with ERAD, contribute to the disposure of misfolded/unfolded or orphan proteins through degradation or secretion. SEL1L (alias SEL1L-A) is a type I transmembrane glycoprotein resident in the endoplasmic reticulum (ER) 2The abbreviations used are: ER, endoplasmic reticulum; ERAD, ER-associated degradation; UPR, unfolded protein response; siRNA, small interfering RNA; DTT, dithiothreitol; RT, reverse transcription; RACE, rapid amplification of cDNA ends; PNGase F, N-glycosidase F; endo H, endoglycosidase H; PBS, phosphate-buffered saline; E2, ubiquitin conjugating protein; E3, ubiquitin ligase. and involved in the recognition and/or dislocation of misfolded/unfolded proteins for ER-associated degradation (ERAD) (1.Mueller B. Lilley B.N. Ploegh H.L. J. Cell Biol. 2006; 175: 261-270Crossref PubMed Scopus (158) Google Scholar, 2.Cattaneo M. Otsu M. Fagioli C. Martino S. Lotti L.V. Sitia R. Biunno I. J. Cell. Physiol. 2008; 215: 794-802Crossref PubMed Scopus (35) Google Scholar, 3.Christianson J.C. Shaler T.A. Tyler R.E. Kopito R. Nat. Cell Biol. 2008; 10: 272-282Crossref PubMed Scopus (397) Google Scholar). ERAD is responsible for the recognition, selection, ubiquitination, and retrotranslocation across the ER membrane of unfolded or misfolded proteins for their degradation by cytosolic proteasome (4.Kostova Z. Wolf D.H. EMBO J. 2003; 22: 2309-2317Crossref PubMed Scopus (363) Google Scholar). It has been recently demonstrated that several well characterized ERAD substrates are cleared by SEL1L-A and its associated E3 ligase, HRD1 (1.Mueller B. Lilley B.N. Ploegh H.L. J. Cell Biol. 2006; 175: 261-270Crossref PubMed Scopus (158) Google Scholar, 2.Cattaneo M. Otsu M. Fagioli C. Martino S. Lotti L.V. Sitia R. Biunno I. J. Cell. Physiol. 2008; 215: 794-802Crossref PubMed Scopus (35) Google Scholar, 3.Christianson J.C. Shaler T.A. Tyler R.E. Kopito R. Nat. Cell Biol. 2008; 10: 272-282Crossref PubMed Scopus (397) Google Scholar). Alternative splicing, a major mechanism for expanding, diversifying, and regulating the repertoire of gene functions (5.Modrek B. Lee C. Nat. Genet. 2002; 30: 13-19Crossref PubMed Scopus (1050) Google Scholar, 6.Gravely B.R. Trends Genet. 2001; 17: 100-107Abstract Full Text Full Text PDF PubMed Scopus (939) Google Scholar), can generate a wide range of structurally and functionally diverse protein isoforms from transcripts encoded at a single gene locus. Notably, both soluble and membrane-associated protein isoforms can be produced by alternative exclusion/inclusion of membrane-spanning regions (7.Lainez B. Fernandez-Real J.M. Romero X. Esplugues E. Cañete J.D. Ricart W. Engel P. Int. Immunol. 2003; 16: 169-177Crossref Scopus (48) Google Scholar, 8.Rose-John S. Heinrich P.C. Biochem. J. 1994; 300: 281-290Crossref PubMed Scopus (686) Google Scholar). Soluble isoforms can acquire new properties and/or act as diffusible signals that agonize or antagonize functions of the membrane-bound variants. Here we report the cloning and functional characterization of SEL1L-B and -C, two SEL1L isoforms described as alternatively spliced SEL1L variants by Biunno et al. (10.Biunno I. Cattaneo M. Orlandi R. Canton C. Biagiotti L. Ferrero S. Barberis M. Pupa S.M. Scarpa A. Ménard S. J. Cell. Physiol. 2006; 208: 23-38Crossref PubMed Scopus (36) Google Scholar) and by AceView (April 2007). SEL1L-C was also identified by Swiss-prot search (Q9UBV2–2). Clark et al. (11.Clark H.F. Gurney A.L. Abaya E. Baker K. Baldwin D. Brush J. Chen J. Chow B. Chui C. Crowley C. Currell B. Deuel B. Dowd P. Eaton D. Foster J. Grimaldi C. Gu Q. Hass P.E. Heldens S. Huang A. Kim H.S. Klimowski L. Jin Y. Johnson S. Lee J. Lewis L. Liao D. Mark M. Robbie E. Sanchez C. Schoenfeld J. Seshagiri S. Simmons L. Singh J. Smith V. Stinson J. Vagts A. Vandlen R. Watanabe C. Wieand D. Woods K. Xie M.H. Yansura D. Yi S. Yu G. Yuan J. Zhang M. Zhang Z. Goddard A. Wood W.I. Godowsk i.P. Gray A. Genome Res. 2003; 13: 2265-2270Crossref PubMed Scopus (279) Google Scholar) reported that cDNA clone AY358651, alias SEL1L-C, encodes a putative secretory product. SEL1L-B and -C are respectively encoded by nine and eight of the twenty-one SEL1L-A exons and lack the SEL1L-A C-terminal transmembrane region but retain respectively four and three of the eleven sel-1-like repeats present in SEL1L-A. Alternative splicing events at the 3′ ends generate a tail of 9 amino acid residues (GIYVSPFTF) in SEL1L-B (10.Biunno I. Cattaneo M. Orlandi R. Canton C. Biagiotti L. Ferrero S. Barberis M. Pupa S.M. Scarpa A. Ménard S. J. Cell. Physiol. 2006; 208: 23-38Crossref PubMed Scopus (36) Google Scholar) and an SRL sequence, variant of the conserved peroxisomal targeting signal SKL, in SEL1L-C (9.Gould S.J. Keller G.A. Hosken N. Wilkinson J. Subramani S. J. Cell Biol. 1989; 108: 1657-1664Crossref PubMed Scopus (886) Google Scholar, 10.Biunno I. Cattaneo M. Orlandi R. Canton C. Biagiotti L. Ferrero S. Barberis M. Pupa S.M. Scarpa A. Ménard S. J. Cell. Physiol. 2006; 208: 23-38Crossref PubMed Scopus (36) Google Scholar). A fibronectin type II domain is conserved in all three SEL1L isoforms (10.Biunno I. Cattaneo M. Orlandi R. Canton C. Biagiotti L. Ferrero S. Barberis M. Pupa S.M. Scarpa A. Ménard S. J. Cell. Physiol. 2006; 208: 23-38Crossref PubMed Scopus (36) Google Scholar, 16.Cattaneo M. Canton C. Alberintini A. Biunno I. Gene (Amst.). 2004; 326: 149-156Crossref PubMed Scopus (18) Google Scholar, 17.Biunno I. Appierto V. Cattaneo M. Leone B.E. Balzano G. Socci C. Saccone S. Letizia A. Della Valle G. Sgaramella V. Genomics. 1997; 46: 284-286Crossref PubMed Scopus (44) Google Scholar). Here we show that SEL1L-B and -C are both responsive to ER stress. Although SEL1L-A is confined to the ER membrane and is involved in the degradation of unassembled Ig-μs chains, both SEL1L-B and -C are secreted and capable of exporting orphan Ig-μs outside cells with substrate specificity. With regard to subcellular distribution, both isoforms localize in secretory and degradative compartments and in areas of cell-cell contact. Although SEL1L-B is mainly associated with membrane profiles, SEL1L-C behaves as a typical soluble glycoprotein, localizing intralumenally in secretory compartments and intercellular spaces, and is also present in peroxisomes. Constructs—SEL1L-B and -C were generated by PCR from normal peripheral blood lymphocytes and verified by sequencing. The Myc-tagged SEL1L-B and -C constructs were generated by cloning the full-length SEL1L-B and -C coding sequence fused with a Myc tag at the 3′ end into the pCDNA3.1Myc-Hys(-)A vector (Invitrogen). SEL1L-B harboring a glycine to glutamic acid substitution at position 312 in the fourth sel-1-like repeat was naturally obtained from bacterial transformants. SEL1L-C deleted of the specific C-terminal peroxisomal domain (SELΔ-Cmyc) was derived from the wild-type SEL1L-C isoform. Vectors driving the expression of μs and of the N-terminal region of SEL1L-A (20–372 amino acids), with or without KDEL motif (named SEL1L 20–372 KDEL and SEL1L 20–372, respectively), were kindly provided by Drs. R. Sitia and J. C. Christianson (2.Cattaneo M. Otsu M. Fagioli C. Martino S. Lotti L.V. Sitia R. Biunno I. J. Cell. Physiol. 2008; 215: 794-802Crossref PubMed Scopus (35) Google Scholar, 3.Christianson J.C. Shaler T.A. Tyler R.E. Kopito R. Nat. Cell Biol. 2008; 10: 272-282Crossref PubMed Scopus (397) Google Scholar), respectively. Vectors driving the expression of the null Hong Kong variants of α1-antitrypsin (HHK and α1-AT Z) were kindly provided by Dr. R. Sifers. Cell Lines, Culture Conditions, and Transfections—Multiple myeloma KMS11 and breast cancer SKBr3 cells were maintained in RPMI containing 10% fetal bovine serum (Euroclone, Celbio, Pero, Italy). Human embryo 293 FT kidney cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Euroclone, Celbio, Pero, Italy). The cells were transiently transfected with the indicated plasmids and siRNAs by Lipofectamine 2000 (Invitrogen) and harvested after 48 and 72 h, as previously described (2.Cattaneo M. Otsu M. Fagioli C. Martino S. Lotti L.V. Sitia R. Biunno I. J. Cell. Physiol. 2008; 215: 794-802Crossref PubMed Scopus (35) Google Scholar). Cells at various times of culture were treated with DTT (2 mm) for 3 h, tunicamycin (2 μg/μl) for 24 h, or cycloheximide (200 μg/μl) for 4, 7, or 15 h, as indicated. RT-PCR—Total RNA was extracted using the SV Total RNA isolation system (Promega, Milan, Italy). RNA was reverse-transcribed with SuperScript™ II reverse transcriptase (Invitrogen) according to the manufacturer's instructions. PCR amplifications were performed with 2 μl of and of (Invitrogen) using the specific SEL1L-A, and at amplification and at amplification SEL1L-C, and at amplification and at amplification and at amplification and and at amplification was performed using the cDNA amplification Celbio, Pero, Italy). RNA was by reverse transcriptase with the and II A A and SEL1L-C, were used for amplification of cDNA PCR at for of for for and 72 for 3 and at 72 for PCR were in cloning (Invitrogen) and and of Cell was an N-terminal of human SEL1L R. Cattaneo M. M. Biunno I. Ménard S. Int. J. Biol. 2002; 17: PubMed Google Scholar). was kindly provided by Dr. L. Ploegh (1.Mueller B. Lilley B.N. Ploegh H.L. J. Cell Biol. 2006; 175: 261-270Crossref PubMed Scopus (158) Google Scholar). and α1-antitrypsin were from was obtained from (Invitrogen). The cells were in containing were by the the were on membranes, with specific and with Milan, Italy). cell were and with on protein The were with with and with of cell cells were and with (Invitrogen) for The were with 10% with and on by N-glycosidase or endoglycosidase cell and from transfected 293 FT cells were by at in for and at for with or without PNGase or endo Celbio, Pero, according to the transiently transfected with the indicated as were on with in for at treated with glycine for at and with for at The subcellular of and were using in and of cellular the ER in The the in the in and the peroxisomal in The were using in with PBS, were using in or in for at The were with in for with a with an was performed on a single using a system The of and signals in 293 FT kidney cells or in cells transfected with pCDNA3.1Myc-Hys(-)A vector the of the for were as and in in for 2 at cells were into 10% in on in at and in were at using an with and and or were by with or or by protein A with from G. and J. The was performed with the and or experiments were performed as described previously (2.Cattaneo M. Otsu M. Fagioli C. Martino S. Lotti L.V. Sitia R. Biunno I. J. Cell. Physiol. 2008; 215: 794-802Crossref PubMed Scopus (35) Google Scholar, L.V. L. C. R. A. L. R. 2002; PubMed Scopus Google Scholar). were with a and was by of on the ER, and and of the areas and membrane were and using a with an system transfected at of were used for was as on cells transfected with pCDNA3.1Myc-Hys(-)A vector were using with at and of SEL1L-B and and sequence we investigated the expression of SEL1L-B and -C in 293 FT human kidney cells and in the human cancer cell KMS11 and of these cell SEL1L-B and -C at SEL1L-A M. Orlandi R. C. P. G. S. Biunno I. Int. J. Biol. PubMed Scopus Google Scholar). In the SEL1L-B and -C signals in PCR with for SEL1L-A. SEL1L-B and -C were in cancer cells to 293 FT in all three SEL1L transcripts were by DTT or that ER stress and activation of the unfolded protein by disulfide and respectively. a well known ER gene M. Y. 2003; PubMed Scopus Google Scholar), was in to both DTT and whereas splicing, was more after DTT The expression of SEL1L-A, and -C was by of SEL1L-A 2 Celbio, Pero, PCR of RNA from SKBr3 cells demonstrated that SEL1L-B and -C from the SEL1L-A M. C. G. L. Scarpa A. M. Biunno I. Cell Biol. 2001; PubMed Scopus Google Scholar) of the that the of SEL1L-B and -C are and to the respectively. that SEL1L-B and -C, at are with SEL1L-A, in cells ER stress. of SEL1L-B and in full-length SEL1L-B and -C generated from peripheral blood lymphocytes were in a Myc-tagged vector and used to transiently we investigated of and to PNGase and endo PNGase all of in the whereas endo of the on ER proteins that not through the demonstrated by the of their and and obtained from of transfected cells were to both PNGase and endo with and at and and The medium of or that were PNGase but endo as in of these that and are secreted glycoproteins. The of and in transfected cells was after of protein with a cycloheximide and of and in cells the of both isoforms, with regard to in the of and secreted proteins were and and after the maintained in was with the more of this to Both isoforms more SEL1L-A, a of 3 (1.Mueller B. Lilley B.N. Ploegh H.L. J. Cell Biol. 2006; 175: 261-270Crossref PubMed Scopus (158) Google Scholar). We investigated of 293 FT transfected with Myc-tagged isoforms, were with and and using in both as a and as a The protein as a both and and with molecular and of residues in disulfide bonds. were obtained using from of and in cells on as demonstrated by lack of splicing and In these and are more the SEL1L-A is more for of and not affect the response; and is engaged in disulfide bonds. of and the subcellular of SEL1L-B and -C in transiently transfected the of Myc-tagged constructs to from SEL1L-A and using and of cellular (ER), and that both and were present in involved in the secretory and degradative as the ER, the and 4, Furthermore, with the peroxisomal was for 4, and of and by to with the subcellular of and in transfected 293 FT localizes on the membrane of the ER identified by on peripheral the membrane and at the of cells of as and and compartments are also with in ER and the are also in in peripheral and on both and intercellular in the of and small identified by is also present in the intercellular and in membrane with peripheral small endoplasmic reticulum; In with that and to the ER and to the with A and However, whereas was associated with membrane profiles, was mainly as of with the typical of were also together with identified by were with In both isoforms were present the membrane in small mainly in areas of cell-cell and at H, and was also in the of in intercellular and in with the identified by The peroxisomal localization was in cells transfected with a deleted of the C-terminal peroxisomal signal the subcellular of and a of the in subcellular compartments was in the and their to the ER for to in the that both and localize in secretory and degradative compartments and suggest that these isoforms, in to participate in other as and cell-cell SEL1L with was recently reported that SEL1L-A unassembled Ig-μs and their in with HRD1 (2.Cattaneo M. Otsu M. Fagioli C. Martino S. Lotti L.V. Sitia R. Biunno I. J. Cell. Physiol. 2008; 215: 794-802Crossref PubMed Scopus (35) Google Scholar). Here Myc-tagged constructs were used to of μs by and by a mutant with glutamic acid to glycine substitution at position 312 in the fourth sel-1-like repeat and by deleted of the peroxisomal domain of 293 FT with μs in with of the Myc-tagged were with and by using in of orphan substrate were by both wild-type and and The of signals in single the of the We the of μs with or with a driving the expression of the SEL1L-A amino acid residues 20–372, with a KDEL signal kindly provided by Dr. J.C. Shaler T.A. Tyler R.E. Kopito R. Nat. Cell Biol. 2008; 10: 272-282Crossref PubMed Scopus (397) Google Scholar). constructs were used because a secreted whereas a signal from and an region of 48 is in the 293 FT cells or in with or were treated with Although not affect substrate with In and are to μs without substrate of μs in the of SEL1L and are secreted and we investigated these isoforms were in the of orphan this we and to other SEL1L variants that are secreted or and with or without in μs in the culture medium in the of the SEL1L and the SEL1L-A J.C. Shaler T.A. Tyler R.E. Kopito R. Nat. Cell Biol. 2008; 10: 272-282Crossref PubMed Scopus (397) Google Scholar). The of the of μs μs as a at and were endo and PNGase to in of of unassembled substrate on the response, as by splicing and activation We investigated and be involved in the of two other well characterized ERAD substrates, the null Hong Kong variant of α1-antitrypsin a known SEL1L-A substrate J.C. Shaler T.A. Tyler R.E. Kopito R. Nat. Cell Biol. 2008; 10: 272-282Crossref PubMed Scopus (397) Google Scholar), and mutant α1-AT 2004; PubMed Scopus Google Scholar). of and was not in cells with these secretory SEL1L constructs these that and, more prominently, of a specific ERAD unassembled Ig-μs chains, but that this not affect and do not affect of two other structurally ERAD substrates, and In this study we SEL1L-B and -C, variants of the SEL1L-A a component of the ERAD that as a substrate recognition by and (1.Mueller B. Lilley B.N. Ploegh H.L. J. Cell Biol. 2006; 175: 261-270Crossref PubMed Scopus (158) Google Scholar, 2.Cattaneo M. Otsu M. Fagioli C. Martino S. Lotti L.V. Sitia R. Biunno I. J. Cell. Physiol. 2008; 215: 794-802Crossref PubMed Scopus (35) Google Scholar, 3.Christianson J.C. Shaler T.A. Tyler R.E. Kopito R. Nat. Cell Biol. 2008; 10: 272-282Crossref PubMed Scopus (397) Google Scholar, Z. Wolf D.H. EMBO J. 2003; 22: 2309-2317Crossref PubMed Scopus (363) Google Scholar). It was previously reported that the SEL1L-A is by the through the M. S. Y. K. Y. Y. PubMed Scopus Google Scholar). Here we show that SEL1L-B and -C are with SEL1L-A at in cells ER stress with UPR, is with the from a ER M. C. G. L. Scarpa A. M. Biunno I. Cell Biol. 2001; PubMed Scopus Google Scholar). In we that the of the these SEL1L isoforms, their cellular the other of the SEL1L isoforms not affect of the SEL1L-A transmembrane region into the ER membrane for both the SEL1L-B and -C the secretory of these SEL1L-B and -C structurally in the of sel-1-like repeats and in the C-terminal characterized by a nine amino tail in SEL1L-B and by a peroxisomal domain in new functional properties and to these Using transient transfection experiments on Myc-tagged constructs combined with several biochemical tests we show that SEL1L-B and -C are secreted and that is more for the SEL1L-C isoform. the other biochemical of disulfide that SEL1L-B to multi-protein both cells and secretion. The that this is not by SEL1L-C that the functionally responsible in the sel-1-like repeat of the SEL1L-B an to on as well as on are to the of the of SEL1L-B and their that both SEL1L-B and -C localize in secretory and degradative However, the SEL1L-B with membrane profiles, in the ER and in be with to membrane-bound the other SEL1L-C is the ER and the typical of soluble The of the two isoforms in the intercellular membrane peripheral and intercellular a in Furthermore, the localization of both isoforms in and compartments be with in sorting and/or degradation or secretion. In this regard a peripheral system involved in the recognition and degradation of structurally proteins that ER has been described M. C. M. J. K. B. N. J. Cell Biol. 2004; Scopus (279) Google Scholar). SEL1L-C, the specific peroxisomal localization a in peroxisomal a that with ERAD in the protein targeting system R. PubMed Scopus Google Scholar). In of the as and an ubiquitin domain to an domain and is to the ERAD and an that from repeats to sel-1-like The in the subcellular of the two isoforms the of SEL1L-B with membrane the intralumenal of to the of localization that are not to of Myc-tagged constructs after show that, SEL1L-A, SEL1L-B and -C do not participate to the degradation of unassembled involved in their secretion. secretory is in SEL1L-B and in the mutant with deleted peroxisomal domain, substrate The secretory by on unassembled substrate be to its to participate to multi-protein disulfide substrate However, of μs is to UPR, that these secreted SEL1L isoforms are to the ER stress induced by unassembled to be specific for because two other ERAD substrates, the null Hong Kong variant of α1-antitrypsin, and mutant α1-AT are not substrate on the sel-1-like In the a secreted sel-1-like repeats, suggest that these repeats be involved in interactions with proteins characterized by J. J. PubMed Scopus Google Scholar). protein SEL1L-B and -C are to as because of their with the protein a secreted sel-1-like L. J. Biol. 2004; Scholar, L. C. 2003; 10: PubMed Scopus Google Scholar). their bacterial also SEL1L isoforms their in to or of misfolded/unfolded or orphan to cells from stress. the localization in secretory and degradative the to unassembled substrate and the to that SEL1L-B and -C contribute to cells by proteins through a by the UPR, has been in the of protein and proteins S. S. S. A. Lewis T.A. Nat. Biol. PubMed Scopus Google Scholar, J. V. R. N. 2006; PubMed Scopus Google Scholar). The of SEL1L isoforms and their associated substrates also through the or by in the in a range of are to on of the various SEL1L In to SEL1L-A, small protein that several with the SEL1L isoforms described molecular and was in cells not of this new is by ER stress. of ER protein expression at several as or has been with of as protein disulfide and are secreted by cancer cells and are in the serum of cancer N. S. J. C. S. L. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, C. S.M. L. Cell 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). are to the of SEL1L-B and -C to their in and their as the reported to the of novel parallel in cells to We Drs. J. C. L. R. and R. Sitia for with