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Secretion of novel SEL1L endogenous variants is promoted by ER stress/UPR via endosomes and shed vesicles in human cancer cells.

Cattaneo M, Lotti LV, Martino S, Alessio M, Conti A, Bachi A, Mariani-Costantini R, Biunno I - PLoS ONE (2011)

Bottom Line: We describe here two novel endogenous variants of the human endoplasmic reticulum (ER) cargo receptor SEL1LA, designated p38 and p28.Biochemical and RNA interference studies in tumorigenic and non-tumorigenic cells indicate that p38 and p28 are N-terminal, ER-anchorless and more stable relative to the canonical transmembrane SEL1LA.P28 is detected only in the poorly differentiated SKBr3 cell line, where it is secreted after ER stress.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biomedical Technologies, National Research Council, Milan, Italy.

ABSTRACT
We describe here two novel endogenous variants of the human endoplasmic reticulum (ER) cargo receptor SEL1LA, designated p38 and p28. Biochemical and RNA interference studies in tumorigenic and non-tumorigenic cells indicate that p38 and p28 are N-terminal, ER-anchorless and more stable relative to the canonical transmembrane SEL1LA. P38 is expressed and constitutively secreted, with increase after ER stress, in the KMS11 myeloma line and in the breast cancer lines MCF7 and SKBr3, but not in the non-tumorigenic breast epithelial MCF10A line. P28 is detected only in the poorly differentiated SKBr3 cell line, where it is secreted after ER stress. Consistently with the presence of p38 and p28 in culture media, morphological studies of SKBr3 and KMS11 cells detect N-terminal SEL1L immunolabeling in secretory/degradative compartments and extracellularly-released membrane vesicles. Our findings suggest that the two new SEL1L variants are engaged in endosomal trafficking and secretion via vesicles, which could contribute to relieve ER stress in tumorigenic cells. P38 and p28 could therefore be relevant as diagnostic markers and/or therapeutic targets in cancer.

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Related in: MedlinePlus

SEL1L and TPD52 immunoprecipitations assays.A. SEL1LA and p28 immunoprecipitation analysis: Left panel: SKBr3 cell lysates (1.4 mg) were immunoprecipitated with monoclonal anti-SEL1L antibody (lane 3), resolved by SDS-PAGE (10%) and probed with monoclonal anti-SEL1L antibody. Lysate aliquots (50 µg, lane 1) were loaded to verify protein expression levels and immunoprecipitation efficiency. Arrows indicate the immunoprecipitated bands, asterisks (*) correspond to heavy and light chains. Absence of signal in controls (lanes 4 and 5) confirms the specificity of the immunoprecipitated bands. Right panel: SKBr3 cell lysates (7.0 mg) were immunoprecipitated with monoclonal anti-SEL1L antibody (lane 4), resolved by SDS-PAGE (10%) and stained with Coomassie brilliant blue. Asterisks (*) correspond to heavy and light chains. The arrow indicates the immunoprecipitated band analyzed by mass spectrometry. Absence of signal in controls (lanes 1 and 3) confirms the specificity of the immunoprecipitated band. B. Analysis of the interaction between SEL1L variants and TPD52: Left panel: SKBr3 cell lysates (1.4 mg) were immunoprecipitated with monoclonal anti-SEL1L antibody (lane 3), resolved by SDS-PAGE (10%) and probed with polyclonal anti-TPD52 antibody. Lysate aliquots (40 µg, lane 4) were loaded to verify protein expression levels and immunoprecipitation efficiency. Lane 5 corresponds to unbound aliquots of the samples loaded in lane 4 (40 µg). The arrow indicates the immunoprecipitated band. Absence of signal in controls (lanes 1 and 2) confirms immunoprecipitation specificity. Right panel: SKBr3 lysates (1.4 mg) were immunoprecipitated with polyclonal anti-TPD52 antibody (lane 3), resolved by SDS-PAGE (10%) and probed with monoclonal anti-SEL1L antibody. Lysate aliquots (40 µg, lane 4) were loaded to verify protein expression levels and immunoprecipitation efficiency. Lane 5 corresponds to unbound aliquots of the samples loaded in lane 3 (40 µg). The membrane was successively re-probed with anti-TPD52 antibody. Arrows indicate the immunoprecipitated bands. Absence of signal in controls (lanes 1 and 2) confirms immunoprecipitation specificity.
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pone-0017206-g006: SEL1L and TPD52 immunoprecipitations assays.A. SEL1LA and p28 immunoprecipitation analysis: Left panel: SKBr3 cell lysates (1.4 mg) were immunoprecipitated with monoclonal anti-SEL1L antibody (lane 3), resolved by SDS-PAGE (10%) and probed with monoclonal anti-SEL1L antibody. Lysate aliquots (50 µg, lane 1) were loaded to verify protein expression levels and immunoprecipitation efficiency. Arrows indicate the immunoprecipitated bands, asterisks (*) correspond to heavy and light chains. Absence of signal in controls (lanes 4 and 5) confirms the specificity of the immunoprecipitated bands. Right panel: SKBr3 cell lysates (7.0 mg) were immunoprecipitated with monoclonal anti-SEL1L antibody (lane 4), resolved by SDS-PAGE (10%) and stained with Coomassie brilliant blue. Asterisks (*) correspond to heavy and light chains. The arrow indicates the immunoprecipitated band analyzed by mass spectrometry. Absence of signal in controls (lanes 1 and 3) confirms the specificity of the immunoprecipitated band. B. Analysis of the interaction between SEL1L variants and TPD52: Left panel: SKBr3 cell lysates (1.4 mg) were immunoprecipitated with monoclonal anti-SEL1L antibody (lane 3), resolved by SDS-PAGE (10%) and probed with polyclonal anti-TPD52 antibody. Lysate aliquots (40 µg, lane 4) were loaded to verify protein expression levels and immunoprecipitation efficiency. Lane 5 corresponds to unbound aliquots of the samples loaded in lane 4 (40 µg). The arrow indicates the immunoprecipitated band. Absence of signal in controls (lanes 1 and 2) confirms immunoprecipitation specificity. Right panel: SKBr3 lysates (1.4 mg) were immunoprecipitated with polyclonal anti-TPD52 antibody (lane 3), resolved by SDS-PAGE (10%) and probed with monoclonal anti-SEL1L antibody. Lysate aliquots (40 µg, lane 4) were loaded to verify protein expression levels and immunoprecipitation efficiency. Lane 5 corresponds to unbound aliquots of the samples loaded in lane 3 (40 µg). The membrane was successively re-probed with anti-TPD52 antibody. Arrows indicate the immunoprecipitated bands. Absence of signal in controls (lanes 1 and 2) confirms immunoprecipitation specificity.

Mentions: To isolate p38 and p28, SKBr3 lysates were immunoprecipitated with monoclonal anti-SEL1L N-terminus, fractionated by SDS-PAGE and either immunoblotted (Figure 6A, left panel) or stained with Coomassie Brillant Blue (Figure 6A, right panel). As shown in Figure 6A, SEL1LA and p28 were immunoprecipitated with different stoichiometric ratios (left panel, lane 3, arrows), but p38, which yielded the most intensely recognized band by immunoblotting, was not recovered in the immunoprecipitates obtained using the same monoclonal antibody. The inability to immunoprecipitate p38 even at small level suggests epitope masking in the native protein, but not in the protein subjected to SDS-PAGE, which could reflect: i. protein-protein interactions; ii. additive post-translational modifications occurring only in the p38 form; iii. nature of the p38 structure. Scaling-up the immunoprecipitations allowed to detect by Coomassie staining a 28 KDa protein band (Figure 6A, right panel, lane 4, arrow), which was subjected to matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Surprisingly, MALDI-TOF MS revealed the presence of peptides pertaining to TPD52 (Table 1), a secreted coiled-coil motif-bearing cancer-associated protein implicated in endosomal trafficking and in secretion via membrane-bound vesicles [51]–[61]. TPD52 was readily detectable in SKBr3 cells (Figure S5A), as expected based on the increased gene copy number reported in this cell line [61]. Blast alignment between the coding sequences of SEL1L and of three alternatively-spliced TPD52 isoforms (accession numbers: P55327-1, P55327-2, P55327-3) did not show similarities (data not shown), the monoclonal anti-SEL1L N-terminus did not appear to recognize the myc/GFP-tagged TPD52 isoform 1 (Figure S5B-C) and, in SKBr3 cells, siRNA silencing of SEL1L did not affect TPD52 protein level (Figure S5D). ER stress/UPR slightly promoted the release of TPD52 in the SKBr3 culture medium (Figure S5E).


Secretion of novel SEL1L endogenous variants is promoted by ER stress/UPR via endosomes and shed vesicles in human cancer cells.

Cattaneo M, Lotti LV, Martino S, Alessio M, Conti A, Bachi A, Mariani-Costantini R, Biunno I - PLoS ONE (2011)

SEL1L and TPD52 immunoprecipitations assays.A. SEL1LA and p28 immunoprecipitation analysis: Left panel: SKBr3 cell lysates (1.4 mg) were immunoprecipitated with monoclonal anti-SEL1L antibody (lane 3), resolved by SDS-PAGE (10%) and probed with monoclonal anti-SEL1L antibody. Lysate aliquots (50 µg, lane 1) were loaded to verify protein expression levels and immunoprecipitation efficiency. Arrows indicate the immunoprecipitated bands, asterisks (*) correspond to heavy and light chains. Absence of signal in controls (lanes 4 and 5) confirms the specificity of the immunoprecipitated bands. Right panel: SKBr3 cell lysates (7.0 mg) were immunoprecipitated with monoclonal anti-SEL1L antibody (lane 4), resolved by SDS-PAGE (10%) and stained with Coomassie brilliant blue. Asterisks (*) correspond to heavy and light chains. The arrow indicates the immunoprecipitated band analyzed by mass spectrometry. Absence of signal in controls (lanes 1 and 3) confirms the specificity of the immunoprecipitated band. B. Analysis of the interaction between SEL1L variants and TPD52: Left panel: SKBr3 cell lysates (1.4 mg) were immunoprecipitated with monoclonal anti-SEL1L antibody (lane 3), resolved by SDS-PAGE (10%) and probed with polyclonal anti-TPD52 antibody. Lysate aliquots (40 µg, lane 4) were loaded to verify protein expression levels and immunoprecipitation efficiency. Lane 5 corresponds to unbound aliquots of the samples loaded in lane 4 (40 µg). The arrow indicates the immunoprecipitated band. Absence of signal in controls (lanes 1 and 2) confirms immunoprecipitation specificity. Right panel: SKBr3 lysates (1.4 mg) were immunoprecipitated with polyclonal anti-TPD52 antibody (lane 3), resolved by SDS-PAGE (10%) and probed with monoclonal anti-SEL1L antibody. Lysate aliquots (40 µg, lane 4) were loaded to verify protein expression levels and immunoprecipitation efficiency. Lane 5 corresponds to unbound aliquots of the samples loaded in lane 3 (40 µg). The membrane was successively re-probed with anti-TPD52 antibody. Arrows indicate the immunoprecipitated bands. Absence of signal in controls (lanes 1 and 2) confirms immunoprecipitation specificity.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040770&req=5

pone-0017206-g006: SEL1L and TPD52 immunoprecipitations assays.A. SEL1LA and p28 immunoprecipitation analysis: Left panel: SKBr3 cell lysates (1.4 mg) were immunoprecipitated with monoclonal anti-SEL1L antibody (lane 3), resolved by SDS-PAGE (10%) and probed with monoclonal anti-SEL1L antibody. Lysate aliquots (50 µg, lane 1) were loaded to verify protein expression levels and immunoprecipitation efficiency. Arrows indicate the immunoprecipitated bands, asterisks (*) correspond to heavy and light chains. Absence of signal in controls (lanes 4 and 5) confirms the specificity of the immunoprecipitated bands. Right panel: SKBr3 cell lysates (7.0 mg) were immunoprecipitated with monoclonal anti-SEL1L antibody (lane 4), resolved by SDS-PAGE (10%) and stained with Coomassie brilliant blue. Asterisks (*) correspond to heavy and light chains. The arrow indicates the immunoprecipitated band analyzed by mass spectrometry. Absence of signal in controls (lanes 1 and 3) confirms the specificity of the immunoprecipitated band. B. Analysis of the interaction between SEL1L variants and TPD52: Left panel: SKBr3 cell lysates (1.4 mg) were immunoprecipitated with monoclonal anti-SEL1L antibody (lane 3), resolved by SDS-PAGE (10%) and probed with polyclonal anti-TPD52 antibody. Lysate aliquots (40 µg, lane 4) were loaded to verify protein expression levels and immunoprecipitation efficiency. Lane 5 corresponds to unbound aliquots of the samples loaded in lane 4 (40 µg). The arrow indicates the immunoprecipitated band. Absence of signal in controls (lanes 1 and 2) confirms immunoprecipitation specificity. Right panel: SKBr3 lysates (1.4 mg) were immunoprecipitated with polyclonal anti-TPD52 antibody (lane 3), resolved by SDS-PAGE (10%) and probed with monoclonal anti-SEL1L antibody. Lysate aliquots (40 µg, lane 4) were loaded to verify protein expression levels and immunoprecipitation efficiency. Lane 5 corresponds to unbound aliquots of the samples loaded in lane 3 (40 µg). The membrane was successively re-probed with anti-TPD52 antibody. Arrows indicate the immunoprecipitated bands. Absence of signal in controls (lanes 1 and 2) confirms immunoprecipitation specificity.
Mentions: To isolate p38 and p28, SKBr3 lysates were immunoprecipitated with monoclonal anti-SEL1L N-terminus, fractionated by SDS-PAGE and either immunoblotted (Figure 6A, left panel) or stained with Coomassie Brillant Blue (Figure 6A, right panel). As shown in Figure 6A, SEL1LA and p28 were immunoprecipitated with different stoichiometric ratios (left panel, lane 3, arrows), but p38, which yielded the most intensely recognized band by immunoblotting, was not recovered in the immunoprecipitates obtained using the same monoclonal antibody. The inability to immunoprecipitate p38 even at small level suggests epitope masking in the native protein, but not in the protein subjected to SDS-PAGE, which could reflect: i. protein-protein interactions; ii. additive post-translational modifications occurring only in the p38 form; iii. nature of the p38 structure. Scaling-up the immunoprecipitations allowed to detect by Coomassie staining a 28 KDa protein band (Figure 6A, right panel, lane 4, arrow), which was subjected to matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Surprisingly, MALDI-TOF MS revealed the presence of peptides pertaining to TPD52 (Table 1), a secreted coiled-coil motif-bearing cancer-associated protein implicated in endosomal trafficking and in secretion via membrane-bound vesicles [51]–[61]. TPD52 was readily detectable in SKBr3 cells (Figure S5A), as expected based on the increased gene copy number reported in this cell line [61]. Blast alignment between the coding sequences of SEL1L and of three alternatively-spliced TPD52 isoforms (accession numbers: P55327-1, P55327-2, P55327-3) did not show similarities (data not shown), the monoclonal anti-SEL1L N-terminus did not appear to recognize the myc/GFP-tagged TPD52 isoform 1 (Figure S5B-C) and, in SKBr3 cells, siRNA silencing of SEL1L did not affect TPD52 protein level (Figure S5D). ER stress/UPR slightly promoted the release of TPD52 in the SKBr3 culture medium (Figure S5E).

Bottom Line: We describe here two novel endogenous variants of the human endoplasmic reticulum (ER) cargo receptor SEL1LA, designated p38 and p28.Biochemical and RNA interference studies in tumorigenic and non-tumorigenic cells indicate that p38 and p28 are N-terminal, ER-anchorless and more stable relative to the canonical transmembrane SEL1LA.P28 is detected only in the poorly differentiated SKBr3 cell line, where it is secreted after ER stress.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biomedical Technologies, National Research Council, Milan, Italy.

ABSTRACT
We describe here two novel endogenous variants of the human endoplasmic reticulum (ER) cargo receptor SEL1LA, designated p38 and p28. Biochemical and RNA interference studies in tumorigenic and non-tumorigenic cells indicate that p38 and p28 are N-terminal, ER-anchorless and more stable relative to the canonical transmembrane SEL1LA. P38 is expressed and constitutively secreted, with increase after ER stress, in the KMS11 myeloma line and in the breast cancer lines MCF7 and SKBr3, but not in the non-tumorigenic breast epithelial MCF10A line. P28 is detected only in the poorly differentiated SKBr3 cell line, where it is secreted after ER stress. Consistently with the presence of p38 and p28 in culture media, morphological studies of SKBr3 and KMS11 cells detect N-terminal SEL1L immunolabeling in secretory/degradative compartments and extracellularly-released membrane vesicles. Our findings suggest that the two new SEL1L variants are engaged in endosomal trafficking and secretion via vesicles, which could contribute to relieve ER stress in tumorigenic cells. P38 and p28 could therefore be relevant as diagnostic markers and/or therapeutic targets in cancer.

Show MeSH
Related in: MedlinePlus