Degradation of Human Thyroperoxidase in the Endoplasmic Reticulum Involves Two Different Pathways Depending on the Folding State of the Protein

Abstract

Human thyroperoxidase (hTPO), a type I transmembrane glycoprotein, plays a key role in thyroid hormone synthesis. In a previous paper (Fayadat, L., Niccoli, P., Lanet, J., and Franc, J. L. (1998) Endocrinology 139, 4277–4285) we established that after the synthesis, only 15–20% of the hTPO molecules were recognized by a monoclonal antibody (mAb15) directed against a conformational structure and that only 2% were able to reach the cell surface. In the present study using pulse-chase experiments in the presence or absence of protease inhibitors followed by immunoprecipitation procedures with monoclonal antibodies recognizing unfolded or partially folded hTPO forms we show that: (i) unfolded hTPO forms are degraded by the proteasome and (ii) partially folded hTPO forms are degraded by other proteases. It was also established upon incubating endoplasmic reticulum (ER) membranes in vitrothat the degradation of the partially folded hTPO was carried out by serine and cysteine integral ER membrane proteases. These data provide valuable insights into the quality control mechanisms whereby the cells get rid of misfolded or unfolded proteins. Moreover, this is the first study describing a protein degradation process involving two distinct degradation pathways (proteasome and ER cysteine/serine proteases) at the ER level, depending on the folding state of the protein. Human thyroperoxidase (hTPO), a type I transmembrane glycoprotein, plays a key role in thyroid hormone synthesis. In a previous paper (Fayadat, L., Niccoli, P., Lanet, J., and Franc, J. L. (1998) Endocrinology 139, 4277–4285) we established that after the synthesis, only 15–20% of the hTPO molecules were recognized by a monoclonal antibody (mAb15) directed against a conformational structure and that only 2% were able to reach the cell surface. In the present study using pulse-chase experiments in the presence or absence of protease inhibitors followed by immunoprecipitation procedures with monoclonal antibodies recognizing unfolded or partially folded hTPO forms we show that: (i) unfolded hTPO forms are degraded by the proteasome and (ii) partially folded hTPO forms are degraded by other proteases. It was also established upon incubating endoplasmic reticulum (ER) membranes in vitrothat the degradation of the partially folded hTPO was carried out by serine and cysteine integral ER membrane proteases. These data provide valuable insights into the quality control mechanisms whereby the cells get rid of misfolded or unfolded proteins. Moreover, this is the first study describing a protein degradation process involving two distinct degradation pathways (proteasome and ER cysteine/serine proteases) at the ER level, depending on the folding state of the protein. thyroperoxidase human thyroperoxidase endoplasmic reticulum Chinese hamster ovary monoclonal antibody carbobenzoxy-l-leucyl-l-leucyl-l-leucinal N-acetyl-Leu-Leu-norleucinal l-1-tosylamido-2-phenylethyl chloromethyl ketone (2S,3S)-trans-epoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester fetal bovine serum phosphate-buffered saline polyacrylamide gel electrophoresis Expre35S35S protein labeling mix Thyroperoxidase (TPO)1is a type I transmembrane heme containing glycoprotein that plays a key role in thyroid hormone synthesis. Although TPO catalyzes both thyroglobulin iodination and hormone synthesis at the apical cell surface of thyroid cells, this enzyme is located mainly in the endoplasmic reticulum (ER) and the perinuclear membrane, and only a small proportion is present at the apical surface (reviewed in Ref. 1.Eckholm R. Mol. Cell. Endocrinol. 1981; 24: 141-163Crossref PubMed Scopus (99) Google Scholar). Kuliawat et al. (2.Kuliawat R. Lisanti M. Arvan P. J. Biol. Chem. 1995; 270: 2478-2482Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and Penel et al. (3.Penel C. Gruffat D. Alquier C. Benoliel A.M. Chabaud O. J. Cell. Physiol. 1998; 174: 160-169Crossref PubMed Scopus (20) Google Scholar) report that no more than 30% of the immunoprecipitated TPO was detected at the cell surface of porcine thyrocytes cultured on porous filters. In a previous study (4.Fayadat L. Niccoli-Sire P. Lanet J. Franc J.L. Endocrinology. 1998; 139: 4277-4285Crossref PubMed Scopus (44) Google Scholar), we proposed a model for the folding, degradation, and intracellular trafficking of human TPO (hTPO) expressed in CHO cells. hTPO was stably expressed in the CHO cell line, and its folding was studied with two monoclonal antibodies: mAb47, which recognizes a linear epitope, and mAb15, which recognizes a conformational epitope present in the mature protein. The results showed that only 15–20% of the hTPO molecules were able to acquire a suitable conformation to be recognized by mAb15 (4.Fayadat L. Niccoli-Sire P. Lanet J. Franc J.L. Endocrinology. 1998; 139: 4277-4285Crossref PubMed Scopus (44) Google Scholar). On the other hand, only a fairly small proportion (∼15%) of the latter were able to reach the plasma membrane. These data indicated that after being synthesized, only 2% of the hTPO molecules were able to exit from the ER and reach the cell surface. In the steady state, the hTPO recovery rate at the cell surface ranged between 10 and 15%. This firmly suggests that the process involved in the folding and intracellular trafficking of this glycoprotein is the same in both thyroid and CHO cells and that the inefficient maturation of the wild type hTPO is an intrinsic characteristic of this molecule. The speed at which the remaining molecules were degraded varied depending on their folding state. The incorrectly folded hTPO was degraded faster than that recognized by mAb15 (half-time 2 h versus 7 h) (4.Fayadat L. Niccoli-Sire P. Lanet J. Franc J.L. Endocrinology. 1998; 139: 4277-4285Crossref PubMed Scopus (44) Google Scholar). “Quality control” is a term that refers to the process whereby conformational maturation is continuously controlled in the ER (5.Kopito R. Cell. 1997; 88: 427-430Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar). The conformational maturation of nascent polypeptides in the ER is facilitated by transient physical interactions with folding catalysts (protein disulfide isomerase, peptidyl isomerase, etc.) and molecular chaperones (BiP, GRP 94, calnexin, etc.). The ER is the site of entry of proteins into the secretory pathway. As a rule, misfolded proteins, folding intermediates, unassembled subunits, and incompletely assembled oligomers remain in the ER and are ultimately targeted by the quality control apparatus responsible for intracellular degradation. In previous studies, we reported that (i) interactions between hTPO and calnexin and calreticulin are of crucial importance to the folding of hTPO (6.Fayadat L. Siffroi-Fernandez S. Lanet J. Franc J.L. Endocrinology. 2000; 141: 959-966Crossref PubMed Scopus (24) Google Scholar) and (ii) the insertion of the heme is essential for the hTPO to be able to exit from the ER (7.Fayadat L. Niccoli-Sire P. Lanet J. Franc J.L. J. Biol. Chem. 1999; 274: 10533-10538Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). It is generally agreed that any misfolded proteins that fail to fulfill the requirements for being transported out of the ER are delivered to cytoplasmic proteasomes to be degraded (8.Bonifacino J.S. Weissman A.M. Annu. Rev. Cell Dev. Biol. 1998; 14: 19-57Crossref PubMed Scopus (536) Google Scholar), although exactly how the decision to degrade misfolded or unassembled membrane proteins is made has not yet been completely elucidated. There are now some evidence, however, that ER proteases may also be involved in the degradation of proteins (9.Heinemann F.S. Ozols J. Mol. Biol. Cell. 1998; 9: 3445-3453Crossref PubMed Scopus (62) Google Scholar, 10.Loo T.W. Clarke D.M. J. Biol. Chem. 1998; 273: 32373-32376Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 11.Lopez D. Del Val M. J. Immunol. 1997; 159: 5769-5772PubMed Google Scholar). In the present study, we focused on the degradative processes underlying the degradation of hTPO, since the speed at which hTPO molecules are degraded varies depending on their folding state. This is the first time the degradation of a wild type protein has been found to involve two distinct pathways at the ER level, depending on its folding state: the cytosolic proteasome pathway and a pathway involving some ER membrane-bound cysteine/serine proteases. The following materials were supplied by Sigma: MG132, N-acetyl-Leu-Leu-norleucinal (ALLN), cycloheximide, emetine, leupeptin, aprotinin, pepstatin A, phosphoramidon, 3,4-dichloroisocoumarin, l-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), chloroquine, brefeldin A, and dithiothreitol. Clastolactacystin β-lactone and (2S,3S)-transepoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester (E64d) were from Calbiochem-Novabiochem. Phenylmethylsulfonyl fluoride was purchased from Roche Molecular Biochemicals. Penicillin and streptomycin were from Life Technologies, Inc.. Geneticin was from Life Technologies, Inc. Expre35S35S protein labeling mix (referred to as 35S(Met+Cys)) was from NEN Life Science Products. Protein A-Sepharose 4B was from Zymed Laboratories Inc. Laboratories (San Francisco, CA). Monoclonal antibodies (mAb) raised against hTPO were a gift from J. Ruf (U38 INSERM, Marseille, France). As described previously (4.Fayadat L. Niccoli-Sire P. Lanet J. Franc J.L. Endocrinology. 1998; 139: 4277-4285Crossref PubMed Scopus (44) Google Scholar), full-length 3.60-kilobase pair hTPO cDNA (kindly provided by B. Rapoport, Cedar-Sinai Research Institute, Los Angeles, CA) was cloned into the eukaryotic transfer vector pcDNA3 and stably transfected into the CHO cell line. After being selected with Geneticin, cells were subcloned to obtain a clone expressing a high level of hTPO (referred to as hTPO-CHO cells). The cells were cultured in Ham's F-12 medium supplemented with 10% FBS, penicillin (100 IU/ml) and streptomycin (100 μg/ml). Cells were preincubated for 2 h in Met- and Cys-free Dulbecco's modified Eagle's medium supplemented with 10% dialyzed FBS and 10 mm sodium butyrate and subsequently pulsed for 30 min in the same culture medium supplemented with 35S(Met+Cys) (10 μCi/ml). After the pulse, the radiolabeling medium was removed, the cell surface was washed twice with 1 ml of Ham's F-12 medium, and cells were then chased for periods ranging between 10 min and 48 h in culture medium supplemented with 5 mm Met and 5 mm Cys. Once the chase was over, cells were kept on ice, washed twice with 2 ml of ice-cold PBS, and scraped off into 600 μl of hTPO-extraction buffer containing 50 mm Tris-HCl, pH 7.4, 0.15 m NaCl, 1% Triton X-100, 0.3% sodium deoxycholate, protease inhibitor mixture, and 100 mmiodoacetamide. The cells were then tumbled for 20 min at 4 °C and centrifuged for 3 min at 10,000 × g. The radiolabeled supernatant obtained was saved and incubated for 2 h at room with mAb15 or mAb47, two directed against These been previously with protein A-Sepharose by incubating at 4 were then by × 10 and washed 4 with 1 ml of hTPO buffer and with 1 ml of The proteins were from the A-Sepharose by for 5 min in the buffer supplemented with and were to hTPO-CHO cells were preincubated for 2 h in Met and Dulbecco's modified Eagle's medium supplemented with 10% dialyzed FBS and 10 mm sodium butyrate and subsequently radiolabeled for 2 h in the same culture medium supplemented with 35S(Met+Cys) (10 μCi/ml). The radiolabeling medium was then removed, and the cell surface was washed twice with ice-cold and scraped off into 2 ml of ice-cold After being centrifuged × 4 the cells were in 2 ml of 10 mm Tris-HCl, pH and incubated for 20 min on were then centrifuged × 5 ER membranes were as described by and L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The was in 1 ml of 10 mm Tris-HCl, pH to a and incubated on for 5 After of a 1 ml of mm in 10 mm Tris-HCl, pH was The cells were with 20 in the 2 ml of was with ml of in 1 mm pH The was with of and and 1 pH were centrifuged for 3 h at 4 °C in an at and ER between and were ER were then washed with 3 of 1 mm for 1 h at in a and in ml of the same The ER were and at ER membranes were on and with ice-cold 10 mm Tris-HCl, pH mm and then incubated at °C for periods of protease inhibitors were to the the of the hTPO buffer × was to the chase The cells were then tumbled for 20 min at 4 °C and centrifuged for 3 min at 10,000 × g. The radiolabeled supernatant obtained was saved and incubated for 2 h at room with mAb15 or mAb47, as previously The proteins were from A-Sepharose by for 5 min in the buffer supplemented with and were to the proteins present in the ER from the integral and proteins of the ER ER as previously indicated were on and with ice-cold mm 10 mm Tris-HCl, pH and then for 3 × 10 on Cells were then centrifuged for 1 h at × and into supernatant and were in mm 10 mm Tris-HCl, pH and then incubated at °C for periods of time with or protease After and were as described between of hTPO molecules in their two were mAb15 and These two were selected a of directed against hTPO J. M. P. Endocrinology. PubMed Scopus Google Scholar). The recognizes a linear epitope R. P. Ruf J. P. B. J. Endocrinol. PubMed Scopus Google Scholar), and has been previously established that this not hTPO this a partially or a folded (4.Fayadat L. Niccoli-Sire P. Lanet J. Franc J.L. Endocrinology. 1998; 139: 4277-4285Crossref PubMed Scopus (44) Google M. P. 1999; PubMed Scopus Google Scholar). The mAb15 recognizes a epitope in the hTPO, which is in of the two of the and is to unfolded hTPO molecules J. M. P. Endocrinology. PubMed Scopus Google Scholar). the mAb15 in immunoprecipitation more than of the hTPO expressed in CHO cells was not were in a pulse-chase the of hTPO recognized by a after the pulse, and no hTPO recognized by mAb15 was immunoprecipitated at this the chase the of hTPO recognized by the that recognized by the mAb15 The of hTPO by mAb15 and by the is not from the results of this or not any between the of hTPO molecules recognized by and then hTPO-CHO cells were for 3 h with and after the the first immunoprecipitation was with mAb15 or mAb47, followed by a using the other of the in which the two were the hTPO immunoprecipitation rate was the same in the of both This that using two is to two distinct of hTPO the unfolded forms recognized by and with the partially completely folded forms recognized by In a previous study (4.Fayadat L. Niccoli-Sire P. Lanet J. Franc J.L. Endocrinology. 1998; 139: 4277-4285Crossref PubMed Scopus (44) Google Scholar), we that a hTPO recognized by mAb15 was not able to exit from the was this not the folded and was not able to the quality control requirements of the The be however, that the present at the cell surface may be to that present in the immunoprecipitation with mAb15 and Cells were incubated for 3 h in Met- and Cys-free medium supplemented with 10% FBS, 10 mm sodium and 10 the of the hTPO was immunoprecipitated with or mAb15 and the supernatant was then immunoprecipitated using mAb15 or were by and the was detected and using a This the from a to the and the to which unfolded recognized by and partially folded hTPO molecules recognized by are degraded and the underlying degradation we pulse-chase on hTPO-CHO cells. the proteasome pathway has been found to the degradation of ER proteins, we focused on the of the proteasome in the degradation of Cells were with or a inhibitor of the with and cycloheximide, the protein synthesis which is also to protein degradation. CHO cells were preincubated for 2 h with or and then chased with or or hTPO was then immunoprecipitated using It was that the unfolded forms recognized by were degraded h) and that the cells with this degradation. results were obtained using not no on the degradation of the pulse-chase experiments to described in 3 a were carried that hTPO was immunoprecipitated at the of the chase using mAb15 3 was that the degradation of the partially folded forms was than that of the forms h as with 3 h) and was to the degradation of the The degradation of the forms not involve the proteasome pathway a pathway by the degradation of the partially folded hTPO forms protein synthesis or the forms were degraded a process that is by cycloheximide, we pulse-chase experiments with protein synthesis emetine, which protein synthesis a The results indicated that the degradation of the forms was by which suggests that the degradation of the forms protein synthesis. were then carried out with a to the degradation involved in degradation and its cells two responsible for the intracellular degradation of the pathway and the pathway. we that partially folded hTPO forms not degradation, we the as to molecules are degraded in the experiments were in which or which the pH level, were to the chase proteases. After the hTPO was immunoprecipitated with mAb15 The that the not the degradation rate of the forms against a being responsible for the degradation of On the for the rate of degradation. a the apparatus was for the intracellular degradation of the forms to be we pulse-chase experiments using brefeldin A, an inhibitor of protein the that a of the proteins into the The that degradation of the forms in the presence of brefeldin that the degradation in a at the ER level not obtain insights into the degradation mechanisms involved in the degradation of the forms and to the of this degradation pulse-chase experiments were carried out in which protease inhibitors were to the chase medium at not the cell The following protease inhibitors were for this leupeptin, aprotinin, pepstatin A, phosphoramidon, and and were after the with 35S(Met+Cys) to the chase medium for 2 h or 48 hTPO was then immunoprecipitated using mAb15 the protease inhibitors (i) leupeptin, aprotinin, and were found to the degradation rate of hTPO immunoprecipitated by mAb15, (ii) pepstatin this degradation for the inhibitors and the inhibitor this degradation at 48 h other inhibitors of cysteine proteases (E64d) and serine proteases no on the degradation rate not this more we pulse-chase experiments using the protease which the degradation of the protease serine protease and cysteine proteases and and which is also to The results previous that the degradation of the forms was by leupeptin, aprotinin, and which suggests that the forms were degraded by cysteine and serine proteases at the ER level ER membrane, or ER that the of leupeptin, aprotinin, and were not since not to any of the of the forms not of leupeptin, aprotinin, and on the degradation of partially folded hTPO hTPO cells were for 30 min with 35S(Met+Cys) and then chased for or with or experiments were in which the same of was to the for for The cells were then scraped off and with the hTPO buffer containing 100 mm was with were by The was detected and with a This the results of an that is of experiments the of the pathway responsible for the degradation of the partially folded hTPO we cell and the ER membrane. After incubating ER we that the degradation of the forms which that the proteases involved were located at the ER membrane level and not in the this degradation process was by and to a by and not by not These data out the that proteasomes to the ER S. R. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) may been responsible for the degradation of the forms in It is however, that the of and were in than in CHO cells 7 the membrane the proteases for the degradation of the partially folded ER membranes were and the ER membranes were by the in degradation of the forms and its rate was in with that in the of the control ER membranes and The degradation of the forms not the presence of proteases. the proteases for the degradation of the partially folded hTPO The results of experiments that some ER membrane-bound cysteine/serine proteases are involved in the degradation of the forms and in It is that a in the forms was in the presence of the periods which may be to the forms being into In a previous study (4.Fayadat L. Niccoli-Sire P. Lanet J. Franc J.L. Endocrinology. 1998; 139: 4277-4285Crossref PubMed Scopus (44) Google Scholar), we described the of two crucial in the folding of the hTPO molecules expressed in CHO cells. 15–20% of the hTPO molecules were able to acquire a suitable structure to be recognized by mAb15, and only 2% were able to reach the cell surface. In the study, we that the rate at which the remaining molecules were degraded varied depending on their folding state The of the hTPO molecules recognized by may been to the of or forms of the as previously found to in the of the and cell molecules S. R. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, S. R. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). the described in the latter two studies, we were to the presence of hTPO molecules not we that the degradation of the unfolded forms was by or MG132, which that molecules degradation we were to or hTPO in the or the not In study (6.Fayadat L. Siffroi-Fernandez S. Lanet J. Franc J.L. Endocrinology. 2000; 141: 959-966Crossref PubMed Scopus (24) Google Scholar), we reported that the of hTPO with calnexin or calreticulin was hTPO was and that this degradation involved the proteasome pathway. The latter data are with the present since that the cells with to a in the proportion of the hTPO molecules that was able to reach the partially folded state and be recognized by In the present study, the degradation of the partially folded forms of hTPO was not by the forms were not degraded by the we the of the proteases involved in their degradation. the of protease inhibitors leupeptin, aprotinin, and the degradation of The degradation of the forms involved cysteine/serine proteases. On the other hand, this degradation process was not by the cells with or brefeldin A, which that the degradation in a that in the of and may been since is a and the cells not be completely to this is a that is to be The results of in carried out with ER membranes indicated that the molecules were degraded and that the degradation was to leupeptin, aprotinin, and These data that the process of in degradation of forms the in in the of the in and in processes are J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). In this protein is degraded by the the in is not by is by cysteine protease experiments with ER membrane showed that a was not for the degradation of the partially folded hTPO forms to These results showed that forms were degraded by some ER membrane-bound cysteine/serine proteases. In the that the degradation of the partially folded forms is by and emetine, two protein synthesis suggests that protein synthesis is for this degradation process to As was to be no on ER in which the of protein synthesis is not is that in the synthesis of the involved in the degradation of forms or that of a to the of this of proteins to degradation been found to be degraded the proteasome pathway (8.Bonifacino J.S. Weissman A.M. Annu. Rev. Cell Dev. Biol. 1998; 14: 19-57Crossref PubMed Scopus (536) Google Scholar), although in of the that a cytosolic protease some of the of the proteasome R. M. J.S. M. 1998; PubMed Scopus Google Scholar, M. M. 1999; PubMed Scopus Google Scholar). On the other has been that (i) is degraded by two pathways in a L. J. Cell Biol. PubMed Scopus (44) Google Scholar), (ii) the degradation pathway of of and is at from that of J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), transmembrane S. 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The term refers to a degradation process that in the ER or in the In this study, was established that depending on its folding state, hTPO is degraded by the cytosolic proteasome pathway or by a degradative involving some membrane-bound cysteine/serine proteases present in the ER This degradation be by the cell to the of proteins and to ER proteins. in data provide valuable insights into the quality control mechanisms whereby the cells get rid of misfolded or unfolded proteins, and to this is the first time a wild type protein has been described that is degraded two distinct degradation pathways and ER cysteine/serine depending on the folding state of the protein. B. for full-length hTPO and J. Ruf for hTPO