[Show abstract][Hide abstract] ABSTRACT: Reliable and long-term expression of transgenes remain significant challenges for gene therapy and biotechnology applications, especially when antibiotic selection procedures are not applicable. In this context, transposons represent attractive gene transfer vectors because of their ability to promote efficient genomic integration in a variety of mammalian cell types. However, expression from genome-integrating vectors may be inhibited by variable gene transcription and/or silencing events. In this study, we assessed whether inclusion of two epigenetic control elements, the human Matrix Attachment Region (MAR) 1-68 and X-29, in a piggyBac transposon vector, may lead to more reliable and efficient expression in CHO cells. We found that addition of the MAR 1-68 at the center of the transposon did not interfere with transposition frequency, and transgene expressing cells could be readily detected from the total cell population without antibiotic selection. Inclusion of the MAR led to higher transgene expression per integrated copy, and reliable expression could be obtained from as few as 2-4 genomic copies of the MAR-containing transposon vector. The MAR X-29-containing transposons was found to mediate elevated expression of therapeutic proteins in polyclonal or monoclonal CHO cell populations using a transposable vector devoid of selection gene. Overall, we conclude that MAR and transposable vectors can be used to improve transgene expression from few genomic transposition events, which may be useful when expression from a low number of integrated transgene copies must be obtained and/or when antibiotic selection cannot be applied.
[Show abstract][Hide abstract] ABSTRACT: Multisubunit protein complexes are assembled in the endoplasmic reticulum (ER). Existing pools of single subunits and assembly intermediates ensure the efficient and rapid formation of complete complexes. While being kinetically beneficial, surplus components must be eliminated to prevent potentially harmful accumulation in the ER. Surplus single chains are cleared by the ubiquitin-proteasome system. However, the fate of not secreted assembly intermediates of multisubunit proteins remains elusive. Here we show by high-resolution double-label confocal immunofluorescence and immunogold electron microscopy that naturally occurring surplus fibrinogen Aα-γ assembly intermediates in HepG2 cells are dislocated together with EDEM1 from the ER to the cytoplasm in ER-derived vesicles not corresponding to COPII-coated vesicles originating from the transitional ER. This route corresponds to the novel ER exit path we have previously identified for EDEM1 (Zuber et al. Proc Natl Acad Sci USA 104:4407-4412, 2007). In the cytoplasm, detergent-insoluble aggregates of fibrinogen Aα-γ dimers develop that are targeted by the selective autophagy cargo receptors p62/SQSTM1 and NBR1. These aggregates are degraded by selective autophagy as directly demonstrated by high-resolution microscopy as well as biochemical analysis and inhibition of autophagy by siRNA and kinase inhibitors. Our findings demonstrate that different pathways exist in parallel for ER-to-cytoplasm dislocation and subsequent proteolytic degradation of large luminal protein complexes and of surplus luminal single-chain proteins. This implies that ER-associated protein degradation (ERAD) has a broader function in ER proteostasis and is not limited to the elimination of misfolded glycoproteins.
No preview · Article · Jan 2013 · Cellular and Molecular Life Sciences CMLS
[Show abstract][Hide abstract] ABSTRACT: The ability to efficiently produce recombinant proteins in a secreted form is highly desirable and cultured mammalian cells such as CHO cells have become the preferred host as they secrete proteins with human-like post-translational modifications. However, attempts to express high levels of particular proteins in CHO cells may consistently result in low yields, even for non-engineered proteins such as immunoglobulins. In this study, we identified the responsible faulty step at the stage of translational arrest, translocation and early processing for such a "difficult-to-express" immunoglobulin, resulting in improper cleavage of the light chain and its precipitation in an insoluble cellular fraction unable to contribute to immunoglobulin assembly. We further show that proper processing and secretion were restored by over-expressing human signal receptor protein SRP14 and other components of the secretion pathway. This allowed the expression of the difficult-to-express protein to high yields, and it also increased the production of an easy-to-express protein. Our results demonstrate that components of the secretory and processing pathways can be limiting, and that engineering of the secretory pathway may be used to improve the secretion efficiency of therapeutic proteins from CHO cells.
Full-text · Article · Jan 2013 · Metabolic Engineering
[Show abstract][Hide abstract] ABSTRACT: Quality control of protein folding represents a fundamental cellular activity. Early steps of protein N-glycosylation involving the removal of three glucose and some specific mannose residues in the endoplasmic reticulum have been recognized as being of importance for protein quality control. Specific oligosaccharide structures resulting from the oligosaccharide processing may represent a glycocode promoting productive protein folding, whereas others may represent glyco-codes for routing not correctly folded proteins for dislocation from the endoplasmic reticulum to the cytosol and subsequent degradation. Although quality control of protein folding is essential for the proper functioning of cells, it is also the basis for protein folding disorders since the recognition and elimination of non-native conformers can result either in loss-of-function or pathological-gain-of-function. The machinery for protein folding control represents a prime example of an intricate interactome present in a single organelle, the endoplasmic reticulum. Here, current views of mechanisms for the recognition and retention leading to productive protein folding or the eventual elimination of misfolded glycoproteins in yeast and mammalian cells are reviewed.
[Show abstract][Hide abstract] ABSTRACT: Little is known about the fate of machinery proteins of the protein quality control and endoplasmic reticulum(ER)-associated degradation (ERAD). We investigated the degradation of the ERAD component EDEM1, which directs overexpressed misfolded glycoproteins to degradation. Endogenous EDEM1 was studied since EDEM1 overexpression not only resulted in inappropriate occurrence throughout the ER but also caused cytotoxic effects. Proteasome inhibitors had no effect on the clearance of endogenous EDEM1 in non-starved cells. However, EDEM1 could be detected by immunocytochemistry in autophagosomes and biochemically in LC3 immuno-purified autophagosomes. Furthermore, influencing the lysosome-autophagy pathway by vinblastine or pepstatin A/E64d and inhibiting autophagosome formation by 3-methyladenine or ATGs short interfering RNA knockdown stabilized EDEM1. Autophagic degradation involved removal of cytosolic Triton X-100-insoluble deglycosylated EDEM1, but not of EDEM1-containing ER cisternae. Our studies demonstrate that endogenous EDEM1 in cells not stressed by the expression of a transgenic misfolded protein reaches the cytosol and is degraded by basal autophagy.
No preview · Article · Apr 2009 · Cellular and Molecular Life Sciences CMLS
[Show abstract][Hide abstract] ABSTRACT: In cells the quality of newly synthesized proteins is monitored in regard to proper folding and correct assembly in the early secretory pathway, the cytosol and the nucleoplasm. Proteins recognized as non-native in the ER will be removed and degraded by a process termed ERAD. ERAD of aberrant proteins is accompanied by various changes of cellular organelles and results in protein folding diseases. This review focuses on how the immunocytochemical labeling and electron microscopic analyses have helped to disclose the in situ subcellular distribution pattern of some of the key machinery proteins of the cellular protein quality control, the organelle changes due to the presence of misfolded proteins, and the efficiency of synthetic chaperones to rescue disease-causing trafficking defects of aberrant proteins.
[Show abstract][Hide abstract] ABSTRACT: Previous studies on the fate of human thyroperoxidase (hTPO) molecules have shown that, after being synthesized, these glycoproteins interact with calnexin and calreticulin and that only some of them are able to acquire a partially folded structure. The aim of the present study was to further investigate the potential role of BiP, another major protein chaperon. Co-immunoprecipitation experiments showed the occurrence of interactions between hTPO and BiP. Pulse-chase studies showed that, when hTPO was expressed in a Chinese hamster ovary cell line overexpressing 5 times more BiP than the parent cells, the rate of hTPO recognized by a monoclonal antibody directed against a conformational structure decreased by 50% after 5 h of chase. Overexpression of the BiP-ATPase mutant G37T also led to a decrease in the correct folding rate of hTPO. When this protein was pulsed in the presence of 35S-(Met + Cys) and the reducing agent dithiotreitol and then chased in a culture medium without dithiothreitol, a 2.5-fold decrease in the correct folding rate was observed in cells overexpressing BiP, whereas co-overexpression of calnexin and Erp57 led to an increase in both the unfolded and partially folded form of hTPO after the pulse step. All of these findings show that BiP and calnexin have opposite effects on the folding behavior of hTPO and that the action of specific molecular chaperones may therefore crucially determine the fate of glycoproteins.
[Show abstract][Hide abstract] ABSTRACT: Human thyroperoxidase (hTPO), the key enzyme involved in thyroid hormone synthesis, is synthesized in the form of a 933-amino acid polypeptide that subsequently undergoes posttranslational modifications such as N- and O-glycosylation and heme fixation. In the present study, it was established that the N-terminal part of hTPO is cleaved during the maturation of the enzyme. In the first set of experiments performed in this study, Chines hamster ovary (CHO) cells transfected with hTPO cDNA generated four different species after deglycosylation, namely a 98-kDa species, which corresponds to the full-length deglycosylated hTPO, and two 94-kDa and one 92-kDa species, which were truncated in the N-terminal parts. The three latter forms were detected only at the cell surface. A proprotein convertase inhibitor prevented these cleavages, and experiments using monensin and brefeldin A showed that they occurred in a post-endoplasmic reticulum compartment. Site-directed mutagenesis studies were performed in which Arg65 was identified as one of the cleavage sites. In the second part of the study, hTPO from human thyroid glands was purified using a monoclonal antibody recognizing the folded form of hTPO. Amino acid determination showed that the N-terminal part of this protein begins at Thr109. This cleavage process differs from that observed in CHO cells. The fact that this hTPO was endoglucosaminidase H-sensitive indicated that the cleavage of the propeptide occurs in the endoplasmic reticulum. To analyze the role of the hTPO prosequence, cDNAs with and without prosequence (Cys15-Lys108) were transfected into CHO cells. hTPO propeptide deletion drastically decreased the proportion of the folded hTPO form, and under these conditions the cell surface activity disappeared completely. These results strongly suggest that the prosequence plays a crucial role as an intramolecular chaperone, facilitating the folding of hTPO.
Full-text · Article · Mar 2005 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: La thyroperoxydase humaine (hTPO) est l’enzyme clé de la biosynthèse des hormones
thyroïdiennes, impliquées dans de nombreux processus biologiques. Cette hémo-
glycoprotéine membranaire de type I est exprimée à la surface apicale des thyrocytes où elle
exerce ses fonctions d’iodation de certains résidus de tyrosine de la thyroglobuline et de
couplage de ces iodotyrosines pour former les hormones thyroïdiennes T3 et T4.
Lors de sa biosynthèse, la hTPO subit des modifications post-transcriptionnelles par
épissage alternatif du précurseur de l’ARNm. Trois isoformes de l’enzyme étaient connues :
la TPO1 (ADNc complet), la TPO2 et la TPO3 engendrées par épissage alternatif des exons
10 et 16 du précurseur de l’ARNm de la TPO. Dans cette thèse, nous avons identifié et
quantifié par PCR, cinq nouvelles isoformes toutes induites par des épissages alternatifs : la
TPO4 (sans exon 14), la TPO5 (sans exon 8), la TPO6 (sans exons 10, 12, 13, 14 et 16), la
TPO2/4 (sans exons 10 et 14), et la TPO2/3 (sans exons 10 et 16). Ces isoformes sont plus ou
moins stables, actives ou non et transportées correctement ou non jusqu’à la membrane
Nous avons également démontré, comme pour d’autres cas de pathologie cancéreuse,
l’augmentation des phénomènes d’épissage alternatif de la hTPO en association avec une
diminution globale du taux d’expression transcriptionnel de la protéine dans les différents
types de cancers thyroïdiens.
La hTPO subit également des modifications co- et post-traductionnelles. Elle interagit
en particulier avec les « protéines chaperons » du réticulum endoplasmique (RE), qui aident
les protéines nouvellement synthétisées à se replier correctement et font partie du “contrôle de
qualité” du RE. On sait que la hTPO est largement retenue au niveau du réticulum
endoplasmique et subissait un processus de dégradation faisant intervenir d’une part le
protéasome et d’autre part des protéases du RE. Le repliement correct de la hTPO nécessite
des interactions avec la calnexine (CNX) et la calreticuline. Dans cette thèse, nous montrons
que la co-surexpression de la CNX et de ERp57, impliquée dans la formation des ponts
disulfures des protéines interagissant avec la CNX, n’augmente pas la proportion des formes
correctement repliées, ce qui suggère l’implication d’autres protéines chaperons et/ou de
catalyseurs de repliement dans le processus de maturation de la hTPO. Nous montrons qu’à
l’inverse de la CNX, l’interaction avec une autre protéine chaperon appelée BiP, diminue le
repliement de la hTPO et entraîne la protéine vers la dégradation, suggérant ainsi que BiP
pourrait être un des senseurs de la dégradation de la hTPO.
Nous avons aussi montré que la thyroperoxydase purifiée à partir de thyroïde humaine
ou exprimée dans les CHO subit un clivage endoprotéolytique dans sa partie N-terminale.
L’enzyme impliquée dans ce clivage appartient vraisemblablement à la famille des protéines
convertases, endoprotéases impliquées dans la maturation de nombreux précurseurs de
pro-récepteurs et glycoprotéines de surface. A l’instar d’une autre protéine de la famille des
peroxydases, la myéloperoxydase humaine, la proséquence de la hTPO agit comme une
protéine chaperon interne en facilitant le repliement correct de la protéine.
Ces résultats éclairent davantage notre connaissance des mécanismes impliqués dans la
maturation de la hTPO et expliquent l’hétérogénéité de la TPO exprimée dans la thyroïde
[Show abstract][Hide abstract] ABSTRACT: The levels of human thyroperoxidase (hTPO) mRNA expression and the rates of hTPO mRNA with alternatively spliced exons 10, 14, and 16 were analyzed in normal, benign, and malignant thyroid tissues (13 normal thyroid tissues, 9 adenomas, 4 papillary carcinomas, 11 follicular variant of papillary carcinomas, 16 minimally invasive follicular carcinomas, 6 widely invasive follicular carcinomas) using a semi-quantitative reverse-transcription polymerase chain reaction procedure. The level of hTPO mRNA decreased in the follicular variant of papillary carcinomas and in minimally invasive follicular carcinomas and was more heterogeneous in the other pathological tissues than in normal tissues. Based on the mean values recorded, the splicing of exons 10 and 16 increased by at least 50% in all the carcinomas, as well as in the benign tissues in the case of exon 10. By contrast, no significant increase was observed in the splicing of exon 14 except in the case of the follicular variant of papillary carcinomas. In conclusion, the results of this study show that the splicing of hTPO increases in benign and malignant thyroid tissues. This event might partly explain the decrease in both the quantity and the level of activity of hTPO observed in thyroid cancer due to the loss of stability of the spliced isoforms. In addition, an increase in the alternative splicing of other mRNAs may contribute to the process of malignancy.
[Show abstract][Hide abstract] ABSTRACT: The human thyroperoxidase (hTPO) gene is composed of 17 exons. The longest complete cDNA sequence determined so far contains a full-length hTPO (TPO1) encoding a 933-amino acid polypeptide. Several mRNA species encoding for hTPO isoforms are present in normal thyroid tissues, including TPO2 with exon 10 deleted and TPOzanelli with exon 16 deleted. In the present study, we established the existence of two new single-spliced transcripts, TPO4 and TPO5, lacking exons 14 and 8, respectively. Upon transfecting the TPO4 cDNA into Chinese hamster ovary cells, it was observed that TPO4 is able to reach the cell surface, is enzymatically active, and is able to be recognized by a panel of 12 monoclonal antibodies directed against hTPO, whereas TPO5 does not fold correctly and is unable to reach the cell surface. In normal tissues, the expression of TPO4 mRNA was examined by performing quantitative reverse transcription PCR. This deleted TPO mRNA amounted to 32 +/- 11% of the total TPO mRNAs. In the same tissues, the TPO2, TPOzanelli, and TPO5 amounted to 35 +/- 12%, 36 +/- 14%, and approximately 10%, respectively. The sum of these four species (not including TPO1) was more than 100%, possibly due to the presence of multispliced mRNAs. This possibility was tested, and three new variants were identified: TPO2/3, lacking exons 10 and 16, TPO2/4, lacking exons 10 and 14, and an unexpected variant, TPO6, corresponding to the deletion of exons 10, 12, 13, 14, and 16. In conclusion, these results indicate the existence of five new transcripts. One of them, TPO4, codes for an enzymatically active protein, whereas TPO5 is unable to fold correctly. The functional significance of the other newly spliced mRNA variants still remains to be elucidated, but these results might help to explain the heterogeneity of the hTPO purified from the thyroid gland.
No preview · Article · Mar 2003 · Journal of Biological Chemistry