Zsuzsa Bebok

Somatic mosaicism for copy number variation in differentiated human tissues

Two major types of genetic variation are known: single nucleotide polymorphisms (SNPs), and a more recently discovered structural variation, involving changes in copy number (CNVs) of kilobase‐ to megabase‐sized chromosomal segments. It is unknown whether CNVs arise in somatic cells, but it is, however, generally assumed that normal cells are genetically identical. We tested 34 tissue samples from three subjects and, having analyzed for each tissue ≤10–6 of all cells expected in an adult human, we observed at least six CNVs, affecting a single organ or one or more tissues of the same subject. The CNVs ranged from 82 to 176 kb, often encompassing known genes, potentially affecting gene function. Our results indicate that humans are commonly affected by somatic mosaicism for stochastic CNVs, which occur in a substantial fraction of cells. The majority of described CNVs were previously shown to be polymorphic between unrelated subjects, suggesting that some CNVs previously reported as germline might represent somatic events, since in most studies of this kind, only one tissue is typically examined and analysis of parents for the studied subjects is not routinely performed. A considerable number of human phenotypes are a consequence of a somatic process. Thus, our conclusions will be important for the delineation of genetic factors behind these phenotypes. Consequently, biobanks should consider sampling multiple tissues to better address mosaicism in the studies of somatic disorders. Hum Mutat 0,1–7, 2008.

A Clinical Inflammatory Syndrome Attributable to Aerosolized Lipid- DNA Administration in Cystic Fibrosis

Immunologic reactivity to lipid-DNA conjugates has traditionally been viewed as less of an issue than with viral vectors. We performed a dose escalation safety trial of aerosolized cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the lower airways of eight adult cystic fibrosis patients, and monitored expression by RT-PCR. The cDNA was complexed to a cationic lipid amphiphile (GL-67) consisting of a cholesterol anchor linked to a spermine head group. CFTR transgene was detected in three patients at 2-7 days after gene administration. Four of the eight patients developed a pronounced clinical syndrome of fever (maximum of 103.3EF), myalgias, and arthralgia beginning within 6 hr of gene administration. Serum IL-6 but not levels of IL-8, IL-1, TNF-alpha, or IFN-gamma became elevated within 1-3 hr of gene administration. No antibodies to the cationic liposome or plasmid DNA were detected. We found that plasmid DNA by itself elicited minimal proliferation of peripheral blood mononuclear cells taken from study patients, but led to brisk immune cell proliferation when complexed to a cationic lipid. Lipid and DNA were synergistic in causing this response. Cellular proliferation was also seen with eukaryotic DNA, suggesting that at least part of the immunologic response to lipid-DNA conjugates is independent of unmethylated (E. coli-derived) CpG sequences that have previously been associated with innate inflammatory changes in the lung.

Aminoglycoside suppression of a premature stop mutation in a Cftr–/– mouse carrying a human CFTR-G542X transgene

Abstract. Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Since ~5% of all mutant CF alleles are stop mutations, it can be calculated that ~10% of CF patients carry a premature stop mutation in at least one copy of the CFTR gene. Certain ethnic groups, such as the Ashkenazi Jewish population, carry a much higher percentage of CF stop mutations. Consequently, a therapeutic strategy aimed at suppressing this class of mutation would be highly desirable for the treatment of this common genetic disease. We have shown previously that aminoglycoside antibiotics can suppress premature stop mutations in the CFTR gene in a bronchial epithelial cell line [Nat Med (1997) 3:1280]. To address whether aminoglycosides can suppress a CFTR premature stop mutation in an animal model, we constructed a transgenic mouse with a null mutation in the endogenous CFTR locus (Cftr–/–) that also expressed a human CFTR-G542X cDNA under control of the intestinal fatty acid binding protein promoter. We then investigated whether the daily administration of the aminoglycoside antibiotics gentamicin or tobramycin could restore the expression of a detectable level of CFTR protein. Immunofluorescence staining of intestinal tissues from Cftr–/–hCFTR-G542X mice revealed that gentamicin treatment resulted in the appearance of hCFTR protein at the apical surface of the glands of treated mice. Weaker staining was also observed in the intestinal glands following tobramycin treatment. Short-circuit current measurements made on intestinal tissues from these mice demonstrated that a significant number of positive cAMP-stimulated transepithelial chloride current measurements could be observed following gentamicin treatment (P=0.008) and a near significant number following tobramycin treatment (P=0.052). When taken together, these results indicate that gentamicin, and to a lesser extent tobramycin, can restore the synthesis of functional hCFTR protein by suppressing the hCFTR-G542X premature stop mutation in vivo.

Failure of cAMP agonists to activate rescued ΔF508 CFTR in CFBE41o– airway epithelial monolayers

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP‐regulated chloride channel. Mutations in the CFTR gene result in cystic fibrosis (CF). The most common mutation, ΔF508, results in endoplasmic reticulum‐associated degradation (ERAD) of CFTR. ΔF508 CFTR has been described as a temperature‐sensitive mutation that can be rescued following growth at 27°C. In order to study the processing and function of wild‐type and rescued ΔF508 CFTR at the cell surface under non‐polarized and polarized conditions, we developed stable cell lines expressing ΔF508 or wild‐type CFTR. CFBE41o– is a human airway epithelial cell line capable of forming high resistance, polarized monolayers when cultured on permeable supports, while HeLa cells are normally grown under non‐polarizing conditions. Immunoprecipitation, cell surface biotinylation, immunofluorescence, and functional assays confirmed the presence of ΔF508 CFTR at the cell surface in both cell lines after incubating the cells for 48 h at 27°C. However, stimulators of wild‐type CFTR such as forskolin, β2‐adrenergic or A2B‐adenosine receptor agonists failed to activate rescued ΔF508 CFTR in CFBE41o– monolayers. Rescued ΔF508 CFTR could be stimulated with genistein independent of pretreatment with cAMP signalling agonists. Interestingly, rescued ΔF508 CFTR in HeLa cells could be efficiently stimulated with either forskolin or genistein to promote Cl– transport. These results indicate that ΔF508 CFTR, when rescued in CFBE41o– human airway epithelial cells, is poorly responsive to signalling pathways known to regulate wild‐type CFTR. Furthermore, the differences in rescue and activation of ΔF508 CFTR in the two cell lines suggest that cell‐type specific differences in ΔF508 CFTR processing are likely to complicate efforts to identify potentiators and/or correctors of the ΔF508 defect.

A synonymous single nucleotide polymorphism in δF508 CFTR alters the secondary structure of the mRNA and the expression of the mutant protein

Recent advances in our understanding of translational dynamics indicate that codon usage and mRNA secondary structure influence translation and protein folding. The most frequent cause of cystic fibrosis (CF) is the deletion of three nucleotides (CTT) from the cystic fibrosis transmembrane conductance regulator (CFTR) gene that includes the last cytosine (C) of isoleucine 507 (Ile507ATC) and the two thymidines (T) of phenylalanine 508 (Phe508TTT) codons. The consequences of the deletion are the loss of phenylalanine at the 508 position of the CFTR protein (ΔF508), a synonymous codon change for isoleucine 507 (Ile507ATT), and protein misfolding. Here we demonstrate that the ΔF508 mutation alters the secondary structure of the CFTR mRNA. Molecular modeling predicts and RNase assays support the presence of two enlarged single stranded loops in the ΔF508 CFTR mRNA in the vicinity of the mutation. The consequence of ΔF508 CFTR mRNA “misfolding” is decreased translational rate. A synonymous single nucleotide variant of the ΔF508 CFTR (Ile507ATC), that could exist naturally if Phe-508 was encoded by TTC, has wild type-like mRNA structure, and enhanced expression levels when compared with native ΔF508 CFTR. Because CFTR folding is predominantly cotranslational, changes in translational dynamics may promote ΔF508 CFTR misfolding. Therefore, we propose that mRNA “misfolding” contributes to ΔF508 CFTR protein misfolding and consequently to the severity of the human ΔF508 phenotype. Our studies suggest that in addition to modifier genes, SNPs may also contribute to the differences observed in the symptoms of various ΔF508 homozygous CF patients.

The Mechanism Underlying Cystic Fibrosis Transmembrane Conductance Regulator Transport from the Endoplasmic Reticulum to the Proteasome Includes Sec61β and a Cytosolic, Deglycosylated Intermediary

Endoplasmic reticulum (ER) degradation pathways can selectively route proteins away from folding and maturation. Both soluble and integral membrane proteins can be targeted from the ER to proteasomal degradation in this fashion. The cystic fibrosis transmembrane conductance regulator (CFTR) is an integral, multidomain membrane protein localized to the apical surface of epithelial cells that functions to facilitate Cl−transport. CFTR was among the first membrane proteins for which a role of the proteasome in ER-related degradation was described. However, the signals that route CFTR to ubiquitination and subsequent degradation are not known. Moreover, limited information is available concerning the subcellular localization of polyubiquitinated CFTR or mechanisms underlying retrograde dislocation of CFTR from the ER membrane to the proteasome either before or after ubiquitination. In the present study, we show that proteasome inhibition with clasto-lactacystin β-lactone (4 μm, 1 h) stabilizes the presence of a deglycosylated CFTR intermediate for up to 5 h without increasing the core glycosylated (band B) form of CFTR. Deglycosylated CFTR is present under the same conditions that result in accumulation of polyubiquitinated CFTR. Moreover, the deglycosylated form of both wild type and ΔF508 CFTR can be found in the cytosolic fraction. Both the level and stability of cytosolic, deglycosylated CFTR are increased by proteasome blockade. During retrograde translocation from the ER to the cytosol, CFTR associates with the Sec61 trimeric complex. Sec61 is the key component of the mammalian co-translational protein translocation system and has been proposed to function as a two way channel that transports proteins both into the ER and back to the cytosol for degradation. We show that the level of the Sec61·CFTR complexes are highest when CFTR degradation proceeds at the greatest rate (approximately 90 min after pulse labeling). Quantities of Sec61·CFTR complexes are also increased by inhibition of the proteasome. Based on these results, we propose a model in which complex membrane proteins such as CFTR are transported through the Sec61 trimeric complex back to the cytosol, escorted by the β subunit of Sec61, and degraded by the proteasome or by other proteolytic systems.

Efficient Intracellular Processing of the Endogenous Cystic Fibrosis Transmembrane Conductance Regulator in Epithelial Cell Lines

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase A-activated chloride channel that resides on the apical surface of epithelial cells. One unusual feature of this protein is that during biogenesis, ∼75% of wild type CFTR is degraded by the endoplasmic reticulum (ER)-associated degradative (ERAD) pathway. Examining the biogenesis and structural instability of the molecule has been technically challenging due to the limited amount of CFTR expressed in epithelia. Consequently, investigators have employed heterologous overexpression systems. Based on recent results that epithelial specific factors regulate both CFTR biogenesis and function, we hypothesized that CFTR biogenesis in endogenous CFTR expressing epithelial cells may be more efficient. To test this, we compared CFTR biogenesis in two epithelial cell lines endogenously expressing CFTR (Calu-3 and T84) with two heterologous expression systems (COS-7 and HeLa). Consistent with previous reports, 20 and 35% of the newly synthesized CFTR were converted to maturely glycosylated CFTR in COS-7 and HeLa cells, respectively. In contrast, CFTR maturation was virtually 100% efficient in Calu-3 and T84 cells. Furthermore, inhibition of the proteasome had no effect on CFTR biogenesis in Calu-3 cells, whereas it stabilized the immature form of CFTR in HeLa cells. Quantitative reverse transcriptase-PCR indicated that CFTR message levels are ∼4-fold lower in Calu-3 than HeLa cells, yet steady-state protein levels are comparable. Our results question the structural instability model of wild type CFTR and indicate that epithelial cells endogenously expressing CFTR efficiently process this protein to post-Golgi compartments.

Enhanced cell-surface stability of rescued DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) by pharmacological chaperones.

Misfolded proteins destined for the cell surface are recognized and degraded by the ERAD [ER (endoplasmic reticulum) associated degradation] pathway. TS (temperature-sensitive) mutants at the permissive temperature escape ERAD and reach the cell surface. In this present paper, we examined a TS mutant of the CFTR [CF (cystic fibrosis) transmembrane conductance regulator], CFTR DeltaF508, and analysed its cell-surface trafficking after rescue [rDeltaF508 (rescued DeltaF508) CFTR]. We show that rDeltaF508 CFTR endocytosis is 6-fold more rapid (approximately 30% per 2.5 min) than WT (wild-type, approximately 5% per 2.5 min) CFTR at 37 degrees C in polarized airway epithelial cells (CFBE41o-). We also investigated rDeltaF508 CFTR endocytosis under two further conditions: in culture at the permissive temperature (27 degrees C) and following treatment with pharmacological chaperones. At low temperature, rDeltaF508 CFTR endocytosis slowed to WT rates (20% per 10 min), indicating that the cell-surface trafficking defect of rDeltaF508 CFTR is TS. Furthermore, rDeltaF508 CFTR is stabilized at the lower temperature; its half-life increases from <2 h at 37 degrees C to >8 h at 27 degrees C. Pharmacological chaperone treatment at 37 degrees C corrected the rDeltaF508 CFTR internalization defect, slowing endocytosis from approximately 30% per 2.5 min to approximately 5% per 2.5 min, and doubled DeltaF508 surface half-life from 2 to 4 h. These effects are DeltaF508 CFTR-specific, as pharmacological chaperones did not affect WT CFTR or transferrin receptor internalization rates. The results indicate that small molecular correctors may reproduce the effect of incubation at the permissive temperature, not only by rescuing DeltaF508 CFTR from ERAD, but also by enhancing its cell-surface stability.

The Unfolded Protein Response (UPR)-activated Transcription Factor X-box-binding Protein 1 (XBP1) Induces MicroRNA-346 Expression That Targets the Human Antigen Peptide Transporter 1 (TAP1) mRNA and Governs Immune Regulatory Genes

Background: The adaptive unfolded protein response (UPR) promotes endoplasmic reticulum (ER) expansion and reduces ER load. Results: UPR-activated XBP1 induces miR-346 expression that targets TAP1. Conclusion: We identify a novel function for XBP1 and an miRNA-mediated pathway for ER load reduction through TAP1. Significance: Novel interventions for protein folding disorders will require an understanding of how microRNAs regulate gene expression during ER stress. To identify endoplasmic reticulum (ER) stress-induced microRNAs (miRNA) that govern ER protein influx during the adaptive phase of unfolded protein response, we performed miRNA microarray profiling and analysis in human airway epithelial cells following ER stress induction using proteasome inhibition or tunicamycin treatment. We identified miR-346 as the most significantly induced miRNA by both classic stressors. miR-346 is encoded within an intron of the glutamate receptor ionotropic delta-1 gene (GRID1), but its ER stress-associated expression is independent of GRID1. We demonstrated that the spliced X-box-binding protein-1 (sXBP1) is necessary and sufficient for ER stress-associated miR-346 induction, revealing a novel role for this unfolded protein response-activated transcription factor. In mRNA profiling arrays, we identified 21 mRNAs that were reduced by both ER stress and miR-346. The target genes of miR-346 regulate immune responses and include the major histocompatibility complex (MHC) class I gene products, interferon-induced genes, and the ER antigen peptide transporter 1 (TAP1). Although most of the repressed mRNAs appear to be indirect targets because they lack specific seeding sites for miR-346, we demonstrate that the human TAP1 mRNA is a direct target of miR-346. The human TAP1 mRNA 3′-UTR contains a 6-mer canonical seeding site for miR-346. Importantly, the ER stress-associated reduction in human TAP1 mRNA and protein levels could be reversed with an miR-346 antagomir. Because TAP function is necessary for proper MHC class I-associated antigen presentation, our results provide a novel mechanistic explanation for reduced MHC class I-associated antigen presentation that was observed during ER stress.

Activation of the Unfolded Protein Response by ΔF508 CFTR

Environmental insults and misfolded proteins cause endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR). The UPR decreases endogenous cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels and protein maturation efficiency. Herein, we investigated the effects of the folding-deficient deltaF508 CFTR on ER stress induction and UPR activation. For these studies, we developed and characterized stable clones of Calu3deltaF cells that express different levels of endogenous wild-type (WT) and recombinant deltaF508 CFTR. We also present a novel RT-PCR-based assay for differential quantification of wild-type CFTR mRNA in the presence of deltaF508 CFTR message. The assay is based on a TaqMan minor groove binding (MGB) probe that recognizes a specific TTT sequence (encoding phenylalanine at position 508 in human CFTR). The MGB probe is extremely specific and sensitive to changes in WT CFTR message levels. In RNA samples that contain both WT and deltaF508 CFTR mRNAs, measurement of WT CFTR mRNA levels (using the MGB probe) and total CFTR mRNA (using commercial primers) allowed us to calculate deltaF508 CFTR mRNA levels. The results indicate that overexpression of deltaF508 CFTR causes ER stress and activates the UPR. UPR activation precedes a marked decrease in endogenous WT CFTR mRNA expression. Furthermore, polarized airway epithelial cell lines are important tools in cystic fibrosis research, and herein we provide an airway epithelial model to study the biogenesis and function of WT and deltaF508 CFTR expressed within the same cell.