Bertrand Kleizen

Introduction of an N-Glycosylation Site Increases Secretion of Heterologous Proteins in Yeasts

ABSTRACT Saccharomyces cerevisiae is often used to produce heterologous proteins that are preferentially secreted to increase economic feasibility. We used N-glycosylation as a tool to enhance protein secretion. Secretion of cutinase, a lipase, and llama VHH antibody fragments by S. cerevisiae orPichia pastoris improved following the introduction of an N-glycosylation site. When we introduced an N-glycosylation consensus sequence in the N-terminal region of a hydrophobic cutinase, secretion increased fivefold. If an N-glycosylation site was introduced in the C-terminal region, however, secretion increased only 1.8-fold. These results indicate that the use of N glycosylation can significantly enhance heterologous protein secretion.

Regulated trafficking of the CFTR chloride channel.

The cystic fibrosis transmembrane conductance regulator (CFTR), the ABC transporter encoded by the cystic fibrosis gene, is localized in the apical membrane of epithelial cells where it functions as a cyclic AMP-regulated chloride channel and as a regulator of other ion channels and transporters. Whereas a key role of cAMP-dependent phosphorylation in CFTR-channel gating has been firmly established, more recent studies have provided clear evidence for the existence of a second level of cAMP regulation, i.e. the exocytotic recruitment of CFFR to the plasma membrane and its endocytotic retrieval. Regulated trafficking of the CFTR Cl- channel has sofar been demonstrated only in a subset of CFTR-expressing cell types. However, with the introduction of more sensitive methods to measure CFTR cycling and submembrane localization, it might turn out to be a more general phenomenon that could contribute importantly to both the regulation of CFTR-mediated chloride transport itself and to the regulation of other transporters and CFTR-modulated cellular functions. This review aims to summarize the present state of knowledge regarding polarized and regulated CFTR trafficking and endosomal recycling in epithelial cells, to discuss present gaps in our understanding of these processes at the cellular and molecular level, and to consider its possible implications for cystic fibrosis.

The primary folding defect and rescue of ΔF508 CFTR emerge during translation of the mutant domain

In the vast majority of cystic fibrosis (CF) patients, deletion of residue F508 from CFTR is the cause of disease. F508 resides in the first nucleotide binding domain (NBD1) and its absence leads to CFTR misfolding and degradation. We show here that the primary folding defect arises during synthesis, as soon as NBD1 is translated. Introduction of either the I539T or G550E suppressor mutation in NBD1 partially rescues ΔF508 CFTR to the cell surface, but only I539T repaired ΔF508 NBD1. We demonstrated rescue of folding and stability of NBD1 from full-length ΔF508 CFTR expressed in cells to isolated purified domain. The co-translational rescue of ΔF508 NBD1 misfolding in CFTR by I539T advocates this domain as the most important drug target for cystic fibrosis.

Small molecules intercept Notch signaling and the early secretory pathway

Notch signaling has a pivotal role in numerous cell-fate decisions, and its aberrant activity leads to developmental disorders and cancer. To identify molecules that influence Notch signaling, we screened nearly 17,000 compounds using automated microscopy to monitor the trafficking and processing of a ligand-independent Notch-enhanced GFP (eGFP) reporter. Characterization of hits in vitro by biochemical and cellular assays and in vivo using zebrafish led to five validated compounds, four of which induced accumulation of the reporter at the plasma membrane by inhibiting γ-secretase. One compound, the dihydropyridine FLI-06, disrupted the Golgi apparatus in a manner distinct from that of brefeldin A and golgicide A. FLI-06 inhibited general secretion at a step before exit from the endoplasmic reticulum (ER), which was accompanied by a tubule-to-sheet morphological transition of the ER, rendering FLI-06 the first small molecule acting at such an early stage in secretory traffic. These data highlight the power of phenotypic screening to enable investigations of central cellular signaling pathways.

Alteration of protein function by a silent polymorphism linked to tRNA abundance

Synonymous single nucleotide polymorphisms (sSNPs) are considered neutral for protein function, as by definition they exchange only codons, not amino acids. We identified an sSNP that modifies the local translation speed of the cystic fibrosis transmembrane conductance regulator (CFTR), leading to detrimental changes to protein stability and function. This sSNP introduces a codon pairing to a low-abundance tRNA that is particularly rare in human bronchial epithelia, but not in other human tissues, suggesting tissue-specific effects of this sSNP. Up-regulation of the tRNA cognate to the mutated codon counteracts the effects of the sSNP and rescues protein conformation and function. Our results highlight the wide-ranging impact of sSNPs, which invert the programmed local speed of mRNA translation and provide direct evidence for the central role of cellular tRNA levels in mediating the actions of sSNPs in a tissue-specific manner.

Correcting CFTR folding defects by small-molecule correctors to cure cystic fibrosis

HighlightsCombining correctors that correct different folding defects improves efficacy.Complete F508del CFTR rescue likely requires full correction of NBD1.All classes of CF‐causing mutations may benefit from correctors.F508del CFTR can be rescued from the ER by proteostasis regulators and amplifiers.The drug development pipeline contains promising CFTR corrector combinations. &NA; Pharmacological intervention to treat the lethal genetic disease cystic fibrosis has become reality, even for the severe, most common folding mutant F508del CFTR. CFTR defects range from absence of the protein, misfolding that leads to degradation rather than cell‐surface localization (such as F508del), to functional chloride‐channel defects on the cell surface. Corrector and potentiator drugs improve cell‐surface location and channel activity, respectively, and combination therapy of two correctors and a potentiator have shown synergy. Several combinations are in the drug‐development pipeline and although the primary defect is not repaired, rescue levels are reaching those resembling a cure for CF. Combination therapy with correctors may also improve functional CFTR mutants and benefit patients on potentiator therapy.

A Sweet Send-Off

Proteins that take too long to fold are tagged with sugar to stop their failed attempts and remove them from the cells folding compartment. [Also see Report by Xu et al.] One-third of all proteins encoded by the human genome enter the cellular secretory pathway. The first compartment, the endoplasmic reticulum (ER), is specialized for protein folding, where newly synthesized polypeptides are guided by chaperones and folding enzymes to assume a final native state. Quality control is imposed when this process fails—misfolded proteins are retained in the ER and eventually degraded, thereby keeping the cell healthy and free of protein “traffic jams.” For proteins that are glycosylated, triage decisions (and their timing) involve mannosidases and mannose-specific lectins that recognize an N-linked glycan (N-linked glycosylation site in which a nitrogen atom has been attached to an amino acid) on the polypeptide chain (1). On page 978 of this issue, Xu et al. (2) find that the folding of nonglycosylated proteins is terminated by a similar triage mechanism that surprisingly involves a mannose residue. This “O-mannosylation” (a sugar molecule is added to an oxygen atom in serine or threonine) may act as a cells timer to stop the lingering of nonglycosylated proteins that simply take too long to fold and remove them from the secretory pathway.

Deciphering the mode of action of clinically relevant next generation c2 corrector compounds GLPG2737 and GLPG3221

The current therapeutic strategy to repair cystic fibrosis-causing defects in the chloride channel CFTR is to develop novel and better correctors (to improve folding) and potentiators (to improve function). Galapagos- AbbVie identified a novel potentiator GLPG1837 by compound screening on mutant CFTR. YFP-halide efflux assays and single channel measurements showed ∼2.5-fold improvement in channel activity by GLPG1837 compared to VX-770 (ivacaftor/Kalydeco) on G551D CFTR (1, 2). GLPG1837 successfully passed the Phase-2 clinical trials and proved to be the first potentiator after VX-770 to show competitive results on G551D patients. To identify potential differences in the mode of actions of these potentiators we studied their effects on CFTR folding and function. Biochemical radiolabeling experiments showed that mutations in the intracellular loop 2 (ICL2) disrupt domain assembly between TMD1 and NBD2, a late folding event in CFTR, but in most cases do not impair CFTR trafficking towards the cell surface. Protease-susceptibility assays showed that VX-770 improved late TMD1 folding of many ICL2 mutations, but GLPG1837 did not. YFP-halide efflux assays showed that these ICL2 mutants had varying effect on channel function, ranging from wild-type-like to function-defective mutants. GLPG1837 restored function of non-CF gating mutant E267K much better than VX-770. Residue E267 in ICL2 electrostatically interacts with K1060 in ICL4 to promote channel opening (3). This indicates that GLPG1837 is more efficient in compensating for this lost interaction. Altogether, our biochemical and functional data suggests that potentiators VX-770 and GLPG1837 have a different mode of action.