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Sébastien Wälchli

Publications and source records attributed to Sébastien Wälchli.

14 recordsLinked to original sources
PubMed2006

Transcriptional targeting of small interfering RNAs into cancer cells.

Small interfering RNAs (siRNAs) are widely used for analyzing gene function and have the potential to be developed into human therapeutics. However, persistent siRNA expression in normal cells may cause toxic side effects. Therefore, the therapeutic applications of RNAi in cancer require either the specific delivery of synthetic siRNAs into cancer cells or the control of siRNA expression. Accordingly, we have developed a cancer-specific vector that expresses siRNAs from the human survivin promoter. A plasmid vector expressing siRNAs under this promoter enabled efficient gene silencing of gene expression in different cancer cell lines. The levels of inhibition were comparable to that obtained with the constitutively active U6 promoter. By contrast to U6 promoter, no significant gene silencing was obtained with the Survivin promoter in normal mammary epithelial cells. Collectively, these data indicate that the survivin promoter is suitable for directing siRNA expression in cancer cells, but not normal cells.

Cell Line, Tumor↗
PubMed2006

The ESCRT-III subunit hVps24 is required for degradation but not silencing of the epidermal growth factor receptor.

The endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are thought to mediate the biogenesis of multivesicular endosomes (MVEs) and endosomal sorting of ubiquitinated membrane proteins. Here, we have compared the importance of the ESCRT-I subunit tumor susceptibility gene 101 (Tsg101) and the ESCRT-III subunit hVps24/CHMP3 for endosomal functions and receptor signaling. Like Tsg101, endogenous hVps24 localized mainly to late endosomes. Depletion of hVps24 by siRNA showed that this ESCRT subunit, like Tsg101, is important for degradation of the epidermal growth factor (EGF) receptor (EGFR) and for transport of the receptor from early endosomes to lysosomes. Surprisingly, however, whereas depletion of Tsg101 caused sustained EGF activation of the mitogen-activated protein kinase pathway, depletion of hVps24 had no such effect. Moreover, depletion of Tsg101 but not of hVps24 caused a major fraction of internalized EGF to accumulate in nonacidified endosomes. Electron microscopy of hVps24-depleted cells showed an accumulation of EGFRs in MVEs that were significantly smaller than those in control cells, probably because of an impaired fusion with lyso-bisphosphatidic acid-positive late endosomes/lysosomes. Together, our results reveal functional differences between ESCRT-I and ESCRT-III in degradative protein trafficking and indicate that degradation of the EGFR is not required for termination of its signaling.

Down-Regulation↗
PubMed2006

EDEM is involved in retrotranslocation of ricin from the endoplasmic reticulum to the cytosol.

The plant toxin ricin is transported retrogradely from the cell surface to the endoplasmic reticulum (ER) from where the enzymatically active part is retrotranslocated to the cytosol, presumably by the same mechanism as used by misfolded proteins. The ER degradation enhancing alpha-mannosidase I-like protein, EDEM, is responsible for directing aberrant proteins for ER-associated protein degradation. In this study, we have investigated whether EDEM is involved in ricin retrotranslocation. Overexpression of EDEM strongly protects against ricin. However, when the interaction between EDEM and misfolded proteins is inhibited by kifunensin, EDEM promotes retrotranslocation of ricin from the ER to the cytosol. Furthermore, puromycin, which inhibits synthesis and thereby transport of proteins into the ER, counteracted the protection seen in EDEM-transfected cells. Coimmunoprecipitation studies revealed that ricin can interact with EDEM and with Sec61alpha, and both kifunensin and puromycin increase these interactions. Importantly, vector-based RNA interference against EDEM, which leads to reduction of the cellular level of EDEM, decreased retrotranslocation of ricin A-chain to the cytosol. In conclusion, our results indicate that EDEM is involved in retrotranslocation of ricin from the ER to the cytosol.

Alkaloids↗
PubMed2006

Transport of ricin from endosomes to the Golgi apparatus is regulated by Rab6A and Rab6A'.

Ricin is transported from early endosomes and/or the recycling compartment to the trans-Golgi network (TGN) and subsequently to the endoplasmic recticulum (ER) before it enters the cytosol and intoxicates cells. We have investigated the role of the Rab6 isoforms in retrograde transport of ricin using both oligo- and vector-based RNAi assays. Ricin transport to the TGN was inhibited by the depletion of Rab6A when the Rab6A messenger RNA (mRNA) levels were reduced by more than 40% and less than 75%. However, when Rab6A mRNA was reduced by more than 75% and Rab6A' mRNA was simultaneously up-regulated, the inhibition of ricin sulfation was abolished, indicating that the up-regulation of Rab6A' may compensate for the loss of Rab6A function. In addition, we found that a near complete depletion of Rab6A' gave approximately 40% reduction in ricin sulfation. The up-regulation of Rab6A mRNA levels did not seem to compensate for the loss of Rab6A' function. The depletion of both Rab6A and Rab6A' gave a stronger inhibition of ricin sulfation than what was observed knocking down the two isoforms separately. In conclusion, both Rab6A and Rab6A' seem to be involved in the transport of ricin from endosomes to the Golgi apparatus.

Endosomes↗
PubMed2005

Shiga toxin regulates its entry in a Syk-dependent manner.

Shiga toxin (Stx) is composed of an A-moiety that inhibits protein synthesis after translocation into the cytosol, and a B-moiety that binds to Gb3 at the cell surface and mediates endocytosis of the toxin. After endocytosis, Stx is transported retrogradely to the endoplasmic reticulum, and then the A-fragment enters the cytosol. In this study, we have investigated whether toxin-induced signaling is involved in its entry. Stx was found to activate Syk and induce rapid tyrosine phosphorylation of several proteins, one protein being clathrin heavy chain. Toxin-induced clathrin phosphorylation required Syk activity, and in cells overexpressing Syk, a complex containing clathrin and Syk could be demonstrated. Depletion of Syk by small interfering RNA, expression of a dominant negative Syk mutant (Syk KD), or treatment with the Syk inhibitor piceatannol inhibited not only Stx-induced clathrin phosphorylation but also endocytosis of the toxin. Also, Golgi transport of Stx was inhibited under all these conditions. In conclusion, our data suggest that Stx regulates its entry into target cells.

Clathrin Heavy Chains↗
PubMed2005

Reconstitution of active diphtheria toxin based on a hexahistidine tagged version of the B-fragment produced to high yields in bacteria.

Diphtheria toxin consists of an A-fragment that inactivates elongation factor 2 and a B-fragment that both binds to the toxin receptor and mediates translocation of the A-fragment across cellular membranes to the cytosol. Several fragments of the toxin and an inactive version of the holotoxin have been expressed in Escherichia coli, but the B-fragment alone has proven difficult to express. Only low levels of expression have been achieved. We have designed a hexahistidine tagged version of a modified diphtheria toxin B-fragment (DT-BHis) that can be expressed to high levels in E. coli. DT-BHis contains the entire diphtheria toxin B-fragment preceded by an alanine and succeeded by a leucine, a glutamic acid and a hexahistidine tag and could be purified in a single step using nickel-coated agarose beads to 85% homogeneity. DT-BHis bound specifically to the diphtheria toxin receptor and was able to compete out the effect of the wild type diphtheria toxin. Furthermore, DT-BHis was able to form pores in cellular membranes in a manner similar to the wild type B-fragment. The high yield makes DT-BHis a suitable tool in studies of diphtheria toxin interaction with cells or liposomes since functional diphtheria toxin was easily formed upon addition of A-fragment. The reconstituted diphtheria toxin showed toxicity in the same range as the wild type.

Animals↗
PubMed2005

Sap-1/PTPRH activity is regulated by reversible dimerization.

Sap-1/PTPRH, a receptor protein tyrosine phosphatase (RPTP), is a ubiquitously expressed enzyme that is upregulated in human gastrointestinal cancers. Using both chemical cross-linkers and co-immunoprecipitation we show that overexpressed full-length Sap-1 is present as a stable homodimer. Unlike a number of adhesion RPTPs which have tandem catalytic domains that are involved in dimerization, Sap-1 has a single catalytic domain, and we show that this domain is not required for Sap-1 dimerization, which is mediated instead by the large extracellular and transmembrane domains. Exposing cells that express the receptor to a reducing environment reversibly disrupts the Sap-1 dimer, suggesting that cysteine bonds play a role in dimer formation/stabilization. The switch between Sap-1 dimers and monomers is accompanied by an increase in catalytic activity as judged by its capacity to dephosphorylate and activate c-src, which we identify as a novel substrate for this phosphatase.

Animals↗
PubMed2005

The disulfide isomerase Grp58 is a protective factor against prion neurotoxicity.

Prion diseases are transmissible neurodegenerative disorders characterized by extensive neuronal apoptosis and accumulation of misfolded prion protein (PrP(SC)). Recent reports indicate that PrP(SC) induces neuronal apoptosis via activation of the endoplasmic reticulum (ER) stress pathway and activation of the ER resident caspase-12. Here, we investigate the relationship between prion replication and induction of ER stress during different stages of the disease in a murine scrapie model. The first alteration observed consists of the upregulation of the ER chaperone of the glucose-regulated protein Grp58, which was detected during the presymptomatic phase and followed closely the formation of PrP(SC). An increase in Grp58 expression correlated with PrP(SC) accumulation at all stages of the disease in different brain areas, suggesting that this chaperone may play an important role in the cellular response to prion infection. Indeed, in vitro studies using N2a neuroblastoma cells demonstrated that inhibition of Grp58 expression with small interfering RNA led to a significant enhancement of PrP(SC) toxicity. Conversely, overexpression of Grp58 protected cells against PrP(SC) toxicity and decreased the rate of caspase-12 activation. Grp58 and PrP were shown to interact by coimmunoprecipitation, observing a higher interaction in cells infected with scrapie prions. Our data indicate that expression of Grp58 is an early cellular response to prion replication, acting as a neuroprotective factor against prion neurotoxicity. Our findings suggest that targeting Grp58 interaction may have applications for developing novel strategies for treatment and early diagnosis of prion diseases.

Analysis of Variance↗
PubMed2005

Golgi vesiculation induced by cholesterol occurs by a dynamin- and cPLA2-dependent mechanism.

It was recently demonstrated that an increase in the cellular cholesterol level leads to vesiculation of the Golgi apparatus. This vesiculation affects the entire Golgi apparatus and is a reversible process. We have now started to elucidate the mechanism behind this cholesterol-induced vesiculation of the Golgi apparatus. Transient transfection of cells with dominant negative mutant constructs of dynamin 1 and 2 inhibited the vesiculation; expression of dynK44A in HeLa cells stably transfected with this construct had the same effect. However, the vesiculation seems to be independent of clathrin, as cholesterol-induced vesiculation still occurred following knock down of clathrin heavy chain in HeLa cells using RNA interference as well as in BHK cells where expression of antisense to clathrin heavy chain had been induced. Importantly, the cPLA2 inhibitor MAFP and the chelator BAPTA-AM that binds cytosolic Ca2+ inhibited the cholesterol-induced vesiculation, suggesting involvement of a cPLA2 that requires cytosolic Ca2+ for translocation to membranes. Furthermore, in response to an increased cellular cholesterol level, an EGFP-cPLA2 fusion protein translocated to the Golgi apparatus. Thus, our results demonstrate that the cholesterol-induced vesiculation of the Golgi apparatus is mediated by a cPLA2- and dynamin-dependent mechanism.

Animals↗
PubMed2003

Probing protein-tyrosine phosphatase substrate specificity using a phosphotyrosine-containing phage library.

Protein tyrosine phosphatases (PTPs) play important, highly dynamic roles in signaling. Currently about 90 different PTP genes have been described. The enzymes are highly regulated at all levels of expression, and it is becoming increasingly clear that substrate specificity of the PTP catalytic domains proper contributes considerably to PTP functionality. To investigate PTP substrate selectivity, we have set up a procedure to generate phage libraries that presents randomized, phosphotyrosine-containing peptides. Phages that expressed suitable substrates were selected by immobilized, substrate-trapping GST-PTP fusion proteins. After multiple rounds of selection, positive clones were confirmed by SPOT analysis, dephosphorylation by wild-type enzyme, and Km determinations. We have identified distinct consensus substrate motifs for PTP1B, Sap-1, PTP-beta, SHP1, and SHP2. Our results confirm substrate specificity for individual PTPs at the peptide level. Such consensus sequences may be useful both for identifying potential PTP substrates and for the development of peptidomimetic inhibitors.

Amino Acid Sequence↗
PubMed2003

Identification of protein tyrosine phosphatases with specificity for the ligand-activated growth hormone receptor.

Protein tyrosine phosphatases (PTPs) play key roles in switching off tyrosine phosphorylation cascades, such as initiated by cytokine receptors. We have used substrate-trapping mutants of a large set of PTPs to identify members of the PTP family that have substrate specificity for the phosphorylated human GH receptor (GHR) intracellular domain. Among 31 PTPs tested, T cell (TC)-PTP, PTP-beta, PTP1B, stomach cancer-associated PTP 1 (SAP-1), Pyst-2, Meg-2, and PTP-H1 showed specificity for phosphorylated GHR that had been produced by coexpression with a kinase in bacteria. We then used GH-induced, phosphorylated GH receptor, purified from overexpressing mammalian cells, in a Far Western-based approach to test whether these seven PTPs were also capable of recognizing ligand-induced, physiologically phosphorylated GHR. In this assay, only TC-PTP, PTP1B, PTP-H1, and SAP-1 interacted with the mature form of the phosphorylated GHR. In parallel, we show that these PTPs recognize very different subsets of the seven GHR tyrosines that are potentially phosphorylated. Finally, mRNA tissue distribution of these PTPs by RT-PCR analysis and coexpression of the wild-type PTPs to test their ability to dephosphorylate ligand-activated GHR suggest PTP-H1 and PTP1B as potential candidates involved in GHR signaling.

Amino Acid Sequence↗
PubMed2003

Mapping of synergistic components of weakly interacting protein-protein motifs using arrays of paired peptides.

Protein-protein recognition usually involves multiple interactions among different motifs that are scattered over protein surfaces. To identify such weak interactions, we have developed a novel double peptide synthesis (DS) method. This method allows us to map protein-protein interactions that involve two linear dis- continuous components from a polypeptide by the use of spatially addressable synergistic pairs of synthetic peptides. The DS procedure is based on the "SPOT" membrane-bound peptide synthesis technique, but to synthesize a mixture of two peptides, it uses both Fmoc (N-(9-fluorenyl)methoxycarbonyl))-alanine and Alloc-alanine at the first cycle. This allows their selective deprotection by either piperidine or tributyltin/palladium treatment, respectively. Using SPOT DS, we confirmed as a proof of principle that Elk-1 Ser(383) phosphorylation by ERK-2 kinase is stimulated by the presence of the Elk-1-docking domain. SPOT DS can also be used to dissect protein-protein motifs that define phosphatase substrate affinity. Using this technique, we identified three new regions in the insulin receptor that stimulate the dephosphorylation of the receptor by protein-tyrosine phosphatase (PTP) 1B and presumably increase the selectivity of PTP for this substrate. These data demonstrate that the SPOT DS technique allows the identification of non-linear weakly interacting protein motifs, which are an important determinant of protein kinase and phosphatase substrate specificity and of protein-protein interactions in general.

Combinatorial Chemistry Techniques↗
PubMed2002

Protein tyrosine phosphatases as drug targets: PTP1B and beyond.

Protein tyrosine phosphatases (PTPs) control signal transduction pathways and have recently emerged as potential drug targets. Inhibition of individual PTPs can result in the activation of therapeutically relevant kinase cascades. This is particularly useful in cases where disease is associated with hormonal resistance, such as insensitivity to insulin or leptin. Currently, PTP1B is being investigated by a number of companies as a promising target for leptin/insulin mimetics and in the treatment of diabetes and obesity. Since all 90-100 PTPs have been identified in the human genome, the challenge now is to identify the function of these enzymes and the therapeutic indications that may exist for specific PTP inhibitors.

Capillary Permeability↗