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A Prochiantz

Publications and source records attributed to A Prochiantz.

At least 19 recordsLinked to original sources
PubMed2000

Cell-penetrating peptides.

The established view in cellular biology dictates that the cellular internalization of hydrophilic macromolecules can only be achieved through the classical endocytosis pathway. However, in the past five years several peptides have been demonstrated to translocate across the plasma membrane of eukaryotic cells by a seemingly energy-independent pathway. These peptides have been used successfully for the intracellular delivery of macromolecules with molecular weights several times greater than their own. Cellular delivery using these cell-penetrating peptides offers several advantages over conventional techniques because it is efficient for a range of cell types, can be applied to cells en masse and has a potential therapeutic application.

Amino Acid Sequence↗
PubMed1999

The amyloid precursor protein interacts with Go heterotrimeric protein within a cell compartment specialized in signal transduction.

The function of the beta-amyloid protein precursor (betaAPP), a transmembrane molecule involved in Alzheimer pathologies, is poorly understood. We recently reported the presence of a fraction of betaAPP in cholesterol and sphingoglycolipid-enriched microdomains (CSEM), a caveolae-like compartment specialized in signal transduction. To investigate whether betaAPP actually interferes with cell signaling, we reexamined the interaction between betaAPP and Go GTPase. In strong contrast with results obtained with reconstituted phospholipid vesicles (Okamoto et al., 1995), we find that incubating total neuronal membranes with 22C11, an antibody that recognizes an N-terminal betaAPP epitope, reduces high-affinity Go GTPase activity. This inhibition is specific of Galphao and is reproduced, in the absence of 22C11, by the addition of the betaAPP C-terminal domain but not by two distinct mutated betaAPP C-terminal domains that do not bind Galphao. This inhibition of Galphao GTPase activity by either 22C11 or wild-type betaAPP cytoplasmic domain suggests that intracellular interactions between betaAPP and Galphao could be regulated by extracellular signals. To verify whether this interaction is preserved in CSEM, we first used biochemical, immunocytochemical, and ultrastructural techniques to unambiguously confirm the colocalization of Galphao and betaAPP in CSEM. We show that inhibition of basal Galphao GTPase activity also occurs within CSEM and correlates with the coimmunoprecipitation of Galphao and betaAPP. The regulation of Galphao GTPase activity by betaAPP in a compartment specialized in signaling may have important consequences for our understanding of the physiopathological functions of betaAPP.

Amino Acid Sequence↗
PubMed1999

Regulation of epidermal bullous pemphigoid antigen 1 (BPAG1) synthesis by homeoprotein transcription factors.

In a recent gene-trap screen, we identified the gene coding for Epidermal Bullous Pemphigoid Antigen 1 (BPAG1) as a putative transcriptional target of Engrailed and of other homeoproteins with a glutamine in position 50 of their homeodomain. We now show that the nuclear addressing of the homeodomains of Engrailed (EnHD) and Antennapedia (AntpHD) upregulates BPAG1e transcription in immortalized human keratinocytes (GMA24FIA) expressing En1. This upregulation is not observed with AntpHD-Q50A, a variant of AntpHD in which a single mutation abolishes its high-affinity binding to target DNA, thus strongly suggesting that BPAG1e upregulation homeodomains reflects their specific recognition of homeoprotein-binding sites in the BPAG1e locus. This is further confirmed by DNase I footprinting and electrophoretic mobility shift assays that reveal, within the cloned BPAG1e promoter, several sites of direct interaction with EnHD and Engrailed. Co-transfection experiments in GMA24FIA human keratinocytes, COS-7 simian fibroblasts, and CHP-100 human neuroepithelial cells show that Engrailed, Hoxa-5, and Hoxc-8 regulate BPAG1e promoter activity and that this regulation is context-dependent. Finally, using a mouse line with LacZ inserted within the En1 locus, we identify the keratinocytes of the ventral paws, including the epithelial cells of the eccrine tubules, as a strong site of En1 expression throughout adulthood. We therefore propose that BPAG1e, a 230 kDa keratin-binding protein expressed in keratinocytes and participating in the maintenance of hemidesmosomes at the dermis-epidermis border, is directly regulated by homeoprotein transcription factors.

Animals↗
PubMed1999

Homeodomain-derived peptides. In and out of the cells.

The internalization of homeodomains and of homeopeptides derived from the third helix of the homeodomain of Antennapedia, a Drosophila transcription factor, is used by some investigators to target exogenous hydrophilic compounds into live cells. In addition to this very practical aspect of drug delivery, translocation across biologic membranes of peptides subsequently addressed to the cell cytoplasm and nucleus raises several questions. A first series of questions pertains to the mechanism of translocation. Thanks to the synthesis of several peptides derived from the third helix of the Antennapedia homeodomain, we began to investigate the mechanism of translocation and we have shown that it is not dependent upon the presence of a chiral receptor and probably involves the formation of inverted micelles. A second series of questions is related to the physiologic significance of the phenomenon. In a first approach, we demonstrated that some full-length homeoproteins are internalized and secreted in vitro. The mechanism of internalization is probably similar to that of the homeodomain or of its third helix, but secretion involves a different mechanism which requires an association with specialized intracellular membranous structures. The existence of specific mechanisms for homeoprotein internalization and secretion suggests that this class of transcription factors may have important signaling properties.

Amino Acid Sequence↗
PubMed1999

A short region of its homeodomain is necessary for engrailed nuclear export and secretion.

Engrailed homeoprotein, a transcription factor involved in midbrain/hindbrain patterning, primarily localizes to the cell nucleus. However, significant amounts of the protein are also found in the cell cytoplasm or associated with membrane microdomains enriched in cholesterol and glycosphingoglycolipids (Joliot, A., Trembleau, A., Raposo, G., Calvet, S., Volovitch, M. and Prochiantz, A. (1997) Development 124, 1865-1875). This non-nuclear localization, observed in vitro and in vivo, led us to investigate the possibility that Engrailed be transferred between nuclear and non-nuclear compartments. Monkey COS-7 cells expressing chick Engrailed-2 (cEN2) were fused with 3T3 mouse fibroblasts and the passage of cEN2 from COS-7 to 3T3 nuclei was followed in the interspecies heterokaryons. We find that, 10 minutes following cell fusion, cEN2 is detected in the 3T3 nuclei of 80% of the heterokaryons demonstrating rapid cEN2 nuclear export. Export from donor nuclei can be saturated and is strongly reduced after deletion of a 11 amino acid-long (&Dgr;)1 sequence present within a slightly larger domain that extends between helices 2 and 3 of the homeodomain and shows strong similarities with leucine-rich nuclear export signals (NES). This putative NES, when fused with a nuclear reporter protein, allows its nuclear export, demonstrating that it is not only necessary but also sufficient for nuclear export and can therefore be considered as a true nuclear export sequence. In an earlier report (Joliot, A., Maizel, A., Rosenberg, D., Trembleau, A., Dupas, S., Volovitch, M. and Prochiantz, A. (1998) Current Biology 8, 856-863), we demonstrated that the (&Dgr;)1 sequence is necessary for the access of cEN2 to the lumen of a membrane compartment and for its intercellular transfer. The present study thus strongly suggests that the regulation of Engrailed nuclear export could play a role not only in Engrailed transcriptional activity but also in its ability to gain access to a secretory compartment.

Amino Acid Sequence↗
PubMed1998

Identification of a signaling pathway activated specifically in the somatodendritic compartment by a heparan sulfate that regulates dendrite growth.

In two earlier reports we demonstrated that natural heparan sulfate, but not dermatan or chondroitin sulfate glycosaminoglycans, stimulate axonal elongation and inhibit dendrite growth in vitro (Lafont et al., 1992). The latter specific effect on dendrite elongation was reproduced by chemically synthesized heparan sulfates and by SR 80037A, a purified sulfated and hexanoylated heparin fragment (Lafont et al., 1994). Adding radioactive SR 80037A to purified neurons demonstrated the existence, at the neuronal surface, of heparan sulfate-specific and saturable binding sites, suggesting that SR 80037A activates specific signal transduction pathways. In the present study, using rat or mouse neurons from the embryonic cortex, we show that SR 80037A signaling involves one or several G-coupled receptor or receptors, small GTPases rhoA and/or rhoC, and one or several PKCs. We also demonstrate that the rapid soma rounding elicited by SR 80037A does not require protein synthesis but that the long-term effect on dendrite initiation requires protein synthesis in a short period after the addition of the heparan sulfate. Finally, by preparing membranes from the somatodendritic or axonal compartments we demonstrate that the identified signaling pathway is activated by SR 80037A primarily in the somatodendritic compartment and is not sensitive to the addition of a dermatan sulfate glycosaminoglycan that does not induce the axonal phenotype by impairing dendrite initiation and elongation.

Adenosine Diphosphate↗
PubMed1998

Identification of a signal sequence necessary for the unconventional secretion of Engrailed homeoprotein.

BACKGROUND: Engrailed-1 and Engrailed-2 are homeoproteins--transcription factors implicated in the morphogenesis of discrete structures. Engrailed proteins have a role in patterning the midbrain-hindbrain region and are expressed in the nuclei of rat embryo midbrain-hindbrain cells. We have previously found that both endogenous and exogenously expressed Engrailed proteins also associate with membrane regions implicated in signal transduction and secretion. Within total membrane fractions, a small proportion of Engrailed--about 5%--is protected against proteinase K proteolysis, suggesting that Engrailed has access to a luminal compartment. Together with our finding that homeodomains and homeoproteins can be internalized by live cells, these observations suggest that Engrailed might act as a polypeptidic messenger. In order to investigate this possibility, we looked to see if Engrailed could be secreted. RESULTS: Engrailed expressed in COS cells can be recovered in abutting primary neurons and this is dependent on a short sequence in its homeodomain distinct from 'classical' secretion signals. This sequence, which overlaps with the sequence necessary for Engrailed internalization and which is highly conserved among homeoproteins, is the first example of an 'unconventional' sequence necessary for secretion. Less than 50% of total intracellular Engrailed is secreted and there is a correlation between secretion and access to the membrane compartment where the protein is protected against proteinase K. CONCLUSIONS: Our results lend weight to the proposal that Engrailed, and possibly other homeoproteins, might act as intercellular polypeptidic messengers.

Animals↗
PubMed1998

Trojan peptides: the penetratin system for intracellular delivery.

Internalization of exogenous macromolecules by live cells provides a powerful approach for studying cellular functions. Understanding the mechanism of transfer from the extracellular milieu to the cytoplasm and nucleus could also contribute to the development of new therapeutic approaches. This article summarizes the unexpected properties of penetratins, a class of peptides with translocating properties and capable of carrying hydrophilic compounds across the plasma membrane. This unique system allows direct targeting of oligopeptides and oligonucleotides to the cytoplasm and nucleus, is non-cell-type specific and highly efficient, and therefore has several applications of potential cell-biology and clinical interest.

Amino Acid Sequence↗
PubMed1997

Association of Engrailed homeoproteins with vesicles presenting caveolae-like properties.

We report here that the homeoproteins Engrailed-1 and Engrailed-2 are present in specific non-nuclear subcellular compartments. Using electron microscopy, we observed that chick-Engrailed-2 expressed in COS-7 cells associates with membrane fractions that are characterized as caveolae. This characterization is based on morphological, biochemical and immunological criteria such as, in particular, the absence of clathrin coat and the presence of caveolin and cholera toxin-binding sites. These data are fully confirmed by subcellular fractionation experiments, which demonstrate that transfected chick-Engrailed-2 is present in low density membrane fractions that are resistant to Triton X-100, enriched in caveolin and solubilized by the addition of a cholesterol-binding detergent, a set of properties highly characteristic of caveolae. The association of Engrailed-2 with specific membrane fractions observed after transfection in COS-7 cells is also observed for endogenous Engrailed-1 and Engrailed-2 expressed at late embryonic stages in the cerebellum and posterior mesencephalon of the rodent. Indeed, the two proteins are present in membrane fractions that bear all the characteristics of microdomains or caveolae-like domains, i.e. Triton X-100 resistance, saponin solubilization, low density on sucrose gradients, enrichment in glycosphingolipid GM1, absence of transmembrane Neural Cell Adhesion Molecule, presence of the glypiated (GPI-anchored) glycoprotein F3/F11 and of the acylated growth-associated protein GAP-43. Finally we demonstrate that part of the membrane-associated Engrailed, either expressed in COS-7 cells or endogenously present in neural tissues, is not accessible to proteolytic enzymes unless the membranes have been permeabilized with detergent. This study suggests that, in addition to their well-known presence in the nucleus, Engrailed proteins are also associated with caveolae-like vesicles that are primarily transported anterogradely into the axon, and that they can get access to a compartment compatible with secretion.

Animals↗
PubMed1997

[Morphologic plasticity as a component of cognition].

Two recent developmental genetics studies suggest an important role for transcription factors of the homeodomain protein class in the morphogenesis of cerebral structures and the form and extent of the various domains that constitute the nervous system (Krumlauf, 1994; Rubenstein and Puelles, 1994). Our team recently took a look at the hypothesis that the same transcription parameters have a function in axonal growth and recognition of paths and targets. It has been shown that cortical or spinal medullary neurons are capable of responding, by a change in axonal growth rate, to regulation of the activity of certain transcription factors (Prochiantz and Théodore, 1995). This demonstration was facilitated by a particular property of the DNA binding region of the transcription factors. This property consists in crossing plasmic membranes and addressing cell nuclei directly (Prochiantz, 1996). The homeodomain proteins are expressed not only during development but also in adulthood. It is possible that their continuous expression or reactivation may be associated with the morphological changes in neurons observed in the mature nervous system. The latter hypothesis will be discussed on the basis of our current knowledge of the expression and role of the transcription factors. The authors will draw some conclusions regarding the possibility of considering these nuclear factors as agents of cerebral plasticity and as targets of potential pharmacological value.

Animals↗
PubMed1996

Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent.

We have recently reported that a 16-amino acid long polypeptide corresponding to the third helix of the DNA binding domain (homeodomain) of Antennapedia, a Drosophila transcription factor, is internalized by cells in culture (Derossi, D., Joliot, A. H., Chassaing, G., and Prochiantz, A.(1994) J. Biol. Chem. 269, 10444-10450). The capture of the homeodomain and of its third helix at temperatures below 10 degrees C raised the problem of the mechanism of internalization. The present demonstration, that a reverse helix and a helix composed of D-enantiomers still translocate across biological membranes at 4 and 37 degrees C strongly suggests that the third helix of the homeodomain is internalized by a receptor-independent mechanism. The finding that introducing 1 or 3 prolines in the structure does not hamper internalization also demonstrates that the alpha-helical structure is not necessary. The data presented are compatible with a translocation process based on the establishment of direct interactions with the membrane phospholipids. The third helix of the homeodomain has been used successfully to address biologically active substances to the cytoplasm and nucleus of cells in culture (Théodore, L., Derossi, D., Chassaing, G., Llirbat, B., Kubes, M., Jordan, P., Chneiweiss, H., Godement, P., and Prochiantz, A.(1995) J. Neurosci. 15, 7158-7167). Therefore, in addition to their physiological implications (Prochiantz, A., and Théodore, L.(1995) BioEssays 17, 39-45), the present results open the way to the molecular design of cellular vectors.

Amino Acid Sequence↗
PubMed1996

Introduction of exogenous antigens into the MHC class I processing and presentation pathway by Drosophila antennapedia homeodomain primes cytotoxic T cells in vivo.

The homeodomain of the Antennapedia molecule (AntpHD) spontaneously crosses cellular membranes and can be used to deliver up to 50 additional amino acids to the cytoplasm. We exploited this approach to deliver antigenic peptides to the MHC class I processing and presentation pathway. AntpHD-based fusion peptides expressing the 170-179 HLA-Cw3 CTL epitope (pCw3) were produced in bacteria. Incubation of these fusion peptides with H-2d target cells resulted in efficient delivery to the cytosol as indicated by protease resistance and confocal microscopy. Moreover, this introduction of an exogenous Ag resulted in sensitization of the cell to lysis by a CTL clone specific for the 170-179 HLA-Cw3-derived peptide. Sensitivity of the Ag processing to brefeldin A but not to chloroquine is consistent with the delivery of AntpHD fusion peptides to the conventional class I-associated processing pathway. Immunization of DBA/2 (H-2d) mice with AntpHD pCw3 fusion peptide in the presence of SDS primed H-2Kd-restricted HLA-Cw3-specific CTL. Similar results were obtained with AntpHD fusion peptides expressing the 147-156 influenza nucleoprotein peptide. The strategy outlined in this paper provides a new approach for introducing molecules into the MHC class I Ag-presenting pathway. This approach has clear relevance to the design of synthetic peptide-based vaccines.

Amino Acid Sequence↗
PubMed1996

The contrasting roles of ICE family proteases and interleukin-1beta in apoptosis induced by trophic factor withdrawal and by copper/zinc superoxide dismutase down-regulation.

We compare here the mechanisms of apoptotic death of PC12 cells induced by down-regulation of Cu2+,Zn2+ superoxide dismutase (SOD1) and withdrawal of trophic support (serum/nerve growth factor). Our previous results indicated that the initiating causes of death are different in each paradigm. However, bcl-2 rescues cells in either paradigm, suggesting common downstream elements to the cell death pathway. To determine whether the ICE [interleukin 1beta converting enzyme] family of proteases, which is required for apoptosis on trophic factor withdrawal, is also required for apoptosis induced by oxidative stress, we have developed a novel peptide inhibitor that mimics the common catalytic site of these enzymes and thereby blocks their access to substrates. This differs from the more usual pseudosubstrate approach to enzyme inhibition. Blockade of ICE family proteases by either this inhibitor or by a permeant competitive ICE family antagonist rescues PC12 cells from apoptotic death following apoptosis induced by down-regulation of SOD1, as well as from trophic factor/nerve growth factor deprivation. SOD1 down-regulation results in an increase in interleukin 1beta (IL- 1beta) production by the cells, and cell death under these conditions can be prevented by either blocking antibodies against IL-1beta or the IL-1 receptor antagonist (IL-1Ralpha). In contrast, trophic factor withdrawal does not increase IL-1beta secretion, and the blocking antibody failed to protect PC12 cells from trophic factor withdrawal, whereas the receptor antagonist was only partially protective at very high concentrations. There were substantial differences in the concentrations of pseudosubstrate inhibitors which rescued cells from SOD1 down-regulation and trophic factor deprivation. These results suggest the involvement of different members of the ICE family, different substrates, or both in the two different initiating causes of cell death.

Amino Acid Sequence↗
PubMed1996

Inhibition of FGF-stimulated phosphatidylinositol hydrolysis and neurite outgrowth by a cell-membrane permeable phosphopeptide.

BACKGROUND: Activated receptor tyrosine kinases bind downstream effector molecules with high affinity. Provided that they can be introduced into cells, peptides corresponding to these high-affinity sites should be able to compete for the interaction and thereby inhibit specific signal transduction cascades. The high-affinity binding site for phospholipase C gamma (PLCgamma) on the activated fibroblast growth factor receptor (FGFR) is centred around the tyrosine at position 766 (766Tyr), and peptides corresponding to this site inhibit PLCgamma binding to the receptor in vitro. A 16 amino-acid peptide from the third helix of the Antennapedia homeodomain protein has recently been shown to be able to act as an internalization vector that can deliver other peptides into cells. Here, we have designed a peptide that contains both the internalization sequence and the FGFR high-affinity binding site for PLCgamma, and tested it in cultures of cerebellar neurons for its ability to inhibit the activation of PLCgamma by basic FGF. RESULTS: The peptide containing the FGFR high-affinity binding site for PLCgamma inhibited phospholipid hydrolysis stimulated by basic FGF with a maximal effect at 1 microg ml-1. Phosphorylation of 766Tyr was required for this effect. The phosphorylated peptide had no effect on phospholipid hydrolysis stimulated by platelet-derived growth factor, neurotrophin-3 and bradykinin. The phosphorylated peptide also inhibited neurite outgrowth stimulated by FGF, but had no effect on neurite outgrowth stimulated by agents that activate the FGFR signal transduction cascade downstream from the activation of PLCgamma. CONCLUSIONS: Cell-permeable peptides can be designed that inhibit the function of receptor tyrosine kinases. In this context we have developed a peptide that prevents the FGFR from activating PLCgamma, and have used this peptide to obtain the first direct evidence that activation of PLCgamma is required for the neurite outgrowth response stimulated by basic FGF.

Amino Acid Sequence↗
PubMed1996

Axonal amyloid precursor protein expressed by neurons in vitro is present in a membrane fraction with caveolae-like properties.

In cortical neurons differentiating in vitro, transmembrane amyloid precursor protein (APP) is distributed in two pools. Whereas the first pool is present in all cell compartments, the second pool is highly enriched in the axon and cell body. In an earlier study we demonstrated that this second pool, referred to as axonal-APP (Ax-APP), is present in the vicinity of the plasma membrane and colocalizes only partially with clathrin (Allinquant, B., Moya, K.L., Bouillot, C., and Prochiantz, A. (1994) J. Neurosci. 14, 6842-6854). In this report, using immunocytochemical and fractionation techniques we demonstrate that Ax-APP is present in microdomains enriched in the glypiated glycoprotein F3. The F3/Ax-APP microdomains are resistant to nonionic detergents and sediment at low density on a sucrose gradient. The two latter properties are reminiscent of those of caveolae, a type of plasmalemmal vesicle found in several cell types, but not previously described in the nervous system due to the absence of caveolin in neurons. The presence of Ax-APP in caveolae-like vesicles raises the possibility that APP serves as a transmembrane signaling molecule for GPI-linked glycoproteins. In addition, our data support new hypotheses on the endocytic pathways leading to the production of the amyloidogenic betaA4 peptide.

Amyloid beta-Protein Precursor↗
PubMed1996

Transcription factor hoxa-5 is taken up by cells in culture and conveyed to their nuclei.

Homeoproteins are transcription factors known to be involved in the early patterning of the nervous system and in lineage decisions. While studying a possible role for homeoproteins at later stages of neuronal differentiation, we observed that the Antennapedia homeodomain is internalized by neurons, translocated to their nuclei, and enhances neurite outgrowth. Studies with mutant homeodomains showed that neurite elongation by post-mitotic vertebrate neurons is regulated by homeoproteins. An intriguing possibility suggested by these results, is that full length homeoproteins might be able to translocate through neuronal membranes. We now report that the entire Hoxa-5 homeoprotein is taken up by fibroblasts and neurons in culture and conveyed to their nuclei. Internalization occurs at 4 and 37 degrees C, and at concentrations as low as 10 pM compatible with a physiological mechanism.

Animals↗
PubMed1996

Getting hydrophilic compounds into cells: lessons from homeopeptides.

The homeodomain of Antennapedia, a Drosophila transcription factor, translocates across biological membranes. Within this 60-amino-acid polypeptide, a shorter sequence of 16 amino acids was identified that can be used as an internalization vector for several types of cargo (i.e. other peptides and oligonucleotides) into the cytoplasm and nucleus of all cell types. This article describes our present understanding of this phenomenon and discusses its potential applications in cell biology.

Amino Acid Sequence↗