Magnesium Status and Stress: The Vicious Circle Concept Revisited.

Magnesium deficiency and stress are both common conditions among the general population, which, over time, can increase the risk of health consequences. Numerous studies, both in pre-clinical and clinical settings, have investigated the interaction of magnesium with key mediators of the physiological stress response, and demonstrated that magnesium plays an inhibitory key role in the regulation and neurotransmission of the normal stress response. Furthermore, low magnesium status has been reported in several studies assessing nutritional aspects in subjects suffering from psychological stress or associated symptoms. This overlap in the results suggests that stress could increase magnesium loss, causing a deficiency; and in turn, magnesium deficiency could enhance the body's susceptibility to stress, resulting in a magnesium and stress vicious circle. This review revisits the magnesium and stress vicious circle concept, first introduced in the early 1990s, in light of recent available data.

Spatio-temporal regulation of EGFR signaling by the Eps15 homology domain-containing protein 3 (EHD3).

The epidermal growth factor (EGF) receptor EGFR is a major receptor tyrosine kinase whose role in gliomagenesis is well established. We have recently identified EHD3 [Eps15 homology (EH) domain-containing protein 3], an endocytic trafficking regulatory protein, as a putative brain tumor suppressor. Here, we investigate the underlying mechanisms, by establishing a novel mechanistic and functional connection between EHD3 and the EGFR signaling pathway. We show that, in response to stimulation with the EGF ligand, EHD3 accelerates the rate of EGFR degradation by dramatically increasing its ubiquitination. As part of this process, EHD3 also regulates EGFR endosomal trafficking by diverting it away from the recycling route into the degradative pathway. Moreover, we found that upon EGF activation, rather than affecting the total MAPK and AKT downstream signaling, EHD3 decreases endosome-based signaling of these two pathways, thus suggesting the contribution of EHD3 in the spatial regulation of EGFR signaling. This function explains the higher sensitivity of EHD3-expressing cells to the growth-inhibitory effects of EGF. In summary, this is the first report supporting a mechanism of EHD3-mediated tumor suppression that involves the attenuation of endosomal signaling of the EGFR oncogene.

Role of the EphB2 receptor in autophagy, apoptosis and invasion in human breast cancer cells.

The Eph and Ephrin proteins, which constitute the largest family of receptor tyrosine kinases, are involved in normal tissue development and cancer progression. Here, we examined the expression and role of the B-type Eph receptor EphB2 in breast cancers. By immunohistochemistry using a progression tissue microarray of human clinical samples, we found EphB2 to be expressed in benign tissues, but strongly increased in cancers particularly in invasive and metastatic carcinomas. Subsequently, we found evidence that EphB2, whose expression varies in established cell breast lines, possesses multiple functions. First, the use of a DOX-inducible system to restore EphB2 function to low expressers resulted in decreased tumor growth in vitro and in vivo, while its siRNA-mediated silencing in high expressers increased growth. This function involves the onset of apoptotic death paralleled by caspases 3 and 9 activation. Second, EphB2 was also found to induce autophagy, as assessed by immunofluorescence and/or immunoblotting examination of the LC3, ATG5 and ATG12 markers. Third, EphB2 also has a pro-invasive function in breast cancer cells that involves the regulation of MMP2 and MMP9 metalloproteases and can be blocked by treatment with respective neutralizing antibodies. Furthermore, EphB2-induced invasion is kinase-dependent and is impeded in cells expressing a kinase-dead mutant EphB2. In summary, we identified a mechanism involving a triple role for EphB2 in breast cancer progression, whereby it regulates apoptosis, autophagy, and invasion.

Ehd3, a regulator of vesicular trafficking, is silenced in gliomas and functions as a tumor suppressor by controlling cell cycle arrest and apoptosis.

EHD3 [Eps15 homology (EH) domain-containing protein 3] is a protein that resides in tubular and vesicular membrane structures and participates in endocytic recycling, although all its functions are unknown. Since Ehd3 is most abundantly expressed in brain tissues, we examined its role in brain cancer progression. Using immunohistochemistry, we report loss of EHD3 expression in gliomas, including low-grade astrocytomas, suggesting that this is an early event in gliomagenesis. EHD3 expression is also very low in most of glioma cell lines tested. In two cell lines, a bisulfite sequencing method identifies promoter hypermethylation as a mechanism of Ehd3 silencing, and its expression was restored by the demethylating agent 5-Azacytidine. Doxycycline-inducible restoration of EHD3 expression to glioma cells decreases their growth and invasiveness and induces cell cycle arrest and apoptosis. Furthermore, shRNA-mediated Ehd3 silencing increases cell growth. Using a xenograft model, we demonstrate Ehd3 growth inhibitory functions in glioma cells in vivo. We suggest that Ehd3 functions as a tumor suppressor gene and loss of its expression is a very common event in gliomas. This is the first study to highlight the importance of a member of the C-terminal EHD proteins in cancer and to link their functions to the cell cycle and apoptosis.

Somatic mutations activating STAT3 in human inflammatory hepatocellular adenomas.

Inflammatory hepatocellular adenomas (IHCAs) are benign liver tumors. 60% of these tumors have IL-6 signal transducer (IL6ST; gp130) mutations that activate interleukin 6 (IL-6) signaling. Here, we report that 12% of IHCA subsets lacking IL6ST mutations harbor somatic signal transducer and activator of transcription 3 (STAT3) mutations (6/49). Most of these mutations are amino acid substitutions in the SH2 domain that directs STAT3 dimerization. In contrast to wild-type STAT3, IHCA STAT3 mutants constitutively activated the IL-6 signaling pathway independent of ligand in hepatocellular cells. Indeed, the IHCA STAT3 Y640 mutant homodimerized independent of IL-6 and was hypersensitive to IL-6 stimulation. This was associated with phosphorylation of tyrosine 705, a residue required for IL-6-induced STAT3 activation. Silencing or inhibiting the tyrosine kinases JAK1 or Src, which phosphorylate STAT3, impaired constitutive activity of IHCA STAT3 mutants in hepatocellular cells. Thus, we identified for the first time somatic STAT3 mutations in human tumors, revealing a new mechanism of recurrent STAT3 activation and underscoring the role of the IL-6-STAT3 pathway in benign hepatocellular tumorigenesis.

Chemistry-based protein modification strategy for endocytic pathway analysis.

BACKGROUND INFORMATION: The integrated analysis of intracellular trafficking pathways is one of the current challenges in the field of cell biology, and functional proteomics has become a powerful technique for the large-scale identification of proteins or lipids and the elucidation of biological processes in their natural contexts. For this, new dynamic strategies must be devised to trace proteins that follow a specific pathway such that their initial and final destinations can be detected by automated means. RESULTS: Here, we report a novel vectorial strategy for trafficking pathway analysis. This strategy is based on a chemical modification of plasma membrane proteins with a bSuPeR (biotinylated sulfation site peptide reagent) and metabolic labelling in the Golgi apparatus, such that plasma membrane proteins that traffic via the retrograde route become detectable in complex mixtures. Efficient synthesis schemes are presented for tailor-made chemical tools that are then applied to the step-by-step validation of the strategy, using a known retrograde cargo protein: the STxB (Shiga toxin B-subunit). bSuPeR modification at the plasma membrane does not affect STxB transport to the Golgi apparatus, where the protein is metabolically labelled, allowing its detection in cell lysates. CONCLUSIONS: Our vectorial concept proposes a new chemical approach for traffic-based profiling of proteins that may prove to be applicable to the analysis of diverse endocytic pathways.

Frequent in-frame somatic deletions activate gp130 in inflammatory hepatocellular tumours.

Inflammatory hepatocellular adenomas are benign liver tumours defined by the presence of inflammatory infiltrates and by the increased expression of inflammatory proteins in tumour hepatocytes. Here we show a marked activation of the interleukin (IL)-6 signalling pathway in this tumour type; sequencing candidate genes pinpointed this response to somatic gain-of-function mutations in the IL6ST gene, which encodes the signalling co-receptor gp130. Indeed, 60% of inflammatory hepatocellular adenomas harbour small in-frame deletions that target the binding site of gp130 for IL-6, and expression of four different gp130 mutants in hepatocellular cells activates signal transducer and activator of transcription 3 (STAT3) in the absence of ligand. Furthermore, analysis of hepatocellular carcinomas revealed that rare gp130 alterations are always accompanied by beta-catenin-activating mutations, suggesting a cooperative effect of these signalling pathways in the malignant conversion of hepatocytes. The recurrent gain-of-function gp130 mutations in these human hepatocellular adenomas fully explains activation of the acute inflammatory phase observed in tumourous hepatocytes, and suggests that similar alterations may occur in other inflammatory epithelial tumours with STAT3 activation.

Retrograde delivery of photosensitizer (TPPp-O-beta-GluOH)3 selectively potentiates its photodynamic activity.

Photodynamic therapy involves administration of a photosensitizing drug and its subsequent activation by visible light of the appropriate wavelength. Several approaches to increasing the specificity of photosensitizers for cancerous tissues and, in particular, through their conjugation to ligands that are directed against tumor-associated antigens have been investigated. Here, we have studied the delivery of the photocytotoxic porphyrin compound TPP(p-O-beta-D-GluOH)3 into tumor cells that overexpress the glycosphingolipid Gb3, using the Gb3-binding nontoxic B-subunit of Shiga toxin (STxB) as a vector. To allow for site-directed chemical coupling, an STxB variant carrying a free sulfhydryl moiety at its C-terminal end has been used. Binding affinity, cellular uptake, singlet oxygen quantum yield, and phototoxicity of the conjugate have been examined. Despite some effect of coupling on both the photophysical properties of TPP(p-O-beta-D-GluOH)3 and the affinity of STxB for its receptor, the conjugate exhibited a higher photocytotoxic activity than the photosensitizer alone and was exquisitely selective for Gb3-expressing tumor cells. Furthermore, our data strongly suggest that STxB-mediated retrograde delivery of the photosensitizer to the biosynthetic/secretory pathway is critical for optimal cytotoxic activity. In conclusion, a strong rationale for using retrograde delivery tools such as STxB in combination with photosensitizing agents for the photodynamic therapy of tumors is presented.

B subunit of Shiga toxin-based vaccines synergize with alpha-galactosylceramide to break tolerance against self antigen and elicit antiviral immunity.

The nontoxic B subunit of Shiga toxin (STxB) targets in vivo Ag to dendritic cells that preferentially express the glycolipid Gb(3) receptor. After administration of STxB chemically coupled to OVA (STxB-OVA) or E7, a polypeptide derived from HPV, in mice, we showed that the addition of alpha-galactosylceramide (alpha-GalCer) resulted in a dramatic improvement of the STxB Ag delivery system, as reflected by the more powerful and longer lasting CD8(+) T cell response observed even at very low dose of immunogen (50 ng). This synergy was not found with other adjuvants (CpG, poly(I:C), IFN-alpha) also known to promote dendritic cell maturation. With respect to the possible mechanism explaining this synergy, mice immunized with alpha-GalCer presented in vivo the OVA(257-264)/K(b) complex more significantly and for longer period than mice vaccinated with STxB alone or mixed with other adjuvants. To test whether this vaccine could break tolerance against self Ag, OVA transgenic mice were immunized with STxB-OVA alone or mixed with alpha-GalCer. Although no CTL induction was observed after immunization of OVA transgenic mice with STxB-OVA, tetramer assay clearly detected specific anti-OVA CD8(+) T cells in 8 of 11 mice immunized with STxB-OVA combined with alpha-GalCer. In addition, vaccination with STxB-OVA and alpha-GalCer conferred strong protection against a challenge with vaccinia virus encoding OVA with virus titers in the ovaries reduced by 5 log compared with nonimmunized mice. STxB combined with alpha-GalCer therefore appears as a promising vaccine strategy to more successfully establish protective CD8(+) T cell memory against intracellular pathogens and tumors.

Syntaxin 16 and syntaxin 5 are required for efficient retrograde transport of several exogenous and endogenous cargo proteins.

Retrograde transport allows proteins and lipids to leave the endocytic pathway to reach other intracellular compartments, such as trans-Golgi network (TGN)/Golgi membranes, the endoplasmic reticulum and, in some instances, the cytosol. Here, we have used RNA interference against the SNARE proteins syntaxin 5 and syntaxin 16, combined with recently developed quantitative trafficking assays, morphological approaches and cell intoxication analysis to show that these SNARE proteins are not only required for efficient retrograde transport of Shiga toxin, but also for that of an endogenous cargo protein - the mannose 6-phosphate receptor - and for the productive trafficking into cells of cholera toxin and ricin. We have found that the function of syntaxin 16 was specifically required for, and restricted to, the retrograde pathway. Strikingly, syntaxin 5 RNA interference protected cells particularly strongly against Shiga toxin. Since our trafficking analysis showed that apart from inhibiting retrograde endosome-to-TGN transport, the silencing of syntaxin 5 had no additional effect on Shiga toxin endocytosis or trafficking from TGN/Golgi membranes to the endoplasmic reticulum, we hypothesize that syntaxin 5 also has trafficking-independent functions. In summary, our data demonstrate that several cellular and exogenous cargo proteins use elements of the same SNARE machinery for efficient retrograde transport between early/recycling endosomes and TGN/Golgi membranes.

Functionally different pools of Shiga toxin receptor, globotriaosyl ceramide, in HeLa cells.

Many studies have investigated the intracellular trafficking of Shiga toxin, but very little is known about the underlying dynamics of its cellular receptor, the glycosphingolipid globotriaosyl ceramide. In this study, we show that globotriaosyl ceramide is required not only for Shiga toxin binding to cells, but also for its intracellular trafficking. Shiga toxin induces globotriaosyl ceramide recruitment to detergent-resistant membranes, and subsequent internalization of the lipid. The globotriaosyl ceramide pool at the plasma membrane is then replenished from internal stores. Whereas endocytosis is not affected in the recovery condition, retrograde transport of Shiga toxin to the Golgi apparatus and the endoplasmic reticulum is strongly inhibited. This effect is specific, as cholera toxin trafficking on GM(1) and protein biosynthesis are not impaired. The differential behavior of both toxins is also paralleled by the selective loss of Shiga toxin association with detergent-resistant membranes in the recovery condition, and comparison of the molecular species composition of plasma membrane globotriaosyl ceramide indicates subtle changes in favor of unsaturated fatty acids. In conclusion, this study demonstrates the dynamic behavior of globotriaosyl ceramide at the plasma membrane and suggests that globotriaosyl ceramide-specific determinants, possibly its molecular species composition, are selectively required for efficient retrograde sorting on endosomes, but not for endocytosis.

Synthesis of globo- and isoglobotriosides bearing a cinnamoylphenyl tag as novel electrophilic thiol-specific carbohydrate reagents.

The galactosyl donor, 4,6-di-O-acetyl-2,3-di-O-benzyl-D-galactopyranosyl trichloroacetimidate, was efficiently coupled with regioselectively benzylated lactoside acceptors under standard conditions to stereoselectively afford the corresponding globotrioside and isoglobotrioside derivatives in very good yields. These glycosides were smoothly functionalized with a 6-(p-cinnamoylphenoxy)-hexyl tether tag as novel electrophilic thiol-specific carbohydrate reagents. Immobilization of the globotrioside conjugate to Thiopropyl Sepharose 6B for purification of B-subunit of Shiga toxin (StxB) and coupling of a model cysteine-containing protein (StxB-Z(n)-Cys) to the isoglobotrioside conjugate were both performed with high efficiency.

Internalized Pseudomonas exotoxin A can exploit multiple pathways to reach the endoplasmic reticulum.

Receptor-mediated internalization to the endoplasmic reticulum (ER) and subsequent retro-translocation to the cytosol are essential sequential processes required for the intoxication of mammalian cells by Pseudomonas exotoxin A (PEx). The toxin binds the alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein. Here, we show that in HeLa cells, PEx recruits a proportion of this receptor to detergent-resistant microdomains (DRMs). Uptake of receptor-bound PEx involves transport steps both directly from early endosomes to the trans-Golgi network (TGN) independently of Rab9 function and from late endosomes to the TGN in a Rab9-dependent manner. Furthermore, treatments that simultaneously perturb both Arf1-dependent and Rab6-dependent retrograde pathways show that PEx can use multiple routes to reach the ER. The Rab6-dependent route has only been described previously for cargo with lipid-sorting signals. These findings suggest that partial localization of PEx within DRM permits a choice of trafficking routes consistent with a model that DRM-associated toxins reach the ER on a lipid-dependent sorting pathway whilst non-DRM-associated PEx exploits the previously characterized KDEL receptor-mediated uptake pathway. Thus, unexpectedly, an ER-directed toxin with a proteinaceous receptor shows promiscuity in its intracellular trafficking pathways, exploiting routes controlled by both lipid- and protein-sorting signals.

The Shiga toxin B-subunit targets antigen in vivo to dendritic cells and elicits anti-tumor immunity.

The non-toxic B-subunit of Shiga toxin (STxB) interacts with the glycolipid Gb3, which is preferentially expressed on dendritic cells (DC) and B cells. After administration of STxB chemically coupled to OVA (STxB-OVA) in mice, we showed that the immunodominant OVA(257-264) peptide restricted by K(b) molecules is specifically presented by CD11c+ CD8alpha- DC, some of them displaying a mature phenotype. Using mice carrying a transgene encoding a diphtheria toxin receptor (DTR) under the control of the murine CD11c promoter, which allows inducible ablation of DC, we showed that DC are required for efficient priming of CTL after STxB-OVA vaccination. Immunization of mice with STxB-OVA induced OVA-specific CD8+ T cells detected ex vivo; these cells were long lasting, since they could be detected even 91 days after the last immunization and were composed of both central and memory T cells. Vaccination of mice with STxB-OVA and STxB coupled to E7, a protein derived from HPV16, inhibited tumor growth in prophylactic and therapeutic experiments. This effect was mainly mediated by CD8+ T cells. STxB therefore appears to be a powerful carrier directly targeting DC in vivo, resulting in a strong and durable CTL response associated with tumor protection.

Measuring retrograde transport to the trans-Golgi network.

The recently described retrograde transport route is a highly selective pathway that allows some internalized molecules to reach the trans-Golgi network from early/recycling endosomes, bypassing the recycling route to the plasma membrane and the late endocytic pathway. The non-toxic receptor-binding B-subunit of bacterial Shiga toxin has played an important role in the discovery and molecular dissection of membrane trafficking at the early/recycling endosomes-TGN interface. This unit describes several recent methods for quantitative biochemical and morphological analysis of retrograde transport. The sulfation assay permits the detection and quantification of cargo protein transport from endosomes to the TGN, describing how sulfation-site peptides can be chemically coupled to cargo proteins. Furthermore, a variant of the sulfation assay on permeabilized cells is presented. The chemical crosslinking theme is extended to horseradish peroxidase for the ultrastructural study of the Shiga toxin-containing early/recycling endosomes by whole mount analysis. Finally, an endocytosis assay describes concomitant analysis of cellular uptake of Shiga toxin and transferrin.

1-[3-(2-[18F]fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione: design, synthesis, and radiosynthesis of a new [18F]fluoropyridine-based maleimide reagent for the labeling of peptides and proteins.

FPyME (1-[3-(2-fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione) was designed as a [(18)F]fluoropyridine-based maleimide reagent for the prosthetic labeling of peptides and proteins via selective conjugation with a thiol (sulfhydryl) function. Its pyridinyl moiety carries the radioactive halogen (fluorine-18) which can be efficiently incorporated via a nucleophilic heteroaromatic substitution, and its maleimido function ensures the efficient alkylation of a free thiol function as borne by cysteine residues. [(18)F]FPyME (HPLC-purified) was prepared in 17-20% non-decay-corrected yield, based on starting [(18)F]fluoride, in 110 min using a three-step radiochemical pathway. The developed procedure involves (1) a high-yield nucleophilic heteroaromatic ortho-radiofluorination on [3-(3-tert-butoxycarbonylaminopropoxy)pyridin-2-yl]trimethylammonium trifluoromethanesulfonate as the fluorine-18 incorporation step, followed by (2) rapid and quantitative TFA-induced removal of the N-Boc-protective group and (3) optimized maleimide formation using N-methoxycarbonylmaleimide. Typically, 4.8-6.7 GBq (130-180 mCi) of radiochemically pure [(18)F]FPyME ([(18)F]-1) could be obtained after semipreparative HPLC in 110 min starting from a cyclotron production batch of 33.3 GBq (900 mCi) of [(18)F]fluoride (overall radiochemical yields, based on starting [(18)F]fluoride: 28-37% decay-corrected). [(18)F]FPyME ([(18)F]-1) was first conjugated with a small model hexapeptide ((N-Ac)KAAAAC), confirming the excellent chemoselectivity of the coupling reaction (CH(2)SH versus CH(2)NH(2)) and then conjugated with two 8-kDa proteins of interest, currently being developed as tumor imaging agents (c-AFIM-0 and c-STxB). Conjugation was achieved in high yields (60-70%, isolated and non-decay-corrected) and used optimized, short-time reaction conditions (a 1/9 (v/v) mixture of DMSO and 0.05 M aq Tris NaCl buffer (pH 7.4) or 0.1 M aq PBS (pH 8), at room temperature for 10 min) and purification conditions (a gel filtration using a Sephadex NAP-10 cartridge or a SuperDex Peptide HR 10/30 column), both compatible with the chemical stability of the proteins and the relatively short half-life of the radioisotope concerned. The whole radiosynthetic procedure, including the preparation of the fluorine-18-labeled reagent, the conjugation with the protein and the final purification took 130-140 min. [(18)F]FPyME ([(18)F]-1) represents a new, valuable, thiol-selective, fluorine-18-labeled reagent for the prosthetic labeling with fluorine-18 of peptides and proteins. Because of its excellent chemoselectivity, [(18)F]FPyME offers an interesting alternative to the use of the nonselective carboxylate and amine-reactive [(18)F]reagents and can therefore advantageously be used for the design and development of new peptide- and protein-based radiopharmaceuticals for PET.

Use of high affinity insulin analogues to assess the functional relationships between insulin receptor trafficking, mitogenic signaling and mRNA expression in rat liver.

We have investigated the functional relationships between insulin receptor (IR) trafficking, mitogenic signaling and mRNA expression in rat liver and primary hepatocytes. The low-K(d) insulin analogues [His(A8),His(B4), Glu(B10),His(B27)]-human insulin (-HI) (the H2-analogue), [Asp(B10)]HI and [Glu(A13),Glu(B10)]HI, were studied in liver parenchymal cells and compared with wild-type HI and epidermal growth factor (EGF), a mitogenic inducer. The extent and duration of IR endocytosis were markedly increased in response to the H2-analogue and [Asp(B10)]HI compared to wild-type HI, but similar to HI after [Glu(A13),Glu(B10)]HI administration. Importantly, the insulin analogues induced a higher and more prolonged tyrosine phosphorylation of the IR-beta subunit in endosomes compared to authentic HI. A low cell-free endosome-lysosome transfer of the internalized IR was only observed in response to HI and H2-analogue injection. Concomitant with the low endosome-lysosome transfer of the intact IR-beta subunit, 47 and 50 kDa fragments of the IR-beta subunit accumulated in lysosomal fractions. Neither HI nor the insulin analogues promoted the endosomal recruitment and tyrosine phosphorylation of Shc, whereas EGF accessed the Shc signaling pathway. Moreover, EGF induced a fast and prolonged activation of Raf-1 and MAP-kinase pathways whereas HI and insulin analogues displayed a moderate and transient effect. Finally, treatment of primary rat hepatocytes with HI and the protease-resistant H2-analogue did not affect the total level and relative expression of isotype A and B of IR mRNA regardless of time of exposure. These results suggest a lack of relationship between IR trafficking, endosomal tyrosine phosphorylation and mitogenic signaling in rat liver in vivo.