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.     

Lipid cosorting mediated by shiga toxin induced tubulation

To maintain cell membrane homeostasis, lipids must be dynamically redistributed during the formation of transport intermediates, but the mechanisms driving lipid sorting are not yet fully understood. Lowering sphingolipid concentration can reduce the bending energy of a membrane, and this effect could account for sphingolipid depletion along the retrograde pathway. However, sphingolipids and cholesterol are enriched along the anterograde pathway, implying that other lipid sorting mechanisms, such as protein-mediated sorting, can dominate. To characterize the influence of protein binding on the lipid composition of highly curved membranes, we studied the interactions of the B-subunit of Shiga toxin (STxB) with giant unilamellar vesicles containing its glycosphingolipid receptor [globotriaosylceramide (Gb3)]. STxB binding induced the formation of tubular membrane invaginations, and fluorescence microscopy images of these highly curved membranes were consistent with co-enrichment of Gb3 and sphingolipids. In agreement with theory, sorting was stronger for membrane compositions close to demixing. These results strongly support the hypothesis that proteins can indirectly mediate the sorting of lipids into highly curved transport intermediates via interactions between lipids and the membrane receptor of the protein.     

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.     

Effects of HIV-1 Nef on retrograde transport from the plasma membrane to the endoplasmic reticulum

HIV-1 Nef protein down-regulates several important immunoreceptors through interactions with components of the intracellular sorting machinery. Nef expression is also known to induce modifications of the endocytic pathway. Here, we analyzed the effects of Nef on retrograde transport, from the plasma membrane to the endoplasmic reticulum using Shiga toxin B-subunit (STxB). Nef expression inhibited access of STxB to the endoplasmic reticulum, but did not modify the surface expression level of STxB receptor, Gb₃, nor its internalization rate as measured with a newly developed assay. Mutation of the myristoylation site or of a di-leucine motif of Nef involved in the interaction with the clathrin adaptor complexes AP1 and AP2 abolished the inhibition of retrograde transport. In contrast, mutations of Nef motifs known to interact with PACS-1, βCOP or a subunit of the v-ATPase did not modify the inhibitory activity of Nef on retrograde transport. Ultrastructural analysis revealed that Nef was present in clusters located on endosomal or Golgi membranes together with internalized STxB. Furthermore, in strongly Nef-expressing cells, STxB accumulated in endosomal structures that labeled with AP1. Our observations show that Nef perturbs retrograde transport between the early endosome and the endoplasmic reticulum. The potential transport steps targeted by Nef are discussed .     

Retromer guides STxB and CD8-M6PR from early to recycling endosomes, EHD1 guides STxB from recycling endosome to Golgi

Retrograde trafficking transports proteins, lipids and toxins from the plasma membrane to the Golgi and endoplasmic reticulum (ER). To reach the Golgi, these cargos must transit the endosomal system, consisting of early endosomes (EE), recycling endosomes, late endosomes and lysosomes. All cargos pass through EE, but may take different routes to the Golgi. Retromer-dependent cargos bypass the late endosomes to reach the Golgi. We compared how two very different retromer-dependent cargos negotiate the endosomal sorting system. Shiga toxin B, bound to the external layer of the plasma membrane, and chimeric CD8-mannose-6-phosphate receptor (CI-M6PR), which is anchored via a transmembrane domain. Both appear to pass through the recycling endosome. Ablation of the recycling endosome diverted both of these cargos to an aberrant compartment and prevented them from reaching the Golgi. Once in the recycling endosome, Shiga toxin required EHD1 to traffic to the TGN, while the CI-M6PR was not significantly dependent on EHD1. Knockdown of retromer components left cargo in the EE, suggesting that it is required for retrograde exit from this compartment. This work establishes the recycling endosome as a required step in retrograde traffic of at least these two retromer-dependent cargos. Along this pathway, retromer is associated with EE to recycling endosome traffic, while EHD1 is associated with recycling endosome to TGN traffic of STxB.     

Shiga toxin B-subunit binds to the chaperone BiP and the nucleolar protein B23

BACKGROUND INFORMATION: In many cell lines, such as HeLa cells, STxB (Shiga toxin B-subunit) is transported from the plasma membrane to the ER (endoplasmic reticulum), via early/recycling endosomes and the Golgi apparatus, bypassing the late endocytic pathway. In human monocyte-derived macrophages and dendritic cells that are not sensitive to Shiga toxin-induced protein biosynthesis inhibition, STxB is not detectably targeted to the retrograde route and is degraded in late endosomes/lysosomes. RESULTS: We have identified B-subunit interacting proteins in HeLa cells and macrophages. In HeLa cells, the ER-localized chaperone BiP (binding protein) was co-immunoprecipitated with the B-subunit. This interaction was not observed in macrophages, consistent with our previous trafficking results. In both cell types, the B-subunit also interacted with the nucleolar protein B23. Consistently, the B-subunit could be detected on nucleoli, suggesting that it could serve to bring the holotoxin to the site of synthesis of its molecular target, rRNA. The nucleolar localization data are critically discussed. CONCLUSION: The interaction of STxB with BiP, involved in the retrotranslocation process to the cytosol and nucleolar B23, as described in this study, might be of relevance for explaining the efficiency of even low doses of Shiga toxin to inactivate cellular ribosomes, and for the use of STxB as a vector for targeting antigens to cytosolic proteasomes of the MHC I-restricted antigen presentation pathway.     

Analysis of Articulation Between Clathrin and Retromer in Retrograde Sorting on Early Endosomes

Clathrin and retromer have key functions for retrograde trafficking between early endosomes and the trans -Golgi network (TGN). Previous studies on Shiga toxin suggested that these two coat complexes operate in a sequential manner. Here, we show that the curvature recognition subunit component sorting nexin 1 (SNX1) of retromer interacts with receptor-mediated endocytosis-8 (RME-8) protein, and that RME-8 and SNX1 colocalize on early endosomes together with a model cargo of the retrograde route, the receptor-binding B-subunit of Shiga toxin (STxB). RME-8 has previously been found to bind to the clathrin uncoating adenosine triphosphatase (ATPase) Hsc70, and we now report that depletion of RME-8 or Hsc70 affects retrograde trafficking at the early endosomes–TGN interface of STxB and the cation-independent mannose 6-phosphate receptor, an endogenous retrograde cargo protein. We also provide evidence that retromer interacts with the clathrin-binding protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) not only via SNX1, as previously published (Chin Raynor MC, Wei X, Chen HQ, Li L. Hrs interacts with sorting nexin 1 and regulates degradation of epidermal growth factor receptor. J Biol Chem 2001;276:7069–7078), but also via the core complex component Vps35. Hrs codistributes at the ultrastructural level with STxB on early endosomes, and interfering with Hrs function using antibodies or mild overexpression inhibits retrograde transport. Our combined data suggest a model according to which the functions in retrograde sorting on early endosomes of SNX1/retromer and clathrin are articulated by RME-8, and possibly also by Hrs.     

Shiga toxin B-subunit sequential binding to its natural receptor in lipid membranes

Shiga toxin B-subunit (STxB), a protein involved in the cell-binding and intracellular trafficking of Shiga holotoxin, binds to a specific glycolipid, the globotriaosyl ceramide (Gb(3)). Tryptophan residues of STxB, located at the protein-membrane interface, allow one to study its interaction with model membranes by means of spectroscopic methods with no need for chemical derivatisation with a fluorophore. The protein emits maximally around 346 nm and a blue shift of about 8 nm, as well as the occurrence of changes in the emission fluorescence intensity spectra, is indicative of insertion and partition into the membrane. However, the interaction seems to take place without pentamer dissociation. Acrylamide quenching experiments confirm tryptophan residues become less exposed to solvent when in the presence of vesicles, and the use of lipophilic probes suggests that they are located in a shallow position near the water/membrane interface. Fluorescence intensity and lifetime measurements upon STxB titration with Gb(3)-containing vesicles suggest a complex STxB/Gb(3) docking mechanism involving static quenching in the later stages. Based on our observations, a model of the protein-membrane interaction is proposed and the STxB membrane partition and binding constants were calculated.     

The epithelial cell cytoskeleton and intracellular trafficking. I. Shiga toxin B-subunit system: retrograde transport,intracellular vectorization,and more

Many intracellular transport routes are still little explored. This is particularly true for retrograde transport between the plasma membrane and the endoplasmic reticulum. Shiga toxin B subunit has become a powerful tool to study this pathway, and recent advances on the molecular mechanisms of transport in the retrograde route and on its physiological function(s) are summarized. Furthermore, it is discussed how the study of retrograde transport of Shiga toxin B subunit allows one to design new methods for the intracellular delivery of therapeutic compounds.     

Rab12 Localizes to Shiga Toxin‐Induced Plasma Membrane Invaginations and Controls Toxin Transport

Several exogenous and endogenous cargo proteins are internalized independently of clathrin, including the bacterial Shiga toxin. The mechanisms underlying early steps of clathrin‐independent uptake remain largely unknown. In this study, we have designed a protocol to obtain gradient fractions containing Shiga toxin internalization intermediates. Using stable isotope labeling with amino acids in cell culture (SILAC) and quantitative mass spectrometry, Rab12 was found in association with these very early uptake carriers. The localization of the GTPase on Shiga toxin‐induced plasma membrane invaginations was shown by fluorescence microscopy in cells transfected with GFP‐Rab12. Furthermore, using a quantitative biochemical assay, it was found that the amount of receptor‐binding B‐subunit of Shiga toxin reaching the trans‐Golgi/TGN membranes was decreased in Rab12‐depleted cells, and that cells were partially protected against intoxication by Shiga‐like toxin 1 under these conditions. These findings demonstrate the functional importance of Rab12 for retrograde toxin trafficking. Among several other intracellular transport pathways, only the steady‐state localizations of TGN46 and cation‐independent mannose‐6‐phosphate receptor were affected. These data thus strongly suggest that Rab12 functions in the retrograde transport route.      

Investigating Endocytic Pathways to the Endoplasmic Reticulum and to the Cytosol Using SNAP‐Trap

Cholera toxin enters cells via an unusual pathway that involves trafficking through endosomes to the endoplasmic reticulum (ER). Whether the toxin induces its own pathway or travels along a physiological retrograde route is not known. To study its trafficking, we labeled cholera toxin B (CTB) or endogenous plasma membrane proteins with a small chemical compound, benzylguanine, which covalently reacts with the protein SNAP‐tag. Using ER‐targeted SNAP‐tag as reporter, we found that transport of CTB to the ER depends on dynamin‐2 and syntaxin 5. Plasma membrane proteins and a fluid‐phase marker added to the medium were also transported to the ER. This flux was not affected by exposing cells to CTB but was inhibited by depleting syntaxin 5 and increased by depleting dynamin‐2. As a control for confined intracellular localization of ER‐targeted SNAP‐tag we used adenovirus‐5, which traffics to endosomes and then escapes into the cytosol. The virus did not react with ER‐targeted SNAP but with cytosolic SNAP. Together, our results establish a new method (SNAP‐trap) to study trafficking of different cargo to the ER and the cytosol and provide evidence for the existence of a constitutive pathway from the cell surface to the ER .     

Intracellular drug delivery: Potential usefulness of engineered Shiga toxin subunit B for targeted cancer therapy

A treasure trove of intracellular cancer drug targets remains hidden behind cell membranes. However, engineered pathogen-derived toxins such as Shiga toxins can deliver small or macromolecular drugs to specific intracellular organelles. After binding to ganglioglobotriaosylceramide (Gb3, CD77), the non-toxic subunit B (StxB) of the Shiga-holotoxin is endocytosed and delivers its payload by a unique retrograde trafficking pathway via the endoplasmic reticulum to the cytosol. This review provides an overview of biomedical applications of StxB-based drug delivery systems in targeted cancer diagnosis and therapy. Biotechnological production of the Stx-material is discussed from the perspective of developing efficacious and safe therapeutics.     

Retrograde Transport: Two (or More) Roads Diverged in an Endosomal Tree?

Some proteins and lipids traffic from the plasma membrane to the trans Golgi network (TGN)/Golgi apparatus and the endoplasmic reticulum, via the retrograde transport route. Endosomes are an obligatory through station. Whether early, recycling and late endosomes all hand off material to the TGN have remained a matter of debate. In this review, we give a short historical overview on how retrograde transport was discovered and explored. We then summarize and critically discuss data that have been put forward in favour of the existence of trafficking interfaces between each of the different endocytic localizations and the TGN. We finally point out some conceptual and technological challenges that will have to be met to establish definite conclusions for each of these scenarios.     

Intracellular targeting of the endoplasmic reticulum/nuclear envelope by retrograde transport may determine cell hypersensitivity to verotoxin via globotriaosyl ceramide fatty acid isoform traffic

The pentameric B subunit of verotoxin (VT) mediates the attachment to cell surface globotriaosyl ceramide (Gb3) to facilitate receptor-mediated endocytosis of the toxin. In highly toxin-sensitive tumor cells, the holotoxin and VT1 B subunit is targeted intracellularly to elements of the endoplasmic reticulum (ER)/nuclear membrane. In less sensitive cells, the toxin is targeted to components of the Golgi apparatus. We have studied two cell systems: the induced VT hypersensitivity of human astrocytoma cell lines cultured in the presence of sodium butyrate (compared to sodium propionate and capronate) and the increased VT sensitivity of multiple drug-resistant mutants as compared to parental human ovarian carcinoma cells. In both cases, a difference in the intracellular retrograde transport of the receptor-bound internalized toxin to the ER/nuclear envelope, as opposed to the Golgi, correlated with a >1,000-fold increase in cell sensitivity to VT. This change in intracellular routing may be due to sorting of Gb3 fatty acid isoforms, since nuclear targeting was found in turn to correlate with the preferential synthesis of Gb3 containing shorter chain (primarily C16) fatty acid species. We propose that the isoform-dependent traffic of Gb3 from the cell surface to the ER/nuclear membrane provides a new signal transduction pathway for Gb3 binding proteins.     

SNAP-tag based proteomics approach for the study of the retrograde route

Proteomics is a powerful technique for protein identification at large scales. A number of proteomics approaches have been developed to study the steady state composition of intracellular compartments. Here, we report a novel vectorial proteomics strategy to identify plasma membrane proteins that undergo retrograde transport to the trans-Golgi network (TGN). This strategy is based on the covalent modification of the plasma membrane proteome with a membrane impermeable benzylguanine derivative. Benzylguanine-tagged plasma membrane proteins that are subsequently targeted to the retrograde route are covalently captured by a TGN-localized SNAP-tagged fusion protein, which allows for their identification. The approach was validated step-by-step using a well explored retrograde cargo protein, the B-subunit of Shiga toxin. It was then extended to the proteomics format. Among other hits we found one of the historically first identified cargo proteins that undergo retrograde transport, which further validated our approach. Most of the other hits were kinases, receptors or transporters. In conclusion, we have pioneered a vectorial proteomics approach that complements traditional methods for the study of retrograde protein trafficking. This approach is of generic nature and could in principle be extended to other endocytic pathways.     

Tumor delivery of ultrasound contrast agents using Shiga toxin B subunit

The present study demonstrates the targeting of ultrasound contrast agents to human xenograft tumors by exploiting the overexpression of the glycolipid Gb3 in neovasculature. To this end, microbubbles were functionalized with a natural Gb3 ligand, the B subunit of the Shiga toxin (STxB). The targeting of Gb3-expressing tumor cells by STxB microbubbles was first shown by flow cytometry and fluorescence microscopy. A significantly higher proportion of STxB microbubbles were associated with Gb3-expressing tumor cells compared to cells in which Gb3 expression was inhibited. Moreover, ultrasonic imaging of culture plates showed a 12 dB contrast enhancement in average backscattered acoustic intensity on the surface of Gb3-expressing cells compared to Gb3-negative cells. Also, a 18 dB contrast enhancement was found in favor of STxB microbubbles compared to unspecific microbubbles. Microbubble signal intensity in subcutaneous tumors in mice was more than twice as high after the injection of STxB-functionalized microbubbles compared to the injection of unspecific microbubbles. These in vitro and in vivo experiments demonstrated that STxB-functionalized microbubbles bind specifically to cells expressing the Gb3 glycolipid. The cell-binding moieties of toxins thus appear as a new group of ligands for angiogenesis imaging with ultrasound.     

Bacterial intracellularly active toxins: Membrane localisation of the active domain

Numerous bacterial toxins exert their activity by inactivating or modulating a specific intracellular host target. For this purpose, these toxins have developed efficient strategies to overcome the different host cell defences including specific binding to cell surface, internalisation, passage through the endosome or plasma membrane, exploiting intracellular trafficking and addressing to intracellular targets. Several intracellularly active toxins deliver an active domain into the cytosol that interacts with a target localised to the inner face of the plasma membrane. Thus, the large clostridial glucosylating toxins (LCGTs) target Rho/Ras‐GTPases, certain virulence factors of Gram negative bacteria, Rho‐GTPases, while Pasteurella multocida toxin (PMT) targets trimeric G‐proteins. Others such as botulinum neurotoxins and tetanus neurotoxin have their substrate on synaptic vesicle membrane. LCGTs, PMT, and certain virulence factors from Vibrio sp. show a particular structure constituted of a four‐helix bundle membrane (4HBM) protruding from the catalytic site that specifically binds to the membrane phospholipids and then trap the catalytic domain at the proximity of the membrane anchored substrate. Structural and functional analysis indicate that the 4HBM tip of the Clostridium sordellii lethal toxin (TcsL) from the LCGT family contain two loops forming a cavity that mediates the binding to phospholipids and more specifically to phosphatidylserine.     

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.     

A Conserved Structural Motif Mediates Retrograde Trafficking of Shiga Toxin Types 1 and 2

Shiga toxin‐producing Escherichia coli (STEC) produce two types of Shiga toxin (STx): STx1 and STx2. The toxin A‐subunits block protein synthesis, while the B‐subunits mediate retrograde trafficking. STEC infections do not have definitive treatments, and there is growing interest in generating toxin transport inhibitors for therapy. However, a comprehensive understanding of the mechanisms of toxin trafficking is essential for drug development. While STx2 is more toxic in vivo, prior studies focused on STx1 B‐subunit (STx1B) trafficking. Here, we show that, compared with STx1B, trafficking of the B‐subunit of STx2 (STx2B) to the Golgi occurs with slower kinetics. Despite this difference, similar to STx1B, endosome‐to‐Golgi transport of STx2B does not involve transit through degradative late endosomes and is dependent on dynamin II, epsinR, retromer and syntaxin5. Importantly, additional experiments show that a surface‐exposed loop in STx2B (β4–β5 loop) is required for its endosome‐to‐Golgi trafficking. We previously demonstrated that residues in the corresponding β4–β5 loop of STx1B are required for interaction with GPP130, the STx1B‐specific endosomal receptor, and for endosome‐to‐Golgi transport. Overall, STx1B and STx2B share a common pathway and use a similar structural motif to traffic to the Golgi, suggesting that the underlying mechanisms of endosomal sorting may be evolutionarily conserved.      

Shiga toxin B-subunit sequential binding to its natural receptor in lipid membranes

Shiga toxin B-subunit (STxB), a protein involved in the cell-binding and intracellular trafficking of Shiga holotoxin, binds to a specific glycolipid, the globotriaosyl ceramide (Gb₃). Tryptophan residues of STxB, located at the protein-membrane interface, allow one to study its interaction with model membranes by means of spectroscopic methods with no need for chemical derivatisation with a fluorophore. The protein emits maximally around 346 nm and a blue shift of about 8 nm, as well as the occurrence of changes in the emission fluorescence intensity spectra, is indicative of insertion and partition into the membrane. However, the interaction seems to take place without pentamer dissociation. Acrylamide quenching experiments confirm tryptophan residues become less exposed to solvent when in the presence of vesicles, and the use of lipophilic probes suggests that they are located in a shallow position near the water/membrane interface. Fluorescence intensity and lifetime measurements upon STxB titration with Gb₃-containing vesicles suggest a complex STxB/Gb₃ docking mechanism involving static quenching in the later stages. Based on our observations, a model of the protein-membrane interaction is proposed and the STxB membrane partition and binding constants were calculated.