Translocation Domain Research Articles

Targeted delivery of a human Bcl-2-specific protein binder effectively induces apoptosis of cancer cells

Bcl-2 family proteins are critical switches to control cell death and survival, and Bcl-2 is a key regulator in pro-survival signaling, causing various diseases including cancers. Bcl-2 has drawn a considerable attention as a potential target for developing a pro-apoptotic agent for cancers. We here present the development of a specific protein binder against human Bcl-2 and its cytosolic delivery to effectively induce apoptosis of cancer cells. The protein binder composed of leucine-rich repeat modules was selected for human Bcl-2, and its binding affinity was increased up to 60 nM through a modular evolution-based approach. The protein binder was efficiently delivered into cancer cells by an intracellular delivery system using a translocation domain from a bacterial exotoxin, resulting in a strong suppression of anti-apoptotic signaling in cancer cells. Our results demonstrate that the human Bcl-2-specific protein binder can act as a potent therapeutic agent for cancers.

Endocytosis, trafficking and exocytosis of intact full-length botulinum neurotoxin type a in cultured rat neurons

Botulinum toxin A (BoNT/A) is a potent neurotoxin that acts primarily by silencing synaptic transmission by blocking neurotransmitter release. BoNT/A comprises a light chain (LC/A) intracellular protease and a heavy chain (HC/A) composed of a receptor binding domain (HCC/A) and a translocation domain (HCN/A) that mediates cell entry. Following entry into the neuron, the disulphide bond linking the two peptide chains is reduced to release the LC/A. To gain better insight into the trafficking and fate of BoNT/A before dissociation we have used a catalytically inactive, non-toxic full-length BoNT/A(0) mutant. Our data confirm that BoNT/A(0) enters cortical neurons both in an activity-dependent manner and via a pathway dependent on fibroblast growth factor receptor 3 (Fgfr3) signalling. We demonstrate that both dynamin-dependent endocytosis and lipid rafts are involved in BoNT/A internalisation and that full-length BoNT/A(0) traffics to early endosomes. Furthermore, while a proportion of BoNT/A remains stable in neurons for 3 days, BoNT/A degradation is primarily mediated by the proteasome. Finally, we demonstrate that a fraction of the endocytosed full-length BoNT/A(0) is capable of exiting the cell to intoxicate other neurons. Together, our data shed new light on the entry routes, trafficking and degradation of BoNT/A, and confirm that trafficking properties previously described for the isolated HCC/A receptor binding domain of are also applicable to the intact, full-length toxin.

Conversion of chemical to mechanical energy by the nucleotide binding domains of ABCB1

P-glycoprotein (ABCB1) is an important component of barrier tissues that extrudes a wide range of chemically unrelated compounds. ABCB1 consists of two transmembrane domains forming the substrate binding and translocation domain, and of two cytoplasmic nucleotide binding domains (NBDs) that provide the energy by binding and hydrolyzing ATP. We analyzed the mechanistic and energetic properties of the NBD dimer via molecular dynamics simulations. We find that MgATP stabilizes the NBD dimer through strong attractive forces by serving as an interaction hub. The irreversible ATP hydrolysis step converts the chemical energy stored in the phosphate bonds of ATP into potential energy. Following ATP hydrolysis, interactions between the NBDs and the ATP hydrolysis products MgADP + Pi remain strong, mainly because Mg2+ forms stabilizing interactions with ADP and Pi. Despite these stabilizing interactions MgADP + Pi are unable to hold the dimer together, which becomes separated by avid interactions of MgADP + Pi with water. ATP binding to the open NBDs and ATP hydrolysis in the closed NBD dimer represent two steps of energy input, each leading to the formation of a high energy state. Relaxation from these high energy states occurs through conformational changes that push ABCB1 through the transport cycle.

Cross-Species Functional Conservation and Possible Origin of the N-Terminal Specificity Domain of Mitochondrial Presequences.

Plants have two endosymbiotic organelles, chloroplast and mitochondrion. Although they have their own genomes, proteome assembly in these organelles depends on the import of proteins encoded by the nuclear genome. Previously, we elucidated the general design principles of chloroplast and mitochondrial targeting signals, transit peptide, and presequence, respectively, which are highly diverse in primary structure. Both targeting signals are composed of N-terminal specificity domain and C-terminal translocation domain. Especially, the N-terminal specificity domain of mitochondrial presequences contains multiple arginine residues and hydrophobic sequence motif. In this study we investigated whether the design principles of plant mitochondrial presequences can be applied to those in other eukaryotic species. We provide evidence that both presequences and import mechanisms are remarkably conserved throughout the species. In addition, we present evidence that the N-terminal specificity domain of presequence might have evolved from the bacterial TAT (twin-arginine translocation) signal sequence.

Fluorescent Study on Tetanus Neurotoxin

Bacteria produce and secrete various toxins under specific circumstances. One class of these bacterial toxins, including the clostridial neurotoxins, are a special class of proteins which begin in a globular, soluble form and convert to an integral membrane protein. The clostridial neurotoxins have a three globular domain structure with each portion responsible for one aspect of its function. During intoxication, it inserts into the endosomal membrane and concurrently translocates a portion of the protein into the mammalian cytosol, where it has toxic activities. The middle 50kDa domain is the translocation domain. This domain is responsible for the switch from globular soluble to integral membrane and translocating a second domain across the endosomal membrane, in response to the acidic low pH of the endosomal lumen. However, the exact residues which enter the membrane are not known. By putting an environmentally sensitive fluorescent probe at various positions and controlling the environment through the addition of membranes and then low pH, the local environment of each position can be monitored. From our studies, we have identified residues which are membrane inserted and further differentiated their depth into the membrane bilayer.

Accurate Determination of Membrane Penetration of Proteins Using Fluorescence Quenching and Molecular Dynamics Simulations

Determination of the depth of membrane penetration provides important information for studies of membrane protein folding and protein-lipid interactions [1,2]. Here we use a combination of molecular dynamics (MD) simulations and depth-dependent fluorescence quenching to calibrate the methodology for extracting quantitative information on membrane penetration. The knowledge of the immersion depth of fluorescence quenchers (e.g., Doxyl or TEMPO spin probe attached to lipid acyl chains or headgroup) is critical for the determination of the transverse location of fluorescence probes attached to proteins and peptides. The question of bilayer penetration of TEMPO moiety, attached to lipid headgroup in TEMPO-PC, has recently came into prominence in two studies of interfacial solvation [3,4]. Here, we re-examine the arguments on TEMPO penetration using the cross-validation of MD simulations and depth-dependent fluorescence-quenching experiments, which confirms that TEMPO in TEMPO-PC penetrates below the level of phosphate groups. The proper analysis of fluorescence quenching requires the use of Tempo position below the level of phosphate groups; and failure to do so will result in substantial systematic errors in determining the penetration of the labeled site on a membrane protein [5]. Finally, we illustrate the application of this methodology by determining membrane penetration of site-selectively labeled mutants of diphtheria toxin translocation domain [6] and apoptotic inhibitor Bcl-xL [7]. [1] A. S. Ladokhin (2014) Biochim. Biophys. Acta 1838, 2289. [2] A. Kyrychenko et al. (2017) Reviews in Fluorescence 2016: Springer, 243-274. [3] C.-Y. Cheng et al. (2015) Biophys J 109, 330. [4] Y. Lee et al. (2016) Biophys J 111, 2481. [5] A. Kyrychenko et al. (2019) J Membr Biol 252, doi:10.1007/s00232-019-00094-1. [6] A. Kyrychenko et al. (2018) J Membr Biol 251, 379. [7] V. Vasquez-Montes et al. (2019) Biochim Biophys Acta 1867, 691.

Crystal structure of botulinum neurotoxin subtype A3 cell binding domain in complex with GD1a co‐receptor ganglioside

Botulinum neurotoxins (BoNTs) are one of the most toxic proteins known to humans. Their molecular structure is comprised of three essential domains—a cell binding domain (HC), translocation domain and catalytic domain (light chain) . The HC domain facilitates the highly specific binding of BoNTs to the neuronal membrane via a dual‐receptor complex involving a protein receptor and a ganglioside. Variation in activity/toxicity across subtypes of serotype A has been attributed to changes in protein and ganglioside interactions, and their implications are important in the design of novel BoNT‐based therapeutics. Here, we present the structure of BoNT/A3 cell binding domain (HC/A3) in complex with the ganglioside GD1a at 1.75 Å resolution. The structure revealed that six residues interact with the three outermost monosaccharides of GD1a through several key hydrogen bonding interactions. A detailed comparison of structures of HC/A3 with HC/A1 revealed subtle conformational differences at the ganglioside binding site upon carbohydrate binding.

The C. difficile toxin B membrane translocation machinery is an evolutionarily conserved protein delivery apparatus

Large Clostridial Toxins (LCTs) are a family of six homologous protein toxins that are implicated in severe disease. LCTs infiltrate host cells using a translocation domain (LCT-T) that contains both cell-surface receptor binding sites and a membrane translocation apparatus. Despite much effort, LCT translocation remains poorly understood. Here we report the identification of 1104 LCT-T homologs, with 769 proteins from bacteria outside of clostridia. Sequences are widely distributed in pathogenic and host-associated species, in a variety of contexts and architectures. Consistent with these homologs being functional toxins, we show that a distant LCT-T homolog from Serratia marcescens acts as a pH-dependent translocase to deliver its effector into host cells. Based on evolutionary footprinting of LCT-T homologs, we further define an evolutionarily conserved translocase region that we show is an autonomous translocase capable of delivering heterologous cargo into host cells. Our work uncovers a broad class of translocating toxins and provides insights into LCT translocation.

Human antibodies neutralizing diphtheria toxin in vitro and in vivo

Diphtheria is an infectious disease caused by Corynebacterium diphtheriae. The bacterium primarily infects the throat and upper airways and the produced diphtheria toxin (DT), which binds to the elongation factor 2 and blocks protein synthesis, can spread through the bloodstream and affect organs, such as the heart and kidneys. For more than 125 years, the therapy against diphtheria has been based on polyclonal horse sera directed against DT (diphtheria antitoxin; DAT). Animal sera have many disadvantages including serum sickness, batch-to-batch variation in quality and the use of animals for production. In this work, 400 human recombinant antibodies were generated against DT from two different phage display panning strategies using a human immune library. A panning in microtiter plates resulted in 22 unique in vitro neutralizing antibodies and a panning in solution combined with a functional neutralization screening resulted in 268 in vitro neutralizing antibodies. 61 unique antibodies were further characterized as scFv-Fc with 35 produced as fully human IgG1. The best in vitro neutralizing antibody showed an estimated relative potency of 454 IU/mg and minimal effective dose 50% (MED50%) of 3.0 pM at a constant amount of DT (4x minimal cytopathic dose) in the IgG format. The targeted domains of the 35 antibodies were analyzed by immunoblot and by epitope mapping using phage display. All three DT domains (enzymatic domain, translocation domain and receptor binding domain) are targets for neutralizing antibodies. When toxin neutralization assays were performed at higher toxin dose levels, the neutralizing capacity of individual antibodies was markedly reduced but this was largely compensated for by using two or more antibodies in combination, resulting in a potency of 79.4 IU/mg in the in vivo intradermal challenge assay. These recombinant antibody combinations are candidates for further clinical and regulatory development to replace equine DAT.

Evaluation of cytotoxic and apoptotic effects of DT386–BR2: A promising anticancer fusion protein

Purpose: In the previous studies, we designed an anticancer immunotoxin containing the catalytic and translocation domains of diphtheria toxin fused to BR2, a buforin II-derived antimicrobial peptide as a cancer-specific cell penetrating peptide, in order to target various cancer cells. The aim of this study was to evaluate the in vitro cytotoxicity of DT386–BR2 against K-562 cells as the most famous cell line for leukemia. Materials and Methods: MTT and flow-cytometry assays were used for determining the cytotoxic effects and cell death mechanism of DT386–BR2, respectively, against K-562 cell line. The recombinant DT386 and synthetic BR2 were used as the negative control in cytotoxicity assay. Results: The results of this study showed a significant reduction in survival of K-562 cells caused by DT386–BR2 as compared with BR2 and DT386 fragments. On the contrary, the flow-cytometry results showed apoptosis induction by DT386–BR2 after 12h in a dose- and time-dependent manner. Conclusion: DT386–BR2 fusion protein can be used for further preclinical studies for determining its pharmacokinetic/pharmacodynamic profiles and evaluating its anticancer efficacy in suitable animal models.

Resolving the Molecular Steps in Clostridial Neurotoxin Light Chain Translocation.

The clostridial neurotoxins (CNTs), botulinum toxin and tetanus toxin, are the most toxic proteins for humans. Neurotoxicity is based upon the specificity of the CNTs for neural host receptors and substrates. CNTs are organized into three domains, a Light Chain (LC) that is a metalloprotease and a Heavy Chain (HC) that has two domains, an N-terminal LC translocation domain (HCN) and a C-terminal receptor binding domain (HCC). While catalysis and receptor binding functions of the CNTs have been developed, our understanding of LC translocation is limited. This is due to the intrinsic complexity of the translocation process and limited tools to assess the step-by-step events in LC translocation. Recently, we developed a novel, cell-based TT-reporter to measure LC translocation as the translocation of a β-lactamase reporter across a vesicle membrane in neurons. Using this approach, we identified a role for a cis-Loop, located within the HCN, in LC translocation. In this commentary, we describe our current understanding of how CNTs mediate LC translocation and place the role of the cis-Loop in the LC translocation process relative to other independent functions that have been implicated in LC translocation. Understanding the basis for LC translocation will enhance the use of CNTs in vaccine development and as human therapies.

New Engineered-Botulinum Toxins Inhibit the Release of Pain-Related Mediators.

Targeted delivery of potent inhibitor of cytokine/pain-mediator into inflammatory or pain-sensing cells is a promising avenue for treating chronic pain, a world-wide major healthcare burden. An unmet need exists for a specific and effective delivery strategy. Herein, we describe a new approach using sortase to site-specifically ligate a non-toxic botulinum neurotoxin D (BoNT/D) core-therapeutic (synaptobrevin-cleaving protease and translocation domains) to cell-specific targeting ligands. An engineered core-therapeutic was efficiently ligated to IL-1β ligand within minutes. The resultant conjugate specifically entered into cultured murine primary macrophages, cleaved synaptobrevin 3 and inhibited LPS/IFN-γ evoked IL-6 release. Likewise, a CGRP receptor antagonist ligand delivered BoNT/D protease into sensory neurons and inhibited K+-evoked substance P release. As cytokines and neuropeptides are major regulators of inflammation and pain, blocking their release by novel engineered inhibitors highlights their therapeutic potential. Our report describes a new and widely-applicable strategy for the production of targeted bio-therapeutics for numerous chronic diseases.

Removal of the translocation domain and the furin cleavage site decreases the relative hepatotoxicity of the targeted antitumor toxins

Targeted toxins are promising anticancer agents that allow selectively destroying cancer cells due to the increased content of onco-specific markers on their surface. The use of such anti-cancer toxins in medicine is mainly hampered by their high non-specific toxicity, in particular, hepatotoxicity. In our work on human cell line, we have shown that the removal of the DARPin-PE40 translocation toxin domain leads to a decrease in hepatoto-xicity. The same effect is also observed when inactivation of the furin cleavage site in the DARPin-PE40 molecule was done. Simultaneous removal of both the translocation domain and the furin cleavage site showed the best results. This toxin modification can be used to create more selective anti-cancer toxins.

Removal of the Translocation Domain and the Furin Cleavage Site Decreases the Relative Hepatotoxicity of the Targeted Antitumor Toxins.

Targeted toxins are promising anticancer agents that allow selectively destroying cancer cells due to the increased content of onco-specific markers on their surface. The use of such anti-cancer toxins in medicine is mainly hampered by their high non-specific toxicity, in particular, hepatotoxicity. In our work on human cell line, we have shown that the removal of the DARPin-PE40 translocation toxin domain leads to a decrease in hepatotoxicity. The same effect is also observed when inactivation of the furin cleavage site in the DARPin-PE40 molecule was done. Simultaneous removal of both the translocation domain and the furin cleavage site showed the best results. This toxin modification can be used to create more selective anti-cancer toxins.

Activation of AMPK promotes thyroid cancer cell migration through its interaction with PKM2 and β-catenin

AMP-activated protein kinase (AMPK) is induced by the exhaustion of cellular energy and activates adaptive alterations in cellular metabolism, which is the basis for cell survival during different environmental stresses. We aimed to investigate the biological functions of AMPK and its molecular mechanism in regulating thyroid cancer (TC) progression. In current study, we found that activation of AMPK by multiple agonists suppresses TC cell proliferation. However, AMPK activation also led to TC cell migration at the same time. Depletion of AMPK abolished the effect of its agonist on cell multiplication and migration. Mechanistic investigations revealed that the impact of AMPK in terms of cell migration is dependent on its nuclear translocation, since site mutation of AMPK in its nuclear translocation domain (K244A) abolished TC cell migration but did not affect the inhibition of cell proliferation by AMPK agonist. Moreover, the nuclear AMPK recruits PKM2 and β-catenin by their interaction, which promotes the transcription of cell migration related genes, including MMP7 and c-Myc. Furthermore, depletion of PKM2/β-catenin abolished the migration effect of AMPK agonists, but did not affect their effects on suppression of cell proliferation. Our results provided a novel function of AMPK in cancer migration, and suggested that a combination of AMPK activation and PKM2 depletion or inhibition can be a new strategy to achieve better therapeutic effects for TC patients.

2590. Streptolysin O Enhances Binding of the Group A Streptococcal NAD+-Glycohydrolase Toxin to Oropharyngeal Keratinocytes

BackgroundStreptolysin O (SLO) and the NAD+-glycohydrolase (NADase) are co-toxins secreted by group A Streptococcus (GAS) that play a significant role in virulence. NADase requires SLO for translocation into the host cell cytoplasm, a process termed cytolysin-mediated translocation (CMT). Recently, we noted that interaction of the two toxins mutually increased their stability. Although NADase is predicted to bind to the host cell surface, this interaction is incompletely understood. Here, we investigate potential mechanisms by which NADase binds to oropharyngeal keratinocytes.MethodsThe amino terminal region of NADase has been implicated in CMT, but the structure of the putative translocation domain has not been characterized. We determined the solution structure of this domain by NMR spectroscopy. We used flow-cytometry and confocal microscopy to investigate whether NADase could interact directly with oropharyngeal keratinocytes. Finally, since we expect that NADase and SLO are co-expressed from the same operon, are secreted in a coordinated fashion, and interact in solution, we tested whether SLO affects NADase binding to host cells.ResultsThe solution structure of the NADase translocation domain revealed a β-sandwich fold with an elongated N-terminal intrinsically disordered region. Structural homology searches (DALI) identified a potential carbohydrate binding module, suggesting the translocation domain could play a role in glycan binding. We also demonstrated by flow-cytometry that purified recombinant NADase toxin is able to independently interact with the cell surface of oropharyngeal keratinocytes. Importantly, interaction with SLO significantly enhanced the association of NADase with the cell surface, resulting in a 5-fold increase of the geometric mean fluorescence intensity.ConclusionThe structure of the NADase translocation domain reveals a potential carbohydrate binding module, which may mediate binding of the toxin to a cell-surface glycan. Binding of NADase to host cells is markedly enhanced by its interaction with SLO. We conclude that interaction of the two toxins contributes to the CMT process by functionally increasing the local concentration of NADase at the cell surface.Disclosures All authors: No reported disclosures.

Triggering Receptor Expressed on Myeloid Cells-1 Inhibitor Targeted to Endothelium Decreases Cell Activation.

TREM-1 (Triggering Receptor Expressed on Myeloid cells-1) is an immunoreceptor expressed on neutrophils, monocytes/macrophages, and endothelial cells. It amplifies the inflammatory response driven by Toll-Like Receptors (TLR) engagement. The pharmacological inhibition of TREM-1 confers protection in several pre-clinical models of acute inflammation. In this study, we aimed to investigate the role of TREM-1 in endothelial cells using a sneaking ligand construct (SLC) inhibiting TREM-1 in the endothelium. The SLC was made of 3 modules: an E-selectin targeting domain, a Pseudomonas aeruginosa exotoxin a translocation domain, and a 7 aa peptide (LSKSLVF) that contains the interaction site between TREM-1 and its adaptor protein DAP-12. SLC peptide was effectively picked up by endothelial cells following LPS stimulation. It decreased LPS induced TREM-1 up-regulation and cell activation, neutrophils extravasation, and improved median survival time during experimental peritonitis in mice. We reported that a targeted endothelial TREM-1 inhibition is able to dampen cell activation and to confer protection during septic shock in mice. The use of such cell-specific, ligand- independent TREM-1 inhibitors deserve further investigations during acute or chronic inflammatory disorders.

Curcumin Blocks Cytotoxicity of Enteroaggregative and Enteropathogenic Escherichia coli by Blocking Pet and EspC Proteolytic Release From Bacterial Outer Membrane.

Pet and EspC are toxins secreted by enteroaggregative (EAEC) and enteropathogenic (EPEC) diarrheagenic Escherichia coli pathotypes, respectively. Both toxins are members of the Serine Protease Autotransporters of Enterobacteriaceae (SPATEs) family. Pet and EspC are important virulence factors that produce cytotoxic and enterotoxic effects on enterocytes. Here, we evaluated the effect of curcumin, a polyphenolic compound obtained from the rhizomes of Curcuma longa L. (Zingiberaceae) on the secretion and cytotoxic effects of Pet and EspC proteins. We found that curcumin prevents Pet and EspC secretion without affecting bacterial growth or the expression of pet and espC. Our results show that curcumin affects the release of these SPATEs from the translocation domain, thereby affecting the pathogenesis of EAEC and EPEC. Curcumin-treated EAEC and EPEC did not induce significant cell damage like the ability to disrupt the actin cytoskeleton, without affecting their characteristic adherence patterns on epithelial cells. A molecular model of docking predicted that curcumin interacts with the determinant residues Asp1018-Asp1019 and Asp1029-Asp1030 of the translocation domain required for the release of Pet and EspC, respectively. Consequently, curcumin blocks Pet and EspC cytotoxicity on epithelial cells by preventing their release from the outer membrane.

A Spectroscopic Study on Secondary Structure and Thermal Unfolding of the Plant Toxin Gelonin Confirms Some Typical Structural Characteristics and Unravels the Sequence of Thermal Unfolding Events.

Gelonin from the Indian plant Gelonium multiflorum belongs to the type I ribosome-inactivating proteins (RIPs). Like other members of RIPs, this toxin glycoprotein inhibits protein synthesis of eukaryotic cells; hence, it is largely used in the construction of immunotoxins composed of cell-targeted antibodies. Lysosomal degradation is one of the main issues in targeted tumor therapies, especially for type I RIP-based toxins, as they lack the translocation domains. The result is an attenuated cytosolic delivery and a decrease of the antitumor efficacy of these plant-derived toxins; therefore, strategies to permit their release from endosomal vesicles or modifications of the toxins to make them resistant to degradation are necessary to improve their efficacy. Using infrared spectroscopy, we thoroughly analyzed both the secondary structure and the thermal unfolding of gelonin. Moreover, by the combination of two-dimensional correlation spectroscopy and phase diagram method, it was possible to deduce the sequence of events during the unfolding, confirming the typical characteristic of the RIP members to denature in two steps, as a sequential loss of tertiary and secondary structure was detected at 58 °C and at 65 °C, respectively. Additionally, some discrepancies in the unfolding process between gelonin and saporin-S6, another type I RIP protein, were detected.

Recent advances with Treg depleting fusion protein toxins for cancer immunotherapy.

T regulatory cells (Tregs) are an important T cell population for immune tolerance, prevention of autoimmune diseases and inhibition of antitumor immunity. The tumor-promoting role played by Tregs in cancer has prompted numerous approaches to develop immunotherapeutics targeting Tregs. One approach to depletion of Treg cells is retargeting the highly potent cytotoxic activity of bacterial toxins. These agents capitalize on the well-characterized bacterial toxins, diphtheria toxin and Pseudomonas aeruginosa exotoxin A-both of which harbor membrane translocation domains and enzymatic domains that catalytically halt protein synthesis within intoxicated eukaryotic cells and act at picomolar or subpicomolar concentrations. In this review, we summarize the preclinical and clinical development of several Treg-depleting cancer immunotherapies based on these two bacterial toxins.