On-demand assembly of macromolecules used for the design and application of targeted secretion inhibitors

Neurological and endocrine pathologies such as acromegalie, Cushing’s disease, and neuropathic pain display disregulated exocytosis. Silencing specific cell populations would thus be invaluable to correct these debilitating disorders. To achieve this goal, we reengineered the Botulinum neurotoxin (BoT), a highly potent pharmaceutical compound capable of inhibiting exocytosis, and fused to it a protein “stapling” domain [1,2]. These peptide motifs, that form an irreversible tetrahelical coiled-coil, are able to link a variety of targeting domains onto the enzyme and thus redirect it towards normally unaffected cells. The conformational diversity of this assembly process greatly supersedes traditional protein expression since multiple targeting domains (homo- and hetero-) can be linked onto one scaffold, larger yields can be produced separately, it permits the combination of solid-phase peptide synthesis with recombinant protein expression, and it can avoid the necessity of an N- to C- translational fusion. With only a few dozen building “blocks” it is possible to construct thousands of different complexes specifically tailored for each purpose as every individual component can be linked onto any other cognate stapling moieties

SNARE based peptide linking as an efficient strategy to retarget botulinum neurotoxin’s enzymatic domain to specific neurons using diverse neuropeptides as targeting domains

Many disease states are caused by miss-regulated neurotransmission. A small fraction of these diseases can currently be treated with botulinum neurotoxin type A (BoNT/A). BoNT/A is composed of three functional domains – the light chain (Lc) is a zinc metalloprotease that cleaves intracellular SNAP25 which inhibits exocytosis, the translocation domain (Td) that enables the export of the light chain from the endosome to the cytosol, and the receptor binding domain (Rbd) that binds to extracellular gangliosides and synaptic vesicle glycoproteins while awaiting internalisation [1]. Current endeavours are directed towards retargeting Bont/A as well as finding safer methods of preparation and administration. Recently, our laboratory has developed a SNARE based linking strategy to recombine non-toxic BoNT/A fragments into a functional protein by simple mixing [2]. This SNARE based linking strategy permits the stepwise assembly of highly stable macromolecular complexes [2,3]. Onto these three SNARE peptides, diverse functional groups can be attached to the N- or C- terminus by direct synthesis and/or by genetic design. To enhance the therapeutic potential of BoNT/A, this method enables the rapid assembly of a large array of neuropeptide-SNAREs to their cognate LcTd-SNARE. A substitution of the Rbd with various neuropeptide sequences permits a large throughput combinatorial assay of LcTd to target new cell types. In this study, we have fused LcTd to 3 different Synaptobrevin sequences; we also use a small protein staple, and 26 different Syntaxin-neuropeptide fusions (permitting the assay of 78 new chimeric LcTd proteins with modified targeting domains). These neuropeptides such as, but not exclusively, somatostatin (SS), vasoactive intestinal peptide, substance P, opioid peptide analogues, Gonadotropin releasing hormone, and Arginine Vasopressin, which natively function through G protein coupled receptors (GPCR) can undergo agonist induced internalisation upon activation. The ability of our new constructs, once endocytosed, to inhibit neurotransmitter release was tested on different neuronal cell lines with immunoblotting of endogenous SNAP25. This cleavage by Lc reflects the ultimate readout of the enzyme’s efficacy, which incorporates the cell surface binding, internalisation kinetics, translocation of the Lc to the cytosol, and finally the enzymatic cleavage of SNAP25. Internalisation of the toxins can also be monitored with confocal microscopy and FACS by the substitution of the staple peptide for a fluorescent homologue. Figure 1 shows that whole boNT/A (upper left) can have its Rbd replaced with SNARE peptides, which will fuse together to form highly stable chimeric proteins with an altered targeting domain (right). Figure 1 also shows 4 different neuropeptide synthaxins in complex, resolved on SDS-PAGE gel (bottom left lanes 1-4, boiled 1’-4’). Fig. 1. SNARE-linked botulinum neurotoxins used for the retargeting of Bont/A. 29

Cleaved intracellular SNARE peptides are implicated in a novel cytotoxicity mechanism of botulinum serotype C

Recent advances in intracellular protein delivery have enabled more in-depth analyses of cellular functions. A specialized family of SNARE proteases, known as Botulinum Neurotoxins, blocks neurotransmitter exocytosis, which leads to systemic toxicity caused by flaccid paralysis. These pharmaceutically valuable enzymes have also been helpful in the study of SNARE functions. As can be seen in Figure 1A, SNARE bundle formation causes vesicle docking at the presynapse. Although these toxins are systemically toxic, no known cytotoxic effects have been reported with the curious exception of the Botulinum serotype C [1]. This enzyme cleaves intracellular SNAP25, as does serotype A and E, but also, exceptionally, cleaves Syntaxin 1. Using an array of lipid and polymer transfection reagents we were able to deliver different combinations of Botulinum holoenzymes into the normally unaffected, Neuro2A, SH-SY5Y, PC12, and Min6 cells to analyze the individual contribution of each SNARE protein and their cleaved peptide products

Unexpected transcellular protein crossover occurs during canonical DNA transfection.

Transfection of DNA has been invaluable for biological sciences, yet the effects upon membrane homeostasis are far from negligible. Here, we demonstrate that Neuro2A cells transfected using Lipofectamine LTX with the fluorescently coupled Botulinum serotype A holoenzyme (EGFP-LcA) cDNA express this SNAP25 protease that can, once translated, escape the transfected host cytosol and become endocytosed into untransfected cells, without its innate binding and translocation domains. Fluorescent readouts revealed moderate transfection rates (30–50%) while immunoblotting revealed a surprisingly total enzymatic cleavage of SNAP25; the transgenic protein acted beyond the confines of its host cell. Using intracellular dyes, no important cytotoxic effects were observed from reagent treatment alone, which excluded the possibility of membrane ruptures, though noticeably, intracellular acidic organelles were redistributed towards the plasma membrane. This drastic, yet frequently unobserved, change in protein permeability and endosomal trafficking following reagent treatment highlights important concerns for all studies using transient transfection

Botulinum neurotoxin type C protease induces apoptosis in differentiated human neuroblastoma cells

Neuroblastomas constitute a major cause of cancer-related deaths in young children. In recent years, a number of translation-inhibiting enzymes have been evaluated for killing neuroblastoma cells. Here we investigated the potential vulnerability of human neuroblastoma cells to protease activity derived from botulinum neurotoxin type C. We show that following retinoic acid treatment, human neuroblastoma cells, SiMa and SH-SY5Y, acquire a neuronal phenotype evidenced by axonal growth and expression of neuronal markers. Botulinum neurotoxin type C which cleaves neuron-specific SNAP25 and syntaxin1 caused apoptotic death only in differentiated neuroblastoma cells. Direct comparison of translation-inhibiting enzymes and the type C botulinum protease revealed one order higher cytotoxic potency of the latter suggesting a novel neuroblastoma-targeting pathway. Our mechanistic insights revealed that loss of ubiquitous SNAP23 due to differentiation coupled to SNAP25 cleavage due to botulinum activity may underlie the apoptotic death of human neuroblastoma cells

Supreme Council of Judges in ensuring the constitutionality of the judiciary\u27s independence

Article reveals the real independence of judicial authority, securing of openness and publicity of its activity and also the legal basis of increasing the effeciency and prestige of judges activity. Also elucidated important aspects of the law “About supreme council of judges of the Republic of Uzbekista