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

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

Binary polypeptide system for permanent and oriented protein immobilization.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: Many techniques in molecular biology, clinical diagnostics and biotechnology rely on binary affinity tags. The existing tags are based on either small molecules (e.g., biotin/streptavidin or glutathione/GST) or peptide tags (FLAG, Myc, HA, Strep-tag and His-tag). Among these, the biotin-streptavidin system is most popular due to the nearly irreversible interaction of biotin with the tetrameric protein, streptavidin. The major drawback of the stable biotin-streptavidin system, however, is that neither of the two tags can be added to a protein of interest via recombinant means (except for the Strep-tag case) leading to the requirement for chemical coupling. RESULTS: Here we report a new immobilization system which utilizes two monomeric polypeptides which self-assemble to produce non-covalent yet nearly irreversible complex which is stable in strong detergents, chaotropic agents, as well as in acids and alkali. Our system is based on the core region of the tetra-helical bundle known as the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex. This irreversible protein attachment system (IPAS) uses either a shortened syntaxin helix and fused SNAP25-synaptobrevin or a fused syntaxin-synaptobrevin and SNAP25 allowing a two-component system suitable for recombinant protein tagging, capture and immobilization. We also show that IPAS is suitable for use with traditional beads and chromatography, planar surfaces and Biacore, gold nanoparticles and for protein-protein interaction in solution. CONCLUSIONS: IPAS offers an alternative to chemical cross-linking, streptavidin-biotin system and to traditional peptide affinity tags and can be used for a wide range of applications in nanotechnology and molecular sciences.Published versio

Cryptochrome proteins regulate the circadian intracellular behavior and localization of PER2 in mouse suprachiasmatic nucleus neurons.

The ∼20,000 cells of the suprachiasmatic nucleus (SCN), the master circadian clock of the mammalian brain, coordinate subordinate cellular clocks across the organism, driving adaptive daily rhythms of physiology and behavior. The canonical model for SCN timekeeping pivots around transcriptional/translational feedback loops (TTFL) whereby PERIOD (PER) and CRYPTOCHROME (CRY) clock proteins associate and translocate to the nucleus to inhibit their own expression. The fundamental individual and interactive behaviors of PER and CRY in the SCN cellular environment and the mechanisms that regulate them are poorly understood. We therefore used confocal imaging to explore the behavior of endogenous PER2 in the SCN of PER2::Venus reporter mice, transduced with viral vectors expressing various forms of CRY1 and CRY2. In contrast to nuclear localization in wild-type SCN, in the absence of CRY proteins, PER2 was predominantly cytoplasmic and more mobile, as measured by fluorescence recovery after photobleaching. Virally expressed CRY1 or CRY2 relocalized PER2 to the nucleus, initiated SCN circadian rhythms, and determined their period. We used translational switching to control CRY1 cellular abundance and found that low levels of CRY1 resulted in minimal relocalization of PER2, but yet, remarkably, were sufficient to initiate and maintain circadian rhythmicity. Importantly, the C-terminal tail was necessary for CRY1 to localize PER2 to the nucleus and to initiate SCN rhythms. In CRY1-null SCN, CRY1Δtail opposed PER2 nuclear localization and correspondingly shortened SCN period. Through manipulation of CRY proteins, we have obtained insights into the spatiotemporal behaviors of PER and CRY sitting at the heart of the TTFL molecular mechanism

Re-Assembled Botulinum Neurotoxin Inhibits CNS Functions without Systemic Toxicity

The therapeutic potential of botulinum neurotoxin type A (BoNT/A) has recently been widely recognized. BoNT/A acts to silence synaptic transmission via specific proteolytic cleavage of an essential neuronal protein, SNAP25. The advantages of BoNT/A-mediated synaptic silencing include very long duration, high potency and localized action. However, there is a fear of possible side-effects of BoNT/A due to its diffusible nature which may lead to neuromuscular blockade away from the injection site. We recently developed a “protein-stapling” technology which allows re-assembly of BoNT/A from two separate fragments. This technology allowed, for the first time, safe production of this popular neuronal silencing agent. Here we evaluated the re-assembled toxin in several CNS assays and assessed its systemic effects in an animal model. Our results show that the re-assembled toxin is potent in inhibiting CNS function at 1 nM concentration but surprisingly does not exhibit systemic toxicity after intraperitoneal injection even at 200 ng/kg dose. This shows that the re-assembled toxin represents a uniquely safe tool for neuroscience research and future medical applications

Assembly of Protein Building Blocks Using a Short Synthetic Peptide

Combining proteins or their defined domains offers new enhanced functions. Conventionally, two proteins are either fused into a single polypeptide chain by recombinant means or chemically cross-linked. However, these strategies can have drawbacks such as poor expression (recombinant fusions) or aggregation and inactivation (chemical cross-linking), especially in the case of large multifunctional proteins. We developed a new linking method which allows site-oriented, noncovalent, yet irreversible stapling of modified proteins at neutral pH and ambient temperature. This method is based on two distinct polypeptide linkers which self-assemble in the presence of a specific peptide staple allowing on-demand and irreversible combination of protein domains. Here we show that linkers can either be expressed or be chemically conjugated to proteins of interest, depending on the source of the proteins. We also show that the peptide staple can be shortened to 24 amino acids still permitting an irreversible combination of functional proteins. The versatility of this modular technique is demonstrated by stapling a variety of proteins either in solution or to surfaces

Heat inactivation of clinical COVID-19 samples on an industrial scale for low risk and efficient high-throughput qRT-PCR diagnostic testing.

We report the development of a large scale process for heat inactivation of clinical COVID-19 samples prior to laboratory processing for detection of SARS-CoV-2 by RT-qPCR. With more than 266 million confirmed cases, over 5.26 million deaths already recorded at the time of writing, COVID-19 continues to spread in many parts of the world. Consequently, mass testing for SARS-CoV-2 will remain at the forefront of the COVID-19 response and prevention for the near future. Due to biosafety considerations the standard testing process requires a significant amount of manual handling of patient samples within calibrated microbiological safety cabinets. This makes the process expensive, effects operator ergonomics and restricts testing to higher containment level laboratories. We have successfully modified the process by using industrial catering ovens for bulk heat inactivation of oropharyngeal/nasopharyngeal swab samples within their secondary containment packaging before processing in the lab to enable all subsequent activities to be performed in the open laboratory. As part of a validation process, we tested greater than 1200 clinical COVID-19 samples and showed less than 1 Cq loss in RT-qPCR test sensitivity. We also demonstrate the bulk heat inactivation protocol inactivates a murine surrogate of human SARS-CoV-2. Using bulk heat inactivation, the assay is no longer reliant on containment level 2 facilities and practices, which reduces cost, improves operator safety and ergonomics and makes the process scalable. In addition, heating as the sole method of virus inactivation is ideally suited to streamlined and more rapid workflows such as 'direct to PCR' assays that do not involve RNA extraction or chemical neutralisation methods