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

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

Effects of Mitochondrial-Targeted Antioxidants on Real-Time Blood Nitric Oxide and Hydrogen Peroxide Release in Acute Hyperglycemia Rats

Acute hyperglycemia in non-diabetic subjects can impair vascularendothelial function, causing decreased endothelium-derived nitric oxide (NO) release and increased reactive oxygen species (ROS), such assuperoxide and hydrogen peroxide (H2O2). Hyperglycemia may induce mitochondrial dysfunction leading to ROS production and exacerbation of vascular endothelial dysfunction. We investigated whether mitochondrial-targeted antioxidants mitigate acute hyperglycemia-induced oxidative stress and reduced blood NO. To test this hypothesis, blood NO or H2O2 levels were measured simultaneously using NO or H2O2 microsensors (100 µm) which were placed into the femoral veins of anesthesized male Sprague-Dawley rats. Acute hyperglycemia was induced by infusion 20% D-glucose intravenously with or without mitochondria-targeted antioxidants (mitoquinone: mitoQ, MW=1714 g/mol, 2.3 mg/Kg; SS-31: (D-Arg)-Dmt-Lys-Phe-Amide, MW=640g/mol, 2.7 mg/Kg) for 3 hours. We found that acute hyperglycemia (200 mg/dL) significantly increased blood H2O2 by 3.0±0.5 M (n=7) and reduced blood NO by 68.0±13.5 nM (n=9) compared to the saline group at end of infusion (both p\u3c0.05). MitoQ significantly attenuated hyperglycemia– induced H2O2 levels by 2.5±0.2 M (n=7) and increased blood NO levels by 59.3±9.7 nM (n=5) (both p\u3c0.05 compared to hyperglycemia). Similarly, SS-31 significantly reduced hyperglycemia-induced blood H2O2 level by 4.0±0.6 M (n=5) and enhanced blood NO levels by 52.8±7.7 nM (n=6) at end of infusion (both p\u3c0.05 compared to hyperglycemia). In summary, acute hyperglycemia induces mitochondria-derived ROS which in turn contribute to vascular endothelial dysfunction. Therefore, mitochondria-targeted antioxidants are useful to attenuate acute hyperglycemia-induced vascular endothelial dysfunction and oxidative stress

The Effects of Protein Kinase C Beta II Peptide Modulation on Superoxide Release in Rat Polymorphonuclear Leukocytes

Phorbol 12-myristate 13-acetate (PMA; a diacylglycerol mimetic) is known to augment polymorphonuclear leukocyte (PMN) superoxide (SO) release via protein kinase C (PKC) activation. However, the role of PKC beta II (βII) mediating this response is not known. It’s known that myristic acid (myr-) conjugation facilitates intracellular delivery of the cargo sequence, and that putative PKCβII activator and inhibitor peptides work by augmenting or attenuating PKCβII translocation to cell membrane substrates (e.g. NOX-2). Therefore, we hypothesize that myr- conjugated PKCβII peptide-activator (N-myr-SVEIWD; myr-PKCβ+) would increase PMA-induced rat PMN SO release, whereas, myr-PKCβII peptide-inhibitor (N-myr-SLNPEWNET; myr-PKCβ-) would attenuate this response compared to non-drug treated controls. Rat PMNs (5x106) were incubated for 15min at 370C in the presence/absence of myr-PKCβ+/- (20 μM) or SO dismutase (SOD;10μg/mL; n=8) as positive control. PMA (100nM) induced PMN SO release was measured spectrophotometrically at 550nm via reduction of ferricytochrome c for 390 sec. PMN SO release increased absorbance to 0.39±0.04 in non-drug treated controls (n=28), and 0.49±0.05 in myr-PKCβ+(n=16). This response was significantly increased from 180 seconds to 240 seconds (p\u3c0.05). By contrast, myr-PKCβ- (0.26±0.03; n=14) significantly attenuated PMA-induced SO release compared to non-drug controls and myr-PKCβ+ (p\u3c0.05). SOD-treated samples showed \u3e90% reduction of PMA-induced SO release and was significantly different from all groups (p\u3c0.01). Cell viability ranged between 94± to 98±2% in all groups as determined by 0.2% trypan blue exclusion. Preliminary results suggest that myr-PKCβ- significantly attenuates PMA-induced SO release, whereas myr-PKCβ+ significantly augments PMA-induced SO release, albeit transiently. Additional dose response and western blot experiments are planned with myr-PKCβ+/- in PMA-induced PMN SO release assays. This research was supported by the Department of Bio-Medical Sciences and the Division of Research at PCOM and by Young Therapeutics, LLC

Protein Kinase C Beta II Peptide Inhibitor Elicits Robust Effects on Attenuating Myocardial Ischemia/Reperfusion Injury

Reperfusion injury contributes to myocardial tissue damage following a heart attack partly due to the generation of reactive oxygen species (ROS) upon cardio-angioplasty. Protein kinase C beta II (PKCβII) inhibition during reperfusion with peptide inhibitor (N-myr-SLNPEWNET; PKCβII-) decreases ROS release and leukocyte infiltration in rat hind-limb and myocardial ischemia/reperfusion (I/R) studies, respectively. However, the role of activating PKCβII during reperfusion has not been previously determined. In this study, we hypothesize that myristoylated (myr)-PKCβII- will decrease infarct size and improve post-reperfused cardiac function compared to untreated controls, whereas PKCβII peptide activator (N-myr-SVEIWD; myr-PKCβII+) will show no improvement compared to control. Myristoylation of PKCβII peptides facilitate their entry into the cell in order to affect PKCβII activity by either augmenting or attenuating its translocation to cell membrane proteins, such as NOX-2. Isolated perfused rat hearts were subjected to global I(30min)/R(50min) and infused with myr-PKCβII+ (20μM; n=9), myr-PKCβII- (20µM; n=8), or plasma (control; n=9) at reperfusion. Hearts were frozen (-20oC), sectioned and stained using 1% triphenyltetrazolium chloride to differentiate necrotic tissue. The measurement of Left ventricular (LV) cardiac function was determined using a pressure transducer and infarct size was calculated as percent dead tissue vs. total heart tissue weight. Myr-PKCβII- significantly improved LV end-diastolic pressure 37±7 mmHg compared to control (58±5; p\u3c0.01) and myr-PKCβII+ (58±4; p\u3c0.01). Myr-PKCβII- significantly reduced infarct size to 14±3% compared to control (26±5%; p\u3c0.01), while myr-PKCβII+ (25±3%) showed no difference. The data indicate that myr-PKCβII- may be a putative treatment to reduce myocardial reperfusion injury when given to heart attack patients during cardio-angioplasty. Future studies are planned to determine infarct size by Image J analysis

Myristoylated protein kinase C beta II peptide inhibitor exerts dose-dependent inhibition of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced leukocyte superoxide release

Protein kinase C (PKC) phosphorylation of leukocyte NADPH oxidase is essential to generatesuperoxide (SO) release. Inhibition of leukocyte SO release attenuates inflammation mediated vascular injury. However, the role of PKC isoforms mediating this response has not been fully elucidated. We hypothesize that PKC beta II (βII) isoform positively regulates leukocyte NADPH oxidase, and that a cell-permeable (myr)-PKC βII peptide inhibitor (N-myr-SLNPEWNET) would dose-dependently attenuate fMLP induced leukocyte SO release. fMLP is a leukocyte chemoattractant cell membrane receptor agonist. We isolated leukocytes by peritoneal lavage from male Sprague-Dawley rats using standard methods. fMLP (1 M)-induced leukocyte SO release was measured for 120 sec spectrophotometrically by reduction of ferricytochrome c in the presence/absence of myr-PKC βII peptide inhibitor (0.2 to 20 M) in 5 x 106 leukocytes. After each assay, cell viability was determined by 0.3% trypan blue exclusion. fMLP-induced leukocyte SO release increased peak absorbance to 0.18±0.03 in controls (n=20). This response was dose-dependently inhibited by myr-PKC βII peptide inhibitor at 0.17±0.05 (0.2 M; n=9), 0.14±0.05 (0.5 M; n=11), 0.1±0.05 (1 M; n=9), 0.05±0.03 (5 M; n=9), 0.04±0.03 (10 M; n=8) and 0.05±0.03 (20 µM; n=7) and was significantly attenuated in the 5 to 20 M range compared to controls (p\u3c0.05). Moreover, cell viability was \u3e 94±1% in all study groups. These results suggest that myr-PKC βII peptide inhibitor dose-dependently inhibits fMLP-induced leukocyte SO release in the 0.2 to 5 M dose-range and these effects are attributed to inhibition of PKC βII isoform