Blueberries are rich in polyphenolic compounds and have shown improvement in cognitive function in several clinical trials. The molecular basis of the neuronal protection of blueberries, however, is not fully understood. The objective of this research is to understand the biochemistry basis of neuronal protection effects of blueberries through their impacts on several enzymes and pathways involved in Alzheimer’s disease (AD) and other neurodegenerative diseases. We examined the inhibition effects of blueberries on the enzymatic activity of cholinesterase (acetylcholinesterase, AChE; and butyrylcholinesterase, BuChE), tyrosinase, and cyclooxygenase-2 (COX-2). The effects of blueberries on the biosynthesis of acetylcholinesterase in a cellular model were also studied. Further, the effect of blueberries on amyloid fibril formation was evaluated. Our results showed that blueberries directly inhibit the enzymatic activity of AChE, BuChE, tyrosinase, and COX-2, with the IC50 at 48 mg/mL, 9 mg/mL, 403 mg/mL, and 12 mg/mL of fresh berry equivalent, respectively. Further, blueberries delay the amyloid fibril formation by 24 h at 39 mg fresh berry/mL. It also reduces the synthesis of acetylcholinesterase synthesis at 19 mg fresh berry/mL in a cellular model. Those results suggested that the neuroprotection effects of blueberries may involve different pathways, including enhancing cholinergic signaling through their effect on cholinesterase, reducing neuroinflammation through inhibition of COX-2, and reducing amyloid formation. Collectively, blueberries may play a vital role in neuronal protection beyond their antioxidant activity and our results provide more molecular mechanisms for their neuroprotective effects, and support blueberries being nutraceutical to improve cognitive function.

The study on botulinum neurotoxins (BoNTs) has rapidly evolved for their structure and functions as opposed to them being poisons or cures. Since their discoveries, the scientific community has come a long way in understanding BoNTs' structure and biological activity. Given its current application as a tool for understanding neurocellular activity and as a drug against over 800 neurological disorders, relevant and sensitive assays have become critical for biochemical, physiological, and pharmacological studies. The natural entry of the toxin being ingestion, it has also become important to examine its mechanism while crossing the epithelial cell barrier. Several techniques and methodologies have been developed, for its entry, pharmacokinetics, and biological activity for identification, and drug efficacy both in vivo and in vitro conditions. However, each of them presents its own challenges. The cell-based assay is a platform that exceeds the sensitivity of mouse bioassay while encompassing all the steps of intoxication including cell binding, transcytosis, endocytosis, translocation and proteolytic activity. In this article we review in detail both the neuronal and nonneuronal based cellular interaction of BoNT involving its transportation, and interaction with the targeted cells, and intracellular activities.

Abstract The study on botulinum neurotoxins (BoNTs) has rapidly evolved for their structure and functions as opposed to them being poisons or cures. Since their discoveries, the scientific community has come a long way in understanding BoNTs’ structure and biological activity. Given its current application as a tool for understanding neurocellular activity and as a drug against over 800 neurological disorders, relevant and sensitive assays have become critical for biochemical, physiological, and pharmacological studies. The natural entry of the toxin being ingestion, it has also become important to examine its mechanism while crossing the epithelial cell barrier. Several techniques and methodologies have been developed, for its entry, pharmacokinetics, and biological activity for identification, and drug efficacy both in vivo and in vitro conditions. However, each of them presents its own challenges. The cell-based assay is a platform that exceeds the sensitivity of mouse bioassay while encompassing all the steps of intoxication including cell binding, transcytosis, endocytosis, translocation and proteolytic activity. In this article we review in detail both the neuronal and nonneuronal based cellular interaction of BoNT involving its transportation, and interaction with the targeted cells, and intracellular activities.

BACKGROUND:A natural product analog, 3-(4-nitrophenyl)-7H-furo[3,2-g]chromen-7-one, which is a nitrophenyl psoralen (NPP) was found to be an effective inhibitor of botulinum neurotoxin type A (BoNT/A).METHODS:In this work, we performed enzyme inhibition kinetics and employed biochemical techniques such as isothermal calorimetry (ITC) and fluorescence spectroscopy as well as molecular modeling to examine the kinetics and binding mechanism of NPP inhibitor with BoNT/A LC.RESULTS:Studies of inhibition mechanism and binding dynamics of NPP to BoNT/A light chain (BoNT/A LC) showed that NPP is a mixed type inhibitor for the zinc endopeptidase activity, implying that at least part of the inhibitor-enzyme binding site may be different from the substrate-enzyme binding site. By using biochemical techniques, we demonstrated NPP forms a stable complex with BoNT/A LC. These observations were confirmed by Molecular Dynamics (MD) simulation, which demonstrates that NPP binds to the site near the active site.CONCLUSION:The NPP binding interferes with BoNT/A LC binding to the SNAP-25, hence, inhibits its cleavage. Based on these results, we propose a modified strategy for designing a molecule to enhance the efficiency of the inhibition against the neurotoxic effect of BoNT.GENERAL SIGNIFICANCE:Insights into the interactions of NPP with BoNT/A LC using biochemical and computational approaches will aid in the future development of effective countermeasures and better pharmacological strategies against botulism.

Gram-positive bacteria are ancient organisms. Many bacteria, including Gram-positive bacteria, produce toxins to manipulate the host, leading to various diseases. While the targets of Gram-positive bacterial toxins are diverse, many of those toxins use a similar mechanism to invade host cells and exert their functions. Clostridial neurotoxins produced by Clostridial tetani and Clostridial botulinum provide a classical example to illustrate the structure–function relationship of bacterial toxins. Here, we critically review the recent progress of the structure–function relationship of clostridial neurotoxins, including the diversity of the clostridial neurotoxins, the mode of actions, and the flexible structures required for the activation of toxins. The mechanism clostridial neurotoxins use for triggering their activity is shared with many other Gram-positive bacterial toxins, especially molten globule-type structures. This review also summarizes the implications of the molten globule-type flexible structures to other Gram-positive bacterial toxins. Understanding these highly dynamic flexible structures in solution and their role in the function of bacterial toxins not only fills in the missing link of the high-resolution structures from X-ray crystallography but also provides vital information for better designing antidotes against those toxins.

Toxins can function both as a harmful and therapeutic molecule, depending on their concentrations. The diversity in their function allows us to ask some very pertinent questions related to their origin and roles: (a) What makes them such effective molecules? (b) Are there evolutionary features encoded within the structures of the toxins for their function? (c) Is structural hierarchy in the toxins important for maintaining their structure and function? (d) Do protein dynamics play a role in the function of toxins? and (e) Do the evolutionary connections to these unique features and functions provide the fundamental points in driving evolution? In light of the growing evidence in structural biology, it would be appropriate to suggest that protein dynamics and flexibility play a much bigger role in the function of the toxin than the structure itself. Discovery of IDPs (intrinsically disorder proteins), multifunctionality, and the concept of native aggregation are shaking the paradigm of the requirement of a fixed three-dimensional structure for the protein’s function. Growing evidence supporting the above concepts allow us to redesign the structure-function aspects of the protein molecules. An evolutionary model is necessary and needs to be developed to study these important aspects. The criteria for a well-defined model would be: (a) diversity in structure and function, (b) unique functionality, and (c) must belong to a family to define the evolutionary relationships. All these characteristics are largely fulfilled by bacterial toxins. Bacterial toxins are diverse and widely distributed in all three forms of life (Bacteria, Archaea and Eukaryotes). Some of the unique characteristics include structural folding, sequence and functional combination of domains, targeting a cellular process to execute their function, and most importantly their flexibility and dynamics. In this work, we summarize certain unique aspects of bacterial toxins, including role of structure in defining toxin function, uniqueness in their enzymatic function, and interaction with their substrates and other proteins. Finally, we have discussed the evolutionary aspects of toxins in detail, which will help us rethink the current evolutionary theories. A careful study, and appropriate interpretations, will provide answers to several questions related to the structure-function relationship of proteins, in general. Additionally, this will also allow us to refine the current evolution theories.

Botulinum neurotoxin (BoNT) is responsible for botulism, a clinical condition resulting in flaccid muscle paralysis and potentially death. The light chain is responsible for its intracellular toxicity through its endopeptidase activity. Available crystal structures of BoNT/A light chains (LCA) are based on various truncated versions (tLCA) of the full-length LCA (fLCA) and do not necessarily reflect the true structure of LCA in solution. The understanding of the mechanism of action, longevity of intoxication, and an improved development of endopeptidase inhibitors are dependent on first having a better insight into the structure of LCA in solution. Using an array of biophysical techniques, we report that the fLCA structure is significantly more flexible than tLCA in solution, which may be responsible for its dramatically higher enzymatic activity. This seems to be achieved by a much stronger, more rapid binding to substrate (SNAP-25) of the fLCA compared to tLCA. These results suggest that the C-terminus of LCA plays a critical role in introducing a flexible structure, which is essential for its biological function. This is the first report of such a massive structural role of the C-terminus of a protein being critical for maintaining a functional state.

Alzheimer's disease (AD) is a neurodegenerative disorder in humans. It is responsible for memory loss and it is a major contributor (60-70 %) for dementia. The definite cause of AD still remains unclear. There are hypothesis that holds acetylcholinesterase and butylcholinesterase - enzymes responsible for hydrolysis of acetylcholine, a neurotransmitter in the human brain into choline and acetate. Acetylcholine plays a significant role in memory boost. Tyrosinase, which catalyzes the oxidation of phenols like tyrosine and dopamine, can produce reactive oxygen and nitrogen species leading to cell death. Blueberries, classified under genus Vaccinium, are known to have anti-oxidant phytonutrients with high levels of anthocyanins and phenolic content that helps in anti-aging. This research primarily focuses on examination of blueberries for the inhibition of acetylcholinesterase and tyrosinase in-vitro and at cellular level. Blueberry samples were extracted to obtain polar compounds and were subsequently identified using LC-MS from Waters ACQUITYTM UPLC-Q-TOF-MS system with a BEH C18 column. The blueberry extract showed strong inhibition on acetylcholinesterase and tyrosinase, with the half maximal inhibitory concentration (IC50) of 94 μg/mL for acetylcholinesterase and IC50 of 90 μg/mL for tyrosinase. The inhibition of acetylcholinesterase by blueberry is uncompetitive in nature. Furthermore, our result demonstrated that blueberry extract inhibited the synthesis of acetylcholinesterase in cellular model.

The long-lasting endopeptidase activity of BoNT is a critical biological activity inside the nerve cell, as it prompts proteolysis of the SNARE proteins, involved in the exocytosis of the neurotransmitter acetylcholine. Thus, the BoNT endopeptidase activity is an appropriate clinical target for designing new small-molecule antidotes against BoNT with the potential to reverse the paralysis syndrome of botulism. In principle, small-molecule inhibitors (SMIs) can gain entry into BoNT-intoxicated cells if they have a suitable octanol-water partition coefficient (log P ) value and other favorable characteristics (P. Leeson, Nature 481:455–456, 2012, https://doi.org/10.1038/481455a ). Several efforts have been made in the past to develop SMIs, but inhibitors effective under in vitro conditions have not in general been effective in vivo or in cellular models (L. M. Eubanks, M. S. Hixon, W. Jin, S. Hong, et al., Proc Natl Acad Sci U S A 104:2602–2607, 2007, https://doi.org/10.1073/pnas.0611213104 ). The difference between the in vitro and cellular efficacy presumably results from difficulties experienced by the compounds in crossing the cell membrane, in conjunction with poor bioavailability and high cytotoxicity. The screened nitrophenyl psoralen (NPP) effectively antagonized BoNT/A in both in vitro and ex vivo assays. Importantly, NPP inhibited the BoNT/A light chain but not other general zinc endopeptidases, such as thermolysin, suggesting high selectivity for its target. Small-molecule (nonpeptidic) inhibitors have better oral bioavailability, better stability, and better tissue and cell permeation than antitoxins or peptide inhibitors.

Botulinum neurotoxins (BoNTs) are Category A agents on the NIAID (National Institute of Allergy and Infectious Diseases) priority pathogen list owing to their extreme toxicity and the relative ease of production. These deadly toxins, in minute quantities (estimated human i.v. lethal dose LD50 of 1–2 ng/kg body weight), cause fatal flaccid paralysis by blocking neurotransmitter release. The current gold standard detection method, the mouse-bioassay, often takes days to confirm botulism. Furthermore, there are no effective antidotes known to reverse the symptoms of botulism, and as a result, patients with severe botulism often require meticulous care during the prolonged paralytic illness. To combat potential bio-terrorism incidents of botulinum neurotoxins, their rapid detection is paramount. Surface plasmon resonance (SPR) is a very sensitive technique to examine bio-molecular interactions. The label-free, real-time analysis, with high sensitivity and low sample consumption makes this technology particularly suitable for detection of the toxin. In this study, we demonstrated the feasibility in an assay with a newly designed SPR instrument for the rapid detection of botulinum neurotoxins. The LOD (limit of detection) of the Newton Photonics (NP) SPR based assay is 6.76 pg/mL for Botulinum Neurotoxin type A Light Chain (BoNT/A LC). We established that the detection sensitivity of the system is comparable to the traditional mouse LD50 bioassay in BoNT/A using this SPR technology.

A double-mutant E224A/E262A full-length botulinum neurotoxin (BoNT) Type A with structural similarity to native BoNT/A but lacking the endopeptidase activity provides an ideal surrogate for testing pharmacokinetics and immunochemical characteristics of BoNT.

Botulinum neurotoxin (BoNT), a category A agent, is the most toxic molecule known to mankind. The endopeptidase activity of light chain domain of BoNT is the cause for the inhibition of the neurotransmitter release and the flaccid paralysis that leads to lethality in botulism. Currently, antidotes are not available to reverse the flaccid paralysis caused by BoNT. In the present study, a non-radioactive-based systematic evolution of ligands by exponential enrichment (SELEX) process is developed by utilizing surface plasmon resonance to monitor the binding enrichment. Two RNA aptamers have been identified as strong binders against light chain of botulinum neurotoxin type A. These two aptamers showed strong inhibition activity on LCA, with IC50 in nanomolar range. Inhibition kinetic studies reveal mid nanomolar K-I and non-competitive nature of their inhibition, suggesting that they have strong potential as antidotes that can reverse the symptom caused by BoNT/A. More importantly, we observed that the 2'-fluorine-pyrimidine-modified RNA aptamers identified here do not change their binding and biological activities. This observation could lead to a cost-effective way for SELEX, by using regular nucleotide during SELEX, and 2'-fluorine-pyrimidine-modified nucleotide for final application to enhance their RNase-resistance.

Botulinum neurotoxins (BoNTs) are the most toxic substances to mankind, and yet, there are no therapeutics available to reverse the paralysis, the deadly symptom of botulism caused by BoNTs. Here we report that RNA aptamer, selected through in vitro process against light chain of botulinum neurotoxin type A (BoNT/A) showed in vivo protection efficacy against botulism. Our results showed that RNA aptamer not only accelerated the recovery of local paralysis induced by BoNT/A through mouse exercise wheel assay, but also significantly delayed the time-to-death through mouse protection assay, and rescued one mouse (out of six) completely intoxicated by BoNT/A. Our results demonstrated the potential using RNA aptamer as antidote against deadly botulism.

Biological toxins can be used by hostile states or terrorists as biological weapons and pose a great threat to public health and homeland security. Yet there are no effective countermeasures for most biological toxins. Aptamers are short single-stranded DNA or RNA oligonucleotides with high affinity and specificity to their targets. They are selected through the entire in vitro process and have many advantages over antibodies, leading them to have great potential for many therapeutic and diagnostic applications. In this chapter, the potential of aptamers as novel countermeasures and detection platform against biological toxins has been reviewed, along with the discussion of four biological toxins: ricin, botulinum neurotoxins (BoNTs), staphylococcal enterotoxin B (SEB), and epsilon toxin of Clostridium perfringens, which are among the highest level of biothreat agents. This approach opens new avenues to combat biothreat and bioterrorism against public health and national security.

The structure-function relationship of Botulinum Neurotoxin (BoNT) proteins is greatly influenced by pH. While the low pH of endosome favors membrane interaction of the heavy chain (HC) for the formation of a membrane channel and translocation of the light chain (LC), the catalytic activity of the LC requires a neutral pH for cleavage of the soluble NSF attachment protein receptor (SNARE) complex in the cytosol. In this study, we monitored secondary structural characteristics of LC, HC and holotoxin at individual pHs 4.5 and 7.2 and at the transition pH4.5 to 7.2 to identify the structural signatures underlying their function. The HC showed higher thermal stability at pH4.5 with a melting temperature (Tm) of 60.4°C. The structural analysis of HC in the presence of liposomes showed no difference in ellipticity with that of HC at pH7.2 at 208 and 222 nm but a 25.2% decrease in ellipticity at 208 nm at acidic pH, indicating low pH-induced structural changes that might facilitate interaction with the membrane. Further, HC showed 18% release of K+ ions from liposomes at pH4.5 as against 6% at neutral pH, reinforcing its role in membrane channel formation. LC on the other hand, showed maximum ellipticity at pH7.2, a condition that is relevant to its endopeptidase activity in the cytosol of the neurons. Also, the similarity in the structures at pH7.2 and transition pH4.5 to 7.2 suggested that the flexibility acquired by the protein at low pH was reversible upon exposure to neutral pH for cleavage of SNARE proteins.

Botulinum neurotoxins (BoNTs) are the most poisonous substances known to mankind, which act on the peripheral nervous system leading to flaccid paralysis. Although co-crystal structure of BoNT/A light chain (LC) reveals some unique features of the biological function of this molecule, structural characteristics in solution reveal its dynamic features, not available through the published crystal structures. In this study, we have examined internal flexibility of this molecule by measuring rotational correlation time as a function of viscosity, using frequency domain fluorescence anisotropy decay technique. Fluorescence anisotropy decay of BoNT/A LC resolved sub-nanosecond local motion (faster component), interpreted as internal flexibility of the molecule was affected significantly with viscosity. Both local and global movements were affected by viscosity, which indicates the accessibility of protein core and flexibility of overall structure. In conclusion, this work demonstrates the presence of flexibility in the internal peptide segments, which appears to play a significant role in BoNT/A LC biological function.

Botulinum neurotoxins (BoNTs) are proteins of great interest not only because of their extreme toxicity but also paradoxically for their therapeutic applications. All the known serotypes (A-G) have varying degrees of longevity and potency inside the neuronal cell. Differential chemical modifications such as phosphorylation and ubiquitination have been suggested as possible mechanisms for their longevity, but the molecular basis of the longevity remains unclear. Since the endopeptidase domain (light chain; LC) of toxin apparently survives inside the neuronal cells for months, it is important to examine the structural features of this domain to understand its resistance to intracellular degradation. Published crystal structures (both botulinum neurotoxins and endopeptidase domain) have not provided adequate explanation for the intracellular longevity of the domain. Structural features obtained from spectroscopic analysis of LCA and LCB were similar, and a PRIME (PReImminent Molten Globule Enzyme) conformation appears to be responsible for their optimal enzymatic activity at 37°C. LCE, on the other hand, was although optimally active at 37°C, but its active conformation differed from the PRIME conformation of LCA and LCB. This study establishes and confirms our earlier finding that an optimally active conformation of these proteins in the form of PRIME exists for the most poisonous poison, botulinum neurotoxin. There are substantial variations in the structural and functional characteristics of these active molten globule related structures among the three BoNT endopeptidases examined. These differential conformations of LCs are important in understanding the fundamental structural features of proteins, and their possible connection to intracellular longevity could provide significant clues for devising new countermeasures and effective therapeutics.

Seven serotypes of botulinum neurotoxins, the most toxic substances known to mankind, are each produced by different strains of Clostridium botulinum along with a group of neurotoxin-associated proteins (NAPs). NAPs play a critical role in the toxicoinfection process of botulism in addition to their role in protecting the neurotoxin from proteolytic digestion in the GI tract as well as from adverse environmental conditions. In this study we have investigated the effect of temperature on the structural and functional stability of BoNT/A complex (BoNT/AC) and BoNT/E complex (BoNT/EC). Although the NAPs in the two complexes are quite different, both groups of NAPs activate the endopeptidase activities of their BoNTs without any need to reduce the disulfide bonds between light and heavy chains of respective BoNTs. BoNT/AC attains optimum enzyme activity at the physiological temperature of 37 degrees C whereas BoNT/EC is maximally active at 45 degrees C, and this is accompanied by conformational alterations in its polypeptide folding at this temperature, leading to favorable binding with its intracellular substrate, SNAP-25, and subsequent cleavage of the latter. BoNT/A in its complex form is found to be structurally more stable against temperature whereas BoNT/E in its complex form is functionally better protected against temperature. Based on the analysis of isolated NAPs we have observed that the structural stability of the BoNT/AC is contributed by the NAPs. In addition to the unique structural conditions in which the enzyme remains active, functional stability of botulinum neurotoxins against temperature plays a critical role in the survival of the agent in cooked food and in food-borne botulism.