scholarly journals Reengineering the Specificity of the Highly Selective Clostridium botulinum Protease via Directed Evolution

2020 ◽  
Author(s):  
Rebekah P. Dyer ◽  
Hariny M. Isoda ◽  
Gabriela S. Salcedo ◽  
Gaetano Speciale ◽  
Madison H. Fletcher ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Therapeutic Potential ◽  
Catalytic Efficiency ◽  
Peptide Bond ◽  
Cellular Protein ◽  
Control Loops ◽  
Cleavage Specificity ◽  
Wide Range ◽  
Botulinum Neurotoxin Serotype A ◽  
Exquisite Specificity

AbstractThe botulinum neurotoxin serotype A (BoNT/A) cuts a single peptide bond in SNAP25, an activity used to treat a wide range of diseases. Reengineering the substrate specificity of BoNT/A’s protease domain (LC/A) could expand its therapeutic applications; however, LC/A’s extended substrate recognition (≈60 residues) challenges conventional approaches. We report a directed evolution method for retargeting LC/A’s substrate and retaining its exquisite specificity. The resultant eight-mutation LC/A (omLC/A) has improved cleavage specificity and catalytic efficiency (1300- and 120-fold, respectively) for SNAP23 versus SNAP25 compared to a previously reported LC/A variant. Importantly, the BoNT/A holotoxin equipped with omLC/A infiltrates neurons and retains its SNAP23 activity. The identification of substrate control loops outside BoNT/A’s active site could guide the design of improved BoNT proteases and inhibitors.One Sentence SummaryDirected evolution of the BoNT/A protease targets a new cellular protein, SNAP23, expanding its therapeutic potential.

scholarly journals Directed evolution improves the catalytic efficiency of TEV protease

2019 ◽  
Author(s):  
Mateo I Sanchez ◽  
Alice Y Ting
Keyword(s):  
Directed Evolution ◽  
Catalytic Properties ◽  
Light Exposure ◽  
Catalytic Efficiency ◽  
Tobacco Etch Virus ◽  
Sequence Specificity ◽  
Tobacco Etch Virus Protease ◽  
Tev Protease ◽  
Wide Range ◽  
Catalytic Rate

AbstractTobacco etch virus protease (TEV) is one of the most widely-used proteases in biotechnology because of its exquisite sequence-specificity. A limitation, however, is its slow catalytic rate. We developed a generalizable yeast-based platform for directed evolution of protease catalytic properties. Protease activity is read out via proteolytic release of a membrane-anchored transcription factor, and we temporally regulate access to TEV’s cleavage substrate using a photosensory LOV domain. By gradually decreasing light exposure time, we enriched faster variants of TEV over multiple rounds of selection. Our S153N mutant (uTEV1Δ), when incorporated into the calcium integrator FLARE, improved the signal/background ratio by 27-fold, and enabled recording of neuronal activity in culture with 60-second temporal resolution. Given the widespread use of TEV in biotechnology, both our evolved TEV mutants and the directed evolution platform used to generate them, could be beneficial across a wide range of applications.

scholarly journals An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Parinthon Nearmnala ◽  
Manutsawee Thanaburakorn ◽  
Watanalai Panbangred ◽  
Pimchai Chaiyen ◽  
Narupat Hongdilokkul
Keyword(s):  
Directed Evolution ◽  
Selection Pressure ◽  
Catalytic Efficiency ◽  
Selection System ◽  
In Vivo Selection ◽  
Highly Efficient ◽  
Highly Active ◽  
Wide Range ◽  
Regulated Gene Expression

AbstractIn vivo selection systems are powerful tools for directed evolution of enzymes. The selection pressure of the systems can be tuned by regulating the expression levels of the catalysts. In this work, we engineered a selection system for laboratory evolution of highly active enzymes by incorporating a translationally suppressing cis repressor as well as an inducible promoter to impart stringent and tunable selection pressure. We demonstrated the utility of our selection system by performing directed evolution experiments using TEM β-lactamase as the model enzyme. Five evolutionary rounds afforded a highly active variant exhibiting 440-fold improvement in catalytic efficiency. We also showed that, without the cis repressor, the selection system cannot provide sufficient selection pressure required for evolving highly efficient TEM β-lactamase. The selection system should be applicable for the exploration of catalytic perfection of a wide range of enzymes.

scholarly journals Implications of FBXW7 in Neurodevelopment and Neurodegeneration: Molecular Mechanisms and Therapeutic Potential

2021 ◽  
Vol 15 ◽  
Author(s):  
Yu Yang ◽  
Xuan Zhou ◽  
Xinpeng Liu ◽  
Ruying Song ◽  
Yiming Gao ◽  
...  
Keyword(s):  
Neurodegenerative Disorders ◽  
Molecular Mechanisms ◽  
Therapeutic Potential ◽  
Ubiquitin Proteasome System ◽  
Cellular Protein ◽  
Protein Homeostasis ◽  
Potential Therapeutic Target ◽  
Cellular Processes ◽  
Wide Range ◽  
Ubiquitin Proteasome

The ubiquitin-proteasome system (UPS) mediated protein degradation is crucial to maintain quantitive and functional homeostasis of diverse proteins. Balanced cellular protein homeostasis controlled by UPS is fundamental to normal neurological functions while impairment of UPS can also lead to some neurodevelopmental and neurodegenerative disorders. Functioning as the substrate recognition component of the SCF-type E3 ubiquitin ligase, FBXW7 is essential to multiple aspects of cellular processes via targeting a wide range of substrates for proteasome-mediated degradation. Accumulated evidence shows that FBXW7 is fundamental to neurological functions and especially implicated in neurodevelopment and the nosogenesis of neurodegeneration. In this review, we describe general features of FBXW7 gene and proteins, and mainly present recent findings that highlight the vital roles and molecular mechanisms of FBXW7 in neurodevelopment such as neurogenesis, myelination and cerebral vasculogenesis and in the pathogenesis of some typical neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease. Additionally, we also provide a prospect on focusing FBXW7 as a potential therapeutic target to rescue neurodevelopmental and neurodegenerative impairment.

p-Sulfonic acid calix[n]arene catalyzed synthesis of bioactive heterocycles: A review

2020 ◽  
Vol 24 ◽  
Author(s):  
Bubun Banerjee ◽  
Gurpreet Kaur ◽  
Navdeep Kaur
Keyword(s):  
Supramolecular Chemistry ◽  
Sulfonic Acid ◽  
Catalytic Efficiency ◽  
The Other ◽  
Reaction Conditions ◽  
Metal Free ◽  
Bioactive Scaffolds ◽  
Wide Range ◽  
Efficient Alternative ◽  
Bioactive Heterocycles

: Metal-free organocatalysts are becoming an important tool for the sustainable developments of various bioactive heterocycles. On the other hand, during last two decades, calix[n]arenes have been gaining considerable attention due to their wide range of applicability in the field of supramolecular chemistry. Recently, sulfonic acid functionalized calix[n] arenes are being employed as an efficient alternative catalyst for the synthesis of various bioactive scaffolds. In this review we have summarized the catalytic efficiency of p-sulfonic acid calix[n]arenes for the synthesis of diverse biologically promising scaffolds under various reaction conditions. There is no such review available in the literature showing the catalytic applicability of p-sulfonic acid calix[n]arenes. Therefore, we strongly believe that this review will surely attract those researchers who are interested about this fascinating organocatalyst.

Isoform-Selective PI3K Inhibitors for Various Diseases

2020 ◽  
Vol 20 (12) ◽  
pp. 1074-1092 ◽  
Author(s):  
Rammohan R.Y. Bheemanaboina
Keyword(s):  
Intracellular Signaling ◽  
Kinase Inhibitors ◽  
Therapeutic Potential ◽  
Type I ◽  
Selective Inhibitors ◽  
Pi3k Inhibitors ◽  
Wide Range ◽  
Lipid Kinases ◽  
Isoform Selectivity

Phosphoinositide 3-kinases (PI3Ks) are a family of ubiquitously distributed lipid kinases that control a wide variety of intracellular signaling pathways. Over the years, PI3K has emerged as an attractive target for the development of novel pharmaceuticals to treat cancer and various other diseases. In the last five years, four of the PI3K inhibitors viz. Idelalisib, Copanlisib, Duvelisib, and Alpelisib were approved by the FDA for the treatment of different types of cancer and several other PI3K inhibitors are currently under active clinical development. So far clinical candidates are non-selective kinase inhibitors with various off-target liabilities due to cross-reactivities. Hence, there is a need for the discovery of isoform-selective inhibitors with improved efficacy and fewer side-effects. The development of isoform-selective inhibitors is essential to reveal the unique functions of each isoform and its corresponding therapeutic potential. Although the clinical effect and relative benefit of pan and isoformselective inhibition will ultimately be determined, with the development of drug resistance and the demand for next-generation inhibitors, it will continue to be of great significance to understand the potential mechanism of isoform-selectivity. Because of the important role of type I PI3K family members in various pathophysiological processes, isoform-selective PI3K inhibitors may ultimately have considerable efficacy in a wide range of human diseases. This review summarizes the progress of isoformselective PI3K inhibitors in preclinical and early clinical studies for anticancer and other various diseases.

scholarly journals Bioactive potential of natural biomaterials: identification, retention and assessment of biological properties

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Kieran Joyce ◽  
Georgina Targa Fabra ◽  
Yagmur Bozkurt ◽  
Abhay Pandit
Keyword(s):  
Tissue Engineering ◽  
Therapeutic Potential ◽  
Biological Properties ◽  
Biological Assessment ◽  
Cross Linking ◽  
Natural Polymers ◽  
Drug Delivery Device ◽  
Biomedical Device ◽  
Wide Range ◽  
Bioactive Potential

AbstractBiomaterials have had an increasingly important role in recent decades, in biomedical device design and the development of tissue engineering solutions for cell delivery, drug delivery, device integration, tissue replacement, and more. There is an increasing trend in tissue engineering to use natural substrates, such as macromolecules native to plants and animals to improve the biocompatibility and biodegradability of delivered materials. At the same time, these materials have favourable mechanical properties and often considered to be biologically inert. More importantly, these macromolecules possess innate functions and properties due to their unique chemical composition and structure, which increase their bioactivity and therapeutic potential in a wide range of applications. While much focus has been on integrating these materials into these devices via a spectrum of cross-linking mechanisms, little attention is drawn to residual bioactivity that is often hampered during isolation, purification, and production processes. Herein, we discuss methods of initial material characterisation to determine innate bioactivity, means of material processing including cross-linking, decellularisation, and purification techniques and finally, a biological assessment of retained bioactivity of a final product. This review aims to address considerations for biomaterials design from natural polymers, through the optimisation and preservation of bioactive components that maximise the inherent bioactive potency of the substrate to promote tissue regeneration.

scholarly journals Protein Engineering Approaches to Enhance Fungal Laccase Production in S. cerevisiae

2021 ◽  
Vol 22 (3) ◽  
pp. 1157
Author(s):  
Pablo Aza ◽  
Felipe de Salas ◽  
Gonzalo Molpeceres ◽  
David Rodríguez-Escribano ◽  
Iñigo de la Fuente ◽  
...  
Keyword(s):  
Protein Engineering ◽  
Directed Evolution ◽  
Signal Peptide ◽  
Laccase Activity ◽  
Laccase Production ◽  
Wide Range ◽  
Mating Factor ◽  
Conserved Amino Acids ◽  
Basidiomycete Fungi

Laccases secreted by saprotrophic basidiomycete fungi are versatile biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the sole requirement. Saccharomyces cerevisiae is a preferred host for engineering fungal laccases. To assist the difficult secretion of active enzymes by yeast, the native signal peptide is usually replaced by the preproleader of S. cerevisiae alfa mating factor (MFα1). However, in most cases, only basal enzyme levels are obtained. During directed evolution in S. cerevisiae of laccases fused to the α-factor preproleader, we demonstrated that mutations accumulated in the signal peptide notably raised enzyme secretion. Here we describe different protein engineering approaches carried out to enhance the laccase activity detected in the liquid extracts of S. cerevisiae cultures. We demonstrate the improved secretion of native and engineered laccases by using the fittest mutated α-factor preproleader obtained through successive laccase evolution campaigns in our lab. Special attention is also paid to the role of protein N-glycosylation in laccase production and properties, and to the introduction of conserved amino acids through consensus design enabling the expression of certain laccases otherwise not produced by the yeast. Finally, we revise the contribution of mutations accumulated in laccase coding sequence (CDS) during previous directed evolution campaigns that facilitate enzyme production.

Targeted chemical nucleases have a wide range of untapped applications in biological fields, including gene therapy

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Metal Complex ◽  
Dna Cleavage ◽  
Therapeutic Potential ◽  
Chemical Reactivity ◽  
Major Groove ◽  
Sequence Recognition ◽  
Wide Range ◽  
Chemical Nucleases

A feasible alternative to state-of-the-art enzymatic nucleases was created by regulating the cleavage activity of metal complexes using (covalent or non-covalent) homing agents. Targeted AMNs, unlike enzymatic nucleases, break DNA by an oxidative mechanism and can therefore permanently knock off genes. Compared to larger enzymatic nucleases, the modest size of the metal complex may aid cellular transfection. Furthermore, the painstaking construction of the sequence-specific probe permits a metal complex to be directed to dsDNA's minor or major groove. To direct the chemical reactivity of several small-molecule compounds to dsDNA's minor groove, covalently bonded polyamide samples were used. PNA and DNA were also used to construct antisense and antigen hybrids, with Watson–Crick or Hoogsteen base pairing with major groove nucleobases giving sequence recognition. Click chemistry created chimeric AMN-TFOs with desirable focused effects and negligible off-target cleavage. Clip-Phen-modified TFOs, 230 polypyridyl-modified TFOs, 232 and intercalating phenanthrene-modified TFOs are three contemporary instances of copper AMN–TFOs. All three systems have distinct advantages in maintaining the desired 2:1 phenthroline/copper ratio for DNA cleavage (clip-Phen TFOs), caging the copper center and facilitating efficient ROS-mediated strand scission (polypyridyl-modified TFO) and improving triplex stability (polypyridyl-modified TFO) (phenanthrene-TFOs). Cerium (IV)/EDTA complexes, recently shown to bind and hydrolytically cleave ssDNA/dsDNA junctions and used in conjunction with PNA to successfully introduce genome changes in vitro and in vivo, are another important class of targeted chemical nucleases. The chemical reactivity and wide flexibility of metal complex design, combined with their coupling to sequence specific samples for directed applications, show that these compounds have a wide range of untapped applications in biological fields such as chemotherapy, protein engineering, DNA footprinting, and gene editing. Parallel advancements in cell and tissue targeting will be essential to maximise their therapeutic potential, either by using specific ligands or creating new targeting modalities.

scholarly journals New Pharmacological Opportunities for Betulinic Acid

Planta Medica ◽  
2017 ◽  
Vol 84 (01) ◽  
pp. 8-19 ◽  
Author(s):  
José Ríos ◽  
Salvador Máñez
Keyword(s):  
Metabolic Syndrome ◽  
Natural Product ◽  
Betulinic Acid ◽  
Therapeutic Potential ◽  
Present Review ◽  
Antitumor Effects ◽  
Pharmacological Activities ◽  
Naturally Occurring ◽  
Wide Range ◽  
Birch Trees

AbstractBetulinic acid is a naturally occurring pentacyclic lupane-type triterpenoid usually isolated from birch trees, but present in many other botanical sources. It is found in different plant organs, both as a free aglycon and as glycosyl derivatives. A wide range of pharmacological activities has been described for this triterpenoid, including antiviral and antitumor effects. In addition, several other interesting properties have been identified in the fields of immunity and metabolism, namely antidiabetic, antihyperlipidemic, and anti-inflammatory activities. Taken together, these latter three properties make betulinic acid a highly interesting prospect for treating metabolic syndrome. The present review focuses on the therapeutic potential of this agent, along with several of its semisynthetic derivatives, which could open new frontiers in the use of natural product-based medicines.

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