Methods in enzymology最新文献

Pathogenic microorganisms, such as viruses, have threatened human health and will continue to contribute to future epidemics and pandemics, highlighting the importance of developing effective diagnostics. To contain viral outbreaks within populations, fast and early diagnosis of infected individuals is essential. Although current standard methods are highly sensitive and specific, like RT-qPCR, some can have slow turnaround times, which can hinder the prevention of viral transmission. The discovery of CRISPR-Cas systems in bacteria and archaea initially revolutionized the world of genome editing. Intriguingly, CRISPR-Cas enzymes also have the ability to detect nucleic acids with high sensitivity and specificity, which sparked the interest of researchers to also explore their potential in diagnosis of viral pathogens. In particular, the CRISPR-Cas13 system has been used as a tool for detecting viral nucleic acids. Cas13's capability to detect both target RNA and non-specific RNAs has led to the development of detection methods that leverage these characteristics through designing specific detection read-outs. Optimization of viral sample collection, amplification steps and the detection process within the Cas13 detection workflow has resulted in assays with high sensitivity, rapid turnaround times and the capacity for large-scale implementation. This review focuses on the significant innovations of various CRISPR-Cas13-based viral nucleic acid detection methods, comparing their strengths and weaknesses while highlighting Cas13's great potential as a tool for viral diagnostics.

Antizyme is a key regulator of polyamine homeostasis, and the biosynthesis of this short-lived protein is induced in response to the increase of the intracellular polyamine concentration. Once synthesized, antizyme inhibits polyamine transport and directs the ODC subunit to the 26S proteasome, that normalize the polyamine pool in the cell. Here we demonstrated that polyamines induce dimerization of full-length mouse antizyme with the formation of (antizyme)₂-polyamine complex. This can be modulated by C-methylated analogues of spermidine and functionally active 2-methylspermidine turned to be a very poor inducer unlike spermidine and its other C-methylated analogues. The protocols for gram-scale synthesis of C-methylated spermidines and for detecting antizyme dimerization using isothermal titration calorimetry and electrophoresis are described.

Phosphonothrixin (PTX) is a herbicidal natural product characterized by the presence of an acetyl group and a phosphonic acid moiety. It is produced by the actinobacterium Saccharothrix sp. ST-888. The biosynthesis of PTX has been elucidated, and the split-gene transketolase PtxB5/6 has been proposed to mediate the key acetyltransferase reaction in a thiamine diphosphate-dependent manner. In this chapter, we describe the identification of the PTX biosynthetic gene cluster in Saccharothrix sp. ST-888 and detail the experimental procedures used to characterize PTX biosynthetic enzymes. In vitro assays with purified recombinant PtxB5/6 demonstrated that the enzyme recognizes hydroxyethyl-thiamine diphosphate (provided by the acetohydroxyacid synthase homolog PtxB7) as a substrate and catalyzes the stereoselective acetylation of (3-hydroxy-2-oxopropyl)phosphonic acid at the C2 position, ultimately resulting in PTX formation. These experiments collectively highlighted a previously unrecognized reaction mechanism driven by the cooperative actions of the split-gene transketolase PtxB5/6 and the acetohydroxyacid synthase homolog PtxB7.

This chapter describes the development and application of a 96-well electrochemical screening plate for the identification of inhibitors of transketolase, a key enzyme of the pentose phosphate pathway, involved in multiple diseases (cancers, neurodegenerative disorders, and infections). This work details the design and manufacturing of printed circuit board (PCB) plates with screen-printed electrodes, optimized for independent electrochemical readout in each well. It describes the electrochemical assay, including electrode quality control, as well as the high-throughput screening of a chemical library of 1360 molecules using an affordable substrate (L-erythrulose). This proof-of-concept is validated with the transketolase of Escherichia coli, as model of bacterial enzyme. Key advantages include speed, universality, and not requiring chromogenic reagents, offset by the need for specialized equipment and strict electrode quality control. Hits are validated by complementary colorimetric assays to confirm results and determine kinetic parameters.

Transketolase (TKT), a key rate-limiting enzyme in the non-oxidative branch of the pentose phosphate pathway, plays a critical role in metabolic processes including nucleotide synthesis and tumorigenesis. Its inhibitors could modulate the enzyme activity and metabolic flux by competitively binding to the cofactor thiamine pyrophosphate (TPP) or allosteric modulatory sites, demonstrating significant potential in drug development for cancer and infectious diseases. In this chapter, we present a systematic evaluation of the binding, affinity and metabolic regulatory activity of a transketolase inhibitor in terms of binding affinity and metabolic regulatory activity. We previously evaluated binding affinity of TKT-inhibitor using two-dimensional (2D) TKT protein biological chromatography, drug affinity responsive target stability assay (DARTS), cellular thermal shift assay (CETSA), surface plasmon resonance analysis (SPR), competitive binding and molecular docking. Moreover, metabolic regulatory activity of a transketolase inhibitor was characterized using spectrophotometric assay and targeted quantitative metabolites analysis, and anti-tumor activity was determined with patient-derived organoids. Notably, several sections of this chapter were originally published in a paper and have been reproduced here for this book.

Transketolases are ubiquitous enzymes that predominantly control the pentose phosphate pathway and are involved in the synthesis of aromatic amino acids, nucleotides and the regulation of oxidative stress. The development of specific inhibitors of human pathogen transketolases could provide a new class of antibiotics. To answer this question, it is necessary to compare human transketolase with those of pathogenic organisms to ensure they are sufficiently different. This chapter presents two protocols for the expression of human and of thirteen transketolases from priority pathogenic organisms, in order to obtain six new experimental structures by X-ray diffraction. Resolution of the electron density maps was performed using in silico models, using detailed protocol for their generation and validation. The experimental structures and models made possible to map and compare for the first time active sites and monomer-monomer interfaces of transketolases. Being at least 50 residues shorter, animal transketolases have evolved differently from those of bacteria, fungi and parasites. The comparison of the monomer-monomer interface also demonstrates that this zone is highly specific to each transketolase, in contrast to their conserved active site. However, in both areas, human transketolase has a significantly higher number of non-covalent bonds than pathogen transketolases, probably to maintain its shorter structure. These observations suggest that pathogen transketolases can be specifically inhibited, particularly targeting the monomer-monomer interface, without affecting human transketolase activity.

Polyamine metabolism in higher eukaryotes is well studied; however, the mechanism of how the polyamines putrescine, spermidine and spermine enter the cell remains unclear. An effective approach to investigate potential players that function in the uptake of polyamines involves using the Drosophila melanogaster imaginal disc assay. Leg imaginal discs dissected from Drosophila melanogaster wandering third star larvae can be assessed for leg development after 18 h of treatment with hormones to induce this process. The protocol described here details how to use genetically manipulated Drosophila melanogaster to test candidate genes involved in the polyamine transport system, how to dissect leg imaginal discs and how to assess the entry of polyamines into the cells of the imaginal disc.

The rise of antibiotic-resistant bacteria poses a critical threat to public health. A key mechanism by which bacteria acquire resistance is through multidrug efflux pumps that expel toxic compounds under antibiotic pressure. Among these, AcrAB-TolC (composed by AcrA, AcrB and TolC, with AcrB belongs to RND family) and MacAB-TolC (composed by MacA, MacB and TolC, with MacB belongs to ABC family) represent two major families of tripartite efflux pump systems in Gram-negative bacteria, each utilizing the same outer membrane channel TolC but differing in their inner membrane components and energization sources. Understanding assembling and functioning mechanism of these pumps requires cellular environment and precise conformational coordination for effective operation. Electron Cryo-tomography (cryoET), in combination with subtomogram averaging, is a unique approach enable direct visualizing macromolecular assemblies within native cellular contexts at subnanometer resolution without any purification, providing critical insights into their in situ architecture, assembly, and function. In this chapter, we present a detailed protocol for the in situ structural characterization of both AcrAB-TolC and MacAB-TolC efflux pumps in Escherichia coli. This unified workflow is broadly applicable to other efflux pumps on other bacterial strains and provides a starting point for studying antibiotic resistance mechanisms.

Multidrug efflux pumps of the Resistance Nodulation-cell Division (RND) superfamily are integral membrane transporters that play a central role in intrinsic and acquired antibiotic resistance in Gram-negative bacteria. Computational approaches have proven invaluable in complementing experimental studies by providing atomistic insight into substrate recognition, transport mechanisms, and inhibitor binding. In this chapter, we provide detailed protocols and tools for most common computational methods applied to RND efflux systems, including homology modelling, molecular docking, all-atom molecular dynamics simulations, and estimation of binding free energy. Each method is presented with practical details on software, input preparation and analysis strategies. Guidelines are included for avoiding common pitfalls and for ensuring reproducibility across computational platforms. Comparisons of the strengths and limitations of these approaches are provided, together with a word of caution on overclaiming results from in silico models without experimental validation. Finally, we discuss the current landscape of computational applications in efflux research illustrating both the opportunities and caveats of these approaches. Together, these methods enable systematic investigation of transporter dynamics, substrate polyspecificity, and inhibition strategies, and can be adapted to other membrane transporters of clinical relevance.

Tripartite efflux pumps are central to multidrug resistance in Gram-negative bacteria, actively extruding antibiotics across the cell envelope. They operate as tripartite complexes spanning the inner and outer membranes, connected by periplasmic adaptors. Here, we describe the in vitro reconstitution of two representative systems into biomimetic environments: the RND-type pump MexAB-OprM from Pseudomonas aeruginosa and the ABC-type pump MacAB-TolC from Escherichia coli. We provide detailed protocols for heterologous expression and purification of individual subunits, followed by their stepwise incorporation into proteoliposomes, nanodiscs, or amphipols. The protocols are adaptable to other Gram-negative multidrug efflux systems and provide a robust platform for dissecting structure-function relationships.