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Molecular Cell Nov 2016Macroautophagy is an intracellular degradation system that utilizes the autophagosome to deliver cytoplasmic components to the lysosome. Measuring autophagic activity is...
Macroautophagy is an intracellular degradation system that utilizes the autophagosome to deliver cytoplasmic components to the lysosome. Measuring autophagic activity is critically important but remains complicated and challenging. Here, we have developed GFP-LC3-RFP-LC3ΔG, a fluorescent probe to evaluate autophagic flux. This probe is cleaved by endogenous ATG4 proteases into equimolar amounts of GFP-LC3 and RFP-LC3ΔG. GFP-LC3 is degraded by autophagy, while RFP-LC3ΔG remains in the cytosol, serving as an internal control. Thus, autophagic flux can be estimated by calculating the GFP/RFP signal ratio. Using this probe, we re-evaluated previously reported autophagy-modulating compounds, performed a high-throughput screen of an approved drug library, and identified autophagy modulators. Furthermore, we succeeded in measuring both induced and basal autophagic flux in embryos and tissues of zebrafish and mice. The GFP-LC3-RFP-LC3ΔG probe is a simple and quantitative method to evaluate autophagic flux in cultured cells and whole organisms.
Topics: Animals; Autophagy; Cysteine Endopeptidases; DNA-Binding Proteins; Embryo, Nonmammalian; Gene Expression Regulation; Genes, Reporter; Green Fluorescent Proteins; HeLa Cells; High-Throughput Screening Assays; Humans; Lysosomes; Mice; Microtubule-Associated Proteins; Molecular Probes; Nuclear Proteins; Phagosomes; Small Molecule Libraries; Spectrometry, Fluorescence; Ubiquitin-Protein Ligases; Zebrafish
PubMed: 27818143
DOI: 10.1016/j.molcel.2016.09.037 -
Biological & Pharmaceutical Bulletin 2017In vivo molecular imaging is the visualization, characterization, and measurement of biological processes at the molecular and cellular levels in humans and other living... (Review)
Review
In vivo molecular imaging is the visualization, characterization, and measurement of biological processes at the molecular and cellular levels in humans and other living systems. Among the methodologies used in in vivo molecular imaging, two methodologies are of great interest from the view of high sensitivity. One is nuclear medical imaging, and distribution and kinetics of a radiolabeled molecular probe are measured using positron emission tomography (PET) and single-photon emission computed tomography (SPECT). The other is optical molecular imaging, and distribution and kinetics of a fluorescent probe are measured using a fluorescent imaging instrument. In this review, the development of imaging probes for these two methodologies is briefly discussed. In nuclear medical molecular imaging, based on structure-activity-biodistribution relationship studies for small molecule and the concept of "functional unit-binding multifunctional molecular probe" containing 3 functional units (target recognition unit, signal-releasing unit, linker unit) for peptides and proteins, we developed radiolabeled probes with high and specific accumulation to the target for neuroreceptors, β-amyloid plaques, and tau aggregates in the brain, tumors, atherosclerotic plaques, pancreatic β-cell, myocardial sympathetic nerves, and so on. We also discuss the progression of molecular imaging toward therapy (radiotheranotics). In in vivo optical molecular imaging, taking into account the characteristics of optical imaging, we designed tumor-specific optical imaging probes with characteristic imaging mechanism, including near-infrared (NIR) fluorescent probes and activatable probes. Furthermore, we developed a photoacoustic imaging probe, which enables highly sensitive and high-resolution imaging in deep tissues.
Topics: Animals; Fluorescent Dyes; Humans; Molecular Imaging; Molecular Probes
PubMed: 28966233
DOI: 10.1248/bpb.b17-00505 -
Cell Chemical Biology Aug 2020Dynamic proteins perform critical roles in cellular machines, including those that control proteostasis, transcription, translation, and signaling. Thus, dynamic... (Review)
Review
Dynamic proteins perform critical roles in cellular machines, including those that control proteostasis, transcription, translation, and signaling. Thus, dynamic proteins are prime candidates for chemical probe and drug discovery but difficult targets because they do not conform to classical rules of design and screening. Selectivity is pivotal for candidate probe molecules due to the extensive interaction network of these dynamic hubs. Recognition that the traditional rules of probe discovery are not necessarily applicable to dynamic proteins and their complexes, as well as technological advances in screening, have produced remarkable results in the last 2-4 years. Particularly notable are the improvements in target selectivity for small-molecule modulators of dynamic proteins, especially with techniques that increase the discovery likelihood of allosteric regulatory mechanisms. We focus on approaches to small-molecule screening that appear to be more suitable for highly dynamic targets and have the potential to streamline identification of selective modulators.
Topics: Allosteric Regulation; CREB-Binding Protein; HSP70 Heat-Shock Proteins; Models, Molecular; Molecular Probes; Protein Binding; Proteins; Small Molecule Libraries
PubMed: 32783965
DOI: 10.1016/j.chembiol.2020.07.019 -
Cell Chemical Biology Apr 2020Ferroptosis is a recently described form of cell death driven by iron-dependent lipid peroxidation. This type of cell death was first observed in response to treatment... (Review)
Review
Ferroptosis is a recently described form of cell death driven by iron-dependent lipid peroxidation. This type of cell death was first observed in response to treatment of tumor cells with a small-molecule chemical probe named erastin. Most subsequent advances in understanding the mechanisms governing ferroptosis involved the use of genetic screens and small-molecule probes. We describe herein the utility and limitations of chemical probes that have been used to analyze and perturb ferroptosis, as well as mechanistic studies of ferroptosis that benefitted from the use of these probes and genetic screens. We also suggest probes for ferroptosis and highlight mechanistic questions surrounding this form of cell death that will be a high priority for exploration in the future.
Topics: Amino Acid Transport System y+; Energy Metabolism; Ferroptosis; Humans; Iron; Lipid Peroxidation; Molecular Probes; Neoplasms; Phospholipid Hydroperoxide Glutathione Peroxidase; Signal Transduction
PubMed: 32294465
DOI: 10.1016/j.chembiol.2020.03.013 -
Cell Chemical Biology Aug 2020
Topics: Drug Discovery; Molecular Probes; Small Molecule Libraries
PubMed: 32822618
DOI: 10.1016/j.chembiol.2020.08.004 -
Theranostics 2022Exploring and understanding the interaction of changes in the activities of various enzymes, such as proteases, phosphatases, and oxidoreductases with tumor invasion,... (Review)
Review
Exploring and understanding the interaction of changes in the activities of various enzymes, such as proteases, phosphatases, and oxidoreductases with tumor invasion, proliferation, and metastasis is of great significance for early cancer diagnosis. To detect the activity of tumor-related enzymes, various molecular probes have been developed with different imaging methods, including optical imaging, photoacoustic imaging (PAI), magnetic resonance imaging, positron emission tomography, and so on. In this review, we first describe the biological functions of various enzymes and the selectively recognized chemical linkers or groups. Subsequently, we systematically summarize the design mechanism of imaging probes and different imaging methods. Finally, we explore the challenges and development prospects in the field of enzyme activity detection. This comprehensive review will provide more insight into the design and development of enzyme activated molecular probes.
Topics: Humans; Molecular Imaging; Molecular Probes; Neoplasms; Optical Imaging; Photoacoustic Techniques; Tomography, X-Ray Computed
PubMed: 35154500
DOI: 10.7150/thno.66676 -
International Journal of Nanomedicine 2020Fluorine-19 (F) magnetic resonance (MR) molecular imaging is a promising noninvasive and quantitative molecular imaging approach with intensive research due to the high... (Review)
Review
Fluorine-19 (F) magnetic resonance (MR) molecular imaging is a promising noninvasive and quantitative molecular imaging approach with intensive research due to the high sensitivity and low endogenous background signal of the F atom in vivo. Perfluorocarbons (PFCs) have been used as blood substitutes since 1970s. More recently, a variety of PFC nanoparticles have been designed for the detection and imaging of physiological and pathological changes. These molecular imaging probes have been developed to label cells, target specific epitopes in tumors, monitor the prognosis and therapy efficacy and quantitate characterization of tumors and changes in tumor microenvironment noninvasively, therefore, significantly improving the prognosis and therapy efficacy. Herein, we discuss the recent development and applications of F MR techniques with PFC nanoparticles in biomedicine, with particular emphasis on ligand-targeted and quantitative F MR imaging approaches for tumor detection, oxygenation measurement, smart stimulus response and therapy efficacy monitoring, et al.
Topics: Animals; Drug Delivery Systems; Fluorine; Fluorocarbons; Humans; Magnetic Resonance Imaging; Molecular Imaging; Molecular Probes; Nanoparticles; Neoplasms; Oxygen
PubMed: 33061385
DOI: 10.2147/IJN.S255084 -
Proceedings of the National Academy of... Aug 2023Trehalose plays a crucial role in the survival and virulence of the deadly human pathogen (). The type I ATP-binding cassette (ABC) transporter LpqY-SugABC is the sole...
Trehalose plays a crucial role in the survival and virulence of the deadly human pathogen (). The type I ATP-binding cassette (ABC) transporter LpqY-SugABC is the sole pathway for trehalose to enter . The substrate-binding protein, LpqY, which forms a stable complex with the translocator SugABC, recognizes and captures trehalose and its analogues in the periplasmic space, but the precise molecular mechanism for this process is still not well understood. This study reports a 3.02-Å cryoelectron microscopy structure of trehalose-bound LpqY-SugABC in the pretranslocation state, a crystal structure of LpqY in a closed form with trehalose bound and five crystal structures of LpqY in complex with different trehalose analogues. These structures, accompanied by substrate-stimulated ATPase activity data, reveal how LpqY recognizes and binds trehalose and its analogues, and highlight the flexibility in the substrate binding pocket of LpqY. These data provide critical insights into the design of trehalose analogues that could serve as potential molecular probe tools or as anti-TB drugs.
Topics: Humans; Cryoelectron Microscopy; Mycobacterium tuberculosis; Trehalose; ATP-Binding Cassette Transporters; Molecular Probes
PubMed: 37603751
DOI: 10.1073/pnas.2307625120 -
Bioconjugate Chemistry Jun 2015Visualization of biological processes and pathologic conditions at the cellular and tissue levels largely relies on the use of fluorescence intensity signals from... (Review)
Review
Visualization of biological processes and pathologic conditions at the cellular and tissue levels largely relies on the use of fluorescence intensity signals from fluorophores or their bioconjugates. To overcome the concentration dependency of intensity measurements, evaluate subtle molecular interactions, and determine biochemical status of intracellular or extracellular microenvironments, fluorescence lifetime (FLT) imaging has emerged as a reliable imaging method complementary to intensity measurements. Driven by a wide variety of dyes exhibiting stable or environment-responsive FLTs, information multiplexing can be readily accomplished without the need for ratiometric spectral imaging. With knowledge of the fluorescent states of the molecules, it is entirely possible to predict the functional status of biomolecules or microevironment of cells. Whereas the use of FLT spectroscopy and microscopy in biological studies is now well-established, in vivo imaging of biological processes based on FLT imaging techniques is still evolving. This review summarizes recent advances in the application of the FLT of molecular probes for imaging cells and small animal models of human diseases. It also highlights some challenges that continue to limit the full realization of the potential of using FLT molecular probes to address diverse biological problems and outlines areas of potential high impact in the future.
Topics: Animals; Fluorescent Dyes; Humans; Microscopy, Fluorescence; Models, Molecular; Molecular Probes; Optical Imaging; Spectrometry, Fluorescence
PubMed: 25961514
DOI: 10.1021/acs.bioconjchem.5b00167 -
Chembiochem : a European Journal of... Apr 2021RNA molecules can fold into complex two- and three-dimensional shapes that are critical for their function. Chemical probes have long been utilized to interrogate RNA... (Review)
Review
RNA molecules can fold into complex two- and three-dimensional shapes that are critical for their function. Chemical probes have long been utilized to interrogate RNA structure and are now considered invaluable resources in the goal of relating structure to function. Recently, the power of deep sequencing and careful chemical probe design have merged, permitting researchers to obtain a holistic understanding of how RNA structure can be utilized to control RNA biology transcriptome-wide. Within this review, we outline the recent advancements in chemical probe design for interrogating RNA structures inside cells and discuss the recent advances in our understanding of RNA biology through the lens of chemical probing.
Topics: DNA Adducts; DNA, Complementary; Molecular Probes; Nucleic Acid Conformation; RNA; RNA, Messenger; Transcriptome
PubMed: 32737940
DOI: 10.1002/cbic.202000340