Optimizing the Protein Fluorescence Reporting System for Somatic Embryogenesis Regeneration Screening and Visual Labeling of Functional Genes in Cotton

Protein fluorescence reporting systems are of crucial importance to in-depth life science research, providing systematic labeling tools for visualization of microscopic biological activities in vivo and revolutionizing basic research. Cotton somatic cell regeneration efficiency is low, causing difficulty in cotton transformation. It is conducive to screening transgenic somatic embryo using the fluorescence reporting system. However, available fluorescence labeling systems in cotton are currently limited. To optimize the fluorescence reporting system of cotton with an expanded range of available fluorescent proteins, we selected 11 fluorescent proteins covering red, green, yellow, and cyan fluorescence colors and expressed them in cotton. Besides mRuby2 and G3GFP, the other nine fluorescent proteins (mCherry, tdTomato, sfGFP, Clover, EYFP, YPet, mVenus, mCerulean, and ECFP) were stably and intensely expressed in transgenic callus and embryo, and inherited in different cotton organs derive from the screened embryo. In addition, transgenic cotton expressing tdTomato appears pink under white light, not only for callus and embryo tissues but also various organs of mature plants, providing a visual marker in the cotton genetic transformation process, accelerating the evaluation of transgenic events. Further, we constructed transgenic cotton expressing mCherry-labeled organelle markers in vivo that cover seven specific subcellular compartments: plasma membrane, endoplasmic reticulum, tonoplast, mitochondrion, plastid, Golgi apparatus, and peroxisome. We also provide a simple and highly efficient strategy to quickly determine the subcellular localization of uncharacterized proteins in cotton cells using organelle markers. Lastly, we built the first cotton stomatal fluorescence reporting system using stomata-specific expression promoters (ProKST1, ProGbSLSP, and ProGC1) to drive Clover expression. The optimized fluorescence labeling system for transgenic somatic embryo screening and functional gene labeling in this study offers the potential to accelerating somatic cell regeneration efficiency and the in vivo monitoring of diverse cellular processes in cotton.

Ubiquitination Destabilizes Protein Sphingosine Kinase 2 to Regulate Glioma Malignancy

Gliomas are the most common and lethal malignant tumor in the central nervous system. The tumor oncogene sphingosine kinase 2 (SphK2) was previously found to be upregulated in glioma tissues and enhance glioma cell epithelial-to-mesenchymal transition through the AKT/β-catenin pathway. Nevertheless, ubiquitination of SphK2 protein has yet to be well elucidated. In this study, mass spectrometry analysis was performed to identify proteins that interacted with SphK2 protein. Co-immunoprecipitation (co-IP) and immunoblotting (IB) were used to prove the specific interaction between SphK2 protein and the neural precursor cell-expressed developmentally downregulated 4-like (NEDD4L) protein. Fluorescence microscopy was used for detecting the distribution of related proteins. Ubiquitylation assay was utilized to characterize that SphK2 was ubiquitylated by NEDD4L. Cell viability assay, flow cytometry assay, and transwell invasion assay were performed to illustrate the roles of NEDD4L-mediated SphK2 ubiquitination in glioma viability, apoptosis, and invasion, respectively. We found that NEDD4L directly interacted with SphK2 and ubiquinated it for degradation. Ubiquitination of SphK2 mediated by NEDD4L overexpression suppressed glioma cell viability and invasion but promoted glioma apoptosis. Knockdown of NEDD4L presented opposite results. Moreover, further results suggested that ubiquitination of SphK2 regulated glioma malignancy via the AKT/β-catenin pathway. in vivo assay also supported the above findings. This study reveals that NEDD4L mediates SphK2 ubiquitination to regulate glioma malignancy and may provide some meaningful suggestions for glioma treatment.

Urea-based amino sugar agent clears murine liver and preserves protein fluorescence and lipophilic dyes

Five established clearing protocols were compared with a modified and simplified method to determine an optimal clearing reagent for three-dimensionally visualizing fluorophores in the murine liver, a challenging organ to clear. We report successful clearing of whole liver lobes by modification of an established protocol (UbasM) using only Ub-1, a urea-based amino sugar reagent, in a simpler protocol that requires only a 24-h processing time. With Ub-1 alone, we observed sufficiently preserved liver tissue structure in three dimensions along with excellent preservation of fluorophore emissions from endogenous protein reporters and lipophilic tracer dyes. This streamlined technique can be used for 3D cell lineage tracing and fluoroprobe-based reporter gene expression to compare various experimental conditions.

Competition between protons and substrate for binding to the major facilitator superfamily multidrug/H+ antiporter MdtM

Proton electrochemical gradient-driven multidrug efflux activity of representatives of the major facilitator superfamily (MFS) of secondary active transporters contributes to antimicrobial resistance of pathogenic bacteria. Integral to the mechanism of these transporters is a proposed competition between substrate and protons for the binding site of the protein. The current work investigated the competition between protons and antimicrobial substrate for binding to the Escherichia coli MFS multidrug/H+ antiporter MdtM by measuring the quench of intrinsic protein fluorescence upon titration of substrate tetraphenylphosphonium into a solution of purified MdtM over a range of pH values between pH 8.8 and 5.9. The results, which revealed that protons inhibit binding of substrate to MdtM in a competitive manner, are consistent with those reported in a study on the related MFS multidrug/H+ antiporter MdfA and provide further evidence that competition for binding between substrate and protons is a general feature of secondary multidrug efflux.

Retinal glial cells under hypoxia possibly function in mammalian myopia by responding to mechanical stimuli, affecting hormone metabolism and peptide secretion

PurposeChanges in the retina and the choroid blood vessels are regularly observed in myopia. The aim of this study is to test if the retinal glial cells, which directly contact blood vessels, play a role in mammalian myopia.MethodWe adapted the common form-deprivation myopia mouse model and used the retina slice and whole-mount immunofluorescence technique to evaluate changes in the morphology, and distribution of retinal glial cells. We then searched the Gene Expression Omnibus database for series on myopia and retinal glial cells (astrocytes and Müller cells). Using review articles and the National Center for Biotechnology Information gene database, we obtained clusters of myopia-related gene lists. By searching SwissTargetPrediction, we collected information on atropine target proteins. We then used online tools to find the Gene Ontology analysis enriched clusters, pathways, and proteins that provide correlative evidence.ResultGlial fibrillary acidic protein fluorescence was observed in mice eyes that were covered and deprived of light for five days compared to uncovered eyes, and the cell morphology became more star-like. From in silico experiments, we identified several pathways and proteins that were common to both myopia and retinal glial cells in hypoxic conditions. The common pathways in human astrocytes represented response to mechanical stimuli, peptide secretion, skeletal system development, and monosaccharide binding. In mouse Müller cells, the pathways represented membrane raft and extracellular structure organisation. The proteins common to myopia and hypoxic glial cells were highly relevant to atropine target proteins.ConclusionRetinal astrocytes and Müller cells under hypoxic conditions contribute to the development of myopia, and may be a valid target for atropine.

Sensing of Proteins by ICD Response of Iron(II) Clathrochelates Functionalized by Carboxyalkylsulfide Groups

Recognition of elements of protein tertiary structure is crucial for biotechnological and biomedical tasks; this makes the development of optical sensors for certain protein surface elements important. Herein, we demonstrated the ability of iron(II) clathrochelates (1–3) functionalized with mono-, di- and hexa-carboxyalkylsulfide to induce selective circular dichroism (CD) response upon binding to globular proteins. Thus, inherently CD-silent clathrochelates revealed selective inducing of CD spectra when binding to human serum albumin (HSA) (1, 2), beta-lactoglobuline (2) and bovine serum albumin (BSA) (3). Hence, functionalization of iron(II) clathrochelates with the carboxyalkylsulfide group appears to be a promising tool for the design of CD-probes sensitive to certain surface elements of proteins tertiary structure. Additionally, interaction of 1–3 with proteins was also studied by isothermal titration calorimetry, protein fluorescence quenching, electrospray ionization mass spectrometry (ESI-MS) and computer simulations. Formation of both 1:1 and 1:2 assemblies of HSA with 1–3 was evidenced by ESI-MS. A protein fluorescence quenching study suggests that 3 binds with both BSA and HSA via the sites close to Trp residues. Molecular docking calculations indicate that for both BSA and HSA, binding of 3 to Site I and to an “additional site” is more favorable energetically than binding to Site II.

Binding Studies of AICAR and Human Serum Albumin by Spectroscopic, Theoretical, and Computational Methodologies

The interactions of small molecule drugs with plasma serum albumin are important because of the influence of such interactions on the pharmacokinetics of these therapeutic agents. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) is one such drug candidate that has recently gained attention for its promising clinical applications as an anti-cancer agent. This study sheds light upon key aspects of AICAR’s pharmacokinetics, which are not well understood. We performed in-depth experimental and computational binding analyses of AICAR with human serum albumin (HSA) under simulated biochemical conditions, using ligand-dependent fluorescence sensitivity of HSA. This allowed us to characterize the strength and modes of binding, mechanism of fluorescence quenching, validation of FRET, and intermolecular interactions for the AICAR–HSA complexes. We determined that AICAR and HSA form two stable low-energy complexes, leading to conformational changes and quenching of protein fluorescence. Stern–Volmer analysis of the fluorescence data also revealed a collision-independent static mechanism for fluorescence quenching upon formation of the AICAR–HSA complex. Ligand-competitive displacement experiments, using known site-specific ligands for HSA’s binding sites (I, II, and III) suggest that AICAR is capable of binding to both HSA site I (warfarin binding site, subdomain IIA) and site II (flufenamic acid binding site, subdomain IIIA). Computational molecular docking experiments corroborated these site-competitive experiments, revealing key hydrogen bonding interactions involved in stabilization of both AICAR–HSA complexes, reaffirming that AICAR binds to both site I and site II.

Analysis of Ion and pH Effects on Iron Response Element (IRE) and mRNA-Iron Regulatory Protein (IRP1) Interactions

Background: Cellular iron uptake, utilization, and storage are tightly controlled through the action of iron regulatory proteins (IRPs). IRPs achieve this control by binding to IREs-mRNA in the 5'- or 3'-end of mRNAs that encode proteins involved in iron metabolism. The interaction of iron regulatory proteins with mRNAs containing an iron responsive element plays a central role in this regulation. The IRE RNA family of mRNA regulatory structures combines absolutely conserved protein binding sites with phylogenetically conserved base pairs that are specific to each IREs and influence RNA/protein stability. Our previous result revealed the binding and kinetics of IRE RNA with IRP1. The aim of the present study is to gain further insight into the differences in protein/RNA stability as a function of pH and ionic strength. Objective: To determine the extent to which the binding affinity and stability of protein/RNA complex was affected by ionic strength and pH. Methods: Fluorescence spectroscopy was used to characterize IRE RNA-IRP protein interaction. Results: Scatchard analysis revealed that the IRP1 protein binds to a single IRE RNA molecule. The binding affinity of two IRE RNA/IRP was significantly changed with the change in pH. The data suggests that the optimum binding of RNA/IRP complex occurred at pH 7.6. Dissociation constant for two IRE RNA/IRP increased with an increase in ionic strength, with a larger effect for FRT IRE RNA. This suggests that numerous electrostatic interactions occur in the ferritin IRE RNA/IRP than ACO2 IRE RNA/IRP complex. Iodide quenching shows that the majority of the tryptophan residues in IRP1 are solvent-accessible, assuming that most of the tryptophan residues contribute to protein fluorescence. Conclusion: The results obtained from this study clearly indicate that IRE RNA/IRP complex is destabilized by the change in pH and ionic strength. These observations suggest that both pH and ion are important for the assembly and stability of the IRE RNA/IRP complex formation.