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571 results on '"James, T."'

3. Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

Author

Zhang, Jianye, Choi, Elliot H, Tworak, Aleksander, Salom, David, Leinonen, Henri, Sander, Christopher L, Hoang, Thanh V, Handa, James T, Blackshaw, Seth, Palczewska, Grazyna, Kiser, Philip D, and Palczewski, Krzysztof

Subjects
Biomedical and Clinical Sciences, Ophthalmology and Optometry, Neurosciences, Eye Disease and Disorders of Vision, Eye, Animals, Cattle, Eye Proteins, Humans, Mice, Models, Molecular, Molecular Structure, RNA-Seq, Receptors, G-Protein-Coupled, Retinal Pigment Epithelium, Retinaldehyde, Stereoisomerism, enzymatic photoisomerization, 11-cis-retinal, 13-cis-retinal, visual chromophore, RPE-retinal G protein-coupled receptor, retinal pigment epithelium-specific 65 kDa protein, visible light, cellular retinaldehyde-binding protein, rhodopsin, cone opsin, G-protein–coupled receptor, retina, retinoid-binding protein, retinol, Chemical Sciences, Biological Sciences, Medical and Health Sciences, Biochemistry & Molecular Biology, Biological sciences, Biomedical and clinical sciences, Chemical sciences
Abstract

Photoisomerization of the 11-cis-retinal chromophore of rod and cone visual pigments to an all-trans-configuration is the initiating event for vision in vertebrates. The regeneration of 11-cis-retinal, necessary for sustained visual function, is an endergonic process normally conducted by specialized enzyme systems. However, 11-cis-retinal also can be formed through reverse photoisomerization from all-trans-retinal. A nonvisual opsin known as retinal pigment epithelium (RPE)-retinal G-protein-coupled receptor (RGR) was previously shown to mediate visual chromophore regeneration in photic conditions, but conflicting results have cast doubt on its role as a photoisomerase. Here, we describe high-level production of 11-cis-retinal from RPE membranes stimulated by illumination at a narrow band of wavelengths. This activity was associated with RGR and enhanced by cellular retinaldehyde-binding protein (CRALBP), which binds the 11-cis-retinal produced by RGR and prevents its re-isomerization to all-trans-retinal. The activity was recapitulated with cells heterologously expressing RGR and with purified recombinant RGR. Using an RGR variant, K255A, we confirmed that a Schiff base linkage at Lys-255 is critical for substrate binding and isomerization. Single-cell RNA-Seq analysis of the retina and RPE tissue confirmed that RGR is expressed in human and bovine RPE and Müller glia, whereas mouse RGR is expressed in RPE but not in Müller glia. These results provide key insights into the mechanisms of physiological retinoid photoisomerization and suggest a novel mechanism by which RGR, in concert with CRALBP, regenerates the visual chromophore in the RPE under sustained light conditions.

Published
2019

4. The selective estrogen receptor modulator raloxifene mitigates the effect of all-trans-retinal toxicity in photoreceptor degeneration

Author

Getter, Tamar, Suh, Susie, Hoang, Thanh, Handa, James T, Dong, Zhiqian, Ma, Xiuli, Chen, Yuanyuan, Blackshaw, Seth, and Palczewski, Krzysztof

Subjects
Biomedical and Clinical Sciences, Ophthalmology and Optometry, Neurosciences, Macular Degeneration, Neurodegenerative, Estrogen, Rare Diseases, Prevention, Eye Disease and Disorders of Vision, Eye, ATP-Binding Cassette Transporters, Alcohol Oxidoreductases, Animals, Female, Male, Mice, Mice, Knockout, Photoreceptor Cells, Vertebrate, Protective Agents, Raloxifene Hydrochloride, Retinal Degeneration, Retinal Pigment Epithelium, Retinaldehyde, Selective Estrogen Receptor Modulators, retina, vitamin A, vision, retinal degeneration, rhodopsin, all-trans-retinal, high-throughput screen, estrogen receptor, selective estrogen receptor modulator, photoreceptor degeneration, blindness, cytotoxicity, cell death, Chemical Sciences, Biological Sciences, Medical and Health Sciences, Biochemistry & Molecular Biology, Biological sciences, Biomedical and clinical sciences, Chemical sciences
Abstract

The retinoid cycle is a metabolic process in the vertebrate retina that continuously regenerates 11-cis-retinal (11-cisRAL) from the all-trans-retinal (atRAL) isomer. atRAL accumulation can cause photoreceptor degeneration and irreversible visual dysfunction associated with incurable blinding retinal diseases, such as Stargardt disease, retinitis pigmentosa (RP), and atrophic age-related macular degeneration (AMD). The underlying cellular mechanisms leading to retinal degeneration remain uncertain, although previous studies have shown that atRAL promotes calcium influx associated with cell apoptosis. To identify compounds that mitigate the effects of atRAL toxicity, here we developed an unbiased and robust image-based assay that can detect changes in intracellular calcium levels in U2OS cells. Using our assay in a high-throughput screen of 2,400 compounds, we noted that selective estrogen receptor modulators (SERMs) potently stabilize intracellular calcium and thereby counteract atRAL-induced toxicity. In a light-induced retinal degeneration mouse model (Abca4 -/- Rdh8 -/-), raloxifene (a benzothiophene-type scaffold SERM) prevented the onset of photoreceptor apoptosis and thus protected the retina from degeneration. The minor structural differences between raloxifene and one of its derivatives (Y 134) had a major impact on calcium homeostasis after atRAL exposure in vitro, and we verified this differential impact in vivo In summary, the SERM raloxifene has structural and functional neuroprotective effects in the retina. We propose that the highly sensitive image-based assay developed here could be applied for the discovery of additional drug candidates preventing photoreceptor degeneration.

Published
2019

7. Quantitative time-course metabolomics in human red blood cells reveal the temperature dependence of human metabolic networks

Author

Yurkovich, James T, Zielinski, Daniel C, Yang, Laurence, Paglia, Giuseppe, Rolfsson, Ottar, Sigurjónsson, Ólafur E, Broddrick, Jared T, Bordbar, Aarash, Wichuk, Kristine, Brynjólfsson, Sigurður, Palsson, Sirus, Gudmundsson, Sveinn, and Palsson, Bernhard O

Subjects
Erythrocytes, Glycolysis, Humans, In Vitro Techniques, Metabolomics, Temperature, computational biology, erythrocyte, metabolism, metabolomics, systems biology, Chemical Sciences, Biological Sciences, Medical and Health Sciences, Biochemistry & Molecular Biology
Abstract

The temperature dependence of biological processes has been studied at the levels of individual biochemical reactions and organism physiology (e.g. basal metabolic rates) but has not been examined at the metabolic network level. Here, we used a systems biology approach to characterize the temperature dependence of the human red blood cell (RBC) metabolic network between 4 and 37 °C through absolutely quantified exo- and endometabolomics data. We used an Arrhenius-type model (Q10) to describe how the rate of a biochemical process changes with every 10 °C change in temperature. Multivariate statistical analysis of the metabolomics data revealed that the same metabolic network-level trends previously reported for RBCs at 4 °C were conserved but accelerated with increasing temperature. We calculated a median Q10 coefficient of 2.89 ± 1.03, within the expected range of 2-3 for biological processes, for 48 individual metabolite concentrations. We then integrated these metabolomics measurements into a cell-scale metabolic model to study pathway usage, calculating a median Q10 coefficient of 2.73 ± 0.75 for 35 reaction fluxes. The relative fluxes through glycolysis and nucleotide metabolism pathways were consistent across the studied temperature range despite the non-uniform distributions of Q10 coefficients of individual metabolites and reaction fluxes. Together, these results indicate that the rate of change of network-level responses to temperature differences in RBC metabolism is consistent between 4 and 37 °C. More broadly, we provide a baseline characterization of a biochemical network given no transcriptional or translational regulation that can be used to explore the temperature dependence of metabolism.

Published
2017

8. A simple and versatile system for the ATP-dependent assembly of chromatin

Author

Khuong, Mai T, Fei, Jia, Cruz-Becerra, Grisel, and Kadonaga, James T

Subjects
Biochemistry and Cell Biology, Bioinformatics and Computational Biology, Genetics, Biological Sciences, Human Genome, Biotechnology, Underpinning research, 1.1 Normal biological development and functioning, Generic health relevance, Adenosine Triphosphatases, Adenosine Triphosphate, Animals, Chromatin, Chromatin Assembly and Disassembly, DNA, Drosophila melanogaster, Histones, Humans, Nucleoplasmins, Nucleosome Assembly Protein 1, Nucleosomes, Transcription Factors, chromatin, chromatin structure, histone, histone chaperone, nucleosome, Chemical Sciences, Medical and Health Sciences, Biochemistry & Molecular Biology, Biological sciences, Biomedical and clinical sciences, Chemical sciences
Abstract

Chromatin is the natural form of DNA in the eukaryotic nucleus and is the substrate for diverse biological phenomena. The functional analysis of these processes ideally would be carried out with nucleosomal templates that are assembled with customized core histones, DNA sequences, and chromosomal proteins. Here we report a simple, reliable, and versatile method for the ATP-dependent assembly of evenly spaced nucleosome arrays. This minimal chromatin assembly system comprises the Drosophila nucleoplasmin-like protein (dNLP) histone chaperone, the imitation switch (ISWI) ATP-driven motor protein, core histones, template DNA, and ATP. The dNLP and ISWI components were synthesized in bacteria, and each protein could be purified in a single step by affinity chromatography. We show that the dNLP-ISWI system can be used with different DNA sequences, linear or circular DNA, bulk genomic DNA, recombinant or native Drosophila core histones, native human histones, the linker histone H1, the non-histone chromosomal protein HMGN2, and the core histone variants H3.3 and H2A.V. The dNLP-ISWI system should be accessible to a wide range of researchers and enable the assembly of customized chromatin with specifically desired DNA sequences, core histones, and other chromosomal proteins.

Published
2017

33. Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

Author

Philip D. Kiser, Thanh Hoang, David Salom, X. Krzysztof Palczewski, Aleksander Tworak, Christopher L. Sander, Jianye Zhang, Henri Leinonen, X. Seth Blackshaw, Grazyna Palczewska, James T. Handa, and Elliot H. Choi

Subjects
0301 basic medicine, Retina, Opsin, 11-cis retinal, Retinal pigment epithelium, genetic structures, 030102 biochemistry & molecular biology, biology, Chemistry, Retinoid binding protein, Photoisomerase, Cell Biology, Biochemistry, eye diseases, Cell biology, 03 medical and health sciences, 030104 developmental biology, medicine.anatomical_structure, Rhodopsin, medicine, biology.protein, sense organs, Molecular Biology, Muller glia
Abstract

Photoisomerization of the 11-cis-retinal chromophore of rod and cone visual pigments to an all-trans-configuration is the initiating event for vision in vertebrates. The regeneration of 11-cis-retinal, necessary for sustained visual function, is an endergonic process normally conducted by specialized enzyme systems. However, 11-cis-retinal also can be formed through reverse photoisomerization from all-trans-retinal. A nonvisual opsin known as retinal pigment epithelium (RPE)-retinal G-protein–coupled receptor (RGR) was previously shown to mediate visual chromophore regeneration in photic conditions, but conflicting results have cast doubt on its role as a photoisomerase. Here, we describe high-level production of 11-cis-retinal from RPE membranes stimulated by illumination at a narrow band of wavelengths. This activity was associated with RGR and enhanced by cellular retinaldehyde-binding protein (CRALBP), which binds the 11-cis-retinal produced by RGR and prevents its re-isomerization to all-trans-retinal. The activity was recapitulated with cells heterologously expressing RGR and with purified recombinant RGR. Using an RGR variant, K255A, we confirmed that a Schiff base linkage at Lys-255 is critical for substrate binding and isomerization. Single-cell RNA-Seq analysis of the retina and RPE tissue confirmed that RGR is expressed in human and bovine RPE and Muller glia, whereas mouse RGR is expressed in RPE but not in Muller glia. These results provide key insights into the mechanisms of physiological retinoid photoisomerization and suggest a novel mechanism by which RGR, in concert with CRALBP, regenerates the visual chromophore in the RPE under sustained light conditions.

Published
2019
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34. The selective estrogen receptor modulator raloxifene mitigates the effect of all-trans-retinal toxicity in photoreceptor degeneration

Author

Susie Suh, Seth Blackshaw, Tamar Getter, Yuanyuan Chen, Xiuli Ma, Zhiqian Dong, Krzysztof Palczewski, James T. Handa, and Thanh Hoang

Subjects
0301 basic medicine, Retinal degeneration, Retina, genetic structures, 030102 biochemistry & molecular biology, Retinal, Cell Biology, Macular degeneration, medicine.disease, Biochemistry, eye diseases, Cell biology, Stargardt disease, 03 medical and health sciences, chemistry.chemical_compound, 030104 developmental biology, medicine.anatomical_structure, chemistry, Selective estrogen receptor modulator, Retinitis pigmentosa, medicine, Raloxifene, sense organs, Molecular Biology, medicine.drug
Abstract

The retinoid cycle is a metabolic process in the vertebrate retina that continuously regenerates 11-cis-retinal (11-cisRAL) from the all-trans-retinal (atRAL) isomer. atRAL accumulation can cause photoreceptor degeneration and irreversible visual dysfunction associated with incurable blinding retinal diseases, such as Stargardt disease, retinitis pigmentosa (RP), and atrophic age-related macular degeneration (AMD). The underlying cellular mechanisms leading to retinal degeneration remain uncertain, although previous studies have shown that atRAL promotes calcium influx associated with cell apoptosis. To identify compounds that mitigate the effects of atRAL toxicity, here we developed an unbiased and robust image-based assay that can detect changes in intracellular calcium levels in U2OS cells. Using our assay in a high-throughput screen of 2,400 compounds, we noted that selective estrogen receptor modulators (SERMs) potently stabilize intracellular calcium and thereby counteract atRAL-induced toxicity. In a light-induced retinal degeneration mouse model (Abca4−/−Rdh8−/−), raloxifene (a benzothiophene-type scaffold SERM) prevented the onset of photoreceptor apoptosis and thus protected the retina from degeneration. The minor structural differences between raloxifene and one of its derivatives (Y 134) had a major impact on calcium homeostasis after atRAL exposure in vitro, and we verified this differential impact in vivo. In summary, the SERM raloxifene has structural and functional neuroprotective effects in the retina. We propose that the highly sensitive image-based assay developed here could be applied for the discovery of additional drug candidates preventing photoreceptor degeneration.

Published
2019
Full Text
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47. Splice variants of cytosolic polyadenylation element–binding protein 2 (CPEB2) differentially regulate pathways linked to cancer metastasis

Author

Margaret A. Park, Grace Lin, James T. DeLigio, and Charles E. Chalfant

Subjects
0301 basic medicine, Gene isoform, Polyadenylation, Regulator, Breast Neoplasms, Biology, Biochemistry, 03 medical and health sciences, Exon, 0302 clinical medicine, Cell Line, Tumor, Humans, Epithelial–mesenchymal transition, Neoplasm Metastasis, Cyclic AMP Response Element-Binding Protein, Molecular Biology, Messenger RNA, Twist-Related Protein 1, Alternative splicing, Nuclear Proteins, Cell Biology, Anoikis, Hypoxia-Inducible Factor 1, alpha Subunit, Neoplasm Proteins, Alternative Splicing, 030104 developmental biology, 030220 oncology & carcinogenesis, RNA splicing, Cancer research, RNA, Female
Abstract

The translational regulator cytosolic polyadenylation element–binding protein 2 (CPEB2) has two isoforms, CPEB2A and CPEB2B, derived by alternative splicing of RNA into a mature form that either includes or excludes exon 4. Previously, we reported that this splicing event is highly dysregulated in aggressive forms of breast cancers, which overexpress CPEB2B. The loss of CPEB2A with a concomitant increase in CPEB2B was also required for breast cancer cells to resist cell death because of detachment (anoikis resistance) and metastasize in vivo. To examine the mechanism by which CPEB2 isoforms mediate opposing effects on cancer-related phenotypes, we used next generation sequencing of triple negative breast cancer cells in which the isoforms were specifically down-regulated. Down-regulation of the CPEB2B isoform inhibited pathways driving the epithelial-to-mesenchymal transition and hypoxic response, whereas down-regulation of the CPEB2A isoform did not have this effect. Examining key nodes of these pathways showed that CPEB2B induced the expression of regulatory DNA trans-factors (e.g. HIF1α and TWIST1). Specifically, CPEB2B functioned as a translational activator of TWIST1 and HIF1α. Functional studies showed that specific down-regulation of either HIF1α or TWIST1 inhibited the ability of CPEB2B to induce the acquisition of anoikis resistance and drive metastasis. Overall, this study demonstrates that CPEB2 alternative splicing is a major regulator of key cellular pathways linked to anoikis resistance and metastasis.

Published
2017
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