Deconstructing and repurposing the light-regulated interplay between Arabidopsis phytochromes and interacting factors

AbstractPhytochrome photoreceptors mediate adaptive responses of plants to red and far-red light. These responses generally entail light-regulated association between phytochromes and other proteins, among them the phytochrome-interacting factors (PIF). The interaction with Arabidopsis thaliana phytochrome B (AtPhyB) localizes to the bipartite APB motif of the A. thaliana PIFs (AtPIF). To address a dearth of quantitative interaction data, we construct and analyze numerous AtPIF3/6 variants. Red-light-activated binding is predominantly mediated by the APB N-terminus, whereas the C-terminus modulates binding and underlies the differential affinity of AtPIF3 and AtPIF6. We identify AtPIF variants of reduced size, monomeric or homodimeric state, and with AtPhyB affinities between 10 and 700 nM. Optogenetically deployed in mammalian cells, the AtPIF variants drive light-regulated gene expression and membrane recruitment, in certain cases reducing basal activity and enhancing regulatory response. Moreover, our results provide hitherto unavailable quantitative insight into the AtPhyB:AtPIF interaction underpinning vital light-dependent responses in plants.

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SUMOylation of phytochrome-B negatively regulates light-induced signaling in Arabidopsis thaliana

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1415260112 ◽

2015 ◽

Vol 112 (35) ◽

pp. 11108-11113 ◽

Cited By ~ 41

Author(s):

Ari Sadanandom ◽

Éva Ádám ◽

Beatriz Orosa ◽

András Viczián ◽

Cornelia Klose ◽

...

Keyword(s):

Molecular Level ◽

Expression Patterns ◽

Red Light ◽

Regulatory Proteins ◽

Light Signaling ◽

Phytochrome B ◽

Sumo Proteases ◽

C Terminus

The red/far red light absorbing photoreceptor phytochrome-B (phyB) cycles between the biologically inactive (Pr, λmax, 660 nm) and active (Pfr; λmax, 730 nm) forms and functions as a light quality and quantity controlled switch to regulate photomorphogenesis in Arabidopsis. At the molecular level, phyB interacts in a conformation-dependent fashion with a battery of downstream regulatory proteins, including PHYTOCHROME INTERACTING FACTOR transcription factors, and by modulating their activity/abundance, it alters expression patterns of genes underlying photomorphogenesis. Here we report that the small ubiquitin-like modifier (SUMO) is conjugated (SUMOylation) to the C terminus of phyB; the accumulation of SUMOylated phyB is enhanced by red light and displays a diurnal pattern in plants grown under light/dark cycles. Our data demonstrate that (i) transgenic plants expressing the mutant phyBLys996Arg-YFP photoreceptor are hypersensitive to red light, (ii) light-induced SUMOylation of the mutant phyB is drastically decreased compared with phyB-YFP, and (iii) SUMOylation of phyB inhibits binding of PHYTOCHROME INTERACTING FACTOR 5 to phyB Pfr. In addition, we show that OVERLY TOLERANT TO SALT 1 (OTS1) de-SUMOylates phyB in vitro, it interacts with phyB in vivo, and the ots1/ots2 mutant is hyposensitive to red light. Taken together, we conclude that SUMOylation of phyB negatively regulates light signaling and it is mediated, at least partly, by the action of OTS SUMO proteases.

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Retrograde induction of phyB orchestrates ethylene-auxin hierarchy to regulate growth

10.1101/2020.01.31.929133 ◽

2020 ◽

Author(s):

Jishan Jiang ◽

Yanmei Xiao ◽

Wei Hu ◽

Hao Chen ◽

Liping Zeng ◽

...

Keyword(s):

Signaling Pathways ◽

Red Light ◽

Fine Tuning ◽

Retrograde Signaling ◽

Phytochrome B ◽

Ethylene Signaling ◽

Adaptive Responses ◽

Hypocotyl Growth ◽

Stress Signal ◽

Ethylene Signaling Pathway

AbstractExquisitely regulated plastid-to-nucleus communication by retrograde signaling pathways is essential for fine-tuning of responses to the prevailing environmental conditions. The plastidial retrograde signaling metabolite methylerythritol cyclodiphosphate (MEcPP) has emerged as a stress signal transduced into a diverse ensemble of response outputs. Here we demonstrate enhanced phytochrome B protein abundance in red-light grown MEcPP-accumulating mutant (ceh1) plant relative to wild-type seedlings. We further establish MEcPP-mediated coordination of phytochrome B with auxin and ethylene signaling pathways, and uncover differential hypocotyl growth of red-light grown seedlings in response to these phytohormones. Genetic and pharmacological interference with ethylene and auxin pathways outline the hierarchy of responses, placing auxin epistatic to the ethylene signaling pathway. Collectively, our finding establishes the key role of a plastidial retrograde metabolite in orchestrating the transduction of a repertoire of signaling cascades, and positions plastids at the zenith of relaying information coordinating external signals and internal regulatory circuitry to secure organismal integrity.Two sentence summaryThe plastidial retrograde metabolite, MEcPP, orchestrates coordination of light and hormonal signaling cascade through induction of phytochrome B abundance and modulation of auxin and ethylene levels for optimal adaptive responses to light environment.

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A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress

Cancers ◽

10.3390/cancers13133317 ◽

2021 ◽

Vol 13 (13) ◽

pp. 3317

Author(s):

Eric Moeglin ◽

Dominique Desplancq ◽

Audrey Stoessel ◽

Christian Massute ◽

Jeremy Ranniger ◽

...

Keyword(s):

Cancer Cells ◽

Mammalian Cells ◽

Chromatin Modification ◽

Replication Stress ◽

Living Cells ◽

Single Step ◽

Dna Double Strand Breaks ◽

Drug Induced ◽

Histone H2ax ◽

C Terminus

Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.

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Racemization-free synthesis of Nα-2-thiophenoyl-phenylalanine-2-morpholinoanilide enantiomers and their antimycobacterial activity

Amino Acids ◽

10.1007/s00726-021-03044-1 ◽

2021 ◽

Author(s):

Lea Mann ◽

Markus Lang ◽

Philipp Schulze ◽

Jan Henrik Halz ◽

René Csuk ◽

...

Keyword(s):

Title Compound ◽

Mammalian Cells ◽

Antimycobacterial Activity ◽

Lead Compound ◽

Coupling Reagents ◽

Hydrogen Bonding Interactions ◽

Amide Coupling ◽

Subsequent Separation ◽

Synthetic Routes ◽

Insight Into

AbstractNα-2-thiophenoyl-d-phenylalanine-2-morpholinoanilide (MMV688845, IUPAC: N-(1-((2-morpholinophenyl)amino)-1-oxo-3-phenylpropan-2-yl)thiophene-2-carboxamide) from the Pathogen Box® library (Medicines for Malaria Ventures, MMV) is a promising lead compound for antimycobacterial drug development. Two straightforward synthetic routes to the title compound starting from phenylalanine or its Boc-protected derivative are reported. Employing Boc-phenylalanine as starting material and the T3P® and PyBOP® amide coupling reagents enables racemization-free synthesis, avoiding the need for subsequent separation of the enantiomers. The crystal structure of the racemic counterpart gives insight into the molecular structure and hydrogen bonding interactions in the solid state. The R-enantiomer of the title compound (derived from d-phenylalanine) exhibits activity against non-pathogenic and pathogenic mycobacterial strains, whereas the S-enantiomer is inactive. Neither of the enantiomers and the racemate of the title compound shows cytotoxicity against various mammalian cells.

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Arabidopsis Phytochrome B Promotes SPA1 Nuclear Accumulation to Repress Photomorphogenesis under Far-Red Light

The Plant Cell ◽

10.1105/tpc.112.107086 ◽

2013 ◽

Vol 25 (1) ◽

pp. 115-133 ◽

Cited By ~ 38

Author(s):

Xu Zheng ◽

Suowei Wu ◽

Huqu Zhai ◽

Peng Zhou ◽

Meifang Song ◽

...

Keyword(s):

Red Light ◽

Nuclear Accumulation ◽

Phytochrome B

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Limitations of Allotopic Expression of Mitochondrial Genes in Mammalian Cells

Genetics ◽

10.1093/genetics/165.2.707 ◽

2003 ◽

Vol 165 (2) ◽

pp. 707-720

Author(s):

Jose Oca-Cossio ◽

Lesley Kenyon ◽

Huiling Hao ◽

Carlos T Moraes

Keyword(s):

Mitochondrial Dna ◽

Mammalian Cells ◽

Mitochondrial Genes ◽

Terminal Segment ◽

Universal Genetic Code ◽

Apocytochrome B ◽

Dna Mutations ◽

C Terminus ◽

Cytoplasmic Compartment ◽

Allotopic Expression

Abstract The possibility of expressing mitochondrial DNA-coded genes in the nuclear-cytoplasmic compartment provides an attractive system for genetic treatment of mitochondrial disorders associated with mitochondrial DNA mutations. In theory, by recoding mitochondrial genes to adapt them to the universal genetic code and by adding a DNA sequence coding for a mitochondrial-targeting sequence, one could achieve correct localization of the gene product. Such transfer has occurred in nature, and certain species of algae and plants express a number of polypeptides that are commonly coded by mtDNA in the nuclear-cytoplasmic compartment. In the present study, allotopic expression of three different mtDNA-coded polypeptides (ATPase8, apocytochrome b, and ND4) into COS-7 and HeLa cells was analyzed. Among these, only ATPase8 was correctly expressed and localized to mitochondria. The full-length, as well as truncated forms, of apocytochrome b and ND4 decorated the periphery of mitochondria, but also aggregated in fiber-like structures containing tubulin and in some cases also vimentin. The addition of a hydrophilic tail (EGFP) to the C terminus of these polypeptides did not change their localization. Overexpression of molecular chaperones also did not have a significant effect in preventing aggregations. Allotopic expression of apocytochrome b and ND4 induced a loss of mitochondrial membrane potential in transfected cells, which can lead to cell death. Our observations suggest that only a subset of mitochondrial genes can be replaced allotopically. Analyses of the hydrophobic patterns of different polypeptides suggest that hydrophobicity of the N-terminal segment is the main determinant for the importability of peptides into mammalian mitochondria.

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The intracellular dynamic of protein palmitoylation

The Journal of Cell Biology ◽

10.1083/jcb.201008160 ◽

2010 ◽

Vol 191 (7) ◽

pp. 1229-1238 ◽

Cited By ~ 198

Author(s):

Christine Salaun ◽

Jennifer Greaves ◽

Luke H. Chamberlain

Keyword(s):

Posttranslational Modifications ◽

Posttranslational Modification ◽

Mammalian Cells ◽

Protein Functionality ◽

Peripheral Membrane Proteins ◽

Protein Palmitoylation ◽

Thioester Bond ◽

Intracellular Sorting ◽

Intracellular Dynamic ◽

Insight Into

S-palmitoylation describes the reversible attachment of fatty acids (predominantly palmitate) onto cysteine residues via a labile thioester bond. This posttranslational modification impacts protein functionality by regulating membrane interactions, intracellular sorting, stability, and membrane micropatterning. Several recent findings have provided a tantalizing insight into the regulation and spatiotemporal dynamics of protein palmitoylation. In mammalian cells, the Golgi has emerged as a possible super-reaction center for the palmitoylation of peripheral membrane proteins, whereas palmitoylation reactions on post-Golgi compartments contribute to the regulation of specific substrates. In addition to palmitoylating and depalmitoylating enzymes, intracellular palmitoylation dynamics may also be controlled through interplay with distinct posttranslational modifications, such as phosphorylation and nitrosylation.

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Modified Rhodopsins From Aureobasidium pullulans Excel With Very High Proton-Transport Rates

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.750528 ◽

2021 ◽

Vol 8 ◽

Author(s):

Sabine Panzer ◽

Chong Zhang ◽

Tilen Konte ◽

Celine Bräuer ◽

Anne Diemar ◽

...

Keyword(s):

Proton Pump ◽

Membrane Trafficking ◽

Mammalian Cells ◽

Aureobasidium Pullulans ◽

Green Light ◽

Maximal Activity ◽

Site Directed Mutagenesis ◽

Proton Pumps ◽

C Terminus ◽

Pump Activity

Aureobasidium pullulans is a black fungus that can adapt to various stressful conditions like hypersaline, acidic, and alkaline environments. The genome of A. pullulans exhibits three genes coding for putative opsins ApOps1, ApOps2, and ApOps3. We heterologously expressed these genes in mammalian cells and Xenopus oocytes. Localization in the plasma membrane was greatly improved by introducing additional membrane trafficking signals at the N-terminus and the C-terminus. In patch-clamp and two-electrode-voltage clamp experiments, all three proteins showed proton pump activity with maximal activity in green light. Among them, ApOps2 exhibited the most pronounced proton pump activity with current amplitudes occasionally extending 10 pA/pF at 0 mV. Proton pump activity was further supported in the presence of extracellular weak organic acids. Furthermore, we used site-directed mutagenesis to reshape protein functions and thereby implemented light-gated proton channels. We discuss the difference to other well-known proton pumps and the potential of these rhodopsins for optogenetic applications.

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Oxygen sensing and molecular adaptation to hypoxia

Physiological Reviews ◽

10.1152/physrev.1996.76.3.839 ◽

1996 ◽

Vol 76 (3) ◽

pp. 839-885 ◽

Cited By ~ 815

Author(s):

H. F. Bunn ◽

R. O. Poyton

Keyword(s):

Transcription Factors ◽

Mammalian Cells ◽

Critical Role ◽

Hypoxia Inducible Factor ◽

Signal Transduction Pathway ◽

Adaptation To Hypoxia ◽

Adaptive Responses ◽

Expression Of Genes ◽

Activation Of Transcription ◽

Activation Of Oxygen

This review focuses on the molecular stratagems utilized by bacteria, yeast, and mammals in their adaptation to hypoxia. Among this broad range of organisms, changes in oxygen tension appear to be sensed by heme proteins, with subsequent transfer of electrons along a signal transduction pathway which may depend on reactive oxygen species. These heme-based sensors are generally two-domain proteins. Some are hemokinases, while others are flavohemoproteins [flavohemoglobins and NAD(P)H oxidases]. Hypoxia-dependent kinase activation of transcription factors in nitrogen-fixing bacteria bears a striking analogy to the phosphorylation of hypoxia inducible factor-1 (HIF-1) in mammalian cells. Moreover, redox chemistry appears to play a critical role both in the trans-activation of oxygen-responsive genes in unicellular organisms as well as in the activation of HIF-1. In yeast and bacteria, regulatory operons coordinate expression of genes responsible for adaptive responses to hypoxia and hyperoxia. Similarly, in mammals, combinatorial interactions of HIF-1 with other identified transcription factors are required for the hypoxic induction of physiologically important genes.

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Chemical carcinogens

Oxford Textbook of Cancer Biology ◽

10.1093/med/9780198779452.003.0007 ◽

2019 ◽

pp. 79-90

Author(s):

David H. Phillips

Keyword(s):

Dna Repair ◽

Cancer Incidence ◽

Mammalian Cells ◽

Human Exposure ◽

Chemical Carcinogens ◽

Regulatory Processes ◽

Genotoxic Carcinogens ◽

The Many ◽

Transformation Methods ◽

Insight Into

Large geographical and temporal differences in cancer incidence indicate that the causes of the majority of cases are a consequence of environmental and lifestyle factors. While many of these remain unknown, around half have known causes, and these include chemicals in air, water, and food, as well as products of industrial processes and of combustion. The major classes of chemical carcinogens and how they were discovered are described. A property shared by many of them is that they, or one or more of their metabolites, are electrophiles that can damage DNA in mammalian cells, leading to cellular responses including DNA repair, cytotoxicity, apoptosis, mutagenesis, and malignant transformation. Methods for predicting the carcinogenicity of new chemicals are part of the regulatory processes for safety assessment, and sensitive methods for monitoring human exposure to carcinogens provide insight into the aetiology of cancer. The mutational signatures that genotoxic carcinogens leave in the tumours they induce provide evidence of the chemicals that have caused them, and the approach has promise for shedding light on the many as-yet-unidentified cases of cancer worldwide.

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