Phytochrome chromophore-deficient mutants

Phytochrome chromophore-deficient mutants have been used as phytochrome-deficient plants to study many aspects of plant development. However, there are still a number of important questions to be resolved concerning both the targets and the phenotypic consequences of these mutations. Recently, progress has been made in our understanding of the molecular basis of the chromophore deficiency in these mutants. Biochemical assays for the committed steps of chromophore synthesis have been developed and used to demonstrate that the pcd1 and yellow-green-2 mutants of pea and tomato, respectively, are unable to synthesize biliverdin IXα from heme while pcd2 and aurea are deficient in phytochromobilin synthase activity. This review focuses on how this information can be used to help understand the basis of other chromophore-deficient mutants, such as the hy1 and hy2 mutants of Arabidopsis, and discusses how the phenotype of chromophore-deficient mutants is related to lesions in the chromophore biosynthesis pathway.     

Crystallographic study of red fluorescent protein eqFP578 and its far-red variant Katushka reveals opposite pH-induced isomerization of chromophore

The wild type red fluorescent protein eqFP578 (from sea anemone Entacmaea quadricolor, λ_ex = 552 nm, λ_em = 578 nm) and its bright far‐red fluorescent variant Katushka (λ_ex = 588 nm, λ_em = 635 nm) are characterized by the pronounced pH dependence of their fluorescence. The crystal structures of eqFP578f (eqFP578 with two point mutations improving the protein folding) and Katushka have been determined at the resolution ranging from 1.15 to 1.85 Å at two pH values, corresponding to low and high level of fluorescence. The observed extinguishing of fluorescence upon reducing pH in eqFP578f and Katushka has been shown to be accompanied by the opposite trans‐cis and cis‐trans chromophore isomerization, respectively. Asn143, Ser158, His197 and Ser143, Leu174, and Arg197 have been shown to stabilize the respective trans and cis fluorescent states of the chromophores in eqFP578f and Katushka at higher pH. The cis state has been suggested as being primarily responsible for the observed far‐red shift of the emission maximum of Katushka relative to that of eqFP578f.     

Influence of the Dietary Protein Deficiency on the Protein Synthetic Activity of Microsome and Soluble Fraction from Muscle and Liver of Rats

Phytochromes are photoreceptors that regulate many aspects of plant growth and development in response to red/far-red light signals from the environment. In this study, we analyzed chromophore ligation and photochromism of missense phytochrome mutants in the Per-Arnt-Sim (PAS)-related domain (PRD). Among the 14 mutants analyzed, the Gly768Asp mutant of Avena phytochrome A showed aberrant photochromism and dark reversion, suggesting that amino acid residues in the C-terminal domain affect the photochemical properties of the photosensory N-terminal domain.     

Visual pigment evolution in Characiformes: The dynamic interplay of teleost whole‐genome duplication,surviving opsins and spectral tuning

Vision represents an excellent model for studying adaptation, given the genotype‐to‐phenotype map that has been characterized in a number of taxa. Fish possess a diverse range of visual sensitivities and adaptations to underwater light, making them an excellent group to study visual system evolution. In particular, some speciose but understudied lineages can provide a unique opportunity to better understand aspects of visual system evolution such as opsin gene duplication and neofunctionalization. In this study, we showcase the visual system evolution of neotropical Characiformes and the spectral tuning mechanisms they exhibit to modulate their visual sensitivities. Such mechanisms include gene duplications and losses, gene conversion, opsin amino acid sequence and expression variation, and A₁/A₂‐chromophore shifts. The Characiforms we studied utilize three cone opsin classes (SWS2, RH2, LWS) and a rod opsin (RH1). However, the characiform's entire opsin gene repertoire is a product of dynamic evolution by opsin gene loss (SWS1, RH2) and duplication (LWS, RH1). The LWS‐ and RH1‐duplicates originated from a teleost specific whole‐genome duplication as well as characiform‐specific duplication events. Both LWS‐opsins exhibit gene conversion and, through substitutions in key tuning sites, one of the LWS‐paralogues has acquired spectral sensitivity to green light. These sequence changes suggest reversion and parallel evolution of key tuning sites. Furthermore, characiforms' colour vision is based on the expression of both LWS‐paralogues and SWS2. Finally, we found interspecific and intraspecific variation in A₁/A₂‐chromophores proportions, correlating with the light environment. These multiple mechanisms may be a result of the diverse visual environments where Characiformes have evolved.     

2‐D mapping of skin chromophores in the spectral range 500 – 700 nm

The multi‐spectral imaging technique has been used for distant mapping of in‐vivo skin chromophores by analyzing spectral data at each reflected image pixel and constructing 2‐D maps of the relative concentrations of oxy‐/deoxy‐haemoglobin and melanin. Instead of using a broad visible‐NIR spectral range, this study focuses on narrowed spectral band 500–700 nm, speeding‐up the signal processing procedure. Regression analysis confirmed that superposition of three Gaussians is optimal analytic approximation for the oxy‐haemoglobin absorption tabular spectrum in this spectral band, while superposition of two Gaussians fits well for deoxy‐haemoglobin absorption and exponential function – for melanin absorption. The proposed approach was clinically tested for three types of in‐vivo skin provocations: ultraviolet irradiance, chemical reaction with vinegar essence and finger arterial occlusion. Spectral range 500–700 nm provided better sensitivity to oxy‐haemoglobin changes and higher response stability to melanin than two reduced ranges 500–600 nm and 530–620 nm. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Understanding the role of Arg96 in structure and stability of green fluorescent protein

Arg96 is a highly conservative residue known to catalyze spontaneous green fluorescent protein (GFP) chromophore biosynthesis. To understand a role of Arg96 in conformational stability and structural behavior of EGFP, the properties of a series of the EGFP mutants bearing substitutions at this position were studied using circular dichroism, steady state fluorescence spectroscopy, fluorescence lifetime, kinetics and equilibrium unfolding analysis, and acrylamide-induced fluorescence quenching. During the protein production and purification, high yield was achieved for EGFP/Arg96Cys variant, whereas EGFP/Arg96Ser and EGFP/Arg96Ala were characterized by essentially lower yields and no protein was produced when Arg96 was substituted by Gly. We have also shown that only EGFP/Arg96Cys possessed relatively fast chromophore maturation, whereas it took EGFP/Arg96Ser and EGFP/Arg96Ala about a year to develop a noticeable green fluorescence. The intensity of the characteristic green fluorescence measured for the EGFP/Arg96Cys and EGFP/Arg96Ser (or EGFP/Arg96Ala) was 5- and 50-times lower than that of the nonmodified EGFP. Intriguingly, EGFP/Arg96Cys was shown to be more stable than EGFP toward the GdmCl-induced unfolding both in kinetics and in the quasi-equilibrium experiments. In comparison with EGFP, tryptophan residues of EGFP/Arg96Cys were more accessible to the solvent. These data taken together suggest that besides established earlier crucial catalytic role, Arg96 is important for the overall folding and conformational stability of GFP.     

Host pigments: potential facilitators of photosynthesis in coral symbioses

Reef‐building corals occur as a range of colour morphs because of varying types and concentrations of pigments within the host tissues, but little is known about their physiological or ecological significance. Here, we examined whether specific host pigments act as an alternative mechanism for photoacclimation in the coral holobiont. We used the coral Montipora monasteriata (Forskål 1775) as a case study because it occurs in multiple colour morphs (tan, blue, brown, green and red) within varying light‐habitat distributions. We demonstrated that two of the non‐fluorescent host pigments are responsive to changes in external irradiance, with some host pigments up‐regulating in response to elevated irradiance. This appeared to facilitate the retention of antennal chlorophyll by endosymbionts and hence, photosynthetic capacity. Specifically, net P_max Chl a^?1 correlated strongly with the concentration of an orange‐absorbing non‐fluorescent pigment (CP‐580). This had major implications for the energetics of bleached blue‐pigmented (CP‐580) colonies that maintained net P_max cm^?2 by increasing P_max Chl a^?1. The data suggested that blue morphs can bleach, decreasing their symbiont populations by an order of magnitude without compromising symbiont or coral health.     

Applications of genetically encoded photosensitizer miniSOG: from correlative light electron microscopy to immunophotosensitizing

Genetically encoded photosensitizers (PSs), e.g. ROS generating proteins, correspond to a novel class of PSs that are highly desirable for biological and medical applications since they can be used in combination with a variety of genetic engineering manipulations allowing for precise spatio‐temporal control of ROS production within living cells and organisms. In contrast to the commonly used chemical PSs, they can be modified using genetic engineering approaches and targeted to particular cellular compartments and cell types. Mini Singlet Oxygen Generator (miniSOG), a small flavoprotein capable of singlet oxygen production upon blue light irradiation, was initially reported as a high contrast probe for correlative light electron microscopy (CLEM) without the need of exogenous ligands, probes or destructive permeabilizing detergents. Further miniSOG was successfully applied for chromophore‐assisted light inactivation (CALI) of proteins, as well as for photo‐induced cell ablation in tissue cultures and in Caenorhabditis elegans. Finally, a novel approach of immunophotosensitizing has been developed, exploiting the specificity of mini‐antibodies or selective scaffold proteins and photo‐induced cytotoxicity of miniSOG, which is particularly promising for selective non‐invasive photodynamic therapy of cancer (PDT) due to the spatial selectivity and locality of destructive action compared to other methods of oncotherapy.

     

Proteomic profiling of the cancer cell secretome: informing clinical research

Introduction: Cancer represents one of the major causes of human deaths. Identification of proteins as biomarkers for early detection of cancer and therapeutic targets for cancer treatment are important issues in precision medicine. Secretome of cancer cells represents the collection of proteins secreted or shed from cancer cells. Proteomic profiling of the cancer cell secretome has been proven to be a convenient and efficient way to discover cancer biomarker and/or therapeutic targets.

Areas covered: There have been numerous reviews describing the history and application of secretome analysis in cancer biomarker/therapeutic target research. The present review focuses on the technological advancement for profiling low-molecular-mass proteins in secretome, the latest information regarding the new candidate biomarkers and molecular mechanisms discovered on the basis of cancer cell secretome analysis, as well as the previously discovered candidate biomarkers that enter into clinical trials.

Expert commentary: Current technologies for protein sample preparation/separation and MS-based protein identification have allowed in-depth analysis of cancer cell secretome. Future efforts should focus on the comprehensiveness of cancer cell secretome, meta-analysis of different secretome datasets and integrated analysis via combining other omics datasets, as well as the incorporation of MS-based biomarker verification pipeline into both preclinical studies and clinical trials.     

Kinetic Analysis of Maturation and Denaturation of DsRed, a Coral-Derived Red Fluorescent Protein

The red fluorescent protein DsRed recently cloned from Discosoma coral, with its significantly red-shifted excitation and emission maxima (558 and 583 nm, respectively), has attracted great interest because of its spectral complementation to other fluorescent proteins, including the green fluorescent protein and its enhanced mutant EGFP. We demonstrated that the much slower DsRed fluorescence development could be described by a three-step kinetic model, in contrast to the fast EGFP maturation, which was fitted by a one-step model. At pH below 5.0 DsRed fluorescence gradually decreased, and the rate and degree of this fluorescence inactivation depended on the pH value. The kinetics of fluorescence inactivation under acidic conditions was fitted by a two-exponential function where the initial inactivation rate was proportional to the fourth power of proton concentration. Subsequent DsRed alkalization resulted in partial fluorescence recovery, and the rate and degree of such recovery depended on the incubation time in the acid. Recovery kinetics had a lag-time and was fitted minimally by three exponential functions. The DsRed absorbance and circular dichroism spectra revealed that the fluorescence loss was accompanied by protein denaturation. We developed a kinetic mechanism for DsRed denaturation that includes consecutive conversion of the initial state of the protein, protonated by four hydrogen ions, to the denatured one through three intermediates. The first intermediate still emits fluorescence, and the last one is subjected to irreversible inactivation. Because of tight DsRed tetramerization we have suggested that obligatory protonation of each monomer results in the fluorescence inactivation of the whole tetramer.