Fluorescent Protein Research Articles

Protein kinase C (PKC) inhibitor Calphostin C activates PKC in a light-dependent manner at high concentrations via the production of singlet oxygen

Calphostin C (Cal-C) is a protein kinase C (PKC) inhibitor that binds to its C1 domain. The aim of the present study was to elucidate the action of Cal-C in addition to PKC inhibition. First, we confirmed that Cal-C at low concentrations (<200 nM) inhibit phorbol ester-induced PKC translocation and G-protein-coupled receptor (GPCR)-mediated PKC activation. Cal-C at higher concentrations (>2 μM) increased intracellular calcium ion concentrations ([Ca2+]i) in a concentration-dependent manner. The origin of this increase is the mobilization of the endoplasmic reticulum (ER), which does not involve GPCR or ryanodine receptors. Cal-C at high concentrations also cause structural changes in the ER, such as the formation of vacuoles and aggregates, and calcium leakage from the ER. At 2 μM, Cal-C translocated a calcium-sensitive PKCα. Studies using a C-kinase activity reporter and a myristoylated alanine-rich protein kinase C substrate fused with green fluorescent protein (GFP) have also revealed that Cal-C at high concentrations activate PKC in living cells. Additionally, the PKC-activating effects of Cal-C were light-dependent. Finally, studies using Si-DMA, an indicator of singlet oxygen, showed that Cal-C at high concentrations generated singlet oxygen, causing structural changes in the ER and leakage of calcium into the cytosol, which triggered PKC activation. This study confirms the novel action of Cal-C, solely considered a PKC inhibitor. Cal-C acted as a PKC inhibitor at low concentrations and a PKC activator at high concentrations by generating singlet oxygen in a light-dependent manner, suggesting that Cal-C can be used in photodynamic therapy.

Extracellular bimolecular fluorescence complementation for investigating membrane protein dimerization: a proof of concept using class B GPCRs.

Bimolecular fluorescence complementation (BiFC) methodology uses split fluorescent proteins to detect interactions between proteins in living cells. To date, BiFC has been used to investigate receptor dimerization by splitting the fluorescent protein between the intracellular portions of different receptor components. We reasoned that attaching these split proteins to the extracellular N-terminus instead may improve the flexibility of this methodology and reduce the likelihood of impaired intracellular signal transduction. As a proof-of-concept we used receptors for calcitonin gene-related peptide, which comprise heterodimers of either the calcitonin or calcitonin receptor-like receptor in complex with an accessory protein (receptor activity-modifying protein 1). We created fusion constructs in which split mVenus fragments were attached to either the C-termini or N-termini of receptor subunits. The resulting constructs were transfected into Cos7 and HEK293S cells, where we measured cAMP production in response to ligand stimulation, cell surface expression of receptor complexes, and BiFC fluorescence. Additionally, we investigated ligand-dependent internalization in HEK293S cells. We found N-terminal fusions were better tolerated with regards to cAMP signaling and receptor internalization. N-terminal fusions also allowed reconstitution of functional fluorescent mVenus proteins, however fluorescence yields were lower than with C-terminal fusion. Our results suggest that BiFC methodologies can be applied to the receptor N-terminus, thereby increasing the flexibility of this approach, and enabling further insights into receptor dimerization.

Sera from Rheumatoid Arthritis Patients Induce Oxidative Stress and Pro-Angiogenic and Profibrotic Phenotypes in Human Endothelial Cells.

Background: Rheumatoid arthritis (RA) is a long-term autoimmune condition marked by persistent inflammation of the joints and various systemic complications, including endothelial dysfunction, atherosclerosis, and pulmonary fibrosis. Oxidative stress is a key contributor to the pathogenesis of RA, potentially exacerbating vascular damage and promoting pro-angiogenic and profibrotic processes. Objective: This study aims to investigate the effects of sera from RA patients on human umbilical vein endothelial cells (HUVECs), focusing on the induction of oxidative stress, endothelial cell proliferation, migration, and collagen type I synthesis. Methods: Twenty-eight serum samples were collected from RA patients and healthy donors (HDs). HUVECs were exposed to these sera, and intracellular reactive oxygen species (ROS) levels were fluorescently detected using H2DCF-DA. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell migration was evaluated through a scratch wound assay, and collagen type I synthesis was measured using a lentiviral vector expressing the green fluorescent protein (GFP) under the control of the human COL1A1 gene promoter. Results: Exposure to RA sera resulted in a significant increase in intracellular ROS levels in HUVECs compared to HD sera, indicating an elevated state of oxidative stress. RA sera also promoted endothelial cell proliferation and migration, suggesting a pro-angiogenic stimulus. Additionally, RA sera significantly increased collagen type I synthesis in HUVECs, implicating a potential role in profibrotic processes associated with RA. Conclusion: The results of this study emphasize the importance of circulating factors in RA sera in promoting oxidative stress, endothelial dysfunction, and pro-angiogenic and profibrotic phenotypes in endothelial cells. These processes may contribute to the vascular and fibrotic complications observed in RA, highlighting the necessity for additional research into focused therapeutic approaches to alleviate these effects.

Nanofat Improves Vascularization and Tissue Integration of Dermal Substitutes without Affecting Their Biocompatibility.

Dermal substitutes require sufficient tissue integration and vascularization to be successfully covered with split-thickness skin grafts. To rapidly achieve this, we provide the proof of principle for a novel vascularization strategy with high translational potential. Nanofat was generated from subcutaneous adipose tissue of green fluorescence protein (GFP)+ C57BL/6J donor mice and seeded onto small samples (4 mm in diameter) of the clinically approved dermal substitute Integra®. These samples and non-seeded controls were then implanted into full-thickness skin defects in the dorsal skinfold chamber of C57BL/6J wild-type mice and analyzed by intravital fluorescence microscopy, histology and immunohistochemistry over a 14-day period. Nanofat-seeded dermal substitutes exhibited an accelerated vascularization, as indicated by a significantly higher functional microvessel density on days 10 and 14 when compared to controls. This was primarily caused by the reassembly of GFP+ microvascular fragments inside the nanofat into microvascular networks. The improved vascularization promoted integration of the implants into the surrounding host tissue, which finally exhibited an increased formation of a collagen-rich granulation tissue. There were no marked differences in the inflammatory host tissue reaction to nanofat-seeded and control implants. These findings demonstrate that nanofat significantly improves the in vivo performance of dermal substitutes without affecting their biocompatibility.

Development of CadR-based cadmium whole cell biosensor for visual detection of environmental Cd2+

BackgroundAs a threat to human health and public health, cadmium (Cd) pollution has received widespread social concern. Our previously constructed CadR-based bacterial whole cell biosensor (WCB) epCadR5 showed high sensitivity and specificity in cadmium detection. However, the application of the sensor is still hindered by the need for laboratory equipment to read the fluorescence signal output. In this study, we aimed to optimizing the sensor to make it available for visual detection of environmental cadmium and simplify the detection process to advance practical application of the sensor. ResultsBy replacing the constitutive promoter with J110, the fluorescence signal output of the sensor was significantly increased and the fluorescence leakage was decreased. In addition, the fluorescence signal output of green fluorescence protein (GFP) was enhanced by the addition of a 5′ untranslated region (5′-UTR) mlcR10. The fluorescence signal output of the WCB is sufficiently robust to be visible and distinguishable to the naked eye, which is of paramount importance for visual detection. The sensor readout can be conveniently recorded by mobile phone camera and quantified. For ease of on-site application, the WCB's visual detection procedures and conditions were further optimized and simplified. The WCB demonstrated good linearity and detection limit (1.81 μg/L) for visual detection of Cd2+ without the assistance of bulky laboratory equipment. For the detection of real environmental samples, the WCB visual detection results were close to those of WCB-flow cytometry (FACS) and graphite furnace atomic absorption spectroscopy (GFAAS). SignificanceIn this work, we developed an easy-to-use, on-site and visual detection biosensor for monitoring environmental Cd2+. It will advance the utilization of cadmium WCBs in practical settings. The optimization and simplification strategy in the study also provide new insights into the visualization of other bacterial biosensors, and will advance the practical application of WCBs.

RNA lipid nanoparticles as efficient in vivo CRISPR-Cas9 gene editing tool for therapeutic target validation in glioblastoma cancer stem cells

In vitro and ex-vivo target identification strategies often fail to predict in vivo efficacy, particularly for glioblastoma (GBM), a highly heterogenous tumor rich in resistant cancer stem cells (GSCs). An in vivo screening tool can improve prediction of therapeutic efficacy by considering the complex tumor microenvironment and the dynamic plasticity of GSCs driving therapy resistance and recurrence. This study proposes lipid nanoparticles (LNPs) as an efficient in vivo CRISPR-Cas9 gene editing tool for target validation in mesenchymal GSCs. LNPs co-delivering mRNA (mCas9) and single-guide RNA (sgRNA) were successfully formulated and optimized facilitating both in vitro and in vivo gene editing. In vitro, LNPs achieved up to 67 % reduction in green fluorescent protein (GFP) expression, used as a model target, outperforming a commercial transfection reagent. Intratumoral administration of LNPs in GSCs resulted in ∼80 % GFP gene knock-out and a 2-fold reduction in GFP signal by day 14. This study showcases the applicability of CRISPR-Cas9 LNPs as a potential in vivo screening tool in GSCs, currently lacking effective treatment. By replacing GFP with a pool of potential targets, the proposed platform presents an exciting prospect for therapeutic target validation in orthotopic GSCs, bridging the gap between preclinical and clinical research.

An ATP detection system based on the enzyme reaction with biotin protein ligase

Adenosine triphosphate (ATP) is the energy currency of all living organisms and can be used as an indicator for cell proliferation and cytotoxicity. In the present work, we have developed a novel ATP detection system by combining the biotinylation reaction from archaeon Sulfolobus tokodaii with fluorescence resonance energy transfer (FRET). In biotinylation from S. tokodaii, an enzyme known as biotin protein ligase (BPL) forms a very stable complex with its product, biotinylated substrate protein (BCCP). Here, BPL and BCCP were fused to the fluorescent proteins Cerulean and Clover, respectively, and ATP detection was accomplished by monitoring the FRET signal between the two fluorescent proteins, since ATP is an essential component for biotinylation and the tight BPL-BCCP complex is formed only after biotinylation. Using this system, we have succeeded in detecting 5 nM of ATP by biotinylation reaction with 50 nM of each fusion protein. Our method has a characteristic that the signal does not decay for at least 2 h after the start of the reaction, unlike in the case of the luminescence-based assay with luciferase commonly used for the ATP detection. Thus, our system allows for ATP detection which is not significantly constrained by measurement timing.

Multiplex Genomic Tagging of Mammalian ATG8s to Study Autophagy

Atg8 proteins play a crucial role in autophagy. There is a single Atg8 isoform in yeast, while mammals have up to seven homologs categorized into LC3s and GABARAPs. The GABARAP subfamily consists of GABARAP, GABARAPL1, and GABARAPL2/GATE16, implicated in various stages along the pathway. However, the intricacies among GABARAP proteins are complex and require a more precise delineation.Here, we introduce a new cellular platform to study autophagy using CRISPR/Cas9-mediated tagging of endogenous genes of the GABARAP subfamily with different fluorescent proteins. This platform allows robust examination of autophagy by flow cytometry of cell populations and monitoring of GABARAP homologs at single-cell resolution using fluorescence microscopy. Strikingly, the simultaneous labeling of the different endogenous GABARAPs allows the identification and isolation of autophagosomes differentially marked by these proteins. Using this system, we found that the different GABARAPs are associated with different autophagosomes. We argue that this new cellular platform will be crucial in studying the unique roles of individual GABARAP proteins in autophagy and other putative cellular processes.

Cooperation between Coagulase and von Willebrand factor binding protein in Staphylococcus aureus fibrin pseudocapsule formation

The major human pathogen Staphylococcus aureus forms biofilms comprising of a fibrin network that increases attachment to surfaces and shields bacteria from the immune system. It secretes two coagulases, Coagulase (Coa) and von Willebrand factor binding protein (vWbp), which hijack the host coagulation cascade and trigger the formation of this fibrin clot. However, it is unclear how Coa and vWbp contribute differently to the localisation and dynamics of clot assembly in growing biofilms.Here, we address this question using high-precision time-resolved confocal microscopy of fluorescent fibrin to establish the spatiotemporal dynamics of fibrin clot formation in functional biofilms. We also use fluorescent fusion proteins to visualise the locations of Coa and vWbp in biofilms using both confocal laser scanning and high resolution highly inclined and laminated optical sheet microscopy. We visualise and quantify the spatiotemporal dynamics of fibrin production during initiation of biofilms in plasma amended with fluorescently labelled fibrinogen.We find that human serum stimulates coagulase production, and that Coa and vWbp loosely associate to the bacterial cell surface. Coa localises to cell surfaces to produce a surface-attached fibrin pseudocapsule but can diffuse from cells to produce matrix-associated fibrin. vWbp produces matrix-associated fibrin in the absence of Coa, and furthermore accelerates pseudocapsule production when Coa is present. Finally, we observe that fibrin production varies across the biofilm. A sub-population of non-dividing cells does not produce any pseudocapsule but remains within the protective extended fibrin network, which could be important for the persistence of S. aureus biofilm infections as antibiotics are more effective against actively growing cells.Our findings indicate a more cooperative role between Coa and vWbp in building fibrin networks than previously thought, and a bet-hedging cell strategy where some cells produce biofilm matrix while others do not, but instead assume a dormant phenotype that could be associated with antibiotic tolerance.

Tolerance and phytoremediation capacity of atrazine and S-metolachlor by two duckweeds.

The phytotoxicity and removal of atrazine and S-metolachlor in sterile duckweed systems were estimated in this study. Herbicides were added at environmentally relevant ranges: 0-400µg/L for atrazine or 0-200µg/L for S-metolachlor in systems with Spirodela polyrhiza or Lemna minor. Toxicity biomarkers, i.e., changes in plant biomass, surface area, chlorophyll fluorescence parameters, pigments, lipid peroxidation, protein concentration, and antioxidative enzyme activities in plants were estimated after 7days. S. polyrhiza (RGRbiomass-EC50 = 164.8µg/L) was more tolerant to atrazine than L. minor (RGRbiomass-EC50 = 101.0µg/L). Atrazine caused damage to photosystem II (ΦM), a reduction in electron transport between PSII and PSI (Φ'M), as well as disruption in energy distribution pathways (decrease in qPrel and increase in UQFrel), most prominently in L. minor. However, L. minor (RGRbiomass-EC50 = 128.9µg/L) was more tolerant to S-metolachlor than S. polyrhiza (RGRbiomass-EC50 = 15.5μg/L). The highest sensitivity of S. polyrhiza to S-metolachlor was attributed to a decrease in absorbed energy used in photochemistry (qPrel) and an increase in lipid peroxidation, indicating that S. polyrhiza plants were experiencing oxidative stress. Residual pesticide analysis in the water after seven days allowed us to conclude that plants were responsible for reducing up to 16.5% of atrazine and 28.7% of S-metolachlor in the duckweed system. S. polyrhiza showed higher atrazine phytoremediation capacity than L. minor. S. polyrhiza was more efficient at an environmentally relevant concentration of S-metolachlor (25μg/L) and L. minor at higher concentrations (200μg/L).

Recombinant Influenza A Viruses Expressing Reporter Genes from the Viral NS Segment.

Studying influenza A viruses (IAVs) requires secondary experimental procedures to detect the presence of the virus in infected cells or animals. The ability to generate recombinant (r)IAV using reverse genetics techniques has allowed investigators to generate viruses expressing foreign genes, including fluorescent and luciferase proteins. These rIAVs expressing reporter genes have allowed for easily tracking viral infections in cultured cells and animal models of infection without the need for secondary approaches, representing an excellent option to study different aspects in the biology of IAV where expression of reporter genes can be used as a readout of viral replication and spread. Likewise, these reporter-expressing rIAVs provide an excellent opportunity for the rapid identification and characterization of prophylactic and/or therapeutic approaches. To date, rIAV expressing reporter genes from different viral segments have been described in the literature. Among those, rIAV expressing reporter genes from the viral NS segment have been shown to represent an excellent option to track IAV infection in vitro and in vivo, eliminating the need for secondary approaches to identify the presence of the virus. Here, we summarize the status on rIAV expressing traceable reporter genes from the viral NS segment and their applications for in vitro and in vivo influenza research.

Study on the Recombinant Human Interferon α1b, α2b, and Gamma Transient Expression and in Vitro Activities in Tobacco.

Interferons (IFNs) are universally acknowledged for their pivotal role in antiviral and anticancer responses. Thus, the primary aim of our study was to explore the expressions of IFN-α1b, α2b, and gamma in tobacco leaves via agrobacterium-mediated transient transformation and investigate their possible activities. Briefly, fusion with green fluorescent protein tags aided in detecting the expressed IFN proteins in the foliar tissues. The genetic constructs encoding these fusion proteins were inserted into the MagnICON plant transient expression vector, followed by transformation into the Agrobacterium strain GV3101. The transformed bacteria were then used to infiltrate tobacco leaves. After post-infiltration, protein expression was confirmed within 72 h via sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the fusion proteins were subsequently purified using high-performance liquid chromatography for identification. Both the antiviral and anticancer potencies of these IFN fusion proteins were evaluated using the WISH/VSV (WISH cells/Vesicular stomatitis virus) microneutralization and MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, respectively. Results indicated robust expression of the targeted IFN genes in plant tissues and significant biological activities against pathogens and cancer cells. Consequently, this study substantiated the viability of producing these therapeutic proteins in plants, potentially revolutionizing the manufacture of interferons biologically.

Structural characterization of green fluorescent protein in the I-state

Green fluorescent protein (GFP) is widely utilized as a fluorescent tag in biochemical fields. Whereas the intermediate (I) state has been proposed in the photoreaction cycle in addition to the A and B states, until now the structure of I has only been estimated by computational studies. In this paper, we report the crystal structures of the I stabilizing variants of GFP at high resolutions where respective atoms can be observed separately. Comparison with the structures in the other states highlights the structural feature of the I state. The side chain of one of the substituted residues, Val203, adopts the gauche- conformation observed for Thr203 in the A state, which is different from the B state. On the other hand, His148 interacts with the chromophore by ordinary hydrogen bonding with a distance of 2.85 Å, while the weaker interaction by longer distances is observed in the A state. Therefore, it was indicated that it is possible to distinguish three states A, B and I by the two hydrogen bond distances Oγ-Thr203···Oη-chromophore and Nδ1-His148···Oη-chromophore. We discuss the characteristics of the I intermediate of wild-type GFP on the bases of the structure estimated from the variant structures by quantum chemical calculations.

A protein blueprint of the diatom CO2-fixing organelle

Diatoms are central to the global carbon cycle. At the heart of diatom carbon fixation is an overlooked organelle called the pyrenoid, where concentrated CO2 is delivered to densely packed Rubisco. Diatom pyrenoids fix approximately one-fifth of global CO2, but the protein composition of this organelle is largely unknown. Using fluorescence protein tagging and affinity purification-mass spectrometry, we generate a high-confidence spatially defined protein-protein interaction network for the diatom pyrenoid. Within our pyrenoid interaction network are 10 proteins with previously unknown functions. We show that six of these form a shell that encapsulates the Rubisco matrix and is critical for pyrenoid structural integrity, shape, and function. Although not conserved at a sequence or structural level, the diatom pyrenoid shares some architectural similarities to prokaryotic carboxysomes. Collectively, our results support the convergent evolution of pyrenoids across the two main plastid lineages and uncover a major structural and functional component of global CO2 fixation.

Lipid nanoparticles as nano-Trojan-horses for siRNA delivery and gene-knockdown

The therapeutic messenger RNA strategies, such as those using small interfering RNAs, take several advantages (versatility, efficiency and selectivity) over plasmid DNA-based strategies. However, the challenge remains to find nanovectors capable of properly loading the genetic material, transporting it through troublesome environments, like a tumoral site, and delivering it into the cytoplasm of target cells. Here, lipid nanoparticles, consisting of a Gemini cationic/neutral helper lipid mixture, are proposed as siRNA nanovector. Cells from cervical and brain cancer overexpressing the green fluorescent protein (GFP) were chosen to analyse the biological response as well as the efficiency and safety of the siRNA-loaded nanovector according to the cell phenotype. Flow cytometry and epifluorescence or confocal microscopy were used to follow the gene knockdown in these overexpressed cells. The effect of the nanovector on cellular proliferation was evaluated with cytotoxicity assays while their potential oxidative stress generation was determined by quantifying the generation of reactive oxygen species. To explore the mechanism of cellular uptake, different inhibitors of endocytic pathways were used during incubation with cells. Finally, nanovectors were incubated in 3D-grown cells (spheroids) to see whether they can penetrate the complex tumoral microenvironments, their efficiency to knockdown GFP expression being monitored by confocal microscopy.

Localization of albumin with correlative super resolution light- and electron microscopy in the kidney

The functioning of vertebrate life relies on renal filtration of surplus fluid and elimination of low-molecular-weight waste products, while keeping serum proteins in the blood. In disease, however, there is leak of serum proteins and tracing them to identify the leaking position within tissue with a nanometer resolution poses a significant challenge. Correlative microscopy integrates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Using chemical tagging of albumin with synthetic fluorophores we achieve protein-specific labeling that preserve their post-embedding fluorescence after high-pressure freezing and freeze-substitution of murine kidney tissue. Using advanced registration techniques for super-resolution correlative light and electron microscopy, we can localize the labeled albumin with a high precision in the x-y plane of electron micrographs and cartograph its distribution. Thereby we can quantify the albumin concentration and measure a linear reduction gradient across the kidney filtration barrier. Our study shows the feasibility of combining different microscopy contrasts for tracing fluorescently labeled protein markers with super resolution in various tissue samples and opens new perspectives for correlative imaging in volume electron microscopy.

Postnatal Wing Morph of Pea Aphids Regulates Hemolymph Trehalose Levels.

Trehalose, a nonreducing disaccharide composed of two glucose molecules, functions as a critical energy source in various insect tissues and organs and is the predominant sugar component of the hemolymph. The pea aphid, Acyrthosiphon pisum, exhibits higher hemolymph trehalose levels than other insects. However, the dynamics of hemolymph trehalose levels throughout its life stages remain unclear owing to the challenges associated with obtaining hemolymph from these small insects. Therefore, this study was conducted to quantify hemolymph trehalose levels in A. pisum using a fluorescent trehalose sensor (Tre-C04), which enhances green fluorescent protein fluorescence through the binding of trehalose to a ligand-binding protein fused to the fluorophore. Trehalose levels were successfully quantified in minimal hemolymph samples from individual aphids, with measurements spanning from the first nymphal stage to the adult stage in both the winged and wingless forms of A. pisum. Hemolymph trehalose levels remained relatively stable throughout the life cycle but exhibited a gradual increase with each developmental stage. Notably, adult winged aphids exhibited significantly higher hemolymph trehalose levels than wingless aphids. Given that wing morph determination occurs early in the nymphal stage, these findings suggest that hemolymph trehalose levels are regulated post-wing morph development. Further investigation of the expression of genes associated with trehalose metabolism revealed that trehalose phosphate synthase 2 levels were downregulated in early-stage wingless adults, whereas insulin-related peptide 5 levels were upregulated in wingless aphids. These findings indicate that A. pisum synthesizes trehalose during the winged adult stage to serve as an energy source for flight.

HERD-1 mediates multiphase condensate immiscibility to regulate small RNA-driven transgenerational epigenetic inheritance.

Biomolecular condensates, such as the nucleolus, stress granules/processing bodies and germ granules, are multiphase assemblages whose formation mechanisms and significance remain poorly understood. Here we identify protein constituents of the spatiotemporally ordered P, Z and M multiphase condensates in Caenorhabditis elegans germ granules using optimized TurboID-mediated proximity biotin labelling. These include 462, 41 and 86 proteins localizing to P, Z and M condensates, respectively, of which 522 were previously unknown protein constituents. Each condensate's proteins are enriched for distinct classes of structured and intrinsically disordered domains, suggesting divergent functions and assembly mechanisms. Through a functional screen, we identify a germ granule protein, HERD-1, which prevents the mixing of P, Z and M condensates. Mixing in herd-1 mutants correlates with disorganization of germline small RNA pathways and prolonged epigenetic inheritance of RNA interference-induced gene silencing. Forced mixing of these condensate components using a nanobody with specific binding activity against green fluorescent protein also extends epigenetic inheritance. We propose that active maintenance of germ granule immiscibility helps to organize and regulate small RNA-driven transgenerational epigenetic inheritance in C. elegans.

Challenges and Solutions for Leave-One-Out Biosensor Design in the Context of a Rugged Fitness Landscape

The leave-one-out (LOO) green fluorescent protein (GFP) approach to biosensor design combines computational protein design with split protein reconstitution. LOO-GFPs reversibly fold and gain fluorescence upon encountering the target peptide, which can be redefined by computational design of the LOO site. Such an approach can be used to create reusable biosensors for the early detection of emerging biological threats. Enlightening biophysical inferences for nine LOO-GFP biosensor libraries are presented, with target sequences from dengue, influenza, or HIV, replacing beta strands 7, 8, or 11. An initially low hit rate was traced to components of the energy function, manifesting in the over-rewarding of over-tight side chain packing. Also, screening by colony picking required a low library complexity, but designing a biosensor against a peptide of at least 12 residues requires a high-complexity library. This double-bind was solved using a “piecemeal” iterative design strategy. Also, designed LOO-GFPs fluoresced in the unbound state due to unwanted dimerization, but this was solved by fusing a fully functional prototype LOO-GFP to a fiber-forming protein, Drosophila ultrabithorax, creating a biosensor fiber. One influenza hemagglutinin biosensor is characterized here in detail, showing a shifted excitation/emission spectrum, a micromolar affinity for the target peptide, and an unexpected photo-switching ability.

Protein P5 of pear chlorotic leaf spot-associated virus is a pathogenic factor that suppresses RNA silencing and enhances virus movement.

Pear chlorotic leaf spot-associated virus (PCLSaV) is a newly described emaravirus that infects pear trees. The virus genome consists of at least five single-stranded, negative-sense RNAs. The P5 encoded by RNA5 is unique to PCLSaV. In this study, the RNA silencing suppression (RSS) activity of P5 and its subcellular localization were determined in Nicotiana benthamiana plants by Agrobacterium tumefaciens-mediated expression assays and green fluorescent protein RNA silencing induction. Protein P5 partially suppressed local RNA silencing, strongly suppressed systemic RNA silencing and triggered reactive oxygen species accumulation. The P5 self-interacted and showed subcellular locations in plasmodesmata, endoplasmic reticulum and nucleus. Furthermore, P5 rescued the cell-to-cell movement of a movement defective mutant PVXΔP25 of potato virus X (PVX) and enhanced the pathogenicity of PVX. The N-terminal 1-89 amino acids of the P5 were responsible for the self-interaction ability and RSS activity, for which the signal peptide at positions 1-19 was indispensable. This study demonstrated the function of an emaravirus protein as a pathogenic factor suppressing plant RNA silencing to enhance virus infection and as an enhancer of virus movement.