scholarly journals Modular Diversity of the BLUF Proteins and Their Potential for the Development of Diverse Optogenetic Tools

2019 ◽  
Vol 9 (18) ◽  
pp. 3924
Author(s):  
Manish Kaushik ◽  
Ramandeep Sharma ◽  
Sindhu Veetil ◽  
Sandeep Srivastava ◽  
Suneel Kateriya
Keyword(s):  
Rational Design ◽  
Enzymatic Catalysis ◽  
Effector Proteins ◽  
Potential Candidate ◽  
Protein Signaling ◽  
Cellular Processes ◽  
Wide Range ◽  
Modular Architectures ◽  
Eukaryotic Origin ◽  
Sensory Photoreceptors

Organisms can respond to varying light conditions using a wide range of sensory photoreceptors. These photoreceptors can be standalone proteins or represent a module in multidomain proteins, where one or more modules sense light as an input signal which is converted into an output response via structural rearrangements in these receptors. The output signals are utilized downstream by effector proteins or multiprotein clusters to modulate their activity, which could further affect specific interactions, gene regulation or enzymatic catalysis. The blue-light using flavin (BLUF) photosensory module is an autonomous unit that is naturally distributed among functionally distinct proteins. In this study, we identified 34 BLUF photoreceptors of prokaryotic and eukaryotic origin from available bioinformatics sequence databases. Interestingly, our analysis shows diverse BLUF-effector arrangements with a functional association that was previously unknown or thought to be rare among the BLUF class of sensory proteins, such as endonucleases, tet repressor family (tetR), regulators of G-protein signaling, GAL4 transcription family and several other previously unidentified effectors, such as RhoGEF, Phosphatidyl-Ethanolamine Binding protein (PBP), ankyrin and leucine-rich repeats. Interaction studies and the indexing of BLUF domains further show the diversity of BLUF-effector combinations. These diverse modular architectures highlight how the organism’s behaviour, cellular processes, and distinct cellular outputs are regulated by integrating BLUF sensing modules in combination with a plethora of diverse signatures. Our analysis highlights the modular diversity of BLUF containing proteins and opens the possibility of creating a rational design of novel functional chimeras using a BLUF architecture with relevant cellular effectors. Thus, the BLUF domain could be a potential candidate for the development of powerful novel optogenetic tools for its application in modulating diverse cell signaling.

Creating a customized intracellular niche: subversion of host cell signaling byLegionellatype IV secretion system effectors

2015 ◽  
Vol 61 (9) ◽  
pp. 617-635 ◽  
Author(s):  
Ernest C. So ◽  
Corinna Mattheis ◽  
Edward W. Tate ◽  
Gad Frankel ◽  
Gunnar N. Schroeder
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Current Knowledge ◽  
Secretion System ◽  
Effector Proteins ◽  
Cellular Processes ◽  
Type Iv ◽  
Open Questions ◽  
Exact Function ◽  
Wide Range

The Gram-negative facultative intracellular pathogen Legionella pneumophila infects a wide range of different protozoa in the environment and also human alveolar macrophages upon inhalation of contaminated aerosols. Inside its hosts, it creates a defined and unique compartment, termed the Legionella-containing vacuole (LCV), for survival and replication. To establish the LCV, L. pneumophila uses its Dot/Icm type IV secretion system (T4SS) to translocate more than 300 effector proteins into the host cell. Although it has become apparent in the past years that these effectors subvert a multitude of cellular processes and allow Legionella to take control of host cell vesicle trafficking, transcription, and translation, the exact function of the vast majority of effectors still remains unknown. This is partly due to high functional redundancy among the effectors, which renders conventional genetic approaches to elucidate their role ineffective. Here, we review the current knowledge about Legionella T4SS effectors, highlight open questions, and discuss new methods that promise to facilitate the characterization of T4SS effector functions in the future.

scholarly journals Selectivity Determinants of RHO GTPase Binding to IQGAPs

2021 ◽  
Vol 22 (22) ◽  
pp. 12596
Author(s):  
Niloufar Mosaddeghzadeh ◽  
Kazem Nouri ◽  
Oliver H. F. Krumbach ◽  
Ehsan Amin ◽  
Radovan Dvorsky ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Rho Gtpase ◽  
Decisive Role ◽  
Effector Proteins ◽  
Iq Motif ◽  
Cellular Processes ◽  
Wide Range ◽  
Switch Regions ◽  
Protein Components ◽  
Biochemical Analyses

IQ motif-containing GTPase-activating proteins (IQGAPs) modulate a wide range of cellular processes by acting as scaffolds and driving protein components into distinct signaling networks. Their functional states have been proposed to be controlled by members of the RHO family of GTPases, among other regulators. In this study, we show that IQGAP1 and IQGAP2 can associate with CDC42 and RAC1-like proteins but not with RIF, RHOD, or RHO-like proteins, including RHOA. This seems to be based on the distribution of charged surface residues, which varies significantly among RHO GTPases despite their high sequence homology. Although effector proteins bind first to the highly flexible switch regions of RHO GTPases, additional contacts outside are required for effector activation. Sequence alignment and structural, mutational, and competitive biochemical analyses revealed that RHO GTPases possess paralog-specific residues outside the two highly conserved switch regions that essentially determine the selectivity of RHO GTPase binding to IQGAPs. Amino acid substitution of these specific residues in RHOA to the corresponding residues in RAC1 resulted in RHOA association with IQGAP1. Thus, electrostatics most likely plays a decisive role in these interactions.

scholarly journals Integration of early disease-resistance phenotyping, histological characterization, and transcriptome sequencing reveals insights into downy mildew resistance in impatiens

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Ze Peng ◽  
Yanhong He ◽  
Saroj Parajuli ◽  
Qian You ◽  
Weining Wang ◽  
...  
Keyword(s):  
Disease Resistance ◽  
Downy Mildew ◽  
Transcriptome Sequencing ◽  
Early Stage ◽  
Economic Losses ◽  
Potential Candidate ◽  
Early Disease ◽  
True Leaf ◽  
Cotyledon Stage ◽  
Wide Range

AbstractDowny mildew (DM), caused by obligate parasitic oomycetes, is a destructive disease for a wide range of crops worldwide. Recent outbreaks of impatiens downy mildew (IDM) in many countries have caused huge economic losses. A system to reveal plant–pathogen interactions in the early stage of infection and quickly assess resistance/susceptibility of plants to DM is desired. In this study, we established an early and rapid system to achieve these goals using impatiens as a model. Thirty-two cultivars of Impatiens walleriana and I. hawkeri were evaluated for their responses to IDM at cotyledon, first/second pair of true leaf, and mature plant stages. All I. walleriana cultivars were highly susceptible to IDM. While all I. hawkeri cultivars were resistant to IDM starting at the first true leaf stage, many (14/16) were susceptible to IDM at the cotyledon stage. Two cultivars showed resistance even at the cotyledon stage. Histological characterization showed that the resistance mechanism of the I. hawkeri cultivars resembles that in grapevine and type II resistance in sunflower. By integrating full-length transcriptome sequencing (Iso-Seq) and RNA-Seq, we constructed the first reference transcriptome for Impatiens comprised of 48,758 sequences with an N50 length of 2060 bp. Comparative transcriptome and qRT-PCR analyses revealed strong candidate genes for IDM resistance, including three resistance genes orthologous to the sunflower gene RGC203, a potential candidate associated with DM resistance. Our approach of integrating early disease-resistance phenotyping, histological characterization, and transcriptome analysis lay a solid foundation to improve DM resistance in impatiens and may provide a model for other crops.

scholarly journals Intracellular Ionic Strength Sensing Using NanoLuc

2021 ◽  
Vol 22 (2) ◽  
pp. 677
Author(s):  
Tausif Altamash ◽  
Wesam Ahmed ◽  
Saad Rasool ◽  
Kabir H. Biswas
Keyword(s):  
Thermal Stability ◽  
Phase Separation ◽  
Drug Discovery ◽  
Ionic Strength ◽  
Cellular Signaling ◽  
Live Cells ◽  
Protein Activity ◽  
Cellular Survival ◽  
Cellular Processes ◽  
Wide Range

Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation, phase separation, and cell volume. It could be altered by changes in the activity of cellular signaling pathways, such as those that impact the activity of membrane-localized ion channels or by alterations in the microenvironmental osmolarity. Therefore, there is a demand for the development of sensitive tools for real-time monitoring of intracellular ionic strength. Here, we developed a bioluminescence-based intracellular ionic strength sensing strategy using the Nano Luciferase (NanoLuc) protein that has gained tremendous utility due to its high, long-lived bioluminescence output and thermal stability. Biochemical experiments using a recombinantly purified protein showed that NanoLuc bioluminescence is dependent on the ionic strength of the reaction buffer for a wide range of ionic strength conditions. Importantly, the decrease in the NanoLuc activity observed at higher ionic strengths could be reversed by decreasing the ionic strength of the reaction, thus making it suitable for sensing intracellular ionic strength alterations. Finally, we used an mNeonGreen–NanoLuc fusion protein to successfully monitor ionic strength alterations in a ratiometric manner through independent fluorescence and bioluminescence measurements in cell lysates and live cells. We envisage that the biosensing strategy developed here for detecting alterations in intracellular ionic strength will be applicable in a wide range of experiments, including high throughput cellular signaling, ion channel functional genomics, and drug discovery.

scholarly journals Genome-wide association study and gene network analyses reveal potential candidate genes for high night temperature tolerance in rice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Raju Bheemanahalli ◽  
Montana Knight ◽  
Cherryl Quinones ◽  
Colleen J. Doherty ◽  
S. V. Krishna Jagadish
Keyword(s):  
Grain Size ◽  
Candidate Genes ◽  
Gene Network ◽  
Genome Wide Association ◽  
Potential Candidate ◽  
Genetic Loci ◽  
Stay Green ◽  
Yield And Quality ◽  
Genome Wide ◽  
Wide Range

AbstractHigh night temperatures (HNT) are shown to significantly reduce rice (Oryza sativa L.) yield and quality. A better understanding of the genetic architecture of HNT tolerance will help rice breeders to develop varieties adapted to future warmer climates. In this study, a diverse indica rice panel displayed a wide range of phenotypic variability in yield and quality traits under control night (24 °C) and higher night (29 °C) temperatures. Genome-wide association analysis revealed 38 genetic loci associated across treatments (18 for control and 20 for HNT). Nineteen loci were detected with the relative changes in the traits between control and HNT. Positive phenotypic correlations and co-located genetic loci with previously cloned grain size genes revealed common genetic regulation between control and HNT, particularly grain size. Network-based predictive models prioritized 20 causal genes at the genetic loci based on known gene/s expression under HNT in rice. Our study provides important insights for future candidate gene validation and molecular marker development to enhance HNT tolerance in rice. Integrated physiological, genomic, and gene network-informed approaches indicate that the candidate genes for stay-green trait may be relevant to minimizing HNT-induced yield and quality losses during grain filling in rice by optimizing source-sink relationships.

scholarly journals Fluorescent thermal shift-based method for detection of NF-κB binding to double-stranded DNA

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Peter D. Leitner ◽  
Ilja Vietor ◽  
Lukas A. Huber ◽  
Taras Valovka
Keyword(s):  
Dna Binding ◽  
Rational Design ◽  
Dissociation Constants ◽  
Inflammatory Diseases ◽  
Dimethyl Fumarate ◽  
Bioinformatic Analysis ◽  
Quantitative Detection ◽  
Regulatory Sequences ◽  
Wide Range ◽  
Inhibitory Drug

AbstractThe nuclear factor kappa B (NF-κB) family of dimeric transcription factors regulates a wide range of genes by binding to their specific DNA regulatory sequences. NF-κB is an important therapeutic target linked to a number of cancers as well as autoimmune and inflammatory diseases. Therefore, effective high-throughput methods for the detection of NF-κB DNA binding are essential for studying its transcriptional activity and for inhibitory drug screening. We describe here a novel fluorescence-based assay for quantitative detection of κB consensus double-stranded (ds) DNA binding by measuring the thermal stability of the NF-κB proteins. Specifically, DNA binding proficient NF-κB probes, consisting of the N-terminal p65/RelA (aa 1–306) and p50 (aa 1–367) regions, were designed using bioinformatic analysis of protein hydrophobicity, folding and sequence similarities. By measuring the SYPRO Orange fluorescence during thermal denaturation of the probes, we detected and quantified a shift in the melting temperatures (ΔTm) of p65/RelA and p50 produced by the dsDNA binding. The increase in Tm was proportional to the concentration of dsDNA with apparent dissociation constants (KD) of 2.228 × 10–6 M and 0.794 × 10–6 M, respectively. The use of withaferin A (WFA), dimethyl fumarate (DMF) and p-xyleneselenocyanate (p-XSC) verified the suitability of this assay for measuring dose-dependent antagonistic effects on DNA binding. In addition, the assay can be used to analyse the direct binding of inhibitors and their effects on structural stability of the protein probe. This may facilitate the identification and rational design of new drug candidates interfering with NF-κB functions.

Experiences with alluvial foundations for earth dams in the Prairie provinces

1979 ◽  
Vol 16 (2) ◽  
pp. 255-271 ◽  
Author(s):  
N. Peters ◽  
K. N. Lamb
Keyword(s):  
Rational Design ◽  
Preliminary Design ◽  
Empirical Relationships ◽  
Design Concepts ◽  
Theoretical Design ◽  
Pore Pressures ◽  
Wide Range ◽  
Thick Layers ◽  
Limited Testing ◽  
Prairie Provinces

The foundations for numerous dams in proglacial and interglacial valleys in the Prairie provinces consist of soft alluvial soils. The deposits are up to 60 m deep, and contain thick layers of clay interspersed with lenses and layers of silt, sand, and gravel.This paper describes the damsite investigation and laboratory testing required, the design methods and construction procedures used, and the foundation performance observed during and after construction. A number of empirical relationships between index tests and physical properties of the soils, which provide useful guidelines for preliminary design, are presented.The design approach has gradually evolved from an empirical design with limited testing to a more rational design based on detailed investigations and thorough instrumentation. Increased reliance is placed on observational apparatus to monitor movements and pore pressures to confirm design assumptions as construction proceeds. The theoretical design is always checked with former designs of dams that have performed satisfactorily.Safe economical dams have been constructed in spite of large deformations and high pore pressures. Two case histories illustrate the wide range in dam design for alluvial foundations. The first shows an older design cross section with modifications required to ensure a stable dam, and the second describes a recently constructed dam that incorporates many of the latest design concepts.

scholarly journals Purification and Characterization of Asparaginase fromPhaseolus vulgarisSeeds

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Saleh A. Mohamed ◽  
Mohamed F. Elshal ◽  
Taha A. Kumosani ◽  
Alia M. Aldahlawi
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Potential Candidate ◽  
Purification And Characterization ◽  
Apparent Homogeneity ◽  
Wide Range ◽  
Optimum Ph ◽  
Glutaminase Activity ◽  
Low Activity

L-asparaginase from bacteria has been used in treatment of acute lymphoblastic leukemia. The aim of this study was to purify and characterize L-asparaginase fromPhaseolus vulgarisseeds instead of microbial sources. L-asparaginase was purified to apparent homogeneity. The enzyme has molecular mass of 79 kDa. The purified asparaginase had very low activity toward a number of asparagine and glutamine analogues. L-asparaginase was free from glutaminase activity. Kinetic parameters, Km andVmax of purified enzyme, were found to be 6.72 mM and 0.16 μM, respectively. The enzyme had optimum pH at 8.0. The enzyme showed high stability at alkaline pH (pH 7.5–9.0) when incubated for up to 24 h. L-asparaginase had the same temperature optimum and thermal stability at 37°C. K+was able to greatly enhance the activity of asparaginase by 150% compared with other metals tested. In conclusion, L-asparaginase showed no glutaminase activity and good stability over a wide range of physiological conditions, and thus it could be used as a potential candidate for treatment of acute lymphoblastic leukemia.

Automation and Management of Design Process of the Main Drive for an Innovative Fruits and Vegetables Washer

2021 ◽  
Vol 27 (1) ◽  
pp. 9-17
Author(s):  
V. P. Bui ◽  
◽  
S. S. Gavruishin ◽  
V. B. Phung ◽  
H. M. Dang ◽  
...  
Keyword(s):  
Design Process ◽  
Rational Design ◽  
Fruits And Vegetables ◽  
Rational Solution ◽  
Quality Criteria ◽  
Pareto Optimal ◽  
Optimal Domain ◽  
Wide Range ◽  
A New Technique ◽  
New Generation

A new technique is described, used by the authors to automate the design process of the main drive of a new generation machine intended for industrial washing of fruits and vegetables. To solve the problem of multi-criteria design, the original approach is proposed that uses interconnected mathematical models describing the dynamic behavior, strength reliability and functional characteristics of the machine in a unified information space. The generalized mathematical model includes 12 controlled parameters, 16 functional constraints, and 3 quality criteria. A genetic algorithm was used to find the space of Pareto-optimal solutions. The situational approach was used to select the final rational solution from a set of solutions belonging to the Pareto-optimal domain. The rational design of option the washer found using the proposed approach is compared with the existing ones. The proposed design methodology can be recommended for the design of a wide range of similar mechanical structures.

scholarly journals The AAA+ ATPase p97, a cellular multitool

2017 ◽  
Vol 474 (17) ◽  
pp. 2953-2976 ◽  
Author(s):  
Lasse Stach ◽  
Paul S. Freemont
Keyword(s):  
Atp Hydrolysis ◽  
Current Status ◽  
Cellular Functions ◽  
Aaa Atpase ◽  
Cellular Processes ◽  
Proteotoxic Stress ◽  
Binding Domains ◽  
Wide Range ◽  
Aaa Atpases ◽  
Substrate Proteins

The AAA+ (ATPases associated with diverse cellular activities) ATPase p97 is essential to a wide range of cellular functions, including endoplasmic reticulum-associated degradation, membrane fusion, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and chromatin-associated processes, which are regulated by ubiquitination. p97 acts downstream from ubiquitin signaling events and utilizes the energy from ATP hydrolysis to extract its substrate proteins from cellular structures or multiprotein complexes. A multitude of p97 cofactors have evolved which are essential to p97 function. Ubiquitin-interacting domains and p97-binding domains combine to form bi-functional cofactors, whose complexes with p97 enable the enzyme to interact with a wide range of ubiquitinated substrates. A set of mutations in p97 have been shown to cause the multisystem proteinopathy inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia. In addition, p97 inhibition has been identified as a promising approach to provoke proteotoxic stress in tumors. In this review, we will describe the cellular processes governed by p97, how the cofactors interact with both p97 and its ubiquitinated substrates, p97 enzymology and the current status in developing p97 inhibitors for cancer therapy.

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