Investigation of the Interaction between the Large and Small Subunits of Potato ADP-Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase), a key allosteric enzyme involved in higher plant starch biosynthesis, is composed of pairs of large (LS) and small subunits (SS). Current evidence indicates that the two subunit types play distinct roles in enzyme function. Recently the heterotetrameric structure of potato AGPase has been modeled. In the current study, we have applied the molecular mechanics generalized born surface area (MM-GBSA) method and identified critical amino acids of the potato AGPase LS and SS subunits that interact with each other during the native heterotetrameric structure formation. We have further shown the role of the LS amino acids in subunit-subunit interaction by yeast two-hybrid, bacterial complementation assay and native gel. Comparison of the computational results with the experiments has indicated that the backbone energy contribution (rather than the side chain energies) of the interface residues is more important in identifying critical residues. We have found that lateral interaction of the LS-SS is much stronger than the longitudinal one, and it is mainly mediated by hydrophobic interactions. This study will not only enhance our understanding of the interaction between the SS and the LS of AGPase, but will also enable us to engineer proteins to obtain better assembled variants of AGPase which can be used for the improvement of plant yield

Nanoengineering InP Quantum Dot-Based Photoactive Biointerfaces for Optical Control of Neurons

Light-activated biointerfaces provide a non-genetic route for effective control of neural activity. InP quantum dots (QDs) have a high potential for such biomedical applications due to their uniquely tunable electronic properties, photostability, toxic-heavy-metal-free content, heterostructuring, and solution-processing ability. However, the effect of QD nanostructure and biointerface architecture on the photoelectrical cellular interfacing remained unexplored. Here, we unravel the control of the photoelectrical response of InP QD-based biointerfaces via nanoengineering from QD to device-level. At QD level, thin ZnS shell growth (∼0.65 nm) enhances the current level of biointerfaces over an order of magnitude with respect to only InP core QDs. At device-level, band alignment engineering allows for the bidirectional photoelectrochemical current generation, which enables light-induced temporally precise and rapidly reversible action potential generation and hyperpolarization on primary hippocampal neurons. Our findings show that nanoengineering QD-based biointerfaces hold great promise for next-generation neurostimulation devices

DNA repair by photolyases

Photolyases belong to the cryptochrome/photolyase protein family (CPF) which perform different functions such as DNA repair, circadian photoreceptor, and transcriptional regulation. Photolyase is a flavoprotein that repairs UV-induced DNA damages of cyclobutane pyrimidine dimer (CPD) and pyrimidine-pyrimidone (6-4) photoproducts using blue-light as an energy source. This enzyme has two chromophores: flavin adenine dinucleotide (FAD) as a cofactor and a photoantenna such as methenyltetrahydrofolate (MTHF). The FAD is essential for catalysis of the DNA repair. The second chromophore absorbs photons from the blue light spectrum and transfers energy to FAD to increase the repair efficiency of the enzyme. Phylogenetic analysis in which amino acid sequences of several hundreds of CPF members are used suggests that they form more classes than we have considered so far. In this chapter, we discussed structure-functions and reaction mechanisms of different classes of photolyases

Identification of novel small molecules targeting core clock proteins to regulate circadian rhythm

© 2021 Elsevier LtdThe circadian rhythms are physiological, biochemical, and behavioral oscillations that cycle every 24 hours to anticipate the daily changes in the external environment. Disruption of the circadian clock in mammals results in increased susceptibility to different types of diseases such as metabolic, mood and sleep disorders and cancer. To this end, different approaches have been taken to find small molecules that have the potential to correct the disrupted circadian clock. In this review, we highlight the recent developments in identifying novel molecules that regulate the activities of the core clock proteins. Finally, we discuss virtual screening-based methods using the crystal structures of core clock proteins for the discovery of small molecules that regulate the circadian rhythm

Insights into Subunit Interactions in the Heterotetrameric Structure of Potato ADP-Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase, a key allosteric enzyme involved in higher plant starch biosynthesis, is composed of pairs of large (LS) and small subunits (SS). Current evidence indicates that the two subunit types play distinct roles in enzyme function. The LS is involved in mainly allosteric regulation through its interaction with the catalytic SS. Recently the crystal structure of the SS homotetramer has been solved, but no crystal structure of the native heterotetrameric enzyme is currently available. In this study, we first modeled the three-dimensional structure of the LS to construct the heterotetrameric enzyme. Because the enzyme has a 2-fold symmetry, six different dimeric (either up-down or side-by-side) interactions were possible. Molecular dynamics simulations were carried out for each of these possible dimers. Trajectories obtained from molecular dynamics simulations of each dimer were then analyzed by the molecular mechanics/Poisson-Boltzmann surface area method to identify the most favorable dimers, one for up-down and the other for side-by-side. Computational results combined with site directed mutagenesis and yeast two hybrid experiments suggested that the most favorable heterotetramer is formed by LS-SS (side-by-side), and LS-SS (up-down). We further determined the order of assembly during the heterotetrameric structure formation. First, side-by-side LS-SS dimers form followed by the up-down tetramerization based on the relative binding free energies

MerR and ChrR mediate blue light induced photo-oxidative stress response at the transcriptional level in Vibrio cholerae

Blue light (BL) is a major environmental factor that affects the physiology, behavior, and infectivity of bacteria as it contributes to the generation of reactive oxygen species (ROS) while increasing photo-oxidative stress in cells. However, precise photo-oxidative response mechanism in non-phototrophic bacteria is yet to be elucidated. In this study, we investigated the effect of BL in Vibrio cholerae by using genetics and transcriptome profiling. Genome-wide analysis revealed that transcription of 6.3% of V. cholerae genes were regulated by BL. We further showed that BL enhances ROS production, which is generated through the oxidative phosphorylation. To understand signaling mechanisms, we generated several knockouts and analyzed their transcriptome under BL exposure. Studies with a double-knockout confirm an anti-sigma factor (ChrR) and putative metalloregulatory-like protein (MerR) are responsible for the genome-wide regulation to BL response in V. cholerae. Collectively, these results demonstrate that MerR-like proteins, in addition to ChrR, are required for V. cholerae to mount an appropriate response against photo-oxidative stress induced by BL. Outside its natural host, V. cholerae can survive for extended periods in natural aquatic environments. Therefore, the regulation of light response for V. cholerae may be a critical cellular process for its survival in these environments

RNA-seq analysis of the transcriptional response to blue and red light in the extremophilic red alga, Cyanidioschyzon merolae

Light is one of the main environmental cues that affects the physiology and behavior of many organisms. The effect of light on genome-wide transcriptional regulation has been well-studied in green algae and plants, but not in red algae. Cyanidioschyzon merolae is used as a model red algae, and is suitable for studies on transcriptomics because of its compact genome with a relatively small number of genes. In addition, complete genome sequences of the nucleus, mitochondrion, and chloroplast of this organism have been determined. Together, these attributes make C. merolae an ideal model organism to study the response to light stimuli at the transcriptional and the systems biology levels. Previous studies have shown that light significantly affects cell signaling in this organism, but there are no reports on its blue light- and red light-mediated transcriptional responses. We investigated the direct effects of blue and red light at the transcriptional level using RNA-seq. Blue and red lights were found to regulate 35 % of the total genes in C. merolae. Blue light affected the transcription of genes involved in protein synthesis while red light specifically regulated the transcription of genes involved in photosynthesis and DNA repair. Blue or red light regulated genes involved in carbon metabolism and pigment biosynthesis. Overall, our data showed that red and blue light regulate the majority of the cellular, cell division, and repair processes in C. merolae

Comparative RNA-seq analysis of the drought-sensitive lentil (Lens culinaris) root and leaf under short- and long-term water deficits

Drought stress is one of the main environmental factors that affects growth and productivity of crop plants, including lentil. To gain insights into the genome-wide transcriptional regulation in lentil root and leaf under short- and long-term drought conditions, we performed RNA-seq on a drought-sensitive lentil cultivar (Lens culinaris Medik. cv. Sultan). After establishing drought conditions, lentil samples were subjected to de novo RNA-seq-based transcriptome analysis. The 207,076 gene transcripts were successfully constructed by de novo assembly from the sequences obtained from root, leaf, and stems. Differentially expressed gene (DEG) analysis on these transcripts indicated that period of drought stress had a greater impact on the transcriptional regulation in lentil root. The numbers of DEGs were 2915 under short-term drought stress while the numbers of DEGs were increased to 18,327 under long-term drought stress condition in the root. Further, Gene Ontology analysis revealed that the following biological processes were differentially regulated in response to long-term drought stress: protein phosphorylation, embryo development seed dormancy, DNA replication, and maintenance of root meristem identity. Additionally, DEGs, which play a role in circadian rhythm and photoreception, were downregulated suggesting that drought stress has a negative effect on the internal oscillators which may have detrimental consequences on plant growth and survival. Collectively, this study provides a detailed comparative transcriptome response of drought-sensitive lentil strain under short- and long-term drought conditions in root and leaf. Our finding suggests that not only the regulation of genes in leaves is important but also genes regulated in roots are important and need to be considered for improving drought tolerance in lentil

Molecular Aspects of Circadian Pharmacology and Relevance for Cancer Chronotherapy

The circadian timing system (CTS) controls various biological functions in mammals including xenobiotic metabolism and detoxification, immune functions, cell cycle events, apoptosis and angiogenesis. Although the importance of the CTS is well known in the pharmacology of drugs, it is less appreciated at the clinical level. Genome-wide studies highlighted that the majority of drug target genes are controlled by CTS. This suggests that chronotherapeutic approaches should be taken for many drugs to enhance their effectiveness. Currently chronotherapeutic approaches are successfully applied in the treatment of different types of cancers. The chronotherapy approach has improved the tolerability and antitumor efficacy of anticancer drugs both in experimental animals and in cancer patients. Thus, chronobiological studies have been of importance in determining the most appropriate time of administration of anticancer agents to minimize their side effects or toxicity and enhance treatment efficacy, so as to optimize the therapeutic ratio. This review focuses on the underlying mechanisms of the circadian pharmacology i.e., chronopharmacokinetics and chronopharmacodynamics of anticancer agents with the molecular aspects, and provides an overview of chronotherapy in cancer and some of the recent advances in the development of chronopharmaceutics