Biology subjects

Upadhyay, A.

Publications and source records attributed to Upadhyay, A..

29 records · Page 2Linked to original sources

bioRxiv2020

Multiple random phosphorylations provide long delays and switches in circadian clocks

Theory predicts that self-sustained oscillations require robust delays and nonlinearities (ultrasensitivity). Delayed negative feedback loops with switch-like inhibition of transcription constitute the core of eukaryotic circadian clock. The kinetics of core clock proteins such as PER2 in mammals and FRQ in Neurospora crassa is governed by multiple phosphorylations. We investigate how multiple, slow and random phosphorylations control delay and molecular switches. We model phosphorylations of intrinsically disordered clock proteins (IDPs) using conceptual models of sequential and distributive phosphorylations. Our models help to understand the underlying mechanisms leading to delays and ultrasensitivity. The model shows temporal and steady state switches for the free kinase and the phosphoprotein. We show that random phosphorylations and sequestration mechanisms allow high Hill coefficients required for self-sustained oscillations.

systems biology↗

bioRxiv2020

Nonallosteric Sirtuin Enzyme Activation: Characterization of Hit Compounds

Mammalian sirtuins (SIRT1-SIRT7) are a family of nicotinamide adenine dinucleotide (NAD+)-dependent protein deacylases that play critical roles in lifespan and age-related diseases. The physiological importance of sirtuins has stimulated intense interest in designing sirtuin-activating compounds. However, except for allosteric activators of SIRT1-catalyzed reactions that are limited to particular substrates, a general framework for the design of sirtuin-activating compounds has been lacking. Recently, we introduced a general mode of sirtuin activation that is distinct from the known modes of enzyme activation, establishing biophysical properties of small molecule modulators that can, in principle, result in enzyme activation for various sirtuins and substrates. Here, we characterize small molecules reported in the literature to activate the SIRT3 enzyme using a variety of computational, biochemical and biophysical techniques including protein-ligand docking, molecular dynamics simulation, nonlinear reaction dynamics simulation, kinetic assays and thermodynamic assays with multiple substrates and protocols. In particular, we identify the mechanism of action of the compound honokiol on the human SIRT3 enzyme, modeling its effect on active site conformational degrees of freedom and demonstrating how it nonallosterically activates the human SIRT3 enzyme under physiologically relevant conditions. We show that honokiol constitutes a hit compound for the design of a new generation of nonallosteric activators that can activate SIRT3 through the proposed mechanism-based mode of activation.

biophysics↗

bioRxiv2020

Mechanism-based enzyme activating compounds

Compared to inhibitors, which constitute the vast majority of todays drugs, enzyme activators have considerable advantages, especially in the context of enzymes that regulate reactive flux through metabolic pathways associated with chronic, age-related diseases and lifespan. Across all families of enzymes, only a dozen or so distinct classes of small molecule activators have been characterized. Enzyme activators that are not based on naturally evolved allosteric mechanisms are much more difficult to design than inhibitors, because enzymatic catalysis has been optimized over billions of years of evolution. Here, we introduce modes of enzyme activation based on the catalytic reaction mechanisms of enzymes for which naturally evolved activators may not exist. We establish biophysical properties of small molecule modulators that are necessary to achieve desired changes in the steady state and non-steady state parameters of these enzymes, including changes in local conformational degrees of freedom conducive to the enhancement of catalytic activity that can be identified through computational modeling of their active sites. We illustrate how the modes of action of several compounds reported to activate enzymes without known allosteric sites may be understood using the framework presented. We also present simulations and new experimental results in support of this framework, including identification of the mechanism of a compound that activates the human SIRT3 enzyme, which does not contain a known allosteric site, under physiologically relevant conditions.

biochemistry↗

bioRxiv2020

Engineering multiple species-like genetic incompatibilities in insects

Speciation constrains the flow of genetic information between populations of sexually reproducing organisms. Gaining control over mechanisms of speciation would enable new strategies to manage wild populations of disease vectors, agricultural pests, and invasive species. Additionally, such control would provide safe biocontainment of transgenes and gene drives. Natural speciation can be driven by pre-zygotic barriers that prevent fertilization or by post-zygotic genetic incompatibilities that render the hybrid progeny inviable or sterile. Here we demonstrate a general approach to create engineered genetic incompatibilities (EGIs) in the model insect Drosophila melanogaster. Our system couples a dominant lethal transgene with a recessive resistance allele. EGI strains that are homozygous for both elements are fertile and fecund when they mate with similarly engineered strains, but incompatible with wild-type strains that lack resistant alleles. We show that EGI genotypes can be tuned to cause hybrid lethality at different developmental life-stages. Further, we demonstrate that multiple orthogonal EGI strains of D. melanogaster can be engineered to be mutually incompatible with wild-type and with each other. Our approach to create EGI organisms is simple, robust, and functional in multiple sexually reproducing organisms.

synthetic biology↗

bioRxiv2020

A Combined Evidence Approach to Prioritize Nipah Virus Inhibitors

Nipah Virus (NiV) came into limelight due to an outbreak in Kerala, India. NiV causes severe disease and death in people with over 75% case fatality rate. It is a public health concern and has the potential to become a global pandemic. Lack of treatment has forced the containment methods to be restricted to isolation and surveillance. WHOs R&D Blueprint list of priority diseases (2018) indicates that there is an urgent need for accelerated research & development for addressing NiV. In the quest for druglike NiV inhibitors (NVIs) a thorough literature search followed by systematic data curation was conducted. Rigorous data analysis was done with curated NVIs for prioritizing druglike compounds. For the same, more than 1800 descriptors of NVIs were computed and comparative analysis was performed with the FDA approved small molecules and antivirals. These compounds were further evaluated through PAINS filter to study their toxicity profile. Simultaneously, compounds were also prioritized based on the robustness of the assays through which they were identified. Our efforts lead to the creation of a well-curated structured knowledgebase of 182 NVIs with 98 small molecule inhibitors. The reported IC50/EC50 values for some of these inhibitors are in the nanomolar range - as low as 0.47 nM. In order to prioritize these inhibitors, we performed several tests and applied filters to identify drug-like non-toxic compounds. Of 98, a few compounds passed DruLito & PAINS filters exhibiting drug-like properties and were also prioritized in an independent screen based only the assay robustness. The NVIs have diverse structural features and offer a wide spectrum of ways in which they can be developed further as druglike molecules. We report a knowledgebase for furthering the development of NVIs. The platform has a diverse set of 98 NVIs of which a few have been prioritized based on a combined evidence strategy. The platform has the provision to submit new inhibitors as and when reported by the community for further enhancement of NiV inhibitor landscape.

bioinformatics↗

bioRxiv2019

Leaderless short ORFs in mycobacteria comprise a translational regulon

Genome-wide transcriptomic analyses have revealed abundant expressed short open reading frames (ORFs) in bacteria. Whether these short ORFs, or the small proteins they encode, are functional remains an open question. One quarter of mycobacterial mRNAs are leaderless, beginning with a 5-AUG or GUG initiation codon. Leaderless mRNAs often encode unannotated short ORFs as the first gene of a polycistronic transcript. Here we show that polycysteine-encoding leaderless short ORFs function as cysteine-responsive attenuators of operonic gene expression. Detailed mutational analysis shows that one polycysteine short ORF controls expression of the downstream genes. Our data indicate that ribosomes stalled in the polycysteine tract block mRNA structures that otherwise sequester the ribosome-binding site of the 3gene. We assessed endogenous proteomic responses to cysteine limitation in Mycobacterium smegmatis using mass spectrometry. Six cysteine metabolic loci having unannotated polycysteine-encoding leaderless short ORF architectures responded to cysteine limitation, revealing widespread cysteine-responsive attenuation in mycobacteria. Individual leaderless short ORFs confer independent operon-level control, while their shared dependence on cysteine ensures a collective response mediated by ribosome pausing. We propose the term ribulon to classify ribosome-directed regulons. Regulon-level coordination by ribosomes on sensory short ORFs illustrates one utility of the many unannotated short ORFs expressed in bacterial genomes.

microbiology↗

bioRxiv2019

Batch cultivation of non-transgenic males suitable for SIT programs

The field performance of Sterile Insect Technique (SIT) is improved by sex-sorting and releasing only the sterile males. This can be accomplished by resource-intensive separation of males from females by morphology. Alternatively, sex-ratio biasing genetic constructs can be used to selectively kill one sex without the need for manual or automated sorting, but the resulting genetically engineered (GE) control agents would be subject to additional governmental regulation. Here we describe and demonstrate a method for the batch production of non-GE males that is applicable for sex-selective production of males suitable for genetic biocontrol programs. This method could be applied to generate the heterogametic sex (XY, or WZ) in any organism with chromosomal sex determination. We observed up to 100% sex-selection with batch cultures of more than 103 individuals. Using a stringent transgene detection assay, we demonstrate the potential of mass rearing of transgene free males.

synthetic biology↗

bioRxiv2019

Muscle-derived Myoglianin regulates Drosophila melanogaster imaginal disc growth

Organ growth and size are finely tuned by intrinsic and extrinsic signaling molecules. In Drosophila, the BMP family member Dpp is produced in a limited set of imaginal disc cells and functions as a classic morphogen to regulate pattern and growth by diffusing throughout imaginal discs. However, the role of TGF{beta}/Activin-like ligands in disc growth control remains ill-defined. Here we demonstrate that Myoglianin (Myo), an Activin family member, and a close homolog of mammalian Myostatin (Mstn), is a muscle-derived extrinsic factor that uses canonical dSmad2 mediated signaling to regulate wing size. We propose that Myo is a myokine that helps mediate an allometric relationship between muscles and their associated appendages.

developmental biology↗

bioRxiv2019

Motoneuron-derived Activinβ regulates Drosophila body size and tissue-scaling during larval growth and adult development

Correct scaling of body and organ size is crucial for proper development and survival of all organisms. Perturbations in circulating hormones, including insulins and steroids, are largely responsible for changing body size in response to both genetic and environmental factors. Such perturbations typically produce adults whose organs and appendages scale proportionately with final size. The identity of additional factors that might contribute to scaling of organs and appendages with body size is unknown. Here we report that loss-of-function mutations in Drosophila Activin{beta} (Act{beta}), a member of the TGF-{beta} superfamily, lead to production of small larvae/pupae and undersized rare adult escapers. Morphometric measurements of escaper adult appendage size (wings, legs), as well as heads, thoraxes, and abdomens, reveal a disproportional reduction in abdominal size compared to other tissues. Similar size measurements of selected Act{beta} mutant larval tissues demonstrate that somatic muscle size is disproportionately smaller when compared to fat body, salivary glands, prothoracic glands, imaginal discs and brain. We also show that Act{beta} control of body size is dependent on canonical signaling through the transcription-factor dSmad2 and that it modulates the growth rate, but not feeding behavior, during the third instar period. Tissue and cell-specific knockdown and overexpression studies reveal that motoneuron derived Act{beta} is essential for regulating proper body size and tissue scaling. These studies suggest that, unlike in vertebrates where Myostatin, and certain other Activin-like factors act as systemic negative regulators of muscle mass, in Drosophila Act{beta} is a positive regulator of muscle mass that is directly delivered to muscles by motoneurons. We discuss the importance of these findings in coordinating proportional scaling of insect muscle mass to appendage size.

developmental biology↗

bioRxiv2019

An inactivation switch enables rhythms in a Neurospora clock model

An autonomous endogenous time-keeping is ubiquitous across many living organisms known as circadian clock when it has a period of about 24 hours. Interestingly, the fundamental design principle with a network of interconnected negative and positive feedback loops is conserved through evolution, although the molecular components differ. Filamentous fungus Neurospora crassa is a well established chrono-genetics model organism to investigate the underlying mechanisms. The core negative feedback loop of the clock of Neurospora is composed of the transcription activator White Collar Complex (WCC) (heterodimer of WC1 and WC2) and the inhibitory element called FFC complex which is made of FRQ (Frequency protein), FRH (Frequency interacting RNA Helicase) and CK1a (Casein kinase 1a). While exploring their temporal dynamics we investigate how limit cycle oscillations arise and how molecular switches support self-sustained rhythms. We develop a mathematical model of 10 variables with 26 parameters to understand the interactions and feedbacks among WC1 and FFC elements in nuclear and cytoplasmic compartments. We performed control and bifurcation analysis to show that our novel model produces robust oscillations with a wild-type period of 22.5 hrs. Our model reveals a switch between WC1 induced transcription and FFC assisted inactivation of WC1. Using the new model we also study possible mechanisms of glucose compensation. A fairly simple model with just 3 non-linearities helps to elucidate clock dynamics revealing a mechanism of rhythms production. The model can further be utilized to study entrainment and temperature compensation.

systems biology↗

bioRxiv2018

Optogenetic and transcriptomic interrogation of enhanced muscle function in the paralyzed mouse whisker pad

The functional state of denervated muscle is a critical factor in the ability to restore movement after injury- or disease-related paralysis. Here we used peripheral optogenetic stimulation and transcriptome profiling in the mouse whisker system to investigate the time course of changes in neuromuscular function following complete unilateral facial nerve transection. While most skeletal muscles rapidly lose functionality after lower motor neuron denervation, optogenetic muscle stimulation of the paralyzed whisker pad revealed sustained increases in the sensitivity, velocity, and amplitude of whisker movements, and reduced fatigability, starting 48 h after denervation. RNA-seq analysis showed distinct regulation of multiple gene families in denervated whisker pad muscles compared to the atrophy-prone soleus, including prominent changes in ion channels and contractile fibers. Together, our results define the unique functional and transcriptomic landscape of denervated facial muscles, and have general implications for restoring movement after neuromuscular injury or disease. New & NoteworthyOptogenetic activation of muscle can be used to non-invasively induce movements and probe muscle function. We used this technique in mice to investigate changes in whisker movements following facial nerve transection. We found unexpectedly enhanced functional properties of whisker pad muscle following denervation, accompanied by unique transcriptomic changes. Our findings highlight the utility of the mouse whisker pad for investigating the restoration of movement after paralysis.

neuroscience↗