Biology subjects

Yadav, R.

Publications and source records attributed to Yadav, R..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

Single session of rTMS enhances brain metastability and intrinsic ignition

BackgroundEmerging evidence support the view that brain stimulation might improve essential tremor (ET) by altering brain networks and facilitating plasticity. Yet, we are still missing a mechanistic explanation of the whole brain dynamics underlying these plasticity defining changes. MethodIn this study, we explored the effect of low-frequency repetitive transcranial magnetic stimulation (rTMS) over left primary motor cortex (L-M1) on functional connectivity dynamics (FCD) in patients with ET. Resting-state fMRI (RsfMRI) was acquired before and after a single session of rTMS in 30 patients with ET and compared with RsfMRI of 20 age and gender matched healthy controls (HCs). We have measured the effect of brain stimulation using network topological re-organization through whole brain integration and segregation, brain stability and capacity of neural propagation through metastability and intrinsic ignition. ResultsPatients with ET had altered FCD measures compared to controls. After a single session rTMS, the brain connectivity measures approached normality and patients with ET revealed significantly higher integration, lower segregation with higher metastability and increased intrinsic ignition. ConclusionBrain metastability and intrinsic ignition measures could be valuable tools in appreciating mechanisms of brain stimulation in ET and other neurological diseases.

neuroscience↗

bioRxiv2022

Identification of a multipotent lung progenitor for lung regeneration

We recently showed that intravenous infusion of mouse or human, fetal or adult lung cells following conditioning of recipient mice leads to lung chimerism within alveolar and bronchiolar lineages, in distinct patches containing both epithelial and endothelial cells. We show here, using R26R-Confetti mice as donors, that these multi-lineage patches are derived from a single lung progenitor. FACS of adult mouse lung cells revealed that the putative patch-forming progenitors co-express the endothelial marker CD31 (PECAM-1) and the epithelial marker CD326 (EPCAM). Transplantation of lung cells from transgenic Cre/lox mice expressing nuclear GFP under the VEcad promoter (VEcad-Cre-nTnG), led to GFP+ patches comprising both GFP+ endothelial and epithelial cells in vivo, and in ex-vivo culture of CD326+CD31+ progenitors. Single cell RNA sequencing of CD326+CD31+ lung cells revealed a subpopulation expressing canonical epithelial and endothelial genes. Such double positive GFP+NKX2.1+SOX17+ cells were also detected by immunohistological staining in lungs of VEcad-Cre-nTnG (expressing nuclear GFP) mice in proximity to blood vessels. These findings provide new insights on lung progenitors and lung development and suggest a potential novel approach for lung regeneration. SummaryWe show in the present study, that multi-lineage regenerative patches in our transplantation model are derived from a single lung progenitor, co-expressing the endothelial marker CD31 and the epithelial marker CD326. These findings provide new insights on lung progenitors and lung development.

cell biology↗

bioRxiv2022

Biochemical and structural insights into SARS-CoV-2 polyprotein processing by Mpro

SARS-CoV-2, a human coronavirus, is the causative agent of the COVID-19 pandemic. Its [~]30 kb RNA genome is translated into two large polyproteins subsequently cleaved by viral papain-like protease and main protease (Mpro/nsp5). Polyprotein processing is essential yet incompletely understood. We studied Mpro-mediated processing of the nsp7-10/11 polyprotein, whose mature products are cofactors of the viral replicase, identifying the order of cleavages as: 1) nsp9-10, 2) nsp8-9/nsp10-11, and 3) nsp7-8. Integrative modeling based on mass spectrometry (including hydrogen-deuterium exchange and cross-linking) and X-ray scattering yielded three-dimensional models of the nsp7-10/11 polyprotein. Our data suggest that the nsp7- 10/11 structure in complex with Mpro strongly resembles the unbound polyprotein, and that both polyprotein conformation and junction accessibility determine the preference and order of cleavages. Finally, we used limited proteolysis assays to characterize the effect of a series of inhibitors/binders on Mpro processing of nsp7-11 and Mpro inhibition using a polyprotein substrate. TeaserWe elucidated the structural basis of order of cleavage of SARS-CoV-2 nsp7-11 polyprotein, with implications for Mpro inhibition.

biochemistry↗

bioRxiv2022

Tissue and cell-type specific molecular and functional signatures of 16p11.2 reciprocal genomic disorder across mouse brain and human neuronal models

Recurrent deletion and duplication of [~]743 kilobases of unique genomic sequence and segmental duplications at chromosome 16p11.2 underlie a reciprocal genomic disorder (RGD; OMIM 611913 and 614671) associated with neurodevelopmental and psychiatric phenotypes, including intellectual disability, autism spectrum disorder (ASD), and schizophrenia (SCZ). To define molecular alterations associated with the 16p11.2 RGD, we performed transcriptome analyses of mice with reciprocal copy number variants (CNVs) of the syntenic chromosome 7qF3 region and human neuronal models derived from isogenic human induced pluripotent stem cells (hiPSCs) carrying CRISPR-engineered CNVs at 16p11.2. Analysis of differentially expressed genes (DEGs) in mouse cortex, striatum, cerebellum and three non-brain tissues, as well as in human neural stem cells and induced glutamatergic neurons revealed that the strongest and most consistent effects occurred within the CNV sequence, with notable instances of differential expression of genes in the immediate vicinity that could reflect position effect. While differential expression of genes outside of chromosome 16p11.2 was largely region, tissue, and cell type-specific, a small but significant minority of such DEGs was shared between brain regions or human cell types. Gene Ontology (GO) enrichment analyses to identify cellular processes dysregulated due to these CNVs found support in select circumstances for terms related to energy metabolism, RNA metabolism, and translation but did not reveal a single universally affected process. Weighted gene co-expression network analysis identified modules that showed significant correlation with reciprocal or individual CNV genotype and better captured shared effects, indicating that energy metabolism, RNA metabolism, translation and protein targeting were disrupted across all three brain regions. The first two of these processes also emerged in the human neural stem cell (NSC) data. A subset of co-expression modules that correlated with CNV genotype revealed significant enrichments for known neurodevelopmental disorder genes, loss-of-function constrained genes, FMRP targets, and chromatin modifiers. Intriguingly, neuronal differentiation of the hiPSCs revealed that both the deletion and duplication CNV resulted in similar deficits in neurite extension and branching and alterations in electrical activity. Finally, generation of cerebral organoid derivatives indicated that the CNVs reciprocally altered the ratio of excitatory and inhibitory GABAergic neurons generated during in vitro neurodevelopment, consistent with a major mechanistic hypothesis for ASD. Collectively, our data suggest that the 16p11.2 RGD involves disruption of multiple biological processes, with a relative impact that is context-specific. Perturbation of individual and multiple genes within the CNV region will be required to dissect single-gene effects, uncover regulatory interactions, and define how each contributes to abnormal neurodevelopment.

genomics↗

bioRxiv2022

Elongated Hypocotyl 5 (HY5) regulates BRUTUS (BTS) to maintain Iron homeostasis in Arabidopsis thaliana.

Iron (Fe) is an essential micronutrient for both plants and animals. Fe limitation significantly reduces crop yield and therefore has adverse impacts on human nutrition. Owing to limited bioavailability of Fe, plants have adapted different strategies that regulate Fe uptake and homeostasis. Particularly, modifications of root growth traits are a key for the survival of plants on Fe-deficient soils. Understanding the molecular basis for these root growth responses will have critical implications for plant breeding. Fe uptake is regulated by a cascade of basic helix-loop-helix (bHLH) transcription factors. In our study, we show that HY5 (Elongated Hypocotyl 5), a member of the basic leucine zipper (bZIP) family of transcription factors plays an important role in the Fe deficiency signalling pathway in Arabidopsis thaliana. The hy5 mutant plants failed to mount an optimum Fe deficiency response and showed severe root growth defects under Fe limitation that could be partially reverted by complementation of hy5 mutant. qRT-PCR analysis revealed that the induction of the genes involved in Fe uptake pathway [FER-like iron deficiency-induced transcription factor (FIT), Ferric Reduction Oxidase 2 (FRO2) and Iron-Regulated Transporter1 (IRT1)] is significantly reduced in the hy5 mutants as compared to the wild-type plants under Fe deficiency. Moreover, we also found that HY5 function is critical for activating the expression of coumarin biosynthesis genes (F6H1, S8H, PDR9 and BGLU42) under Fe deficiency. Interestingly, our results showed that HY5 acts as a negative regulator of BRUTUS (BTS) which is known to negatively regulate Fe deficiency response. Chromatin Immunoprecipitation followed by qPCR revealed direct binding of HY5 to the promoter of BTS. Altogether, our results showed that HY5 plays an important role in regulation of Fe deficiency responses in Arabidopsis.

plant biology↗

bioRxiv2022

A diet-independent zebrafish model for NAFLD recapitulates patient lipid profiles and offers a system for small molecule screening

Non-alcoholic Fatty Liver Disease (NAFLD) or pathological hepatic lipid overload, is considered to affect obese individuals. However, NAFLD in lean individuals is prevalent, especially in South Asian population. The pathophysiology of lean NAFLD is not well understood and most animal models of NAFLD use the high-fat diet paradigm. To bridge this gap, we have developed a diet-independent model of NAFLD in zebrafish. We have previously showed that chronic systemic inflammation causes metabolic changes in the liver leading to hepatic fat accumulation in an IL6 overexpressing (IL6-OE) zebrafish model. In the present study, we compared the hepatic lipid composition of adult IL6-OE zebrafish to the controls and found an accumulation of saturated triacylglycerols and a reduction in the unsaturated triacylglycerol species reminiscent of NAFLD patients. Zebrafish is an ideal system for chemical genetic screens. We tested whether the hepatic lipid accumulation in the IL6-OE is responsive to chemical treatment. We found that PPAR-gamma agonist Rosiglitazone, known to reduce lipid overload in the high fat diet models of NAFLD, could ameliorate the fatty liver phenotype of the IL6-OE fish. Rosiglitazone treatment reduced the accumulation of saturated lipids and showed a concomitant increase in unsaturated TAG species in our inflammation-induced NAFLD model. Our observations suggest that the IL6-OE model can be effective for small molecule screening to identify compounds that can reverse hepatic lipid accumulation, especially relevant to lean NAFLD.

pharmacology and toxicology↗

bioRxiv2022

Predicting Protein Function and Orientation on a Gold Nanoparticle Surface Using a Residue-Based Affinity Scale

The orientation adopted by proteins on nanoparticle surfaces determines the nanoparticles bioactivity and its interactions with living systems. Here, we present a residue-based affinity scale for predicting protein orientation on citrate-gold nanoparticles (AuNPs). Competitive binding between protein variants accounts for thermodynamic and kinetic aspects of adsorption in this scale. For hydrophobic residues, the steric considerations dominate, whereas electrostatic interactions are critical for hydrophilic residues. The scale rationalizes the well-defined binding orientation of the small GB3 protein, and it subsequently predicts the orientation and active site accessibility of two enzymes on AuNPs. Additionally, our approach accounts for the AuNP-bound activity of five out of six additional enzymes from the literature. The model developed here enables high-throughput predictions of protein behavior on nanoparticles, and it enhances our understanding of protein orientation in the biomolecular corona, which should greatly enhance the performance and safety of nanomedicines used in vivo.

biophysics↗

bioRxiv2022

Arabidopsis PFA-DSP-type phosphohydrolases target specific inositol pyrophosphate messengers

Inositol pyrophosphates are signaling molecules containing at least one phosphoanhydride bond that regulate a wide range of cellular processes in eukaryotes. With a cyclic array of phosphate esters and diphosphate groups around myo-inositol, these molecular messengers possess the highest charge density found in nature. Recent work deciphering inositol pyrophosphate biosynthesis in Arabidopsis revealed important functions of these messengers in nutrient sensing, hormone signaling and plant immunity. However, despite the rapid hydrolysis of these molecules in plant extracts, very little is known about the molecular identity of the phosphohydrolases that convert these messengers back to their inositol polyphosphate precursors. Here, we investigate whether Arabidopsis Plant and Fungi Atypical Dual Specificity Phosphatases (PFA-DSP1-5) catalyze inositol pyrophosphate phosphohydrolase activity. We find that recombinant proteins of all five Arabidopsis PFA-DSP homologs display phosphohydrolase activity with a high specificity for the 5-{beta}-phosphate of inositol pyrophosphates. We further show that heterologous expression of Arabidopsis PFA-DSP1-5 rescues wortmannin-sensitivity and deranged inositol pyrophosphate homeostasis caused by the deficiency of the PFA-DSP-type inositol pyrophosphate phosphohydrolase Siw14 in yeast. Heterologous expression in Nicotiana benthamiana leaves provided evidence that Arabidopsis PFA-DSP1 also displays 5-{beta}-phosphate specific inositol pyrophosphate phosphohydrolase activity in planta. Our findings lay the biochemical basis and provide the genetic tools to uncover the roles of inositol pyrophosphates in plant physiology and plant development.

plant biology↗

bioRxiv2022

Transcriptional and functional consequences of alterations to MEF2C and its topological organization in neuronal models

Point mutations and structural variants directly disrupting the coding sequence of MEF2C have been associated with a spectrum of neurodevelopmental disorders (NDDs), while recent studies have also implicated altered noncoding regulation of MEF2C expression in NDDs. However, the impact of haploinsufficiency of MEF2C on neurodevelopmental pathways and synaptic processes is not well understood, nor are the complex mechanisms that govern regulation of MEF2C. To explore the transcriptional and functional changes associated with coding and noncoding structural variants, we generated an allelic series of 204 isogenic iPSC-derived neuronal cell lines harboring CRISPR-engineered mutations that directly delete predominant isoforms of MEF2C, as well as deletions to the boundaries of topologically associating domains (TADs) and chromatin loops encompassing MEF2C. We then performed systematic profiling of mutation-specific alterations to transcriptional signatures, regulatory interactions, chromatin contacts, and electrophysiological effects. Our analyses reveal that direct deletion of MEF2C causes differential expression of genes enriched for neurodevelopmental and synaptic-associated pathways, accompanied by a significant reduction in synaptic firing and synchrony in neurons. By contrast, we observe robust buffering against MEF2C regulatory disruption upon deletion of a distal 5q14.3 TAD and loop boundary; however, homozygous loss of proximal loop boundary resulted in significant down-regulation of MEF2C expression and significantly reduced electrophysiological activity that was comparable to direct MEF2C disruption. Collectively, our findings demonstrate the functional impact of MEF2C haploinsufficiency in human-derived neural models and highlight the complex interactions of gene regulation and chromatin topology that challenge a priori regulatory predictions of structural variant disruption to three-dimensional genome organization.

genomics↗

bioRxiv2022

Tissue-specific and repeat length-dependent somatic instability of the X-linked dystonia parkinsonism-associated CCCTCT repeat

X-linked dystonia-parkinsonism (XDP) is a progressive adult-onset neurodegenerative disorder caused by insertion of a SINE-VNTR-Alu (SVA) retrotransposon in the TAF1 gene. The SVA retrotransposon contains a CCCTCT hexameric repeat tract of variable length, whose length is inversely correlated with age at onset. This places XDP in a broader class of repeat expansion diseases, characterized by the instability of their causative repeat mutations. Here, we observe similar inverse correlations between CCCTCT repeat length with age at onset and age at death and no obvious correlation with disease duration. To gain insight into repeat instability in XDP we performed comprehensive quantitative analyses of somatic instability of the XDP CCCTCT repeat in blood and in seventeen brain regions from affected males. Our findings reveal repeat length-dependent and expansion-based instability of the XDP CCCTCT repeat, with greater levels of expansion in brain than in blood. The brain exhibits regional-specific patterns of instability that are broadly similar across individuals, with cerebellum exhibiting low instability and cortical regions exhibiting relatively high instability. The spectrum of somatic instability in the brain includes a high proportion of moderate repeat length changes of up to 5 repeats, as well as expansions of [~]20->100 repeats and contractions of [~]20-40 repeats at lower frequencies. Comparison with HTT CAG repeat instability in postmortem Huntingtons disease brains reveals similar brain region-specific profiles, indicating common trans-acting factors that contribute to the instability of both repeats. Analyses in XDP brains of expansion of a different SVA-associated CCCTCT located in the LIPG gene, and not known to be disease-associated, reveals repeat length-dependent expansion at overall lower levels relative to the XDP CCCTCT repeat, suggesting that expansion propensity may be modified by local chromatin structure. Together, the data support a role for repeat length-dependent somatic expansion in the process(es) driving the onset of XDP and prompt further investigation into repeat dynamics and the relationship to disease.

genetics↗

bioRxiv2021

Structural insight into the dual function of LbpB in mediating Neisserial pathogenesis

Lactoferrin binding protein B (LbpB) is a lipoprotein present on the surface of Neisseria that has been postulated to serve dual functions during pathogenesis in both iron acquisition from lactoferrin, and in providing protection against the cationic antimicrobial peptide lactoferricin. Here, we present the structures of LbpB from N. meningitidis and N. gonorrhoeae in complex with human holo-lactoferrin, forming a 1:1 complex and confirmed by SEC-SAXS. LbpB consists of N- and C-lobes with the N-lobe interacting extensively with the C-lobe of lactoferrin. Our structures provides insight into LbpBs preference towards holo-lactoferrin, and our mutagenesis and binding studies show that lactoferrin and lactoferricin bind independently. Our studies provide the molecular details for how LbpB serves to capture and preserve lactoferrin in an iron-bound state for delivery to the membrane transporter LbpA for iron piracy, and as an antimicrobial peptide sink to evade host immune defenses.

biophysics↗

bioRxiv2021

Dystonia-specific mutations in THAP1 alter transcription of genes associated with neurodevelopment and myelin

Dystonia is a neurologic disorder associated with an increasingly large number of variants in many genes, resulting in characteristic disturbances in volitional movement. Dissecting the relationships between these mutations and their functional outcomes is a critical step in understanding the key pathways that drive dystonia pathogenesis. Here we established a pipeline for characterizing an allelic series of dystonia-specific mutations in isogenic induced pluripotent stem cells (iPSCs). We used this strategy to investigate the molecular consequences of variation in THAP1, which encodes a transcription factor that has been linked to neural differentiation. Multiple pathogenic mutations that have been associated with dystonia cluster within distinct THAP1 functional domains and are predicted to alter its DNA binding properties and/or protein interactions differently, yet the relative impact of these varied changes on molecular signatures and neural deficits is unclear. To determine the effects of these mutations on THAP1 transcriptional activity, we engineered an allelic series of eight mutations in a common iPSC background and differentiated these lines into a panel of near-isogenic neural stem cells (n = 94 lines). Transcriptome profiling of these neural derivatives followed by joint analysis of the most robust individual signatures across mutations identified a convergent pattern of dysregulated genes functionally related to neurodevelopment, lysosomal lipid metabolism, and myelin. Based on these observations, we examined mice bearing Thap1-disruptive alleles and detected significant changes in myelin gene expression and reduction of myelin structural integrity relative to tissue from control mice. These results suggest that deficits in neurodevelopment and myelination are common consequences of dystonia-associated THAP1 mutations and highlight the potential role of neuron-glial interactions in the pathogenesis of dystonia.

genetics↗

bioRxiv2021

Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

Coronavirus (CoV) non-structural proteins (nsps) assemble to form the replication-transcription complex (RTC) responsible for viral RNA synthesis. nsp7 and nsp8 are important cofactors of the RTC, as they interact and regulate the activity of RNA-dependent RNA polymerase (RdRp) and other nsps. To date, no structure of full-length SARS-CoV-2 nsp7:nsp8 complex has been published. Current understanding of this complex is based on structures from truncated constructs or with missing electron densities and complexes from related CoV species with which SARS-CoV-2 nsp7 and nsp8 share upwards of 90% sequence identity. Despite available structures being solved using crystallography and cryo-EM representing detailed snapshots of the nsp7:nsp8 complex, it is evident that the complex has a high degree of structural plasticity. However, relatively little is known about the conformational dynamics of the complex and how it assembles to interact with other nsps. Here, the solution-based structural proteomic techniques, hydrogen-deuterium exchange mass spectrometry (HDX-MS) and crosslinking mass spectrometry (XL-MS), illuminate the structural dynamics of the SARS-CoV-2 full-length nsp7:nsp8 complex. The results presented from the two techniques are complementary and validate the interaction surfaces identified from the published three-dimensional heterotetrameric crystal structure of SARS-CoV-2 truncated nsp7:nsp8 complex. Furthermore, mapping of XL-MS data onto higher order complexes suggests that SARS-CoV-2 nsp7 and nsp8 do not assemble into a hexadecameric structure as implied by the SARS-CoV full-length nsp7:nsp8 crystal structure. Instead our results suggest that the nsp7:nsp8 heterotetramer can dissociate into a stable dimeric unit that might bind to nsp12 in the RTC without altering nsp7-nsp8 interactions.

biophysics↗

bioRxiv2021

Distribution of Huntington's disease Haplogroups in Indian population

Huntingtons disease (HD), a rare neurodegenerative disorder, is inherited in an autosomal dominant manner, and caused by a pathological trinucleotide expansion at exon1 of the HTT locus. Previous studies have described the haplogroups at the HTT locus that can explain the differences in prevalence of HD. We have selected three informative SNPs (rs762855, rs3856973 and rs4690073) to study these haplogroups in an Indian sample. Our results show that the genotype frequencies are significantly different between cases and controls for these SNPs. More than 90% of both cases and controls belong to Haplogroup A which is the predominant European haplogroup.

neuroscience↗

bioRxiv2020

Emergence and expansion of highly infectious spike:D614G mutant SARS-CoV-2 in central India

COVID 19 has emerged as global pandemic with largest damage to the economy and human psyche. The genomic signature deciphered during the ongoing pandemic period is valuable to understand the virus evolutionary patterns and spread across the globe. Increased availability of genome information of circulating strain in our country will enable to generate selective details in virulent and non virulent markers to prophylaxis and therapeutic interventions. The first case of SARS CoV-2 was detected in Chambal region of Madhya Pradesh state in mid of March 2020 followed by multiple introduction events and expansion of COVID-19 cases within 3 months in this region. We analyzed around 5000 COVID -19 suspected samples referred to Defence Research and Development Establishment, Gwalior, Madhya Pradesh. A total of 136 cases were found positive over a span of three months period this includes virus introduction to region and further spread. Whole genome sequences employing Oxford nanopore technology were deciphered for 26 SARS-CoV-2 circulating in 10 different districts in Madhya Pradesh State of India. The region witnessed index cases with multiple travel history responsible for introduction of COVID-19 followed by remarkable expansion of virus. The genome wide substitutions including in important viral proteins were observed. The detailed phylogenetic analysis revealed the circulating SARS-CoV-2 clustered in multiple clades A2a, A4 and B. The cluster wise segregation was observed suggesting multiple introduction links and evolution of virus in the region. This is the first comprehensive details of whole genome sequence analysis from central India region, which will add genome wide knowledge towards diagnostic and therapeutic interventions.

molecular biology↗

bioRxiv2020

Chronic exposure to IL6 leads to deregulation of glycolysis and fat accumulation in the zebrafish liver

BACKGROUND AND AIMS Inflammation is a constant in Non-Alcoholic Fatty Liver Disease (NAFLD) and is usually considered a consequence. We propose that inflammation can be a cause for NAFLD. Obesity is strongly associated with (NAFLD), but not always. NAFLD in lean individuals is more common in certain populations, especially Asian-Indians. Lean healthy Indians also have a higher basal circulating IL6 suggesting a link with inflammation. We propose that inflammation-induced fatty liver could be relevant for studying obesity-independent NAFLD. Commonly used high-fat diet-induced NAFLD animal models are not ideal for testing this hypothesis.APPROACH AND RESULTS In this study we used a transgenic zebrafish with chronic systemic overexpression of human IL6 (IL6-OE) and found accumulation of triglyceride in the liver. We performed comparative transcriptomics and proteomics on the IL6-OE liver and found an expression signature distinct from the diet-based NAFLD models. We discovered a deregulation of glycolysis/gluconeogenesis pathway, especially a robust down regulation of the glycolytic enzyme aldolase b in the IL6-OE liver. Metabolomics of the IL6-OE liver showed accumulation of hexose monophosphates and their derivatives, which can act as precursors for triglyceride synthesis. Patients with the genetic disease Hereditary Fructose Intolerance (HFI) caused by ALDOLASE B deficiency also have a higher propensity to develop fatty liver disease.CONCLUSIONS Our study demonstrates a causative role for inflammation in intrahepatic lipid accumulation. Further, our results suggest that IL6-driven repression of glycolysis/gluconeogenesis, specifically aldolase b, may be a novel mechanism for development of fatty liver, especially in obesity-independent NAFLD.Competing Interest StatementThe authors have declared no competing interest.List of AbbreviationsNAFLDNon-Alcoholic Fatty Liver DiseaseIL6Interleukin 6OEOverexpressionHFIHigh Fructose IntoleranceNASHNon-Alcoholic SteatohepatitisSLESystemic Lupus ErythematosusIBDInflammatory Bowel DiseaseRARheumatoid ArthritisHFDHigh Fat dietqRT-PCRQuantitative Real-Time Polymerase Chain ReactionPPARPeroxisome Proliferator-Activated ReceptorDHAPDihydroxyacetone PhosphateView Full Text

molecular biology↗

bioRxiv2020

Conserved tryptophan mutation disrupts structure and function of immunoglobulin domain revealing unusual tyrosine fluorescence

Immunoglobulin (Ig) domains are the most prevalent protein domain structure and share a highly conserved folding pattern; however, this structural family of proteins is also the most diverse in terms of biological roles and tissue expression. Ig domains vary significantly in amino acid sequence but share a highly conserved tryptophan in the hydrophobic core of this beta-stranded protein. Palladin is an actin binding and bundling protein that has five Ig domains and plays an important role in normal cell adhesion and motility. Mutation of the core tryptophan in one Ig domain of palladin has been identified in a pancreatic cancer cell line, suggesting a crucial role for this sole tryptophan in palladin Ig domain structure, stability, and function. We found that actin binding and bundling was not completely abolished with removal of this tryptophan despite a partially unfolded structure and significantly reduced stability of the mutant Ig domain as shown by circular dichroism investigations. In addition, this mutant palladin domain displays a tryptophan-like fluorescence attributed to an anomalous tyrosine emission at 345 nm. Our results indicate that this emission originates from a tyrosinate that may be formed in the excited ground state by proton transfer to a nearby glutamyl residue. Furthermore, this study emphasizes the importance of tryptophan in protein structural stability and illustrates how tyrosinate emission contributions may be overlooked during the interpretation of the fluorescence properties of proteins. SHORT ABSTRACTThis study explores the functional and structural consequences of a point mutation in palladin, an Ig domain protein first identified in a pancreatic tumor cancer cell line. While exploring the consequences of mutating this conserved tryptophan in the hydrophobic core of the most prevalent domain structure found in proteins, an anomalous tyrosine fluorescence phenomenon was exposed.

biochemistry↗

bioRxiv2019

Discovery of dual inhibitors of KPC-3 and KPC-15 of Klebsiella pneumoniae - a ligand and structure based virtual screening study

BackgroundThe development of carbapenem resistance against Klebsiella pneumoniae is a situation of grave concern and requires urgent attention. Among the KPC produced by K.pneumoniae, KPC-3, and KPC-15, play a significant role in the development of resistance to carbapenem. Materials and methodsThe binding sites of KPC-3 and KPC-15 were predicted by the COACH server. Drug-like ligands from ZINC were then screened by ligand-based drug screening (LBVS) by keeping Relebactam as a template. The top 50,000 selected ligands were then screened by structure-based virtual screening using idock. For keeping an account of the dual inhibitors stability in complex with KPC-3 and KPC-15, MDS were carried out for each complex. ResultsBased on consensus weighted ranks, the top 3 ligands with the dual inhibitory property are ZINC76060350 (consensus weighted rank - 1.5), ZINC05528590 (2), ZINC72290395 (3.5). All the top 3 dual inhibitors have a reasonable probability of passing through the blood-brain barrier. The RDKit and Morgan fingerprint scores between Relebactam and the top three ligands were 0.24, 0.22, 0.23, and 0.26, 0.19, 0.25, respectively (showing only 20% similarity). The MD simulation result revealed good binding stability of ligand ZINC05528590 with both KPC-3 and KPC-15, whereas ligand ZINC76060350 showed good binding stability to KPC-3. ConclusionThe ligand ZINC05528590 could be taken forward to develop a new drug against a multi-resistant- Klebsiella pneumoniae infection. At the same time, ZINC76060350 can be considered to develop a new drug against KPC-15 resistant Klebsiella pneumoniae.

bioinformatics↗