Biology subjects

Singh, S.

Publications and source records attributed to Singh, S..

At least 469 records · Page 26Linked to original sources

bioRxiv2020

Interaction of 14-3-3I and CDPK1 mediates the growth of human malaria parasite

Scaffold proteins play pivotal role as modulators of cellular processes by operating as multipurpose conformation clamps. 14-3-3 proteins are gold-standard scaffold modules that recognize phosphoSer/Thr (pS/pT) containing conserved motifs of target proteins and confer conformational changes leading to modulation of their functional parameters. Modulation in functional activity of kinases has been attributed to their interaction with 14-3-3 proteins. Herein, we have characterized Plasmodium falciparum 14-3-3 and its interaction with key kinase of the parasite, Calcium-Dependent Protein Kinase 1 (CDPK1) by performing various analytical biochemistry and biophysical assays. Towards this, we annotated PF3D7_0818200 as 14-3-3 isoform I through extensive phylogenetic and comparative sequence analysis. Molecular dynamics simulation studies indicated that phosphoSer64 present in CDPK1 polypeptide sequence (61KLGpS64) behaves as canonical Mode I-type (RXXpS/pT) consensus 14-3-3 binding motif, mediating the interaction. The protein-protein interaction was validated in vitro with ELISA and SPR, which confirmed that CDPK1 interacts with 14-3-3I in a phosphorylation dependent manner, with binding affinity constant of 670 {+/-} 3.6 nM. The interaction of 14-3-3I with CDPK1 was validated with well characterized optimal 14-3-3 recognition motifs: ARSHpSYPA and RLYHpSLPA as CDPK1 mimetics, by simulation studies and ITC. Further, interaction antagonizing peptidomimetics showed growth inhibitory impact on the parasite indicating crucial physiological role of 14-3-3/CDPK1 interaction. Overall, this study characterizes 14-3-3I as a scaffold protein in the malaria parasite and unveils CDPK1 as its previously unidentified target. This sets a precedent for the rational design of 14-3-3 based PPI inhibitors by utilizing 14-3-3 recognition motif peptides, as a potential antimalarial strategy.

cell biology↗

bioRxiv2019

A toolbox for automated video analysis of rodents engaged in string-pulling: Phenotyping motor behavior of mice for sensory, whole-body and bimanual skilled hand function

String-pulling in rodents (rats and mice) is a task in which animals make hand-over-hand movements to spontaneously reel in a string with or without a food reward attached to its end. The task elicits bilateral skilled hand movements for which rodents require little training. The task is suitable for phenotyping physiology and pathophysiology of sensorimotor integration in rodent models of neurological and motor disorders. Because a rodent stands in the same location and its movements are repetitive, the task lends itself to quantification of topographical and kinematic parameters for on-line tactile tracking of the string, skilled hand movements for grasping, and rhythmical bilateral forearm movements to advance the string. Here we describe a Matlab(R) based software with a graphical user interface to assist researchers in analyzing the video record of string pulling. The software allows global characterization of position and motion using optical flow estimation, descriptive statistics, principal component, and independent component analyses as well as temporal measures of Fano factor, entropy, and Higuchi fractal dimension. Based on image segmentation and object tracking heuristic algorithms, the software also allows independent tracking of the body, ears, nose, and forehands for estimation of kinematic parameters such as body length, body angle, head roll, head yaw, head pitch, movement paths and speed of hand movement. The utility of the task and that of the software is presented by describing mouse strain characteristics in string-pulling behavior of two strains of mice, C57BL/6 and Swiss Webster. Postural and skilled hand kinematic differences that characterize the strains highlight the utility of the task and assessment methods for phenotypic and neurological analysis of healthy and rodent models of diseases such as Parkinsons, Huntingtons, Alzheimers and other neurological and motor disorders. Significance statementMouse models are used to investigate the physiology and pathophysiology of motor deficits observed in human neurological conditions, for testing substances for therapeutic drug development, and to investigate the role of neural systems and their genetic basis in the expression of behavior. Behavioral tasks involving unconditioned and natural behavior can provide rich insights into motor performance in animal models and analyses can be aided by the automated processing of video data for reliable quantification and high throughput.

neuroscience↗

bioRxiv2019

Geldanamycin inhibits Jumonji histone lysine demethylase KDM4 and targets chimeric transcription factor PAX3-FOXO1

Histone lysine demethylases (KDMs) are emerging as therapeutic targets in cancer. Development of potent KDM inhibitors may provide additional options for epigenomics-oriented therapies. Using a Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) functional demethylation assay, in combination with a high-content immunofluorescence imaging phenotypic screen, Matrix-Assisted Laser Desorption/Ionization-Fourier Transform Ion Cyclotron Resonance mass spectrometry (MALDI-FTICR MS) and Amplified Luminescent Proximity Homogeneous Assay (ALPHA), we identified geldanamycin, an inhibitor of heat shock protein 90 (Hsp90), as a novel inhibitor of JmjC-domain containing demethylases such as KDM4B. We further found that geldanamycin can destabilize the PAX3-FOXO1 fusion oncoprotein, an Hsp90 client, which is a driver of clinically unfavorable alveolar rhabdomyosarcoma (aRMS). We then hypothesized that dual inhibition of PAX3-FOXO1 and epigenetic modifiers of aRMS would have synergistic antitumor activity. We repurposed the geldanamycin analog 17-DMAG to target aRMS and found that 17-DMAG significantly delays tumor growth, extends survival in xenograft mouse models, and inhibits expression of PAX3-FOXO1 targets and multiple oncogenic pathways including MYC, E2F and NOTCH. In addition, the combination of 17-DMAG with conventional chemotherapy or the bromodomain inhibitor JQ1 significantly enhances therapeutic efficacy. In summary, we have identified geldanamycin and 17-DMAG as dual KDM/Hsp90 inhibitors and 17-DMAG is efficacious against PAX3-FOXO1-driven rhabdomyosarcoma.

cancer biology↗

bioRxiv2019

A versatile high throughput screening platform for plant metabolic engineering highlights the major role of ABI3 in lipid metabolism regulation

Traditional functional genetic studies in crops are time-consuming, complicated and cannot be readily scaled up. The reason is that mutant or transformed crops need to be generated to study the effect of gene modifications on specific traits of interest. However, many crop species have a complex genome and a long generation time. As a result, it usually takes several months to over a year to obtain desired mutants or transgenic plants, which represents a significant bottleneck in the development of new crop varieties. To overcome this major issue, we are currently establishing a versatile plant genetic screening platform, amenable to high throughput screening in almost any crop species, with a unique workflow. This platform combines protoplast transformation and fluorescence-activated cell sorting. Here we show that tobacco protoplasts can accumulate high levels of lipids if transiently transformed with genes involved in lipid biosynthesis and can be sorted based on lipid content. Hence, protoplasts can be used as a predictive tool for plant lipid engineering. Using this newly established strategy, we demonstrate the major role of ABI3 in plant lipid accumulation. We anticipate that this workflow can be applied to numerous highly valuable metabolic traits other than storage lipid accumulation. This new strategy represents a significant step towards screening complex genetic libraries, in a single experiment and in a matter of days, as opposed to years by conventional means.

plant biology↗

bioRxiv2019

The RGG Domain of the Yeast Nucleolin Nsr1 Is Required for the Genome Instability Associated with Co-transcriptionally Formed G4 DNA

A significant increase in genome instability is associated with the conformational shift of a guanine-run-containing DNA strand into the four-stranded G-quadruplex (G4) DNA. The mechanism underlying the recombination and genome rearrangements following the formation of G4 DNA in vivo has been difficult to elucidate but has become better clarified by the identification and functional characterization of several key G4 DNA-binding proteins. Mammalian nucleolin NCL is a highly specific G4 DNA-binding protein with a well-defined role in the transcriptional regulation of genes with associated G4 DNA-forming sequence motifs at their promoters. The consequence of the in vivo interaction between G4 DNA and nucleolin in respect to the genome instability has not been previously investigated. We show here that G4 DNA-binding is a conserved function in the yeast nucleolin Nsr1. Furthermore, we demonstrate that the Nsr1-G4 DNA complex formation results in replication obstruction and is a major factor in inducing the genome instability associated with the co-transcriptionally formed G4 DNA in the yeast genome. The G4-associated genome instability and the G4 DNA-binding in vivo requires the arginine-glycine-glycine (RGG) repeats located at the C-terminus of the Nsr1 protein. Nsr1 with the deletion of RGG domain supports normal cell growth and is sufficient for its pre-rRNA processing function. However, the truncation of RGG domain of Nsr1 significantly weakens its interaction with G4 DNA in vitro and in vivo and restores unhindered replication, overall resulting in a sharp reduction in the G4-associated genome instability. Our data suggest that the interaction between Nsr1 with the intact RGG repeats and G4 DNA impairs genome stability by precluding the access of G4-resolving proteins and obstructing replication. AUTHOR SUMMARYGenome instability is uniquely elevated at sequences containing multiple runs of guanines, which can fold into the unusual, four-stranded G-quadruplex (G4) DNA. In this study, we report a novel finding that a highly conserved G4 DNA binding protein Nsr1 can elevate the rate of recombination and chromosomal rearrangement occurring at a G4 DNA-forming sequence in the genome of Saccharomyces cerevisiae. The elevated genome instability requires the C-terminally located RGG domain of Nsr1, which supports the high-affinity interaction between the protein and G4 DNA. The connection between G4-specific genome instability and the function of Nsr1 to form stable complex with G4 DNA led to the hypothesis that the high-affinity Nsr1-G4 DNA complexes can become a barrier to replication. We demonstrate here that the presence of Nsr1 in fact slows the replication past a G4 DNA-containing genomic site and that the RGG domain is required to facilitate such replication block.

genetics↗

bioRxiv2019

Plasmodium falciparum growth is regulated by Sphingosine 1 phosphate produced by Host Erythrocyte Membrane Sphingosine kinase 1

Sphingosine-1-phosphate (S1P) a bioactive lipid is produced in its primary reservoir, erythrocytes by an enzyme Sphingosine kinase-1 (SphK-1). The activation of such kinases and the subsequent S1P generation and secretion in the blood serum represent a major regulator of many cellular signaling cascades. Orthologue of sphingosine kinases 1 and 2 (SphK-1 and 2) that catalyze the phosphorylation of sphingosine generating S1P are not present in malaria parasite. The malaria parasite, Plasmodium falciparum, is an intracellular obligatory organism that reside in the human erythrocyte during its blood stage life cycle and orchestrates many metabolic interactions with host for its survival. Given the regulatory role of S1P, we targeted host SphK-1 by a generic pharmacological inhibitor N,N-Dimethyl-sphingosine (DMS) and analyzed growth of intra-erythrocytic parasite. We found that reducing S1P levels by inhibiting host SphK-1 activity led to halted parasite growth and ultimately cell death. Reduced intracellular S1P levels were attributed to decreased glycolysis marked by the low uptake of glucose by parasite and by less production of lactate, a byproduct of glycolysis. Reduced glycolysis was mediated by decrease translocation of the glycolytic enzyme, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to the cytosol of infected erythrocytes and cell death. Knocking down of erythrocyte SphK-1 is not lethal to the host and being a host encoded enzyme, targeting it with safe and specific drugs will not lead to the problem of resistance; thus, SphK-1 represents a potent target for the development of therapeutics against intra-erythrocytic P. falciparum.\n\nAuthor SummaryErythrocytes membrane enzyme Sphingosine kinase-1 (SphK-1) produces Sphingosine-1-phosphate (S1P) a bioactive lipid by phosphorylation of Sphingosine (Sph). S1P generated by activation of SphK is prosurvival signal and regulate cell growth. The malaria parasite, Plasmodium falciparum, is an intracellular obligatory pathogen that reside in erythrocyte during its blood stage life cycle and orchestrates many metabolic interactions with its host erythrocytes for survival. Orthologue of SphK-1/ 2 are not present in malaria parasite, therefore treatment with SphK inhibitor targeted host SphK-1 and led to reduced S1P level. The reduction in host S1P led to halted parasite growth and cell death. Furthermore, reduced erythrocyte S1P levels led to decreased glycolysis marked by the low uptake of glucose by parasite and by less production of lactate. Erythrocyte SphK-1 being a host encoded enzyme, is resistance safe and represents a potent target for the development of therapeutics against intra-erythrocytic P. falciparum.

cell biology↗

bioRxiv2019

Cell-substrate adhesion drives Scar/WAVE activation and phosphorylation, which controls pseudopod lifetime

The Scar/WAVE complex is the principal catalyst of pseudopod and lamellipod formation. Here we show that Scar/WAVEs proline-rich domain is polyphosphorylated after the complex is activated. Treatments that stop activation block phosphorylation in both Dictyostelium and mammalian cells. This implies that phosphorylation modulates pseudopods after they have been formed, rather than controlling whether a protrusion is initiated. Unexpectedly, activation-dependent phosphorylation is not promoted by chemotactic signalling, or by signal-dependent kinases such as ERKs, but is greatly stimulated by cell:substrate adhesion. Scar/WAVE that has been mutated to be either unphosphorylatable or phosphomimetic is activated normally, and rescues the phenotype of scar- cells, demonstrating that phosphorylation is dispensible for activation and actin regulation. However, pseudopods and patches of Scar/WAVE complex recruitment last substantially longer in unphosphorylatable mutants, altering cell polarisation and the efficiency of migration. We conclude that pseudopod engagement with substratum is more important than extracellular signals at regulating Scar/WAVEs activity, and that phosphorylation acts as a timer, restricting pseudopod lifetime by promoting Scar/WAVE turnover.

cell biology↗

bioRxiv2019

Anti-inflammatory Role of Curcumin in LPS Treated A549 cells at Global Proteome level and on Mycobacterial infection.

A diet derived agent Curcumin (Diferuloylmethane), demonstrated its clinical application in inflammation, infection and cancer conditions. Nevertheless, its impact on the proteome of epithelial cells of non-small cell lung carcinoma (NSCLC) is yet to be explored. We employed a stable isotope labeling method for cell culture (SILAC) based relative quantitative proteomics and informatics analysis to comprehend global proteome change in A549 cells treated with curcumin and/or Lipopolysaccharide (LPS). Pretreated A549 cells were infected with Mycobacterium tuberculosis H37Rv strain to monitor bacterial load. With exposure to curcumin and LPS, out of the 1492 identified proteins, 305 and 346 proteins showed deregulation respectively. The expression of BID and AIFM1 mitochondrial proteins which play critical role in apoptotic pathway were deregulated in curcumin treated cells. Higher mitochondria intensity was observed in curcumin treated A549 cells than LPS treatment. Simultaneous treatment of curcumin and LPS neutralized the effect of LPS. Curcumin and/or LPS pretreated A549 cells infected with H37Rv, showed successful bacterial internalization. LPS treated A549 cells after infection showed increased bacterial load than curcumin compared to non-treated infected cells. However, simultaneous treatment of curcumin and LPS neutralized the effect of LPS. This study generated molecular evidence to deepen our understanding of the anti-inflammatory role of curcumin and may be useful to identify molecular targets for drug discovery.

biochemistry↗

bioRxiv2019

A synthetic metabolic network for physicochemical homeostasis

One of the grand challenges in chemistry is the construction of functional out-of-equilibrium networks, which are typical of living cells. Building such a system from molecular components requires control over the formation and degradation of the interacting chemicals and homeostasis of the internal physical-chemical conditions. The provision and consumption of ATP lies at the heart of this challenge. We report the in vitro construction in vesicles of a pathway for sustained ATP production that is maintained away from equilibrium by control of energy dissipation. We maintain a constant level of ATP with varying load on the system. The pathway enables us to control the transmembrane fluxes of osmolytes and to demonstrate basic physicochemical homeostasis. Our work demonstrates metabolic energy conservation and cell volume regulatory mechanisms in a cell-like system at a level of complexity minimally needed for life.

synthetic biology↗

bioRxiv2019

Monoclonal IgM antibodies raised against Candida albicans Hyr1 provide cross-kingdom protection against Gram negative bacteria

Recent years have seen an unprecedented rise in the incidence of multidrug resistant (MDR) Gram negative bacteria (GNB) such as Acinetobacter and Klebsiella species. In view of the shortage of novel drugs in the pipeline, alternative strategies to prevent and treat infections by GNB are urgently needed. Previously, we have reported that the C. albicans hyphal-regulated protein Hyr1 shares striking 3D structural homology with cell surface proteins of A. baumannii; and active or passive vaccination with rHyr1p-N or anti-Hyr1p polyclonal antibody, respectively; protect mice from Acinetobacter infections. Here, we show that monoclonal antibodies (mAb) generated against Hyr1p, bind to the surface of Acinetobacter as well as K. pneumoniae. The anti-Hyr1 mAb also block damage to primary endothelial cells by the bacteria, and protect mice from lethal pulmonary infections mediated by A. baumannii and K. pneumoniae. Our current studies emphasize the potential of harnessing Hyr1p mAb as a cross-kingdom immunotherapeutic strategy against MDR GNB.

immunology↗

bioRxiv2019

Reach and Grasp Altered in Pantomime String-Pulling: A Test of the Action/Perception Theory in a Bilateral Reaching Task

The action/perception theory of cortical organization is supported by the finding that pantomime hand movements of reaching and grasping are different from real movements. Frame-by-frame video analysis and MATLAB(R) based tracking examined real/pantomime differences in a bilaterally movement, string-pulling, pulling down a rope with hand-over-hand movements. Sensory control of string-pulling varied from visually-direct when cued, visually-indirect when non cued and somatosensory controlled in the absence of vision. Cued grasping points were visual tracked and the pupils dilated in anticipation of the grasp, but when noncued, visual tracking and pupil responses were absent. In real string-pulling, grasping and releasing the string featured an arpeggio movement in which the fingers close and open in the sequence 5 through 1 (pinki first, thumb last); in pantomime, finger order was reversed, 1 through 5. In real string-pulling, the hand is fully opened and closed to grasp and release; in pantomime, hand opening was attenuated and featured a gradual opening centered on the grasp. The temporal structure of arm movements in real string-pulling featured up-arm movements that were faster than down-arm movement. In pantomime, up/down movements had similar speed. In real string-pulling, up/down arm movements were direct and symmetric; in pantomime, they were more circular and asymmetric. That pantomime string-pulling featured less motoric and temporal complexity than real string-pulling is discussed in relation to the action/perception theory and in relation to the idea that pantomimed string-pulling may feature the substitution of gestures for real movement.\n\nSignificant StatementMost laboratory studies investigating hand movements made by humans feature single hand movements, the current study presents a novel string-pulling task to study bimanual coordination of left and right hands in real and pantomime conditions. The results show that pantomime string-pulling featured less motoric and temporal complexity than real string-pulling. These findings are relevant to the contemporary theory of action and perception that the dorsal stream (parietal cortex) is related to actions and the ventral stream (temporal cortex) is related to perception.

neuroscience↗

bioRxiv2019

NaV1.1 and NaV1.6 selective compounds reduce the behavior phenotype in a novel zebrafish model for Dravet Syndrome

Dravet syndrome is caused by dominant loss-of-function mutations in SCN1A which cause reduced activity of Nav1.1 leading to lack of neuronal inhibition. On the other hand, gain-of-function mutations in SCN8A can lead to a severe epileptic encephalopathy subtype by over activating NaV1.6 channels. These observations suggest that Nav1.1 and Nav1.6 represent two opposing sides of the neuronal balance between inhibition and activation. Here, we hypothesize that Dravet syndrome may be treated by either enhancing Nav1.1 or reducing Nav1.6 activity. To test this hypothesis we generated and characterized a novel DS zebrafish model and tested new compounds that selectively activate or inhibit the human NaV1.1 or NaV1.6 channel respectively. We used CRISPR/Cas9 to generate two separate Scn1Lab knockout lines as an alternative to previous knock-down models. Using an optimized locomotor assay, spontaneous burst movements were detected that were unique to Scn1Lab knockouts and disappear when introducing human SCN1A mRNA. Besides the behavioral phenotype, Scn1Lab knockouts show sudden, electrical discharges in the brain that indicate epileptic seizures in zebrafish. Scn1Lab knockouts showed increased sensitivity to the convulsant pentylenetetrazole and a reduction in whole organism GABA levels. Drug screenings further validated a Dravet syndrome phenotype. We tested the NaV1.1 activator AA43279 and our newly synthesized NaV1.6 inhibitors MV1369 and MV1312 in the Scn1Lab knockouts. Both type of compounds significantly reduced the number of burst movements. Our results show that selective inhibition of NaV1.6 could be just as efficient as selective activation of NaV1.1 and these approaches could prove to be novel potential treatment strategies for Dravet syndrome and other (genetic) epilepsies. Compounds tested in zebrafish however, should always be further validated in other model systems, preferably human derived.

genetics↗

bioRxiv2019

Super-resolution imaging reveals the evolution of higher-order chromatin folding in early carcinogenesis

Aberrant chromatin structure is a hallmark in cancer cells and has long been used for clinical diagnosis of cancer. However, underlying higher-order chromatin folding during malignant transformation remains elusive, due to the lack of molecular scale resolution. Using optimized stochastic optical reconstruction microscopy (STORM) for pathological tissue (PathSTORM), we uncovered a gradual decompaction and fragmented higher-order chromatin folding throughout all stages of carcinogenesis in multiple tumor types, even prior to the tumor formation. Our integrated imaging, genomic, and transcriptomic analyses reveal the functional consequences in enhanced formation of transcription factories, spatial juxtaposition with relaxed nanosized chromatin domains and impaired genomic stability. We also demonstrate the potential of imaging higher-order chromatin decompaction to detect high-risk precursors that cannot be distinguished by conventional pathology. Taken together, our findings reveal the gradual decompaction and fragmentation of higher-order chromatin structure as an enabling characteristic in early carcinogenesis to facilitate malignant transformation, which may improve cancer diagnosis, risk stratification, and prevention.\n\nSIGNIFICANCEGenomic DNA is folded into a higher-order structure that regulates transcription and maintains genomic stability. Although much progress has been made on understanding biochemical characteristics of epigenetic modifications in cancer, the higher-order folding of chromatin structure remains largely unknown. Using optimized super-resolution microscopy, we uncover de-compacted and fragmented chromatin folding in tumor initiation and stepwise progression in multiple tumor types, even prior to the presence of tumor cells. This study underlines the significance of unfolding higher-order chromatin structure as an enabling characteristic to promote tumorigenesis, which may facilitate the development and evaluation of new preventive strategies. The potential of imaging higher-order chromatin folding to improve cancer detection and risk stratification is demonstrated by detecting high-risk precursors that cannot be distinguished by conventional pathology.

cancer biology↗

bioRxiv2019

Histopathological study of different organs of charles foster strain rat under the exposure of Pueraria tuberosa.

The present study was undertaken to investigate the safe doses of Pueraria tuberosa water extract (PTWE) on different organs. The OECD guidelines 407 of repeated toxicity was followed with respect to the selection of dose and days for different organs. The selected doses of PTWE were 250, 500, 1000 and 2000 mg/kg b wt for 7, 14, 21 and 28 days. Haematoxylin and eosin staining was used to study the morphological alterations in heart, intestine, testis, adrenal gland and spleen. In the present study, no adverse alterations in cardiac fibers of the heart, size and shapes in crypts and villi of intestine, seminiferous tubules and spermatozoa count in testis, three zones of adrenal gland, and spleen were seen in all treated groups of PTWE. There were no adverse morphological alterations found in described organs. The PTWE are safe at 1000 mg/kg b wt. up to 28 days and 2000 mg/ kg b. wt up to 21 days, respectively.

pharmacology and toxicology↗

bioRxiv2019

Long-term experimental evolution reveals purifying selection on piRNA-mediated control of transposable element expression

Transposable elements (TEs) are an almost universal constituent of eukaryotic genomes. In animals, Piwi-interacting small RNAs (piRNAs) and repressive chromatin often play crucial roles in preventing TE transcription and thus restricting TE activity. Nevertheless, TE content varies widely across eukaryotes and the dynamics of TE activity and TE silencing across evolutionary time is poorly understood. Here we used experimentally evolved populations of C. elegans to study the dynamics of TE expression over 400 generations. The experimental populations were evolved at three different population sizes to manipulate the efficiency of natural selection versus genetic drift. We demonstrate increased TE expression relative to the ancestral population, with the largest increases occurring in the smallest populations. We show that the transcriptional activation of TEs within active regions of the genome is associated with failure of piRNA-mediated silencing, whilst desilenced TEs in repressed chromatin domains retain small RNAs. Additionally, we find that the sequence context of the surrounding region influences the propensity of TEs to lose silencing through failure of small RNA-mediated silencing. Together, our results show that natural selection in C. elegans is responsible for maintaining low levels of TE expression, and provide new insights into the epigenomic features responsible.

molecular biology↗

bioRxiv2019

Absence of Replication fork associated factor CTF4 and F-box motif Encoding Gene SAF1 leads to reduction in Cell Size and Stress Tolerance Phenotype in S. cerevisiae

Chromosome transmission fidelity factor, Ctf4 in S. cerevisiae associates with replication fork and helps in the sister chromatid cohesion. At the replication fork, Ctf4 links DNA helicase with the DNA polymerase. The absence of Ctf4 invokes replication checkpoint in the cells. The Saf1 of S.cerevisiae interacts with Skp1 of SCF-E3 ligase though F box-motif and ubiquitinates the adenine deaminase Aah1 during phase transition due to nutrient stress. The genetic interaction between the CTF4 and SAF1 has not been studied. Here we report genetic interaction between CTF4 and SAF1 which impacts the growth fitness and response to stress. The single and double gene deletions of SAF1 and CTF4 were constructed in the BY4741 genetic background. The strains were tested for growth on rich media and media containing stress causing agents. The saf1{Delta}ctf4{Delta} cells with reduced cell size showed the fastest growth phenotype on YPD medium when compared with the saf1{Delta}, ctf4{Delta}, and WT. The saf1{Delta}ctf4{Delta} cells also showed the tolerance to MMS, NaCl, Glycerol, SDS, Calcofluor white, H2O2, DMSO, Benomyl, and Nocodazole when compared with the saf1{Delta}, ctf4{Delta}, and WT cells. However, saf1{Delta}ctf4{Delta} cells showed the sensitivity to HU when compared with WT and saf1{Delta}. Based on these observations we suggest that SAF1 and CTF4 interact genetically to regulate the cell size, growth and stress response.

genetics↗

bioRxiv2019

The plasma Factor XIII heterotetrameric complex structure: unexpected unequal pairing within a symmetric complex

Factor XIII (FXIII) is a predominant determinant of clot stability, strength, and composition. Plasma FXIII circulates as a pro-transglutaminase with 2 catalytic A subunits and 2 carrier-protective B subunits in a heterotetramer (FXIII-A2B2). FXIII-A2 and -B2 subunits are synthesized separately and then assembled in plasma. Following proteolytic activation by thrombin and calcium-mediated dissociation of the B-subunits, activated FXIII (FXIIIa) covalently cross-links fibrin, promoting clot stability. The zymogen and active states of the FXIII-A subunits have been structurally characterized; however, the structure of FXIII-B subunits and the FXIII-A2B2 complex have remained elusive. Using integrative hybrid approaches including atomic force microscopy, cross-linking mass spectrometry, and computational approaches, we have constructed the first all-atom model of the FXIII-A2B2 complex. We also used molecular dynamic simulations in combination with isothermal titration calorimetry to characterize FXIII-A2B2 assembly, activation, and dissociation. Our data reveal unequal pairing of individual subunit monomers in an otherwise symmetric complex, and suggest this unusual structure is critical for both assembly and activation of this complex. Our findings enhance understanding of mechanisms associating FXIII-A2B2 mutations with disease and have important implications for the rational design of molecules to alter FXIII assembly and/or activity to reduce bleeding and thrombotic complications.

molecular biology↗

bioRxiv2019

Neuronal TLR4 signaling enhances AMPA currents and drives posttraumatic epileptogenesis

Traumatic brain injury is a major risk factor for acquired epilepsies and understanding the mechanisms underlying the early pathophysiology could yield viable therapeutic targets. Growing evidence indicates a role for inflammatory signaling in modifying neuronal excitability and promoting epileptogenesis. Here, we identify that signaling through an innate immune receptor, toll-like receptor 4 (TLR4), in neurons, augments calcium-permeable AMPA receptor (CP-AMPAR) currents in the hippocampal dentate gyrus after brain injury. Blocking TLR4 signaling in vivo shortly after brain injury reduced dentate network excitability and seizure susceptibility. When blocking of TLR4 signaling after injury was delayed, however, this treatment failed to reduce post-injury seizure susceptibility. Further, TLR4 signal blocking was less efficacious in limiting seizure susceptibility when AMPAR currents, downstream targets of TLR4 signaling, were transiently enhanced. Paradoxically, blocking TLR4 signaling augmented both network excitability and seizure susceptibility in uninjured controls. Despite the differential effect on seizure susceptibility, TLR4 antagonism suppressed cellular inflammatory responses after injury without impacting sham controls. These findings demonstrate that independently of glia, the immune receptor TLR4 directly regulates post-traumatic neuronal excitability. Moreover, the TLR4-dependent early increase in dentate excitability is causally associated with epileptogenesis. Identification and selective targeting of the mechanisms underlying the aberrant TLR4-mediated increase in CP-AMPAR signaling after injury may prevent epileptogenesis after traumatic brain injury. Graphical AbstractO_ST_ABSSummary of interactions between TLR4 signaling and brain injury on network excitability and epileptogenesisC_ST_ABSGraphic illustration of the effect of injury and early TLR4 antagonist treatment on early network excitability and the long-term network state. The schematic neurons include TLR4 and AMPAR subunit expression profiles in the acute phase of sham or brain injury. The corresponding early effects on network excitability are depicted by schematic population response traces (inset on upper left). Note the increase in excitability of the uninjured neuron after TLR4 antagonism without changes in AMPAR expression. Note also the increase in TLR4, calcium permeable AMPARs and population excitability after injury and its reduction by TLR4 antagonist treatment. Ampakine enhancement of excitability during TLR4 antagonism is illustrated. The early phase responses and manipulations (including injury, treatments, and molecular responses) are superimposed on a two-tone color-coded network state topology where green indicates low-normal network excitability, ensuring network stability and low risk for epilepsy (Inset on upper right). Note the correspondence between early excitability state (population response profile) and long-term seizure susceptibility and the effects of pharmacological manipulations.

neuroscience↗