Biology subjects

Kapoor, S.

Publications and source records attributed to Kapoor, S..

24 records · Page 2Linked to original sources

bioRxiv2021

Constitutive high expression of NOXA sensitizes human embryonic stem cells for rapid cell death

Human embryonic stem (hES) cells are highly sensitive to apoptotic stimuli such as DNA damage, which allows for rapid elimination of mutated cells during development. However, the mechanisms that maintain hES cells in the primed apoptotic state are not completely known. Key activators of apoptosis, the BH3-only proteins, are present at low levels in most cell types. In contrast, hES cells have constitutive high levels of the BH3-only protein, NOXA. We examined the importance of NOXA for enabling apoptosis in hES cells. hES cells deleted for NOXA showed remarkable protection against multiple apoptotic stimuli. NOXA was constitutively localized to the mitochondria, where it interacted with MCL1. Strikingly, inhibition of MCL1 in NOXA knockout cells was sufficient to sensitize these cells to DNA damage, and subsequently, cell death. Our study demonstrates, an essential function of constitutive high levels of NOXA in hES cells is to effectively antagonize MCL1 to permit rapid apoptosis. Significance statementHuman embryonic stem (hES) cells give rise to the entire organism, hence understanding how these cells regulate their survival and death is important. These cells undergo rapid death in response to DNA damage thereby removing mutated cells from the developing embryo. We focused on identifying the mechanism underlying the sensitivity of these cells to DNA damage. We discovered that the protein NOXA is essential for cell death in hES cells. Further, the crucial function of NOXA is to neutralize high levels of antiapototic protein, MCL1, thus enabling hES cells to respond rapidly to DNA damage.

cell biology↗

bioRxiv2020

Sca1+ cells as direct isolate (ex vivo) versus in vitro cultured exhibit differential proteomic signatures in murine skeletal muscle

Stem cell antigen-1 (Sca-1) is a glycosyl-phosphatidylinositol-anchored membrane protein that is expressed in a sub-population of muscle stem and progenitor cell types. Reportedly, Sca-1 regulates the myogenic property of myoblasts and Sca-1-/- mice exhibited defective muscle regeneration. Although the role of Sca-1 in muscle development and maintenance is well-acknowledged, molecular composition of muscle derived Sca-1+ cells is not characterized. Here, we applied a high-resolution mass spectrometry-based workflow to characterize the proteomic landscape of mouse hindlimb skeletal muscle derived Sca-1+ cells. Furthermore, we characterized the impact of the cellular microenvironments on the proteomes of Sca-1+ cells. The proteome component of freshly isolated (ex vivo) Sca-1+ cells was compared with that of Sca-1+ cells expanded in cell culture (in vitro). The analysis revealed significant differences in the protein abundances in the two conditions reflective of their functional variations. The identified proteins were enriched in various biological pathways. Notably, we identified proteins related to myotube differentiation, myotube cell development and myoblast fusion. We also identified a panel of cell surface marker proteins that can be leveraged in future to enrich Sca-1+ cells using combinatorial strategies. Comparative analysis implicated the activation of various pathways leading to increased protein synthesis under in vitro condition. We report here the most comprehensive proteome map of Sca-1+ cells that provides insights into the molecular networks operative in Sca-1+ cells. Importantly, through our work we generated the proteomic blueprint of protein abundances significantly altered in Sca-1+ cells under ex vivo and in vitro conditions.

systems biology↗

bioRxiv2020

Structural characterization of Myxococcus xanthus MglC, a component of polarity control system, and its interactions with MglB

Myxococcus xanthus displays two types of motilities i.e. Social (S) and Adventurous (A). The pole-to-pole reversals of these motility regulator proteins is the key to this process. Here, we determined ~1.85 [A] resolution crystal structure of MglC, which revealed that despite sharing <9% sequence identity, both MglB and MglC adopt Regulatory Light Chain 7 (RLC7) family fold. Interestingly, MglC is structurally unique compared to the other known RLC7 family proteins having ~30{degrees}-40{degrees} shift in the orientation of functionally important 2 helix. Using isothermal titration calorimetry and gel filtration chromatography, we show that MglC binds MglB in 2:4 stoichiometry with submicromolar range dissociation constant. Using combination of small angle X-ray scattering and molecular docking studies, we show that MglBC complex is formed by MglC homodimer sandwiched between two homodimers of MglB. In BriefKapoor et al. report the crystal structure of Myxococcus xanthus MglC, a Roadblock Light Chain 7 (RLC7) family protein, involved in polarity reversal. The structure reveals a distinct orientation of 2 helix compared to other RLC7 proteins. They also demonstrate that MglC binds a GTPase activating protein, MglB, with submicromolar range dissociation constant. HighlightsO_LIMglC adopts RLC7 fold and has distinct structural features. C_LIO_LIMglC interacts MglB to form a stable complex having submicromolar range dissociation constant. C_LIO_LIMglC homodimer is sandwiched between two MglB homodimers to form a 2:4 stoichiometric complex. C_LI

biophysics↗

bioRxiv2020

A series of dual-reporter vectors for ratiometric analysis of protein abundance in plants

Ratiometric reporter systems enable comparisons of the abundance of a protein of interest, or "target," relative to a reference protein. Both proteins are encoded on a single transcript but are separated during translation. This arrangement bypasses the potential for discordant expression that can arise when the target and reference proteins are encoded by separate genes. We generated a set of 18 Gateway-compatible vectors termed pRATIO that combine a variety of promoters, fluorescent and bioluminescent reporters, and 2A "self-cleaving" peptides. These constructs are easily modified to produce additional combinations or introduce new reporter proteins. We found that mScarlet-I provides the best signal-to-noise ratio among several fluorescent reporter proteins during transient expression experiments in Nicotiana benthamiana. Firefly and Gaussia luciferase also produce high signal-to-noise in N. benthamiana. As proof of concept, we used this system to investigate whether degradation of the receptor KAI2 after karrikin treatment is influenced by its subcellular localization. KAI2 is normally found in the cytoplasm and the nucleus of plant cells. In N. benthamiana, karrikin-induced degradation of KAI2 was only observed when it was retained in the nucleus. These vectors are tools to easily monitor in vivo the abundance of a protein that is transiently expressed in plants, and will be particularly useful for investigating protein turnover in response to different stimuli.

plant biology↗

bioRxiv2019

Anastrozole mediated modulation of mitochondrial activity by inhibition of mitochondrial permeability transition pore opening: An initial perspective

BackgroundMitochondrial permeability transmembrane pore [mPTP] plays a vital role in alteration of the structure and function of mitochondria. Cyclophillin D is a mitochondrial protein that regulates mPTP function and a known drug target for therapeutic studies involving mitochondria. While aromatase inhibitors role on mPTP has been previously studied, the role of anastrazole on mPTP is not completely elucidated.\n\nMethodsThe role of anastrozole in modulating the mPTP was evaluated by docking and molecular dynamics using human cyclophillin D data. Peripheral blood mononuclear cells [PBMCs] of patients with mitochondrial disorders and healthy controls were treated with anastrazole and evaluated for mean fluorescence by flow cytometer. Spectrophotometry was employed for total ATP level estimation.\n\nFindingsAnastrozole - cyclophillin D complex is more stable when compared to cyclosporine A - cyclophillin D. Anastrozole performed better than cyclosporine in inhibiting mPTP pore. Additional effects included reduction in mitochondrial swelling and mitochondrial membrane depolarization, decreased super oxide generations, caspase 3 intrinsic activity and cellular apoptosis levels and increase in ATP levels.\n\nInterpretationThese results highlights the potency of anastrozole as a promising agent in ameliorating the phenotype by inhibiting the opening of mPTP pore. However, larger functional studies are required to validate the efficacy of this molecule as a therapeutic agent in mitochondrial disorders.

molecular biology↗

bioRxiv2018

Centrosome Aurora A gradient ensures a single PAR-2 polarity axis by regulating RhoGEF ECT-2 localization in C. elegans embryos

The proper establishment of the cell polarity is essential for development and morphogenesis. In the Caenorhabditis elegans one-cell embryo, a centrosome localized signal provides spatial information that is responsible for generating a single polarity axis. It is hypothesized that such a signal causes local inhibition of cortical actomyosin network in the vicinity of the centrosome. This pivotal event initiates symmetry breaking to direct partitioning of the partition defective proteins (PARs) in the one-cell embryo. However, the molecular nature of the centrosome regulated signal that impinges on the posterior cortex to bring upon cortical anisotropy in the actomyosin network and to promote polarity establishment remains elusive. Here, we discover that Aurora A kinase (AIR-1 in C. elegans) is essential for proper cortical contractility in the one-cell embryo. Loss of AIR-1 causes pronounced cortical contractions on the entire embryo surface during polarity establishment phase, and this creates more than one PAR-2 polarity axis. Moreover, we show that in the absence of AIR-1, centrosome positioning becomes dispensable in dictating the PAR-2 polarity axis. Interestingly, we identify that Rho Guanine Exchange Factor (GEF) ECT-2 acts downstream to AIR-1 to control excess contractility and notably AIR-1 loss affects ECT-2 cortical localization and thereby polarity establishment. Overall, our study unravels a novel insight whereby an evolutionarily conserved kinase Aurora A inhibits promiscuous PAR-2 domain formation and ensures singularity in the polarity establishment axis.

cell biology↗