Biology subjects

Kang, C.

Publications and source records attributed to Kang, C..

5 recordsLinked to original sources

bioRxiv2018

Gαq sensitizes TRPM8 to inhibition by PI(4,5)P2 depletion upon receptor activation

Activation of G-protein coupled receptors (GPCRs) was proposed to inhibit the cold and menthol sensitive Transient Receptor Potential Melastatin 8 (TRPM8) channels via direct binding of Gq to the channel. It is well documented that TRPM8 requires the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2 or PIP2] for activity. It was claimed however that a decrease in cellular levels of this lipid does not contribute to channel inhibition upon receptor activation. Here we show that supplementing the whole cell patch pipette with PI(4,5)P2 reduced inhibition of TRPM8 by activation of Gq-coupled receptors in mouse dorsal root ganglion (DRG) neurons. Activation of the same receptors induced Phospholipase C (PLC) activation and decreased plasma membrane PI(4,5)P2 levels in these neurons. PI(4,5)P2 also reduced inhibition of TRPM8 by activation of heterologously expressed Gq-coupled muscarinic M1 receptors. Co-expression of a constitutively active Gq protein that does not couple to PLC inhibited TRPM8 activity, and in cells expressing this protein decreasing PI(4,5)P2 levels using a voltage sensitive 5-phosphatase induced a stronger inhibition of TRPM8 activity than in control cells. Our data indicate that PI(4,5)P2 depletion plays an important role in TRPM8 inhibition upon GPCR activation, and Gq inhibits the channel by reducing its apparent affinity for PI(4,5)P2 and thus sensitizes the channel to inhibition by decreasing PI(4,5)P2 levels.

neuroscience↗

bioRxiv2018

The EVcouplings Python framework for coevolutionary sequence analysis

SummaryCoevolutionary sequence analysis has become a commonly used technique for de novo prediction of the structure and function of proteins, RNA, and protein complexes. This approach requires extensive computational pipelines that integrate multiple tools, databases, and data processing steps. We present the EVcouplings framework, a fully integrated open-source application and Python package for coevolutionary analysis. The framework enables generation of sequence alignments, calculation and evaluation of evolutionary couplings (ECs), and de novo prediction of structure and mutation effects. The application has an easy to use command line interface to run workflows with user control over all analysis parameters, while the underlying modular Python package allows interactive data analysis and rapid development of new workflows. Through this multi-layered approach, the EVcouplings framework makes the full power of coevolutionary analyses available to entry-level and advanced users.\n\nAvailabilityhttps://github.com/debbiemarkslab/evcouplings\n\nContactsander.research@gmail.com, debbie@hms.harvard.edu

bioinformatics↗

bioRxiv2018

Substrate channeling in oxylipin biosynthesis through a protein complex in the plastid envelope of Arabidopsis thaliana

Oxygenated membrane fatty acid derivatives dubbed oxylipins play important roles in the plants defense against biotic and abiotic cues. Plants challenged by insect pests, for example, synthesize a blend of different defense compounds that, amongst others, comprise volatile aldehydes and jasmonic acid (JA). Because all oxylipins are derived from the same pathway, we asked how their synthesis might be regulated and focused on two closely related, atypical cytochrome P450 enzymes designated CYP74A and CYP74B, i.e., allene oxide synthase (AOS) and hydroperoxide lyase (HPL). Both enzymes compete for the same substrate but give rise to different products. While the final product of the AOS branch is JA, those of the HPL branch comprise volatile aldehydes and alcohols. AOS and HPL are plastid envelope enzymes in Arabidopsis thaliana but accumulate at different locations. Biochemical experiments identified AOS as constituent of complexes also containing lipoxygenase 2 (LOX2) and allene oxide cyclase (AOC), which catalyze consecutive steps in JA precursor biosynthesis, while excluding the concurrent HPL reaction. Based on published X-ray data, the structure of this complex could be modelled and amino acids involved in catalysis and subunit interactions identified. Genetic studies identified the microRNA 319 (miR319)-regulated clade of TCP (TEOSINTE BRANCHED/CYCLOIDEA/PCF) transcription factor genes and CORONATINE INSENSITIVE 1 (COI1) to control JA production through the AOS-LOX2-AOC2 complex. Together, our results define a molecular branch point in oxylipin biosynthesis that allows fine-tuning the plants defense machinery in response to biotic and abiotic stimuli.

cell biology↗

bioRxiv2018

Building superfast muscles: insights from molecular parallelism in fast-twitch muscle proteins in echolocating mammals

Detecting associations between genomic changes and phenotypic differences is fundamental to understanding how phenotypes evolved. By systematically screening for parallel amino acid substitutions, we detected known as well as novel cases (Strc, Tecta, Cabp2) of parallelism between echolocating bats and toothed whales in proteins that could contribute to high frequency hearing adaptations. Interestingly, our screen also showed that echolocating mammals exhibit an unusually high number of parallel substitutions in fast-twitch muscle fiber proteins. Both bats and dolphins produce an extremely rapid call rate when homing in on their prey, which was shown in bats to be powered by specialized superfast muscles. We show that these genes with parallel substitutions (Casq1, Atp2a1, Myh2, Myl1) are expressed in the superfast sound-producing muscle of bats. Furthermore, we found that the calcium storage protein calsequestrin 1 of bats and dolphins functionally converged in its ability to form calcium-sequestering polymers at lower calcium concentrations, which may contribute to rapid calcium transients required for superfast muscle physiology. The proteins that our genomic screen detected could be involved in the convergent evolution of vocalization in echolocating mammals by potentially contributing to both rapid Ca2+ transients and increased shortening velocities in superfast muscles.\n\nAbbreviations

evolutionary biology↗

bioRxiv2017

SWELL1 is a glucose sensor required for β-cell excitability and insulin secretion

Insulin secretion from the pancreatic {beta}-cell initiated by activation of voltage-gated Ca2+ channels (VGCC) to trigger Ca2+-mediated insulin vesicle fusion with the {beta}-cell plasma membrane. The firing of VGCC depends on the {beta}-cell membrane potential, which is in turn mediated by the balance of depolarizing (excitatory) and hyperpolarizing (inhibitory) ionic currents1-3. While much attention has focused on inhibitory potassium currents4-10 there is little knowledge about the excitatory currents required to depolarize the {beta}-cell, including the molecular identity of these excitatory currents3. Here we show that SWELL1 (LRRC8a) mediates a swell-activated, depolarizing chloride current (ICl,SWELL) in {beta}-cells. Hypotonic and glucose-stimulated {beta}-cell swelling activates SWELL1-mediated ICl,SWELL and this is required for both glucose-stimulated and hypotonic swell-mediated activation of VGCC-dependent intracellular calcium signaling in {beta}-cells. SWELL1 KO MIN6 cells and {beta}-cell targeted SWELL1 KO murine islets exhibit significantly impaired glucose-stimulated insulin secretion, with preserved insulin content in vitro. Tamoxifen-inducible {beta}-cell targeted SWELL1 KO mice have normal fasting insulin levels but display markedly impaired glucose-stimulated insulin secretion. Our results reveal a physiological role for SWELL1 as a glucose sensor - linking glucose-mediated {beta}-cell swelling to SWELL1-dependent activation of VGCC-triggered calcium signaling, and highlights SWELL1-mediated \"swell-secretion\" coupling as required for glucose-stimulated insulin secretion.

cell biology↗