Disulphide Bridges of Phospholipase C of Chlamydomonas reinhardtii Modulates Lipid Interaction and Dimer Stability

BACKGROUND: Phospholipase C (PLC) is an enzyme that plays pivotal role in a number of signaling cascades. These are active in the plasma membrane and triggers cellular responses by catalyzing the hydrolysis of membrane phospholipids and thereby generating the secondary messengers. Phosphatidylinositol-PLC (PI-PLC) specifically interacts with phosphoinositide and/or phosphoinositol and catalyzes specific cleavage of sn-3- phosphodiester bond. Several isoforms of PLC are known to form and function as dimer but very little is known about the molecular basis of the dimerization and its importance in the lipid interaction. PRINCIPAL FINDINGS: We herein report that, the disruption of disulphide bond of a novel PI-specific PLC of C. reinhardtii (CrPLC) can modulate its interaction affinity with a set of phospholipids and also the stability of its dimer. CrPLC was found to form a mixture of higher oligomeric states with monomer and dimer as major species. Dimer adduct of CrPLC disappeared in the presence of DTT, which suggested the involvement of disulphide bond(s) in CrPLC oligomerization. Dimer-monomer equilibrium studies with the isolated fractions of CrPLC monomer and dimer supported the involvement of covalent forces in the dimerization of CrPLC. A disulphide bridge was found to be responsible for the dimerization and Cys7 seems to be involved in the formation of the disulphide bond. This crucial disulphide bond also modulated the lipid affinity of CrPLC. Oligomers of CrPLC were also captured in in vivo condition. CrPLC was mainly found to be localized in the plasma membrane of the cell. The cell surface localization of CrPLC may have significant implication in the downstream regulatory function of CrPLC. SIGNIFICANCE: This study helps in establishing the role of CrPLC (or similar proteins) in the quaternary structure of the molecule its affinities during lipid interactions

Cellular localization of CrPLC.

<p>(A) CrPLC localizes in the plasma membrane of <i>C. reinhardtii</i> cell as shown by green channel (B) Magnified image of a single cell. (C) Plasma membrane traced by using FM4-64 tracker dye is shown in red. (D) Merged image represent the overlay, where green and red channels are merged together to confirm CrPLC localization in the plasma membrane. (E) Cells visualized in DIC mode of light microscopy. (F and G) Immunolocalization with pre-immune serum and auto-fluorescence served as negative control.</p

Homology analysis of PI-PLC isozymes from different organisms.

<p>Sequences of Phospholipase C from different organisms including <i>Chlamydomonas reinhatdtii</i> (CrPLC), <i>Drosophila melanogaster</i> (DmPLC), <i>Arabidopsis thaliana</i> (AtPLC), <i>Homo sapiens</i> (HsPLC), and <i>Mus musculus</i> (MmPLC) were compared. Identical amino acid residues are represented in white with black background and residues with greater than 85% similarity in sequences are highlighted in black with grey background. Residues present in the X–Y domain and catalytic domain are indicated by black and grey solid bars respectively. Catalytically important and conserved amino acid residues for substrate binding are marked by solid black circles.</p

CrPLC dimerizes both <i>in vitro</i> and <i>in vivo</i>.

<p>(A) Immunoblotting of crosslinked (Glutaraldehyde; GA) dimer and monomer peak fractions. Numbers above each lane are the elution volume in ml with 70 ml (dimer peak fraction) and 82 ml (monomer peak fraction). (B) Cellular detection of CrPLC dimer and monomer species by glutaraldehyde (GA) crosslinking of <i>Chlamydomonas</i> total cell protein (Cr TCL) followed by immunoblotting. (C) Immunoblot analysis of CrPLC dimer and monomer peak fractions of size exclusion chromatography in both presence (reducing) and absence (non-reducing) of dithiothreitol (DTT). (D) Comparative chromatogram of recombinant CrPLC performed under reducing condition using 5 mM DTT (black) and in non-reducing condition (red). (E) Cellular detection of CrPLC in <i>Cr</i> TCL under reducing and non-reducing conditions. Dimer and Monomer in the blots are marked as D and M respectively.</p

Quaternary structures of recombinant CrPLC.

<p>(A) Size exclusion chromatogram of recombinant CrPLC. Straight line depicts the calibration of standard molecular weight marker including, thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.35 kDa). (B) Size-exclusion profile of CrPLC of various concentrations. Elution profiles of CrPLC corresponding to various concentrations of recombinant CrPLC (5, 10 and 50 µM) were recorded using analytical grade size exclusion chromatography column and shown in different line patterns as mentioned in the inset of figure. (C and D) Size exclusion chromatogram of reloaded monomer and dimer fractions using preparatory grade chromatography column are shown in black, overlaid with chromatogram of CrPLC (dotted line).</p

Novel Modular Rhodopsins from Green Algae Hold Great Potential for Cellular Optogenetic Modulation Across the Biological Model Systems

Light-gated ion channel and ion pump rhodopsins are widely used as optogenetic tools and these can control the electrically excitable cells as (1) they are a single-component system i.e., their light sensing and ion-conducting functions are encoded by the 7-transmembrane domains and, (2) they show fast kinetics with small dark-thermal recovery time. In cellular signaling, a signal receptor, modulator, and the effector components are involved in attaining synchronous regulation of signaling. Optical modulation of the multicomponent network requires either receptor to effector encoded in a single ORF or direct modulation of the effector domain through bypassing all upstream players. Recently discovered modular rhodopsins like rhodopsin guanylate cyclase (RhoGC) and rhodopsin phosphodiesterase (RhoPDE) paves the way to establish a proof of concept for utilization of complex rhodopsin (modular rhodopsin) for optogenetic applications. Light sensor coupled modular system could be expressed in any cell type and hence holds great potential in the advancement of optogenetics 2.0 which would enable manipulating the entire relevant cell signaling system. Here, we had identified 50 novel modular rhodopsins with variant domains and their diverse cognate signaling cascades encoded in a single ORF, which are associated with specialized functions in the cells. These novel modular algal rhodopsins have been characterized based on their sequence and structural homology with previously reported rhodopsins. The presented novel modular rhodopsins with various effector domains leverage the potential to expand the optogenetic tool kit to regulate various cellular signaling pathways across the diverse biological model systems.https://doi.org/10.3390/life1011025

Novel Modular Rhodopsins from Green Algae Hold Great Potential for Cellular Optogenetic Modulation Across the Biological Model Systems

Light-gated ion channel and ion pump rhodopsins are widely used as optogenetic tools and these can control the electrically excitable cells as (1) they are a single-component system i.e., their light sensing and ion-conducting functions are encoded by the 7-transmembrane domains and, (2) they show fast kinetics with small dark-thermal recovery time. In cellular signaling, a signal receptor, modulator, and the effector components are involved in attaining synchronous regulation of signaling. Optical modulation of the multicomponent network requires either receptor to effector encoded in a single ORF or direct modulation of the effector domain through bypassing all upstream players. Recently discovered modular rhodopsins like rhodopsin guanylate cyclase (RhoGC) and rhodopsin phosphodiesterase (RhoPDE) paves the way to establish a proof of concept for utilization of complex rhodopsin (modular rhodopsin) for optogenetic applications. Light sensor coupled modular system could be expressed in any cell type and hence holds great potential in the advancement of optogenetics 2.0 which would enable manipulating the entire relevant cell signaling system. Here, we had identified 50 novel modular rhodopsins with variant domains and their diverse cognate signaling cascades encoded in a single ORF, which are associated with specialized functions in the cells. These novel modular algal rhodopsins have been characterized based on their sequence and structural homology with previously reported rhodopsins. The presented novel modular rhodopsins with various effector domains leverage the potential to expand the optogenetic tool kit to regulate various cellular signaling pathways across the diverse biological model systems

The Sialoside-Binding Pocket of SARS-CoV-2 Spike Glycoprotein Structurally Resembles MERS-CoV

COVID-19 novel coronavirus (CoV) disease caused by severe acquired respiratory syndrome (SARS)-CoV-2 manifests severe lethal respiratory illness in humans and has recently developed into a worldwide pandemic. The lack of effective treatment strategy and vaccines against the SARS-CoV-2 poses a threat to human health. An extremely high infection rate and multi-organ secondary infection within a short period of time makes this virus more deadly and challenging for therapeutic interventions. Despite high sequence similarity and utilization of common host-cell receptor, human angiotensin-converting enzyme-2 (ACE2) for virus entry, SARS-CoV-2 is much more infectious than SARS-CoV. Structure-based sequence comparison of the N-terminal domain (NTD) of the spike protein of Middle East respiratory syndrome (MERS)-CoV, SARS-CoV, and SARS-CoV-2 illustrate three divergent loop regions in SARS-CoV-2, which is reminiscent of MERS-CoV sialoside binding pockets. Comparative binding analysis with host sialosides revealed conformational flexibility of SARS-CoV-2 divergent loop regions to accommodate diverse glycan-rich sialosides. These key differences with SARS-CoV and similarity with MERS-CoV suggest an evolutionary adaptation of SARS-CoV-2 spike glycoprotein reciprocal interaction with host surface sialosides to infect host cells with wide tissue tropism.https://doi.org/10.3390/v1209090

The Sialoside-Binding Pocket of SARS-CoV-2 Spike Glycoprotein Structurally Resembles MERS-CoV

COVID-19 novel coronavirus (CoV) disease caused by severe acquired respiratory syndrome (SARS)-CoV-2 manifests severe lethal respiratory illness in humans and has recently developed into a worldwide pandemic. The lack of effective treatment strategy and vaccines against the SARS-CoV-2 poses a threat to human health. An extremely high infection rate and multi-organ secondary infection within a short period of time makes this virus more deadly and challenging for therapeutic interventions. Despite high sequence similarity and utilization of common host-cell receptor, human angiotensin-converting enzyme-2 (ACE2) for virus entry, SARS-CoV-2 is much more infectious than SARS-CoV. Structure-based sequence comparison of the N-terminal domain (NTD) of the spike protein of Middle East respiratory syndrome (MERS)-CoV, SARS-CoV, and SARS-CoV-2 illustrate three divergent loop regions in SARS-CoV-2, which is reminiscent of MERS-CoV sialoside binding pockets. Comparative binding analysis with host sialosides revealed conformational flexibility of SARS-CoV-2 divergent loop regions to accommodate diverse glycan-rich sialosides. These key differences with SARS-CoV and similarity with MERS-CoV suggest an evolutionary adaptation of SARS-CoV-2 spike glycoprotein reciprocal interaction with host surface sialosides to infect host cells with wide tissue tropism