MOESM1 of Localization and dimer stability of a newly identified microbial rhodopsin from a polar, non-motile green algae

Additional file 1. Recombinant CsR expressed in E. coli were probed with anti-Penta His Ab and anti-CsR Ab

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

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

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

COVID-19 ORF3a Viroporin-Influenced Common and Unique Cellular Signaling Cascades in Lung, Heart, and the Brain Choroid Plexus Organoids with Additional Enriched MicroRNA Network Analyses for Lung and the Brain Tissues

Tissue-specific implications of SARS-CoV-2-encoded accessory proteins are not fully understood. SARS-CoV-2 infection can severely affect three major organsthe heart, lungs, and brain. We analyzed SARS-CoV-2 ORF3a interacting host proteins in these three major organs. Furthermore, we identified common and unique interacting host proteins and their targeting miRNAs (lung and brain) and delineated associated biological processes by reanalyzing RNA-seq data from the brain (COVID-19-infected/uninfected choroid plexus organoid study), lung tissue from COVID-19 patients/healthy subjects, and cardiomyocyte cells-based transcriptomics analyses. Our in silico studies showed ORF3a interacting proteins could vary depending upon tissues. The number of unique ORF3a interacting proteins in the brain, lungs, and heart were 10, 7, and 1, respectively. Though common pathways influenced by SARS-CoV-2 infection were more, unique 21 brain and 7 heart pathways were found. One unique pathway for the heart was negative regulation of calcium ion transport. Reported observations of COVID-19 patients with a history of hypertension taking calcium channel blockers (CCBs) or dihydropyridine CCBs had an elevated rate of intubation or increased rate of intubation/death, respectively. Also, the likelihood of hospitalization of chronic CCB users with COVID-19 was greater in comparison to long-term angiotensin-converting enzyme inhibitors/angiotensin receptor blockers users. Further studies are necessary to confirm this. miRNA analysis of ORF3a interacting proteins in the brain and lungs revealed 3 of 37 brain miRNAs and 1 of 25 lung miRNAs with high degree and betweenness indicating their significance as hubs in the interaction network. Our study could help in identifying potential tissue-specific COVID-19 drug/drug repurposing targets

COVID-19 ORF3a Viroporin-Influenced Common and Unique Cellular Signaling Cascades in Lung, Heart, and the Brain Choroid Plexus Organoids with Additional Enriched MicroRNA Network Analyses for Lung and the Brain Tissues

Tissue-specific implications of SARS-CoV-2-encoded accessory proteins are not fully understood. SARS-CoV-2 infection can severely affect three major organsthe heart, lungs, and brain. We analyzed SARS-CoV-2 ORF3a interacting host proteins in these three major organs. Furthermore, we identified common and unique interacting host proteins and their targeting miRNAs (lung and brain) and delineated associated biological processes by reanalyzing RNA-seq data from the brain (COVID-19-infected/uninfected choroid plexus organoid study), lung tissue from COVID-19 patients/healthy subjects, and cardiomyocyte cells-based transcriptomics analyses. Our in silico studies showed ORF3a interacting proteins could vary depending upon tissues. The number of unique ORF3a interacting proteins in the brain, lungs, and heart were 10, 7, and 1, respectively. Though common pathways influenced by SARS-CoV-2 infection were more, unique 21 brain and 7 heart pathways were found. One unique pathway for the heart was negative regulation of calcium ion transport. Reported observations of COVID-19 patients with a history of hypertension taking calcium channel blockers (CCBs) or dihydropyridine CCBs had an elevated rate of intubation or increased rate of intubation/death, respectively. Also, the likelihood of hospitalization of chronic CCB users with COVID-19 was greater in comparison to long-term angiotensin-converting enzyme inhibitors/angiotensin receptor blockers users. Further studies are necessary to confirm this. miRNA analysis of ORF3a interacting proteins in the brain and lungs revealed 3 of 37 brain miRNAs and 1 of 25 lung miRNAs with high degree and betweenness indicating their significance as hubs in the interaction network. Our study could help in identifying potential tissue-specific COVID-19 drug/drug repurposing targets

COVID-19 ORF3a Viroporin-Influenced Common and Unique Cellular Signaling Cascades in Lung, Heart, and the Brain Choroid Plexus Organoids with Additional Enriched MicroRNA Network Analyses for Lung and the Brain Tissues

Tissue-specific implications of SARS-CoV-2-encoded accessory proteins are not fully understood. SARS-CoV-2 infection can severely affect three major organsthe heart, lungs, and brain. We analyzed SARS-CoV-2 ORF3a interacting host proteins in these three major organs. Furthermore, we identified common and unique interacting host proteins and their targeting miRNAs (lung and brain) and delineated associated biological processes by reanalyzing RNA-seq data from the brain (COVID-19-infected/uninfected choroid plexus organoid study), lung tissue from COVID-19 patients/healthy subjects, and cardiomyocyte cells-based transcriptomics analyses. Our in silico studies showed ORF3a interacting proteins could vary depending upon tissues. The number of unique ORF3a interacting proteins in the brain, lungs, and heart were 10, 7, and 1, respectively. Though common pathways influenced by SARS-CoV-2 infection were more, unique 21 brain and 7 heart pathways were found. One unique pathway for the heart was negative regulation of calcium ion transport. Reported observations of COVID-19 patients with a history of hypertension taking calcium channel blockers (CCBs) or dihydropyridine CCBs had an elevated rate of intubation or increased rate of intubation/death, respectively. Also, the likelihood of hospitalization of chronic CCB users with COVID-19 was greater in comparison to long-term angiotensin-converting enzyme inhibitors/angiotensin receptor blockers users. Further studies are necessary to confirm this. miRNA analysis of ORF3a interacting proteins in the brain and lungs revealed 3 of 37 brain miRNAs and 1 of 25 lung miRNAs with high degree and betweenness indicating their significance as hubs in the interaction network. Our study could help in identifying potential tissue-specific COVID-19 drug/drug repurposing targets

COVID-19 ORF3a Viroporin-Influenced Common and Unique Cellular Signaling Cascades in Lung, Heart, and the Brain Choroid Plexus Organoids with Additional Enriched MicroRNA Network Analyses for Lung and the Brain Tissues

Tissue-specific implications of SARS-CoV-2-encoded accessory proteins are not fully understood. SARS-CoV-2 infection can severely affect three major organsthe heart, lungs, and brain. We analyzed SARS-CoV-2 ORF3a interacting host proteins in these three major organs. Furthermore, we identified common and unique interacting host proteins and their targeting miRNAs (lung and brain) and delineated associated biological processes by reanalyzing RNA-seq data from the brain (COVID-19-infected/uninfected choroid plexus organoid study), lung tissue from COVID-19 patients/healthy subjects, and cardiomyocyte cells-based transcriptomics analyses. Our in silico studies showed ORF3a interacting proteins could vary depending upon tissues. The number of unique ORF3a interacting proteins in the brain, lungs, and heart were 10, 7, and 1, respectively. Though common pathways influenced by SARS-CoV-2 infection were more, unique 21 brain and 7 heart pathways were found. One unique pathway for the heart was negative regulation of calcium ion transport. Reported observations of COVID-19 patients with a history of hypertension taking calcium channel blockers (CCBs) or dihydropyridine CCBs had an elevated rate of intubation or increased rate of intubation/death, respectively. Also, the likelihood of hospitalization of chronic CCB users with COVID-19 was greater in comparison to long-term angiotensin-converting enzyme inhibitors/angiotensin receptor blockers users. Further studies are necessary to confirm this. miRNA analysis of ORF3a interacting proteins in the brain and lungs revealed 3 of 37 brain miRNAs and 1 of 25 lung miRNAs with high degree and betweenness indicating their significance as hubs in the interaction network. Our study could help in identifying potential tissue-specific COVID-19 drug/drug repurposing targets

Effect of metal ions on the intrinsic fluorescence of PdeM.

<p>(A) Effect of Mn²⁺ and, (B) Effect of Effect of Fe³⁺ concentrations on the intrinsic tryptophan fluorescence of the purified PdeM protein. Saturation curves were generated from the intrinsic fluorescence data by plotting the change in fluorescence intensity at 340 nm as a function of increasing concentrations of (C) Mn²⁺ and (D) Fe³⁺.</p