Journal: Immunogenetics

Article Title: Comparative genetics of a highly divergent DRB microsatellite in different macaque species

doi: 10.1007/s00251-008-0333-z

Figure Lengend Snippet: Phylogenetic tree of mtDNA sequences coding for parts of the 12S rRNA of cynomolgus macaques. Brackets indicate the different geographic clusters. The tree was rooted with selected rhesus macaque sequences from India, China, and Burma. mtDNA sequences without accession numbers have been published recently or have now been submitted to EMBL/GeneBank database. Juanita, Rastafa, Alfa, and Sayonara are representatives of the four Cynomolgus families, and Cyn81 and Cyn83 are the two unrelated animals analyzed in this study ( shadowed )

Article Snippet: Multiple sequence alignments of the 12S rRNA sequences were obtained of newly generated mtDNA sequences (accession numbers see above) together with published mtDNA sequences of animals of documented origin ( ; ) using MacVector v9.5.2 (MacVector, Inc., Cambridge, UK).

Techniques:

Journal: Biochemistry

Article Title: Biochemical and spectroscopic characterization of a radical SAM enzyme involved in the formation of a peptide thioether crosslink

doi: 10.1021/acs.biochem.6b00145

Figure Lengend Snippet: (A) The mature nisin peptide has thioether crosslinks between Cys and dehydroalanine (Dha) and dehydrobutyrine (Dhb) residues. (B) The sactipeptide subtilosin A is shown, highlighting the three thioether crosslinks. In its fully mature form, subtilosin A is circularized. The stereochemistry at the three attachment sites is shown in red. The sactipeptides are distinct from the lanthipeptides in that the thioether crosslinks are formed to the Cα of the peptide. (C) Sequence logo for the SCIFF peptides showing the conserved C-terminal sequence. The figure was generated by aligning 100 SCIFF sequences selected from Interpro family IPR023975 using the Clustal Omega61 multiple sequence alignment and visualized by Weblogo.62,63

Article Snippet: The figure was generated by aligning 100 SCIFF sequences selected from Interpro family IPR023975 using the Clustal Omega61 multiple sequence alignment and visualized by Weblogo.62,63.

Techniques: Sequencing, Generated

Journal: iScience

Article Title: Mieap forms membrane-less organelles involved in cardiolipin metabolism

doi: 10.1016/j.isci.2024.108916

Figure Lengend Snippet: Mieap forms mitochondrial biomolecular condensates (A) Live-cell imaging and the 3D reconstruction showing the spatial relationship between the Mi-BCs (EGFP-Mieap) and the mitochondrial outer membranes visualized with mApple-TOMM20. The A549 cells were co-infected with Ad-EGFP-Mieap and Ad-mApple-TOMM20. Left: a cell image. Right: higher magnification of the area indicated by the dashed square in the left panel and a line-scan of fluorescence intensities along the dashed arrow. Scale bars, 10 μm (left panel) and 2 μm (right panel). See also . (B) Super-resolution images showing the spatial relationship between Mi-BCs (EGFP-Mieap) and mitochondrial outer membranes visualized with mApple-TOMM20. Scale bar, 1 μm. (C) z stack images of the cell in (A). Scale bar, 10 μm. (D) 3D reconstruction of the cell shown in (A). See also .

Article Snippet: Multiple sequence alignment for Mieap orthologs was performed using Genetyx ver.

Techniques: Live Cell Imaging, Infection, Fluorescence

Journal: iScience

Article Title: Mieap forms membrane-less organelles involved in cardiolipin metabolism

doi: 10.1016/j.isci.2024.108916

Figure Lengend Snippet: Mieap is an IDR-containing protein that has a potential to drive LLPS (A) Phylogenetic spread of Mieap orthologs annotated with OrthoDB v10. Red sectors indicate present species. Light blue sectors indicate missing species. (B) Proportion of amino acid residues in each domain of the Mieap protein. (C) Multiple sequence alignment for Mieap orthologs in representative eukaryotes. Black and gray boxes indicate 100% and 80% identical residues among eukaryotes, respectively. Blue letters indicate IDRs annotated by VL3-BA. Orange letters indicate coiled-coil regions annotated by COILS. (D) Schematic of the domain structure of Mieap. The dashed vertical line indicates the boundary of gross hydrophilic and hydrophobic halves, separated by IDR3 and the adjacent structured region. Asterisks indicate clusters of positively charged residues. (E) Sequence analyses of Mieap protein. VL3-BA prediction of IDRs on the amino acid sequence of Mieap, in which bold lines indicate IDRs; DisMeta, meta-prediction of IDRs on the amino acid sequence of Mieap; COILS; coiled-coil regions annotated on the amino acid sequence of Mieap using a 21-residue sliding window; Hydro, hydrophobicity of Mieap using a 9-residue sliding window; NCPR, the linear net charge per residue of Mieap using a 5-residue sliding window.

Article Snippet: Multiple sequence alignment for Mieap orthologs was performed using Genetyx ver.

Techniques: Sequencing, Residue

Journal: iScience

Article Title: Mieap forms membrane-less organelles involved in cardiolipin metabolism

doi: 10.1016/j.isci.2024.108916

Figure Lengend Snippet: Material state and dynamics of Mi-BCs and phase-separation of the mitochondrial phospholipid cardiolipin by Mi-BCs (A) EGFP-Mieap and the three deletion-mutant forms. The schematic indicates wild-type (WT) and three deletion mutants (ΔCC, Δ275, and Δ496) of EGFP-Mieap protein. Numbers indicate amino acid residues. (B) Normalized average fluorescence recovery in the FRAP experiment. EGFP-Mieap, EGFP-Mieap ΔCC, EGFP-Mieap Δ275, and EGFP-Mieap Δ496 were expressed in A549 cells to generate condensates by infection with Ad-EGFP-Mieap, Ad-EGFP-Mieap ΔCC, Ad-EGFP-Mieap Δ275, and Ad-EGFP-Mieap Δ496, respectively. Each condensate was subjected to spot-bleaching using a 488-nm laser at 10% laser power with 11.6 μs/μm exposure time and followed up for 60 s n = 15 condensates for each construct. Data shown are means ± SD. (C) Normalized average fluorescence recovery in the FRAP experiment with weaker laser exposure as in (B). Laser power was weakened to 1.4% and exposure time was shortened to 1.4 μs/μm. Observation duration was expanded to 15 min after photobleaching entire condensates. n = 10 condensates for each construct. Data shown are means ± SD. (D–F) Screening of the mitochondrial molecules involved in phase-separation by Mi-BCs. Ad-EGFP-BNIP3 (D), Ad-EGFP-NIX (E), and Ad-AcGFP1-Mito (F) were co-infected with Ad-Mieap and Ad-TagRFP-T-Mieap in A549 cells. Whether each mitochondrial fluorescence probe is phase-separated by Mi-BCs was examined with live-cell imaging analysis in A549 cells. EGFP-BNIP3 (D), EGFP-NIX (E), and AcGFP-mito (F) were not incorporated into Mi-BCs. Lower right: line-scan of fluorescence intensities along the dashed arrow. Scale bars, 10 μm. See also . (G) Live-cell imaging showing phase-separation of CL by Mi-BCs. CL was visualized by 10-nonylacridine orange bromide (NAO) in A549 cells. A549 cells were infected with Ad-Mieap and Ad-TagRFP-T-Mieap, and subsequently treated with NAO (200 nM). NAO was incorporated into Mi-BCs. Lower right: line-scan of fluorescence intensities along the dashed arrow. Scale bar, 10 μm. See also . (H) Lipid-binding analysis of GST-tagged Mieap protein. GST-Mieap or GST was incubated with membranes on which increasing amounts of CL, phosphatidylcholine (PC), and phosphatidylethanolamine (PE), ranging from 0 to 667 pmol, were spotted. Protein-lipid interactions were visualized using an anti-Mieap antibody and/or anti-GST antibody, as indicated.

Article Snippet: Multiple sequence alignment for Mieap orthologs was performed using Genetyx ver.

Techniques: Mutagenesis, Fluorescence, Infection, Construct, Live Cell Imaging, Binding Assay, Incubation

Journal: iScience

Article Title: Mieap forms membrane-less organelles involved in cardiolipin metabolism

doi: 10.1016/j.isci.2024.108916

Figure Lengend Snippet: Mi-BCs are possible membrane-less organelles involved in CL metabolism (A) Quantitative assessment of total CL by mass spectrometric analysis. Uninfected A549 cells or cells infected with Ad-Mieap were subjected to mass spectrometric analysis for CL. Data shown are means ± SE (n = 4). ∗p < 0.05, two tailed paired t test. (B) Quantitative and rate assessments of CL species by mass spectrometric analysis. A549 cells were analyzed as described in (A). Absolute values of selected CL species are shown as amounts of substance per cell (left panel). Relative values of selected CL species are shown as % of total CL (right panel). Data shown are means ± SE (n = 4). ∗p < 0.05, ∗∗p < 0.01, two tailed paired t test. (C) The conventional CL metabolic pathway. PA, phosphatidic acid; CDP-DG: cytidine diphosphate diacylglycerol; PGP, phosphatidylglycerophosphate; PG, phosphatidylglycerol; CL N , nascent cardiolipin; CL M , mature cardiolipin. (D–L) Live-cell imaging showing specific phase-separation of CL metabolic enzymes by Mi-BCs. A549 cells were transfected with pEGFP-TAMM41 (D), EGFP-PGS1 (E), EGFP-PTPMT1 (F), EGFP-CRLS1 (G), EGFP-PLA2G6 (H), EGFP-TAZ (I), EGFP-PRELI (J), EGFP-LONP1 (K) and EGFP-PLD6 (L), and subsequently infected with Ad-Mieap and Ad-TagRFP-T-Mieap. EGFP-TAMM41 (D), EGFP-PGS1 (E), EGFP-PTPMT1 (F), EGFP-CRLS1 (G), EGFP-PLA2G6 (H), EGFP-TAZ (I), and EGFP-PRELI (J) were incorporated into the Mieap-depleted phase of Mi-BCs. In contrast, EGFP-LONP1 (K) and EGFP-PLD6 (L) were not incorporated into either the Mieap-containing phase or the Mieap-depleted phase of Mi-BCs. Lower right: line-scan of fluorescence intensities along the dashed arrow. See also .

Article Snippet: Multiple sequence alignment for Mieap orthologs was performed using Genetyx ver.

Techniques: Membrane, Infection, Two Tailed Test, Live Cell Imaging, Transfection, Fluorescence

Journal: iScience

Article Title: Mieap forms membrane-less organelles involved in cardiolipin metabolism

doi: 10.1016/j.isci.2024.108916

Figure Lengend Snippet: Mieap protein is highly concentrated in mitochondrial BCs via its C-terminal hydrophobic region (A and B) 3D reconstruction showing the spatial relationship between Mi-BCs and mitochondrial inner membrane visualized with EGFP-TAMM41 (A) or NAO (B). See also , , and . (C–H) 3D reconstruction showing the spatial relationship between BCs formed by ΔCC, Δ275, or Δ496, and mitochondrial inner membrane visualized with EGFP-TAMM41 (C–E) or NAO (F–H). See also , , and . HeLa cells were transfected with pEGFP-TAMM41 to visualize mitochondrial inner membrane, and subsequently infected with Ad-TagRFP-T-Mieap WT (A), Ad-TagRFP-T-Mieap ΔCC (C), Ad-TagRFP-T-Mieap Δ275 (D), or Ad-TagRFP-T-Mieap Δ496 (E) to form BCs. A549 cells were infected with Ad-TagRFP-T-Mieap WT (B), Ad-TagRFP-T-Mieap ΔCC (F), Ad-TagRFP-T-Mieap Δ275 (G), or Ad-TagRFP-T-Mieap Δ496 (H) to form BCs, and after BCs were formed, mitochondrial inner membrane was visualized by NAO. (I) Partitioning behavior of EGFP-Mieap WT, ΔCC, Δ275, or Δ496 protein in condensates and cytoplasm, displayed in violin plot. A549 cells were infected with Ad-EGFP-Mieap WT, ΔCC, Δ275, or Δ496 to generate condensates, and after BCs were formed, the intensity ratio (IR) of condensates and cytoplasm was measured. n = 40 cells for each construct in A549 cells. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, two tailed Student’s t test. When the data were visualized using violin plots, boxplots were overlaid. The center line in the box indicates the median. The bottom and top of the box indicate the 25 th and 75 th percentiles. The whiskers extend 1.5 times the interquartile range (IQR) from the top and bottom of the box unless the minimum and maximum values are within the IQR. The values which fall above or below the whiskers are plotted individually as outliers.

Article Snippet: Multiple sequence alignment for Mieap orthologs was performed using Genetyx ver.

Techniques: Membrane, Transfection, Infection, Construct, Two Tailed Test

Journal: iScience

Article Title: Mieap forms membrane-less organelles involved in cardiolipin metabolism

doi: 10.1016/j.isci.2024.108916

Figure Lengend Snippet: Both N- and C-terminal regions of Mieap are required to generate the multi-phase structure of Mi-BCs (A–D) Comparison of phase-separating behaviors on the CL metabolic enzyme, TAMM41 (EGFP-TAMM41), between BCs formed by TagRFP-T-Mieap WT (A), ΔCC (B), Δ275 (C), and Δ496 (D). Right: line-scan of fluorescence intensities along the dashed arrow. Scale bars, 10 μm. (E–H) CL metabolic enzymes wet the interface in the Mieap-depleted phase of Mi-BCs. Distributions of the CL metabolic enzymes, EGFP-PGS1 (E, F) and EGFP-TAMM41 (G, H), in the Mieap-depleted phase of Mi-BCs are shown. Lower right: line-scan of fluorescence intensities along the dashed arrow. Scale bars, 2 μm. (I–L) CL metabolic enzymes wet the interface in the Mieap Δ496-depleted phase of Δ496-BCs. Distributions of the CL metabolic enzymes, EGFP-PGS1 (I, J) and EGFP-TAMM41 (K, L), in the Mieap Δ496-depleted phase of BCs formed by the Δ496 mutant are shown. Lower right: line-scan as in (E–H). Scale bars, 2 μm. HeLa cells were transfected with pEGFP-TAMM41 (A–D, G, H, K, and L) or pEGFP-PGS1 (E, F, I, and J), and subsequently infected with Ad-TagRFP-T-Mieap WT (A, E–H), Ad-TagRFP-T-Mieap ΔCC (B), Ad-TagRFP-T-Mieap Δ275 (C), or Ad-TagRFP-T-Mieap Δ496 (D, I–J). After BCs were formed, the relationship between EGFP-TAMM41 and each mutant BCs was analyzed (A–D), or the Mieap-depleted phase was analyzed on wetting of EGFP-PGS1 (E, F, I, J) or EGFP-TAMM41 (G, H, K, and L).

Article Snippet: Multiple sequence alignment for Mieap orthologs was performed using Genetyx ver.

Techniques: Comparison, Fluorescence, Mutagenesis, Transfection, Infection

Journal: iScience

Article Title: Mieap forms membrane-less organelles involved in cardiolipin metabolism

doi: 10.1016/j.isci.2024.108916

Figure Lengend Snippet: Mieap contributes to mitochondrial quality control by promoting CL metabolism (A) Oxygen consumption rates (OCR) of LS174T-cont and Mieap-KD cells under normal conditions calculated with a flux analyzer. Data are shown as means ± SD (n = 9). (B) Mitochondrial ATP production rates of LS174T-cont and Mieap-KD cells under normal conditions calculated with a flux analyzer, using a Seahorse XF real-time ATP rate assay. Data are shown as means ± SD (n = 9). (C) Morphology of mitochondria of LS174T-cont and Mieap-KD cells with transmission electron microscopy (TEM). Scale bars, 2 μm. (D) Ratio of crista area per mitochondrial section of LS174T-cont and Mieap-KD cells. Quantitative data were obtained from cont mitochondria (n = 197) and Mieap-KD mitochondria (n = 329) in TEM images and displayed in a violin plot. (E) Quantitative assessment of total CL by mass spectrometric analysis. LS174T cells with (Cont) and without (Mieap-KD) endogenous Mieap expressions were subjected to mass spectrometric analysis. Data shown are means ± SE (n = 6). (F) Quantitative and rate assessments of CL species by mass spectrometric analysis. LS174T cells were analyzed as described in (E). Absolute values of selected CL species are shown as the amount of substance per cell (left panel). Relative values of selected CL species are shown as % of total CL (right panel). Data shown are means ± SE (n = 6). (G) The kinetic profile of the OCR using the Seahorse XF Real-Time ATP rate assay in HCT116 cells infected with Ad-Mieap or Ad-empty. (H–J) Quantitative assessment of OCR (H), mitochondrial ATP production rates (I), and total ATP production rates (J) of the HCT116 cells as in (G). Data are shown as means ± SD (n = 9). (K) Morphology of kidney mitochondria of Mieap +/+ and Mieap −/− mice with TEM. Scale bars, 1 μm (upper panels) and 200 nm (lower panels). (L) Ratios of crista area per mitochondrial section of Mieap +/+ and Mieap −/− mouse kidneys. Quantitative data were obtained from Mieap +/+ kidney mitochondria (n = 190) and Mieap −/− kidney mitochondria (n = 234) in TEM images and displayed in a violin plot. (M) Morphology of liver mitochondria of Mieap +/+ and Mieap −/− mice with TEM. Scale bars, 1 μm (upper panels) and 200 nm (lower panels). (N) Ratios of crista area per mitochondrial section of Mieap +/+ and Mieap −/− mouse livers. Quantitative data were obtained from Mieap +/+ liver mitochondria (n = 146) and Mieap −/− liver mitochondria (n = 134) in TEM images and displayed in a violin plot. (A, B, D, H-J, L, N) ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, two tailed Student’s t test. (E, F) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, two tailed paired t test. When the data were visualized using violin plots, boxplots were overlaid. The center line in the box indicates the median. The bottom and top of the box indicate the 25 th and 75 th percentiles. The whiskers extend 1.5 times the interquartile range (IQR) from the top and bottom of the box unless the minimum and maximum values are within the IQR. The values which fall above or below the whiskers are plotted individually as outliers.

Article Snippet: Multiple sequence alignment for Mieap orthologs was performed using Genetyx ver.

Techniques: Control, Transmission Assay, Electron Microscopy, Infection, Two Tailed Test

Journal: iScience

Article Title: Mieap forms membrane-less organelles involved in cardiolipin metabolism

doi: 10.1016/j.isci.2024.108916

Figure Lengend Snippet: Mieap prevents obesity by maintaining cristae structures of BAT (A–C) Body weights of Mieap +/+ , Mieap +/− , and Mieap −/− mice (7–130 weeks of age) (A), (44–104 weeks of age) (B), and (53–62 weeks of age) (C). Data shown are means ± SE. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, two tailed Student’s t test. (D) Morphology of brown adipose tissue (BAT) mitochondria of Mieap +/+ and Mieap −/− mice with TEM. Scale bars, 1 μm. (E) The ratio of crista area per mitochondrial section of Mieap +/+ and Mieap −/− mice BAT. Quantitative data were obtained from Mieap +/+ BAT mitochondria (n = 181) and Mieap −/− BAT mitochondria (n = 129) in TEM images and displayed in a violin plot. ∗∗∗∗p < 0.0001, two tailed Student’s t test. The center line in the box indicates the median. When the data were visualized using violin plots, boxplots were overlaid. The bottom and top of the box indicate the 25 th and 75 th percentiles. The whiskers extend 1.5 times the interquartile range (IQR) from the top and bottom of the box unless the minimum and maximum values are within the IQR. The values which fall above or below the whiskers are plotted individually as outliers. (F) A hypothetical model for mitochondrial quality control via the Mieap-MLOs-CL metabolism axis.

Article Snippet: Multiple sequence alignment for Mieap orthologs was performed using Genetyx ver.

Techniques: Two Tailed Test, Control

Journal: iScience

Article Title: Mieap forms membrane-less organelles involved in cardiolipin metabolism

doi: 10.1016/j.isci.2024.108916

Figure Lengend Snippet: Hypothetical models for Mieap-mediated sequential enzymatic reactions in CL metabolism (A) Hypothetical model of biosurfactant activity of Mieap. Mieap (green) exists in the Mieap-containing phase (lipid phase) (black), as a “scaffold” protein and/or as a potential “biosurfactant.” At the boundary between the surfaces of Mi-BCs (aqueous phase) and the Mieap-containing phase (lipid phase) or between the Mieap-containing phase (lipid phase) and the Mieap-depeleted phase (aqueous phase), the hydrophilic N-terminal end of Mieap always faces the aqueous phase at the boundary. (B and C) Hypothetical model for Mieap-mediated sequential enzymatic reactions in CL metabolism. Black areas indicate the Mieap-containing phase (lipid phase) containing CL and Mieap. Gray areas indicate the Mieap-depeleted (aqueous phase) containing enzymes. Sequential reactions occur at the interface between the surface of Mi-BCs (aqueous phase) and the Mieap-containing phase (lipid phase) (B) or between the Mieap-containing phase (lipid phase) and the Mieap-containing phase (aqueous phase) (C). Once Mieap (green) stably interacts with PA via its C-terminal region, one of the enzymes transiently and weakly interacts with the N-terminal region of Mieap. When Mieap interacts with TAMM41, PA is converted to CDP-DG. Such reactions between biosynthetic enzymes and corresponding substrates could be repeated until mature CL is produced. Concentration of enzymes and substrates at Mi-BC surfaces, segregation of enzymes and substrates into distinct sub-compartments of Mi-BCs, interfacial catalysis, and biosurfactant activity of Mieap may enable efficient sequential reactions for CL metabolism. PA, phosphatidic acid; CDP-DG: cytidine diphosphate diacylglycerol; PGP, phosphatidylglycerophosphate; PG, phosphatidylglycerol; CLOX, oxidized cardiolipin; CLN, nascent cardiolipin; CLM, mature cardiolipin.

Article Snippet: Multiple sequence alignment for Mieap orthologs was performed using Genetyx ver.

Techniques: Activity Assay, Stable Transfection, Produced, Concentration Assay

Journal: iScience

Article Title: Mieap forms membrane-less organelles involved in cardiolipin metabolism

doi: 10.1016/j.isci.2024.108916

Figure Lengend Snippet:

Article Snippet: Multiple sequence alignment for Mieap orthologs was performed using Genetyx ver.

Techniques: Virus, Recombinant, Plasmid Preparation, SYBR Green Assay, Membrane, Western Blot, PCR Cloning, Mutagenesis, Generated, Software, Cider Assay