negative control stable cell line  (Genechem)

Journal: bioRxiv

Article Title: BOLA3 and NFU1 link mitoribosome iron-sulfur cluster assembly to multiple mitochondrial dysfunctions syndrome

doi: 10.1101/2023.05.27.542581

Figure Lengend Snippet: ( A ) Mitoribosome cryo-EM reconstitution (PDB: 7qi4) in which the pairs of proteins that coordinate a [2Fe-2S] cluster are indicated. The right-side panel shows the mS25-bS16m pair magnified, and the residues involved in [2Fe-2S] cluster coordination are indicated. mS25 residues are shown in orange, and bS16m are shown in yellow. The [2Fe-2S] cluster is depicted as 4 blue spheres. Figures were prepared in Pymol and Adobe Photoshop. ( B, F ) Steady-state levels of mS25, bS16m, additional mitoribosome markers (mS40 and uL10m), and a marker of mitochondrial protein synthesis (complex IV subunit COX1) were assessed by immunoblotting in HEK293T wild-type cells, and mS25 -KO ( B ) or bS16m -KO cells ( F ) reconstituted with an empty vector (EV), or plasmids expressing the corresponding wild-type protein (WT), or variants carrying mutations in the indicated [2Fe-2S] cluster coordinating cysteines. ACTIN was used as a loading control. ( C, H ) Mitochondrial protein synthesis following 35 S-methionine incorporation into newly synthesized mitochondrial proteins in the presence of emetine to inhibit cytoplasmic protein synthesis. ACTIN was used as a loading control. ( D, J ) Sucrose gradient sedimentation analyses of mtSSU (bS16m, mS25, and mS27) and mtLSU markers (uL10m) in mitochondria purified from the indicated cell lines and extracted in the presence of 100 mM KCl, 20 mM MgCl 2 , and 0.5% digitonin. ( E, K ) KCN-sensitive endogenous cell respiration for indicated cell lines measured polarographically. The bar graphs represent the average ± SD of three independent experiments. Black dots represent individual data points. Two-tailed unpaired t -test, ** p < 0.01; *** p < 0.001; n.s., not significant. ( G, I ) The graphs show the densitometry (average ± SD) of three independent experiments as in panel F ( G ) or H ( I ). Two-tailed unpaired t -test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. In panel I, PS indicates protein synthesis.

Article Snippet: To create stable human bS16m -KO HEK293T cell lines, two gRNA vectors and one linear donor were obtained from OriGene Technologies (KN405837).

Techniques: Cryo-EM Sample Prep, Marker, Western Blot, Plasmid Preparation, Expressing, Synthesized, Sedimentation, Purification, Two Tailed Test

Journal: bioRxiv

Article Title: BOLA3 and NFU1 link mitoribosome iron-sulfur cluster assembly to multiple mitochondrial dysfunctions syndrome

doi: 10.1101/2023.05.27.542581

Figure Lengend Snippet: ( A ) Mitochondrial protein synthesis following 35 S-methionine incorporation into newly synthesized mitochondrial proteins in the presence of emetine to inhibit cytoplasmic protein synthesis. Wild-type HEK293T cells were treated with 0, 0.25, or 0.50 mM H 2 O 2 for 3 h. ACTIN served as a loading control. The right-side graphs represent the quantification of the signal in a digitalized image using the Adobe Acrobat Histogram tool. The data for COX1, ND2, and ATP6, normalized by ACTIN and expressed as the fraction of untreated cells, are presented. ( B ) Reverse thiol trapping approach to detect the native cysteine residues inaccessible to a cell-permeable alkylating compound (2-iodoacetamide, IAA) that upon denaturation with SDS (sodium dodecyl sulfate) and full reduction with TCEP (tris(2-carboxyethyl)phosphine) are bound to AMS (4-acetamido-4’-maleimidylstilbene-2,2’-disulfonic acid), which adds ∼540 Da per thiol group. ( C and F) Cryo-EM structures of the mS25-bL16m pair of proteins that coordinate a [2Fe-2S] cluster in the mtSSU (PDB: 7qi4) ( C ), and the mtSSU assembly factor METTL17 that harbors a [4Fe-4S] cluster (PDB: 8csp) ( F ) The [Fe-S] cluster-coordinating cysteine residues are indicated in red, and all additional cysteines in each protein are depicted in black. The Fe-S clusters are shown (not to scale) as blue spheres. Figures were prepared in Pymol and Adobe Photoshop. ( D and G) Reverse thiol trapping of mS25 and bS16m ( D ) or METTL17 (G) in mitochondria isolated from WT HEK293T cells, treated or not with 1 mM H 2 O 2 for 3 h. ( E and H) The bar graphs represent the number of AMS-bound cysteines (unavailable to IAA binding in organello ). The data are the average of two independent experiments. Figures are representative of two independent repetitions with similar results.

Article Snippet: To create stable human bS16m -KO HEK293T cell lines, two gRNA vectors and one linear donor were obtained from OriGene Technologies (KN405837).

Techniques: Synthesized, Cryo-EM Sample Prep, Isolation, Binding Assay

Journal: bioRxiv

Article Title: BOLA3 and NFU1 link mitoribosome iron-sulfur cluster assembly to multiple mitochondrial dysfunctions syndrome

doi: 10.1101/2023.05.27.542581

Figure Lengend Snippet: ( A ) Volcano plots displaying differentially expressed proteins between HEK293T cells treated with siNT (non-targeting) and si GLRX5 , si BOLA3 , si ISCA1, or si NFU1 for 9 days. The y -axis displays the log10 (P values), and the x -axis displays the Log2 (fold-change value). The horizontal grey line represents the 10 -1.3 threshold on the p values, while the vertical lines show thresholds of ± 1.2-fold changes. The raw Multiplex TMT-MS/MS data have been uploaded to PRIDE repository, with ref# 1-20230106-165123. ( B ) Heat map showing the levels of mtSSU and mtLSU proteins in si GLRX5 , si BOLA3 , si ISCA1, and si NFU1 cells, normalized by siNT-treated HEK293T cells after 9 days of silencing. The map represents the average of four independent experiments. ( C ) The schematic depicts the approach followed to assess 55 Fe incorporation into mitoribosomes. The bar graph represents the average ± SD of three independent experiments. Grey dots represent individual data points. Two-tailed unpaired t -test, * p < 0.05; **** p < 0.0001 ( D ) Mitochondrial protein synthesis following 35 S-methionine incorporation into newly synthesized mitochondrial proteins in the presence of emetine to inhibit cytoplasmic protein synthesis. ACTIN was used as a loading control. The bar graph represents the average of two independent experiments. Black dots represent individual data points. ( E ) Schematic showing the delivery of Fe-S clusters in mammalian cells, highlighting the GLRX5-BOLA3 pathway involved in [2Fe-2S] cluster delivery to the mitoribosome, and the ISCA1-NFU1 node that targets a [4Fe-4S] cluster to the mtSSU assembly factor METTL17.

Article Snippet: To create stable human bS16m -KO HEK293T cell lines, two gRNA vectors and one linear donor were obtained from OriGene Technologies (KN405837).

Techniques: Multiplex Assay, Tandem Mass Spectroscopy, Two Tailed Test, Synthesized