1. TLR-activated repression of Fe-S cluster biogenesis drives a metabolic shift and alters histone and tubulin acetylation
by Wing-Hang Tong, Nunziata Maio, De-Liang Zhang, Erika M. Palmieri, Hayden Ollivierre, Manik C. Ghosh, Daniel W. McVicar, and Tracey A. Rouault
BloodAdv
Volume 2(10):
May 22, 2018
©2018 by American Society of Hematology

2. Wing-Hang Tong et al. Blood Adv 2018;2:1146-1156
©2018 by American Society of Hematology

3. Reduced expression of Fe-S cluster biogenesis factors impairs the regeneration/repair of Fe-S proteins involved in mitochondrial oxidative metabolism.
Reduced expression of Fe-S cluster biogenesis factors impairs the regeneration/repair of Fe-S proteins involved in mitochondrial oxidative metabolism. (A) Levels of lipoyl-PDH E2, SDHB, NDUFS1, and FECH in A549 cells decreased in response to silencing of NFS1 and ISCU. (B) Levels of SDHB and NDUFS1 decreased in CRISPR/Cas9 HSC20-knockout HEK293 cell mitochondrial lysates (crRNA HSC20). A scramble CRISPR RNA was used as control (crRNA NT CTRL). (C) Levels of lipoyl-PDH E2, lipoyl-αKGDH E2, SDHB, and NDUFS1 decreased in response to silencing of FXN. (D) Exposure of RAW264.7 cells to LPS and IFN-γ led to a decrease in RCI and RCII activity. (E) Activation of RAW264.7 cells with LPS or LPS/IFN-γ led to decreased mRNA levels of the Fe-S cluster biogenesis factors Nfs1, Iscu, Hscb, Fxn, and Lyrm4 (Isd11). (F) Protein levels of NFS1, ISCU, HSC20, ISD11, SDHB, and FECH and lipoylation of PDH E2 and αKGDH E2 decreased in RAW264.7 cells treated with LPS and IFN-γ. (G) Transcript levels of Iscu, Hsc20, Nfs1, Glrx5, and Lyrm4 decreased in peritoneal macrophages stimulated ex vivo with LPS and IFN-γ. (H) Protein levels of ISCU, NFS1, and HSC20 decreased in peritoneal macrophages stimulated ex vivo with LPS and IFN-γ. Actin or VDAC1 were used as loading controls. All data presented are representative of ≥3 independent experiments. NT, nontargeting.
Wing-Hang Tong et al. Blood Adv 2018;2:
©2018 by American Society of Hematology

4. Repression of Fe-S cluster biogenesis factors amplifies NO-induced damage of Fe-S proteins.
Repression of Fe-S cluster biogenesis factors amplifies NO-induced damage of Fe-S proteins. Exposure of RAW264.7 cells to LPS and IFN-γ led to rapid activations of the Akt, mTOR, HIF-1α, and NF-κB signaling pathways (A) and lactate production (B), followed by an induction of iNOS (A) and a concomitant decrease in the levels of NFS1, ISCU, SDHB, and FECH, aconitase activity, and lipoyl-PDH E2 (C). (D) HeLa cells transfected with NT siRNA or NFS1 siRNA were treated with 400 μM DEA-NO for 1 hour and then washed and incubated in fresh medium for up to 6 hours. In-gel assays indicated that aconitase activity recovered poorly in NFS1-depleted cells compared with control cells. (E) RCII activity and levels of SDHB, FECH, lipoyl-PDH, and lipoyl-KGDH were largely rescued by cotransfection of RAW264.7 cells with pCMV-NFS1-F/M, pCMV-ISCU-F/M, and pCMV-HSC20. In contrast, substitution of ISCU with a construct that expressed an inactive ISCU mutant (ISCU C138S) failed to restore RCII activity and the levels of SDHB, FECH, lipoyl-PDH, and lipoyl-KGDH. **P < .004.
Wing-Hang Tong et al. Blood Adv 2018;2:
©2018 by American Society of Hematology

5. Oxidative and nitrative stress and NF-κB signaling contribute to repression of Fe-S cluster biogenesis, whereas inhibition of TLR-driven glycolysis reduces the repression of NFS1 and ISCU. (A-B) ISCU levels decreased when RAW264.7 cells were exposed to 95% oxygen (A) or menadione (B).
Oxidative and nitrative stress and NF-κB signaling contribute to repression of Fe-S cluster biogenesis, whereas inhibition of TLR-driven glycolysis reduces the repression of NFS1 and ISCU. (A-B) ISCU levels decreased when RAW264.7 cells were exposed to 95% oxygen (A) or menadione (B). (C) ISCU levels decreased when RAW264.7 cells were exposed to the NO donor (NOC-18). (D) Low glucose concentrations in the media mitigated the LPS/IFN-γ– or poly(I:C)/IFN-γ–induced suppression of NFS1 and ISCU and partially rescued lipoyl-PDH E2 and FECH levels and aconitase activity. (E) Repression of NFS1 was mitigated by pretreating RAW264.7 cells with MLN120B, an inhibitor of NF-κB signaling, for 1 hour before the addition of LPS/IFN-γ. (F) Levels of NFS1, SDHB, and NDUFS1 decreased in HeLa cells in response to TNF and IFN-γ.
Wing-Hang Tong et al. Blood Adv 2018;2:
©2018 by American Society of Hematology

6. Restriction of Fe-S cluster biogenesis impairs PDHc and ELP3 function, resulting in decreased overall histone acetylation.
Restriction of Fe-S cluster biogenesis impairs PDHc and ELP3 function, resulting in decreased overall histone acetylation. (A-B) Knockdown of NFS1 (A) or PDH E2 (B) resulted in decreased histone acetylation (ac-histones) in HeLa cells. (C-D) Knockdown of NFS1 (C) or ISCU (D) reduced levels of the acetyltransferase ELP3. (E) Knockdown of FXN decreased lipoylation of PDH E2 and αKGDH E2, decreased ELP3 levels, and decreased acetylation of histones. (F) Histone acetylation decreased in RAW264.7 cells activated with LPS/IFN-γ. (G) Acetate supplementation mitigated the LPS/IFN-γ–induced decrease in histone acetylation. (H) Treatment of HeLa cells with the iron chelator deferoxamine (Dfo) decreased levels of FXN, ISCU, NFS1, ISD11, lipoyl-PDH E2, ELP3, and SDHB, decreased overall histone acetylation, and increased H3K9me3 modifications.
Wing-Hang Tong et al. Blood Adv 2018;2:
©2018 by American Society of Hematology

7. Restriction of Fe-S cluster biogenesis increases expression of the acetyltransferase MEC17 and induces hyperacetylation of tubulin.
Restriction of Fe-S cluster biogenesis increases expression of the acetyltransferase MEC17 and induces hyperacetylation of tubulin. (A) LPS/IFN-γ activation of RAW264.7 cells increased acetylated α-tubulin (ac-tubulin) levels. (B-C) Levels of the cytosolic acetyltransferase MEC17 and tubulin acetylation increased in response to knockdown of FXN (B) or ISCU (C). (D) Schematic illustrating how restriction of the Fe-S cluster biogenesis machinery may increase (blue) or decrease (red) key metabolites and proteins important in metabolic and epigenetic remodeling.
Wing-Hang Tong et al. Blood Adv 2018;2:
©2018 by American Society of Hematology