Figure 3: BOLA3 controls Fe-S integrity and mitochondrial complex activity as well as mitochondrial lipoate-containing 2-oxoacid dehydrogenases.

(A) Knockdown of BOLA3 to PAECs compromised Fe-S integrity, as assessed by lentiviral delivery of Fe-S-dependent (glutaredoxin 2, GRX2) vs. Fe-S-independent (GCN4) fluorescent sensors. (B) By immunoblot and densitometry, expression of NDUFV2 (doublet band) and SDHB, representative mitochondrial Complex I and II components respectively, was decreased by BOLA3 knockdown in normoxia (Nx) and hypoxia (Hx); however, hypoxia alone did not robustly alter their expression. (C) Conversely, forced lentiviral expression of BOLA3 compared with GFP control increased NDUFV2 and SDHB expression in normoxia and hypoxia. (D-E) In PAECs, hypoxia-dependent Fe-S-dependent Complex I activity was down-regulated by BOLA3 knockdown (D) and conversely rescued by forced BOLA3 expression (E). (F) PAEC expression of lipoate-containing pyruvate dehydrogenase (PDH) was decreased by BOLA3 knockdown in normoxia and was phenocopied by hypoxic exposure. (G) In correlation, PDH activity was decreased by BOLA3 knockdown in normoxia and by hypoxic exposure; BOLA3 knockdown in combination with hypoxia further decreased PDH activity. (H) In the setting of increased lactate dehydrogenase (LDHA) and pyruvate dehydrogenase kinase 1 (PDK1) expression in hypoxia, by RT-qPCR, BOLA3 knockdown in PAECs increased LDHA and PDK1 in normoxia and hypoxia. (I-J) Conversely, BOLA3 overexpression increased lipoate-containing PDH (I) and increased PDH activity in normoxia and hypoxia (J). (K) BOLA3 overexpression blunted the upregulation of LDHA and PDK1 specifically in hypoxia. In all panels, mean expression of control groups (si-NC, Nx+si-NC, Nx+LV-GFP) was normalized to fold change of 1, to which all relevant samples were compared (N = 3/group). Data represent the mean ± SEM (*P < 0.05, **P < 0.01).