Small cell lung cancer (SCLC) remains a highly lethal malignancy with limited therapeutic advances. Although high-dose vitamin C (Vc) exhibits anti-tumor activity in various cancers, its therapeutic potential and underlying resistance mechanisms in SCLC remain poorly understood. Here, we identified heterogeneous responses to Vc among SCLC cell lines and found that Vc-resistant cells acquire enhanced antioxidant capacity to tolerate Vc-induced oxidative stress. Through transcriptomic, multi-omics, and functional analyses, we identified glucose-6-phosphate dehydrogenase (G6PD) as a critical determinant of Vc resistance. Mechanistically, Vc activates the p-AKT/p-GSK3β/NRF2 signaling axis in resistant cells, leading to NRF2-dependent transcriptional upregulation of G6PD and activation of antioxidant adaptation programs. Integrated analyses of transcriptomic, proteomic, single-cell, and spatial transcriptomic datasets further supported the involvement of the NRF2-G6PD axis in oxidative stress adaptation in SCLC. Importantly, pharmacological inhibition of G6PD with RRx-001 restored Vc sensitivity and synergistically suppressed SCLC growth in vitro and in vivo. Collectively, our findings reveal a previously unrecognized mechanism underlying Vc resistance in SCLC and demonstrate that co-targeting G6PD provides a potential strategy to enhance the therapeutic efficacy of Vc.
Protein chimeras enable new biological functions by combining otherwise independent protein elements, but identifying insertion sites that preserve host protein function remains largely empirical. Here, we introduce CRISPR-guided protospacer adjacent motif (PAM) scanning, a scalable strategy for mapping chimera-permissive sites directly in living cells. PAM scanning uses CRISPR-addressable positions across coding sequences to systematically install functional payloads and measure insertion tolerance. In yeast, PAM accessibility provided near-comprehensive coverage across proteins of diverse size and function. We first applied this approach across an entire G protein-coupled receptor (GPCR) signaling pathway, generating 91 chimeras containing inserted protease recognition sequences to map insertion tolerance and site-specific biophysical protection from a protease probe by surrounding protein structure and binding partners. Cognate protease cleavage revealed how this protection varied with protease inhibition and size. We then used PAM scanning to identify bifunctional GPCR chimeras that retained receptor signaling while supporting fluorescent or luminescent reporters. Screening human adenosine A2A and melatonin MTNR1A receptors in yeast identified functional mNeonGreen and NanoLuc insertion sites that retained ligand-dependent signaling following translation to human cells. Together, these studies establish PAM scanning as a general framework for mapping insertional tolerance and biophysical protection in living cells, accelerating functional chimera engineering, and experimentally validating generative chimera design.
Mitochondria act as dynamic signaling hubs intertwining cellular metabolism and environmental perception in fungi. Here, we elucidate the assembly of the red-light sensing phytochrome (FphA) at the mitochondrial outer membrane in Alternaria alternata. Phytochrome photoconversion strictly relies on biliverdin biosynthesis by heme oxygenases. While mammalian and plant heme oxygenases localize to the endoplasmic reticulum or chloroplasts, the fungal system uniquely repurposes the outer mitochondrial membrane. We demonstrate that the fungal heme oxygenases HoxA and HoxB form a heteromeric complex integrated into the membrane via mitochondrial import machinery components Mim1 and Mim2. At the mitochondrial surface, only HoxA binds directly to FphA. Genomic screening identified Bifunctional Cytochrome P450 Reductase/Ferredoxin A (BcfA) as the essential physiological electron donor. Quantitative kinetics established that BcfA operates as an asymmetric hub, displaying higher affinity for HoxB than HoxA. HoxB functions as a high-affinity kinetic tether locking the donor at the membrane boundary. Consequently, CRISPR/Cas9-mediated deletion of bcfA disrupts red-light signalling. Thus, a multi-protein mitochondrial complex evolved in fungi to wire light perception through spatial compartmentalization.
Liver endothelial cell (LEC)-derived bone morphogenetic proteins (BMP)2 and BMP6 coordinately regulate hepatocyte hepcidin production to control systemic iron homeostasis, but the mechanisms are uncertain. BMPs are homodimeric or heterodimeric proteins that are generated as precursors comprised of a non-signaling prodomain and an active ligand released by proteolytic cleavage. Although prodomains do not signal, they play pleiotropic roles in ligand generation and activity. Here, we used LEC- and hepatocyte-derived cell cultures to explore how BMP2 and BMP6 prodomains and proteolytic cleavage impact homodimer formation, heterodimer formation, and hepcidin regulation. We show that when co-expressed in LECs, BMP2 and BMP6 form heterodimers, which more potently induce hepcidin versus homodimers. Proteolytic cleavage of BMP2 precursors at two furin consensus sites is required to generate fully active BMP2/6 heterodimers, although cleavage at either site generates partially active heterodimers. Uncleavable BMP6 precursors fail to generate fully active BMP2/6 heterodimers and dominant negatively inhibit BMP2 homodimer formation. We also show that in LECs, cognate BMP2 and BMP6 prodomains are essential for homodimer and heterodimer formation. Finally, both prodomains remain noncovalently associated with BMP2/6 heterodimers after cleavage and secretion. BMP6 prodomains also remain associated with BMP6 homodimers, whereas the association of BMP2 prodomains with BMP2 homodimers is influenced by the presence of the linker region between the cleavage sites. Our findings suggest that BMP2/6 heterodimers are likely the primary endogenous ligand that regulates hepcidin and iron homeostasis, and both BMP2 and BMP6 prodomains are critical to functional BMP2/6 formation in LECs.
Pyk2 and Src are non-receptor tyrosine kinases that assemble into a complex to promote mutual activation. The Pyk2 FERM domain mediates autoinhibition by direct interaction with the kinase. Src autoinhibition is maintained by intramolecular interactions of the SH3 and SH2 domains with internal ligands, including an inhibitory C-terminal phosphotyrosine. FERM disengagement permits Pyk2 autophosphorylation of the FERM-kinase linker to generate a scaffolding site for Src recruitment. Although Src-mediated phosphorylation of the Pyk2 activation loop is well-established, the mechanism by which complex formation activates Src remains unclear. We reconstituted defined phosphorylation and regulatory states of both kinases, combining site-directed mutagenesis with phosphosite-resolved activity profiling to dissect the reciprocal regulation. Src phosphorylates both Pyk2 activation-loop tyrosines by a random, distributive mechanism with negative cooperativity between the sites. Although activated Pyk2 productively phosphorylates the Src activation loop, Pyk2-Src complex formation does not significantly increase activation loop phosphorylation above the rate of Src autophosphorylation alone. Conversely, autoinhibited Src strictly requires Pyk2 scaffolding engagement to productively phosphorylate Pyk2. Pyk2 therefore relieves Src autoinhibition by presenting competing scaffolding sites to disengage intramolecular SH3 and SH2 conformational constraints. The results distinguish activation loop phosphorylation from the conformational competence required for substrate phosphorylation. We propose that autophosphorylated Pyk2 functions principally as a conformational activator and scaffolding platform, opening Src for phosphatase-mediated removal of the inhibitory C-terminal phosphosite and committing both Pyk2 and Src to downstream substrate phosphorylation.
