Hypoxia is a hallmark of solid tumors that promotes cell growth, survival, and metastasis and confers resistance to chemo and radiotherapies. Hypoxic responses are largely mediated by the transcription factors hypoxia-inducible factor 1α (HIF-1α) and HIF-2α. Our work demonstrates that HIF-2α is essential for colorectal cancer (CRC) progression. However, targeting hypoxic cells is difficult, and tumors rapidly acquire resistance to inhibitors of HIF-2α. To overcome this limitation, we performed a small molecule screen to identify HIF-2α–dependent vulnerabilities. Several known ferroptosis activators and dimethyl fumarate (DMF), a cell-permeable mitochondrial metabolite derivative, led to selective synthetic lethality in HIF-2α–expressing tumor enteroids. Our work demonstrated that HIF-2α integrated 2 independent forms of cell death via regulation of cellular iron and oxidation. First, activation of HIF-2α upregulated lipid and iron regulatory genes in CRC cells and colon tumors in mice and led to a ferroptosis-susceptible cell state. Second, via an iron-dependent, lipid peroxidation–independent pathway, HIF-2α activation potentiated ROS via irreversible cysteine oxidation and enhanced cell death. Inhibition or knockdown of HIF-2α decreased ROS and resistance to oxidative cell death in vitro and in vivo. Our results demonstrated a mechanistic vulnerability in cancer cells that were dependent on HIF-2α that can be leveraged for CRC treatment.

Overproduction of reactive oxygen species and compromised antioxidant defenses perturb intracellular redox homeostasis and is associated with a myriad of human diseases as well as with the natural process of aging. Hydrogen sulfide (H₂S), which is biosynthesized by organisms ranging from bacteria to man, influences a broad range of physiological functions. A highly touted molecular mechanism by which H₂S exerts its cellular effects is via post-translational modification of the thiol redox proteome, converting cysteine thiols to persulfides, in a process referred to as protein persulfidation. The physiological relevance of this modification in the context of specific signal transmission pathways remains to be rigorously established, while a general protective role for protein persulfidation against hyper-oxidation of the cysteine proteome is better supported. A second mechanism by which H₂S modulates redox homeostasis is via remodeling the redox metabolome, targeting the electron transfer chain and perturbing the major redox nodes i.e. CoQ/CoQH₂, NAD⁺/NADH and FAD/FADH₂. The metabolic changes that result from H₂S-induced redox changes fan out from the mitochondrion to other compartments. In this review, we discuss recent developments in elucidating the roles of H₂S and its oxidation products on redox homeostasis and its role in protecting the thiol proteome.

Parkinson's disease (PD) is a multifactorial malady and the second most common neurodegenerative disorder, characterized by loss of dopaminergic neurons in the midbrain. A hallmark of PD pathology is the formation of intracellular protein inclusions, termed Lewy bodies (LBs). Recent MS studies have shown that OTU deubiquitinase ubiquitin aldehyde-binding 1 (OTUB1), a deubiquitinating enzyme of the OTU family, is enriched together with α-synuclein in LBs from individuals with PD and is also present in amyloid plaques associated with Alzheimer's disease. In the present study, using mammalian cell cultures and a PD mouse model, along with CD spectroscopy, atomic force microscopy, immunofluorescence-based imaging, and various biochemical assays, we demonstrate that after heat-induced protein aggregation, OTUB1 reacts strongly with both anti-A11 and anti-osteocalcin antibodies, detecting oligomeric, prefibrillar structures or fibrillar species of amyloidogenic proteins, respectively. Further, recombinant OTUB1 exhibited high thioflavin-T and Congo red binding and increased β-sheet formation upon heat induction. The oligomeric OTUB1 aggregates were highly cytotoxic, characteristic of many amyloid proteins. OTUB1 formed inclusions in neuronal cells and co-localized with thioflavin S and with α-synuclein during rotenone-induced stress. It also co-localized with the disease-associated variant pS129-α-synuclein in rotenone-exposed mouse brains. Interestingly, OTUB1 aggregates were also associated with severe cytoskeleton damage, rapid internalization inside the neuronal cells, and mitochondrial damage, all of which contribute to neurotoxicity. In conclusion, the results of our study indicate that OTUB1 may contribute to LB pathology through its amyloidogenic properties.

Ubiquinol or coenzyme Q (CoQ) is a lipid-soluble electron carrier in the respiratory chain and an electron acceptor for various enzymes in metabolic pathways that intersect at this cofactor hub in the mitochondrial inner membrane. The reduced form of CoQ is an antioxidant, which protects against lipid peroxidation. In this study, we have optimized a UV-detected HPLC method for CoQ analysis from biological materials, which involves a rapid single-step extraction into n-propanol followed by direct sample injection onto a column. Using this method, we have measured the oxidized, reduced, and total CoQ pools and monitored shifts in the CoQ redox status in response to cell culture conditions and bioenergetic perturbations. We find that hypoxia or sulfide exposure induces a reductive shift in the intracellular CoQ pool. The effect of hypoxia is, however, rapidly reversed by exposure to ambient air. Interventions at different loci in the electron transport chain can induce sizeable redox shifts in the oxidative or reductive direction, depending on whether they are up- or downstream of complex III. We have also used this method to confirm that CoQ levels are higher and more reduced in murine heart versus brain. In summary, the availability of a convenient HPLC-based method described herein will facilitate studies on CoQ redox dynamics in response to environmental, nutritional, and endogenous alterations.

The host–microbiome interface is rich in metabolite exchanges and exquisitely sensitive to diet. Hydrogen sulfide (H ₂ S) is present at high concentrations at this interface and is a product of both microbial and host metabolism. The mitochondrial enzyme, sulfide quinone oxidoreductase (SQOR), couples H ₂ S detoxification to oxidative phosphorylation; its inherited deficiency presents as Leigh disease. Since an estimated two-thirds of systemic H ₂ S metabolism originates in the gut, it raises questions as to whether impaired sulfide clearance in this compartment contributes to disease and whether it can be modulated by dietary sulfur content. In this study, we report that SQOR deficiency confined to murine intestinal epithelial cells perturbs colon bioenergetics that is reversed by antibiotics, revealing a significant local contribution of microbial H ₂ S to host physiology. We also find that a 2.5-fold higher methionine intake, mimicking the difference between animal and plant proteins, synergizes with intestinal SQOR deficiency to adversely impact colon architecture and alter microbiome composition. In serum, increased thiosulfate, a biomarker of H ₂ S oxidation, reveals that intestinal SQOR deficiency combined with higher dietary methionine affects sulfide metabolism globally and perturbs energy metabolism as indicated by higher ketone bodies. The mice exhibit lower exploratory locomotor activity while brain MRI reveals an atypical reduction in ventricular volume, which is associated with lower aquaporin 1 that is important for cerebrospinal fluid secretion. Our study reveals the dynamic interaction between dietary sulfur intake and sulfide metabolism at the host–microbe interface, impacting gut health, and the potential for lower dietary methionine intake to modulate pathology.

Oxygen (O₂) sensing by hypoxia-inducible factor (HIF) is a principal mechanism by which aerobic organisms adjust cellular energy metabolism and adapt to O₂ limitation. In this study, we show that hydrogen sulfide (H₂S), a product of host and microbial metabolism, profoundly influences the threshold for HIF-dependent hypoxia sensing by increasing intracellular O₂. The dose-dependent destabilization of HIF by H₂S is inversely correlated with sulfide quinone oxidoreductase, which oxidizes sulfide in the mitochondrion. Hypoxia sensors provide a semiquantitative estimate of the magnitude of H₂S-induced perturbation. Thus, the O₂ concentration in cells grown in a 2% O₂ atmosphere is sensed as 5% or 15% O₂ in the presence of 25 or 100 ppm H₂S, respectively. Sustained exposure to H₂S elicits the hallmarks of hyperoxia-associated cytotoxicity, including loss of iron-sulfur proteins in cellular and murine models. H₂S thus emerges as a powerful regulator of O₂ sensing and signaling with possible implications for dysregulation in O₂ toxicity diseases.