Methanobactin (Mbn) is a ribosomally synthesized and post-translationally modified peptide (RiPP) natural product that binds Cu(I) with high affinity. The copper-chelating thioamide/oxazolone groups in Mbn are installed on the precursor peptide MbnA by the core enzyme complex, MbnBC, which includes the multinuclear non-heme iron-dependent oxidase (MNIO) MbnB and its RiPP recognition element-containing partner protein MbnC. For the extensively characterized Mbn biosynthetic gene cluster (BGC) from the methanotroph Methylosinus trichosporium OB3b, the tailoring aminotransferase MbnN further modifies MbnA after leader sequence cleavage by an unknown mechanism. Here we detail methods to express and purify M. trichosporium OB3b MbnBC and MbnN along with protocols for assessing MbnA modification by MbnBC and MbnN aminotransferase activity. In addition, we describe crystallization and structure determination of MbnBC. These procedures can be adapted for other MNIOs and partner proteins encoded in Mbn and Mbn-like BGCs. Furthermore, these methods provide a first step toward in vitro biosynthesis of Mbns and related natural products as potential therapeutics.

UNASSIGNED: This research aims to establish a copper homeostasis-related gene signature for predicting the prognosis of epithelial ovarian cancer and to investigate its underlying mechanisms.
UNASSIGNED: We mainly constructed the copper homeostasis-related gene signature by LASSO regression analysis. Then multiple methods were used to evaluate the independent predictive ability of the model and explored the mechanisms.
UNASSIGNED: The 15-copper homeostasis-related gene (15-CHRG) signature was successfully established. Utilizing an optimal cut-off value of 0.35, we divided the training dataset into high-risk and low-risk subgroups. Kaplan-Meier analysis revealed that survival times for the high-risk subgroup were significantly shorter than those in the low-risk group (P < .05). Additionally, the Area Under the Curve (AUC) of the 15-CHRG signature achieved 0.822 at 1 year, 0.762 at 3 years, and 0.696 at 5 years in the training set. COX regression analysis confirmed the 15-CHRG signature as both accurate and independent. Gene set enrichment (GSEA), Kyoto Encyclopedia of Gene and Genome (KEGG) and Gene Ontology (GO) analysis showed that there were significant differences in apoptosis, p53 pathway, protein synthesis, hydrolase and transport-related pathways between high-risk group and low-risk group. In tumor immune cell (TIC) analysis, the increased expression of resting mast cells was positively correlated with the risk score.
UNASSIGNED: Consequently, the 15-CHRG signature shows significant potential as a method for accurately predicting clinical outcomes and treatment responses in patients with epithelial ovarian cancer.

BACKGROUND: Obesity and the forkhead box O1(FOXO1) affect the survival of breast cancer patients, but the underlying mechanism remains unclear. We aimed to investigate the role of FOXO1 in obesity-associated-breast cancer.
METHODS: We screened 383 breast disease patients from the first affiliated hospital with Nanjing Medical University in 2020. We performed wound healing, transwell, matrigel assays to assess the metastatic ability of cancer cells. We adopted mRNAs sequencing to select the differentially expressed transcripts in breast cancer. We applied immunohistochemistry, western blot, tissue microarrays to assess the level of FOXO1 and epithelial-mesenchymal transition (EMT) pathways. We conducted bioinformatic analysis to investigate interactions between FOXO1 and miR-135b. We used fluorescence in situ hybridization, RT-qPCR to confirm the characteristics of circCNIH4. We conducted luciferase reporter assay, rescue experiments to investigate interactions between circCNIH4 and miR-135b.
RESULTS: Obesity was positively correlated with the incidence and progression of breast cancer. Adipocytes enhanced the migration of breast cancer and attenuated the effects of FOXO1. MiR-135b was a binding gene of FOXO1 and was regulated by circCNIH4. CircCNIH4 exhibited antitumor activity in vitro and in vivo.
CONCLUSIONS: Adipocytes might accelerate the progression of breast cancer by modulating FOXO1/miR-135b/ circCNIH4 /EMT axis and regulating copper homeostasis.

Cuproptosis, a newly characterized form of regulated cell death driven by copper accumulation, has emerged as a significant mechanism underlying various non-cancerous diseases. This review delves into the complex interplay between copper metabolism and the pathogenesis of conditions such as Wilson\'s disease (WD), neurodegenerative disorders, and cardiovascular pathologies. We examine the molecular mechanisms by which copper dysregulation induces cuproptosis, highlighting the pivotal roles of key copper transporters and enzymes. Additionally, we evaluate the therapeutic potential of copper chelation strategies, which have shown promise in experimental models by mitigating copper-induced cellular damage and restoring physiological homeostasis. Through a comprehensive synthesis of recent advancements and current knowledge, this review underscores the necessity of further research to translate these findings into clinical applications. The ultimate goal is to harness the therapeutic potential of targeting cuproptosis, thereby improving disease management and patient outcomes in non-cancerous conditions associated with copper dysregulation.

Copper homeostasis is critical to the functioning of the brain, and its breakdown is linked with many brain diseases. Copper is also known to interact with the negatively charged lipid, phosphatidylserine (PS), as well as α-synuclein, an aggregation-prone protein enriched in the synapse, which plays a role in synaptic vesicle docking and fusion. However, the interplay between copper, PS lipid, and α-synuclein is not known. Herein, we report a detailed and predominantly kinetic study of the interactions among these three components pertinent to copper homeostasis and neurotransmission. We found that synaptic vesicle-mimicking small unilamellar vesicles (SUVs) can sequester any excess free Cu2+ within milliseconds, and bound Cu2+ on SUVs can be reduced to Cu+ by GSH at a nearly constant rate under physiological conditions. Moreover, we revealed that SUV-bound Cu2+ does not affect the binding between wild-type α-synuclein and SUVs but affect that between N-terminal acetylated α-synuclein and SUVs. In contrast, Cu2+ can effectively displace both types of α-synuclein from the vesicles. Our results suggest that synaptic vesicles may mediate copper transfer in the brain, while copper could participate in synaptic vesicle docking to the plasma membrane via its regulation of the interaction between α-synuclein and synaptic vesicle.

The copper efflux regulator (CueR) is a classical member of the MerR family of metalloregulators and is common in gram-negative bacteria. Through its C-terminal effector-binding domain, CueR senses cytoplasmic copper ions to regulate the transcription of genes contributing to copper homeostasis, an essential process for survival of all cells. In this chapter, we review the regulatory roles of CueR in the model organism Escherichia coli and the mechanisms for CueR in copper binding, DNA recognition, and interplay with RNA polymerase in regulating transcription. In light of biochemical and structural analyses, we provide molecular details for how CueR represses transcription in the absence of copper ions, how copper ions mediate CueR conformational change to form holo CueR, and how CueR bends and twists promoter DNA to activate transcription. We also characterize the functional domains and key residues involved in these processes. Since CueR is a representative member of the MerR family, elucidating its regulatory mechanisms could help to understand the CueR-like regulators in other organisms and facilitate the understanding of other metalloregulators in the same family.

Copper (Cu) redox state has been an important issue in biology and toxicology research, but many research gaps remain to be explored due to the limitations in the detecting techniques. Herein, the regulation of Cu homeostasis, including absorption, translocation, utilization, storage, and elimination behavior is discussed. Cuproptosis, a newly identified type of cell death caused by excessive Cu accumulation, which results in the aggregation of DLAT protein or the loss of Fe-S cluster and finally proteotoxic stress, is reviewed. Several longstanding mysteries of diseases such as Wilson disease and toxic effects, may be attributed to cuproptosis. Furthermore, we review the advanced detection methods and application of Cu(I) and Cu(II), especially the in-situ imaging techniques such as XANES, and chemosensors. Most of the existing studies using these detection techniques focus on the bioaccumulation and toxicity of Cu(I) and Cu(II) in cells and aquatic organisms. Finally, it will be important to identify the roles of Cu(I) and Cu(II) in the growth, development, and diseases of organisms, as well as the relationship between bioaccumulation and toxicity of Cu(I) and Cu(II) in cellular and aquatic toxicology.

Cu is an essential micronutrient for various physiological processes in almost all human cell types. Given the critical role of Cu in a wide range of cellular processes, the local concentrations of Cu and the cellular distribution of Cu transporter proteins in the lung are essential for maintaining a steady-state internal environment. Dysfunctional Cu metabolism or regulatory pathways can lead to an imbalance in Cu homeostasis in the lungs, affecting both acute and chronic pathological processes. Recent studies have identified a new form of Cu-dependent cell death called cuproptosis, which has generated renewed interest in the role of Cu homeostasis in diseases. Cuproptosis differs from other known cell death pathways. This occurs through the direct binding of Cu ions to lipoylated components of the tricarboxylic acid cycle during mitochondrial respiration, leading to the aggregation of lipoylated proteins and the subsequent downregulation of Fe-S cluster proteins, which causes toxic stress to the proteins and ultimately leads to cell death. Here, we discuss the impact of dysregulated Cu homeostasis on the pathogenesis of various respiratory diseases, including asthma, chronic obstructive pulmonary disease, idiopathic interstitial fibrosis, and lung cancer. We also discuss the therapeutic potential of targeting Cu. This study highlights the intricate interplay between copper, cellular processes, and respiratory health. Copper, while essential, must be carefully regulated to maintain the delicate balance between necessity and toxicity in living organisms. This review highlights the need to further investigate the precise mechanisms of copper interactions with infections and immune inflammation in the context of respiratory diseases and explore the potential of therapeutic strategies for copper, cuproptosis, and other related effects.

Bacteria use specialized proteins, like transcription factors, to rapidly control metal ion balance. CueR is a Gram-negative bacterial copper regulator. The structure of E. coli CueR complexed with Cu(I) and DNA was published, since then many studies have shed light on its function. However, P. aeruginosa CueR, which shows high sequence similarity to E. coli CueR, has been less studied. Here, we applied room-temperature electron paramagnetic resonance (EPR) measurements to explore changes in dynamics of P. aeruginosa CueR in dependency of copper concentrations and interaction with two different DNA promoter regions. We showed that P. aeruginosa CueR is less dynamic than the E. coli CueR protein and exhibits much higher sensitivity to DNA binding as compared to its E. coli CueR homolog. Moreover, a difference in dynamical behavior was observed when P. aeruginosa CueR binds to the copZ2 DNA promoter sequence compared to the mexPQ-opmE promoter sequence. Such dynamical differences may affect the expression levels of CopZ2 and MexPQ-OpmE proteins in P. aeruginosa. Overall, such comparative measurements of protein-DNA complexes derived from different bacterial systems reveal insights about how structural and dynamical differences between two highly homologous proteins lead to quite different DNA sequence-recognition and mechanistic properties.

Candida albicans is a human colonizer and also an opportunistic yeast occupying different niches that are mostly hypoxic. While hypoxia is the prevalent condition within the host, the machinery that integrates oxygen status to tune the fitness of fungal pathogens remains poorly characterized. Here, we uncovered that Snf5, a subunit of the chromatin remodeling complex SWI/SNF, is required to tolerate antifungal stress particularly under hypoxia. RNA-seq profiling of snf5 mutant exposed to amphotericin B and fluconazole under hypoxic conditions uncovered a signature that is reminiscent of copper (Cu) starvation. We found that under hypoxic and Cu-starved environments, Snf5 is critical for preserving Cu homeostasis and the transcriptional modulation of the Cu regulon. Furthermore, snf5 exhibits elevated levels of reactive oxygen species and an increased sensitivity to oxidative stress principally under hypoxia. Supplementing growth medium with Cu or increasing gene dosage of the Cu transporter CTR1 alleviated snf5 growth defect and attenuated reactive oxygen species levels in response to antifungal challenge. Genetic interaction analysis suggests that Snf5 and the bona fide Cu homeostasis regulator Mac1 function in separate pathways. Together, our data underlined a unique role of SWI/SNF complex as a potent regulator of Cu metabolism and antifungal stress under hypoxia.