Intervertebral disc degeneration (IDD) is an age-related disease and is responsible for low back pain. Oxidative stress-induced cell death plays a fundamental role in IDD pathogenesis. Cuproptosis is a recently discovered form of programmed cell death dependent on copper availability. Whether cuproptosis is involved in IDD progression remains unknown. Herein, we established in vitro and in vivo models to investigate cuproptosis in IDD and the mechanisms by which oxidative stress interacts with copper sensitivity in nucleus pulposus cells (NPCs). We found that ferredoxin-1 (FDX1) content increased in both rat and human degenerated discs. Sublethal oxidative stress on NPCs led to increased FDX1 expression, tricarboxylic acid (TCA) cycle-related proteins lipoylation and aggregation, and cell death in the presence of Cu2+ at physiological concentrations, while FDX1 knockdown inhibited cell death. Since copper homeostasis is involved in copper-induced cytotoxicity, we investigated the role of copper transport-related proteins, including importer (CTR1) and efflux pumps (ATPase transporter, ATP7A, and ATP7B). CTR1 and ATP7A content increased under oxidative stress, and blocking CTR1 reduced oxidative stress/copper-induced TCA-related protein aggregation and cell death. Moreover, oxidative stress promoted the expression of specific protein 1 (SP1) and SP1-mediated CTR1 transcription. SP1 inhibition decreased cell death rates, preserved disc hydration, and alleviated tissue degeneration. This suggests that oxidative stress upregulates FDX1 expression and copper flux through promoting SP1-mediated CTR1 transcription, leading to increased TCA cycle-related protein aggregation and cuproptosis. This study highlights the importance of cuproptosis in IDD progression and provides a promising therapeutic target for IDD treatment.

Volatile compounds, such as nitric oxide and ethylene gas, play a vital role as signaling molecules in organisms. Ethylene is a plant hormone that regulates a wide range of plant growth, development, and responses to stress and is perceived by a family of ethylene receptors that localize in the endoplasmic reticulum. Constitutive Triple Response 1 (CTR1), a Raf-like protein kinase and a key negative regulator for ethylene responses, tethers to the ethylene receptors, but undergoes nuclear translocation upon activation of ethylene signaling. This ER-to-nucleus trafficking transforms CTR1 into a positive regulator for ethylene responses, significantly enhancing stress resilience to drought and salinity. The nuclear trafficking of CTR1 demonstrates that the spatiotemporal control of ethylene signaling is essential for stress adaptation. Understanding the mechanisms governing the spatiotemporal control of ethylene signaling elements is crucial for unraveling the system-level regulatory mechanisms that collectively fine-tune ethylene responses to optimize plant growth, development, and stress adaptation.

The Arabidopsis (Arabidopsis thaliana) constitutive triple response1-10 (ctr1-10) mutant produces a reduced level of CTR1 protein and exhibits a weak ctr1 mutant phenotype. Sequence analysis revealed highly active translation of the upstream open reading frame (uORF) at the extended 5\'-UTR of the ctr1-10 mRNA, resulting from T-DNA insertion. Enhancer screening for ctr1-10 isolated the fragile histidine triad-1 (fhit-1) mutation. The fhit-1 ctr1-10 mutant phenotypically resembled strong ctr1 mutants and barely produced CTR1, and the fhit-1 mutation reduced the translation efficiency of ctr1-10 but not that of CTR1 mRNA. The human (Homo sapiens) Fhit that involves tumorigenesis and genome instability has the in vitro dinucleotide 5\',5\'″-P1, P3-triphosphate hydrolase activity, and expression of the human HsFHIT or the hydrolase-defective HsFHITH96N transgene reversed the fhit-1 ctr1-10 mutant phenotype and restored CTR1 levels. Genetic editing that in situ disrupts individual upstream ATG codons proximal to the ctr1-10 mORF elevated CTR1 levels in ctr1-10 plants independent of FHIT. EUKARYOTIC INITIATION FACTOR3G (eIF3G), which is involved in translation and reinitiation, interacted with FHIT, and both were associated with the polysome. We propose that FHIT resumes early terminated ctr1-10 mORF translation in the face of active and complex uORF translation. Our study unveils a niche that may lead to investigations on the molecular mechanism of Fhit-like proteins in translation reinitiation. The biological significance of FHIT-regulated translation is discussed.

Endothelial dysfunction is a common complication of diabetes mellitus (DM) and contributes to the high incidence and mortality of cardiovascular and cerebrovascular diseases. Aberrant epigenetic regulation under diabetic conditions, including histone modifications, DNA methylation, and non-coding RNAs (ncRNAs) play key roles in the initiation and progression of diabetic vascular complications. ASH2L, a H3K4me3 regulator, triggers genetic transcription, which is critical for physiological and pathogenic processes. In this study we investigated the role of ASH2L in mediating diabetic endothelial dysfunction. We showed that ASH2L expression was significantly elevated in vascular tissues from diabetic db/db mice and in rat aortic endothelial cells (RAECs) treated with high glucose medium (11 and 22 mM). Knockdown of ASH2L in RAECs markedly inhibited the deteriorating effects of high glucose, characterized by reduced oxidative stress and inflammatory responses. Deletion of endothelial ASH2L in db/db mice by injection of an adeno-associated virus (AAV)-endothelial specific system carrying shRNA against Ash2l (AAV-shAsh2l) restored the impaired endothelium-dependent relaxations, and ameliorated DM-induced vascular dysfunction. We revealed that ASH2L expression activated reductase STEAP4 transcription in vitro and in vivo, which consequently elevated Cu(I) transportation into ECs by the copper transporter CTR1. Excess copper produced by STEAP4-mediated copper uptake triggered oxidative stress and inflammatory responses, resulting in endothelial dysfunction. Our results demonstrate that hyperglycemia triggered ASH2L-STEAP4 axis contributes to diabetic endothelial dysfunction by modulating copper uptake into ECs and highlight the therapeutic potential of blocking the endothelial ASH2L in the pathogenesis of diabetic vascular complications.

Protein-protein interactions play a crucial role in driving cellular processes and enabling appropriate physiological responses in organisms. The plant hormone ethylene signaling pathway is complex and regulated by the spatiotemporal regulation of its signaling molecules. Constitutive Triple Response 1 (CTR1), a key negative regulator of the pathway, regulates the function of Ethylene-Insensitive 2 (EIN2), a positive regulator of ethylene signaling, at the endoplasmic reticulum (ER) through phosphorylation. Our recent study revealed that CTR1 can also translocate from the ER to the nucleus in response to ethylene and positively regulate ethylene responses by stabilizing EIN3. To gain further insights into the role of CTR1 in plants, we used TurboID-based proximity labeling and mass spectrometry to identify the proximal proteomes of CTR1 in Nicotiana benthamiana. The identified proximal proteins include known ethylene signaling components, as well as proteins involved in diverse cellular processes such as mitochondrial respiration, mRNA metabolism, and organelle biogenesis. Our study demonstrates the feasibility of proximity labeling using the N. benthamiana transient expression system and identifies the potential interactors of CTR1 in vivo, uncovering the potential roles of CTR1 in a wide range of cellular processes.

High salinity stress promotes plant ethylene biosynthesis and triggers the ethylene signalling response. However, the precise mechanism underlying how plants transduce ethylene signalling in response to salt stress remains largely unknown. In this study, we discovered that SALT OVERLY SENSITIVE 2 (SOS2) inhibits the kinase activity of CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) by phosphorylating the 87th serine (S87). This phosphorylation event activates the ethylene signalling response, leading to enhanced plant salt resistance. Furthermore, through genetic analysis, we determined that the loss of CTR1 or the gain of SOS2-mediated CTR1 phosphorylation both contribute to improved plant salt tolerance. Additionally, in the sos2 mutant, we observed compromised proteolytic processing of ETHYLENE INSENSITIVE 2 (EIN2) and reduced nuclear localization of EIN2 C-terminal fragments (EIN2-C), which correlate with decreased accumulation of ETHYLENE INSENSITIVE 3 (EIN3). Collectively, our findings unveil the role of the SOS2-CTR1 regulatory module in promoting the activation of the ethylene signalling pathway and enhancing plant salt tolerance.

Copper is a critical element for eukaryotic life involved in numerous cellular functions, including redox balance, but is toxic in excess. Therefore, tight regulation of copper acquisition and homeostasis is essential for cell physiology and survival. Here, we identify a different regulatory mechanism for cellular copper homeostasis that requires the presence of an endogenous retroviral envelope glycoprotein called Refrex1. We show that cells respond to elevated extracellular copper by increasing the expression of Refrex1, which regulates copper acquisition through interaction with the main copper transporter CTR1. Downmodulation of Refrex1 results in intracellular copper accumulation leading to reactive oxygen species (ROS) production and subsequent apoptosis, which is prevented by copper chelator treatment. Our results show that Refrex1 has been co-opted for its ability to regulate copper entry through CTR1 in order to limit copper excess, redox imbalance, and ensuing cell death, strongly suggesting that other endogenous retroviruses may have similar metabolic functions among vertebrates.

Cuproplasia, or copper-dependent cell proliferation, has been observed in varieties of solid tumors along with aberrant copper homeostasis. Several studies reported good response of patients to copper chelator assisted neoadjuvant chemotherapy, however, the internal target molecules are still undetermined. Unravel copper-associated tumor signaling would be valuable to forge new links to translate biology of copper into clinical cancer therapies. We evaluated the significance of high-affinity copper transporter-1 (CTR1) by bioinformatic analysis, and in 19 pairs of clinical specimens. Then, with the help of gene interference and chelating agent, enriched signaling pathways were identified by KEGG analysis and immunoblotting. Accompanying biological capability of pancreatic carcinoma-associated proliferation, cell cycle, apoptosis, and angiogenesis were investigated. Furthermore, a combination of mTOR inhibitor and CTR1 suppressor has been assessed in xenografted tumor mouse models. Hyperactive CTR1 was investigated in pancreatic cancer tissues and proven to as the key point of cancer copper homeostasis. Intracellular copper deprivation induced by CTR1 gene knock-down or systematic copper chelation by tetrathiomolybdate suppressed proliferation and angiogenesis of pancreatic cancer cell. PI3K/AKT/mTOR signaling pathway was suppressed by inhibiting the activation of p70(S6)K and p-AKT, and finally inhibited mTORC1 and mTORC2 after copper deprivation. Additionally, CTR1 gene silencing successfully improved the anti-cancer effect of mTOR inhibitor rapamycin. Our study reveals that CTR1 contributes to pancreatic tumorigenesis and progression, by up-regulating the phosphorylation of AKT/mTOR signaling molecules. Recovering copper balance by copper deprivation addresses as promising strategy for improved cancer chemotherapy.

Organoids are a three-dimensional (3D) culture system that simulate actual organs. Therefore, tumor organoids are expected to predict precise response to chemotherapy in patients. However, to date, few studies have studied the drug responses in organoids of malignant mesothelioma (MM). The poor prognosis of MM emphasizes the importance of establishing a protocol for generating MM-organoid for research and clinical use. Here, we established murine MM organoids from p53+/- or wild-type C57BL/6 strain by intraperitoneal injection either with crocidolite or carbon nanotube. Established MM-organoids proliferated in Matrigel as spheroids. Subcutaneous injection assays revealed that the MM-organoids mimicked actual tissue architecture and maintained the original histological features of the primary MM. RNA sequencing and pathway analyses revealed that the significant expressional differences between the 2D- and 3D-culture systems were observed in receptor tyrosine kinases, including IGF1R and EGFR, glycosylation and cholesterol/steroid metabolism. MM-organoids exhibited a more sensitive response to cisplatin through stable plasma membrane localization of a major cisplatin transporter, copper transporter 1/Slc31A1 (Ctr1) in comparison to 2D-cultures, presumably through glycosylation and lipidation. The Matrigel culture system facilitated the localization of CTR1 on the plasma membrane, which simulated the original MMs and the subcutaneous xenografts. These results suggest that the newly developed protocol for MM-organoids is useful to study strategies to overcome chemotherapy resistance to cisplatin.

UNASSIGNED: Osteosarcoma (OS) is a malignant bone tumor mainly affecting children and young adolescents. Cisplatin is a first-line chemotherapy drug for OS, however, drug resistance severely limits the survival of OS. Nevertheless, cellular factors in cisplatin resistance for OS remain obscure. In this study, the function and potential mechanism of p53 in cisplatin absorption were explored in OS cells.
UNASSIGNED: The CRISPR-Cas9 gene editing technology was performed to obtain p53 gene knock-out U2OS cells. The p53 over-expression 143B cell line was established by lentivirus-mediated virus infection. Moreover, the functions of p53 and CTR1 in cisplatin absorption were assessed by inductively coupled plasma mass spectrometry (ICP-MS) through CTR1 over-expression and knock-down. Further, the DNA binding activity of SP1 on CTR1 gene promoter was determined by dual-luciferase assay and chromatin immunoprecipitation (ChIP) assay. The functional regulation of p53 on SP1 was studied by nucleocytoplasmic separation assay and electrophoretic mobility shift assay (EMSA). The interaction between p53 and SP1 was verified by Co-Immunoprecipitation assay.
UNASSIGNED: Under cisplatin treatment, p53 knock-out promoted CTR1 expression and cisplatin uptake, while p53 overexpression inhibited CTR1 expression and cisplatin uptake. Moreover, p53 regulated CTR1 level not by binding to CTR1 promoter directly but by suppressing the nuclear translocation of transcription factor specificity protein 1 (SP1). It was verified that SP1 is directly bound with CTR1 promoter. SP1 overexpression stimulated CTR1 expression, and SP1 knock-down attenuated CTR1 expression.
UNASSIGNED: The p53 might function as a negative regulator in CTR1 mediated cisplatin absorption, and the p53-SP1-CTR1 axis is a target for cisplatin resistance.