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EMBO Molecular Medicine Dec 2023Triple-negative breast cancer (TNBC) often develops resistance to single-agent treatment, which can be circumvented using targeted combinatorial approaches. Here, we...
Triple-negative breast cancer (TNBC) often develops resistance to single-agent treatment, which can be circumvented using targeted combinatorial approaches. Here, we demonstrate that the simultaneous inhibition of LOXL2 and BRD4 synergistically limits TNBC proliferation in vitro and in vivo. Mechanistically, LOXL2 interacts in the nucleus with the short isoform of BRD4 (BRD4S), MED1, and the cell cycle transcriptional regulator B-MyB. These interactions sustain the formation of BRD4 and MED1 nuclear transcriptional foci and control cell cycle progression at the gene expression level. The pharmacological co-inhibition of LOXL2 and BRD4 reduces BRD4 nuclear foci, BRD4-MED1 colocalization, and the transcription of cell cycle genes, thus suppressing TNBC cell proliferation. Targeting the interaction between BRD4S and LOXL2 could be a starting point for the development of new anticancer strategies for the treatment of TNBC.
Topics: Humans; Amino Acid Oxidoreductases; Bromodomain Containing Proteins; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Mediator Complex Subunit 1; Nuclear Proteins; Transcription Factors; Triple Negative Breast Neoplasms; Animals
PubMed: 37937685
DOI: 10.15252/emmm.202318459 -
Redox Biology Nov 2023We recently reported a previously unknown salutary role for xanthine oxidoreductase (XOR) in intravascular heme overload whereby hepatocellular export of XOR to the...
We recently reported a previously unknown salutary role for xanthine oxidoreductase (XOR) in intravascular heme overload whereby hepatocellular export of XOR to the circulation was identified as a seminal step in affording protection. However, the cellular signaling and export mechanisms underpinning this process were not identified. Here, we present novel data showing hepatocytes upregulate XOR expression/protein abundance and actively release it to the extracellular compartment following exposure to hemopexin-bound hemin, hemin or free iron. For example, murine (AML-12 cells) hepatocytes treated with hemin (10 μM) exported XOR to the medium in the absence of cell death or loss of membrane integrity (2.0 ± 1.0 vs 16 ± 9 μU/mL p < 0.0001). The path of exocytosis was found to be noncanonical as pretreatment of the hepatocytes with Vaculin-1, a lysosomal trafficking inhibitor, and not Brefeldin A inhibited XOR release and promoted intracellular XOR accumulation (84 ± 17 vs 24 ± 8 hemin vs 5 ± 3 control μU/mg). Interestingly, free iron (Fe and Fe) induced similar upregulation and release of XOR compared to hemin. Conversely, concomitant treatment with hemin and the classic transition metal chelator DTPA (20 μM) or uric acid completely blocked XOR release (p < 0.01). Our previously published time course showed XOR release from hepatocytes likely required transcriptional upregulation. As such, we determined that both Sp1 and NF-kB were acutely activated by hemin treatment (∼2-fold > controls for both, p < 0.05) and that silencing either or TLR4 with siRNA prevented hemin-induced XOR upregulation (p < 0.01). Finally, to confirm direct action of these transcription factors on the Xdh gene, chromatin immunoprecipitation was performed indicating that hemin significantly enriched (∼5-fold) both Sp1 and NF-kB near the transcription start site. In summary, our study identified a previously unknown pathway by which XOR is upregulated via SP1/NF-kB and subsequently exported to the extracellular environment. This is, to our knowledge, the very first study to demonstrate mechanistically that XOR can be specifically targeted for export as the seminal step in a compensatory response to heme/Fe overload.
Topics: Animals; Mice; Xanthine Dehydrogenase; Hemin; Iron; NF-kappa B; Heme; Hepatocytes
PubMed: 37703667
DOI: 10.1016/j.redox.2023.102866 -
Nature Communications Jul 2023Chemotherapy-induced cardiac damage remains a leading cause of death amongst cancer survivors. Anthracycline-induced cardiotoxicity is mediated by severe mitochondrial...
Chemotherapy-induced cardiac damage remains a leading cause of death amongst cancer survivors. Anthracycline-induced cardiotoxicity is mediated by severe mitochondrial injury, but little is known about the mechanisms by which cardiomyocytes adaptively respond to the injury. We observed the translocation of selected mitochondrial tricarboxylic acid (TCA) cycle dehydrogenases to the nucleus as an adaptive stress response to anthracycline-cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes and in vivo. The expression of nuclear-targeted mitochondrial dehydrogenases shifts the nuclear metabolic milieu to maintain their function both in vitro and in vivo. This protective effect is mediated by two parallel pathways: metabolite-induced chromatin accessibility and AMP-kinase (AMPK) signaling. The extent of chemotherapy-induced cardiac damage thus reflects a balance between mitochondrial injury and the protective response initiated by the nuclear pool of mitochondrial dehydrogenases. Our study identifies nuclear translocation of mitochondrial dehydrogenases as an endogenous adaptive mechanism that can be leveraged to attenuate cardiomyocyte injury.
Topics: Humans; Cardiotoxicity; Heart Diseases; Induced Pluripotent Stem Cells; Antibiotics, Antineoplastic; Anthracyclines; Topoisomerase II Inhibitors; Oxidoreductases; Myocytes, Cardiac; Doxorubicin
PubMed: 37468519
DOI: 10.1038/s41467-023-40084-5 -
Blood Advances Nov 2023Patients with relapsed or refractory T-cell acute lymphoblastic leukemia (T-ALL) have a poor prognosis with few therapeutic options. With the goal of identifying novel...
Patients with relapsed or refractory T-cell acute lymphoblastic leukemia (T-ALL) have a poor prognosis with few therapeutic options. With the goal of identifying novel therapeutic targets, we used data from the Dependency Map project to identify dihydroorotate dehydrogenase (DHODH) as one of the top metabolic dependencies in T-ALL. DHODH catalyzes the fourth step of de novo pyrimidine nucleotide synthesis. Small molecule inhibition of DHODH rapidly leads to the depletion of intracellular pyrimidine pools and forces cells to rely on extracellular salvage. In the absence of sufficient salvage, this intracellular nucleotide starvation results in the inhibition of DNA and RNA synthesis, cell cycle arrest, and, ultimately, death. T lymphoblasts appear to be specifically and exquisitely sensitive to nucleotide starvation after DHODH inhibition. We have confirmed this sensitivity in vitro and in vivo in 3 murine models of T-ALL. We identified that certain subsets of T-ALL seem to have an increased reliance on oxidative phosphorylation when treated with DHODH inhibitors. Through a series of metabolic assays, we show that leukemia cells, in the setting of nucleotide starvation, undergo changes in their mitochondrial membrane potential and may be more highly dependent on alternative fuel sources. The effect on normal T-cell development in young mice was also examined to show that DHODH inhibition does not permanently damage the developing thymus. These changes suggest a new metabolic vulnerability that may distinguish these cells from normal T cells and other normal hematopoietic cells and offer an exploitable therapeutic opportunity. The availability of clinical-grade DHODH inhibitors currently in human clinical trials suggests a potential for rapidly advancing this work into the clinic.
Topics: Humans; Animals; Mice; Dihydroorotate Dehydrogenase; Oxidoreductases Acting on CH-CH Group Donors; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Enzyme Inhibitors; T-Lymphocytes; Nucleotides
PubMed: 37648673
DOI: 10.1182/bloodadvances.2023010337 -
The EMBO Journal Dec 2023Ceramide synthases (CerS) catalyze ceramide formation via N-acylation of a sphingoid base with a fatty acyl-CoA and are attractive drug targets for treating numerous...
Ceramide synthases (CerS) catalyze ceramide formation via N-acylation of a sphingoid base with a fatty acyl-CoA and are attractive drug targets for treating numerous metabolic diseases and cancers. Here, we present the cryo-EM structure of a yeast CerS complex, consisting of a catalytic Lac1 subunit and a regulatory Lip1 subunit, in complex with C26-CoA substrate. The CerS holoenzyme exists as a dimer of Lac1-Lip1 heterodimers. Lac1 contains a hydrophilic reaction chamber and a hydrophobic tunnel for binding the CoA moiety and C26-acyl chain of C26-CoA, respectively. Lip1 interacts with both the transmembrane region and the last luminal loop of Lac1 to maintain the proper acyl chain binding tunnel. A lateral opening on Lac1 serves as a potential entrance for the sphingoid base substrate. Our findings provide a template for understanding the working mechanism of eukaryotic ceramide synthases and may facilitate the development of therapeutic CerS modulators.
Topics: Ceramides; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Oxidoreductases; Membrane Proteins
PubMed: 37953642
DOI: 10.15252/embj.2023114889 -
Biochimica Et Biophysica Acta.... Jun 2024Iron-sulfur clusters serve as indispensable cofactors within proteins across all three domains of life. Fe/S clusters emerged early during the evolution of life on our... (Review)
Review
Iron-sulfur clusters serve as indispensable cofactors within proteins across all three domains of life. Fe/S clusters emerged early during the evolution of life on our planet and the biogeochemical cycle of sulfur is one of the most ancient and important element cycles. It is therefore no surprise that Fe/S proteins have crucial roles in the multiple steps of microbial sulfur metabolism. During dissimilatory sulfur oxidation in prokaryotes, Fe/S proteins not only serve as electron carriers in several steps, but also perform catalytic roles, including unprecedented reactions. Two cytoplasmic enzyme systems that oxidize sulfane sulfur to sulfite are of particular interest in this context: The rDsr pathway employs the reverse acting dissimilatory sulfite reductase rDsrAB as its key enzyme, while the sHdr pathway utilizes polypeptides resembling the HdrA, HdrB and HdrC subunits of heterodisulfide reductase from methanogenic archaea. Both pathways involve components predicted to bind unusual noncubane Fe/S clusters acting as catalysts for the formation of disulfide or sulfite. Mapping of Fe/S cluster machineries on the sulfur-oxidizing prokaryote tree reveals that ISC, SUF, MIS and SMS are all sufficient to meet the Fe/S cluster maturation requirements for operation of the sHdr or rDsr pathways. The sHdr pathway is dependent on lipoate-binding proteins that are assembled by a novel pathway, involving two Radical SAM proteins, namely LipS1 and LipS2. These proteins coordinate sulfur-donating auxiliary Fe/S clusters in atypical patterns by three cysteines and one histidine and act as lipoyl synthases by jointly inserting two sulfur atoms to an octanoyl residue. This article is part of a Special Issue entitled: Biogenesis and Function of Fe/S proteins.
Topics: Oxidation-Reduction; Sulfur; Iron-Sulfur Proteins; Bacterial Proteins; Archaea; Oxidoreductases
PubMed: 38631440
DOI: 10.1016/j.bbamcr.2024.119732 -
Journal of Nanobiotechnology May 2024Various clinical symptoms of digestive system, such as infectious, inflammatory, and malignant disorders, have a profound impact on the quality of life and overall... (Review)
Review
Various clinical symptoms of digestive system, such as infectious, inflammatory, and malignant disorders, have a profound impact on the quality of life and overall health of patients. Therefore, the chase for more potent medicines is both highly significant and urgent. Nanozymes, a novel class of nanomaterials, amalgamate the biological properties of nanomaterials with the catalytic activity of enzymes, and have been engineered for various biomedical applications, including complex gastrointestinal diseases (GI). Particularly, because of their distinctive metal coordination structure and ability to maximize atom use efficiency, single-atom nanozymes (SAzymes) with atomically scattered metal centers are becoming a more viable substitute for natural enzymes. Traditional nanozyme design strategies are no longer able to meet the current requirements for efficient and diverse SAzymes design due to the diversification and complexity of preparation processes. As a result, this review emphasizes the design concept and the synthesis strategy of SAzymes, and corresponding bioenzyme-like activities, such as superoxide dismutase (SOD), peroxidase (POD), oxidase (OXD), catalase (CAT), and glutathione peroxidase (GPx). Then the various application of SAzymes in GI illnesses are summarized, which should encourage further research into nanozymes to achieve better application characteristics.
Topics: Humans; Gastrointestinal Diseases; Nanostructures; Animals; Enzymes; Superoxide Dismutase; Catalase; Catalysis; Glutathione Peroxidase
PubMed: 38796465
DOI: 10.1186/s12951-024-02569-3 -
Nature Communications Sep 2023Rieske oxygenases use a Rieske-type [2Fe-2S] cluster and a mononuclear iron center to initiate a range of chemical transformations. However, few details exist regarding...
Rieske oxygenases use a Rieske-type [2Fe-2S] cluster and a mononuclear iron center to initiate a range of chemical transformations. However, few details exist regarding how this catalytic scaffold can be predictively tuned to catalyze divergent reactions. Therefore, in this work, using a combination of structural analyses, as well as substrate and rational protein-based engineering campaigns, we elucidate the architectural trends that govern catalytic outcome in the Rieske monooxygenase TsaM. We identify structural features that permit a substrate to be functionalized by TsaM and pinpoint active-site residues that can be targeted to manipulate reactivity. Exploiting these findings allowed for custom tuning of TsaM reactivity: substrates are identified that support divergent TsaM-catalyzed reactions and variants are created that exclusively catalyze dioxygenation or sequential monooxygenation chemistry. Importantly, we further leverage these trends to tune the reactivity of additional monooxygenase and dioxygenase enzymes, and thereby provide strategies to custom tune Rieske oxygenase reaction outcomes.
Topics: Oxygenases; Mixed Function Oxygenases; Dioxygenases; Catalysis; Culture
PubMed: 37730711
DOI: 10.1038/s41467-023-41428-x -
Nature Communications Jul 2023Protein-S-glutathionylation is a post-translational modification involving the conjugation of glutathione to protein thiols, which can modulate the activity and...
Protein-S-glutathionylation is a post-translational modification involving the conjugation of glutathione to protein thiols, which can modulate the activity and structure of key cellular proteins. Glutaredoxins (GLRX) are oxidoreductases that regulate this process by performing deglutathionylation. However, GLRX has five cysteines that are potentially vulnerable to oxidative modification, which is associated with GLRX aggregation and loss of activity. To date, GLRX cysteines that are oxidatively modified and their relative susceptibilities remain unknown. We utilized molecular modeling approaches, activity assays using recombinant GLRX, coupled with site-directed mutagenesis of each cysteine both individually and in combination to address the oxidizibility of GLRX cysteines. These approaches reveal that C8 and C83 are targets for S-glutathionylation and oxidation by hydrogen peroxide in vitro. In silico modeling and experimental validation confirm a prominent role of C8 for dimer formation and aggregation. Lastly, combinatorial mutation of C8, C26, and C83 results in increased activity of GLRX and resistance to oxidative inactivation and aggregation. Results from these integrated computational and experimental studies provide insights into the relative oxidizability of GLRX's cysteines and have implications for the use of GLRX as a therapeutic in settings of dysregulated protein glutathionylation.
Topics: Animals; Cysteine; Glutaredoxins; Glutathione; Mammals; Oxidation-Reduction; Proteins
PubMed: 37507364
DOI: 10.1038/s41467-023-39664-2 -
Advanced Science (Weinheim,... Mar 2024Various forms of programmed cell death (PCD) exhibit distinct characteristics depending on their specific molecular mechanisms, and there are interactions among these...
Various forms of programmed cell death (PCD) exhibit distinct characteristics depending on their specific molecular mechanisms, and there are interactions among these different forms. Ferroptosis, which is related to autophagy and apoptosis, has an unknown potential interaction with pyroptosis. This study revealed a mutually antagonistic relationship between ferroptosis and pyroptosis, with 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) playing a key role in their interaction. It is found that HMGCR predominantly localized to mitochondria during ferroptosis but shifted to the endoplasmic reticulum following treatment with a pyroptosis inducer. Furthermore, this study demonstrated that BRCC36 (BRCA1/BRCA2-containing complex subunit 36) deubiquitinated HMGCR in a manner dependent on deubiquitinating enzyme (DUB) activity, and inhibited ferroptosis and promoted pyroptosis. Moreover, as an oncogene in hepatocellular carcinoma (HCC), BRCC36 promoted cancer cell proliferation, migration, invasion, and tumor growth. Thiolutin, an inhibitor of BRCC36, effectively suppressed the interaction between BRCC36 and HMGCR, leading to the inhibition of HCC growth. Therefore, targeting BRCC36 can offer a novel and promising therapeutic strategy for HCC treatment. In conclusion, these findings provide new theoretical evidence for further characterizing tumor heterogeneity and offer new molecular targets for the diagnosis and treatment of HCC.
Topics: Humans; Pyroptosis; Carcinoma, Hepatocellular; Oxidoreductases; Ferroptosis; Liver Neoplasms; Hydroxymethylglutaryl CoA Reductases
PubMed: 38178583
DOI: 10.1002/advs.202304263