Autophagy aggravates multi-walled carbon nanotube-induced ferroptosis by suppressing PGC-1 dependent-mitochondrial biogenesis in lung epithelial cells.

Multi-walled carbon nanotube (MWCNT) induced respiratory toxicity has become a growing concern, with ferroptosis emerging as a novel mechanism implicated in various respiratory diseases. However, whether ferroptosis is involved in MWCNT-elicited lung injury and the underlying molecular mechanisms warrant further exploration. In this study, we found that MWCNT-induced ferroptosis is autophagy-dependent, contributing to its cellular toxicity. Inhibiting of autophagy by pharmacological inhibitors 3-MA or ATG5 gene knockdown significantly attenuated MWCNT-induced ferroptosis, concomitant with rescued mitochondrial biogenesis. Rapamycin, the autophagy agonist, exacerbated the mitochondrial damage and MWCNT-induced ferroptosis. Moreover, lentivirus-mediated overexpression of PGC-1α inhibited ferroptosis, while inhibition of PGC-1α aggravated ferroptosis. In summary, our study unveils ferroptosis as a novel mechanism underlying MWCNT-induced respiratory toxicity, with autophagy promoting MWCNTs-induced ferroptosis by hindering PGC-1α-dependent mitochondrial biogenesis.

Highly Selective Electrocatalytic CO2 Conversion to Tailored Products through Precise Regulation of Hydrogenation and C-C Coupling.

The electrochemical reduction reaction of carbon dioxide (CO2RR) into valuable products offers notable economic benefits and contributes to environmental sustainability. However, precisely controlling the reaction pathways and selectively converting key intermediates pose considerable challenges. In this study, our theoretical calculations reveal that the active sites with different states of copper atoms (1-3-5-7-9) play a pivotal role in the adsorption behavior of the *CHO critical intermediate. This behavior dictates the subsequent hydrogenation and coupling steps, ultimately influencing the formation of the desired products. Consequently, we designed two model electrocatalysts comprising Cu single atoms and particles supported on CeO2. This design enables controlled *CHO intermediate transformation through either hydrogenation with *H or coupling with *CO, leading to a highly selective CO2RR. Notably, our selective control strategy tunes the Faradaic efficiency from 61.1% for ethylene (C2H4) to 61.2% for methane (CH4). Additionally, the catalyst demonstrated a high current density and remarkable stability, exceeding 500 h of operation. This work not only provides efficient catalysts for selective CO2RR but also offers valuable insights into tailoring surface chemistry and designing catalysts for precise control over catalytic processes to achieve targeted product generation in CO2RR technology.

Modulation of copper sites in porphyrin metal-organic frameworks for electrochemical ascorbic acid sensing.

Two metal-organic frameworks (MOFs) with different Cu-centered coordination structures were synthesized. By introducing 4,4-bipyridine as a linker in the Cu-MOFs, we have discovered that Cu-O, instead of Cu-N, is the active site with higher electrocatalytical activity towards ascorbic acid, which is essential to understand and develop Cu-based ascorbic acid sensors.

Stress granule-localized USP8 potentiates cGAS-mediated type I interferonopathies through deubiquitination of DDX3X.

Cyclic GMP-AMP synthase (cGAS) undergoes liquid-liquid phase separation (LLPS) to trigger downstream signaling upon double-stranded DNA (dsDNA) stimulation, and the condensed cGAS colocalizes with stress granules (SGs). However, the molecular mechanism underlying the modulation of cGAS activation by SGs remains elusive. In this study, we show that USP8 is localized to SGs upon dsDNA stimulation and potentiates cGAS-stimulator of interferon genes (STING) signaling. A USP8 inhibitor ameliorates pathological inflammation in Trex1-/- mice. Systemic lupus erythematosus (SLE) databases indicate a positive correlation between USP8 expression and SLE. Mechanistic study shows that the SG protein DDX3X promotes cGAS phase separation and activation in a manner dependent on its intrinsic LLPS. USP8 cleaves K27-linked ubiquitin chains from the intrinsically disordered region (IDR) of DDX3X to enhance its condensation. In conclusion, we demonstrate that USP8 catalyzes the deubiquitination of DDX3X to facilitate cGAS condensation and activation and that inhibiting USP8 is a promising strategy for alleviating cGAS-mediated autoimmune diseases.

Manganese induces neuronal apoptosis by activating mTOR signaling pathway in vitro and in vivo.

Manganese (Mn) is a well-known environmental pollutant and occupational toxicant that causes neurotoxicity, which present as neurodegenerative-like symptoms. However, the mechanism of Mn-induced neuronal injury remains unclear. In this research, we explored the mechanism of Mn-induced neurotoxicity, focusing on the mTOR signaling pathway. A plasmid expressing a short hairpin RNA (shRNA) targeting mTOR (shRNA-mTOR) was transfected into N27 cells in vitro, and rapamycin was used as an mTOR inhibitor in vivo to block the mTOR signaling pathway. Cells were treated with different concentrations of manganese (II) chloride (MnCl2). We found that Mn induced cell injury and apoptosis and markedly upregulated the expression of mTOR pathway-related proteins. The phosphorylation of 4E-BP1, S6K1, Akt and SGK1 was markedly decreased after blocking mTOR, and cell apoptosis was also reduced. Furthermore, the mTOR-specific inhibitor rapamycin restored learning and memory abilities in vivo. This research highlights that inhibiting mTOR might be useful for preventing Mn-induced neurodegenerative-like disorders.

Targeting GDP-Dissociation Inhibitor Beta (GDI2) with a Benzo[a]quinolizidine Library to Induce Paraptosis for Cancer Therapy.

Inducing paraptosis, a nonapoptotic form of cell death, has great therapeutic potential in cancer therapy, especially for drug-resistant tumors. However, the specific molecular target(s) that trigger paraptosis have not yet been deciphered yet. Herein, by using activity-based protein profiling, we identified the GDP-dissociation inhibitor beta (GDI2) as a manipulable target for inducing paraptosis and uncovered benzo[a]quinolizidine BQZ-485 as a potent inhibitor of GDI2 through the interaction with Tyr245. Comprehensive target validation revealed that BQZ-485 disrupts the intrinsic GDI2-Rab1A interaction, thereby abolishing vesicular transport from the endoplasmic reticulum (ER) to the Golgi apparatus and initiating subsequent paraptosis events including ER dilation and fusion, ER stress, the unfolded protein response, and cytoplasmic vacuolization. Based on the structure of BQZ-485, we created a small benzo[a]quinolizidine library by click chemistry and discovered more potent GDI2 inhibitors using a NanoLuc-based screening platform. Leveraging the engagement of BQZ-485 with GDI2, we developed a selective GDI2 degrader. The optimized inhibitor (+)-37 and degrader 21 described in this study exhibited excellent in vivo antitumor activity in two GDI2-overexpressing pancreatic xenograft models, including an AsPc-1 solid tumor model and a transplanted human PDAC tumor model. Altogether, our findings provide a promising strategy for targeting GDI2 for paraptosis in the treatment of pancreatic cancers, and these lead compounds could be further optimized to be effective chemotherapeutics.

Structural insights into FSP1 catalysis and ferroptosis inhibition.

Ferroptosis suppressor protein 1 (FSP1, also known as AIMF2, AMID or PRG3) is a recently identified glutathione-independent ferroptosis suppressor1-3, but its underlying structural mechanism remains unknown. Here we report the crystal structures of Gallus gallus FSP1 in its substrate-free and ubiquinone-bound forms. The structures reveal a FAD-binding domain and a NAD(P)H-binding domain, both of which are shared with AIF and NADH oxidoreductases4-9, and a characteristic carboxy-terminal domain as well. We demonstrate that the carboxy-terminal domain is crucial for the catalytic activity and ferroptosis inhibition of FSP1 by mediating the functional dimerization of FSP1, and the formation of two active sites located on two sides of FAD, which are responsible for ubiquinone reduction and a unique FAD hydroxylation respectively. We also identify that FSP1 can catalyze the production of H2O2 and the conversion of FAD to 6-hydroxy-FAD in the presence of oxygen and NAD(P)H in vitro, and 6-hydroxy-FAD directly inhibits ferroptosis in cells. Together, these findings further our understanding on the catalytic and ferroptosis suppression mechanisms of FSP1 and establish 6-hydroxy-FAD as an active cofactor in FSP1 and a potent radical-trapping antioxidant in ferroptosis inhibition.

Enhanced Proton Transfer in Proton-Exchange Membranes with Interconnected and Zwitterion-Functionalized Covalent Porous Material Structures.

Excellent proton-conductive accelerators are indispensable for efficient proton-exchange membranes (PEMs). Covalent porous materials (CPMs), with adjustable functionalities and well-ordered porosities, show much promise as effective proton-conductive accelerators. In this study, an interconnected and zwitterion-functionalized CPM structure based on carbon nanotubes and a Schiff-base network (CNT@ZSNW-1) is constructed as a highly efficient proton-conducting accelerator by in situ growth of SNW-1 onto carbon nanotubes (CNTs) and subsequent zwitterion functionalization. A composite PEM with enhanced proton conduction is acquired by integrating CNT@ZSNW-1 with Nafion. Zwitterion functionalization offers additional proton-conducting sites and promotes the water retention capacity. Moreover, the interconnected structure of CNT@ZSNW-1 induces a more consecutive arrangement of ionic clusters, which significantly relieves the proton transfer barrier of the composite PEM and increases its proton conductivity to 0.287 S cm-1 under 95 % RH at 90 °C (about 2.2 times that of the recast Nafion, 0.131 S cm-1 ). Furthermore, the composite PEM displays a peak power density of 39.6 mW cm-2 in a direct methanol fuel cell, which is significantly higher than that of the recast Nafion (19.9 mW cm-2 ). This study affords a potential reference for devising and preparing functionalized CPMs with optimized structures to expedite proton transfer in PEMs.

Cobalt Nanoparticles Anchored on N-Doped Porous Carbon Derived from Yeast for Enhanced Electrocatalytic Oxygen Reduction Reaction.

Biomass-derived carbon materials have received extensive attention for use in high-performance electrocatalysts. In this study, a highly efficient electrocatalyst is developed with Co nanoparticles anchored on N-doped porous carbon material (CoNC) by using yeast as a biomass precursor through a facial activation and pyrolysis process. CoNC exhibits comparable catalytic activity with commercial 20 % Pt/C for the oxygen reduction reaction (ORR) with a half-wave potential of 0.854 V. A home-made primary Zn-air battery exhibited an open circuit potential of 1.45 V and a peak power density of 188 mW cm-2 . Moreover, the discharge voltage of the primary battery maintained at a stable value up to 9 days. The enhanced performance of CoNC was probably ascribed to its high content of pyridinic-N and graphitic-N species, extra Co loading and porous structure, which provided sufficient active sites and channels to promote mass/electron transfer for ORR. This work provides a promising strategy to develop an efficient non-noble metal carbon-based electrocatalyst for fuel cells and metal-air batteries.

CD-NTase family member MB21D2 promotes cGAS-mediated antiviral and antitumor immunity.

cGAS/DncV-like nucleotidyltransferase (CD-NTase) family members are immune sensors that synthesize diverse nucleotide signals to initiate antiviral response in bacteria and animals. As a founding member of CD-NTase enzyme, cGAS has been identified as a key sensor for cytoplasmic DNA and type I interferons (IFNs) signaling in metazoan. However, the functions of other metazoan CD-NTases remain enigmatic. Here, we showed that Mab-21 domain-containing protein 2 (MB21D2), another member of the CD-NTase family, plays a positive role in modulating the cGAS-STING signaling in myeloid cells. Deficiency of MB21D2 in THP-1 cells or mice macrophages led to impaired production of type I interferon upon DNA stimulation. Consistently, Mb21d2-/- mice showed more susceptible to infection with DNA virus and faster growth of melanoma, compared to its counterparts. Mechanistically, MB21D2 specially bound with the N-terminal of cGAS, facilitated its liquid phase condensation and DNA-binding activity, leading to the enhanced production of cGAMP and subsequent IFN-ß production. Thus, our findings unveiled that the CD-NTase family member MB21D2 contributes to host antiviral and antitumor responses by enhancing cGAS activation.

Regulation of cGAS activity by RNA-modulated phase separation.

Cyclic GMP-AMP synthase (cGAS) is a double-stranded DNA (dsDNA) sensor that functions in the innate immune system. Upon binding dsDNA, cGAS and dsDNA form phase-separated condensates in which cGAS catalyzes the synthesis of 2'3'-cyclic GMP-AMP that subsequently triggers a STING-dependent, type I interferon (IFN-I) response. Here, we show that cytoplasmic RNAs regulate cGAS activity. We discover that RNAs do not activate cGAS but rather promote phase separation of cGAS in vitro. In cells, cGAS colocalizes with RNA and forms complexes with RNA. In the presence of cytoplasmic dsDNA, RNAs colocalize with phase-separated condensates of cGAS and dsDNA. Further in vitro assays showed that RNAs promote the formation of cGAS-containing phase separations and enhance cGAS activity when the dsDNA concentration is low. Cotransfection of RNA with a small amount of dsDNA into THP1 cells significantly enhances the production of the downstream signaling molecule interferon beta (IFNB). This enhancement can be blocked by a cGAS-specific inhibitor. Thus, cytoplasmic RNAs could regulate cGAS activity by modulating the formation of cGAS-containing condensates.

Combinatorial Biosynthesis of Oxidized Combamides Using Cytochrome P450 Enzymes from Different Polycyclic Tetramate Macrolactam Pathways.

Polycyclic tetramate macrolactams (PoTeMs) are a family of natural products containing a tetramic acid moiety and a polycyclic system. Due to the valuable biological activities of different PoTeMs and the genetic simplicity of their biosynthetic genes, it is highly desirable to manipulate the biosynthesis of PoTeMs by swapping modification genes between different pathways. Herein, by combining the cytochrome P450 (CYP) enzymes from different PoTeM pathways with the combamides' biosynthetic genes, the new combamides G (3), I (5), and J (6) along with the known combamides B (1), D (2), and H (4) were identified from the recombinant strains. Combamides G (3), H (4), and J (6) displayed cytotoxic activity against human cancer cell lines. Furthermore, our results demonstrated for the first time the substrate specificity of the PoTeM-related CYPs in vivo, which will facilitate the engineered biosynthesis of other PoTeMs in the future.

The coupled reaction catalyzed by EchB and EchC lead to the formation of the common 2',3',5'-trihydroxy-benzene core in echosides biosynthesis.

p-Terphenyls represent a unique family of aromatic natural products generated by nonribosomal peptide synthetase-like (NRPS-like) enzyme. After formation of p-terphenyl skeleton, tailoring modifications will give rise to structural diversity and various biological activities. Here we demonstrated a two-enzyme (EchB, a short-chain dehydrogenase/reductase (SDR), and EchC, a nuclear transport factor 2 (NTF2)-like dehydratase) participated transformation from dihydroxybenzoquinone core to 2',3',5'-trihydroxy-benzene in the biosynthesis of echosides. Beginning with polyporic acid as substrate, successive steps of reduction-dehydration-reduction cascade catalyzed by EchB-EchC-EchB were concluded after in vivo gene disruption and in vitro bioassay experiments. These findings demonstrated a conserved synthesis pathway of 2',3',5'-trihydroxy-p-terphenyls in bacteria, such as Actinomycetes and Burkholderia. The parallel pathway in fungi has yet to be explored.

Anterior gradient 2 increases long-chain fatty acid uptake via stabilizing FABP1 and facilitates lipid accumulation.

Anterior gradient 2 (AGR2), a protein disulfide isomerase (PDI), is a well-established oncogene. Here, we found that Agr2-/- mice had a decreased fat mass and hepatic and serum lipid levels compared with their wild-type littermates after fasting, and exhibited reduced high-fat diet (HFD)-induced fat accumulation. Transgenic mice overexpressing AGR2 (Agr2/Tg) readily gained fat weight on a HFD but not a normal diet. Proteomic analysis of hepatic samples from Agr2-/- mice revealed that depletion of AGR2 impaired long-chain fatty acid uptake and activation but did not affect de novo hepatic lipogenesis. Further investigations led to the identification of several effector substrates, particularly fatty acid binding protein-1 (FABP1) as essential for the AGR2-mediated effects. AGR2 was coexpressed with FABP1, and knockdown of AGR2 resulted in a reduction in FABP1 stability. Physical interactions of AGR2 and FABP1 depended on the PDI motif in AGR2 and the formation of a disulfide bond between these two proteins. Overexpression of AGR2 but not a mutant AGR2 protein lacking PDI activity suppressed lipid accumulation in cells lacking FABP1. Moreover, AGR2 deficiency significantly reduced fatty acid absorption in the intestine, which might be resulted from decreased fatty acid transporter CD36 in mice. These findings demonstrated a novel role of AGR2 in fatty-acid uptake and activation in both the liver and intestine, which contributed to the AGR2-mediated lipid accumulation, suggesting that AGR2 is an important regulator of whole-body lipid metabolism and down-regulation of AGR2 may antagonize the development of obesity.

Efficient preparation of c-di-AMP at gram-scale using an immobilized Vibrio cholerae dinucleotide cyclase DncV.

Cyclic di-AMP is a bacterial nucleotide second messenger and evaluated as a potential vaccine adjuvant candidate. Here, we report a practical and economical enzymatic method for gram-scale preparation of c-di-AMP using an immobilized Vibrio cholerae dinucleotide cyclase DncV. The method mainly includes four steps: preparation of DncV-immobilized resin, enzymatic synthesis of c-di-AMP, purification using macroporous absorption resin SP207, and desiccation using rotary evaporation and lyophilization. Enzymatic synthesis is the most critical step, and almost all substrate ATP was converted to c-di-AMP under an optimum condition in which 300 mL of 300 mM NH4Ac/NH3 pH 9.5 buffer supplemented with 20 mM MnCl2, 10 mM ATP and 4 mL of DncV-immobilized resin containing ∼19 mg DncV were incubated at 30 °C overnight. After purification, up to 1 g of the diammonium salt of c-di-AMP with weight purity of ≥98% was obtained as white powder, which corresponds to an overall yield of ∼80% based on the ATP input into the reaction. The method is easily performed in laboratory to prepare c-di-AMP on a gram scale and could be used in industry on a large scale.

Expression of the Clifednamide Biosynthetic Pathway in Streptomyces Generates 27,28-seco-Derivatives.

Polycyclic tetramate macrolactams (PoTeMs) are a group of hybrid PK-NRP natural products having a variable set of carbocyclic rings, a conserved assembly pathway, and diverse bioactivities. We report here the identification of seven new PoTeMs, clifednamides D-J (3-9), along with the known clifednamides A (1) and B (2) through rational pathway refactoring and heterologous expression. Remarkably, clifednamides D (3), G (6), and H (7) feature an unprecedented 27,28-seco skeleton. The cytotoxic activities of compounds 1-9 indicated that the hydroxy group of C-25, the methyl group of C-30, the inner five-membered ring, and the intact macrocycle are all critical for the activities. Meanwhile, the cytochrome P450 enzyme CftS023A and the hydroxylase CftS023E involved in oxidative tailoring of clifednamides were found to decorate the fused 5-6 bicyclic intermediates. Accordingly, the biosynthetic pathway for clifednamides was proposed.

Highly Efficient Preparation of Cyclic Dinucleotides via Engineering of Dinucleotide Cyclases in Escherichia coli.

Cyclic dinucleotides (CDNs) are widely used secondary signaling molecules in bacterial and mammalian cells. The family of CDNs includes c-di-GMP, c-di-AMP and two distinct versions of hybrid cGAMPs. Studies related to these CDNs require large doses that are relatively expensive to generate by current methods. Here we report what to our knowledge is the first feasible microbial-based method to prepare these CDNs including c-di-GMP, 3'3'-cGAMP and 2'3'-cGAMP. The method mainly includes two parts: producing high yield of CDNs by engineering the overexpression of the proper dinucleotide cyclases (DNCs) and other related proteins in Escherichia coli, and purifying the bacteria-produced CDNs by a unified and simple process involving a STING affinity column, macroporous adsorption resin and C18 reverse-phase liquid chromatography. After purification, we obtained the diammonium salts of c-di-GMP, 3'3'-cGAMP and 2'3'-cGAMP with weight purity of >99, >96, >99% and in yields of >68, >26, and >82 milligrams per liter of culture, respectively. This technological platform enables the production of CDNs from cheaper material, provides a sustainable source of CDNs for scientific investigation, and can easily be further developed to prepare CDNs on a large scale for industry.

Pro-metastatic activity of AGR2 interrupts angiogenesis target bevacizumab efficiency via direct interaction with VEGFA and activation of NF-κB pathway.

Anterior gradient 2 (AGR2), an endoplasmic reticulum (ER)-resident protein-disulfide isomerase (PDI), is associated with cancer development and malignant progression. Here, we show that high level of AGR2 promotes the aggressive phenotype of prostate cancer (PCa) mouse models developed by either patient-derived xenografts or surgical intra-prostate implantation of PCa cells, associated with enrichment of the blood vessel network in tumor tissues. Angiogenesis markers VEGFR2 and CD34, accompanied with the invasive marker Vimentin, were predominantly stained in metastatic liver tissues. Secreted AGR2 was defined to enhance VEGFR2 activity as evidenced by physical interaction of purified recombinant human AGR2 (rhAGR2) with rhVEGFA through the formation of a disulfide bond. Mutant or deleted thioredoxin motif in rhAGR2 was also unable to bind to rhVEGFA that led to the significant abolishment in the vessel formation, but partially affecting the aggressive process, implicating alternative mechanisms are required for AGR2-conferring metastasis. Cytosolic AGR2 contributed to cell metastasis ascribed to its stabilizing effect on p65 protein, which subsequently activated the NF-κB and facilitated epithelial to mesenchymal transition (EMT). Importantly, GSH and cabozantinib, but not bevacizumab, effectively blocked the pro-angiogenic effect of rhAGR2 in vitro and in vivo, providing evidence that secreted AGR2 acts as a predictive biomarker for selection of angiogenesis-targeting therapeutic drugs based on its levels in the circular system.

Downregulation of reticulocalbin-1 differentially facilitates apoptosis and necroptosis in human prostate cancer cells.

Reticulocalbin 1 (RCN1), an endoplasmic reticulum (ER)-resident Ca2+ -binding protein, is dysregulated in cancers, but its pathophysiological roles are largely unclear. Here, we demonstrate that RCN1 is overexpressed in clinical prostate cancer (PCa) samples, associated with cyclin B, not cyclin D1 expression, compared to that of benign tissues in a Chinese Han population. Downregulation of endogenous RCN1 significantly suppresses PCa cell viability and arrests the cell cycles of DU145 and LNCaP cells at the S and G2/M phases, respectively. RCN1 depletion causes ER stress, which is evidenced by induction of GRP78, activation of PERK and phosphorylation of eIF2α in PCa cells. Remarkably, RCN1 loss triggers DU145 cell apoptosis in a caspase-dependent manner but mainly causes necroptosis in LNCaP cells. An animal-based analysis confirms that RCN1 depletion suppresses cell proliferation and promotes cell death. Further investigations reveal that RCN1 depletion leads to elevation of phosphatase and tensin homolog (PTEN) and inactivation of AKT in DU145 cells. Silencing of PTEN partially restores apoptotic cells upon RCN1 loss. In LNCaP cells, predominant activation of CaMKII is important for necroptosis in response to RCN1 depletion. Thus, RCN1 may promote cell survival and serve as a useful target for cancer therapy.

Hyper-acetylation contributes to the sensitivity of chemo-resistant prostate cancer cells to histone deacetylase inhibitor Trichostatin A.

Therapeutic agents are urgently needed for treating metastatic castration-refractory prostate cancer (mCRPC) that is unresponsive to androgen deprivation and chemotherapy. Our screening assays demonstrated that chemotherapy-resistant prostate cancer (PCa) cells are more sensitive to HDAC inhibitors than paired sensitive PCa cells, as demonstrated by cell proliferation and apoptosis in vitro and in vivo. Kinetic study revealed that TSA-induced apoptosis was significantly dependent on enhanced transcription and protein synthesis in an early stage, which subsequently caused ER stress and apoptosis. ChIP analysis indicated that TSA increased H4K16 acetylation, promoting ER stress gene transcription. The changes in Ac-H4K16, ATF3 and ATF4 were also validated in TSA-treated animals. Further study revealed the higher enzyme activity of HDACs and an increase in acetylated proteins in resistant cells. The higher nucleocytoplasmic acetyl-CoA in resistant cells was responsible for elevated acetylation status of protein and a more vigorous growth state. These results strongly support the pre-clinical application of HDAC inhibitors for treating chemotherapy-resistant mCRPC.