ABSTRACT
Carbon dioxide (CO2) emissions constitute the primary contribution to global climate change. Synthetic CO2 fixation represents an exceptionally appealing and sustainable method for carbon neutralization. Unlike the limitations of chemical catalysis, biological CO2 fixation displays high selectivity and the ability to operate under mild conditions. The superfamily of amidohydrolases has demonstrated the ability to synthesize a range of aromatic monocarboxylic acids. However, there is a scarcity of reported carboxylases capable of synthesizing aromatic dicarboxylic acids. Among these, 4-hydroxyisophthalic acid holds significant potential for applications across various fields, yet no enzyme has been reported for its synthesis. In this study, we developed for the first time that exhibits starting activity in fixing CO2 to synthesize 4-hydroxyisophthalic acid. Furthermore, we have devised a computational strategy that effectively enhances the catalytic activity of this enzyme. A focused library comprising only 13 variants was generated. Experimental validation confirmed a threefold improvement in the carboxylation activity of the optimal variant (L47M). The computational enzyme design strategy proposed in this paper demonstrates broad applicability in developing carboxylases for synthesizing other aromatic dicarboxylic acids. This lays the groundwork for leveraging biocatalysis in industrial synthesis for CO2 fixation.
Subject(s)
Carbon Dioxide , Phthalic Acids , Carbon Dioxide/chemistry , Carbon Dioxide/metabolism , Phthalic Acids/chemistry , BiocatalysisABSTRACT
This study investigated the mechanism by which fucoxanthin acts as a novel ferroptosis inducer to inhibit tongue cancer. The MTT assay was used to detect the inhibitory effects of fucoxanthin on SCC-25 human tongue squamous carcinoma cells. The levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), and total iron were measured. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to assess glutathione peroxidase 4 (GPX4), nuclear factor erythroid 2-related factor 2 (Nrf2), Keap1, solute carrier family 7 member 11 (SLC7A11), transferrin receptor protein 1 (TFR1), p53, and heme oxygenase 1 (HO-1) expression. Molecular docking was performed to validate interactions. Compared with the control group, the activity of fucoxanthin-treated SCC-25 cells significantly decreased in a dose- and time-dependent manner. The levels of MMP, GSH, and SOD significantly decreased in fucoxanthin-treated SCC-25 cells; the levels of ROS, MDA, and total iron significantly increased. mRNA and protein expression levels of Keap1, GPX4, Nrf2, and HO-1 in fucoxanthin-treated cells were significantly decreased, whereas levels of TFR1 and p53 were significantly increased, in a concentration-dependent manner. Molecular docking analysis revealed that binding free energies of fucoxanthin with p53, SLC7A11, GPX4, Nrf2, Keap1, HO-1, and TFR1 were below -5 kcal/mol, primarily based on active site hydrogen bonding. Our findings suggest that fucoxanthin can induce ferroptosis in SCC-25 cells, highlighting its potential as a treatment for tongue cancer.
Subject(s)
Ferroptosis , Heme Oxygenase-1 , Molecular Docking Simulation , NF-E2-Related Factor 2 , Phospholipid Hydroperoxide Glutathione Peroxidase , Xanthophylls , Humans , NF-E2-Related Factor 2/metabolism , Ferroptosis/drug effects , Xanthophylls/pharmacology , Xanthophylls/chemistry , Heme Oxygenase-1/metabolism , Heme Oxygenase-1/genetics , Cell Line, Tumor , Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , Tongue Neoplasms/drug therapy , Tongue Neoplasms/metabolism , Tongue Neoplasms/pathology , Receptors, Transferrin/metabolism , Membrane Potential, Mitochondrial/drug effects , Kelch-Like ECH-Associated Protein 1/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Amino Acid Transport System y+/metabolism , Amino Acid Transport System y+/genetics , Superoxide Dismutase/metabolism , Down-Regulation/drug effects , Antigens, CDABSTRACT
Pancreatoblastoma (PB) is a rare malignant pancreatic epithelial tumor that mostly occurs in children and occasionally occurs in adults. The tumor has acinar cell differentiation and squamous corpuscles/squamous epithelial islands, which are frequently separated by fibrous bundles. Familial adenomatous polyposis (FAP) is an autosomal dominant inherited disease characterized by the presence of numerous adenomatous polyps in the colon and rectum. Cases of pancreatoblastoma combined with familial adenomatous polyposis (FAP) are rarely reported. A review of a rare case of adult pancreatoblastoma with atypical histological morphology combined with familial adenomatous polyposis is presented herein. In this case, the patient was first diagnosed with familial adenomatous polyposis and subsequently found to have pancreatoblastoma 1 year and 3 months later. This suggests pancreatoblastoma may occur in patients with familial adenomatous polyposis or a family history of the condition, indicating a possible association between the two tumors. Therefore, pancreatoblastoma should be included in a differential diagnosis for FAP patients with a pancreatic mass. The final diagnosis of pancreatoblastoma depends on the pathological diagnosis. Acinar-like cells and squamous corpuscles/squamous epithelial cell islands under light microscopy are the key diagnostic points. This case report also can improve the awareness of clinicians, radiologists, and pathologists on the presence of rare tumor-adult pancreatoblastoma in patients with familial adenomatous polyposis.
ABSTRACT
Thermally stable proteins find extensive applications in industrial production, pharmaceutical development, and serve as a highly evolved starting point in protein engineering. The thermal stability of proteins is commonly characterized by their melting temperature (Tm). However, due to the limited availability of experimentally determined Tm data and the insufficient accuracy of existing computational methods in predicting Tm, there is an urgent need for a computational approach to accurately forecast the Tm values of thermophilic proteins. Here, we present a deep learning-based model, called DeepTM, which exclusively utilizes protein sequences as input and accurately predicts the Tm values of target thermophilic proteins on a dataset consisting of 7790 thermophilic protein entries. On a test set of 1550 samples, DeepTM demonstrates excellent performance with a coefficient of determination (R2) of 0.75, Pearson correlation coefficient (P) of 0.87, and root mean square error (RMSE) of 6.24â¯â. We further analyzed the sequence features that determine the thermal stability of thermophilic proteins and found that dipeptide frequency, optimal growth temperature (OGT) of the host organisms, and the evolutionary information of the protein significantly affect its melting temperature. We compared the performance of DeepTM with recently reported methods, ProTstab2 and DeepSTABp, in predicting the Tm values on two blind test datasets. One dataset comprised 22 PET plastic-degrading enzymes, while the other included 29 thermally stable proteins of broader classification. In the PET plastic-degrading enzyme dataset, DeepTM achieved RMSE of 8.25â¯â. Compared to ProTstab2 (20.05â¯â) and DeepSTABp (20.97â¯â), DeepTM demonstrated a reduction in RMSE of 58.85% and 60.66%, respectively. In the dataset of thermally stable proteins, DeepTM (RMSE=7.66â¯â) demonstrated a 51.73% reduction in RMSE compared to ProTstab2 (RMSE=15.87â¯â). DeepTM, with the sole requirement of protein sequence information, accurately predicts the melting temperature and achieves a fully end-to-end prediction process, thus providing enhanced convenience and expediency for further protein engineering.
ABSTRACT
Recently developed enzymes for the depolymerization of polyethylene terephthalate (PET) such as FAST-PETase and LCC-ICCG are inhibited by the intermediate PET product mono(2-hydroxyethyl) terephthalate (MHET). Consequently, the conversion of PET enzymatically into its constituent monomers terephthalic acid (TPA) and ethylene glycol (EG) is inefficient. In this study, a protein scaffold (1TQH) corresponding to a thermophilic carboxylesterase (Est30) was selected from the structural database and redesigned in silico. Among designs, a double variant KL-MHETase (I171K/G130L) with a similar protein melting temperature (67.58 °C) to that of the PET hydrolase FAST-PETase (67.80 °C) exhibited a 67-fold higher activity for MHET hydrolysis than FAST-PETase. A fused dual enzyme system comprising KL-MHETase and FAST-PETase exhibited a 2.6-fold faster PET depolymerization rate than FAST-PETase alone. Synergy increased the yield of TPA by 1.64 fold, and its purity in the released aromatic products reached 99.5%. In large reaction systems with 100 g/L substrate concentrations, the dual enzyme system KL36F achieved over 90% PET depolymerization into monomers, demonstrating its potential applicability in the industrial recycling of PET plastics. Therefore, a dual enzyme system can greatly reduce the reaction and separation cost for sustainable enzymatic PET recycling.
Subject(s)
Hydrolases , Polyethylene Terephthalates , Hydrolases/chemistry , Polyethylene Terephthalates/chemistry , Polyethylene Terephthalates/metabolism , Hydrolysis , Carboxylesterase , Plastics/chemistryABSTRACT
2D magnetic materials have been of interest due to their unique long-range magnetic ordering in the low-dimensional regime and potential applications in spintronics. Currently, most studies are focused on strippable van der Waals magnetic materials with layered structures, which typically suffer from a poor stability and scarce species. Spinel oxides have a good environmental stability and rich magnetic properties. However, the isotropic bonding and close-packed nonlayered crystal structure make their 2D growth challenging, let alone the phase engineering. Herein, a phase-controllable synthesis of 2D single-crystalline spinel-type oxides is reported. Using the van der Waals epitaxy strategy, the thicknesses of the obtained tetragonal and hexagonal manganese oxide (Mn3 O4 ) nanosheets can be tuned down to 7.1 nm and one unit cell (0.7 nm), respectively. The magnetic properties of these two phases are evaluated using vibrating-sample magnetometry and first-principle calculations. Both structures exhibit a Curie temperature of 48 K. Owing to its ultrathin geometry, the Mn3 O4 nanosheet exhibits a superior ultraviolet detection performance with an ultralow noise power density of 0.126 pA Hz-1/2 . This study broadens the range of 2D magnetic semiconductors and highlights their potential applications in future information devices.
ABSTRACT
Mitochondrial biogenesis is the process of generating new mitochondria to maintain cellular homeostasis. Here, we report that viruses exploit mitochondrial biogenesis to antagonize innate antiviral immunity. We found that nuclear respiratory factor-1 (NRF1), a vital transcriptional factor involved in nuclear-mitochondrial interactions, is essential for RNA (VSV) or DNA (HSV-1) virus-induced mitochondrial biogenesis. NRF1 deficiency resulted in enhanced innate immunity, a diminished viral load, and morbidity in mice. Mechanistically, the inhibition of NRF1-mediated mitochondrial biogenesis aggravated virus-induced mitochondrial damage, promoted the release of mitochondrial DNA (mtDNA), increased the production of mitochondrial reactive oxygen species (mtROS), and activated the innate immune response. Notably, virus-activated kinase TBK1 phosphorylated NRF1 at Ser318 and thereby triggered the inactivation of the NRF1-TFAM axis during HSV-1 infection. A knock-in (KI) strategy that mimicked TBK1-NRF1 signaling revealed that interrupting the TBK1-NRF1 connection ablated mtDNA release and thereby attenuated the HSV-1-induced innate antiviral response. Our study reveals a previously unidentified antiviral mechanism that utilizes a NRF1-mediated negative feedback loop to modulate mitochondrial biogenesis and antagonize innate immune response.
Subject(s)
Antiviral Agents , Organelle Biogenesis , Animals , Mice , DNA, Mitochondrial/genetics , Immunity, Innate , Nuclear Respiratory Factor 1/geneticsABSTRACT
Ectopic lipid deposition and mitochondrial dysfunction are common etiologies of obesity and metabolic disorders. Excessive dietary uptake of saturated fatty acids (SFAs) causes mitochondrial dysfunction and metabolic disorders, while unsaturated fatty acids (UFAs) counterbalance these detrimental effects. It remains elusive how SFAs and UFAs differentially signal toward mitochondria for mitochondrial performance. We report here that saturated dietary fatty acids such as palmitic acid (PA), but not unsaturated oleic acid (OA), increase lysophosphatidylinositol (LPI) production to impact on the stability of the mitophagy receptor FUNDC1 and on mitochondrial quality. Mechanistically, PA shifts FUNDC1 from dimer to monomer via enhanced production of LPI. Monomeric FUNDC1 shows increased acetylation at K104 due to dissociation of HDAC3 and increased interaction with Tip60. Acetylated FUNDC1 can be further ubiquitinated by MARCH5 for proteasomal degradation. Conversely, OA antagonizes PA-induced accumulation of LPI, and FUNDC1 monomerization and degradation. A fructose-, palmitate-, and cholesterol-enriched (FPC) diet also affects FUNDC1 dimerization and promotes its degradation in a non-alcoholic steatohepatitis (NASH) mouse model. We thus uncover a signaling pathway that orchestrates lipid metabolism with mitochondrial quality.
Subject(s)
Fatty Acids , Mitophagy , Mice , Animals , Fatty Acids/metabolism , Dimerization , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Membrane Proteins/metabolismABSTRACT
OBJECTIVE: To explore the clinicopathological features of solitary fibrous tumors (SFTs) of the lung. METHODS: We collected the clinical data of 52 patients with SFTs of the lung confirmed by pathology, and summarized the clinical, radiological, and morphological features, the immunophenotypes, and the prognosis of SFTs. RESULTS: Fifty-two cases of SFTs of the lung were enrolled in this study, including 51 cases of borderline and 1 case of malignancy, 22 males and 30 females. The average onset age was 52.7 years. The lower lobe of the left lung was the preferred site of SFTs, accounting for 30.77% (16/52). Chest CT showed regular and well-demarcated soft tissue density mass, and the tumor size of most cases (46/52, 88.46%) was 1-10 cm. Morphological features: The distribution of tumor cells showed sparse and dense areas. Tumor cells were spindle-shaped, in whorls or hemangiopericytoma-like conformation. Atypia, mitotic figures, and necrosis were found. Immunohistochemistry showed positive expression of CD34, STAT6, Vimentin, BCL2, and CD99. Ki-67 was ≤ 5% in borderline SFTs and >20% in a malignant SFT. CONCLUSIONS: Solitary fibrous tumors of the lung occur more frequently in middle-aged and elderly people, and there is no significant difference in gender. The lower lobe of the left lung is the preferred site of SFTs. The size of most SFTs is 1-10 cm. Chest CT shows morphologically regular and well-demarcated soft tissue density mass. Pathologically, SFTs of the lung are mostly borderline and occasionally malignant. Immunohistochemistry shows the positive expression of CD34, STAT6, Vimentin, BCL2, and CD99.
Subject(s)
Severe Fever with Thrombocytopenia Syndrome , Solitary Fibrous Tumors , Aged , Middle Aged , Male , Female , Humans , Vimentin , Biomarkers, Tumor/analysis , Solitary Fibrous Tumors/genetics , Solitary Fibrous Tumors/metabolism , Solitary Fibrous Tumors/pathology , Lung/pathology , Proto-Oncogene Proteins c-bcl-2ABSTRACT
Computational enzyme design has been successfully applied to identify new alternatives to natural enzymes for the biosynthesis of important compounds. However, the moderate catalytic activities of de novo designed enzymes indicate that the modeling accuracy of current computational enzyme design methods should be improved. Here, high-throughput molecular dynamics simulations were used to enhance computational enzyme design, thus allowing the identification of variants with higher activities in silico. Different time schemes of high-throughput molecular dynamics simulations were tested to identify the catalytic features of evolved Kemp eliminases. The 20 × 1 ns molecular dynamics simulation scheme was sufficiently accurate and computationally viable to screen the computationally designed massive variants of Kemp elimination enzymes. The developed hybrid computational strategy was used to redesign the most active Kemp eliminase, HG3.17, and five variants were generated and experimentally confirmed to afford higher catalytic efficiencies than that of HG3.17, with one double variant (D52Q/A53S) exhibiting a 55% increase. The hybrid computational enzyme design strategy is general and computationally economical, with which we anticipate the efficient creation of practical enzymes for industrial biocatalysis.
Subject(s)
Enzymes , Molecular Dynamics Simulation , Biocatalysis , Catalysis , Enzymes/metabolism , Protein EngineeringABSTRACT
In mammalian cells, the Golgi apparatus serves as the central hub for membrane trafficking. Notably, the membrane trafficking and Golgi integrity are tightly regulated by reversible post-translational modifications, such as glycosylation, phosphorylation and ubiquitination. Nonetheless, how the Golgi apparatus responses to stress to ensure appropriate membrane assembly and distribution of cargo is poorly understood. The Golgi resident protein ATG9A is the only multi-spanning membrane protein in the ATG family and has been demonstrated to traffic through the plasma membrane, endosomes and Golgi to deliver materials for the initiation of macroautophagy/autophagy. Our recent work reveals a noncanonical function of ATG9A for Golgi dynamics and identifies a pathway for sensing Golgi stress via the MARCHF9-ATG9A axis.
Subject(s)
Autophagy , Vesicular Transport Proteins , Animals , Vesicular Transport Proteins/metabolism , Protein Transport , Membrane Proteins/metabolism , Golgi Apparatus/metabolism , Autophagy-Related Proteins/metabolism , Mammals/metabolismABSTRACT
ATG9A is a highly conserved membrane protein required for autophagy initiation. It is trafficked from the trans-Golgi network (TGN) to the phagophore to act as a membrane source for autophagosome expansion. Here, we show that ATG9A is not just a passenger protein in the TGN but rather works in concert with GRASP55, a stacking factor for Golgi structure, to organize Golgi dynamics and integrity. Upon heat stress, the E3 ubiquitin ligase MARCH9 is promoted to ubiquitinate ATG9A in the form of K63 conjugation, and the nondegradable ubiquitinated ATG9A disperses from the Golgi apparatus to the cytoplasm more intensely, accompanied by inhibiting GRASP55 oligomerization, further resulting in Golgi fragmentation. Knockout of ATG9A or MARCH9 largely prevents Golgi fragmentation and protects Golgi functions under heat and other Golgi stresses. Our results reveal a noncanonical function of ATG9A for Golgi dynamics and suggest the pathway for sensing Golgi stress via the MARCH9/ATG9A axis.
Subject(s)
Autophagosomes , Golgi Apparatus , Autophagosomes/metabolism , Autophagy , Autophagy-Related Proteins/metabolism , Golgi Apparatus/metabolism , Protein Transport , Ubiquitin/metabolism , trans-Golgi Network/metabolismABSTRACT
DBC1 has been characterized as a key regulator of physiological and pathophysiological activities, such as DNA damage, senescence, and tumorigenesis. However, the mechanism by which the functional stability of DBC1 is regulated has yet to be elucidated. Here, we report that the ubiquitination-mediated degradation of DBC1 is regulated by the E3 ubiquitin ligase SIAH2 and deubiquitinase OTUD5 under hypoxic stress. Mechanistically, hypoxia promoted DBC1 to interact with SIAH2 but not OTUD5, resulting in the ubiquitination and subsequent degradation of DBC1 through the ubiquitin-proteasome pathway. SIAH2 knockout inhibited tumor cell proliferation and migration, which could be rescued by double knockout of SIAH2/CCAR2. Human tissue microarray analysis further revealed that the SIAH2/DBC1 axis was responsible for tumor progression under hypoxic stress. These findings define a key role of the hypoxia-mediated SIAH2-DBC1 pathway in the progression of human breast cancer and provide novel insights into the metastatic mechanism of breast cancer.
Subject(s)
Breast Neoplasms , Adaptor Proteins, Signal Transducing/metabolism , Breast/metabolism , Breast Neoplasms/pathology , Female , Humans , Hypoxia/metabolism , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
A preorganization oriented computational strategy for de novo enzyme design based on computational enzyme design tool PRODA was developed and demonstrated by the creation of Kemp elimination enzymes. A pre-organized active site model of proton transfer from carbon with a low energy barrier was proposed and then anchored into the scaffold 3AOF, the endoglucanase from Thermotoga maritima, which was selected from the protein structural database. The low-energy amino acid sequences at the binding pocket to stabilize the catalytic productive geometry were computationally generated via the iterative protein redesign and molecular dynamics simulation. The designed variant (3AOF-KE03) bearing 17 mutations was experimentally confirmed to afford catalytic activity (kcat/KM=14.04M-1s-1) towards Kemp elimination, with measured rate (kcat=0.033s-1) enhancement of up to 104-fold. This computational strategy is general, and we anticipate the creation of a wide range of artificial enzymes to catalyze reactions with industrial significance in the future.
Subject(s)
Molecular Dynamics Simulation , Protons , Catalysis , Catalytic Domain , Enzymes/metabolismABSTRACT
Lignin is a highly complex phenolic polymer which is essential for plants, but also makes it difficult for industrial processing. Engineering lignin by introducing relatively labile linkages into the lignin backbone can render it more amenable to chemical depolymerization. It has been reported that introducing a feruloyl-coenzyme A monolignol transferase from Angelica sinensis (AsFMT) into poplar could incorporate monolignol ferulate conjugates (ML-FAs) into lignin polymers, suggesting a promising way to manipulate plants for readily deconstructing. FMT catalyzes a reaction between monolignols and feruloyl-CoA to produce ML-FAs and free CoA-SH. However, the mechanisms of substrate specificity and catalytic process of FMT remains poorly understood. Here we report the structure of AsFMT, which adopts a typical fold of BAHD acyltransferase family. Structural comparisons with other BAHD homologs reveal several unique structural features of AsFMT, different from those of the BAHD homologs. Further molecular docking studies showed that T375 in AsFMT may function as an oxyanion hole to stabilize the reaction intermediate and also proposed a role of H278 in the binding of the nucleophilic hydroxyl group of monolignols. Together, this study provides important structural insights into the reactions catalyzed by AsFMT and will shed light on its future application in lignin engineering.
Subject(s)
Acyl Coenzyme A/chemistry , Aldehyde Oxidoreductases/chemistry , Angelica/enzymology , Oxidoreductases/chemistry , Catalysis , Catalytic Domain , Crystallography, X-Ray , Lignin/chemistry , Molecular Docking Simulation , Protein Binding , Protein Conformation , Protein Domains , Substrate Specificity , Transferases/metabolism , UltracentrifugationABSTRACT
The transcription factor EB (TFEB) plays a critical role in autophagy induction and lysosomal biogenesis by orchestrating the expression of autophagy- and lysosome-related genes. In response to a series of stresses such as nutrient starvation, TFEB translocates from the cytoplasm to the nucleus, where it exerts its regulatory function. The activity of TFEB is tightly regulated by multiple phosphorylation and acetylation sites. Methods that rely on the analysis of posttranslational modification as a proxy for TFEB activation are often misleading. Here, we elaborate on protocols for monitoring nuclear translocation of TFEB by fluorescence microscopy.
Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Lysosomes , Autophagy , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Cell Nucleus/genetics , Cell Nucleus/metabolism , Lysosomes/metabolism , Phosphorylation , Protein TransportABSTRACT
A humanized antiTolllike receptor 4 (TLR4) monoclonal antibody (mAb) was previously produced using phage antibody library technology, and it was found that the mAb could effectively ameliorate lipopolysaccharide (LPS)induced damage in macrophages. The present study investigated the protective effects exerted by the humanized antiTLR4 mAb against LPSinduced acute kidney injury (AKI), as well as the underlying mechanisms. Female C57BL/6 mice were randomly divided into four groups (n=8 per group): i) Control; ii) LPS; iii) LPS + humanized antiTLR4 mAb (1 µg/g); and iv) LPS + humanized antiTLR4 mAb (10 µg/g). Serum creatinine, blood urea nitrogen, IL6, TNFα and IL1ß levels were then examined, followed by renal pathology assessment, immunohistochemical staining, reverse transcriptionquantitative PCR and western blotting to assess apoptosis/survival/inflammationrelated molecules and kidney injury molecule (KIM)1. The humanized antiTLR4 mAb successfully ameliorated LPSinduced AKI and renal pathological damage. The humanized antiTLR4 mAb also dosedependently suppressed LPSinduced elevations in serum IL6, TNFα and IL1ß, and decreased the renal expression levels of myeloid differentiation primary response 88 (MyD88), IKKα/ß, IκB, p65 and KIM1. Compared with the LPS group, renal Bax and KIM1 expression levels were significantly downregulated, and Bcl2 expression was notably upregulated by the humanized antiTLR4 mAb. Moreover, the humanized antiTLR4 mAb also significantly decreased the protein expression levels of MyD88, phosphorylated (p)IKKα/ß, pIκB and pp65 in the renal tissues compared with the LPS group. Therefore, the present study indicated that the antiinflammatory effects of the humanized antiTLR4 mAb against LPSrelated AKI in mice were mediated via inhibition of the TLR4/NFκB signaling pathway.
Subject(s)
Acute Kidney Injury/drug therapy , Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Monoclonal, Humanized/therapeutic use , NF-kappa B/antagonists & inhibitors , Protective Agents/pharmacology , Protective Agents/therapeutic use , Toll-Like Receptor 4/immunology , Acute Kidney Injury/chemically induced , Acute Kidney Injury/pathology , Animals , Cytokines/blood , Disease Models, Animal , Female , Hepatitis A Virus Cellular Receptor 1/metabolism , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , I-kappa B Proteins/genetics , I-kappa B Proteins/metabolism , Lipopolysaccharides/toxicity , Mice, Inbred C57BL , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Signal Transduction/drug effects , Transcription Factor RelA/genetics , Transcription Factor RelA/metabolism , bcl-2-Associated X Protein/metabolismABSTRACT
BACKGROUND/AIMS: Diabetic nephropathy (DN) is one of the main causes of end-stage kidney disease worldwide. Emerging studies have suggested that its pathogenesis is distinct from nondiabetic renal diseases in many aspects. However, it still lacks a comprehensive understanding of the unique molecular mechanism of DN. METHODS: A total of 255 Affymetrix U133 microarray datasets (Affymetrix, Santa Calra, CA, USA) of human glomerular and tubulointerstitial tissues were collected. The 22 215 Affymetrix identifiers shared by the Human Genome U133 Plus 2.0 and U133A Array were extracted to facilitate dataset pooling. Next, a linear model was constructed and the empirical Bayes method was used to select the differentially expressed genes (DEGs) of each kidney disease. Based on these DEG sets, the unique DEGs of DN were identified and further analyzed using gene ontology and pathway enrichment analysis. Finally, the protein-protein interaction networks (PINs) were constructed and hub genes were selected to further refine the results. RESULTS: A total of 129 and 1251 unique DEGs were identified in the diabetic glomerulus (upregulated n = 83 and downregulated n = 203) and the diabetic tubulointerstitium (upregulated n = 399 and downregulated n = 874), respectively. Enrichment analysis revealed that the DEGs in the diabetic glomerulus were significantly associated with the extracellular matrix, cell growth, regulation of blood coagulation, cholesterol homeostasis, intrinsic apoptotic signaling pathway and renal filtration cell differentiation. In the diabetic tubulointerstitium, the significantly enriched biological processes and pathways included metabolism, the advanced glycation end products-receptor for advanced glycation end products signaling pathway in diabetic complications, the epidermal growth factor receptor (EGFR) signaling pathway, the FoxO signaling pathway, autophagy and ferroptosis. By constructing PINs, several nodes, such as AGR2, CSNK2A1, EGFR and HSPD1, were identified as hub genes, which might play key roles in regulating the development of DN. CONCLUSIONS: Our study not only reveals the unique molecular mechanism of DN but also provides a valuable resource for biomarker and therapeutic target discovery. Some of our findings are promising and should be explored in future work.
ABSTRACT
Erythropoietin (EPO) drives erythropoiesis and is secreted mainly by the kidney upon hypoxic or anemic stress. The paucity of EPO production in renal EPO-producing cells (REPs) causes renal anemia, one of the most common complications of chronic nephropathies. Although mitochondrial dysfunction is commonly observed in several renal and hematopoietic disorders, the mechanism by which mitochondrial quality control impacts renal anemia remains elusive. In this study, we showed that FUNDC1, a mitophagy receptor, plays a critical role in EPO-driven erythropoiesis induced by stresses. Mechanistically, EPO production is impaired in REPs in Fundc1-/- mice upon stresses, and the impairment is caused by the accumulation of damaged mitochondria, which consequently leads to the elevation of the reactive oxygen species (ROS) level and triggers inflammatory responses by up-regulating proinflammatory cytokines. These inflammatory factors promote the myofibroblastic transformation of REPs, resulting in the reduction of EPO production. We therefore provide a link between aberrant mitophagy and deficient EPO generation in renal anemia. Our results also suggest that the mitochondrial quality control safeguards REPs under stresses, which may serve as a potential therapeutic strategy for the treatment of renal anemia.
Subject(s)
Anemia/prevention & control , Erythropoietin/metabolism , Gene Expression Regulation , Kidney Diseases/prevention & control , Membrane Proteins/genetics , Mitochondrial Proteins/genetics , Mitophagy/genetics , Animals , Erythropoiesis/genetics , Erythropoiesis/physiology , Erythropoietin/analysis , Erythropoietin/genetics , Kidney Diseases/classification , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mitochondrial Proteins/metabolism , Mitophagy/physiology , Reactive Oxygen SpeciesABSTRACT
Mitophagy is an essential cellular autophagic process that selectively removes superfluous and damaged mitochondria, and it is coordinated with mitochondrial biogenesis to fine tune the quantity and quality of mitochondria. Coordination between these two opposing processes to maintain the functional mitochondrial network is of paramount importance for normal cellular and organismal metabolism. However, the underlying mechanism is not completely understood. Here we report that PGC-1α and nuclear respiratory factor 1 (NRF1), master regulators of mitochondrial biogenesis and metabolic adaptation, also transcriptionally upregulate the gene encoding FUNDC1, a previously characterized mitophagy receptor, in response to cold stress in brown fat tissue. NRF1 binds to the classic consensus site in the promoter of Fundc1 to upregulate its expression and to enhance mitophagy through its interaction with LC3. Specific knockout of Fundc1 in BAT results in reduced mitochondrial turnover and accumulation of functionally compromised mitochondria, leading to impaired adaptive thermogenesis. Our results demonstrate that FUNDC1-dependent mitophagy is directly coupled with mitochondrial biogenesis through the PGC-1α/NRF1 pathway, which dictates mitochondrial quantity, quality, and turnover and contributes to adaptive thermogenesis.