The sulfhydryl group of Cys138 of rusticyanin from Acidithiobacillus ferrooxidans is crucial for copper binding

Rusticyanin is a small blue copper protein isolated from Acidithiobacillus ferrooxidans with extreme acid stability and redox potential. The protein is thought to be a principal component in the iron respiratory electron transport chain in this microorganism, but its exact role in electron transfer remains controversial. The gene of rusticyanin was cloned then overexpressed in Escherichia coli, the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. It was reported that Cys138, His85 and His143 were important residues for copper binding, but the significance of Cys138 was not verified so far. We constructed the mutant expression plasmids of these three residues using site-directed mutagenesis. Mutant proteins were expressed in E. coli and purified with a nickel metal affinity column. The EPR and atomic absorption spectroscopy results confirmed that Cys138 was crucial for copper binding. Removal of the sulfhydryl group of Cys138 resulted in copper loss. Mutations of His85 and His143 showed little effect on copper binding.     

Preparation and characterization of recombinant protein ScFv(CD11c)-TRP2 for tumor therapy from inclusion bodies in Escherichia coli

Dendritic cells (DCs)-based immunotherapy represents an approach to the prevention and treatment of cancers. Targeting antigens to receptors on DCs can be expected to enhance immune response. We have constructed an expression vector pET32a(+)-ScFv(CD11c)-TRP2 based on a single-chain antibody fragment (ScFv) that targets the high affinity receptor CD11c which is expressed on murine DCs. The 3'-terminal end of the ScFv was ligated to the gene for MHC class I molecule-recognized peptide from mouse tyrosine-related protein 2 (TRP2). Using this vector, we have expressed and purified ScFv(CD11c)-TRP2, a fusion protein that could target TRP2 peptide to CD11c on DCs in vivo to elicit anti-tumor responses. This fusion protein was expressed in inclusion bodies in Escherichia coli BL21(DE3) and was refolded and purified on-column effectively by immobilized metal affinity chromatography using His-tag. Flow cytometry assays showed the specific binding ability of ScFv(CD11c)-TRP2 to DCs, which could be blocked by a hamster anti-mouse CD11c produced by N418 hybridoma. Further studies demonstrated that ScFv(CD11c)-targeted TRP2 peptide processed by DCs was capable of stimulating T cells proliferation. Thus, this fusion protein provides a basis for further research in cancer therapy in vivo.     

Cordycepin enhances hyperthermia-induced apoptosis and cell cycle arrest by modulating the MAPK pathway in human lymphoma U937 cells

Molecular Biology Reports - Hyperthermia induces cancer cell death. However, the cytotoxic effect of hyperthermia is not sufficient. Cordycepin can also induce apoptosis in cancer cells and enhance...     

Synthesis and Glycogen Phosphorylase Inhibitory Activity of the Conjugates of 5-Chloroindoledicarboxylic Acid and Diazepine Derivatives

Russian Journal of Bioorganic Chemistry - In this work, three novel amide derivatives with chloroindole as the core were synthesized through different experimental schemes. Subsequently, biological...     

Mammalian STE20-like Kinase 1 Knockdown Attenuates TNFα-Mediated Neurodegenerative Disease by Repressing the JNK Pathway and Mitochondrial Stress

Neuroinflammation has been acknowledged as a primary factor contributing to the pathogenesis of neurodegenerative disease. However, the molecular mechanism underlying inflammation stress-mediated neuronal dysfunction is not fully understood. The aim of our study was to explore the influence of mammalian STE20-like kinase 1 (Mst1) in neuroinflammation using TNFα and CATH.a cells in vitro. The results of our study demonstrated that the expression of Mst1 was dose-dependently increased after TNFα treatment. Interestingly, knockdown of Mst1 using siRNA transfection significantly repressed TNFα-induced neuronal death. We also found that TNFα treatment was associated with mitochondrial stress, including mitochondrial ROS overloading, mitochondrial permeability transition pore (mPTP) opening, mitochondrial membrane potential reduction, and mitochondrial pro-apoptotic factor release. Interestingly, loss of Mst1 attenuated TNFα-triggered mitochondrial stress and sustained mitochondrial function in CATH.a cells. We found that Mst1 modulated mitochondrial homeostasis and cell viability via the JNK pathway in a TNFα-induced inflammatory environment. Inhibition of the JNK pathway abolished TNFα-mediated CATH.a cell death and mitochondrial malfunction, similar to the results obtained via silencing of Mst1. Taken together, our results indicate that inflammation-mediated neuronal dysfunction is implicated in Mst1 upregulation, which promotes mitochondrial stress and neuronal death by activating the JNK pathway. Accordingly, our study identifies the Mst1–JNK-mitochondria axis as a novel signaling pathway involved in neuroinflammation.

     

Modulation of miR-34a in curcumin-induced antiproliferation of prostate cancer cells

Curcumin is a phytochemical which exhibits significant inhibitory effect in multiple cancers including prostate cancer. MicroRNA-34a (miR-34a) was found to be a master tumor suppressor miRNA and regulated the growth of cancer cells. To date, however, the role of miR-34a in the anticancer action of curcumin against prostate cancer has been rarely reported. In the present study, we showed that curcumin altered the expression of cell cycle-related genes (cyclin D1, PCNA, and p21) and inhibited the proliferation of prostate cancer cells. Furthermore, we found that curcumin significantly upregulated the expression of miR-34a, along with the downregulated expression of β-catenin and c-myc in three prostate cancer cell lines. Inhibition of miR-34a activated β-catenin/c-myc axis, altered cell cycle-related genes expression and significantly suppressed the antiproliferation effect of curcumin in prostate cancer cells. Findings from this study revealed that miR-34a plays an important role in the antiproliferation effect of curcumin in prostate cancer.     

Bone marrow–derived mesenchymal stem cells–derived exosomes prevent oligodendrocyte apoptosis through exosomal miR-134 by targeting caspase-8

Ischemic stroke causes severe brain damage and remains one of the leading causes of morbidity and mortality worldwide. The microRNA-134 (miR-134) is involved in regulating the process of ischemia injury in neural cells and brain with ischemia stroke. The role of miR-134 in ischemic stroke remains poorly understood. The purpose of the current study was to investigate the effect of bone marrow–derived mesenchymal stem cells (BMSCs)-derived exosomal miR-134 on rat oligodendrocytes (OLs) apoptosis and its underlying mechanism of action. The results demonstrated that levels of miR-134 in BMSCs-exosome decreased but increased incaspase-8 after oxygen-glucose deprivation (OGD) treatment. Exosomal miR-134 significantly inhibited apoptosis by decreasing caspase-8 expression and activity in OGD-treated group cultured with BMSCs-exosome and OLs. In addition, the miR-134 mimics decreased caspase-8 expression in OGD-treated OLs, whereas miR-134 inhibitors exacerbated the changes in the expression of the procaspase-8 and caspase-8 cleaved product proteins caused by OGD. The caspase-8 knockdown using caspase-8 small interfering RNA decreased OLs apoptosis, reversing the improvements that the miR-134 inhibited cells apoptosis by targeting caspase-8. Taken together, these results demonstrated that BMSCs-derived exosomes suppressed OLs apoptosis through exosomal miR-134 by negatively regulating the caspase-8-dependent apoptosis pathway and may, therefore, be a novel potential therapeutic target for ischemic stroke treatment.     

LncRNA CRNDE promotes the epithelial-mesenchymal transition of hepatocellular carcinoma cells via enhancing the Wnt/β-catenin signaling pathway

Colorectal neoplasia differentially expressed (CRNDE) is a significantly upregulated long noncoding RNA in hepatocellular carcinoma (HCC). CRNDE could promote cell proliferation, migration, and invasion, while its molecular mechanisms were still largely unclear. In this study, we investigated the expression and function of CRNDE. CRNDE was significantly upregulated in tumor tissues compared with adjacent normal tissues. In vitro, we revealed that knockdown of CRNDE inhibited cell proliferation, migration, and cell invasion capacities in HCC. Animal studies indicated that CRNDE knockdown represses both growth and metastasis of HCC tumors in vivo. Moreover, knockdown of CRNDE suppressed the cell epithelial-mesenchymal transition (EMT) process by increasing the expression of E-cadherin and ZO-1, whereas, decreasing the expression of N-cadherin, slug, twist, and vimentin in HCC cells. We also revealed that knockdown of CRNDE suppressed the Wnt/β-catenin signaling in HCC. Thus, CRNDE could modulate EMT of HCC cells and knockdown of CRNDE impaired the mesenchymal properties. CRNDE increased invasion of HCC cells might be through activating the Wnt/β-catenin signaling pathway.     

CPT1A‐mediated succinylation of S100A10 increases human gastric cancer invasion

Gastric cancer (GC) is a malignancy of the lining of the stomach and is prone to distant metastasis, which involves a variety of complex molecules. The S100 proteins are a family of calcium‐binding cytosolic proteins that possess a wide range of intracellular and extracellular functions and play pivotal roles in the invasion and migration of tumour cells. Among these, S100A10 is known to be overexpressed in GC. Lysine succinylation, a recently identified form of protein post‐translational modification, is an important regulator of cellular processes. Here, we demonstrated that S100A10 was succinylated at lysine residue 47 (K47), and levels of succinylated S100A10 were increased in human GC. Moreover, K47 succinylation of S100A10 was stabilized by suppression of ubiquitylation and subsequent proteasomal degradation. Furthermore, carnitine palmitoyltransferase 1A (CPT1A) was found to function as a lysine succinyltransferase that interacts with S100A10. Succinylation of S100A10 is regulated by CPT1A, while desuccinylation is regulated by SIRT5. Overexpression of a succinylation mimetic mutant, K47E S100A10, increased cell invasion and migration. Taken together, this study reveals a novel mechanism of S100A10 accumulation mediated by succinylation in GC, which promotes GC progression and is regulated by the succinyltransferase CPT1A and SIRT5‐mediated desuccinylation.