The GNAQ T96S Mutation Affects Cell Signaling and Enhances the Oncogenic Properties of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC), the most common malignant tumor in the liver, grows and metastasizes rapidly. Despite advances in treatment modalities, the five-year survival rate of HCC remains less than 30%. We sought genetic mutations that may affect the oncogenic properties of HCC, using The Cancer Genome Atlas (TCGA) data analysis. We found that the GNAQ T96S mutation (threonine 96 to serine alteration of the Gαq protein) was present in 12 out of 373 HCC patients (3.2%). To examine the effect of the GNAQ T96S mutation on HCC, we transfected the SK-Hep-1 cell line with the wild-type or the mutant GNAQ T96S expression vector. Transfection with the wild-type GNAQ expression vector enhanced anchorage-independent growth, migration, and the MAPK pathways in the SK-Hep-1 cells compared to control vector transfection. Moreover, cell proliferation, anchorage-independent growth, migration, and the MAPK pathways were further enhanced in the SK-Hep-1 cells transfected with the GNAQ T96S expression vector compared to the wild-type GNAQ-transfected cells. In silico structural analysis shows that the substitution of the GNAQ amino acid threonine 96 with a serine may destabilize the interaction between the regulator of G protein signaling (RGS) protein and GNAQ. This may reduce the inhibitory effect of RGS on GNAQ signaling, enhancing the GNAQ signaling pathway. Single nucleotide polymorphism (SNP) genotyping analysis for Korean HCC patients shows that the GNAQ T96S mutation was found in only one of the 456 patients (0.22%). Our data suggest that the GNAQ T96S hotspot mutation may play an oncogenic role in HCC by potentiating the GNAQ signal transduction pathway.     

剩余污泥资源化提取蛋白质方法的优选

剩余污泥中蛋白质的资源化利用是目前研究的热点,污泥预处理则是实现污泥中蛋白质释放的重要途径。为了进一步提高剩余污泥中蛋白质的溶出效果,选取热碱预处理、超声碱联合预处理、溶菌酶预处理对污泥进行溶胞,以蛋白质提取浓度为主要指标进行参数优化,并利用等电点法对粗提取蛋白进行纯化回收。结果表明：溶胞效果热碱预处理（pH值13、温度140℃、时间1.5 h,2 062.98 mg/L）>超声碱联合预处理（497.76 mg/L）>溶菌酶预处理（269.95 mg/L）,且在pH值为3时热碱预处理蛋白质纯化回收率可达62.42%。试验结果表明：热碱预处理在提取效果方面较另外两种方法优势明显,具有良好的利用前景。     

Screening of Inhibitors Targeting Heat Shock Protein 47 Involved in the Development of Idiopathic Pulmonary Fibrosis

Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is causally related to fibrotic diseases, including idiopathic pulmonary fibrosis. The identification of Compounds that interfere with the HSP47-collagen interaction is essential for the development of relevant therapeutics. Herein, we prepared human HSP47 as a soluble fusion protein expressed in E. coli and established an assay system for HSP47 inhibitor screening. We screened a natural and synthetic Compound library established at Nagasaki University. Among 1023 Compounds, 13 exhibited inhibitory activity against human HSP47, of which three inhibited its function in a dose-dependent manner. Epigallocatechin-3-O-gallate, one of these three Compounds, is a typical polyphenol Compound derived from tea leaves. Structurally related Compounds were synthesized and examined for their activity, revealing a hydroxyl group at A-ring position 5 as important for its activity. The present findings provide valuable insight for the development of natural product-derived therapeutics for fibrotic diseases, including idiopathic pulmonary fibrosis.     

Amyloidogenic Properties of Peptides Derived from the VHL Tumor Suppressor Protein

The von Hippel-Lindau tumor suppressor protein (pVHL) is involved in maintaining cellular oxygen homeostasis through the regulated degradation of HIF-α. The intrinsically disordered nature of pVHL makes it prone to aggregation that impairs its function, and this is further aggravated in mutant versions of the protein, thus promoting tumor development. By using in silico analysis, we predicted six peptide fragments from pVHL to be amyloidogenic. This was verified for two of the peptides by biophysical approaches, which demonstrated self-assembly and formation of β-sheet-rich aggregates, which, under transmission electron microscopy, atomic force microscopy, and X-ray diffraction, displayed typical fibrillar amyloid characteristics. These motifs may serve as proxies for exploring the nature of pVHL aggregation.     

Effects of Edible Insect Tenebrio molitor Larva Fermentation Extract as a Substitute Protein on Hepatosteatogenesis and Proteomic Changes in Obese Mice Induced by High-Fat Diet

Mealworms (Tenebrio molitor larva) are an edible insect and a protein-rich food; however, research on mealworms as a substitute protein is insufficient. In this study, mealworm fermentation extract (TMP) was assessed as a replacement for soy protein (SP) in a control diet (CON) or a high-fat diet (HFD) of mice for 12 weeks. TMP substitution reduced body weight, body weight gain, body fat mass (perirenal and mesenteric), fat size, glucose intolerance, and insulin resistance compared to the HFD-SP group. TMP alleviated hepatic steatosis (lipid contents and lipid droplets) in high-fat-fed mice and down-regulated the PPARγ, CD36, and DGAT2 gene levels. Proteomic analysis showed that a HFD for 12 weeks up-regulated 20 proteins and down-regulated 17 proteins in mice fed SP. On the other hand, TMP reversed the protein profiles. TMP significantly down-regulated KHK, GLO1, ATP5H, SOD, and DDAH1 and up-regulated DLD, Mup1, CPS1, Ces3b, PDI, and HYOU1 compared to the HFD-SP group. These proteins are involved in the glucose, lipid, and amino acid metabolism, as well as in oxidative stress and endoplasmic reticulum stress. Thus, substituting SP for TMP helped improve HFD-induced obesity, steatosis, and insulin resistance in mice. These results suggest that TMP is a potential substitute for commonly used protein sources.     

TRPM7-Mediated Calcium Transport in HAT-7 Ameloblasts

TRPM7 plays an important role in cellular Ca²⁺, Zn²⁺ and Mg²⁺ homeostasis. TRPM7 channels are abundantly expressed in ameloblasts and, in the absence of TRPM7, dental enamel is hypomineralized. The potential role of TRPM7 channels in Ca²⁺ transport during amelogenesis was investigated in the HAT-7 rat ameloblast cell line. The cells showed strong TRPM7 mRNA and protein expression. Characteristic TRPM7 transmembrane currents were observed, which increased in the absence of intracellular Mg²⁺ ([Mg²⁺]_i), were reduced by elevated [Mg²⁺]_i, and were inhibited by the TRPM7 inhibitors NS8593 and FTY720. Mibefradil evoked similar currents, which were suppressed by elevated [Mg²⁺]_i, reducing extracellular pH stimulated transmembrane currents, which were inhibited by FTY720. Naltriben and mibefradil both evoked Ca²⁺ influx, which was further enhanced by the acidic intracellular conditions. The SOCE inhibitor BTP2 blocked Ca²⁺ entry induced by naltriben but not by mibefradil. Thus, in HAT-7 cells, TRPM7 may serves both as a potential modulator of Orai-dependent Ca²⁺ uptake and as an independent Ca²⁺ entry pathway sensitive to pH. Therefore, TRPM7 may contribute directly to transepithelial Ca²⁺ transport in amelogenesis.     

Evidence for a Negative Correlation between Human Reactive Enamine-Imine Intermediate Deaminase A (RIDA) Activity and Cell Proliferation Rate: Role of Lysine Succinylation of RIDA

Reactive intermediate deaminase (Rid) proteins are enzymes conserved in all domains of life. UK114, a mammalian member of RidA subfamily, has been firstly identified as a component of liver perchloric acid-soluble proteins (L-PSP). Although still poorly defined, several functions have been attributed to the mammalian protein UK114/RIDA, including the reactive intermediate deamination activity. The expression of UK114/RIDA has been observed in some tumors, arousing interest in this protein as an evaluable tumor marker. However, other studies reported a negative correlation between UK114/RIDA expression, tumor differentiation degree and cell proliferation. This work addressed the question of UK114/RIDA expression in human non-tumor HEK293 cell lines and in some human tumor cell lines. Here we reported that human RIDA (hRIDA) was expressed in all the analyzed cell line and subjected to lysine (K-)succinylation. In HEK293, hRIDA K-succinylation was negatively correlated to the cell proliferation rate and was under the control of SIRT5. Moreover, K-succinylation clearly altered hRIDA quantification by immunoblotting, explaining, at least in part, some discrepancies about RIDA expression reported in previous studies. We found that hRIDA was able to deaminate reactive enamine-imine intermediates and that K-succinylation drastically reduced deaminase activity. As predicted by in silico analysis, the observed reduction of deaminase activity has been related to the drastic alterations of hRIDA structure inferred by K-succinylation. The role of hRIDA and the importance of its K-succinylation in cell metabolism, especially in cancer biology, have been discussed.     

High-Fat Diet Leads to Reduced Protein O-GlcNAcylation and Mitochondrial Defects Promoting the Development of Alzheimer’s Disease Signatures

The disturbance of protein O-GlcNAcylation is emerging as a possible link between altered brain metabolism and the progression of neurodegeneration. As observed in brains with Alzheimer’s disease (AD), flaws of the cerebral glucose uptake translate into reduced protein O-GlcNAcylation, which promote the formation of pathological hallmarks. A high-fat diet (HFD) is known to foster metabolic dysregulation and insulin resistance in the brain and such effects have been associated with the reduction of cognitive performances. Remarkably, a significant role in HFD-related cognitive decline might be played by aberrant protein O-GlcNAcylation by triggering the development of AD signature and mitochondrial impairment. Our data support the impairment of total protein O-GlcNAcylation profile both in the brain of mice subjected to a 6-week high-fat-diet (HFD) and in our in vitro transposition on SH-SY5Y cells. The reduction of protein O-GlcNAcylation was associated with the development of insulin resistance, induced by overfeeding (i.e., defective insulin signaling and reduced mitochondrial activity), which promoted the dysregulation of the hexosamine biosynthetic pathway (HBP) flux, through the AMPK-driven reduction of GFAT1 activation. Further, we observed that a HFD induced the selective impairment of O-GlcNAcylated-tau and of O-GlcNAcylated-Complex I subunit NDUFB8, thus resulting in tau toxicity and reduced respiratory chain functionality respectively, highlighting the involvement of this posttranslational modification in the neurodegenerative process.     

Deciphering the Binding Interactions between Acinetobacter baumannii ACP and β-ketoacyl ACP Synthase III to Improve Antibiotic Targeting Using NMR Spectroscopy

Fatty acid synthesis is essential for bacterial viability. Thus, fatty acid synthases (FASs) represent effective targets for antibiotics. Nevertheless, multidrug-resistant bacteria, including the human opportunistic bacteria, Acinetobacter baumannii, are emerging threats. Meanwhile, the FAS pathway of A. baumannii is relatively unexplored. Considering that acyl carrier protein (ACP) has an important role in the delivery of fatty acyl intermediates to other FAS enzymes, we elucidated the solution structure of A. baumannii ACP (AbACP) and, using NMR spectroscopy, investigated its interactions with β-ketoacyl ACP synthase III (AbKAS III), which initiates fatty acid elongation. The results show that AbACP comprises four helices, while Ca²⁺ reduces the electrostatic repulsion between acid residues, and the unconserved F47 plays a key role in thermal stability. Moreover, AbACP exhibits flexibility near the hydrophobic cavity entrance from D59 to T65, as well as in the α₁α₂ loop region. Further, F29 and A69 participate in slow exchanges, which may be related to shuttling of the growing acyl chain. Additionally, electrostatic interactions occur between the α₂ and α₃-helix of ACP and AbKAS III, while the hydrophobic interactions through the ACP α₂-helix are seemingly important. Our study provides insights for development of potent antibiotics capable of inhibiting A. baumannii FAS protein–protein interactions.