Team Work in Protein Processing

The problem of resistance to therapy in prostate cancer (PCa) is multifaceted. Key determinants of drug resistance include tumor burden and growth kinetics, tumor heterogeneity, physical barriers, immune system and microenvironment, undruggable cancer drivers, and consequences of therapeutic pressures. With regard to the fundamental importance of the androgen receptor (AR) in all stages of PCa from tumorigenesis to progression, AR is postulated to have a continued critical role in castration-resistant prostate cancer (CRPC). Suppression of AR signaling mediated by the full-length AR (AR-FL) is the therapeutic goal of all AR-directed therapies. However, AR-targeting agents ultimately lead to AR aberrations that promote PCa progression and drug resistance. Among these AR aberrations, androgen receptor variant 7 (AR-V7) is gaining attention as a potential predictive marker for as well as one of the resistance mechanisms to the most current anti-AR therapies in CRPC. Meanwhile, development of next-generation drugs that directly or indirectly target AR-V7 signaling is urgently needed. In the present review of the current literature, we have summarized the origin, alternative splicing, expression induction, protein conformation, interaction with coregulators, relationship with AR-FL, transcriptional activity, and biological function of AR-V7 in PCa development and therapeutic resistance. We hope this review will help further understand the molecular origin, expression regulation, and role of AR-V7 in the progression of PCa and provide insight into the design of novel selective inhibitors of AR-V7 in PCa treatment.

Quorum-sensing (QS) systems exist universally in bacteria to regulate multiple biological functions. Klebsiella pneumoniae, an industrially important bacterium that produces bio-based chemicals such as 2,3-butanediol and acetoin, can secrete a furanosyl borate diester (AI-2) as the signalling molecule mediating a QS system, which plays a key regulatory role in the biosynthesis of secondary metabolites. In this study, the molecular regulation and metabolic functions of a QS system in K. pneumoniae were investigated. The results showed that after the disruption of AI-2-mediated QS by the knockout of luxS, the production of acetoin, ethanol and acetic acid were relatively lower in the K. pneumoniae mutant than in the wild type bacteria. However, 2,3-butanediol production was increased by 23.8% and reached 54.93 g/L. The observed enhancement may be attributed to the improvement of the catalytic activity of 2,3-butanediol dehydrogenase (BDH) in transforming acetoin to 2,3-butanediol. This possibility is consistent with the RT-PCR-verified increase in the transcriptional level of budC, which encodes BDH. These results also demonstrated that the physiological metabolism of K. pneumoniae was adversely affected by a QS system. This effect was reversed through the addition of synthetic AI-2. This study provides the basis for a QS-modulated metabolic engineering study of K. pneumoniae.

The retinal circadian clock is crucial for optimal regulation of retinal physiology and function, yet its cellular location in mammals is still controversial. We used laser microdissection to investigate the circadian profiles and phase relations of clock gene expression and Period gene induction by light in the isolated outer (rods/cones) and inner (inner nuclear and ganglion cell layers) regions in wild-type and melanopsin-knockout (Opn 4 (-/-) ) mouse retinas. In the wild-type mouse, all clock genes are rhythmically expressed in the photoreceptor layer but not in the inner retina. For clock genes that are rhythmic in both retinal compartments, the circadian profiles are out of phase. These results are consistent with the view that photoreceptors are a potential site of circadian rhythm generation. In mice lacking melanopsin, we found an unexpected loss of clock gene rhythms and of the photic induction of Per1-Per2 mRNAs only in the outer retina. Since melanopsin ganglion cells are known to provide a feed-back signalling pathway for photic information to dopaminergic cells, we further examined dopamine (DA) synthesis in Opn 4 (-/-) mice. The lack of melanopsin prevented the light-dependent increase of tyrosine hydroxylase (TH) mRNA and of DA and, in constant darkness, led to comparatively high levels of both components. These results suggest that melanopsin is required for molecular clock function and DA regulation in the retina, and that Period gene induction by light is mediated by a melanopsin-dependent, DA-driven signal acting on retinal photoreceptors.

Na+/H+ exchangers (NHE), also called antiporters, are vital transmembrane transporters involved in multiple cellular functions including the regulation of intracellular pH, the control of cell volume and transepithelial ion transport. These transporters are highly regulated by a remarkably wide variety of stimuli which can modulate their expression level and activity. Five isoforms of Na+/H+ exchangers have been cloned and characterized to date; they define a new gene family of vertebrate transporters. These isoforms share the same overall structure but exhibit differences with respect to amiloride-sensitivity, cellular localization, kinetic variables, regulation by various stimuli and plasma membrane targeting in polarized epithelial cells. Biochemical techniques and molecular genetics tools provide the means of analyzing these transporters at the molecular level. The purpose of this manuscript is to give an overview of the main features of the Na+/H+ exchangers with emphasis on recent advances in comprehension of the structure-function relationship and regulation mechanisms of the ubiquitous isoform: NHE-1.

Molecular cloning, gene manipulation, gene expression, protein function, and gene regulation all depend on the introduction of nucleic acids into target cells. Multiple methods have been developed to facilitate such delivery including instrument based microinjection and electroporation, biological methods such as transduction, and chemical methods such as calcium phosphate precipitation, cationic polymers, and lipid based transfection, also known as lipofection. Here we report attempts to lipofect sea urchin coelomocytes using DOTAP lipofection reagent packaged with a range of molecules including fluorochromes, in addition to expression constructs, amplicons, and RNA encoding GFP. DOTAP has low cytotoxicity for coelomocytes, however, lipofection of a variety of molecules fails to produce any signature of success based on results from fluorescence microscopy and flow cytometry. While these results are negative, it is important to report failed attempts so that others conducting similar research do not repeat these approaches. Failure may be the outcome of elevated ionic strength of the coelomocyte culture medium, uptake and degradation of lipoplexes in the endosomal-lysosomal system, failure of the nucleic acids to escape the endosomal vesicles and enter the cytoplasm, and difficulties in lipofecting primary cultures of phagocytic cells. We encourage others to build on this report by using our information to optimize lipofection with a range of other approaches to work towards establishing a successful method of transfecting adult cells from marine invertebrates.

research aspects including molecular structures of CSK and CHK, major signaling pathways such as apoptosis and integrin signaling in cancers affected by CSK and CHK and comprehensive overviews of CSK and CHK highlighting their regulation, molecular targets and functional roles.In addition to the general features of phosphorylating substrates, CSK and CHK have unique structural motifs determining their substrate specificity and enzymatic efficiencies.CSK and CHK are also subject to regulation by different regulators and/or post-translational modifications. To understand the roles of CSK and CHK and their functional differences in inactivating SFKs, Sun et al. have reviewed the structure-function relationship of CSK and CHK with an emphasis on catalysis, substrate recognition, and the interactions between domain and domain/key phosphorylation site. This review not only summarizes the current understanding of CSK/CHK enzymology but also sheds light on the general mechanism of regulation and catalysis of protein tyrosine kinases. This will be helpful in bolstering protein phosphorylation as a key methodology for the development of anti-cancer therapeutics.Apoptosis, a form of programmed cell death, plays a key role in multiple biological processes (Capela E Silva et al., 2023). The extrinsic pathway is initiated through activation of death receptors (such as Fas, DR4, DR5) engaged by their ligands (such as FasL and Apo2L/TRAIL) which ultimately triggers the activation of caspase 8. The intrinsic pathway is initiated through the activation of the Bcl-2 family of pro-apoptotic proteins such as Bax/Bak in response to pro-apoptotic stimuli. Activation of Bax/Bak leads to increased permeability of the mitochondrial membrane and eventually the activation of effector caspases. These cysteine-aspartic proteases subsequently cleave proteins resulting in cellular demise. Fortner et al. provide an overview of the apoptosis mechanisms induced by CSK.CSK suppresses MAPK, STAT3 and PI3K signaling respectively, all of which are involved in promoting cell survival through upregulation of pro-survival molecules such as Myc and Bcl-2, and downregulation of pro-apoptotic molecules such as Bim and FOXO. The Srcdependent pro-apoptotic effect of CSK has been further confirmed by a novel Src inhibitor (Abdelall et al., 2022). Therefore, targeting key molecules in these pathways to promote apoptosis could be a strategy in the development of potential treatment of diseases such as cancer (Tang et al., 2023).As a part of the focal adhesion, integrins can sense and send extracellular signals to the cytosol to regulate different processes such as cell migration, invasion, proliferation, and survival. Therefore, integrins play crucial roles in cancer progression and metastasis.Maldonado et al. reviewed how CSK regulates integrin signaling through SFKs. These authors focuse on the crucial role of integrins in sensing and responding to mechanical changes in the surrounding environment by emphasizing their mechanosensing and mechanotransduction functions (Hamidi et al., 2018, Koudelková et al., 2021). They have also discussed the crosstalk among CSK, integrins, and growth factor receptors suggesting that they could become potential therapeutic targets.Besides the above specific topic focuses, Zhu et al. have provided overviews of how CSK and CHK are regulated, their molecular targets, and novel biological functions involved. Among many interesting new features of CSK/CHK, it's noteworthy that CHK not only phosphorylates SFKs to inactivate them, CHK also serves as a non-enzymatic inhibitor for many Src family members; Unlike CSK which is expressed ubiquitously, expression of CHK is limited in several tissues; Novel targets of CHK including SHPS-1, paxillin, and synuclein have been identified. Therefore, CHK may complement or strengthen CSK's effect in inhibiting SFKs in certain tissues. CHK silencing due to DNA methylation (Zhu et al, 2021;Chüeh et al., 2021) in certain cancer types suggests that CHK epigenetics could serve as a therapeutic or diagnostic candidate. On the other hand, in addition to the established CSK-SFK axis, the recent discoveries of novel CSK targets such as MITA (Gao et al., 2020) and novel regulators of CSK such as SPOP (Tawaratsumida et al., 2022), and the unveiled pathology of many diseases newly reported to be associated with CSK warrant further exploration of CSK.In conclusion, the "Regulation and Dysfunction of CSK and CHK" research topic highlights the recent discoveries of molecular structure, targets, regulation and biological function of CSK/CHK. This not only furthered our understanding in basic research but also paved the way for the development of new therapies and diagnosis.

Arginase (ARG) is a binuclear manganese-containing metalloenzyme that can convert L-arginine to L-ornithine and urea and plays a key role in the urea cycle. It also mediates different cellular functions and processes such as proliferation, senescence, apoptosis, autophagy, and inflammatory responses in various cell types. In mammals, there are two isoenzymes, ARG-1 and ARG-2; they are functionally similar, but their coding genes, tissue distribution, subcellular localization, and molecular regulation are distinct. In recent decades, the abnormal expression of ARG-1 or ARG-2 has been reported to be increasingly linked to a variety of diseases, including cardiovascular disease, inflammatory bowel disease, Alzheimer's disease, and cancer. Therefore, considering the current relevance of this topic and the need to address the growing demand for new and more potent ARG inhibitors in the context of various diseases, this review was conceived. We will provide an overview of all classes of ARG inhibitors developed so far including compounds of synthetic, natural, and semisynthetic origin. For the first time, the synthesis protocol and optimized reaction conditions of each molecule, including those reported in patent applications, will be described. For each molecule, its inhibitory activity in terms of IC50 towards ARG-1 and ARG-2 will be reported specifying the type of assay conducted.

Ex Vivo Generation Of Functional Plasmacytoid and Myeloid Dendritic Cells Is Strongly Promoted By The Aryl Hydrocarbon Receptor Antagonist Stemregenin 1

BackgroundRight ventricular (RV) angiogenesis has been associated with adaptive myocardial remodeling in pulmonary hypertension (PH), though molecular regulators are poorly defined. Endothelial cell VEGFR-2 is considered a “master regulator” of angiogenesis in other models, and the small molecule VEGF receptor tyrosine kinase inhibitor SU5416 is commonly used to generate PH in rodents. We hypothesized that SU5416, through direct effects on cardiac endothelial cell VEGFR-2, would attenuate RV angiogenesis in a murine model of PH.MethodsC57 BL/6 mice were exposed to chronic hypoxia (CH-PH) to generate PH and stimulate RV angiogenesis. SU5416 (20 mg/kg) or vehicle were administered at the start of the CH exposure, and weekly thereafter. Angiogenesis was measured after one week of CH-PH using a combination of unbiased stereological measurements and flow cytometry-based quantification of myocardial endothelial cell proliferation. In complementary experiments, primary cardiac endothelial cells from C57 BL/6 mice were exposed to recombinant VEGF (50 ng/mL) or grown on Matrigel in the presence of SU5416 (5 μM) or vehicle.ResultSU5416 directly inhibited VEGF-mediated ERK phosphorylation, cell proliferation, and Kdr transcription, but not Matrigel tube formation in primary murine cardiac endothelial cells in vitro. SU5416 did not inhibit CH-PH induced RV angiogenesis, endothelial cell proliferation, or RV hypertrophy in vivo, despite significantly altering the expression profile of genes involved in angiogenesis.ConclusionsThese findings demonstrate that SU5416 directly inhibited VEGF-induced proliferation of murine cardiac endothelial cells but does not attenuate CH-PH induced RV angiogenesis or myocardial remodeling in vivo.

BACKGROUNDDNA damage is one of the critical contributors to the occurrence and development of some cancers. APEX1 and APEX2 are the most important molecules in the DNA damage, and APEX1 has been identified as a diagnostic and prognostic biomarker in liver hepatocellular carcinoma (LIHC). However, the expression of APEX2 and its functional mechanisms in LIHC are still unclear.AIMTo examine the expression of APEX2 and the potential mechanism network in LIHC.METHODSWe conducted a pan-cancer analysis of the expression of APEX1 and APEX2 using the interactive TIMER tool. GEO datasets, including GSE14520, GSE22058, and GSE64041, were used to compare the APEX2 expression level in tumor tissues and adjacent non-tumor tissues. Then, we calculated the 5-year survival rate according to the web-based Kaplan-Meier analysis. We included the TCGA liver cancer database in GSEA analysis based on the high and low APEX2 expression, showing the potential mechanisms of APEX2 in LIHC. After that, we conducted Pearson correlation analysis using GEPIA2. Next, we performed quantitative polymerase chain reaction (qPCR) assay to examine the APEX2 levels in normal liver cell line LO2 and several liver cancer cell lines, including HepG2, Huh7, SMMC7721, and HCCLM3. APEX2 in HCCLM3 cells was knocked down using small interfering RNA. The role of APEX2 in cell viability was confirmed using CCK-8. Dual-luciferase reporter assay was performed to examine the promoter activity of CCNB1 and MYC.RESULTSAPEX1 and APEX2 are both highly expressed in the tumor tissues of BLCA, BRCA, CHOL, COAD, ESCA, HNSC, LIHC, LUAD, LUSC, READ, and STAD. APEX2 overexpression in LIHC was validated using GSE14520, GSE22058, and GSE64041 datasets. The survival analysis showed that LIHC patients with high expression of APEX2 had a lower overall survival rate, even in the AJCC T1 patients. High level of APEX2 could indicate a lower overall survival rate in patients with or without viral hepatitis. The GSEA analysis identified that kinetochore and spindle microtubules are the two main cellular components of APEX2 in GO Ontology. APEX2 was also positively associated with molecular function regulation of chromosome segregation and DNA replication. The results of KEGG analysis indicated that APEX2 expression was positively correlated with cell cycle pathway and pro-oncogenic MYC signaling. Pearson correlation analysis showed that APEX2 had a significant positive correlation with CCNB1 and MYC. APEX2 level was higher in liver cancer cell lines than in normal liver LO2 cells. Small interfering RNA could knock down the APEX2 expression in HCCLM3 cells. Knockdown of APEX2 resulted in a decrease in the viability of HCCLM3 cells as well as the expression and promoter activity of CCNB1 and MYC.CONCLUSIONAPEX2 is overexpressed in LIHC, and the higher APEX2 level is associated with a worse prognosis in overall survival. APEX2 is closely involved in the biological processes of chromosome segregation and DNA replication. APEX2 expression is positively correlated with the pro-oncogenic pathways. Knockdown of APEX2 could inhibit the cell viability and CCNB1 and MYC pathways, suggesting that APEX2 is an oncogene in LIHC, which could be a potential pharmaceutic target in the anti-tumor therapy.

Label-free based comparative proteomic analysis of whey proteins between different milk yields of Dezhou donkey

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Cells contain a multitude of different RNAs, which are highly versatile biomolecules that have crucial functions in almost all cellular processes. Furthermore, the inter-relationship between RNA and proteins is essential, as ribonucleoprotein complexes are responsible for the execution and control of fundamental cellular processes. RNA helicases are nucleoside triphosphate-dependent RNA-binding proteins capable of remodelling RNAs and ribonucleoprotein complexes and are, consequently, a key driving force in almost all aspects of RNA metabolism and are expressed not only in all three domains of life but also in some viruses. DHX proteins are a diverse group of Superfamily 2 RNA helicases with shared structural features that display a wide array of RNA remodelling activities in a multitude of cellular processes. Consequently, strict spatial, temporal, and catalytic regulation of DHX proteins is essential in cells, and the dysregulation of helicase activity is often associated with tumorigenesis and disease. However, the cellular function and mode of regulation of several DHX proteins remain elusive. The objective of this study was to broaden the understanding of the functional and regulatory repertoires of DHX proteins. To this end, a toolbox was established for exploring DHX protein functions in the cellular context. The toolbox included verified siRNAs, stably transfected cell lines for inducible expression of tagged DHX proteins, and tested antibodies. The usefulness of this toolbox was exemplified by the functional characterisation of DHX40. Analysis of DHX40 protein interactions by immunoprecipitation and mass spectrometry revealed that DHX40 interacts with several proteins implicated in regulating LINE-1 retrotransposition. Biochemical analysis using recombinantly expressed and purified proteins showed that DHX40 is an RNAdependent ATPase that is stimulated by the putative prolyl isomerase PPIL4, a novel protein cofactor identified in this study. DHX40 was confirmed to be a substrate of the deubiquitinase USP7, and mutagenesis analysis was used to investigate the interaction of DHX40 with the E3 ligase TRIM27. Comprehensive analysis of the RNA interactome by the crosslinking and analysis of cDNA (CRAC) approach revealed that DHX40 interacts with LINE-1 RNAs in HEK293, and complementary differential expression analysis showed that LINE-1 RNAs are upregulated upon depletion of DHX40. Finally, a cell-based LINE-1 retrotransposition assay showed that DHX40 and PPIL4 are suppressors of LINE-1 retrotransposition in HEK293 cells.

PDF HTML阅读 XML下载 导出引用 引用提醒 鲤lipea基因表达的时空特征及与脂肪沉积相关性分析 DOI: 作者: 作者单位: 1. 上海海洋大学, 农业农村部淡水水产种质资源重点实验室, 上海 201306;2. 中国水产科学研究院黑龙江水产研究所, 淡水鱼类育种国家地方联合工程实验室, 黑龙江 哈尔滨 150070 作者简介: 党红阳(1995-),女,硕士研究生,研究方向为水产生物技术.E-mail:danghongyang95@163.com 通讯作者: 中图分类号: S965 基金项目: 国家自然科学基金项目（31302174）；中国水产科学研究院基本科研业务费（2015C04XK01）；国家水产种质资源共享服务平台项目（2018DKA30470）. Temporal-spatial characteristics of lipea gene expression in Cyprinus carpio and its correlation with fat deposition Author: Affiliation: 1. Key Laboratory of Freshwater Fishery Germplasm Resource, Ministry of Agriculture and Rural Affairs;Shanghai Ocean University, Shanghai 201306, China;2. National Local Joint Engineering Laboratory for Freshwater Fish Breeding, Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:本文旨在克隆鲤（）基因，探究其时空表达特征及其与脂肪沉积的关系，以期为鲤脂肪沉积分子调控机制研究奠定理论基础。本实验采用RACE技术克隆获得鲤基因，其cDNA全长为3379 bp，包括230 bp的5'非翻译区（5'-UTR）、1067 bp的3'非翻译区（3'-UTR）和编码693个氨基酸的开放阅读框。多序列比对显示，鲤Oncorhynchus mykiss）的氨基酸序列相似性分别为88.17%、64.94%和63.35%，不同物种间序列差异主要集中在C-端的多肽序列上。蛋白质结构分析显示该蛋白含有3个功能域分别具有脂肪酶活性、乙酰基水解酶活性和水解酶活性。应用实时荧光定量PCR技术分析了在各组织中均有表达，腹腔脂肪中相对表达量最高，其次是腹部肌肉，血液中最少。随着胚胎的发育，表达量与背部肌肉脂肪含量呈正相关，但差异不显著（基因对鲤腹腔脂肪沉积可能具有抑制作用。本实验结果可为进一步解析鲤基因的功能和基因调控脂肪沉积分子机制研究提供参考。 Abstract:The objectives of this study were to investigate the expression pattern of Cyprinus carpio), to lay the foundation for studying and understanding the molecular regulation mechanism of fat deposition in common carp. In this study, a full-length cDNA sequence of gene in common carp was cloned by reverse transcription PCR and rapid-amplification of cDNA ends (RACE) technology, and the expression pattern of gene in different tissues of common carp and during its different developmental stages. cDNA of the gene of common carp was 3379 bp, with an open reading frame of 2079 bp, 5' untranslated region was 230 bp, 3' untranslated region was 1067 bp, encoding 693 amino acids. Multiple sequences alignment revealed that the amino acid sequences of Paralichthys olivaceus, and , respectively. It was determined that the sequence differences were mainly concentrated on the C-terminal polypeptide sequence between different species, and the structural analysis of proteins showed that the protein contains three functional domains with lipase activity, acetyl hydrolase activity, and hydrolase activity, respectively. Real-time quantitative PCR showed that the gene was expressed in all tested tissues, and the relative expression level was the highest in visceral fat, followed by abdominal muscles, and lowest in the blood. Its expression declined during the development of embryo; it was the highest in the 8-cell stage, followed by the late blastula stage. It was the lowest in the feeding period. The mRNA expression in different fat tissues negatively correlated with adipose tissue in the abdominal cavity; the highest and the lowest expression levels differed significantly (<0.01). The expression also positively correlated with dorsal muscle but it was not significant ( gene highlights its function and molecular regulation mechanism of fat development in common carp. 参考文献 相似文献 引证文献

Regulatory T cells (Treg) are essential to maintain immune homeostasis and prevent autoimmune disorders. While the function and molecular regulation of Foxp3+CD4+ Tregs are well established, much of CD8+ Treg biology remains to be revealed. Here, we will review the heterogenous subsets of CD8+ T cells have been named “CD8+ Treg” and mainly focus on CD122hiLy49+CD8+ Tregs present in naïve mice. CD122hiLy49+CD8+ Tregs, which depends on transcription factor Helios and homeostatic cytokine IL-15, have been established as a non-redundant regulator of germinal center (GC) reaction. Recently, we have demonstrated that TGF-β (Transforming growth factor-β) and transcription factor Eomes (Eomesodermin) are essential for the function and homeostasis of CD8+ Tregs. In addition, we will discuss several open questions regarding the differentiation, function and true identity of CD8+ Tregs as well as a brief comparison between two regulatory T cell subsets critical to control GC reaction, namely CD4+ TFR (follicular regulatory T cells) and CD8+ Tregs.