Intracellular Part Research Articles

Differential Transcriptomics Analysis of IPEC-J2 Cells Single or Coinfected With Porcine Epidemic Diarrhea Virus and Transmissible Gastroenteritis Virus.

Porcine epidemic diarrhea (PED) and transmissible gastroenteritis (TGE) caused by porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are two highly contagious intestinal diseases in the swine industry worldwide. Notably, coinfection of TGEV and PEDV is common in piglets with diarrhea-related diseases. In this study, intestinal porcine epithelial cells (IPEC-J2) were single or coinfected with PEDV and/or TGEV, followed by the comparison of differentially expressed genes (DEGs), especially interferon-stimulated genes (ISGs), between different groups via transcriptomics analysis and real-time qPCR. The antiviral activity of swine interferon-induced transmembrane protein 3 (sIFITM3) on PEDV and TGEV infection was also evaluated. The results showed that DEGs can be detected in the cells infected with PEDV, TGEV, and PEDV+TGEV at 12, 24, and 48 hpi, and the number of DEGs was the highest at 24 hpi. The DEGs are mainly annotated to the GO terms of protein binding, immune system process, organelle part, and intracellular organelle part. Furthermore, 90 ISGs were upregulated during PEDV or TGEV infection, 27 of which were associated with antiviral activity, including ISG15, OASL, IFITM1, and IFITM3. Furthermore, sIFITM3 can significantly inhibit PEDV and TGEV infection in porcine IPEC-J2 cells and/or monkey Vero cells. Besides, sIFITM3 can also inhibit vesicular stomatitis virus (VSV) replication in Vero cells. These results indicate that sIFITM3 has broad-spectrum antiviral activity.

Ligand-Dependent Modulation of the Dynamics of Intracellular Loops Dictates Functional Selectivity of 5-HT2AR

The serotonin 2A receptor (5-HT2AR) subtype of the G protein-coupled receptor (GPCR) family is involved in a plethora of neuromodulatory functions (e.g., neurogenesis, sleep, and cognitive processes). 5-HT2AR is the target of pharmacologically distinct classes of ligands, binding of which either activate or inactivate the receptor. Although high-resolution structures of 5-HT2AR as well as several other 5-HT GPCRs provided snapshots of both active and inactive conformational states, these structures, representing a truncated form of the receptor, cannot fully explain the mechanism of conformational transitions during their function. Importantly, biochemical studies have suggested the importance of intracellular loops in receptor functions. In our previous study, a model of the ligand-free form of 5-HT2AR with the third intracellular loop (ICL3) has been meticulously built. Here, we have investigated the functional regulation of 5-HT2AR with intact intracellular loops in ligand-free and five distinct ligand-bound configurations using unbiased atomistic molecular dynamics (MD) simulations. The selected ligands belong to either of the full, partial, or inverse agonist classes, which exert distinct pharmacological responses. We have observed significant structural, dynamic, and thermodynamic differences within ligand-bound complexes. Our results revealed, for the first time, that either activation or inactivation of the receptor upon specific ligand binding is primarily achieved through conformational transitions of its second and third intracellular loops (ICL2 and ICL3). A remarkable allosteric cross-talk between the ligand-binding site and the distal intracellular parts of the receptor, where binding of a specific ligand thermodynamically controls (either stabilizes or destabilizes) the intracellular region, consisting of crucial dynamic elements ICL2 and ICL3, and differential conformational transitions of these loops determine ligand-dependent functional selectivity.

Differential Expression and Bioinformatics Analysis of tRF/tiRNA in Endometriosis Patients.

Background Endometriosis (EMs) is a benign chronic condition that tends to recur in women of childbearing age, with an incidence of approximately 10%. It is a multifactorial disease for which the pathogenesis is currently unclear. This study is aimed at investigating the expression and clinical significance of tRNA-derived small RNA (tsRNA), a novel noncoding small RNA with potential regulatory functions, in endometriosis. Methods The tRF/tiRNA expression profiles in endometrial tissues from three pairs of endometriosis patients and controls were detected by tRF&tiRNA PCR microarray technology and then verified by quantitative real-time polymerase chain reaction (qPCR). The target genes and target sites of TRF396, tiRNA-5030-GlnTTG-3, TRF308, and TRF320 were predicted by miRanda, and the network diagram of their interaction with miRNA was drawn. The impact of tRNA-derived fragments on the pathogenesis of endometriosis was analyzed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Results Two upregulated and 19 downregulated tRNA-derived fragments were identified. The qRT-PCR results of 2 upregulated and 2 downregulated RNA-derived fragments were consistent with the RNA Seq data. The OR2B4 gene related to TRF396, the DGAT1 gene related to tiRNA-5030-GlnTTG-3, the KLF16 gene of TRF308, and the RNF213 gene of TRF320 had significant correlations. Gene Ontology and pathway analysis showed that the target genes of TRF396 and tiRNA-5030-GlnTTG-3 were mainly involved in the intrinsic components of the membrane and the overall composition of the membrane in cell components; molecular functions mainly involve olfactory conduction and G protein-coupled receptor activity. In the biological process, it was mainly involved in the detection of sensory stimuli. The target genes of TRF308 and TRF320 were mainly involved in the intracellular part; molecular functions are mainly related to DNA binding transcription factor activity and protein binding and mainly related to biological regulation of biological processes. Pathway analysis showed that the RAP1 signaling pathway and the AXON GUIDANCE signaling pathway may participate in the progression of endometriosis. Conclusion The differential expression of tRF/tiRNA in endometriosis may be related to the pathogenesis of endometriosis. Furthermore, tRF/tiRNA may be a biomarker for the diagnosis and treatment of EMs in the future.

Screening and preliminary identification of long non-coding RNAs critical for osteogenic differentiation of human umbilical cord mesenchymal stem cells

ABSTRACT Human umbilical cord mesenchymal stem cells (hUCMSCs) are attractive therapeutic cells for tissue engineering to treat bone defects. However, how the cells can differentiate into bone remains unclear. Long non-coding RNAs (lncRNAs) are non-coding RNAs that participate in many biological processes, including stem cell differentiation. In this study, we investigated the profiles and functions of lncRNAs in the osteogenic differentiation of hUCMSCs. We identified 343 lncRNAs differentially expressed during osteogenic differentiation, of which 115 were upregulated and 228 were downregulated. We further analyzed these lncRNAs using bioinformatic analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. GO and KEGG pathway analysis showed that ‘intracellular part’ and ‘Phosphatidylinositol signaling system’ were the most correlated molecular function and pathway, respectively. We selected the top 10 upregulated lncRNAs to construct six competing endogenous RNA networks. We validated the impact of the lncRNA H19 on osteogenic differentiation by overexpressing it in hUCMSCs. Overall, our results pave the way to detailed studies of the molecular mechanisms of hUCMSC osteogenic differentiation, and they provide a new theoretical basis to guide the therapeutic application of hUCMSCs.

Unraveling the allosteric activation of GPCR using metadynamics and deep learning

Allosteric signaling in G-protein-coupled receptors (GPCRs) involve binding of a ligand in the extracellular region which induces conformational changes in the intracellular part and coupling to G proteins. This triggers a cascade of signaling that results in cellular responses. Therefore, to design drugs that modulate the GPCR function, there is an urgent need to understand the conformational pathways in GPCR activation and inactivation and their dynamical behavior. Importantly, the atomic details of the allosteric pathway between the active site and the ligand binding site can help design better drugs that can disrupt or induce specific allosteric pathways.

Integrated Analysis of Long Non-Coding RNA -mRNA Profile and Validation in Diabetic Cataract

ABSTRACTPurposeTo illustrate the expression profile of lncRNAs and mRNAs in diabetic cataract (DC) and explore their potential role in the pathogenesis of DC.MethodsLncRNA and mRNA microarray were conducted using the lens epithelium of DC patients (n = 3) and controls from eye bank (n = 3). Further, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were employed to unveil their underlying biological function and signaling pathway. The co-expression network was constructed to explore the relationship between lncRNAs and mRNAs. Then, lens epithelial cells (LECs) were cultured in vitro under high glucose (HG) and normal glucose (NG) groups to further verified the expression profiles of mRNAs. At last, quantitative RT-PCR (qRT-PCR) was performed to confirm the microarray results for the aberrantly expressed lncRNAs, and to unveil the expression prolife of mRNAs in LECs.ResultsEight thousand three hundred and twenty-six lncRNAs and 3303 mRNAs were dysregulated in DC. GO analysis unveiled that the upregulated mRNAs, highest enrichment score of the GO term belonged to oxidation-reduction process, while the highest for downregulated mRNAs went to protein lipidation. In the CC category, the most significant terms for upregulated and downregulated mRNAs appeared in intracellular part and intrinsic component of membrane, respectively. As to MF category, the most significant term for upregulated mRNAs was protein binding, and for downregulated mRNAs was catecholamine binding. KEGG pathways analysis demonstrated that the major enrichment score of pathways in upregulated mRNAs included Glycolysis/Gluconeogenesis, lysosome, carbon metabolism and AGE-RAGE signaling pathway. For the downregulated mRNAs, the glycosphingolipid biosynthesis-lacto and neolacto series pathway was included. Three lncRNAs (LINC01508, MAFA-AS1, MIAT) and the Sirtuin 2 are positively related in the co-expression network.ConclusionsThis study provided an overview of the lncRNAs and mRNAs expression, and suggesting that several aberrantly expressed lncRNAs might participate in the pathogenesis of DC.

Probing Allosteric Regulation Mechanism of W7.35 on Agonist-Induced Activity for μOR by Mutation Simulation.

The residue located at 15 positions before the most conserved residue in TM7 (7.35 of Ballesteros-Weinstein number) plays important roles in ligand binding and the receptor activity for class A GPCRs. Nevertheless, its regulation mechanism has not been clearly clarified in experiments, and some controversies also exist for its impact on μ-opioid receptors (μOR) bound by agonists. Thus, we chose the μ-opioid receptor (μOR) of class A GPCRs as a representative and utilized a microsecond accelerated molecular dynamics simulation (aMD) coupled with a protein structure network (PSN) to explore the effect of W3187.35 on its functional activity induced by the agonist endomorphin2 mainly by a comparison of the wild system and its W7.35A mutant. When endomorphin2 binds to the wild-type μOR, TM6 in μOR moves outward to form an open intracellular conformation that is beneficial to accommodating the β-arrestin transducer, rather than the G-protein transducer due to the clash with the α5 helix of G-protein, thus acting as a β-arrestin biased agonist. However, the W318A mutation induces the intracellular part of μOR to form a closed state, which disfavors coupling with either G-protein or β-arrestin. The allosteric pathway analysis further reveals that the binding of endomorphin2 to the wild-type μOR transmits more activation signals to the β-arrestin binding site while the W318A mutation induces more structural signals to transmit to common binding residues of the G protein and β-arrestin. More interestingly, the residue at the 7.35 position regulates the shortest allosteric pathway in indirect ways by influencing the interactions between other ligand-binding residues and endomorphin2. W2936.48 and F2896.44 are important for regulating the different activities of μOR induced either by the agonist or by the mutation. Y3367.53, F3438.50, and D3408.47 play crucial roles in activating the β-arrestin biased signal induced by the agonist endomorphin2, while L1583.43 and V2866.41 devote important contributions to the change in the activity of endomorphin2 from the β-arrestin biased agonist to the antagonist upon the W318A mutation.

Microarray analysis of genes with differential expression of m6A methylation in lung cancer.

Purpose: N6-methyladenosine (m6A) is among the most abundant mRNA modifications in eukaryote. The aim of the present study was to investigate function of m6A mRNA methylation in lung cancer and the underlying mechanism.Methods: Microarray analysis was performed to detect the differences in RNA expression between cancerous and adjacent non-cancerous tissue samples. The target mRNAs were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Hierarchical clustering of RNAs was conducted to identify distinct m6A methylation or expression patterns between the samples.Results: In the present study, some differentially expressed genes (DEGs) of mRNAs were identified, including up-regulated secret phosphoprotein 1 (SPP1) and down-regulated pRB. Functional enrichment analysis revealed that while differential hypermethylation was related to cell cycle, intracellular part and protein binding, the main pathway involved herpes simplex virus 1 infection related to down-regulated AKT, Araf1 and BCL2A1. In the meantime, sexual reproduction, cohesin complex and protein C-terminus binding was functionally linked to differential hypomethylation, while fluid shear stress and atherosclerosis were identified as the main pathways related to up-regulated GST and CNP.Conclusions: We showed that lung cancer development involved differential expression of SPP1 and pRB mRNA, as well as m6A mRNA methylation in AKT, APAF1, BCL2A1, GST and CNP genes.

Mining RNA-Seq Data to Depict How Penicillium digitatum Shapes Its Transcriptome in Response to Nanoemulsion.

Penicillium digitatum is the most severe pathogen that infects citrus fruits during storage. It can cause fruit rot and bring significant economic losses. The continuous use of fungicides has resulted in the emergence of drug-resistant strains. Consequently, there is a need to develop naturally and efficiently antifungal fungicides. Natural antimicrobial agents such as clove oil, cinnamon oil, and thyme oil can be extracted from different plant parts. They exhibited broad-spectrum antimicrobial properties and have great potential in the food industry. Here, we exploit a novel cinnamaldehyde (CA), eugenol (EUG), or carvacrol (CAR) combination antifungal therapy and formulate it into nanoemulsion form to overcome lower solubility and instability of essential oil. In this study, the antifungal activity evaluation and transcriptional profile of Penicillium digitatum exposed to compound nanoemulsion were evaluated. Results showed that compound nanoemulsion had a striking inhibitory effect on P. digitatum in a dose-dependent manner. According to RNA-seq analysis, there were 2,169 differentially expressed genes (DEGs) between control and nanoemulsion-treated samples, including 1,028 downregulated and 1,141 upregulated genes. Gene Ontology (GO) analysis indicated that the DEGs were mainly involved in intracellular organelle parts of cell component: cellular respiration, proton transmembrane transport of biological process, and guanyl nucleotide-binding molecular function. KEGG analysis revealed that metabolic pathway, biosynthesis of secondary metabolites, and glyoxylate and dicarboxylate metabolism were the most highly enriched pathways for these DEGs. Taken together, we can conclude the promising antifungal activity of nanoemulsion with multiple action sites against P. digitatum. These outcomes would deepen our knowledge of the inhibitory mechanism from molecular aspects and exploit naturally, efficiently, and harmlessly antifungal agents in the citrus postharvest industry.

Genome Wise Identification and Phylogenetic Analyses of NOTCH1 Gene

The NOTCH gene encode transmembrane receptor. It play a vital role in several process stem cell maintenance and differentiation during embryonic and adult development. When ligand bind at a specific part intracellular part of NOTCH receptor is cleaved and translocate to the nucleus from where it can bind to transcription site. NOTCH activity can promotes tissue growth and cancer in some conditions but they also suppress tumors formation in others. Their gene structure show the amount of introns and exons by a dimensions structure of NOTCH gene. (Powell, Passmore et al. 1998)Various tools or database such as Mega7, Pfam and Gene structure and display server are used to analyze their phylogeny and their chromosome positions gene structure and introns and exons. Further studies are made to target the NOTCH pathway on growth and cancer suppressor.

Analysis of Age-Related Circular RNA Expression Profiles in Mesenchymal Stem Cells of Rat Bone Marrow.

As multicellular organisms age, they undergo a reduction in tissue and organ function. Researchers have put forward a theory that stem cell aging is the main factor responsible for decreased tissue and organ function. The adult stem cells guarantee the maintenance and repair of adult tissues and organs. Among adult stem cells, mesenchymal stem cells (MSCs) are emerging as hopeful candidates for cell-based therapy of numerous diseases. In recent years, high-throughput sequencing technologies have evolved to identify circular RNAs (circRNAs) associated with an increasing number of diseases, such as cancer and age-related diseases. It has been reported that circRNAs can compete with microRNAs (miRNAs) to affect the stability or translation of target RNAs and further regulate gene expression at the transcriptional level. However, the role of circRNAs expressed in MSCs in aging mechanisms has not yet been deciphered. The aim of this study was to explore and analyze the expression profiles of age-related circRNAs in MSCs. In this study, bone marrow MSCs were extracted from aged and young rats and analyzed using high-throughput sequencing and bioinformatics. The reliability of high-throughput RNA sequencing was verified by quantitative real-time polymerase chain reaction. The most important circRNA functions and pathways were further selected by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomics (KEGG) analysis. Age-related circRNAs were found in the circrNA–miRNA–mRNA interaction network. The results of high-throughput sequencing showed that 4,229 circRNAs were involved in age-related senescence of MSCs. Compared with the young group, there were 29 differentially expressed circRNAs in the aged group, of which four were upregulated and 25 were downregulated. GO analysis covered three domains: biological process (BP), cellular component (CC), and molecular function (MF). The terms assigned to the BP domain were cellular metabolic processes and cellular macromolecule metabolic processes. The identified CC terms were intracellular and intracellular part, and the identified MF terms were binding and protein binding. The top five KEGG pathways were mitophagy–animal–Rattus norvegicus, prostate cancer–Rattus norvegicus, pathways in cancer–Rattus norvegicus, lysosome–Rattus norvegicus, and autophagy–animal–Rattus norvegicus. Altogether, circRNAs may play a major role in age-related MSC senescence. This study provides new mechanistic insights into MSC senescence, possibly leading to novel therapeutic strategies for age-related diseases.

Effects of CSF3R mutations on Myeloid Differentiation and Proliferation of Hematopoietic Cells of Congenital Neutropenia Patients

Over the last 25 years, with the clinical approval of recombinant human granulocyte colony-stimulating factor (rhG-CSF) remarkable progress has been achieved in the therapy of severe congenital neutropenia (CN). Nevertheless, CN patients have a high rate of transformation to myelodysplasia (MDS) or acute myeloid leukemia (AML). This risk is especially high in the group of patients who harbor acquired G-CSFR mutations, suggesting that these mutations are involved in the leukemogenesis. Deep sequencing of the intracellular part of G-CSFR allowed us to identify a subset of CN patients with a high mutant allele frequency (MAF) of G-CSFR mutations in the granulocytic compartment. We performed CSF3R mutation analysis of myeloid colonies from colony forming unit (CFU) assay of BM and CD34+ samples of 3 ELANE -CN patients, in which the percentage of granulocytes with G-CSFR mutation varied between 22.6% and 81%. Average number of sequenced colonies per CFU sample was 18. Intriguingly, we found that only cell clones with WT G-CSFR were grown in CFU assay. We hypothesized that lack of G-CSFR mutant clones in CFU assay is due to abnormal signaling downstream of truncated G-CSFR. To compare myeloid differentiation of G-CSFR mutant and WT G-CSFR hematopoietic cells, we generated ELANE -CN patient-derived induced pluripotent stem cells (CN-iPSCs) and performed CRISPR/Cas9 genome editing of CSF3R . Using 2 different sgRNAs specific for the intracellular region of G-CSFR, we introduced nucleotide indels at amino acid positions 740 and 735 (NP_000751.1) in CN-iPSCs. Subsequently, homozygous and heterozygous CN-iPSCs clones with truncated distal part of cytoplasmic domain of G-CSF receptor and subsequent loss of 3 out of 4 conserved tyrosine residues were generated.We compared myeloid differentiation of healthy donor iPSCs (HD-iPSCs), CN-iPSCs and CN-iPSCs with heterozygous and homozygous truncated G-CSF receptor (G-CSFR*HET CN-iPSCs and G-CSFR*HOM CN-iPSCs, respectively). Absolute cell count during myeloid differentiation (18-32 day) of HD-iPSCs, G-CSFR*HET CN-iPSCs and G-CSFR*HOM CN-iPSCs didn't differ significantly (p>0.05). In contrast, absolute cell count was found to be elevated from day 25 to day 32 of differentiation when G-CSFR*HET CN-iPSCs and G-CSFR*HOM CN-iPSCs hematopoietic progenitors were compared with CN-iPSCs-derived hematopoietic cells (p<0.05). FACS analysis of CN-iPSCs derived hematopoietic progenitors with and without truncated G-CSF receptor using a panel of hematopoietic markers (KDR, CD34, CD235a, CD41a, CD45 and CD33) showed no difference in percentage of early progenitors (CD34+/KDR+), E/MK precursors (CD41a+CD235a+/ CD45-), CD34+/CD45+ and CD34+/CD43+ cells. Intriguingly, we observed increase in percentage of CD34+/CD43+ (p<0.01), CD33+/CD45+ (p<0.05) and CD41a+CD235a+/ CD45- (p<0.05) cells at day 14 of myeloid differentiation of G-CSFR*HET CN-iPSCs and G-CSFR*HOM CN-iPSCs versus HD-iPSc. As expected, relative number CD14+ and CD16+ cells were not changed upon myeloid differentiation of hematopoietic progenitors with mutant G-CSFR but relative levels of CD15+ and CD15+/CD16+ were negatively affected by homozygous and heterozygous G-CSFR mutations. Next, CFU assay of hematopoietic progenitors derived from G-CSFR*HET CN-iPSCs and G-CSFR*HOM CN-iPSCs produced markedly less colonies than HD- and CN-iPSCs derived hematopoietic cells. Cell morphology analysis also confirmed the reduced number of mature granulocytes seen after myeloid differentiation of G-CSFR*HET CN-iPSCs and G-CSFR*HOM CN-iPSCs. Finally, reprogramming of BM cells from CN patients into iPSCs followed by mutation analysis of single cell derived iPSCs clones allowed us to identify iPSCs with naturally acquired G-CSFR mutations. Preliminary results revealed reduced myeloid differentiation of these cells, which is in line with data generated from G-CSFR*HET CN-iPSCs and G-CSFR*HOM CN-iPSCs.Using a combination of patient CN-iPSCs and CRISPR/Cas9 gene editing we were able to recapitulate main features of G-CSFR mutant versus G-CSFR WT CN cells - defective neutrophil differentiation and increased proliferation of human hematopoietic progenitors. Since virtually all studies of truncated G-CSFR receptor signaling have been performed in murine cytokine-responsive myeloid cell lines, our approach is more suitable to explore human aberrant G-CSF receptor signaling in depth. DisclosuresNo relevant conflicts of interest to declare.

Rhesus Macaque Activating Killer Immunoglobulin-Like Receptors Associate With Fc Receptor Gamma (FCER1G) and Not With DAP12 Adaptor Proteins Resulting in Stabilized Expression and Enabling Signal Transduction.

Activating killer cell immunoglobulin-like receptors (KIR) in macaques are thought to be derived by genetic recombination of the region encoding the transmembrane and intracellular part of KIR2DL4 and a KIR3D gene. As a result, all macaque activating KIR possess a positively charged arginine residue in the transmembrane region. As human KIR2DL4 associates with the FCER1G (also called Fc receptor-gamma, FcRγ) adaptor, we hypothesized that in contrast to human and great ape the activating KIRs of macaques associate with FcRγ instead of DAP12. By applying co-immunoprecipitation of transfected as well as primary cells, we demonstrate that rhesus macaque KIR3DS05 indeed associates with FcRγ and not with DAP12. This association with FcRγ results in increased and substantially stabilized surface expression of KIR3DS05. In addition, we demonstrate that binding of specific ligands of KIR3DS05, Mamu-A1*001 and A1*011, resulted in signal transduction in the presence of FcRγ in contrast to DAP12.

Features of MOG required for recognition by patients with MOG antibody-associated disorders.

Antibodies to myelin oligodendrocyte glycoprotein (MOG-Abs) define a distinct disease entity. Here we aimed to understand essential structural features of MOG required for recognition by autoantibodies from patients. We produced the N-terminal part of MOG in a conformationally correct form; this domain was insufficient to identify patients with MOG-Abs by ELISA even after site-directed binding. This was neither due to a lack of lipid embedding nor to a missing putative epitope at the C-terminus, which we confirmed to be an intracellular domain. When MOG was displayed on transfected cells, patients with MOG-Abs recognized full-length MOG much better than its N-terminal part with the first hydrophobic domain (P < 0.0001). Even antibodies affinity-purified with the extracellular part of MOG recognized full-length MOG better than the extracellular part of MOG after transfection. The second hydrophobic domain of MOG enhanced the recognition of the extracellular part of MOG by antibodies from patients as seen with truncated variants of MOG. We confirmed the pivotal role of the second hydrophobic domain by fusing the intracellular part of MOG from the evolutionary distant opossum to the human extracellular part; the chimeric construct restored the antibody binding completely. Further, we found that in contrast to 8-18C5, MOG-Abs from patients bound preferentially as F(ab')2 rather than Fab. It was previously found that bivalent binding of human IgG1, the prominent isotype of MOG-Abs, requires that its target antigen is displayed at a distance of 13-16 nm. We found that, upon transfection, molecules of MOG did not interact so closely to induce a Förster resonance energy transfer signal, indicating that they are more than 6 nm apart. We propose that the intracellular part of MOG holds the monomers apart at a suitable distance for bivalent binding; this could explain why a cell-based assay is needed to identify MOG-Abs. Our finding that MOG-Abs from most patients require bivalent binding has implications for understanding the pathogenesis of MOG-Ab associated disorders. Since bivalently bound antibodies have been reported to only poorly bind C1q, we speculate that the pathogenicity of MOG-Abs is mostly mediated by other mechanisms than complement activation. Therefore, therapeutic inhibition of complement activation should be less efficient in MOG-Ab associated disorders than in patients with antibodies to aquaporin-4 .

A Three-Dimensional Model of the Yeast Transmembrane Sensor Wsc1 Obtained by SMA-Based Detergent-Free Purification and Transmission Electron Microscopy.

The cell wall sensor Wsc1 belongs to a small family of transmembrane proteins, which are crucial to sustain cell integrity in yeast and other fungi. Wsc1 acts as a mechanosensor of the cell wall integrity (CWI) signal transduction pathway which responds to external stresses. Here we report on the purification of Wsc1 by its trapping in water-soluble polymer-stabilized lipid nanoparticles, obtained with an amphipathic styrene-maleic acid (SMA) copolymer. The latter was employed to transfer tagged sensors from their native yeast membranes into SMA/lipid particles (SMALPs), which allows their purification in a functional state, i.e., avoiding denaturation. The SMALPs composition was characterized by fluorescence correlation spectroscopy, followed by two-dimensional image acquisition from single particle transmission electron microscopy to build a three-dimensional model of the sensor. The latter confirms that Wsc1 consists of a large extracellular domain connected to a smaller intracellular part by a single transmembrane domain, which is embedded within the hydrophobic moiety of the lipid bilayer. The successful extraction of a sensor from the yeast plasma membrane by a detergent-free procedure into a native-like membrane environment provides new prospects for in vitro structural and functional studies of yeast plasma proteins which are likely to be applicable to other fungi, including plant and human pathogens.

Differential expression of transfer RNA-derived small RNAs in IgA nephropathy.

Background:IgA nephropathy (IgAN) is one of the most common forms of primary glomerulonephritis. Recent studies have indicated that small noncoding RNAs, such as tRNA-derived small RNAs (tsRNAs), might be novel biomarkers for glomerulonephritis. We therefore investigated the potential roles and possible functions of the tsRNAs in IgAN.Method:Peripheral blood mononuclear cells (PBMCs) were extracted from blood samples of the patients with IgAN and healthy control groups. The expression profiles of tsRNAs were assessed by small RNA sequencing (RNA-Seq) in PBMCs of the IgAN and control groups. Dysregulated tsRNAs were selected for validation by quantitative real-time polymerase chain reaction (qRT-PCR). Target gene prediction and enrichment were performed by bioinformatics analysis.Results:The results revealed that 143 significantly upregulated and 202 significantly downregulated tsRNAs were differentially altered in the IgAN group compared with the control group. Five upregulated tsRNAs (tRF-Val-AAC-007, tRF-Ala-AGC-063, tRF-Gln-CTG-010, tRF-Tyr-GTA-011 and tRF-Thr-AGT-007) and 3 downregulated tsRNAs (tiRNA-Val-TAC-004, tRF-Gly-CCC-005 and tRF-His-GTG-006) were selected for validation by qRT-PCR; the results were consistent with the sequencing data. Gene Ontology (GO) analysis revealed that the target genes predicted by upregulated tsRNAs were mostly enriched in “nucleic acid metabolic process," “intracellular part," and “ion binding," whereas the target genes predicted by downregulated tsRNAs were mostly enriched in “regulation of cellular component organization," “membrane-bound organelle," and “ion binding." Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the target genes predicted by upregulated tsRNAs were mostly enriched in “herpes simplex virus 1 infection," whereas the target genes predicted by downregulated tsRNAs were mostly enriched in “circadian rhythmConclusions:The present study confirmed the differential expression of tsRNAs in patients with IgAN, and these dysregulated tsRNAs might be novel potential targets for the diagnosis and treatment of IgAN.

Exploring GPCR-arrestin interfaces with genetically encoded crosslinkers.

β‐arrestins (βarr1 and βarr2) are ubiquitous regulators of G protein‐coupled receptor (GPCR) signaling. Available data suggest that β‐arrestins dock to different receptors in different ways. However, the structural characterization of GPCR‐arrestin complexes is challenging and alternative approaches to study GPCR‐arrestin complexes are needed. Here, starting from the finger loop as a major site for the interaction of arrestins with GPCRs, we genetically incorporate non‐canonical amino acids for photo‐ and chemical crosslinking into βarr1 and βarr2 and explore binding topologies to GPCRs forming either stable or transient complexes with arrestins: the vasopressin receptor 2 (rhodopsin‐like), the corticotropin‐releasing factor receptor 1, and the parathyroid hormone receptor 1 (both secretin‐like). We show that each receptor leaves a unique footprint on arrestins, whereas the two β‐arrestins yield quite similar crosslinking patterns. Furthermore, we show that the method allows defining the orientation of arrestin with respect to the GPCR. Finally, we provide direct evidence for the formation of arrestin oligomers in the cell.

Potential Inhibitors of Protein Tyrosine Phosphatase (PTP1B) Enzyme: Promising Target for Type-II Diabetes Mellitus.

There has been growing interest in the development of highly potent and selective protein tyrosine phosphatase (PTP1B) inhibitors for the past 2-3 decades. Though most PTPs share a common active site motif, the interest in selective inhibitors, particularly against PTP1B is increasing to discover new chemical entities as antidiabetic agents. In the current paradigm to find potent and selective PTP1B inhibitors, which is currently considered as one of the best validated biological targets for non-insulin-dependent diabetic and obese individuals, resistance to insulin due to decreased sensitivity of the insulin receptor is a pathological factor and is also genetically linked, causing type II diabetes. Insulin receptor sensitization is performed by a signal transduction mechanism via a selective protein tyrosine phosphatase (PTP1B). After the interaction of insulin with its receptor, autophosphorylation of the intracellular part of the receptor takes place, turning it into an active kinase (sensitization). PTP1B is involved in the desensitization of the receptor by dephosphorylation. PTP1b inhibitors delay the receptor desensitization, prolonging insulin effect and making PTP1B as a drug target for the treatment of diabetes II. Therefore, it has become a major target for the discovery of potent drugs for the treatment of type II diabetes and obesity. An attempt has been made in the present study to discuss the latest design and discovery of protein tyrosine phosphatase (PTP1B) inhibitors. Many PTP1B inhibitors such as diaminopyrroloquinazoline, triazines, pyrimido triazine derivatives, 2-(benzylamino)-1-phenylethanol, urea, acetamides and piperazinylpropanols, phenylsulphonamides and phenylcarboxamide, benzamido, arylcarboxylic acid derivatives, arylsupfonyl derivatives, thiazoles, isothiozolidiones and thiazolodinones have been discussed, citing the disease mechanisms. The reader will gain an overview of the structure and biological activity of recently developed PTPs inhibitors. The co-crystallized ligands and the screened inhibitors could be used as a template for the further design of potent congeners.

Abstract 2228: PD1/CTLA4 humanized mice - A great model for pre-clinical toxicology and efficacy evaluation of macromolecular drugs

Abstract Immune checkpoint antibodies have conferred huge cancer immunotherapeutic effect (CITE). Besides, severe immunotherapy related adverse events (irAE) occurred in combination therapy such as anti-PD1 combines with anti-CTLA4 antibodies greatly limit the development of cancer therapy. Animal models which can recapitulate both clinical irAE and CITE will be benefit for developing safer immune-checkpoint-targeting reagents. However, no animal models were found can be used for preclinical safety evaluation of drugs up to now. Given this fact, PD1/CTLA4 double-humanized mice models were established in both BALB/c (BALB/c-hPD1/hCTLA4) and C57BL/6 (B6-hPD1/hCTLA4) background, which by replacing the mouse extracellular domain of CTLA4 and PD1 with human segments while retaining mouse intact intracellular part, respectively. Our data showed that strong CITE against anti-hCTLA4 treatment observed in models from both BALB/c and B6 background. Apparent inhibition of tumor growth also emerged on subcutaneously engrafted CT26 tumor in BALB/c-hPD1/hCTLA4 mice by anti-PD1/CTLA4 bispecific antibody treatment. Meanwhile, severe irAEs which have been observed in clinical patients, such as joint swelling, skin inflammation, heart damage and infiltration of inflammatory cells in different tissues and organs could be reproduced in BALB/c-hPD1/hCTLA4 mice. Consistent with previous reports, we could not observe similar irAEs under drugs treatment at the same dose in C57BL/6-hPD1/hCTLA4 mice. Our data indicated that adult BALB/c-hPD1/hCTLA4 mice, but not C57BL/6-hPD1/hCTLA4 mice, were more sensitive to irAEs. Taken together, adult PD1/CTLA4 humanized mice in BALB/c background can not only be used to screen humanized antibodies, but also have tremendous values for preclinical safety evaluation. Citation Format: Cunxiang Ju, Mingkun Zhang, Rui Mao, Ma Xiuying, Jin Tang, Shuai Li, Jingjing Wang, Hongyan Sun, Jing Zhao, Xiang Gao. PD1/CTLA4 humanized mice - A great model for pre-clinical toxicology and efficacy evaluation of macromolecular drugs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2228.

Dimeric states of transmembrane domains of insulin and IGF-1R receptors: Structures and possible role in activation

Despite the biological significance of insulin signaling, the molecular mechanisms of activation of the insulin receptor (IR) and other proteins from its family remain elusive. Current hypothesis on signal transduction suggests ligand-triggered structural changes in the extracellular domain followed by transmembrane (TM) domains closure and dimerization leading to trans-autophosphorylation and kinase activity in intracellular segments of the receptor. Using NMR spectroscopy, we detected dimerization of isolated TM segments of IR in different membrane-mimicking environments and observed multiple signals of NH groups of protein backbone possibly corresponding to several dimer conformations. Taking available experimental data as constraints, several atomistic models of dimeric TM domains of IR and insulin-like growth factor 1 (IGF-1R) receptors were elaborated. Molecular dynamics simulations of IR ectodomain revealed noticeable collective movements potentially responsible for closure of the C-termini of FnIII-3 domains and spatial approaching of TM helices upon insulin-induced receptor activation. In addition, we demonstrated that the intracellular part of the receptor does not impose restrictions on the positioning of TM helices in the membrane. Finally, we used two independent structure prediction methods to generate a series of dimer conformations followed by their cluster analysis and dimerization free energy estimation to select the best dimer models. Biological relevance of the later was further tested via comparison of the hydrophobic organization of TM helices for both wild-type receptors and their mutants. Based on these data, the ability of several segments from other proteins to functionally replace IR and/or IGF-1R TM domains was explained.