Photoactivatable Cross-linking Research Articles

KDM4C represses liver fibrosis by regulating H3K9me3 methylation of ALKBH5 and m6A methylation of snail1 mRNA.

We aimed to disclose the molecular mechanism of snail1 in liver fibrosis. Carbon tetrachloride (CCl4) was used to induce a liver fibrosis model in mice whereby serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were evaluated, and liver pathological alternations were assessed. Rat hepatic stellate cells (HSC-T6) were irritated with transforming growth factor (TGF)-β1, followed by assessment of cell viability and migration. The levels of snail1, ALKBH5, and lysine specific demethylase 4C (KDM4C) were quantified by immunohistochemistry, western blot, or reverse transcription-quantitative polymerase chain reaction, in addition to α-smooth muscle actin (SMA), anti-collagen type I α1 (COL1A1), vimentin, and E-cadherin. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation and RNA stability were evaluated to determine the relationship between ALKBH5 and snail1. Changes in KDM4C-bound ALKBH5 promoter and enrichment of histone H3 lysine 9 trimethylation (H3K9me3) at the ALKBH5 promoter were determined using chromatin immunoprecipitation. In fibrosis mice, snail1 was upregulated while ALKBH5 and KDM4C were downregulated. KDM4C overexpression reduced serum ALT and AST levels, liver injury, and α-SMA, COL1A1 and VIMENTIN expressions but increased E-cadherin expression. However, the aforementioned trends were reversed by concurrent overexpression of snail1. In HSC-T6 cells exposed to TGF-β1, ALKBH5 overexpression weakened cell viability and migration, downregulated α-SMA, COL1A1 and VIMENTIN, upregulated E-CADHERIN, and decreased m6A modification of snail1 and its mRNA stability. KDM4C increased ALKBH5 expression by lowering H3K9me3 level, but inhibited HSC-T6 cell activation by regulating the ALKBH5/snail1 axis. KDM4C decreases H3K9me3 methylation to upregulate ALKBH5 and subsequently inhibits snail1, ultimately impeding liver fibrosis.

4D-printing of high-temperature shape-memory polymers based on polyimide, N,N-dimethylacrylamide and photoactive cross-linkers

Thermally responsive shape memory polyimides (PIs) have attracted significant attention in the high-tech fields like aerospace industry. At the same time creating of materials and approaches to the formation of three-dimensional (3D) shape memory structures containing PIs remains an urgent task. In this work we have developed new photopolymeric compositions (PPCs) based on high-performance readily soluble PI, reactive solvent (N,N-dimethylacrylamide) and photoactive cross-linker (bisphenol A ethoxylate diacrylate (BAEDA) or tris[2-(acryloyloxy)ethyl] isocyanurate (TAI)). The resulting PPCs were used to form 3D-structures using LCD 3D-printing. It has been found that the nature of the cross-linker and post-processing temperature have a significant impact on the thermal and mechanical properties of the materials. The tensile strength has the maximum value after the thermal post-curing at 250°С for 1 h (83.3 ± 7.4 MPa and 60.8 ± 4.8 MPa for PPCs with TAI and BAEDA, respectively). Moreover, 4D-printed PI-containing materials exhibit an excellent shape memory performance at recovery temperatures >150 °C, with shape fixity ratio >99% and shape recovery ratio up to 95.6%.

FOXM1 Participates in Scleral Remodeling in Myopia by Upregulating APOA1 Expression Through METTL3/YTHDF2.

Apolipoprotein A1 (APOA1) is a potential crucial protein and treatment goal for pathological myopia in humans. This study set out to discover the function of APOA1 in scleral remodeling in myopia and its underlying mechanisms. A myopic cell model was induced using hypoxia. Following loss- and gain-of function experiments, the expression of the myofibroblast transdifferentiation-related and collagen production-related factors Forkhead box M1 (FOXM1), APOA1, and methyltransferase-like 3 (METTL3) in the myopic cell model was examined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting. The proliferation and apoptosis were determined by Cell Counting Kit-8 assay and flow cytometry, respectively. Chromatin immunoprecipitation (ChIP) was employed to examine FOXM1 enrichment in the METTL3 promoter, methylated RNA immunoprecipitation (Me-RIP) to examine the N6-methyladenosine (m6A) modification level of APOA1, and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to examine the binding between METTL3 and APOA1. Hypoxia-induced human scleral fibroblasts (HSFs) had high APOA1 and FOXM1 expression and low METTL3 expression. FOXM1 knockdown elevated METTL3 expression and downregulated APOA1 expression. FOXM1 was enriched in METTL3 promoter. APOA1 or FOXM1 knockdown or METTL3 overexpression reversed the hypoxia-induced elevation in vinculin, paxillin, and α-smooth muscle actin (α-SMA) levels and apoptosis and the reduction in collagen, type I, alpha 1 (COL1A1) level and cell proliferation in HSFs. METTL3 or YTH N6-methyladenosine RNA binding protein F2 (YTHDF2) knockdown or APOA1 overexpression reversed the impacts of FOXM1 knockdown on vinculin, paxillin, α-SMA, and COL1A1 expression and cell proliferation and apoptosis. FOXM1 elevated the m6A methylation level of APOA1 by repressing METTL3 transcription and enhanced APOA1 mRNA stability and transcription by reducing the YTHDF2-recognized m6A methylated transcripts.

Optimized infrared photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (IR-PAR-CLIP) protocol identifies novel IGF2BP3-interacting RNAs in colon cancer cells.

The conserved family of RNA-binding proteins (RBPs), IGF2BPs, plays an essential role in posttranscriptional regulation controlling mRNA stability, localization, and translation. Mammalian cells express three isoforms of IGF2BPs: IGF2BP1-3. IGF2BP3 is highly overexpressed in cancer cells, and its expression correlates with a poor prognosis in various tumors. Therefore, revealing its target RNAs with high specificity in healthy tissues and in cancer cells is of crucial importance. Photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) identifies the binding sites of RBPs on their target RNAs at nucleotide resolution in a transcriptome-wide manner. Here, we optimized the PAR-CLIP protocol to study RNA targets of endogenous IGF2BP3 in a human colorectal carcinoma cell line. To this end, we first established an immunoprecipitation protocol to obtain highly pure endogenous IGF2BP3-RNA complexes. Second, we modified the protocol to use highly sensitive infrared (IR) fluorescent dyes instead of radioactive probes to visualize IGF2BP3-crosslinked RNAs. We named the modified method "IR-PAR-CLIP." Third, we compared RNase cleavage conditions and found that sequence preferences of the RNases impact the number of the identified IGF2BP3 targets and introduce a systematic bias in the identified RNA motifs. Fourth, we adapted the single adapter circular ligation approach to increase the efficiency in library preparation. The optimized IR-PAR-CLIP protocol revealed novel RNA targets of IGF2BP3 in a human colorectal carcinoma cell line. We anticipate that our IR-PAR-CLIP approach provides a framework for studies of other RBPs.

Methyltransferase-like 3 facilitates the stem cell properties of esophageal cancer by upregulating patched homolog 1 via N6-methyladenosine methylation.

Patched homolog 1 (PTCH1) has been proven to facilitate cell proliferation and self-renewal in esophageal cancer (EC). The present study intended to exploit the influence of PTCH1 on EC cells and the potential mechanisms. PTCH1 and methyltransferase-like 3 (METTL3) expression were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot in EC cell lines. Following the loss- and gain-of-function assays, cell proliferation was examined by cell counting kit (CCK)-8 and clone formation assays, invasion and migration by Transwell and scratch assays, and the sphere-forming ability of stem cells by cell sphere-forming assay. The expression of stemness genes NANOG homeobox protein (NANOG), octamer-binding transcription factor 4 (Oct4), and sex-determining region Y-box 2 (SOX2) was detected by Western blot. Methylated RNA immunoprecipitation (Me-RIP) assay was performed to test N6-methyladenosine (m6A) modification levels of PTCH1 mRNA, RIP and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) assays to assess the binding of METTL3 to PTCH1, and actinomycin D treatment to examine PTCH1 mRNA stability. A xenograft tumor model in nude mice was established for further in vivo verification. PTCH1 and METTL3 expression was high in EC cells. Knockdown of METTL3 reduced m6A level and stability of PTCH1 mRNA. Knockdown of PTCH1 or METTL3 declined invasion, proliferation, migration, and NANOG, Oct4, and SOX2 levels in EC cells, and reduced sphere-forming abilities of EC stem cells. Overexpression of PTCH1 abolished the suppressive effect of METTL3 knockdown on EC cells in vitro. METTL3 knockdown repressed tumor growth in nude mice, which was negated by further overexpressing PTCH1. METTL3 facilitated growth and stemness of EC cells via upregulation of PTCH1 expression by enhancing PTCH1 m6A modification.NEW & NOTEWORTHY PTCH1 has been proved to facilitate cell proliferation and self-renewal in esophageal cancer. We studied the upstream regulation mechanism of PTCH1 by METTL3 through m6A modification. Our results provide a new target and theoretical basis for the treatment of esophageal cancer.

Photoradical-Mediated Catalyst-Independent Protein Cross-Link with Unusual Fluorescence Properties.

Photo-actively modified natural amino acids have served as lucrative probes for precise mapping of the dynamics, interaction networks, and turnover of cytosolic proteins both in vivo and ex vivo. In our attempts to extend the utility of photoreactive reporters to map the molecular characteristics of vital membrane proteins, we carried out site-selective incorporation of 7-fluoro-indole in the human mitochondrial outer membrane protein VDAC2 (voltage-dependent anion channel isoform 2), with the aim of generating Trp-Phe/Tyr cross-links. Prolonged irradiation at 282 nm provided us with a surprisingly unusual fluorophore that displayed sizably red-shifted excitation (λex-max =280 nm→360 nm) and emission (λem-max =330 nm→430 nm) spectra that was reversible with organic solvents. By measuring the kinetics of the photo-activated cross-linking with a library of hVDAC2 variants, we demonstrate that formation of this unusual fluorophore is kinetically retarded, independent of tryptophan, and is site-specific. Using other membrane (Tom40 and Sam50) and cytosolic (MscR and DNA Pol I) proteins, we additionally show that formation of this fluorophore is protein-independent. Our findings reveal the photoradical-mediated accumulation of reversible tyrosine cross-links, with unusual fluorescent properties. Our findings have immediate applications in protein biochemistry and UV-mediated protein aggregation and cellular damage, opening avenues for formulating therapeutics that prolong cell viability in humans.

A smart pathogen detector engineered from intracellular hydrogelation of DNA-decorated macrophages

Bacterial infection is a major threat to global public health, which urgently requires useful tools to rapidly analyze pathogens in the early stages of infection. Herein, we develop a smart macrophage (Mø)-based bacteria detector, which can recognize, capture, enrich and detect different bacteria and their secreted exotoxins. We transform the fragile native Møs into robust gelated cell particles (GMøs) using photo-activated crosslinking chemistry, which retains membrane integrity and recognition capacity for different microbes. Meanwhile, these GMøs equipped with magnetic nanoparticles and DNA sensing elements can not only respond to an external magnet for facile bacteria collection, but allow the detection of multiple types of bacteria in a single assay. Additionally, we design a propidium iodide-based staining assay to rapidly detect pathogen-associated exotoxins at ultralow concentrations. Overall, these nanoengineered cell particles have broad applicability in the analysis of bacteria, and could potentially be used for the management and diagnosis of infectious diseases.

RNA binding protein PRRC2B mediates translation of specific mRNAs and regulates cell cycle progression.

Accumulating evidence suggests that posttranscriptional control of gene expression, including RNA splicing, transport, modification, translationand degradation, primarily relies on RNA binding proteins (RBPs). However, the functions of many RBPs remain understudied. Here, we characterized the function of a novel RBP, Proline-Rich Coiled-coil 2B (PRRC2B). Through photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation and sequencing (PAR-CLIP-seq), we identified transcriptome-wide CU- or GA-rich PRRC2B binding sites near the translation initiation codon on a specific cohort of mRNAs in HEK293T cells. These mRNAs, including oncogenes and cell cycle regulators such as CCND2 (cyclin D2), exhibited decreased translation upon PRRC2B knockdown as revealed by polysome-associated RNA-seq, resulting in reduced G1/S phase transition and cell proliferation. Antisense oligonucleotides blocking PRRC2B interactions with CCND2 mRNA decreased its translation, thus inhibiting G1/S transition and cell proliferation. Mechanistically, PRRC2B interactome analysis revealed RNA-independent interactions with eukaryotic translation initiation factors 3 (eIF3) and 4G2 (eIF4G2). The interaction with translation initiation factors is essential for PRRC2B function since the eIF3/eIF4G2-interacting defective mutant, unlike wild-type PRRC2B, failed to rescue the translation deficiency or cell proliferation inhibition caused by PRRC2B knockdown. Altogether, our findings reveal that PRRC2B is essential for efficiently translating specific proteins required for cell cycle progression and cell proliferation.

Upregulation of UBR1 m6A methylation by METTL14 inhibits autophagy in spinal cord injury.

Objective: Gene Expression Omnibus database shows significantly downregulated expression of ubiquitin protein ligase E3 component N-recognin 1 (UBR1) in spinal cord injury (SCI). In this study, we investigated the mechanism of action of UBR1 in SCI.Methods: Following the establishment of SCI models in rats and PC12 cells, Basso-Beattie-Bresnahan score and H&E and Nissl staining were used to evaluate SCI. The localization of NeuN/LC3 and the expression of LC3II/I, Beclin-1, and p62 were detected to assess autophagy. The expression of Bax, Bcl-2, and cleaved caspase-3 was detected and TdT-mediated dUTP-biotin nick end-labelling staining was employed to determine the changes in apoptosis. The N(6)-methyladenosine (m6A) modification level of UBR1 was analyzed by methylated RNA immunoprecipitation, and the binding of METTL14 and UBR1 mRNA was analysed by photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation.Results: UBR1 was poorly expressed, and METTL14 was highly expressed in rat and cell models of SCI. UBR1 overexpression or METTL14 knockdown enhanced motor function in rats with SCI. Moreover, this modification increased Nissl bodies and autophagy and inhibited apoptosis in the spinal cord of SCI rats. METTL14 silencing reduced the m6A modification level of UBR1 and enhanced UBR1 expression. Importantly, UBR1 knockdown nullified METTL14 knockdown -induced autophagy promotion and apoptosis reduction.Conclusion: The METTL14-catalyzed m6A methylation of UBR1 promoted apoptosis and inhibited autophagy in SCI.

A multidisciplinary approach to the identification of the protein-RNA connectome in double-stranded RNA virus capsids.

How multi-segmented double-stranded RNA (dsRNA) viruses correctly incorporate their genomes into their capsids remains unclear for many viruses, including Bluetongue virus (BTV), a Reoviridae member, with a genome of 10 segments. To address this, we used an RNA-cross-linking and peptide-fingerprinting assay (RCAP) to identify RNA binding sites of the inner capsid protein VP3, the viral polymerase VP1 and the capping enzyme VP4. Using a combination of mutagenesis, reverse genetics, recombinant proteins and in vitro assembly, we validated the importance of these regions in virus infectivity. Further, to identify which RNA segments and sequences interact with these proteins, we used viral photo-activatable ribonucleoside crosslinking (vPAR-CL) which revealed that the larger RNA segments (S1-S4) and the smallest segment (S10) have more interactions with viral proteins than the other smaller segments. Additionally, using a sequence enrichment analysis we identified an RNA motif of nine bases that is shared by the larger segments. The importance of this motif for virus replication was confirmed by mutagenesis followed by virus recovery. We further demonstrated that these approaches could be applied to a related Reoviridae member, rotavirus (RV), which has human epidemic impact, offering the possibility of novel intervention strategies for a human pathogen.

MiR-199a-3p Induces Mesenchymal to Epithelial Transition of Keratinocytes by Targeting RAP2B

Cutaneous squamous cell carcinoma (CSCC) is an epidermal skin cancer that evolves from normal epidermis along several pre-malignant stages. Previously we found specific miRNAs alterations in each step along these stages. miR-199a-3p expression decreases at the transition to later stages. A crucial step for epithelial carcinoma cells to acquire invasive capacity is the disruption of cell-cell contacts and the gain of mesenchymal motile phenotype, a process known as epithelial-to-mesenchymal transition (EMT). This study aims to study the role of decreased expression of miR-199a-3p in keratinocytes' EMT towards carcinogenesis. First, we measured miR-199a-3p in different stages of epidermal carcinogenesis. Then, we applied Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) assay to search for possible biochemical targets of miR-199a-3p and verified that Ras-associated protein B2 (RAP2B) is a bona-fide target of miR-199a-3p. Next, we analyzed RAP2B expression, in CSCC biopsies. Last, we evaluated possible mechanisms leading to decreased miR-199a-3p expression. miR-199a-3p induces a mesenchymal to epithelial transition (MET) in CSSC cells. Many of the under-expressed genes in CSCC overexpressing miR-199a-3p, are possible targets of miR-199a-3p and play roles in EMT. RAP2B is a biochemical target of miR-199a-3p. Overexpression of miR-199a-3p in CSCC results in decreased phosphorylated focal adhesion kinase (FAK). In addition, inhibiting FAK phosphorylation inhibits EMT marker genes' expression. In addition, we proved that DNA methylation is part of the mechanism by which miR-199a-3p expression is inhibited. However, it is not by the methylation of miR-199a putative promoter. These findings suggest that miR-199a-3p inhibits the EMT process by targeting RAP2B. Inhibitors of RAP2B or FAK may be effective therapeutic agents for CSCC.

HuR-dependent expression of Wisp1 is necessary for TGFβ-induced cardiac myofibroblast activity

Cardiac fibrosis is regulated by the activation and phenotypic switching of quiescent cardiac fibroblasts to active myofibroblasts, which have extracellular matrix (ECM) remodeling and contractile functions which play a central role in cardiac remodeling in response to injury. Here, we show that expression and activity of the RNA binding protein HuR is increased in cardiac fibroblasts upon transformation to an active myofibroblast. Pharmacological inhibition of HuR significantly blunts the TGFβ-dependent increase in ECM remodeling genes, total collagen secretion, in vitro scratch closure, and collagen gel contraction in isolated primary cardiac fibroblasts, suggesting a suppression of TGFβ-induced myofibroblast activation upon HuR inhibition. We identified twenty-four mRNA transcripts that were enriched for HuR binding following TGFβ treatment via photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP). Eleven of these HuR-bound mRNAs also showed significant co-expression correlation with HuR, αSMA, and periostin in primary fibroblasts isolated from the ischemic-zone of infarcted mouse hearts. Of these, WNT1-inducible signaling pathway protein-1 (Wisp1; Ccn4), was the most significantly associated with HuR expression in fibroblasts. Accordingly, we found Wisp1 expression to be increased in cardiac fibroblasts isolated from the ischemic-zone of mouse hearts following ischemia/reperfusion, and confirmed Wisp1 expression to be HuR-dependent in isolated fibroblasts. Finally, addition of exogenous recombinant Wisp1 partially rescued myofibroblast-induced collagen gel contraction following HuR inhibition, demonstrating that HuR-dependent Wisp1 expression plays a functional role in HuR-dependent MF activity downstream of TGFβ. In conclusion, HuR activity is necessary for the functional activation of primary cardiac fibroblasts in response to TGFβ, in part through post-transcriptional regulation of Wisp1.

Lipids mediate supramolecular outer membrane protein assembly in bacteria.

β Barrel outer membrane proteins (OMPs) cluster into supramolecular assemblies that give function to the outer membrane (OM) of Gram-negative bacteria. How such assemblies form is unknown. Here, through photoactivatable cross-linking into the Escherichia coli OM, coupled with simulations, and biochemical and biophysical analysis, we uncover the basis for OMP clustering in vivo. OMPs are typically surrounded by an annular shell of asymmetric lipids that mediate higher-order complexes with neighboring OMPs. OMP assemblies center on the abundant porins OmpF and OmpC, against which low-abundance monomeric β barrels, such as TonB-dependent transporters, are packed. Our study reveals OMP-lipid-OMP complexes to be the basic unit of supramolecular OMP assembly that, by extending across the entire cell surface, couples the requisite multifunctionality of the OM to its stability and impermeability.

Features of Luminescent Properties of Alginate Aerogels with Rare Earth Elements as Photoactive Cross-Linking Agents.

Luminescent aerogels based on sodium alginate cross-linked with ions of rare earth elements (Eu3+, Tb3+, Sm3+) and containing phenanthroline, thenoyltrifluoroacetone, dibenzoylmethane, and acetylacetone as ligands introduced into the matrix during the impregnation of alginate aerogels (AEG), were obtained for the first time in a supercritical carbon dioxide medium. The impregnation method used made it possible to introduce organically soluble sensitizing ligands into polysaccharide matrices over the entire thickness of the sample while maintaining the porous structure of the aerogel. It is shown that the pore size and their specific area are 150 nm and 270 m2/g, respectively. Moreover, metal ions with content of about 23 wt.%, acting as cross-linking agents, are uniformly distributed over the thickness of the sample. In addition, the effect of sensitizing ligands on the luminescence intensity of cross-linked aerogel matrices is considered. The interaction in the resulting metal/ligand systems is unique for each pair, which is confirmed by the detection of broad bands with individual positions in the luminescence excitation spectra of photoactive aerogels.

Control of immediate early gene expression by CPEB4-repressor complex-mediated mRNA degradation

BackgroundCytoplasmic polyadenylation element-binding protein 4 (CPEB4) is known to associate with cytoplasmic polyadenylation elements (CPEs) located in the 3′ untranslated region (UTR) of specific mRNAs and assemble an activator complex promoting the translation of target mRNAs through cytoplasmic polyadenylation.ResultsHere, we find that CPEB4 is part of an alternative repressor complex that mediates mRNA degradation by associating with the evolutionarily conserved CCR4-NOT deadenylase complex. We identify human CPEB4 as an RNA-binding protein (RBP) with enhanced association to poly(A) RNA upon inhibition of class I histone deacetylases (HDACs), a condition known to cause widespread degradation of poly(A)-containing mRNA. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis using endogenously tagged CPEB4 in HeLa cells reveals that CPEB4 preferentially binds to the 3′UTR of immediate early gene mRNAs, at G-containing variants of the canonical U- and A-rich CPE located in close proximity to poly(A) sites. By transcriptome-wide mRNA decay measurements, we find that the strength of CPEB4 binding correlates with short mRNA half-lives and that loss of CPEB4 expression leads to the stabilization of immediate early gene mRNAs. Akin to CPEB4, we demonstrate that CPEB1 and CPEB2 also confer mRNA instability by recruitment of the CCR4-NOT complex.ConclusionsWhile CPEB4 was previously known for its ability to stimulate cytoplasmic polyadenylation, our findings establish an additional function for CPEB4 as the RNA adaptor of a repressor complex that enhances the degradation of short-lived immediate early gene mRNAs.

A method to map the interaction network of the nuclear lamina with genetically encoded photo-crosslinkers in vivo.

Lamins are intermediate filaments that assemble in a meshwork at the inner nuclear periphery of metazoan cells. The nuclear periphery fulfils important functions by providing stability to the nuclear membrane, connecting the cytoskeleton with chromatin, and participating in signal transduction. Mutations in lamins interfere with these functions and cause severe, phenotypically diverse diseases collectively referred to as laminopathies. The molecular consequences of these mutations are largely unclear but likely include alterations in lamin-protein and lamin-chromatin interactions. These interactions are challenging to study biochemically mainly because the lamina is resistant to high salt and detergent concentrations and co-immunoprecipitation are susceptible to artefacts. Here, we used genetic code expansion to install photo-activated crosslinkers to capture direct lamin-protein interactions in vivo. Mapping the Ig-fold of laminC for interactions, we identified laminC-crosslink products with laminB1, LAP2, and TRIM28. We observed significant changes in the crosslink intensities between laminC mutants mimicking different phosphorylation states. Similarly, we found variations in laminC crosslink product intensities comparing asynchronous cells and cells synchronized in prophase. This method can be extended to other laminC domains or other lamins to reveal changes in their interactome as a result of mutations or cell cycle stages.

Abstract 1397: A phenotypic-based drug discovery approach against tumors with MTAP loss

Abstract The Methylthioadenosine phosphorylase (MTAP) gene is in chromosome 9q21 location, which includes p16/CDKNA and is homozygously deleted in about 15% of all cancers, including a large proportion of gliomas, mesotheliomas and T cell lymphomas. MTAP cleaves methylthioadenosine (MTA), a byproduct of polyamine synthesis, to regenerate methionine and adenine. Several functional genomics screens have identified the downstream genes MAT2A and PRMT5 as synthetically lethal with MTAP loss. These two targets have been the subject of several drug discovery efforts. Our approach was based on a forward chemical genetic approach, in which we employed two isogenic cell lines, PANC1, with MTAP loss and PAM cells where MTAP is re-introduced. A library of over 200,000 compounds was screened for hits that affected the viability of PANC1 cell, whereas spared PAM cells. One hit series was found to reproducibly inhibit the proliferation of PANC1 cells with EC50s in the low single digit uM range and one order of selectivity over PAM cells. Phenotype-based optimization resulted in improved analogs, with prototype MTB-22252 exhibiting an EC50 of 47nM in PANC1 cells and 15-fold selectivity. An affinity probe was designed based on an analog of MTB-22252 bearing a biotin moiety via a linker and an azide group for photoactivatable cross-linking with potential interacting proteins. This probe maintained cellular activity and selectivity and it was used in an MS-proteomics experiment in PANC1 cells. The top protein identified by this experiment showed high enrichment over DMSO control and was effectively competed out by MTB-22252. MTB2252 and analogs thermo-stabilized this protein by up to 50C in PANC1 cells. PANC1 cells expressed high levels of this protein, whereas insensitive PAM cell did not express it at all. In additional cell line pairs, sensitivity to MTB-2252 correlated well with the expression levels of this protein. Reintroduction of MTAP in cells lacking MTAP and this new protein, resulted in loss of expression of the new protein, suggesting a regulatory connection. In a wider cell panel of over 130 cell lines, there was correlation of sensitivity with MTAP loss and even better correlation with the expression levels of this protein. Some of the most sensitive cell lines included several colorectal, glioma and hematologic cancer cell lines. In vivo evaluation of MTB-9655 against some of the most sensitive tumor cell lines is on-going. Citation Format: Andreas Goutopoulos, Iris Amanati, Avital Hay-Koren, Ali Fattaey, Philippe Nakache, Dimitri Kovalerchik, Dikla Paz, Simone Botti, Hanna Oppenheimer, Omri Erez, Irit Fainer. A phenotypic-based drug discovery approach against tumors with MTAP loss [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1397.

Essential roles of exosome and circRNA_101093 on ferroptosis desensitization in lung adenocarcinoma.

BackgroundResistance to ferroptosis, a regulated cell death caused by iron‐dependent excessive accumulation of lipid peroxides, has recently been linked to lung adenocarcinoma (LUAD). Intracellular antioxidant systems are required for protection against ferroptosis. The purpose of the present study was to investigate whether and how extracellular system desensitizes LUAD cells to ferroptosis.MethodsEstablished human lung fibroblasts MRC‐5, WI38, and human LUAD H1650, PC9, H1975, H358, A549, and H1299 cell lines, tumor and matched normal adjacent tissues of LUAD, and plasma from healthy individuals and LUAD patients were used in this study. Immunohistochemistry and immunoblotting were used to analyze protein expression, and quantitative reverse transcription‐PCR was used to analyze mRNA expression. Cell viability, cell death, and the lipid reactive oxygen species generation were measured to evaluate the responses to ferroptosis. Exosomes were observed using transmission electron microscope. The localization of arachidonic acid (AA) was detected using click chemistry labeling followed by confocal microscopy. Interactions between RNAs and proteins were detected using RNA pull‐down, RNA immunoprecipitation and photoactivatable ribonucleoside‐enhanced crosslinking and immunoprecipitation methods. Proteomic analysis was used to investigate RNA‐regulated proteins, and metabolomic analysis was performed to analyze metabolites. Cell‐derived xenograft, patient‐derived xenograft, cell‐implanted intrapulmonary LUAD mouse models and plasma/tissue specimens from LUAD patients were used to validate the molecular mechanism.ResultsPlasma exosome from LUAD patients specifically reduced lipid peroxidation and desensitized LUAD cells to ferroptosis. A potential explanation is that exosomal circRNA_101093 (cir93) maintained an elevation in intracellular cir93 in LUAD to modulate AA, a poly‐unsaturated fatty acid critical for ferroptosis‐associated increased peroxidation in the plasma membrane. Mechanistically, cir93 interacted with and increased fatty acid‐binding protein 3 (FABP3), which transported AA and facilitated its reaction with taurine. Thus, global AA was reduced, whereas N‐arachidonoyl taurine (NAT, the product of AA and taurine) was induced. Notably, the role of NAT in suppressing AA incorporation into the plasma membrane was also revealed. In pre‐clinical in vivo models, reducing exosome improved ferroptosis‐based treatment.ConclusionExosome and cir93 are essential for desensitizing LUAD cells to ferroptosis, and blocking exosome may be helpful for future LUAD treatment.

ALKBH5 promotes the progression of infantile hemangioma through regulating the NEAT1/miR-378b/FOSL1 axis.

Our work aims to investigate long non-coding RNA (lncRNA) N6-methyladenosine (m6A) modification and its role in infantile hemangioma (IH). ThemRNA and protein expression levels were assessed using quantitative real-time polymerase chain reaction, western blot and immunohistochemistry. Me-RIP assay was performed to evaluate lncRNA NEAT1 m6A levels. Cell proliferation, migration and invasion were evaluated using cell counting kit-8 assay, transwell migration and invasion assay, respectively. Photo-activatable ribonucleoside-enhanced crosslinking and immunoprecipitation assay was conducted to verify the binding relationship between lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) and ALKBH5 (an RNA demethylase). The binding relationship between lncRNA NEAT1, microRNA (miR)-378b and FOS-like antigen 1 (FOSL1) was verified using dual-luciferase reporter gene assay and/or RNA immunoprecipitation assay. ALKBH5, lncRNA NEAT1 and FOLS1 expression was elevated in IH tissues, while miR-378b was downregulated. ALKBH5 knockdown suppressed cell proliferation, migration and invasion of IH cells, while promoting cell apoptosis. ALKBH5 promoted lncRNA NEAT1 expression by reducing the m6A modification of lncRNA NEAT1. In addition, miR-378b was the target of lncRNA NEAT1, and its overexpression reversed the promotion effect of lncRNA NEAT1 overexpression on IH cell tumor-like behaviors. Moreover, FOLS1 was the target of miR-378b, and its overexpression reversed the inhibitory effect of miR-378b overexpression on IH cell tumor-like behaviors in vitro. ALKBH5 might have great potential as therapeutic target for IH, since ALKBH5 silencing suppressed IH progression by regulation of the NEAT1/miR-378b/FOSL1 axis.

Identification and Characterization of a New Splicing Variant of Prokineticin 2.

Prokineticin 2 (PROK2) is a secreted bioactive peptide that regulates a variety of biological responses via two GPCRs, the prokineticin receptors (PROKRs). The aim of this study was to characterize a new alternatively spliced product of the prok2 gene consisting of four exons. The 40-amino acid peptide, designated PROK2C, is encoded by exon 1 and exon 4, and its expression was detected in the hippocampus and spinal cord of mice. PROK2C was expressed in a heterologous system, Pichia pastoris, and its binding specificity to the amino-terminal regions of PROKR1 and PROKR2 was investigated by GST pull-down experiments. In addition, the introduction of the unnatural amino acid p-benzoyl-L-phenylalanine using amber codon suppression technology demonstrated the role of tryptophan at position 212 of PROKR2 for PROK2C binding by photoactivatable cross-linking. The functional significance of this new isoform was determined in vivo by nociceptive experiments, which showed that PROK2C elicits strong sensitization of peripheral nociceptors to painful stimuli. In order to analyze the induction of PROK2C signal transduction, STAT3 and ERK phosphorylation levels were determined in mammalian CHO cells expressing PROKR1 and PROKR2. Our data show by in vivo and in vitro experiments that PROK2C can bind and activate both prokineticin receptors.