Stable Isotope Labeling With Amino Acids In Cell Culture Research Articles

Cathepsin D inhibition during neuronal differentiation selectively affects individual proteins instead of overall protein turnover

Cathepsin D (CTSD) is a lysosomal aspartic protease and its inherited deficiency causes a severe pediatric neurodegenerative disease called neuronal ceroid lipofuscinosis (NCL) type 10. The lysosomal dysfunction in the affected patients leads to accumulation of undigested lysosomal cargo especially in none-dividing cells, such as neurons, resulting in death shortly after birth. To explore which proteins are mainly affected by the lysosomal dysfunction due to CTSD deficiency, Lund human mesencephalic (LUHMES) cells, capable of inducible dopaminergic neuronal differentiation, were treated with Pepstatin A. This inhibitor of “acidic” aspartic proteases caused accumulation of acidic intracellular vesicles in differentiating LUHMES cells. Pulse-chase experiments involving stable isotope labelling with amino acids in cell culture (SILAC) with subsequent mass-spectrometric protein identification and quantification were performed. By this approach, we studied the degradation and synthesis rates of 695 and 680 proteins during early and late neuronal LUHMES differentiation, respectively. Interestingly, lysosomal bulk proteolysis was not altered upon Pepstatin A treatment. Instead, the protease inhibitor selectively changed the turnover of individual proteins. Especially proteins belonging to the mitochondrial energy supply system were differentially degraded during early and late neuronal differentiation indicating a high energy demand as well as stress level in LUHMES cells treated with Pepstatin A.

The MYC-Regulated RNA-Binding Proteins hnRNPC and LARP1 Are Drivers of Multiple Myeloma Cell Growth and Disease Progression and Negatively Predict Patient Survival

Multiple myeloma (MM) is a malignant plasma cell disorder in which the MYC oncogene is frequently dysregulated. Due to its central role, MYC has been proposed as a drug target; however, the development of a clinically applicable molecule modulating MYC activity remains an unmet challenge. Consequently, an alternative is the development of therapeutic options targeting proteins located downstream of MYC. Therefore, we aimed to identify undescribed MYC-target proteins in MM cells using Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and mass spectrometry. We revealed a cluster of proteins associated with the regulation of translation initiation. Herein, the RNA-binding proteins Heterogeneous Nuclear Ribonucleoprotein C (hnRNPC) and La Ribonucleoprotein 1 (LARP1) were predominantly downregulated upon MYC depletion. CRISPR-mediated knockout of either hnRNPC or LARP1 in conjunction with redundant LARP family proteins resulted in a proliferative disadvantage for MM cells. Moreover, high expression levels of these proteins correlate with high MYC expression and with poor survival and disease progression in MM patients. In conclusion, our study provides valuable insights into MYC’s role in translation initiation by identifying hnRNPC and LARP1 as proliferation drivers of MM cells and as both predictive factors for survival and disease progression in MM patients.

The PRMT5/WDR77 complex restricts hepatitis E virus replication.

Hepatitis E virus (HEV) is one of the main pathogenic agents of acute hepatitis in the world. The mechanism of HEV replication, especially host factors governing HEV replication is still not clear. Here, using HEV ORF1 trans-complementation cell culture system and HEV replicon system, combining with stable isotope labelling with amino acids in cell culture (SILAC) and mass spectrometry (MS), we aimed to identify the host factors regulating HEV replication. We identified a diversity of host factors associated with HEV ORF1 protein, which were putatively responsible for viral genomic RNA replication, in these two cell culture models. Of note, the protein arginine methyltransferase 5 (PRMT5)/WDR77 complex was identified in both cell culture models as the top hit. Furthermore, we demonstrated that PRMT5 and WDR77 can specifically inhibit HEV replication, but not other viruses such as HCV or SARS-CoV-2, and this inhibition is conserved among different HEV strains and genotypes. Mechanistically, PRMT5/WDR77 can catalyse methylation of ORF1 on its R458, impairing its replicase activity, and virus bearing R458K mutation in ORF1 relieves the restriction of PRMT5/WDR77 accordingly. Taken together, our study promotes more comprehensive understanding of viral infections but also provides therapeutic targets for intervention.

Mass-Spectrometric Evaluation of the African Swine Fever Virus-Induced Host Shutoff Using Dynamic Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC).

African swine fever is a viral disease of swine caused by the African swine fever virus (ASFV). Currently, ASFV is spreading over the Eurasian continent and threatening global pig husbandry. One viral strategy to undermine an efficient host cell response is to establish a global shutoff of host protein synthesis. This shutoff has been observed in ASFV-infected cultured cells using two-dimensional electrophoresis combined with metabolic radioactive labeling. However, it remained unclear if this shutoff was selective for certain host proteins. Here, we characterized ASFV-induced shutoff in porcine macrophages by measurement of relative protein synthesis rates using a mass spectrometric approach based on stable isotope labeling with amino acids in cell culture (SILAC). The impact of ASFV infection on the synthesis of >2000 individual host proteins showed a high degree of variability, ranging from complete shutoff to a strong induction of proteins that are absent from naïve cells. GO-term enrichment analysis revealed that the most effective shutoff was observed for proteins related to RNA metabolism, while typical representatives of the innate immune system were strongly induced after infection. This experimental setup is suitable to quantify a virion-induced host shutoff (vhs) after infection with different viruses.

Identification of potential HDAC11 deacylase substrates by affinity pulldown MS.

Lysine fatty acylation is a protein posttranslational modification (PTM) that has been linked to various important biological processes. HDAC11, the sole member of class IV of histone deacetylases (HDACs), has been shown to have high lysine defatty-acylase activity. In order to better understand the functions of lysine fatty acylation and its regulation by HDAC11, it is important to identify the physiological substrates of HDAC11. This can be achieved through profiling the interactome of HDAC11 using a stable isotope labeling with amino acids in cell culture (SILAC) proteomics strategy. Here we describe a detailed method on using SILAC to identify the interactome of HDAC11. This method can be similarly used to identify the interactome, and thus potential substrates, of other PTM enzymes.

Flotillin-2 regulates epidermal growth factor receptor activation, degradation by Cbl-mediated ubiquitination, and cancer growth

Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in various cancers. The ubiquitin ligase Cbl (Casitas B-lineage lymphoma proto-oncogene) regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. Here, we used Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry (MS) to compare Cbl complexes with or without epidermal growth factor (EGF) stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In PC9 and H441 cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by EGFR inhibitor erlotinib. CRISPR knockout (KO) of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and endosomal trafficking. Furthermore, we determined that FLOT2 interacted with both Cbl and EGFR. EGFR downregulation upon FLOT2 loss was Cbl-dependent, as co-knockdown of Cbl and Cbl-b restored EGFR levels. Additionally, FLOT2 overexpression decreased EGFR signaling and growth. Overexpression of wild type (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induced EGFR-dependent proliferation and anchorage-independent growth. Lastly, FLOT2 KO increased tumor formation and tumor volume in nude mice and NSG mice, respectively. Together, these data demonstrated that FLOT2 negatively regulated EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation, leading to reduced proliferation in vitro and in vivo.

Stable Isotope Labeling by Amino Acids and Bioorthogonal Noncanonical Amino Acid Tagging in Cultured Primary Neurons.

Cultured primary neurons are a well-established model for the study of neuronal function. Conventional stable isotope labeling with amino acids in cell culture (SILAC) requires nearly complete metabolic labeling of proteins and therefore is difficult to apply to cultured primary neurons, which do not divide in culture. In a multiplex SILAC strategy, two different sets of heavy amino acids are used for labeling cells for the different experimental conditions. This allows for straightforward SILAC quantitation using partially labeled cells because the two cell populations are always equally labeled. When combined with bioorthogonal noncanonical amino acid tagging (BONCAT), it allows for comparative proteomic analysis of de novo protein synthesis. Here we describe protocols that utilize the multiplex SILAC labeling strategy for primary cultured neurons to study steady-state and nascent proteomes.

Phosphotyrosine Profiling Using SILAC.

Tyrosine phosphorylation on proteins is an important posttranslational modification that regulates various processes in cells. Mass spectrometry-based phosphotyrosine profiling can reveal tyrosine kinase signaling activity in cells. Using quantitative proteomics strategies such as stable isotope labeling with amino acids in cell culture (SILAC) allows comparison of tyrosine kinase signaling activity across two to -three different conditions. In this book chapter, we discuss the reagents required and a step-by-step protocol to carry out phosphotyrosine profiling using SILAC.

SILAC-based quantitative proteomics and microscopy analysis of cancer cells treated with the N-glycolyl GM3-specific anti-tumor antibody 14F7.

Cancer immunotherapy represents a promising approach to specifically target and treat cancer. The most common mechanisms by which monoclonal antibodies kill cells include antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity and apoptosis, but also other mechanisms have been described. 14F7 is an antibody raised against the tumor-associated antigen NeuGc GM3, which was previously reported to kill cancer cells without inducing apoptotic pathways. The antibody was reported to induce giant membrane lesions in tumor cells, with apparent changes in the cytoskeleton. Here, we investigated the effect of humanized 14F7 on HeLa cells using stable isotope labeling with amino acids in cell culture (SILAC) in combination with LC-MS and live cell imaging. 14F7 did not kill the HeLa cells, however, it caused altered protein expression (MS data are available via ProteomeXchange with identifier PXD024320). Several cytoskeletal and nucleic-acid binding proteins were found to be strongly down-regulated in response to antibody treatment, suggesting how 14F7 may induce membrane lesions in cells that contain higher amounts of NeuGc GM3. The altered expression profile identified in this study thus contributes to an improved understanding of the unusual killing mechanism of 14F7.

Coisolation of Peptide Pairs for Peptide Identification and MS/MS-Based Quantification.

Stable-isotope labeling with amino acids in cell culture (SILAC)-based metabolic labeling is a widely adopted proteomics approach that enables quantitative comparisons among a variety of experimental conditions. Despite its quantitative capacity, SILAC experiments analyzed with data-dependent acquisition (DDA) do not fully leverage peptide pair information for identification and suffer from undersampling compared to label-free proteomic experiments. Herein, we developed a DDA strategy that coisolates and fragments SILAC peptide pairs and uses y-ions for their relative quantification. To facilitate the analysis of this type of data, we adapted the Comet sequence database search engine to make use of SILAC peptide paired fragments and developed a tool to annotate and quantify MS/MS spectra of coisolated SILAC pairs. This peptide pair coisolation approach generally improved expectation scores compared to the traditional DDA approach. Fragment ion quantification performed similarly well to precursor quantification in the MS1 and achieved more quantifications. Lastly, our method enables reliable MS/MS quantification of SILAC proteome mixtures with overlapping isotopic distributions. This study shows the feasibility of the coisolation approach. Coupling this approach with intelligent acquisition strategies has the potential to improve SILAC peptide sampling and quantification.

SILAC-based quantitative proteomics to investigate the eicosanoid associated inflammatory response in activated macrophages

BackgroundMacrophages play a central role in inflammation by phagocytosing invading pathogens, apoptotic cells and debris, as well as mediating repair of tissues damaged by trauma. In order to do this, these dynamic cells generate a variety of inflammatory mediators including eicosanoids such as prostaglandins, leukotrienes and hydroxyeicosatraenoic acids (HETEs) that are formed through the cyclooxygenase, lipoxygenase and cytochrome P450 pathways. The ability to examine the effects of eicosanoid production at the protein level is therefore critical to understanding the mechanisms associated with macrophage activation.ResultsThis study presents a stable isotope labelling with amino acids in cell culture (SILAC) -based proteomics strategy to quantify the changes in macrophage protein abundance following inflammatory stimulation with Kdo2-lipid A and ATP, with a focus on eicosanoid metabolism and regulation. Detailed gene ontology analysis, at the protein level, revealed several key pathways with a decrease in expression in response to macrophage activation, which included a promotion of macrophage polarisation and dynamic changes to energy requirements, transcription and translation. These findings suggest that, whilst there is evidence for the induction of a pro-inflammatory response in the form of prostaglandin secretion, there is also metabolic reprogramming along with a change in cell polarisation towards a reduced pro-inflammatory phenotype.ConclusionsAdvanced quantitative proteomics in conjunction with functional pathway network analysis is a useful tool to investigate the molecular pathways involved in inflammation.

Abstract P3097: Post-transcriptional Mechanisms Of FTO-regulated Cardiac Contractility

Background: N6-methyladenosine (m6A), the most abundant and functionally relevant mRNA modification, is reversibly demethylated by FTO, an m6A eraser and a primary regulator of m6A in the heart. In failing hearts, decreased FTO increases m6A, critically reducing cardiomyocyte contraction. FTO overexpression improved cardiac contraction and attenuated remodeling. Here, we investigate post-transcriptional and translational mechanisms of FTO-regulation of cardiomyocyte contraction in vitro and in vivo. We hypothesize that FTO directly binds to selective cardiac contractile mRNAs at their m6A site and affects their nuclear export, polysome binding, cytoplasmic availability, and protein expression. Methods and Results: We performed immunostaining to examine the intracellular location and expression of FTO under different pathological stimuli in human ventricular cardiomyocyte cell line (AC16 cells), rat primary cardiomyocytes and mouse and human hearts. FTO was detected in both cytoplasmic and nuclear compartments, and was significantly reduced in pathological hearts compared to healthy controls. MeRIP (immunoprecipitation of m6A-RNA) sequencing analysis uncovered that FTO targets different sets of cardiac mRNAs in the cytoplasm and nucleus of cardiomyocytes. Knocking down FTO increased nuclear retention of cardiac mRNAs, which was reversed with FTO overexpression. Immunoprecipitation experiments demonstrated that FTO directly binds to cardiac mRNAs. To determine the effect of FTO on translation of select cardiac mRNAs, we isolated the polysome fraction from the cytoplasm of AC16 cells. Interestingly, our stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry analyses suggested increased expression of cardiac contractile proteins with FTO overexpression. Conclusion: We demonstrate that FTO positively regulates post-transcriptional nuclear export, polysome binding of cardiac contractile mRNAs, and regulates their translation under pathological stimuli. By combining experimental and predictive models in human and mice, we have characterized the transcriptome-proteome molecular interplay regulating cardiac function.

Lagovirus Non-structural Protein p23: A Putative Viroporin That Interacts With Heat Shock Proteins and Uses a Disulfide Bond for Dimerization.

The exact function(s) of the lagovirus non-structural protein p23 is unknown as robust cell culture systems for the Rabbit haemorrhagic disease virus (RHDV) and other lagoviruses have not been established. Instead, a range of in vitro and in silico models have been used to study p23, revealing that p23 oligomerizes, accumulates in the cytoplasm, and possesses a conserved C-terminal region with two amphipathic helices. Furthermore, the positional homologs of p23 in other caliciviruses have been shown to possess viroporin activity. Here, we report on the mechanistic details of p23 oligomerization. Site-directed mutagenesis revealed the importance of an N-terminal cysteine for dimerization. Furthermore, we identified cellular interactors of p23 using stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics; heat shock proteins Hsp70 and 110 interact with p23 in transfected cells, suggesting that they ‘chaperone’ p23 proteins before their integration into cellular membranes. We investigated changes to the global transcriptome and proteome that occurred in infected rabbit liver tissue and observed changes to the misfolded protein response, calcium signaling, and the regulation of the endoplasmic reticulum (ER) network. Finally, flow cytometry studies indicate slightly elevated calcium concentrations in the cytoplasm of p23-transfected cells. Taken together, accumulating evidence suggests that p23 is a viroporin that might form calcium-conducting channels in the ER membranes.

Posiphen Reduces the Levels of Huntingtin Protein through Translation Suppression.

Posiphen tartrate (Posiphen) is an orally available small molecule that targets a conserved regulatory element in the mRNAs of amyloid precursor protein (APP) and α-synuclein (αSYN) and inhibits their translation. APP and αSYN can cause neurodegeneration when their aggregates induce neurotoxicity. Therefore, Posiphen is a promising drug candidate for neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. Posiphen’s safety has been demonstrated in three independent phase I clinical trials. Moreover, in a proof of concept study, Posiphen lowered neurotoxic proteins and inflammatory markers in cerebrospinal fluid of mild cognitive impaired patients. Herein we investigated whether Posiphen reduced the expression of other proteins, as assessed by stable isotope labeling with amino acids in cell culture (SILAC) followed by mass spectrometry (MS)-based proteomics. Neuroblastoma SH-SY5Y cells, an in vitro model of neuronal function, were used for the SILAC protein profiling response. Proteins whose expression was altered by Posiphen treatment were characterized for biological functions, pathways and networks analysis. The most significantly affected pathway was the Huntington’s disease signaling pathway, which, along with huntingtin (HTT) protein, was down-regulated by Posiphen in the SH-SY5Y cells. The downregulation of HTT protein by Posiphen was confirmed by quantitative Western blotting and immunofluorescence. Unchanged mRNA levels of HTT and a comparable decay rate of HTT proteins after Posiphen treatment supported the coclusion that Posiphen reduced HTT via downregulation of the translation of HTT mRNA. Meanwhile, the downregulation of APP and αSYN proteins by Posiphen was also confirmed. The mRNAs encoding HTT, APP and αSYN contain an atypical iron response element (IRE) in their 5′-untranslated regions (5′-UTRs) that bind iron regulatory protein 1 (IRP1), and Posiphen specifically bound this complex. Conversely, Posiphen did not bind the IRP1/IRE complex of mRNAs with canonical IREs, and the translation of these mRNAs was not affected by Posiphen. Taken together, Posiphen shows high affinity binding to the IRE/IRP1 complex of mRNAs with an atypical IRE stem loop, inducing their translation suppression, including the mRNAs of neurotoxic proteins APP, αSYN and HTT.

Comprehensive Workflow of Mass Spectrometry-based Shotgun Proteomics of Tissue Samples.

Recent advances in mass spectrometry have resulted in deep proteomicanalysis along with the generation ofrobust andreproducible datasets. However, despite the considerable technical advancements, sample preparation from biospecimens such as patient blood, CSF, and tissue still poses considerable challenges. For identifying biomarkers, tissue proteomics often provides an attractive sample source to translate the research findings from the bench to the clinic. It can reveal potential candidate biomarkers for early diagnosis of cancer and neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, etc. Tissue proteomics also yields a wealth of systemic information based on the abundance of proteins and helps to address interesting biological questions. Quantitative proteomics analysis can be grouped into two broad categories: a label-based and a label-free approach. In the label-based approach, proteins or peptides are labeled using stable isotopes such as SILAC (stable isotope labeling with amino acids in cell culture) or by chemical tags such as ICAT (isotope-coded affinity tags), TMT (tandem mass tag) or iTRAQ (isobaric tag for relative and absolute quantitation). Label-based approaches have the advantage of more accurate quantitation of proteins and using isobaric labels, multiple samples can be analyzed in a single experiment. The label-free approach provides a cost-effective alternative to label-based approaches. Hundreds of patient samples belonging to a particular cohort can be analyzed and compared with other cohorts based on clinical features. Here, we have described an optimized quantitative proteomics workflow for tissue samples using label-free and label-based proteome profiling methods, which is crucial for applications in life sciences, especially biomarker discovery-based projects.

H3K36me3 and PSIP1/LEDGF associate with several DNA repair proteins, suggesting their role in efficient DNA repair at actively transcribing loci.

Background: Trimethylation at histone H3 at lysine 36 (H3K36me3) is associated with expressed gene bodies and recruit proteins implicated in transcription, splicing and DNA repair. PC4 and SF2 interacting protein (PSIP1/LEDGF) is a transcriptional coactivator, possesses an H3K36me3 reader PWWP domain. Alternatively spliced isoforms of PSIP1 binds to H3K36me3 and suggested to function as adaptor proteins to recruit transcriptional modulators, splicing factors and proteins that promote homology-directed repair (HDR), to H3K36me3 chromatin. Methods: We performed chromatin immunoprecipitation of H3K36me3 followed by quantitative mass spectrometry (qMS) to identify proteins associated with H3K36 trimethylated chromatin in mouse embryonic stem cells (mESCs). We also performed stable isotope labelling with amino acids in cell culture (SILAC) followed by qMS for a longer isoform of PSIP1 (PSIP/p75) and MOF/KAT8 in mESCs and mouse embryonic fibroblasts ( MEFs). Furthermore, immunoprecipitation followed by western blotting was performed to validate the qMS data. DNA damage in PSIP1 knockout MEFs was assayed by a comet assay. Results: Proteomic analysis shows the association of proteins involved in transcriptional elongation, RNA processing and DNA repair with H3K36me3 chromatin. Furthermore, we show DNA repair proteins like PARP1, gamma H2A.X, XRCC1, DNA ligase 3, SPT16, Topoisomerases and BAZ1B are predominant interacting partners of PSIP /p75. We further validated the association of PSIP/p75 with PARP1, hnRNPU and gamma H2A.X and also demonstrated accumulation of damaged DNA in PSIP1 knockout MEFs. Conclusions: In contrast to the previously demonstrated role of H3K36me3 and PSIP/p75 in promoting homology-directed repair (HDR), our data support a wider role of H3K36me3 and PSIP1 in maintaining the genome integrity by recruiting proteins involved in DNA damage response pathways to the actively transcribed loci.

Systematic Analysis of the Impact of R-Methylation on RBPs-RNA Interactions: A Proteomic Approach.

RNA binding proteins (RBPs) bind RNAs through specific RNA-binding domains, generating multi-molecular complexes known as ribonucleoproteins (RNPs). Various post-translational modifications (PTMs) have been described to regulate RBP structure, subcellular localization, and interactions with other proteins or RNAs. Recent proteome-wide experiments showed that RBPs are the most representative group within the class of arginine (R)-methylated proteins. Moreover, emerging evidence suggests that this modification plays a role in the regulation of RBP-RNA interactions. Nevertheless, a systematic analysis of how changes in protein-R-methylation can affect globally RBPs-RNA interactions is still missing. We describe here a quantitative proteomics approach to profile global changes of RBP-RNA interactions upon the modulation of type I and II protein arginine methyltransferases (PRMTs). By coupling the recently described Orthogonal Organic Phase Separation (OOPS) strategy with the Stable Isotope Labelling with Amino acids in Cell culture (SILAC) and pharmacological modulation of PRMTs, we profiled RNA-protein interaction dynamics in dependence of protein-R-methylation. Data are available via ProteomeXchange with identifier PXD024601.

H3K36me3 and PSIP1/LEDGF associate with several DNA repair proteins, suggesting their role in efficient DNA repair at actively transcribing loci

Background: Trimethylation at histone H3 at lysine 36 (H3K36me3) is associated with expressed gene bodies and recruit proteins implicated in transcription, splicing and DNA repair. PC4 and SF2 interacting protein (PSIP1/LEDGF) is a transcriptional coactivator, possesses an H3K36me3 reader PWWP domain. Alternatively spliced isoforms of PSIP1 binds to H3K36me3 and suggested to function as adaptor proteins to recruit transcriptional modulators, splicing factors and proteins that promote homology-directed repair (HDR), to H3K36me3 chromatin. Methods: We performed chromatin immunoprecipitation of H3K36me3 followed by quantitative mass spectrometry (qMS) to identify proteins associated with H3K36 trimethylated chromatin in mouse embryonic stem cells (mESCs). We also performed stable isotope labelling with amino acids in cell culture (SILAC) followed by qMS for a longer isoform of PSIP1 (PSIP/p75) and MOF/KAT8 in mESCs and mouse embryonic fibroblasts ( MEFs). Furthermore, immunoprecipitation followed by western blotting was performed to validate the qMS data. DNA damage in PSIP1 knockout MEFs was assayed by a comet assay. Results: Proteomic analysis shows the association of proteins involved in transcriptional elongation, RNA processing and DNA repair with H3K36me3 chromatin. Furthermore, we show DNA repair proteins like PARP1, gamma H2A.X, XRCC1, DNA ligase 3, SPT16, Topoisomerases and BAZ1B are predominant interacting partners of PSIP /p75. We further validated the association of PSIP/p75 with PARP1, hnRNPU and gamma H2A.X and also demonstrated accumulation of damaged DNA in PSIP1 knockout MEFs. Conclusions: In contrast to the previously demonstrated role of H3K36me3 and PSIP/p75 in promoting homology-directed repair (HDR), our data support a wider role of H3K36me3 and PSIP1 in maintaining the genome integrity by recruiting proteins involved in DNA damage response pathways to the actively transcribed loci.

Duck Tembusu Virus Infection Promotes the Expression of Duck Interferon-Induced Protein 35 to Counteract RIG-I Antiviral Signaling in Duck Embryo Fibroblasts.

Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that has caused a substantial drop in egg production and severe neurological disorders in domestic waterfowl. Several studies have revealed that viral proteins encoded by DTMUV antagonize host IFN-mediated antiviral responses to facilitate virus replication. However, the role of host gene expression regulated by DTMUV in innate immune evasion remains largely unknown. Here, we utilized a stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics analysis of DTMUV-infected duck embryo fibroblasts (DEFs) to comprehensively investigate host proteins involved in DTMUV replication and innate immune response. A total of 250 differentially expressed proteins were identified from 2697 quantified cellular proteins, among which duck interferon-induced protein 35 (duIFI35) was dramatically up-regulated due to DTMUV infection in DEFs. Next, we demonstrated that duIFI35 expression promoted DTMUV replication and impaired Sendai virus-induced IFN-β production. Moreover, duIFI35 was able to impede duck RIG-I (duRIG-I)-induced IFN-β promoter activity, rather than IFN-β transcription mediated by MDA5, MAVS, TBK1, IKKϵ, and IRF7. Importantly, we found that because of the specific interaction with duIFI35, the capacity of duRIG-I to recognize double-stranded RNA was significantly impaired, resulting in the decline of duRIG-I-induced IFN-β production. Taken together, our data revealed that duIFI35 expression stimulated by DTMUV infection disrupted duRIG-I-mediated host antiviral response, elucidating a distinct function of duIFI35 from human IFI35, by which DTMUV escapes host innate immune response, and providing information for the design of antiviral drug.

A Direct Comparison of the In Vivo Efficacy and in Vitro Signaling of Chimeric Antigen Receptors and Endogenous T Cell Receptors

The adoptive transfer of chimeric antigen receptor (CAR) expressing T cells targeting the CD19 antigen induces remission in 70-90% of pediatric patients with relapsed and/or refractory acute lymphoblastic leukemia (ALL). Despite initial enthusiasm, emerging data indicate the durability of remission is limited in a substantial number of patients, therefore, addressing relapse will be critical for increasing the clinical efficacy of CAR T cell therapy. CARs are synthetic receptors designed to redirect the antigen specificity of T cells while recapitulating the T cell activating properties of the endogenous T cell receptor (TCR). Clinical and pre-clinical observations have demonstrated that CAR T cells have inferior sensitivity to low levels of antigen and have diminished persistence in vivo compared to T cells responding to TCR activation. These observations suggest the CAR does not fully replicate the signaling of a TCR which subsequently leads to suboptimal T cell function which may underlie the inability of CAR T cell therapy to induce durable remissions in a significant number of patients.In these studies, we directly compared the in vivo and in vitro consequences of CAR versus TCR activation, as well as the signal transduction of each of these receptors. To accomplish this, we created a novel, syngeneic model system in which a 2nd generation CAR is introduced into OTI TCR transgenic T cells to generate a population of T cells (CAR-OTI cells) in which each cell recognizes the CD19 antigen via its CAR as well as the ovalbumin (OVA) antigen via its TCR, thus providing a model in which the function and signaling of each receptor could be studied within a uniform population of T cells. To stimulate either the CAR or the TCR, we generated antigen-expressing cell lines from the pre-B ALL cell line, E2aPbx, that expressed either the cognate antigen for the CAR (CD19), the TCR (OVA) or both. These cell lines allowed us to stimulate either receptor in a model which preserved the properties of the cell-to-cell interactions expected in vivo . We validated the activity of each receptor in this model, by demonstrating similar in vitro degranulation, proliferation and target cell lysis in CAR-OTI cells upon co-incubation with CAR-antigen or TCR-antigen positive E2aPbx cells. Despite the similarities seen in vitro, CAR-OTI cells adoptively transferred into mice with established E2aPbx leukemia demonstrated enhanced expansion at 4 (p<0.001) and 6 days (p<0.01) post-infusion in TCR-activated CAR-OTI cells, relative to CAR-activated CAR-OTI cells. Likewise, clearance of leukemia from the bone marrow (p<0.001) and survival (p<0.01) were each superior in mice in which CAR-OTI cells were activated through the TCR, relative to the CAR. These results demonstrate the significant difference in in vivo behavior and efficacy of CAR- versus TCR-mediated T cell activation, and suggest these differences may result from differences in the signals generated by each receptor.Using our model of CAR-OTI cells and E2aPbx cells expressing either the CAR- or TCR-antigen, we interrogated proteins in the canonical TCR signal transduction cascade after CAR or TCR activation. We demonstrated that CAR activation resulted in increased and prolonged phosphorylation of Zap70, a kinase central to canonical TCR signal transduction. Despite the increased activation of Zap70, the CAR signal demonstrated diminished activation of the MAPK family members, Erk (p<0.001) and p38 (p<0.05), which correlated with the diminished in vivo efficacy seen in this model. Furthermore, this model was used to define the global phosphoproteome of CAR- and TCR-activated CAR-OTI cells by a Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) methodology in conjunction with mass spectrometry (MS). Our SILAC-MS analysis demonstrated multiple differences in the signal transduction cascade of CARs relative to TCRs, including proteins of the signalosome as well as the NFAT and NF-kB pathways. These data demonstrate that a CAR generates an “altered” signal, relative to that of a TCR, correlating with diminished in vivo expansion and efficacy in the treatment of established leukemia. These findings underscore the need to better understand the biology of chimeric antigen receptors as a means of rationally altering their design to improve their clinical effectiveness in mediating durable remissions in patients with relapsed/refractory ALL. DisclosuresNo relevant conflicts of interest to declare.