Biology subjects

Tsichlis, P. N.

Publications and source records attributed to Tsichlis, P. N..

9 recordsLinked to original sources

bioRxiv2024

Identification of TAP2 protein variants resistant to inhibition by the HSV1 ICP47 protein

Herpes Simplex Virus 1 evades the host immune system by expressing a protein, ICP47, that binds to and inhibits the heterodimeric Transporter Associated with Antigen Processing (TAP). We screened a library of 1786 variants in TAP2, one of the components of the TAP heterodimer, and identified 39 variants that were resistant to inhibition by ICP47. Of these 39 variants, five were individually tested, and three (T257I, S274H, and T244R) were confirmed to be significantly resistant to inhibition by ICP47. This resistance to inhibition did not extend to the Epstein Barr Virus BNLF2a protein, another viral factor known to inhibit antigen presentation by targeting TAP. These three residues localize close to the binding site of ICP47, on the 3D structure of TAP, but only Ser274 is spatially close to the antigenic peptide binding site of TAP. These results functionally resolve the TAP2 residues required for peptide binding from those required for ICP47 binding and identify TAP2 residues whose targeting with small molecule inhibitors could effectively prevent Herpes virus downregulation of antigen processing. AUTHOR SUMMARYT cells detect virus infected cells via viral peptides in complex with the major histocompatibility complex type I (MHC-I) on the cell surface. Herpes Simplex Virus 1 expression of the ICP47 protein allows the virus to evade detection and clearance of infected cells by host T cells. ICP47 binds to the TAP1/TAP2 protein complex and prevents the loading of antigenic peptides onto the MHC-I protein complex and thereby blocks the antigen processing. In this study, we screened for and identified variants of TAP2 with single amino acid substitutions that are no longer inhibited by ICP47. TAP2 proteins with the identified amino acid substitutions functioned normally in antigen processing even in the presence of ICP47. Results from this study create the potential strategy for drugs that target the identified portions of the TAP2 protein and block ICP47 inhibition of antigen processing while retaining normal TAP1/TAP2 function and thus suggest a new antiviral therapeutic approach for herpes virus infections.

immunology↗

bioRxiv2024

Multiplexed functional analysis of TAP2 variants in regulating MHC-I cell surface abundance reveals overexpression of PLK1 downregulates antigen presentation.

The abundance of MHC-I on the cell surface depends on its association with antigenic peptides transported to the endoplasmic reticulum by the Transporter Associated with Antigen Processing (TAP), a peptide channel composed of TAP1 and TAP2. We functionally screened over 1400 TAP2 variants for effects on MHC-I cell surface abundance. Amino-acid substitutions of loss of function (LOF) variants clustered in the NTP-binding domain and along the TAP1-TAP2 binding interface, suggesting that these variants interfered with TAP conformational changes associated with peptide transport. Some LOF variants carried potential phosphomimetic substitutions; one such substitution at Ser251 was embedded in a sequence context consistent with the phosphorylation motif of PLK kinases. Inhibition of PLK kinases increased MHC-I surface expression, while overexpression of PLK1 in cells decreased MHC-I in wild type TAP2, but not in TAP2-S251A-expressing cells. Importantly, site-specific phosphorylation of TAP2 in human tumor samples correlated with alterations of gene expression in the cell cycle and antigen presentation pathways, consistent with the notion that phosphorylation downregulates antigenic peptide transport in human tumors. These data strongly support the hypothesis that in cancer cells, TAP2 phosphorylation by PLK1, and perhaps other kinases, can downregulate the antigen presentation process.

cancer biology↗

bioRxiv2024

KDM2B is required for ribosome biogenesis and its depletion unequally affects mRNA translation

KDM2B is a JmjC domain lysine demethylase, which promotes cell immortalization, stem cell self-renewal and tumorigenesis. Here we employed a multi-omics strategy to address its role in ribosome biogenesis and mRNA translation. These processes are required to sustain cell proliferation, an important cancer hallmark. Contrary to earlier observations, KDM2B promotes ribosome biogenesis by stimulating the transcription of genes encoding ribosome biogenesis factors and ribosomal proteins, particularly those involved in the biogenesis of the 40S ribosomal subunits. Knockdown of KDM2B impaired the assembly of the small and large subunit processomes, as evidenced by specific defects in pre-ribosomal RNA processing. The final outcome was a decrease in the rate of ribosome assembly and in the abundance of ribosomes, and inhibition of mRNA translation. The inhibition of translation was distributed unequally among mRNAs with different features, suggesting that mRNA-embedded properties influence how mRNAs interpret ribosome abundance. This study identified a novel mechanism contributing to the regulation of translation and provided evidence for a rich biology elicited by a pathway that depends on KDM2B, and perhaps other regulators of translation.

cancer biology↗

bioRxiv2023

Transcriptional regulation of amino acid metabolism by KDM2B, in the context of ncPRC1.1 and in concert with MYC and ATF4

IntroductionKDM2B encodes a JmjC domain-containing histone lysine demethylase, which functions as an oncogene in several types of tumors, including TNBC. This study was initiated to address the cancer relevance of the results of our earlier work, which had shown that overexpression of KDM2B renders mouse embryonic fibroblasts (MEFs) resistant to oxidative stress by regulating antioxidant mechanisms. MethodsWe mainly employed a multi-omics strategy consisting of RNA-Seq, quantitative TMT proteomics, Mass-spectrometry-based global metabolomics, ATAC-Seq and ChIP-seq, to explore the role of KDM2B in the resistance to oxidative stress and intermediary metabolism. These data and data from existing patient datasets were analyzed using bioinformatic tools, including exon-intron-split analysis (EISA), FLUFF and clustering analyses. The main genetic strategy we employed was gene silencing with shRNAs. ROS were measured by flow cytometry, following staining with CellROX and various metabolites were measured with biochemical assays, using commercially available kits. Gene expression was monitored with qRT-PCR and immunoblotting, as indicated. ResultsThe knockdown of KDM2B in basal-like breast cancer cell lines lowers the levels of GSH and sensitizes the cells to ROS inducers, GSH targeting molecules, and DUB inhibitors. To address the mechanism of GSH regulation, we knocked down KDM2B in MDA-MB-231 cells and we examined the effects of the knockdown, using a multi-omics strategy. The results showed that KDM2B, functioning in the context of ncPRC1.1, regulates a network of epigenetic and transcription factors, which control a host of metabolic enzymes, including those involved in the SGOC, glutamate, and GSH metabolism. They also showed that KDM2B enhances the chromatin accessibility and expression of MYC and ATF4, and that it binds in concert with MYC and ATF4, the promoters of a large number of transcriptionally active genes, including many, encoding metabolic enzymes. Additionally, MYC and ATF4 binding sites were enriched in genes whose accessibility depends on KDM2B, and analysis of a cohort of TNBCs expressing high or low levels of KDM2B, but similar levels of MYC and ATF4 identified a subset of MYC targets, whose expression correlates with the expression of KDM2B. Further analyses of basal-like TNBCs in the same cohort, revealed that tumors expressing high levels of all three regulators exhibit a distinct metabolic signature that carries a poor prognosis. ConclusionsThe present study links KDM2B, ATF4, and MYC in a transcriptional network that regulates the expression of multiple metabolic enzymes, including those that control the interconnected SGOC, glutamate, and GSH metabolic pathways. The co-occupancy of the promoters of many transcriptionally active genes, by all three factors, the enrichment of MYC binding sites in genes whose chromatin accessibility depends on KDM2B, and the correlation of the levels of KDM2B with the expression of a subset of MYC target genes in tumors that express similar levels of MYC, suggest that KDM2B regulates both the expression and the transcriptional activity of MYC. Importantly, the concerted expression of all three factors also defines a distinct metabolic subset of TNBCs with poor prognosis. Overall, this study identifies novel mechanisms of SGOC regulation, suggests novel KDM2B-dependent metabolic vulnerabilities in TNBC, and provides new insights into the role of KDM2B in the epigenetic regulation of transcription. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=122 HEIGHT=200 SRC="FIGDIR/small/548031v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1bac894org.highwire.dtl.DTLVardef@1c6977eorg.highwire.dtl.DTLVardef@103d73corg.highwire.dtl.DTLVardef@127b1ff_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsThe knockdown of KDM2B in basal-like breast cancer cell lines lowers the levels of GSH and sensitizes the cells to ROS inducers, GSH targeting molecules, and DUB inhibitors. KDM2B regulates intermediary metabolism by targeting the expression of a host of metabolic enzymes, including those in the SGOC, glutamate, and GSH metabolism. KDM2B enhances chromatin accessibility of MYC and ATF4 and promotes their expression. MYC and ATF4 binding sites are enriched in the promoters of genes whose accessibility depends on KDM2B. KDM2B functioning in the context of ncPRC1.1, binds the promoters of transcriptionally active genes, including those encoding KDM2B-regulated metabolic enzymes, in concert with MYC and ATF4. Basal-like TNBCs expressing high levels of all three regulators, exhibit a distinct metabolic signature that is associated with poor prognosis.

cancer biology↗

bioRxiv2021

Overexpression of the SETD2 WW domain inhibits the phosphor-IWS1/SETD2 interaction and the oncogenic AKT/IWS1 RNA splicing program.

The authors have withdrawn their manuscript. While attempting to reproduce the data on the alternative splicing of exon 2 of U2AF2, they observed that the proposed splicing mechanism could not give rise to a functional U2AF2 protein. In addition, they observed evidence of manipulation in the electropherogram of the splicing junction between exons 1 and 3 and in the primary data on which this electropherogram was based, which were deposited in Mendeley by the first author. These observations raise questions on the integrity of the reported results. In light of this information, the authors have no confidence in the key findings of the paper, and therefore, do not wish it to be cited. If you have any questions, please contact the corresponding author.

cancer biology↗

bioRxiv2020

IWS1 phosphorylation by AKT kinase controls the nucleocytoplasmic export of type I IFNs and the sensitivity of lung adenocarcinoma cells to oncolytic viral infection, through U2AF2 RNA splicing.

cancer biology↗

bioRxiv2020

IWS1 phosphorylation promotes cell proliferation and predicts poor prognosis in EGFR mutant lung adenocarcinoma patients, through the cell cycle-regulated U2AF2 RNA splicing.

cancer biology↗

bioRxiv2020

The inhibition of KDM2B promotes the differentiation of basal-like breast cancer cells via the posttranslational destabilization of SLUG.

KDM2B is a JmjC domain H3K36me2/H3K36me1 demethylase, which immortalizes cells in culture and contributes to the biology of both embryonic and adult stem cells, including cancer stem cells. Here we show that the silencing of KDM2B activates a tyrosine kinase receptor-dependent paracrine mechanism, which results in the downregulation of SNAI2 (SLUG), SNAI1 (SNAIL) and SOX9, which also contribute to the biology of stem and progenitor cells. The downregulation of these molecules is posttranscriptional and in the case of SNAI2-encoded SLUG, it is due to calpain-dependent proteolytic degradation. SLUG abundance in normally growing cells is under the homeostatic control of GSK3, which phosphorylates SLUG and tags it for proteasomal degradation. The paracrine mechanism activated by KDM2B depletion, activates FGFR1 and EGFR family members, and blocks the homeostatic SLUG degradation by inactivating GSK3. This, however, sensitizes SLUG to classical calpains, which are also activated in KDM2B-depleted cells via Ca2+ influx and calpastatin downregulation. The switch in SLUG degradation pathways, results in the rapid degradation of SLUG and the differentiation of breast cancer stem cells, revealing an unexpected mechanism of stem cell regulation by a lysine demethylase. HIGHLIGHTSO_LIGSK3 phosphorylates SLUG and promotes its homeostatic proteasomal degradation C_LIO_LIKDM2B depletion results in GSK3 inactivation, Ca2+ upregulation and calpain activation. C_LIO_LIDownregulation of SLUG phosphorylation by GSK3, sensitizes SLUG to calpain activation. C_LIO_LIGSK3 inactivation and Ca2+ upregulation are due to an RTK-dependent paracrine mechanism C_LI O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/109819v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1cee15corg.highwire.dtl.DTLVardef@12d757corg.highwire.dtl.DTLVardef@17c2dd4org.highwire.dtl.DTLVardef@1a4dcef_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2020

PHENSIM: Phenotype Simulator

Despite the unprecedented growth in our understanding of cell biology, it still remains challenging to connect it to experimental data obtained with cells and tissues physiopathological status under precise circumstances. This knowledge gap often results in difficulties in designing validation experiments, which are usually labor-intensive, expensive to perform, and hard to interpret. Here we propose PHENSIM, a computational tool using a systems biology approach in order to simulate how cell phenotypes are affected by the activation/inhibition of one or multiple biomolecules and does so by exploiting signaling pathways. Our tools applications include predicting the outcome of drug administration, knockdown experiments, gene transduction, and exposure to exosomal cargo. Importantly, PHENSIM enables the user to make inferences on well-defined cell lines and includes pathway maps from three different model organisms. To assess our approachs reliability, we built a benchmark from transcriptomics data gathered from NCBI GEO and performed four case studies on known biological experiments. Our results show high prediction accuracy, thus highlighting the capabilities of this methodology. PHENSIM standalone Java application is available at https://github.com/alaimos/phensim, along with all data and source codes for benchmarking. A web-based user interface is accessible at https://phensim.atlas.dmi.unict.it/.

bioinformatics↗