Biology subjects

Fesus, L.

Publications and source records attributed to Fesus, L..

6 recordsLinked to original sources

bioRxiv2025

The transglutaminase 2 interactome in HUVECs suggests its participation in an RNA-binding protein network

Human transglutaminase 2 (TG2) is a multifunctional protein that exhibits various protein-modifying catalytic and protein-protein interaction properties. HUVECs express high levels of TG2, and identification of its interacting partners may provide mechanistic insight into its role in endothelial functions such as adhesion, migration, angiogenesis, and NO homeostasis. For this purpose, two approaches were employed. Firstly, bacterially expressed, site-specifically biotinylated recombinant TG2 was mixed with HUVEC extract to isolate interacting partners by affinity chromatography. Secondly, endogenous TG2 was silenced, and to ensure high antibody affinity, a triple Flag-tagged transgenic TG2-expressing HUVEC line was created, allowing stable binding to anti-Flag antibody-coated agarose for isolating TG2-associated protein complexes. Altogether, 170 and 356 TG2-associated proteins were identified, respectively, with 86 proteins overlapping between the two approaches. The most enriched GO Molecular Functions of cellularly assembled TG2-interacting proteins confirm TG2s involvement in cell-cell and cell-extracellular matrix communication, adhesion, cytoskeleton organisation, and exosome secretion. Stabilising the catalytically inactive closed TG2 conformation reduced the number of TG2 interactors, whereas stabilising its open form increased the number of associated membrane transporters. Significant enrichment of RNA-binding proteins associated with TG2 was observed in all experiments, and a 42% overlap between the TG2 interactome and previously identified RNA-binding proteins in HUVEC was noted. Considering the recently recognised RNA-binding ability of TG2, our results suggest that TG2 participates in post-transcriptional regulations as a central hub within the network of RNA-binding proteins. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/672361v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@169329corg.highwire.dtl.DTLVardef@179d5d4org.highwire.dtl.DTLVardef@1c58f9aorg.highwire.dtl.DTLVardef@156def0_HPS_FORMAT_FIGEXP M_FIG Cellularly assembled TG2-associated proteins were isolated and identified from endogenous TG2-silenced (eTG2-KD) and transgenic, high-affinity antibody, triple Flag-tagged TG2 (3F-TG2) expressing HUVECs under untreated conditions and after NC9 or GTP{gamma}S inhibitor treatments, which stabilise its open or closed conformation, respectively. TG2 interactome analysis suggests that it acts as a central hub in an RNA-binding protein (RBP) network. C_FIG

molecular biology↗

bioRxiv2024

Deciphering Species-Specific and Conserved Regulation of Human UCP1 Gene Expression: Insights from Predicted Homeodomain Family Transcription Factor Binding Sites

Adipocyte thermogenesis is a promising target for treating metabolic disorders, but its regulatory mechanisms remain unclear. This study investigates transcription factors (TFs) and regulatory elements that may control the human UCP1 gene, which is essential for thermogenesis and the formation of the adipocyte phenotype. Using the Eukaryota Promoter Database, we performed computational analyses of the UCP1, UCP2 and UCP3 promoter sequences in humans, mice and rats to identify conserved and species-specific elements. We also used transcriptome data from human neck-derived adipocytes and databases (Contra v3, ChiPBase, TFlink, AdipoNet, ISMARA, etc.) to narrow potential regulatory TFs. Our results show that mouse and rat UCP1 enhancers lack large segments, primarily due to the insertion of repetitive elements that are already lost in some clades. We identified key TFs such as PPARA, PPARG, THR, RARE, RXR, JUN, TFAP2 and SREBF1 as general regulators of UCPs. Additionally, human-specific UCP1 regulatory hotspots (e.g. 5-TCTAATTAGA-3) recognised by homeodomain TFs (e.g. EN1, PAX4, HOXA5 and PRRX2) and NFIL3 were detected. Phylogenetically conserved regulatory elements suggest common TFs in human UCP1 paralogues (MAX, MYCN, MNT, HES1) and cross-species (POU6F1). These results improve our understanding of thermogenic adipocyte development and provide new therapeutic targets for metabolic diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/594487v2_ufig1.gif" ALT="Figure 1"> View larger version (82K): org.highwire.dtl.DTLVardef@1fe65a3org.highwire.dtl.DTLVardef@c2c6e5org.highwire.dtl.DTLVardef@1900abborg.highwire.dtl.DTLVardef@1b09883_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2022

Transglutaminase 2 associated with PI3K and PTEN in a putative membrane-bound signalosome platform blunts cell death

Atypically expressed transglutaminase 2 (TG2) has been identified as a poor prognostic factor in a variety of cancers. In this study, we evaluated the contribution of TG2 to the prolonged cell survival of differentiated acute promyelocytic leukaemia (APL) cells in response to the standard treatment with combined retinoic acid (ATRA) and arsenic trioxide (ATO). We report that one advantage of ATRA + ATO treatment compared to ATRA alone diminishes the amount of activated and non-activated CD11b/CD18 and CD11c/CD18 cell surface integrin receptors. These changes suppress ATRA-induced TG2 docking on the cytosolic part of CD18 {beta}2-integrin subunits and reduce cell survival. In addition, TG2 overexpresses and hyperactivates the phosphatidylinositol-3-kinase (PI3K), phospho-AKT S473, and phospho-mTOR S2481 signalling axis. mTORC2 acts as a functional switch between cell survival and death by promoting the full activation of AKT. We show that TG2 presumably triggers the formation of a signalosome platform, hyperactivates downstream mTORC2-AKT signalling, which in turn phosphorylates and inhibits the activity of FOXO3, a key pro-apoptotic transcription factor. In contrast, the absence of TG2 restores basic phospho-mTOR S2481, phospho-AKT S473, PI3K, and PTEN expression and activity, thereby sensitising APL cells to ATO-induced cell death. We conclude, that atypically expressed TG2 may serve as a hub, facilitating signal transduction via signalosome formation by the CD18 subunit with both PI3K hyperactivation and PTEN inactivation through the PI3K PTEN cycle in ATRA-treated APL cells.

cancer biology↗

bioRxiv2021

Biochemical characterisation of human transglutaminase 4

Transglutaminases are protein modifying enzymes involved in physiological and pathological processes with potent therapeutic possibilities. Human TG4, also called prostate transglutaminase, is frequently associated with pathological symptoms and particularly with cancer invasiveness. Although rodent TG4 is well characterised, bio-chemical characteristics of human TG4 that could help the understanding of its way of action are not published. First, we analysed proteomics databases and found that TG4 protein is present in human tissues beyond the prostate. Then, we studied in vitro the transamidase activity of human TG4 and its regulation using the microtiter plate method. Human TG4 has low transamidase activity which prefers slightly acidic pH and a reducing environment. It is enhanced by submicellar concentrations of SDS suggesting that membrane proximity is an important regulatory event. Human TG4 does not bind GTP as tested by GTP-agarose and BODIPY-FL-GTP{gamma}S binding, and its proteolytic activation by dispase or when expressed in AD-293 cells was not observed either. We identified several potential human TG4 glutamine donor substrates in the AD-293 cell extract by biotin-pentylamine incorporation and mass spectrometry. Several of these potential substrates are involved in cell-cell interaction, adhesion and proliferation, suggesting that human TG4 could become an anticancer therapeutic target.

biochemistry↗

bioRxiv2021

Regulatory Modules of Human Thermogenic Adipocytes: Functional Genomics of Meta-Analyses Derived Marker-Genes

Recently, ProFAT and BATLAS scores have been offered to determine thermogenic status of adipocytes using expression pattern of brown and white marker-genes. In this work, we investigated the functional context of these genes. Although the two meta-analyses based marker-gene lists have little overlap, their enriched pathways show strong coincides suggesting they may better characterize adipocytes. We demonstrate that functional genomics of the annotated genes in common pathways enables an extended analysis of thermogenesis regulation, generates testable hypotheses supported by experimental results in human adipocytes with different browning potential and may lead to more global conclusions than single-state studies. Our results imply that different biological processes shape brown and white adipocytes with presumed transitional states. We propose that the thermogenic adipocyte phenotype require both repression of whitening and induction of browning. These simultaneous actions and hitherto unnoticed regulatory modules, such as the exemplified HIF1A that may directly act at UCP1 promoter, can set new direction in obesity research. HighlightsO_LIIntegrated pathways better characterize brown adipocytes than marker-genes C_LIO_LIDifferent processes shape the brown and white adipocyte phenotypes C_LIO_LIThermogenic phenotype may require simultaneous repression of whitening and induction of browning C_LIO_LIProtein network analyses reveals unnoticed regulatory modules of adipocyte phenotype C_LIO_LIHIF1A may regulate thermogenesis by direct control of UCP1 gene-expression C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/428057v2_ufig1.gif" ALT="Figure 1"> View larger version (75K): org.highwire.dtl.DTLVardef@b4c730org.highwire.dtl.DTLVardef@813b58org.highwire.dtl.DTLVardef@5f3398org.highwire.dtl.DTLVardef@344eb5_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

Transcriptome analysis of browning human neck area adipocytes reveals strong influence by the FTO intronic SNP variant in addition to tissue and PPARγ specific regulation

Brown adipocytes, abundant in deep-neck (DN) area in humans, are thermogenic with anti-obesity potential. FTO pro-obesity rs1421085 T-to-C SNP shifts differentiation program towards white adipocytes in subcutaneous fat. Human adipose-derived stromal cells were obtained from subcutaneous neck (SC) and DN fat of 9 donors, of which 3-3 carried risk-free (T/T), heterozygous or obesity-risk (C/C) FTO genotypes. They were differentiated to white and brown (long-term PPAR{gamma} stimulation) adipocytes, then global RNA sequencing was performed and differentially expressed genes (DEGs) were compared. DN and SC progenitors had similar adipocyte differentiation potential but differed in DEGs. DN adipocytes displayed higher browning features according to ProFAT or BATLAS scores and characteristic DEG patterns revealing associated pathways which were highly expressed (thermogenesis, interferon, cytokine, retinoic acid, with UCP1 and BMP4 as prominent network stabilizers) or downregulated (particularly extracellular matrix remodelling) compared to SC ones. Part of DEGs in either DN or SC browning was PPAR{gamma}-dependent. Presence of the FTO obesity-risk allele suppressed the expression of mitochondrial and thermogenesis genes with a striking resemblance between affected pathways and those appearing in ProFAT and BATLAS, underlining the importance of metabolic and mitochondrial pathways in thermogenesis. Among overlapping regulatory influences which determine browning and thermogenic potential of neck adipocytes, FTO genetic background has a so far not recognized prominence.

cell biology↗