Biology subjects

Kiraly, R.

Publications and source records attributed to Kiraly, R..

3 recordsLinked to original sources

bioRxiv2024

Transglutaminase 2 is an RNA-binding protein: Experimental verification and characterisation of a novel transglutaminase feature

Transglutaminase 2 (TG2) is a uniquely versatile protein with diverse catalytic activities, such as transglutaminase, protein disulfide isomerase, GTPase, protein kinase, and participates in several biological processes. According to information available in the RBP2GO database, TG2 can be an RNA-binding protein (RBP). RBPs participate in posttranscriptional gene expression regulation, influencing RNAs function, while RNA molecules can also modulate RBPs biological activity. Our goal was to confirm this novel character of TG2 in human umbilical cord vein endothelial cells (HUVEC), which physiologically express TG2. First, UV cross-linked RNA-protein complexes were isolated from immortalised HUVEC using orthogonal organic phase separation. Compared with the RBP2GO database, mass spectrometry identified 392 potential RBPs, including TG2 and 20 novel, endothelium-related RBPs. Total RNA from HUVEC pulled down recombinant human TG2. Complex formation between TG2 and a 43-mer RNA molecule with a secondary structure as well as a homo-oligomeric single-stranded poly(dG), but not poly(dA), could be observed in magnetic RNA-protein pull-down experiments. Experiments with TG2 inhibitors NC9 and GTP{gamma}S, which stabilise its open and closed conformation, respectively, revealed that the open conformation of the enzyme favoured RNA-binding. Biolayer interferometry revealed a high binding affinity between TG2 and RNA with a KD value of 88 nM. We propose that superficial residues on the catalytic core and C-terminal {beta}-barrel domains, being in a hidden position in the closed TG2, are involved in RNA binding. Our study demonstrates TG2s previously uncharacterised RNA-binding ability, opening new avenues for understanding its multi-functionality. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/591323v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@3c7acaorg.highwire.dtl.DTLVardef@a165f2org.highwire.dtl.DTLVardef@2c0293org.highwire.dtl.DTLVardef@15d3d81_HPS_FORMAT_FIGEXP M_FIG C_FIG Transglutaminase 2 (TG2) is a unique multifunctional protein demonstrating large conformational changes. It has various transglutaminase and other catalytic and non-catalytic activities which show conformation dependency. Our study has characterised a novel, open conformation-related biological activity of TG2 and its RNA-binding ability.

molecular 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

Irisin stimulates the release of CXCL1 from differentiating human subcutaneous and deep-neck derived adipocytes via upregulation of NFκB pathway

Thermogenic brown and beige adipocytes play an important role in combating obesity. Recent studies in rodents and humans have indicated that these adipocytes release cytokines, termed "batokines". Irisin was discovered as a polypeptide regulator of beige adipocytes released by myocytes, primarily during exercise. We performed global RNA sequencing on adipocytes derived from human subcutaneous and deep-neck precursors, which were differentiated in the presence or absence of irisin. Irisin did not exert an effect on the expression of characteristic thermogenic genes, while upregulated genes belonging to various cytokine signaling pathways. Out of the several upregulated cytokines, CXCL1, the highest upregulated, was released throughout the entire differentiation period, and predominantly by differentiated adipocytes. Deep-neck area tissue biopsies also showed a significant release of CXCL1 during 24 hours irisin treatment. Gene expression data indicated upregulation of the NF[kcy]B pathway upon irisin treatment, which was validated by an increase of p50 and decrease of I[kcy]B protein level, respectively. Continuous blocking of the NF{kappa}B pathway, using a cell permeable inhibitor of NF{kappa}B nuclear translocation, significantly reduced CXCL1 release. The released CXCL1 exerted a positive effect on the adhesion of endothelial cells. Together, our findings demonstrate that irisin stimulates the release of a novel "batokine", CXCL1, via upregulation of NF{kappa}B pathway in neck area derived adipocytes, which might play an important role in improving tissue vascularization.

cell biology↗