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Identification of Transglutaminase 2 associated proteins in HUVEC

ABSTRACT: TG2 is physiologically expressed in HUVEC cells, contributing to the maintenance of cardiovascular homeostasis, but the mechanistic details remain unclear. Identification of TG2-associated proteins can provide a deeper view of TG2's role in endothelial cells. We have applied two approaches: First, a site-specifically N-terminally biotinylated recombinant TG2 was produced in the Rosetta 2 strain, and the Neutravidin-agarose resin was used to isolate TG2-associated proteins from the immortalised HUVEC cell extract. Second, we have developed an endogenous TG2-silenced, N-terminally triple-Flag-tagged transgenic TG2-expressing HUVEC cell line, and intracellularly assembled TG2-associated protein complexes were isolated using anti-Flag agarose resin. To reveal the conformation-dependent interactions of TG2, NC9 (24 hours treatment before lysis) and GTPgammaS (during CoIP) were applied to stabilise open or closed TG2, respectively. (One acidic and a denaturing elution were applied in the case of each sample from the appropriate resin. The eluted sample was run into an SDS-polyacrylamide gel till the top of the separation gel, and the protein-containing part of the gel was cut based on Coomassie (Page-Blue) staining. The proteins in the submitted gel pieces were digested with trypsin overnight (37 C degree). After digestion, the samples were dried to a volume of 0.01 ml. The protein samples were prepared for MS analysis using the Easy nLC1200 (Thermo Scientific) nanouplc-Orbitrap Fusion (Thermo Scientific) MS/MS system. The results were imported into the Scaffold 5.0.1 software. In the Scaffold software, the Exclusive Unique Peptide Count display option and a protein threshold of 1.0% false discovery rate (FDR), along with a peptide threshold of 0.1% FDR, were applied, requiring a minimum of one identified peptide for each protein. The fragmentation table of each detected unique peptide was meticulously checked to verify that the detected peptide is a genuine hit. For this verification, the fragmentation table must include at least four B or Y ion series. The experiments were repeated at least 3 times, and the hits obtained from each repetition were aggregated, after which the control non-specifically bound protein hits were deducted.)

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Robert Kiraly

PROVIDER: MSV000099919 | MassIVE | Sat Nov 15 13:29:00 GMT 2025

SECONDARY ACCESSION(S): PXD070779

REPOSITORIES: MassIVE

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Project description:Rhodococcus jostii RHA1 is a catabolically versatile soil actinomycete that can utilize a wide range of organic compounds as growth substrates including steroids. To globally assess the adaptation of the protein composition in the membrane fraction to steroids, the membrane proteomes of RHA1 grown on each of cholesterol and cholate were compared to pyruvate-grown cells using gel-free SIMPLE-MudPIT technology. Label-free quantification by spectral counting revealed 59 significantly regulated proteins, many of them present only during growth on steroids. Cholesterol and cholate induced distinct sets of steroid-degrading enzymes encoded by paralogous gene clusters, consistent with transcriptomic studies. CamM and CamABCD, two systems that take up cholate metabolites, were found exclusively in cholate-grown cells. Similarly, 9 of the 10 Mce4 proteins of the cholesterol uptake system were found uniquely in cholesterol-grown cells. Bioinformatic tools were used to construct a model of Mce4 transporter within the RHA1 cell envelope. Finally, comparison of the membrane and cytoplasm proteomes indicated that several steroid-degrading enzymes are membrane-associated. The implications for the degradation of steroids by actinomycetes, including cholesterol by the pathogen Mycobacterium tuberculosis , are discussed. Bioinformatics and data processing: All database searches were performed using SEQUEST algorithm, embedded in BioworksTM (Rev. 3.3.1, Thermo Fisher Scientific Inc., Waltham, MA), with a RHA1 database containing 9145 sequences. Only tryptic peptides with up to two missed cleavages were accepted. No fixed modifications were considered. Oxidation of methionine was permitted as variable modification. The mass tolerance for precursor ions was set to 10 ppm; the mass tolerance for fragment ions was set to 1 amu. For protein identification a threshold for deltaCn (0.08) and for XCorr values was defined, depending on the peptide charge (>2.5 (+2); > 3.5 (+3)). A protein was considered identified if at least two different peptides met these criteria. To assess the false discovery rate (FDR) of protein identification, a database with reversed protein sequences was searched retaining the search parameters and filter criteria. The FDR was calculated by dividing the absolute number of hits from the reversed database through the sum of hits from both database searches (reversed database and original database). Using the stringent criteria described above, no reversed database hit was found; therefore, the FDR was 0% in all measurements. This low error rate can be attributed to the additional demand for two different peptide matches per protein, which eliminated all otherwise observed protein hits against the decoy database.

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Identification of Transglutaminase 2 associated proteins in HUVEC

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