Anti-FLAG tag antibody

Hydrogen sulfide (H2S) is thought to signal through protein S-sulfuration (persulfidation; S-sulfhydration) in both mammalian systems and bacteria. We previously profiled proteome S-sulfuration in Staphylococcus aureus (S. aureus) and identified two thioredoxin-like proteins, designated TrxP and TrxQ, that were capable of reducing protein persulfides as a potential regulatory mechanism. In this study, we further characterize TrxP, TrxQ and the canonical thioredoxin, TrxA, by identifying candidate protein substrates in S. aureus cells using a mechanism-based profiling assay where we trap mixed disulfides that exist between the attacking cysteine of a FLAG-tagged Trx and a persulfidated cysteine on the candidate substrate protein in cells. Largely non-overlapping sets of four, 32 and three candidate cellular substrates were detected for TrxA, TrxP, and TrxQ, respectively, many of which were previously identified as global proteome S-sulfuration targets including for example, pyruvate kinase, PykA. Both TrxA (k(cat) = 0.13 s(-1)) and TrxP (k(cat) = 0.088 s(-1)) are capable of reducing protein persulfides on PykA, a model substrate detected as a candidate substrate of TrxP; in contrast, TrxQ shows lower activity (k(cat) = 0.015 s(-1)). This work reveals that protein S-sulfuration, central to H2S and reactive sulfur species (RSS) signaling, may impact cellular activities and appears to be regulated in S. aureus largely by TrxP under conditions of sulfide stress.

Epithelial to mesenchymal transition (EMT) is an important prerequisite for metastasis to secondary organs. Interleukin-like EMT inducer (ILEI) protein has been shown to translationally upregulated during EMT and metastatic progression as a consequence of aberrant TGF-beta signaling. Our initial evaluation of FAM3C (encoding ILEI) and ILEI expression in normal prostate (PCS-440-010) and prostate cancer cell lines (DU145, LNCaP, and PC3) revealed detectable protein expression in only LNCaP cell line even though all cell lines tested had comparable FAM3C expression. Given that PC3 and DU145 cell lines did not have detectable ILEI expression hinted at additional level of regulation of ILEI expression. Treatment with MG-132 resulted in robust detection of ILEI in the PCS-440-010, PC3 and DU145 cell lines, suggesting that at least in these cell lines, ILEI is actively degraded by the proteasome. Mass spectrometric analysis of FLAG immunoprecipitates of untreated and MG-132 treated FLAG-ILEI transfected cells indicated that UBE4A and UBE3C ubiquitin ligases were interacting with ILEI. Ectopic overexpression of UBE4A, but not UBE3C, resulted in destabilization of ILEI in LNCaP cells, whereas RNAi-mediated silencing of UBE4A in PCS-440-010, PC3 and DU145 cell lines resulted in robust accumulation of ILEI, indicating UBE4A as the cognate ubiquitin ligase for ILEI. Co-immunoprecipitation experiments established direct interaction of endogenous ILEI and UBE4A. Furthermore, co-immunoprecipitation of FLAG-tagged ILEI in cells co-transfected with either HA-UBE4A or HA-UBE3C revealed robust polyubiquitinated smear of ILEI in cells transfected with UBE4A, but not UBE3C, thus confirming UBE4A as the ubiquitin ligase for ILEI degradation. Ectopic overexpression of UBE4A, but not UBE3C, in cells was downregulated in vitro migration and invasion in these cells. Cumulatively, our data reveals a novel post-translational regulatory mechanism of regulating ILEI1 expression, a protein required for metastatic progression in prostate cancer cells. (C) 2016