Peptide affinity analysis of proteins that bind to an unstructured NH<sub>2</sub>-terminal region of the osmoprotective transcription factor NFAT5

DuMond, Jenna F.; Ramkissoon, Kevin; Zhang, Xue; Yokoyama, Izumi; Wang, Xujing; Eguchi, Koji; Gao, Shaojian; Mukoyama, Masashi; Burg, Maurice B.; Ferraris, Joan D.

doi:10.1152/physiolgenomics.00110.2015

Cited by 8 publications

(23 citation statements)

References 57 publications

(91 reference statements)

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“…All of these findings suggested that HuR may play a crucial role in maintaining ECM homeostasis in NP cells, a function of HuR that has yet to receive much attention and may have important implications for cancer cell metastasis and tissue fibrosis. Recent data suggest that HuR binds to TonEBP, an osmosensitive transcription factor TonEBP (NFAT5) that is one of the important regulators of ECM homeostasis in the NP niche [58][59][60][61][62]. Considering that HuR does not stabilize Hif-1α in NP cells, it is plausible that HuR's actions on ECM could occur through regulation of TonEBP activity or other key transcription factors.…”

Section: Discussionmentioning

confidence: 99%

RNA binding protein HuR regulates extracellular matrix gene expression and pH homeostasis independent of controlling HIF-1α signaling in nucleus pulposus cells

et al. 2019

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Nucleus pulposus (NP) cells reside in the hypoxic niche of the intervertebral disc. Studies have demonstrated that RNA-binding protein HuR modulates hypoxic signaling in several cancers, however, its function in the disc is unknown. HuR did not show cytoplasmic translocation in hypoxia and its silencing did not alter levels of Hif-1α or HIF-targets in NP cells. RNA-Sequencing data revealed that important extracellular matrix-related genes including several collagens, MMPs, aggrecan, Tgf-β3 and Sdc4 were regulated by HuR. Further analysis of HuR-silenced NP cells confirmed that HuR maintained expression of these matrix genes. We confirmed decreased levels of secreted collagen I and Sdc4 and increased pro-MMP13 in HuR-knockdown cells. In addition, messenger ribonucleoprotein immunoprecipitation demonstrated HuR binding to Tgf-β3 and Sdc4 mRNAs. Interestingly, while HuR bound to Hif-1α and Vegf mRNAs, it was clear that compensatory mechanisms sustained their expression when HuR was silenced. Noteworthy, despite the presence of multiple HuR-binding sites and reported interaction in other cell types, HuR showed no binding to Pgk1, Eno1, Pdk1 and Pfkfb3 in NP cells. Metabolic studies showed a significant decrease in the extracellular acidification rate (ECAR) and mitochondrial oxygen consumption rate (OCR) and acidic pH in HuR-silenced NP cells, without appreciable change in total OCR. These changes were likely due to decreased Ca12 expression in HuR silenced cells. Taken together, our study demonstrates for the first time that HuR regulates extracellular matrix (ECM) and pH homeostasis of NP cells and has important implications in the maintenance of intervertebral disc health.

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Section: Discussionmentioning

confidence: 99%

RNA binding protein HuR regulates extracellular matrix gene expression and pH homeostasis independent of controlling HIF-1α signaling in nucleus pulposus cells

et al. 2019

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“…extract, 232 in the nuclear extract, and 41 in both. We previously identified 152 of the 351 proteins in our study (9) of the NH 2-terminal region of NFAT5.…”

Section: Proteins Associated With the Nfat5 Peptide Baitsmentioning

confidence: 87%

“…The present experiments used peptide baits directed at a COOHterminal intrinsically disordered region of NFAT5 to identify proteins that associate with that region. Other than that, the experimental and analytical methods are largely the same as those used in our previous paper directed at the NH 2-terminal intrinsically disordered region of NFAT5 (9) and are briefly summarized as follows:…”

Section: Methodsmentioning

confidence: 99%

“…Previous experiments by coimmunoprecipitation identified many regulatory proteins that associate physically with NFAT5. We previously used coimmunoprecipitation (6) and aptamer-based affinity chromatography coupled to proteomic MS (9) to identify proteins that associate with NFAT5. Using aptamer-based affinity chromatography, we previously identified 467 proteins that associate with an unstructured NH 2 -terminal region of NFAT5 that contains its nuclear localization domain (9).…”

mentioning

confidence: 99%

“…We previously used coimmunoprecipitation (6) and aptamer-based affinity chromatography coupled to proteomic MS (9) to identify proteins that associate with NFAT5. Using aptamer-based affinity chromatography, we previously identified 467 proteins that associate with an unstructured NH 2 -terminal region of NFAT5 that contains its nuclear localization domain (9). The purpose of the present study was to identify protein preys in the unstructured COOH-terminal region of NFAT5 that contains its transactivating domain.…”

mentioning

confidence: 99%

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Peptide affinity analysis of proteins that bind to an unstructured region containing the transactivating domain of the osmoprotective transcription factor NFAT5

DuMond

Zhang

Yokoyama

et al. 2016

Physiological Genomics

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NFAT5 is a transcription factor originally identified because it is activated by hypertonicity and that activation increases expression of genes that protect against the adverse effects of the hypertonicity. However, its targets also include genes not obviously related to tonicity. The transactivating domain of NFAT5 is contained in its COOH-terminal region, which is predicted to be unstructured. Unstructured regions are common in transcription factors particularly in transactivating domains where they can bind co-regulatory proteins essential to their function. To identify potential binding partners of NFAT5 from either cytoplasmic or nuclear HEK293 cell extracts, we used peptide affinity chromatography followed by mass spectrometry. Peptide aptamer-baits consisted of overlapping 20 amino acid peptides within the predicted COOH-terminal unstructured region of NFAT5. We identify a total of 351 unique protein preys that associate with at least one COOH-terminal peptide bait from NFAT5 in either cytoplasmic or nuclear extracts from cells incubated at various tonicities (NaCl varied). In addition to finding many proteins already known to associate with NFAT5, we found many new ones whose function suggest novel aspects of NFAT5 regulation, interaction, and function. Relatively few of the proteins pulled down by peptide baits from NFAT5 are generally involved in transcription, and most, therefore, are likely to be specifically related to the regulation of NFAT5 or its function. The novel associated proteins are involved with cancer, effects of hypertonicity on chromatin, development, splicing of mRNA, transcription, and vesicle trafficking.

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RNY3 modulates cell proliferation and IL13 mRNA levels in a T lymphocyte model: a possible new epigenetic mechanism of IL-13 regulation

et al. 2022

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Allergic asthma is the most common type of asthma. It is characterized by TH2 cell–driven inflammation in which interleukin-13 (IL-13) plays a pivotal role. Cytoplasmic RNAs (Y-RNAs), a variety of non-coding RNAs that are dysregulated in many cancer types, are also differentially expressed in patients with allergic asthma. Their function in the development of the disease is still unknown. We investigated the potential role of RNY3 RNA (hY3) in the TH2 cell inflammatory response using the Jurkat cell line as a model. hY3 expression levels were modulated to mimic the upregulation effect in allergic disease. We evaluated the effect of hY3 over cell stimulation and the expression of the TH2 cytokine IL13. Total RNA was isolated and retrotranscribed, and RNA levels were assessed by qPCR. In Jurkat cells, hY3 levels increased upon stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. When transfecting with high levels of hY3 mimic molecules, cell proliferation rate decreased while IL13 mRNA levels increased upon stimulation compared to stimulated control cells. Our results show the effect of increased hY3 levels on cell proliferation and the levels of IL13 mRNA in Jurkat cells. Also, we showed that hY3 could act over other cells via exosomes. This study opens up new ways to study the potential regulatory function of hY3 over IL-13 production and its implications for asthma development.

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Peptide affinity analysis of proteins that bind to an unstructured NH₂-terminal region of the osmoprotective transcription factor NFAT5

Cited by 8 publications

References 57 publications

RNA binding protein HuR regulates extracellular matrix gene expression and pH homeostasis independent of controlling HIF-1α signaling in nucleus pulposus cells

RNA binding protein HuR regulates extracellular matrix gene expression and pH homeostasis independent of controlling HIF-1α signaling in nucleus pulposus cells

Peptide affinity analysis of proteins that bind to an unstructured region containing the transactivating domain of the osmoprotective transcription factor NFAT5

RNY3 modulates cell proliferation and IL13 mRNA levels in a T lymphocyte model: a possible new epigenetic mechanism of IL-13 regulation

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Peptide affinity analysis of proteins that bind to an unstructured NH2-terminal region of the osmoprotective transcription factor NFAT5

Cited by 8 publications

References 57 publications

RNA binding protein HuR regulates extracellular matrix gene expression and pH homeostasis independent of controlling HIF-1α signaling in nucleus pulposus cells

RNA binding protein HuR regulates extracellular matrix gene expression and pH homeostasis independent of controlling HIF-1α signaling in nucleus pulposus cells

Peptide affinity analysis of proteins that bind to an unstructured region containing the transactivating domain of the osmoprotective transcription factor NFAT5

RNY3 modulates cell proliferation and IL13 mRNA levels in a T lymphocyte model: a possible new epigenetic mechanism of IL-13 regulation

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Peptide affinity analysis of proteins that bind to an unstructured NH₂-terminal region of the osmoprotective transcription factor NFAT5