Characterization of the potential role for RNA-binding protein FUS/TLS in DNA damage response: A quantitative proteomic approach

FUS/TLS (fused in sarcoma/translocated in liposarcoma) is a ubiquitously expressed RNA-binding protein of the hnRNP family, that has been discovered as fused to transcription factors, through chromosomal translocations, in several human sarcomas and found in protein aggregates in neurons of patients with an inherited form of Amyotrophic Lateral Sclerosis (ALS) [1]. To date, FUS/TLS has been implicated in a variety of cellular processes such as gene expression control, transcriptional regulation, pre-mRNA splicing and miRNA processing [2]. In addition, some evidences link FUS/TLS to genome stability control and DNA damage response. In fact, mice lacking FUS/TLS are hypersensitive to ionizing radiation (IR) and show high levels of chromosome instability and in response to double-strand breaks, FUS/TLS gets phosphorylated by the protein kinase ATM [3,4,5]. Furthermore, the inducible depletion of FUS/TLS in a neuroblastoma cell line (SH-SY5Y FUS/TLS TET-off iKD) subjected to genotoxic stress (IR) resulted in an increased phosphorylation of γH2AX respect to control cells, suggesting an higher activation of the DNA damage response. The study aims to investigate the specific role of FUS/TLS in DNA damage response through the characterization of the proteomic profile of SH-SY5Y FUS/TLS iKD cells subjected to DNA damage stress, by mass spectrometry-based quantitative proteomics (e.g. SILAC). Preliminary results of mass spectrometric identification of FUS/TLS interacting proteins in HEK293 cells, expressing a recombinant flag-tagged FUS/TLS protein, highlighted the interactions with several proteins involved in DNA damage response, such as DNA-PK, XRCC-5/-6, and ERCC-6, raising the possibilities that FUS/TLS is involved in this pathway, even thou its exact role still need to be addressed.

Characterization of the potential role for RNA-binding protein FUS in DNA damage response: A quantitative proteomic approach

FUS/TLS (fused in sarcoma/translocated in liposarcoma) is a ubiquitously expressed protein of the hnRNP family, that has been discovered as fused to transcription factors in several human sarcomas and found in protein aggregates in neurons of patients with an inherited form of Amyotrophic Lateral Sclerosis [Vance C. et al., 2009]. FUS is a 53 kDa nuclear protein that contains structural domains, such as a RNA Recognition Motif (RRM) and a zinc finger motif, that give to FUS the ability to bind to both RNA and DNA sequences. It has been implicated in a variety of cellular processes, such as pre-mRNA splicing, miRNA processing, gene expression control and transcriptional regulation [Fiesel FC. and Kahle PJ., 2011]. Moreover, some evidences link FUS to genome stability control and DNA damage response: mice lacking FUS are hypersensitive to ionizing radiation (IR) and show high levels of chromosome instability and, in response to double-strand breaks, FUS is phosphorylated by the protein kinase ATM [Kuroda M. et al., 2000; Hicks GG. et al., 2000; Gardiner M. et al., 2008]. Furthermore, preliminary results of mass spectrometric identification of FUS interacting proteins in HEK293 cells, expressing a recombinant flag-tagged FUS protein, highlighted the interactions with proteins involved in DNA damage response, such as DNA-PK, XRCC-5/-6, and ERCC-6, raising the possibilities that FUS is involved in this pathway, even though its role still needs to be clarified. This study aims to investigate the biological roles of FUS in human cells and in particular the putative role in DNA damage response through the characterization of the proteomic profile of the neuroblastoma cell line SH-SY5Y upon FUS inducible depletion, by a quantitative proteomic approach. The SH-SY5Y cell line that will be used in this study expresses, in presence of tetracycline, a shRNA that targets FUS mRNA, leading to FUS protein depletion (SH-SY5Y FUS iKD cells). To quantify changes in proteins expression levels a SILAC strategy (Stable Isotope Labeling by Amino acids in Cell culture) will be conducted on SH-SY5Y FUS iKD cells and a control SH-SY5Y cell line (that expresses a mock shRNA) and the relative changes in proteins levels will be evaluated after five and seven days upon FUS depletion, by nanoliquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS) and bioinformatics analysis. Preliminary experiments demonstrated that the SH-SY5Y FUS iKD cells, when subjected to genotoxic stress (high dose of IR), upon inducible depletion of FUS, showed a increased phosphorylation of gH2AX with respect to control cells, suggesting an higher activation of the DNA damage response.

Nck-interacting Ste20 kinase couples Eph receptors to c-Jun N-terminal kinase and integrin activation.

The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module. NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck. To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR). EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems. In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2. EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1. Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62(dok), RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein. Tyrosine-phosphorylated p62(dok) most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck. We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1. Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs.

miRNA-26b Overexpression in Ulcerative Colitis-associated Carcinogenesis

BACKGROUND Longstanding ulcerative colitis (UC) bears a high risk for development of UC-associated colorectal carcinoma (UCC). The inflammatory microenvironment influences microRNA expression, which in turn deregulates target gene expression. microRNA-26b (miR-26b) was shown to be instrumental in normal tissue growth and differentiation. Thus, we aimed to investigate the impact of miR-26b in inflammation-associated colorectal carcinogenesis. METHODS Two different cohorts of patients were investigated. In the retrospective group, a tissue microarray with 38 samples from 17 UC/UCC patients was used for miR-26b in situ hybridization and quantitative reverse transcription polymerase chain reaction analyses. In the prospective group, we investigated miR-26b expression in 25 fresh-frozen colon biopsies and corresponding serum samples of 6 UC and 15 non-UC patients, respectively. In silico analysis, Ago2-RNA immunoprecipitation, luciferase reporter assay, quantitative reverse transcription polymerase chain reaction examination, and miR-26b mimic overexpression were employed for target validation. RESULTS miR-26b expression was shown to be upregulated with disease progression in tissues and serum of UC and UCC patients. Using miR-26b and Ki-67 expression levels, an UCC was predicted with high accuracy. We identified 4 novel miR-26b targets (DIP1, MDM2, CREBBP, BRCA1). Among them, the downregulation of the E3 ubiquitin ligase DIP1 was closely related to death-associated protein kinase stabilization along the normal mucosa-UC-UCC sequence. In silico functional pathway analysis revealed that the common cellular pathways affected by miR-26b are highly related to cancerogenesis and the development of gastrointestinal diseases. CONCLUSIONS We suggest that miR-26b could serve as a biomarker for inflammation-associated processes in the gastrointestinal system. Because miR-26b expression is downregulated in sporadic colon cancer, it could discriminate between UCC and the sporadic cancer type.

Sterile-filtered saliva is a strong inducer of IL-6 and IL-8 in oral fibroblasts.

OBJECTIVES Saliva has been implicated to support oral wound healing, a process that requires a transient inflammatory reaction. However, definitive proof that saliva can provoke an inflammatory response remained elusive. MATERIALS AND METHODS We investigated the ability of freshly harvested and sterile-filtered saliva to cause an inflammatory response of oral fibroblasts and epithelial cells. The expression of cytokines and chemokines was assessed by microarray, RT-PCR, immunoassays, and Luminex technology. The involvement of signaling pathways was determined by Western blot analysis and pharmacologic inhibitors. RESULTS We report that sterile-filtered whole saliva was a potent inducer of IL-6 and IL-8 in fibroblasts from the gingiva, the palate, and the periodontal ligament, but not of oral epithelial cells. This strong inflammatory response requires nuclear factor-kappa B and mitogen-activated protein kinase signaling. The pro-inflammatory capacity is heat stable and has a molecular weight of <40 kDa. Genome-wide microarrays and Luminex technology further revealed that saliva substantially increased expression of other inflammatory genes and various chemokines. To preclude that the observed pro-inflammatory activity is the result of oral bacteria, sterile-filtered parotid saliva, collected under almost aseptic conditions, was used and also increased IL-6 and IL-8 expression in gingiva fibroblasts. The inflammatory response was, furthermore, independent of MYD88, an adapter protein of the Toll-like receptor signaling pathway. CONCLUSIONS We conclude that saliva can provoke a robust inflammatory response in oral fibroblasts involving the classical nuclear factor-kappa B and mitogen-activated protein kinase signaling pathway. CLINICAL RELEVANCE Since fibroblasts but not epithelial cells show a strong inflammatory response, saliva may support the innate immunity of defect sites exposing the oral connective tissue.

106: Synthetic preimplantation factor (sPIF*) promotes neuroprotection by modulating PKA/PKC kinases

OBJECTIVE: Survivors of premature birth suffer from long term disabilities. Synthetic PreImplantation Factor (sPIF*) modulates inflammatory responses and reverses neuroinflammation. Proteinkinase A (PKA) and protein kinase C (PKC) are crucial signaling molecules. PKA up-regulates IL-10 and brain-derived neurotrophic factor (BDNF) expression, which exert neuroprotective effects. Anti-apoptotic phosphorylation of Bad is mediated by PKA. PKC phosphorylates GAP-43, a marker for neuronal plasticity and structural recovery. We explored sPIF protective role in neuronal (N2a) cells and in a rat model of encephalopathy of prematurity. *proprietary. STUDY DESIGN: Cells were subjected to LPS and treated with sPIF or scrambled sPIF. Neonatal rats (postnatal day 3: P3) were subjected to LPS, ligation of carotid artery, and hypoxia (8% O2, 65min; n¼ 30). sPIF (0.75mg/kg twice daily) was injected (P6-13) and brains harvested at P13. sPIF’s potential and mechanisms were evaluated using immunohistochemistry, ELISA, Western Blot, and qRT-PCR. Data were analyzed using two-tailed Student’s t-test. P<0.05 wasconsidered statistically significant. RESULTS: In vitro sPIF increased PKA/PKC activity in time dependent manner (Fig. 1A). sPIF induced higher IL-10, BDNF, and GAP-43 and lower CASP3, BAD, and TNF-a mRNA levels (Fig. 1B,C). sPIF increased pGap-43/Gap-43 and decreased pBad/Bad ratio while decreasing Bad (Fig. 1 D,E). In brain tissue sPIF treatment resulted in rescued neuronal number (NeuN positive cells) and reduced apoptosis (Casp-3 positive cells) with decreased glial (Iba-1 positive cells) activation (Fig. 2A,B). The Iba-1 morphology changed from predominantly amoeboid to ramified state. Additionally sPIF increased IL-10 mRNA levels (Fig. 2C) and pGap-43/Gap-43 ratio (Fig. 2D). CONCLUSION: sPIF modulates PKA/PKC pathways reducing apoptosis and inflammatory responses while increasing neuronal plasticity and survival. The identified PKA/PKC regulatory axis strengthens the potential of sPIF in reducing the burden of prematurity.

Post-translational modifications of voltage-gated sodium channels in chronic pain syndromes.

In the peripheral sensory nervous system the neuronal expression of voltage-gated sodium channels (Navs) is very important for the transmission of nociceptive information since they give rise to the upstroke of the action potential (AP). Navs are composed of nine different isoforms with distinct biophysical properties. Studying the mutations associated with the increase or absence of pain sensitivity in humans, as well as other expression studies, have highlighted Nav1.7, Nav1.8, and Nav1.9 as being the most important contributors to the control of nociceptive neuronal electrogenesis. Modulating their expression and/or function can impact the shape of the AP and consequently modify nociceptive transmission, a process that is observed in persistent pain conditions. Post-translational modification (PTM) of Navs is a well-known process that modifies their expression and function. In chronic pain syndromes, the release of inflammatory molecules into the direct environment of dorsal root ganglia (DRG) sensory neurons leads to an abnormal activation of enzymes that induce Navs PTM. The addition of small molecules, i.e., peptides, phosphoryl groups, ubiquitin moieties and/or carbohydrates, can modify the function of Navs in two different ways: via direct physical interference with Nav gating, or via the control of Nav trafficking. Both mechanisms have a profound impact on neuronal excitability. In this review we will discuss the role of Protein Kinase A, B, and C, Mitogen Activated Protein Kinases and Ca++/Calmodulin-dependent Kinase II in peripheral chronic pain syndromes. We will also discuss more recent findings that the ubiquitination of Nav1.7 by Nedd4-2 and the effect of methylglyoxal on Nav1.8 are also implicated in the development of experimental neuropathic pain. We will address the potential roles of other PTMs in chronic pain and highlight the need for further investigation of PTMs of Navs in order to develop new pharmacological tools to alleviate pain.

Effect of mannitol and cold treatments on phosphate uptake and protein phosphorylation in Lemna minor (L.) plants

This report is aimed at elucidating the effect of mannitol and cold treatments on P uptake and protein phosphorylation in Lemna minor plants. Duckweed p lants were incu bated in the presence of [32P]or [33P]Pi in half-strength phosphate deprived E-medium under constant light regime for 1.5 h. Total plant protein extracts (pellet and supernatant) were then prepared and subjected to IEF x SDS-PAGE. To analyse the effect of the stresses on P uptake and protein labelling, Lemna minor plants were preincubated with 0.1, 0.5 mol · L-1 mannitol and at 4°C respectively, for 4 hours, before adding labelled orthophosphate. The results show that the general protein phosphorylation (including LHCII) is related to the level of P uptake. Radioactive phosphate incorporation is stimulated by a low concentration of mannitol (0.1 mol · L-1) but reduced by 0.5 mol · L-1 mannitol and cold stress in planta. The labelling into proteins is affected neither when stresses were applied to the plants after incubation with labelled orthophosphate, nor after in vitro protein phosphorylation. This indicates that general protein kinase activities in vivo are strictly limited by P uptake. A marked accumulation of soluble hexoses (mainly sucrose, glucose, and fructose) is observed under imposed stress, suggesting that the inhibition of P uptake in response to hyperosmotic and cold stresses is mediated by sugar accumulation in situ. However, metabolisable sugars like glucose did not alter the entry of phosphate at concentrations of 0.5 mol · L-1, showing that the chemical nature of the osmoticum influences P uptake.

Antibodies against major histocompatibility complex class II antigens directly inhibit the growth of T cells infected with Theileria parva without affecting their state of activation

We have analyzed the effect of antibodies (Abs) directed against major histocompatibility complex (MHC) class II Abs on the proliferation of Theileria parva-infected (Tpi) T cells. Anti-MHC class II Abs exert a direct effect on Tpi T cells causing an acute block in their proliferation. The inhibition does not involve apoptosis and is also entirely reversible. The rapid arrest of DNA synthesis caused by anti-MHC class II Abs is not due to interference with the state of activation of the T cells since the transcriptional activator NF-kappa B remains activated in arrested cells. In addition, interleukin 2 (IL-2), IL-2R, and c-myc gene expression are also unaffected. By analyzing the cell-cycle phase distribution of inhibited cells, it could be shown that cells in all phases of the cell cycle are inhibited. The signal transduction pathway that results in inhibition was shown to be independent of protein kinase C and extracellular Ca2+. Tyrosine kinase inhibitors, however, partly reduced the level of inhibition and, conversely, phosphatase inhibitors enhanced it. The possible relevance of this phenomenon in other systems is discussed.