Unknown Cellular Proteins Research Articles

Abstract 454: Identification and characterization of tumor-associated antigens (TAAs) and anti-TAAs autoantibodies as biomarkers in immunodiagnosis of human osteosarcoma by serological proteome analysis

Abstract Osteosarcoma (OS) is the most common highly malignant primary solid bone-tumor. Despite its relatively low incidence rate among overall cancers, it remains one of the most harmful primary malignant tumors in childhood and adolescence. Although some tumor markers like mutant p53 can be potentially used as biomarker to detect OS, the sensitivity and specificity are not optimal, especially for early diagnosis. The establishment of a methodology to identify patient with early stage of OS remains to be investigated. There has been a growing interest in using serum autoantibodies against tumor-associated antigens (TAAs) as serological cancer biomarkers, which stems from the notion that anti-TAA antibodies are “sensors” or “reporters” of molecular events associated with tumorigenesis. The objective of this study is to identify and characterize the targeted TAAs as biomarkers in OS, and further to analyze the frequency and specificity of autoantibodies in OS patients. In this initial study, we have tested 35 sera from OS patients, 12 sera from Osteochondroma (OC) patients and 32 age-matched normal human sera (NHS), for the presence of autoantibodies to the TAAs from extracted protein antigens from U2-OS culture cells in 1-D Western blot and by indirect immunofluorescence (IIF). Autoantibodies were detected in 34 of 35 (97.1%) sera from patients with OS, which were significantly higher than that in NHS (12.5%, 4/32). In contrast, there is no significate association between OC and NHS group, which implies that the OS sera may encompass more specific autoantibodies than OC sera. Interestingly, 35% (7/20), 25% (5/20), 20% (4/20) and 35% (7/20) OS sera were identified by 1D Western blotting analysis containing antibodies against unknown cellular protein antigens around 35 kD, 45 kD, 55 kD and 60 kD respectively. Additionally, no reactivity with the 45 kD and 55 kD protein was detected in 32 NHS. In the further study, these cellular proteins targeted by serum antibodies in OS will be identified by using serological proteome analysis (SERPA) approach. This preliminary data supports that autoimmune responses to certain cellular proteins maybe a by-product in the transformation to OS, and further studies of novel targeted proteins in OS may provide insights into how these proteins might be involved in malignancy. Key Words: Osteosarcoma (OS), Tumor-associated antigens (TAAs), Serological proteome analysis (SERPA), Cancer early detection, Immunodiagnosis Citation Format: Jitian Li, Manyu Huang, Manli Luo, Pei Li, Wen Xie, Zongchang Han, Wuyin Li, Jianying Zhang. Identification and characterization of tumor-associated antigens (TAAs) and anti-TAAs autoantibodies as biomarkers in immunodiagnosis of human osteosarcoma by serological proteome analysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 454.

Identification of Cellular Proteins Interacting with the Retroviral Restriction Factor SAMHD1

Human and mouse SAMHD1 proteins block human immunodeficiency virus type 1 (HIV-1) infection in noncycling human monocytic cells by reducing the intracellular deoxynucleoside triphosphate (dNTP) concentrations. Phosphorylation of human SAMHD1 at threonine 592 (T592) by cyclin-dependent kinase 1 (CDK1) and cyclin A2 impairs its HIV-1 restriction activity, but not the dNTP hydrolase activity, suggesting that dNTP depletion is not the sole mechanism of SAMHD1-mediated HIV-1 restriction. Using coimmunoprecipitation and mass spectrometry, we identified and validated two additional host proteins interacting with human SAMHD1, namely, cyclin-dependent kinase 2 (CDK2) and S-phase kinase-associated protein 2 (SKP2). We observed that mouse SAMHD1 specifically interacted with cyclin A2, cyclin B1, CDK1, and CDK2. Given the role of these SAMHD1-interacting proteins in cell cycle progression, we investigated the regulation of these host proteins by monocyte differentiation and activation of CD4+ T cells and examined their effect on the phosphorylation of human SAMHD1 at T592. Our results indicate that primary monocyte differentiation and CD4+ T-cell activation regulate the expression of these SAMHD1-interacting proteins. Furthermore, our results suggest that, in addition to CDK1 and cyclin A2, CDK2 phosphorylates T592 of human SAMHD1 and thereby regulates its HIV-1 restriction function. SAMHD1 is the first dNTP triphosphohydrolase found in mammalian cells. Human and mouse SAMHD1 proteins block HIV-1 infection in noncycling cells. Previous studies suggested that phosphorylation of human SAMHD1 at threonine 592 by CDK1 and cyclin A2 negatively regulates its HIV-1 restriction activity. However, it is unclear whether human SAMHD1 interacts with other host proteins in the cyclin A2 and CDK1 complex and whether mouse SAMHD1 shares similar cellular interacting partners. Here, we identify five cell cycle-related host proteins that interact with human and mouse SAMHD1, including three previously unknown cellular proteins (CDK2, cyclin B1, and SKP2). Our results demonstrate that several SAMHD1-interacting cellular proteins regulate phosphorylation of SAMHD1 and play an important role in HIV-1 restriction function. Our findings help define the role of these cellular interacting partners of SAMHD1 that regulate its HIV-1 restriction function.

Rapid induction of the Growth Hormone Gene Transcription by Glucocorticoids In Vitro: Possible Involvement of Membrane Glucocorticoid Receptors and Phosphatidylinositol 3‐Kinase Activation

The regulation of transcription of the growth hormone (GH) gene by glucocorticoids was studied in MtT/S cells, a cell line derived from an oestrogen-induced mammotrophic tumour in the rat, and in the primary culture of the anterior pituitary gland of adult mice. The levels of the GH heteronuclear RNA (GH hnRNA), which are mainly determined by the transcription rate, increased by 25-fold during 24 h in response to dexamethasone (DEX; 1 μM) in MtT/S cells that were cultured in the medium containing charcoal-stripped serum for 7 days. The stimulatory effect of DEX on the GH hnRNA levels was detectable as early as 30 min. This rapid effect of DEX did not require on-going protein synthesis, whereas it was considered that DEX requires the presence of unknown cellular proteins produced independently of DEX stimulation. By contrast, on-going protein synthesis was required for DEX action when incubated for 6 h, as has been observed in the previous studies. The specific inhibitor of glucocorticoid receptor, RU486, inhibited both rapid (30 min) and delayed (6 h) the effects of glucocorticoids on GH hnRNA levels. Membrane impermeable glucocorticoid, corticosterone-bovine serum albumin conjugate (CSBSA), was found to have effects similar to those of DEX and free corticosterone (CS), suggesting that glucocorticoids regulate GH gene transcription at least in part through the membrane bound receptors. From pharmacological studies, it was suggested that phosphatidylinositol 3-kinase (PI3K) activation is involved in the rapid effects but not in the delayed effects of glucocorticoids. This also suggests that the delayed effects of glucocorticoids depend on mechanisms other than the activation of PI3-kinase. Finally, both rapid and delayed effects of CS and CSBSA were observed not only in MtT/S cells, but also in the mouse pituitary cells in primary culture. Therefore, it is possible that the membrane initiated action of glucocorticoids is involved in the regulation of GH transcription in normal pituitary cells, as well as in pituitary tumour cells.

Epithelial Inducible Nitric-oxide Synthase Is an Apical EBP50-binding Protein That Directs Vectorial Nitric Oxide Output

Nitric oxide (NO), produced via inducible NO synthase (iNOS), can modulate polarized epithelial processes such as solute transport. Given the high reactivity of NO, we hypothesized that optimal NO regulation of polarized epithelial functions is achieved through compartmentalization of iNOS, allowing local NO delivery to its molecular targets. Here, we show that iNOS localizes to the apical domain of epithelial cells within a submembranous protein complex tightly bound to cortical actin. We further show that iNOS can bind to the apical PDZ protein, EBP50 (ezrin-radixin-moesin-binding phosphoprotein 50), an interaction that is dependent on the last three COOH-terminal amino acids of iNOS, SAL, but requires the presence of additional unknown cellular proteins. Mutation of these three COOH-terminal residues abolishes the iNOS-EBP50 interaction and disrupts the apical association of iNOS in transfected cells, showing that this COOH-terminal motif is essential for the correct localization of iNOS in epithelial cells. Apically localized iNOS directs vectorial NO production at the apical proximal tubule epithelial cell surface. These studies define human epithelial iNOS as an apical EBP50-binding protein and suggest that the physical association of iNOS with EBP50 might allow precise NO modulation of EBP50-associated protein functions.

Active dissociation of the fluorescent dye Hoechst 33342 from DNA in a living cell: who could do it?

It is assumed that DNA in mammalian cells is a dynamic conformationally unstable system. This instability provides the cell with a mechanism for dissociating a large number of substances that bind tightly but not covalently to DNA. Among these is the fluorescent dye Hoechst 33342, which binds to DNA in the minor groove. We have selected cell lines with a high capability for active dissociation of Hoechst 33342. Comparative protein analysis of these lines by means of two-dimensional (2-D) electrophoresis was performed. Cell and nuclear proteins were analyzed from these and normal strains. A few proteins with significantly changed quantities have been found. The preliminary search of the 2-D database allowed us to identity some known and unknown cellular proteins that could participate in active dissociation of the dye from DNA.

H10 RNA-binding Proteins Specifically Expressed in the Rat Brain

During brain maturation, histone H1(0) accumulates in both nerve and glial cells. The expression of this "linker" histone, the role of which still remains unclear, is a complex process, having both transcriptional and post-transcriptional regulatory components. In particular, the expression of H1(0) in rat cortical neurons is regulated mainly at the post-transcriptional level, and unknown cellular proteins are likely to affect H1(0) mRNA stability and/or translation. In looking for such factors, we tested the ability of rat brain extracts to protect H1(0) RNA probe from degradation by T1 RNase. The results reported here demonstrate that rat brain contains at least one major (p40) and two minor (p110 and p70) binding factors, specific for H1(0) RNA, all of which are much more or exclusively expressed in adult rat brain, when compared with other tissues. The binding of the factors is confined to a portion of the 3'-untranslated region (3'-UTR), which is highly conserved among murine and human H1(0) mRNAs. These findings suggest that the proteins identified play a critical role in regulating the expression of H1(0) histone in the brain of mammals.

The cellular transcription factor SP1 and an unknown cellular protein are required to mediate Rep protein activation of the adeno-associated virus p19 promoter.

Control of adeno-associated virus (AAV) transcription from the three AAV promoters (p5, p19, and p40) requires the adenovirus E1a protein and the AAV nonstructural (Rep) proteins. The Rep proteins have been shown to repress the AAV p5 promoter yet facilitate activation of the p19 and p40 promoters during a productive infection. To elucidate the mechanism of promoter regulation by the AAV Rep proteins, the cellular factors involved in mediating Rep activation of the p19 promoter were characterized. A series of protein-DNA binding experiments using extracts derived from uninfected HeLa cells was performed to identify cellular factors that bind to the p19 promoter. Electrophoretic mobility shift assays, DNase I protection analyses, and UV cross-linking experiments demonstrated specific interactions with the cellular factor SP1 (or an SP1-like protein) at positions -50 and -130 relative to the start of p19 transcription. Additionally, an unknown cellular protein (cellular AAV activating protein [cAAP]) with an approximate molecular mass of 34 kDa was found to interact with a CArG-like element at position -140. Mutational analysis of the p19 promoter suggested that the SP1 site at -50 and the cAAP site at -140 were necessary to mediate Rep activation of p19. Antibody precipitation experiments demonstrated that Rep-SP1 protein complexes can exist in vivo. Although Rep was demonstrated to interact with p19 DNA directly, the affinity of Rep binding was much lower than that seen for the Rep binding elements within the terminal repeat and the p5 promoter. Furthermore, the interaction of purified Rep68 with the p19 promoter in vitro was negligible unless purified SP1 was also added to the reaction. Thus, the ability of Rep to transactivate the p19 promoter is likely to involve SP1-Rep protein contacts that facilitate Rep interaction with p19 DNA.

Enzymatic Properties of Overexpressed HBV-Mevalonate Kinase Fusion Proteins and Mevalonate Kinase Proteins in the Human Hepatoma Cell Line PLC/PRF/5

We have previously reported a case of integration of HBV sequences in the 5′-noncoding region of the gene for mevalonate kinase (Mk) (EC.2.7.1.36) in the human hepatoma cell line PLC/PRF/5, resulting in overexpression of viral-cellular fusion transcripts and enhanced intracellular levels of the enzyme. Here, we present an evaluation of the functionalities of Mk and HBV/Mk fusion proteins derived from viral-cellular fusion- and Mk-transcripts, some of which lack 156 bp in the Mk coding region as a result of a differential splicing process. cDNA clones with a full-length Mk-ORF produce proteins which can metabolize mevalonate to its monophosphorylated form. Our results suggest that the enhanced and inappropriate expression of Mk may lead to increased metabolism of mevalonate and phosphorylation of hitherto unknown cellular proteins. This consequence of HBV-DNA insertion could thus be related to the activation of proteins that may be relevant in oncogenesis.