Comparison of the baculovirus-insect cell and Pichia pastoris heterologous systems for the expression of the human tumor suppressor protein RNASET2

We report the expression of recombinant RNASET2, the only human member of the Rh/T2/S family of acid ribonucleases, in the yeast Pichia pastoris and the baculovirus-insect cell heterologous systems. In both models, the yield of recombinant protein was comparable and ranged between 5 mg/L (for a catalytically impaired mutant version of RNASET2) and 30 mg/L for the wild-type protein. Thus, the produced protein version rather than the expression system used appears to influence protein yield after optimization of culture conditions. The recombinant protein was found to undergo heterogeneous glycosylation in both systems, particularly in P. pastoris. Most importantly, the wild-type protein purified from both systems was found to be catalytically competent. The expression of recombinant RNASET2 in both systems will allow the implementation of functional assays in vivo and in vitro to better define the antioncogenic properties of this member of the Rh/T2/S ribonuclease family.     

Structure and activity of the only human RNase T2

Mutations in the gene of human RNase T2 are associated with white matter disease of the human brain. Although brain abnormalities (bilateral temporal lobe cysts and multifocal white matter lesions) and clinical symptoms (psychomotor impairments, spasticity and epilepsy) are well characterized, the pathomechanism of RNase T2 deficiency remains unclear. RNase T2 is the only member of the Rh/T2/S family of acidic hydrolases in humans. In recent years, new functions such as tumor suppressing properties of RNase T2 have been reported that are independent of its catalytic activity. We determined the X-ray structure of human RNase T2 at 1.6 Å resolution. The α+β core fold shows high similarity to those of known T2 RNase structures from plants, while, in contrast, the external loop regions show distinct structural differences. The catalytic features of RNase T2 in presence of bivalent cations were analyzed and the structural consequences of known clinical mutations were investigated. Our data provide further insight into the function of human RNase T2 and may prove useful in understanding its mode of action independent of its enzymatic activity.     

RNASET2--an autoantigen in anaplastic large cell lymphoma identified by protein array analysis

Characterising tumour-associated antigens (TAAs) not only represents an important approach to the identification of new diagnostic/prognostic markers, but can also provide information on disease processes and additional potential therapeutic targets. Preliminary screening of a protein macroarray, containing more than 12,000 different proteins, with sera from anaplastic lymphoma kinase (ALK)-negative and ALK-positive anaplastic large cell lymphoma (ALCL) patients identified ribonuclease and tumour suppressor protein Ribonuclease T2 (RNASET2), phosphatase lipid phosphate phosphatase-related protein type 3 (LPPR3) and apoptotic adaptor molecule Fas-associating protein (FADD) as ALK-negative ALCL-associated TAAs. Further validation of these observations was confirmed using the ALCL sera in reverse ELISAs. The circulating anti-RNASET2 autoantibodies present in ALCL patients' sera also recognised eukaryotically expressed RNASET2 protein. RNASET2 expression was then investigated in normal tissues and in lymphomas to explore its clinical potential. RNASET2 protein and mRNA levels showed highest expression in the spleen, leucocytes and pancreas. RNASET2 protein expression was not restricted to ALK-negative ALCL (81%), being expressed in ALK-positive ALCL (65%) as well as in a number of other lymphomas. The immunological recognition of RNASET2, its expression in ALCL and other lymphomas together with its known tumourigenic properties suggest that further studies on this autoantigen are warranted.     

FBXO6-mediated RNASET2 ubiquitination and degradation governs the development of ovarian cancer

RNASET2 (Ribonuclease T2) functions as a tumor suppressor in preventing ovarian tumorigenesis. However, the mechanisms underlying the regulation of RNASET2 protein are completely unknown. Here we identified the F-box protein FBXO6, a substrate recognition subunit of an SCF (Skp1-Cul1-F-box protein) complex, as the ubiquitin E3 ligase for RNASET2. We found that the interaction between FBXO6 and RNASET2 induced RNASET2 instability through the ubiquitin-mediated proteasome degradation pathway. FBXO6 promoted K48-dependent ubiquitination of RNASET2 via its FBA domain. Through analysis of the TCGA dataset, we found that FBXO6 was significantly increased in ovarian cancer tissues and the high expression of FBXO6 was related to the poor overall survival (OS) of ovarian cancer patients at advanced stages. An inverse correlation between the protein levels of FBXO6 and RNASET2 was observed in clinic ovarian cancer samples. Depletion of FBXO6 promoted ovarian cancer cells proliferation, migration, and invasion, which could be partially reversed by RNASET2 silencing. Thus, our data revealed a novel FBXO6-RNASET2 axis, which might contribute to the development of ovarian cancer. We propose that inhibition of FBXO6 might represent an effective therapeutic strategy for ovarian cancer treatment.Subject terms: Oncogenes, Ubiquitin ligases     

Stress-induced RNASET2 overexpression mediates melanocyte apoptosis via the TRAF2 pathway in vitro

The recent genome-wide association study identified a link between vitiligo and genetic variants in the ribonuclease T2 (RNASET2) gene; however, the functional roles of RNASET2 in vitiligo pathogenesis or in melanocyte apoptosis have yet to be determined. The current study was designed to investigate the vitiligo-related expression pattern of RNASET2 and its molecular function involving apoptosis-related signaling proteins and pathways. The results showed overexpression of RNASET2 in epidermis specimens from 40 vitiligo patients compared with that from matched healthy controls. In addition, in vitro analyses indicated that overexpression of RNASET2 was inducible in cultured primary human melanocytes and keratinocytes by stress conditions, that is, exposure to UV irradiation, hydrogen peroxide, and inflammatory factors, respectively, and led to increased cell apoptosis via the tumor necrosis factor receptor-associated factor 2 (TRAF2)–caspases pathway through the physical interaction of RNASET2 with TRAF2. Thus, RNASET2 may contribute to vitiligo pathogenesis by inhibiting TRAF2 expression and, as such, RNASET2 may represent a potential therapeutic target of vitiligo.     

Ginsenoside Rh2(S) induces the differentiation and mineralization of osteoblastic MC3T3-E1 cells through activation of PKD and p38 MAPK pathways

As part of the search for biologically active anti-osteoporotic agents that enhance differentiation and mineralization of osteoblastic MC3T3-E1 cells, we identified the ginsenoside Rh2(S), which is an active component in ginseng. Rh2(S) stimulates osteoblastic differentiation and mineralization, as manifested by the up-regulation of differentiation markers (alkaline phosphatase and osteogenic genes) and Alizarin Red staining, respectively. Rh2(S) activates p38 mitogen-activated protein kinase (MAPK) in time- and concentration-dependent manners, and Rh2(S)-induced differentiation and mineralization of osteoblastic cells were totally inhibited in the presence of the p38 MAPK inhibitor, SB203580. In addition, pretreatment with Go6976, a protein kinase D (PKD) inhibitor, significantly reversed the Rh2(S)-induced p38 MAPK activation, indicating that PKD might be an upstream kinase for p38 MAPK in MC3T3-E1 cells. Taken together, these results suggest that Rh2(S) induces the differentiation and mineralization of MC3T3-E1 cells through activation of PKD/p38 MAPK signaling pathways, and these findings provide a molecular basis for the osteogenic effect of Rh2(S).     

The human polo-like kinase, PLK, regulates cdc2/cyclin B through phosphorylation and activation of the cdc25C phosphatase

Entry into mitosis by mammalian cells is triggered by the activation of the cdc2/cyclin B holoenzyme. This is accomplished by the specific dephosphorylation of key residues by the cdc25C phosphatase. The polo-like kinases are a family of serine/threonine kinases which are also implicated in the control of mitotic events, but their exact regulatory mechanism is not known. Recently, a Xenopus homologue, PLX1, was reported to phosphorylate and activate cdc25, leading to activation of cdc2/cyclin B. Jurkat T leukemia cells were chemically arrested and used to verify that PLK protein expression and its phosphorylation state is regulated with respect to cell cycle phase (i.e., protein is undetectable at G1/S, accumulates at S phase and is modified at G2/M). Herein, we show for the first time that endogenous human PLK protein immunoprecipitated from the G2/M-arrested Jurkat cells directly phosphorylates human cdc25C. In addition, we demonstrate that recombinant human (rh) PLK also phosphorylates rhcdc25C in a time- and concentration-dependent manner. Phosphorylation of endogenous cdc25C and recombinant cdc25C by PLK resulted in the activation of the phosphatase as assessed by dephosphorylation of cdc2/cyclin B. These data are the first to demonstrate that human PLK is capable of phosphorylating and positively regulating human cdc25C activity, allowing cdc25C to dephosphorylate inactive cdc2/cyclin B. As this event is required for cell cycle progression, we define at least one key regulatory mode of action for human PLK in the initiation of mitosis.     

HER2 cytoplasmic domain generates normal mitogenic and transforming signals in a chimeric receptor.

We have investigated the biological function of an unidentified human growth factor, the ligand of the putative HER2 receptor, by characterizing the signalling properties of its receptor. HER2 (or c-erbB-2), the human homolog of the rat neu proto-oncogene, encodes a transmembrane glycoprotein of the tyrosine kinase family that appears to play an important role in human breast carcinoma. Since a potential ligand for HER2 has not yet been identified, it has been difficult to analyze the biochemical properties and biological function of this cell surface protein. For this reason, we replaced the HER2 extracellular domain with the closely related ligand binding domain sequences of the epidermal growth factor (EGF) receptor, and examined the ligand-induced biological signalling potential of this chimeric HER1-2 protein. This HER1-2 receptor is targetted to the cell surface of transfected NIH 3T3 cells, forms high and low affinity binding sites, and generates normal mitogenic and cell transforming signals upon interaction with EGF or TGF alpha. The constitutive activation of wild-type HER2 in transfected NIH 3T3 cells suggests the possibility that these cells synthesize the as yet unidentified HER2 ligand and activate HER2 by an autocrine mechanism.     

Localization of the Rh50-like protein to the contractile vacuole in Dictyostelium

The human Rhesus (Rh) family consists of three polytopic membrane proteins present at the cell surface of red blood cells. Although Rh proteins are essential for the expression of the blood group system their biological function remains unclear. In this study, the gene encoding a protein homologous to Rh50 in Dictyostelium discoideum was sequenced. The Rh50-like protein was localized to the contractile vacuole, the organelle responsible for maintenance of osmotic equilibrium within the cell. However, Rh50-like-deficient mutants in which the Rh50-like gene was disrupted did not appear to exhibit a phenotype related to osmoregulation. Nevertheless, these mutants may provide a valuable tool for studying the function of the rhesus protein.     

人参皂苷单体Rh2对人鼻咽癌CNE-2S细胞增殖及凋亡的影响

目的:探讨人参皂苷单体Rh2对人鼻咽癌CNE-2S细胞增殖及凋亡的影响。方法:将生长在对数期的人鼻咽癌CNE-2S细胞分为空白对照组、阴性对照组和实验组。对照组常规培养,阴性对照组采用含有DMSO的培养液培养,实验组在对照组细胞的基础上加入不同浓度人参皂苷单体Rh2处理。采用MTT法测定细胞增殖,PI单染流式细胞术分析各时期细胞所占百分比,Annexin V-PI双染流式细胞仪检测细胞的凋亡情况。结果:与阴性对照组相比,实验组各浓度下的Rh2对CNE-2S细胞均具有显著的增殖抑制作用(P0.05),且随着Rh2浓度的增加而呈现增强的趋势,其中浓度为12.5 mg·L-1 Rh2增值抑制率最低,浓度为100 mg·L-1Rh2增值抑制率最高。不同浓度人参皂苷单体Rh2 G0/G1期细胞分布显著高于阴性对照组(P0.001),且G2/M、S期细胞比例显著低于阴性对照组(P0.01),且随着人参皂苷单体Rh2浓度的增加作用呈现增强的趋势(P0.05);不同浓度的Rh2单体作用24h,CNE-2S细胞早期、晚期凋亡率及总凋亡率均较阴性对照组明显增高(P0.001),并且在Rh2单体浓度为100 mg·L-1时,凋亡率最高。结论:人参皂苷单体Rh2对人鼻咽癌CNE-2S细胞增殖及凋亡具有显著的影响,并且可能对单体Rh2的浓度存在依懒性。     

Cell cycle-specific UNG2 phosphorylations regulate protein turnover, activity and association with RPA

Human UNG2 is a multifunctional glycosylase that removes uracil near replication forks and in non-replicating DNA, and is important for affinity maturation of antibodies in B cells. How these diverse functions are regulated remains obscure. Here, we report three new phosphoforms of the non-catalytic domain that confer distinct functional properties to UNG2. These are apparently generated by cyclin-dependent kinases through stepwise phosphorylation of S23, T60 and S64 in the cell cycle. Phosphorylation of S23 in late G1/early S confers increased association with replication protein A (RPA) and replicating chromatin and markedly increases the catalytic turnover of UNG2. Conversely, progressive phosphorylation of T60 and S64 throughout S phase mediates reduced binding to RPA and flag UNG2 for breakdown in G2 by forming a cyclin E/c-myc-like phosphodegron. The enhanced catalytic turnover of UNG2 p-S23 likely optimises the protein to excise uracil along with rapidly moving replication forks. Our findings may aid further studies of how UNG2 initiates mutagenic rather than repair processing of activation-induced deaminase-generated uracil at Ig loci in B cells.     

TCR/CD3-Induced activation and binding of Emt/Itk to linker of activated T cell complexes: requirement for the Src homology 2 domain

Expressed in mast and T cells/inducible T cell tyrosine kinase (Emt/Itk), a Tec family protein tyrosine kinase, is critical for the development and activation of T lymphocytes. The mechanism through which Emt/Itk mediates its effector functions is poorly understood. In this study, we show that the Emt/Itk Src homology 2 (SH2) domain is critical for the transphosphorylation and activation of Emt/Itk catalytic activity that is mediated by TCR/CD3 engagement. Furthermore, we find that the Emt/Itk SH2 domain is essential for the formation of TCR/CD3-inducible Emt/Itk-LAT complexes, whereas the SH3 domain and catalytic activity are not required. The Emt/Itk-linker of activated T cells (LAT) complexes are biologically important because Jurkat T cells with deficient LAT expression (JCaM2) fail to increase Emt/Itk tyrosine phosphorylation upon TCR/CD3 stimulation. Confocal microscopy reveals that in activated cells, LAT complexes colocalize with TCR/CD3. The present data suggest that upon TCR/CD3 engagement, the Emt/Itk SH2 domain mediates the formation of a molecular complex containing Emt/Itk, LAT, and TCR/CD3; this complex is essential for Emt/Itk activation and function.     

The chemoattractant receptor-like protein C5L2 binds the C3a des-Arg77/acylation-stimulating protein

The orphan receptor C5L2 has recently been described as a high affinity binding protein for complement fragments C5a and C3a that, unlike the previously described C5a receptor (CD88), couples only weakly to G(i)-like G proteins (Cain, S. A., and Monk, P. N. (2002) J. Biol. Chem. 277, 7165-7169). Here we demonstrate that C5L2 binds the metabolites of C4a and C3a, C4a des-Arg(77), and C3a des-Arg(77) (also known as the acylation-stimulating protein or ASP) at a site distinct from the C5a binding site. The binding of these metabolites to C5L2 does not stimulate the degranulation of transfected rat basophilic leukemia cells either through endogenous rat G proteins or when co-transfected with human G(alpha 16). C3a des-Arg(77)/ASP and C3a can potently stimulate triglyceride synthesis in human skin fibroblasts and 3T3-L1 preadipocytes. Here we show that both cell types and human adipose tissue express C5L2 mRNA and that the human fibroblasts express C5L2 protein at the cell surface. This is the first demonstration of the expression of C5L2 in cells that bind and respond to C3a des-Arg(77)/ASP and C3a. Thus C5L2, a promiscuous complement fragment-binding protein with a high affinity site that binds C3a des-Arg(77)/ASP, may mediate the acylation-stimulating properties of this peptide.     

The squamous cell carcinoma antigen 2 inhibits the cysteine proteinase activity of a major mite allergen, Der p 1

The squamous cell carcinoma antigens 1 (SCCA1) and SCCA2 belong to the ovalbumin-serpin family. Although SCCA1 and SCCA2 are closely homologous, these two molecules have distinct properties; SCCA1 inhibits cysteine proteinases such as cathepsin K, L, and S, whereas SCCA2 inhibits serine proteinases such as cathepsin G and human mast cell chymase. Although several intrinsic target proteinases for SCCA1 and SCCA2 have been found, the biological roles of SCCA1 and SCCA2 remain unknown. A mite allergen, Der p 1, is one of the most immunodominant allergens and also acts as a cysteine proteinase probably involved in the pathogenesis of allergic diseases. We have recently shown that both SCCA1 and SCCA2 are induced by two related Th2-type cytokines, IL-4 and IL-13, in bronchial epithelial cells and that SCCA expression is augmented in bronchial asthma patients. In this study, we explored the possibility that SCCA proteins target Der p 1, and it turned out that SCCA2, but not SCCA1, inhibited the catalytic activities of Der p 1. We furthermore analyzed the inhibitory mechanism of SCCA2 on Der p 1. SCCA2 contributed the suicide substrate-like mechanism without formation of a covalent complex, causing irreversible impairment of the catalytic activity of Der p 1, as SCCA1 does on papain. In addition, resistance to cleavage by Der p 1 also contributed to the inhibitory mechanism of SCCA2. These results suggest that SCCA2 acts as a cross-class serpin targeting an extrinsic cysteine proteinase derived from house dust mites and that it may have a protective role against biological reactions caused by mites.     

Phylogenetic conservation of the regulatory and functional properties of the Vav oncoprotein family

Vav proteins are phosphorylation-dependent GDP/GTP exchange factors for Rho/Rac GTPases. Despite intense characterization of mammalian Vav proteins both biochemically and genetically, there is little information regarding the conservation of their biological properties in lower organisms. To approach this issue, we have performed a characterization of the regulatory, catalytic, and functional properties of the single Vav family member of Drosophila melanogaster. These analyses have shown that the intramolecular mechanisms controlling the enzyme activity of mammalian Vav proteins are already present in Drosophila, suggesting that such properties have been set up before the divergence between protostomes and deuterostomes during evolution. We also show that Drosophila and mammalian Vav proteins have similar catalytic specificities. As a consequence, Drosophila Vav can trigger oncogenic transformation, morphological change, and enhanced cell motility in mammalian cells. Gain-of-function studies using transgenic flies support the implication of this protein in cytoskeletal-dependent processes such as embryonic dorsal closure, myoblast fusion, tracheal development, and the migration/guidance of different cell types. These results highlight the important roles of Vav proteins in the signal transduction pathways regulating cytoskeletal dynamics. Moreover, they indicate that the foundations for the regulatory and enzymatic activities of this protein family have been set up very early during evolution.     

Structural identification of 2'- and 3'-O-acetyl-ADP-ribose as novel metabolites derived from the Sir2 family of beta -NAD+-dependent histone/protein deacetylases

The Sir2 (silent information regulator 2) family of histone/protein deacetylases has been implicated in a wide range of biological activities, including gene silencing, life-span extension, and chromosomal stability. Their dependence on beta-NAD(+) for activity is unique among the known classes of histone/protein deacetylase. Sir2 enzymes have been shown to couple substrate deacetylation and beta-NAD(+) cleavage to the formation of O-acetyl-ADP-ribose, a newly described metabolite. To gain a better understanding of the catalytic mechanism and of the biological implications of producing this molecule, we have performed a detailed enzymatic and structural characterization of O-acetyl-ADP-ribose. Through the use of mass spectrometry, rapid quenching techniques, and NMR structural analyses, 2'-O-acetyl-ADP-ribose and 3'-O-acetyl-ADP-ribose were found to be the solution products produced by the Sir2 family of enzymes. Rapid quenching approaches under single-turnover conditions identified 2'-O-acetyl-ADP-ribose as the enzymatic product, whereas 3'-O-acetyl-ADP-ribose was formed by intramolecular transesterification after enzymatic release into bulk solvent, where 2'- and 3'-O-acetyl-ADP-ribose exist in equilibrium (48:52). In addition to (1)H and (13)C chemical shift assignments for each regioisomer, heteronuclear multiple-bond correlation spectroscopy was used to assign unambiguously the position of the acetyl group. These findings are highly significant, because they differ from the previous conclusion, which suggested that 1'-O-acetyl-ADP-ribose was the solution product of the reaction. Possible mechanisms for the generation of 2'-O-acetyl-ADP-ribose are discussed.     

Distinct Expression Patterns of ICK/MAK/MOK Protein Kinases in the Intestine Implicate Functional Diversity

ICK/MRK (intestinal cell kinase/MAK-related kinase), MAK (male germ cell-associated kinase), and MOK (MAPK/MAK/MRK-overlapping kinase) are closely related serine/threonine protein kinases in the protein kinome. The biological functions and regulatory mechanisms of the ICK/MAK/MOK family are still largely elusive. Despite significant similarities in their catalytic domains, they diverge markedly in the sequence and structural organization of their C-terminal non-catalytic domains, raising the question as to whether they have distinct, overlapping, or redundant biological functions. In order to gain insights into their biological activities and lay a fundamental groundwork for functional studies, we investigated the spatio-temporal distribution patterns and the expression dynamics of ICK/MAK/MOK protein kinases in the intestine. We found that ICK/MAK/MOK proteins display divergent expression patterns along the duodenum-to-colon axis and during postnatal murine development. Furthermore, they are differentially partitioned between intestinal epithelium and mesenchyme. A significant increase in the protein level of ICK, but not MAK, was induced in human primary colon cancer specimens. ICK protein level was up-regulated whereas MOK protein level was down-regulated in mouse intestinal adenomas as compared with their adjacent normal intestinal mucosa. These data suggest distinct roles for ICK/MAK/MOK protein kinases in the regulation of intestinal neoplasia. Taken together, our findings demonstrate that the expressions of ICK/MAK/MOK proteins in the intestinal tract can be differentially and dynamically regulated, implicating a significant functional diversity within this group of protein kinases.