Differential regulation of p53 function by the N-terminal ΔNp53 and Δ113p53 isoforms in zebrafish embryos

Background  

The p53 protein family coordinates stress responses of cells and organisms. Alternative promoter usage and/or splicing of p53 mRNA gives rise to at least nine mammalian p53 proteins with distinct N- and C-termini which are differentially expressed in normal and malignant cells. The human N-terminal p53 variants contain either the full-length (FL), or a truncated (ΔN/Δ40) or no transactivation domain (Δ133) altogether. The functional consequences of coexpression of the different p53 isoforms are poorly defined. Here we investigated functional aspects of the zebrafish ΔNp53 ortholog in the context of FLp53 and the zebrafish Δ133p53 ortholog (Δ113p53) coexpressed in the developing embryo.     

Apoptin induces apoptosis by changing the equilibrium between the stability of TAp73 and ΔNp73 isoforms through ubiquitin ligase PIR2

Apoptin, a protein derived from the chicken anaemia virus, induces cell death in various cancer cells but shows little or no cytotoxicity in normal cells. The mechanism of apoptin-induced cell death is currently unknown but it appears to induce apoptosis independent of p53 status. Here we show that p73, a p53 family member, is important in apoptin-induced apoptosis. In p53 deficient and/or mutated cells, apoptin induced the expression of TAp73 leading to the induction of apoptosis. Knockdown of p73 using siRNA resulted in a significant reduction in apoptin-induced cytotoxicity. The p53 and p73 pro-apoptotic target PUMA plays an important role in apoptin-induced cell death as knockdown of PUMA significantly reduced cell sensitivity to apoptin. Importantly, apoptin expression resulted in a marked increase in TAp73 protein stability. Investigation into the mechanisms of TAp73 stability showed that apoptin induced the expression of the ring finger domain ubiquitin ligase PIR2 which is involved in the degradation of the anti-apoptotic ?Np73 isoform. Collectively, our results suggest a novel mechanism of apoptin-induced apoptosis through increased TAp73 stability and induction of PIR2 resulting in the degradation of ?Np73 and activation of pro-apoptotic targets such as PUMA causing cancer cell death.     

WITHDRAWN: Expression of p53, p63, and p73 isoforms in squamous cell carcinoma and adenocarcinoma of esophagus☆,☆☆

Diabetic microangiopathy is often observed in diabetic patients, but there is little evidence regarding the relationship between post-prandial glycemia or insulinemia and the incidence of diabetic microangiopathy. In this study, to elucidate the relationship between post-prandial glycemia (or insulinemia) and diabetic microangiopathy, we performed a cross-sectional study of 232 subjects with type 2 diabetes mellitus who were not being treated with insulin injections. A multiple regression analysis showed that post-prandial hyperglycemia independently correlated with the incidence of diabetic retinopathy and neuropathy. Post-prandial hyperglycemia also correlated, although not independently, with the incidence of diabetic nephropathy. In addition, interestingly, post-prandial hypoinsulinemia independently correlated with the incidence of diabetic retinopathy, although not correlated with diabetic neuropathy or nephropathy. In conclusion, post-prandial hyperglycemia, rather than fasting glycemia or hemoglobin A1c levels, is an important predictor of the incidence of diabetic microangiopathy in Japanese type 2 diabetic patients.     

Effect of ionophores on the processing of theβ-amyloid precursor protein in different cell lines

Summary 1. Alzheimer's disease is characterized by the deposition in the brain of extracellular amyloid plaques and vascular deposits consisting mostly of amyloid-peptide (A). A, a polypeptide of 39–43 amino acids (M _r, 4 kDa), is derived proteolytically from a family of proteins of 695–770 amino acids (M _r, 110–140 kDa) called-amyloid precursor protein (APP).2.APP, an integral membrane glycoprotein, is extensively posttranslationally modified within the endoplasmic reticulum (ER) and various Golgi compartments.APP is cleaved by proteases in either the trans-Golgi network or the post-Golgi apparatus and then secreted as a truncated soluble form into the conditioned media of cultured cells and cerebrospinal fluid samples from human subjects.APP can be processed either by an antiamyloidogenic secretory pathway or by an endosomal/lysosomal pathway.3. I studied the effect of two ionophores on the processing ofAPP in cultured cells. Monensin and, in some cases, ammonium chloride increase the intracellular accumulation ofAPP in several cell lines and may alter its processing. Monensin, which had the most consistent effects, also inhibited secretion ofAPP in a differentiated (growth factor mediated) cell line. Nigericin, with greater K⁺ selectivity, was less able to alter the accumulation and possible processing of the protein.4. These results suggest that the increase in the accumulation of intracellularAPP observed after treating cells with ionophores has some specificity. The selective effect of these ionophores on the metabolism ofAPP may provide a model system to analyze the pathways for studying maturation, secretion, and degradation ofAPP.     

Distinct subcellular localization of three isoforms of insulinoma-associated protein 2β in neuroendocrine tissues

AimsInsulinoma-associated protein 2β (IA-2β) is considered to play a significant role in regulated secretion. Recent studies have shown that the mouse brain expresses three major isoforms of IA-2β, named IA-2β60, IA-2β64, and IA-2β71. In this study, we analyzed the tissue-, cell- and organelle-specific distributions of IA-2β isoforms in mice.Main methodsTo localize IA-2β expression in mouse tissues and cells, western blot and immunohistochemical analyses were carried out. The subcellular distribution of IA-2β isoforms was assessed by sedimentation of mouse brain homogenates in a discontinuous sucrose density gradient.Key findingsIA-2β60 was abundant in the cerebrum, cerebellum, medulla oblongata, pancreas, adrenal gland, and pituitary, and in the muscular and mucosal layers of the digestive organs. In contrast, the expression of IA-2β64 and IA-2β71 was restricted to the cerebrum, cerebellum, medulla oblongata, and pituitary, and the muscular layers of the digestive organs. Immunohistochemical analysis of mouse pancreatic islets revealed that pancreatic beta cells expressed IA-2β60 exclusively, whereas alpha and delta cells expressed all three isoforms. By the sedimentation of mouse brain homogenates, it was shown that IA-2β64 and IA-2β71 were co-localized with IA-2 on secretory granules, but were absent from synaptic vesicles (SVs). On the other hand, IA-2β60 was co-localized with synaptophisin on SVs, but was absent from secretory granules.SignificanceThe tissue-, cell- and organelle-specific distributions of IA-2β isoforms suggest that IA-2β60 has a role in secretion from SVs, whereas IA-2β64 and IA-2β71 are involved in secretion from secretory granules.     

Regulation of biosynthesis and intracellular localization of rice and tobacco homologues of nucleosome assembly protein 1

The nucleosome assembly protein 1 (NAP1) is considered to be a conserved histone chaperone, facilitating the assembly of nucleosomes in all eukaryotes. However, studies in yeast and animal cells also indicated that NAP1 proteins have diverse functions likely independent of nucleosome-assembly activity. Here, we describe the isolation and characterization of cDNAs encoding NAP1-like proteins from the monocotyledon rice ( Oryza sativa L.) and the dicotyledon tobacco ( Nicotiana tabacum L.). Northern-blot analysis demonstrated that the two rice NAP1-like genes are predominantly expressed in stem tissues such as root and shoot apical meristems as well as in young flowers. During the cell cycle, all four tobacco NAP1-like genes are highly expressed, with one of them showing a slightly increased expression at the G1/S transition. These results are consistent with a role for plant NAP1-like proteins in cell division. In vitro binding assays revealed that different NAP1-like proteins bind, with distinct relative binding strengths, to different classes of histone. Intracellular localization analyses showed that some NAP1-like proteins could be targeted into the nucleus whereas others are exclusively cytoplasm-localized. It is thus likely that different plant NAP1-like proteins have distinct functions in vivo. Plant NAP1-like proteins were observed to concentrate around the metaphase plate and in the phragmoplast, suggesting a role in mitotic events and cytokinesis.     

Differential requirement for the N-terminal catalytic domain of the DNA polymerase ε p255 subunit in the mitotic cell cycle and the endocycle

In Drosophila, the 255kDa catalytic subunit (dpolεp255) and the 58kDa subunit of DNA polymerase ε (dpolεp58) have been identified. The N-terminus of dpolεp255 carries well-conserved six DNA polymerase subdomains and five 3'→5' exonuclease motifs as observed with Polε in other species. We here examined roles of dpolεp255 during Drosophila development using transgenic fly lines expressing double stranded RNA (dsRNA). Expression of dpolεp255 dsRNA in eye discs induced a small eye phenotype and inhibited DNA synthesis, indicating a role in the G1-S transition and/or S-phase progression of the mitotic cycle. Similarly, expression of dpolεp255 dsRNA in the salivary glands resulted in small size and endoreplication defects, demonstrating a critical role in endocycle progression. In the eye disc, defects induced by knockdown of dpolεp255 were rescued by overexpression of the C-terminal region of dpolεp255, indicating that the function of this non-catalytic domain is conserved between yeast and Drosophila. However, this was not the case for the salivary gland, suggesting that the catalytic N-terminal region is crucial for endoreplication and its defect cannot be complemented by other DNA polymerases. In addition, several genetic interactants with dpolεp255 including genes related to DNA replication such as RFC, DNA primase, DNA polη, Mcm10 and Psf2 and chromatin remodeling such as Iswi were also identified.     

Hempseed protein hydrolysates’ effects on the proliferation and induced oxidative stress in normal and cancer cell lines

Food proteins from different sources can provide beneficial effects on human health by releasing the bioactive peptides that are integral part of their native structure. In this study, we tested the biological potential of hempseed protein hydrolysates (HPHs) obtained from hempseed cake protein isolate. The HPHs were prepared by enzyme hydrolysis using three different proteases of microbial origin: Alcalase®, Neutrase® and Protamex®. The antioxidant activity of the obtained hydrolysates was determined by oxygen radical absorbance capacity (ORAC) assay, while the proliferative effects on normal (HaCaT) and cancer (HeLa) cells were determined by the CellTiter 96^® AQ_ueous One Solution Reagent (MTS) assay. HPHs showed dose-dependent antiproliferative effects on HeLa cells and stimulatory effects on the proliferation of HaCaT cells. HPH obtained by Neutrase^® (HPH-N) showed the highest antioxidant activity expressed as an ORAC value. The protective effect of HPH-N on H₂O₂-induced oxidative stress in normal and cancer cells was evaluated and 1 mg/mL of HPH-N significantly reduced the formation of intracellular reactive oxygen species (ROS) in both cell lines. The obtained results indicate the benefits of HPHs as potential natural antioxidants for the food industry and contribute to the growing trend of utilizing hempseed by-products.

     

Interaction of angio-associated migratory cell protein with the TPα and TPβ isoforms of the human thromboxane A₂ receptor

In humans, thromboxane (TX) A₂ signals through the TPα and TPβ isoforms of its G-protein coupled TXA₂ receptor (TP) to mediate a host of (patho)physiologic responses. Herein, angio-associated migratory cell protein (AAMP) was identified as a novel interacting partner of both TPα and TPβ through an interaction dependent on common (residues 312-328) and unique (residues 366-392 of TPβ) sequences within their carboxyl-terminal (C)-tail domains. While the interaction was constitutive in mammalian cells, agonist-stimulation of TPα/TPβ led to a transient dissociation of AAMP from immune complexes which coincided with a transient redistribution of AAMP from its localization in an intracellular fibrous network. Although the GTPase RhoA is a downstream effector of both AAMP and the TPs, AAMP did not influence TP-mediated RhoA or vice versa. Small interfering RNA (siRNA)-mediated disruption of AAMP expression decreased migration of primary human coronary artery smooth muscle cells (1° hCoASMCs). Moreover, siRNA-disruption of AAMP significantly impaired 1° hCoASMC migration in the presence of the TXA₂ mimetic U46619 but did not affect VEGF-mediated cell migration. Given their roles within the vasculature, the identification of a specific interaction between TPα/TPβ and AAMP is likely to have substantial functional implications for vascular pathologies in which they are both implicated.     

Immunohistochemical localization of the α and β subunits of S-100 protein in pleomorphic adenoma of the salivary glands

The immunohistochemical expression of the alpha and beta subunits of S-100 protein in reactive, modified and transformed of myoepithelial cells, salivary pleomorphic was investigated using monoclonal antibodies. With S-100 alpha, normal salivary glands showed strong staining in serous acinar cells and moderate to slight staining in ductal segments, and with S-100 beta staining was slight or negative in acinar cells, but strong in nerve fibres. In pleomorphic salivary adenomas, the immunohistochemical distribution of S-100 alpha and beta proteins indicated great variation in the tumour cells. Some neoplastic cells gave similar staining for both S-100 alpha and beta, others were strongly positive for S-100 alpha and stained only slightly for S-100 beta, or vice versa. Yet other cells were positive for S-100 alpha and negative for S-100 beta, or vice versa. Pleomorphic salivary adenomas were classified both by histopathological criteria and by their staining pattern for S-100 alpha and beta proteins. Great heterogeneity in S-100 alpha and beta protein expression was found in individual tumour cells of both ductal and myoepithelial origin, and no regular pattern was identified. The cellular origin of salivary pleomorphic adenomas is discussed in terms of S-100 alpha and beta protein immunohistochemistry. Pleomorphic adenoma cells may be transformed from reserve cells into tumour cells displaying biologic properties of myoepithelial cells, ductal cells, or a mixture of both.     

The effect of ethylene on the cell-type-specific and intracellular localization of β-1,3-glucanase and chitinase in tobacco leaves

We have studied the effect of ethylene on the localization of the basic isoforms of glucan endo-1,3--glucosidase (-1,3-glucanase, EC 3.2.1.39) and endo-chitinase (chitinase, EC 3.2.1.14) in leaves of Nicotiana tabacum L. cv. Havana 425. Comparisons of the enzyme contents of the lower epidermis of the leaf, leaf expiants with the lower epidermis removed, and intercellular wash fluid indicate that both enzymes are localized inside epidermal cells of untreated leaves. Ethylene treatment (20 l·l^-1, 4d) induced a marked -10- to 30-fold-coordinated accumulation of the enzymes. This was due primarily to induction of the basic isoforms inside chlorenchyma cells of the leaf interior. The localization of basic -1,3-glucanase was confirmed by immunofluorescence histochemistry and immunogold cytochemistry. Immunolabelling was confined to electron-dense bodies of the cell vacuole. No extracellular immunolabelling was detected in control or ethylene-treated leaves. We conclude that ethylene changes the cell-type-specific distribution but not the intracellular compartmentation of the two enzymes. These results support the generalization that basic isoforms of chitinase and -1,3-glucanase are intracellular whereas the acidic isoforms are secreted into the extracellular space.Abbreviations IgG immunoglobulin G - IWF intercellular wash fluid - PBS 0.14 M NaCl, 0.1 M K₂HPO₄, pH 7.5 - TMV tobacco mosaic virus We thank Monique Seldran and Alfred Milani for expert technical help, Patricia Ahl-Goy, Ciba-Geigy, AG, Basel for supplying IWF from TMV-infected leaves, and our colleagues Thomas Boller and Lilian Sticher for their comments and criticism.     

Where in the cell is SIRT3?--functional localization of an NAD+-dependent protein deacetylase

The ability to sense and respond to nutritional cues is among the most fundamental processes that support life in living organisms. At the cellular level, a number of biochemical mechanisms have been proposed to mediate cellular glucose sensing. These include ATP-sensitive potassium channels, AMP-activated protein kinase, activation of PKC (protein kinase C), and flux through the hexosamine pathway. Less well known is how cellularly heterogenous organs couple nutrient availability to prioritization of cell autonomous functions and appropriate growth of the entire organ. Yet what is clear is that when such mechanisms fail or become inappropriately active they can lead to dire consequences such as diabetes, metabolic syndromes, cardiovascular diseases and cancer. In this issue of the Biochemical Journal, Anagnostou and Shepherd report the identification of an important link between cellular glucose sensing and the Wnt/beta-catenin signalling pathway in macrophages. Their data strongly indicate that the Wnt/beta-catenin pathway of Wnt signalling is responsive to physiological concentrations of nutrients but also suggests that that this system could be inappropriately activated in the diabetic (hyperglycaemic) or other metabolically compromised pathological states. This opens the exciting possibility that organ-selective modulation of Wnt signalling may become an attractive therapeutic target to treat these diseases.     

Revealing the complementation of ferredoxin by cytochrome b 5 in the Spirulina-∆6-desaturation reaction by N-terminal fusion and co-expression of the fungal-cytochrome b 5 domain and Spirulina-∆6-acyl-lipid desaturase

Spirulina-acyl-lipid desaturases are integral membrane proteins found in thylakoid and plasma membranes. These enzymes catalyze the fatty acid desaturation process of Spirulina to yield γ-linolenic acid (GLA) as the final desaturation product. It has been reported that the cyanobacterial desaturases use ferredoxin as an electron donor, whereas the acyl-lipid desaturase in plant cytoplasm and the acyl-CoA desaturase of animals and fungi use cytochrome b ₅. The low level of ferredoxin present in Escherichia coli cells leads to an inability to synthesize GLA when the cells are transformed with the Spirulina-∆⁶ desaturase, desD, and grown in the presence of the reaction substrate, linoleic acid. In this study, Spirulina-∆⁶ desaturase, encoded by the desD gene, was N-terminally fused and co-expressed with the cytochrome b ₅ domain from Mucor rouxii. The product, GLA, made heterologously in E. coli and Saccharomyces cerevisiae, was then detected and analyzed. The results revealed the production of GLA by Spirulina-∆⁶ desaturase fused or co-expressed with cytochrome b ₅ in E. coli cells, in which GLA production by this gene cannot occur in the absence of cytochrome b ₅. Moreover, the GLA production ability in the E. coli host cells was lost after the single substitution mutation was introduced to H52 in the HPGG motif of the cytochrome b ₅ domain. These results revealed the complementation of the ferredoxin requirement by the fusion or co-expression of the fungal-cytochrome b ₅ domain in the desaturation process of Spirulina-∆⁶ desaturase. Furthermore, the free form of cytochrome b ₅ domain can also enhance GLA production by the Spirulina-desD gene in yeast cells.