The Fe65 Adaptor Protein Interacts through Its PID1 Domain with the Transcription Factor CP2/LSF/LBP1

The neural protein Fe65 possesses three putative protein-protein interaction domains: one WW domain and two phosphotyrosine interaction/phosphotyrosine binding domains (PID1 and PID2); the most C-terminal of these domains (PID2) interacts in vivo with the Alzheimer's beta-amyloid precursor protein, whereas the WW domain binds to Mena, the mammalian homolog of Drosophila-enabled protein. By the interaction trap procedure, we isolated a cDNA clone encoding a possible ligand of the N-terminal PID/PTB domain of Fe65 (PID1). Sequence analysis of this clone revealed that this ligand corresponded to the previously identified transcription factor CP2/LSF/LBP1. Co-immunoprecipitation experiments demonstrated that the interaction between Fe65 and CP2/LSF/LBP1 also takes place in vivo between the native molecules. The localization of both proteins was studied using fractionated cellular extracts. These experiments demonstrated that the various isoforms of CP2/LSF/LBP1 are differently distributed among subcellular fractions. At least one isoform, derived from alternative splicing (LSF-ID), is present outside the nucleus; Fe65 was found in both fractions. Furthermore, transfection experiments with an HA-tagged CP2/LSF/LBP1 cDNA demonstrated that Fe65 interacts also with the nuclear form of CP2/LSF/LBP1. Considering that the analysis of Fe65 distribution in fractionated cell extracts demonstrated that this protein is present both in nuclear and non-nuclear fractions, we examined the expression of Fe65 deletion mutants in the two fractions. This analysis allowed us to observe that a small region N-terminal to the WW domain is phosphorylated and is necessary for the presence of Fe65 in the nuclear fraction

Fe65 Is Not Involved in the Platelet-derived Growth Factor-induced Processing of Alzheimer's Amyloid Precursor Protein, Which Activates Its Caspase-directed Cleavage

Abstract The proteolytic processing of the precursor of the β-amyloid peptides (APP) is believed to be a key event in the pathogenesis of Alzheimer's disease. This processing is activated through a pathway involving the PDGF receptor, Src, and Rac1. In this paper, we demonstrate that this pathway specifically acts on APP and requires the YENPTY motif present in the APP cytosolic domain. Considering that several results indicate that the adaptor proteins interacting with this domain affect the processing of APP, we examined their possible involvement in the PDGF-induced pathway. By using an APP-Gal4 reporter system, we observed that the overexpression of Fe65 activates APP-Gal4 cleavage, whereas X11 stabilizes APP. Although mDab1 and Jip1 have no effect, Shc induces a strong activation of APP cleavage, and the contemporary exposure of cells to PDGF causes a dramatic cooperative effect. The analysis of point mutations of the APP YENPTY motif indicates that Fe65 and PDGF function through different mechanisms. In fact, Fe65 requires the integrity of APP695 Tyr682 residue, whereas PDGF effect is dependent upon the integrity of Asn684. Furthermore, the mutation of Asp664 of APP, which is the target site for the caspase-directed APP cleavage, strongly decreases the effect of Fe65. This suggests that Fe65 activates the cleavage of APP by caspases, and in fact, caspase inhibitor Z-VEVD decreases the APP cleavage induced by Fe65. On the contrary, the effects of Shc overexpression, like those of PDGF, are completely absent in the presence of compound X and require the integrity of the Asn684 residue of APP695. The involvement of Shc in the pathway regulating APP processing is confirmed by the effects of constitutively active and dominant negative mutants of Src and Rac1

Comparative proteomic expression profile in all-trans retinoic acid differentiated neuroblastoma cell line

Neuroblastoma (NB) is an infant tumor which frequently differentiates into neurons. We used twodimensional differential in-gel electrophoresis (2D-DIGE) to analyze the cytosolic and nuclear protein expression patterns of LAN-5 cells following neuronal differentiating agent all-trans-retinoic acid treatment. We identified several candidate proteins, from which Gβ2 and Prefoldin 3 may have a role on NB development. These results strength the use of proteomics to discover new putative protein targets in cancer. Keywords

Signal transduction through tyrosine-phosphorylated C-terminal fragments of amyloid precursor protein via an enhanced interaction with Shc/Grb2 adaptor proteins in reactive astrocytes of Alzheimer's disease brain.

The proteolytic processing of amyloid precursor protein (APP) through the formation of membrane-bound C-terminal fragments (CTFs) and of soluble beta-amyloid peptides likely influences the development of Alzheimer's disease (AD). We show that in human brain a subset of CTFs are tyrosine-phosphorylated and form stable complexes with the adaptor protein ShcA. Grb2 is also part of these complexes, which are present in higher amounts in AD than in control brains. ShcA immunoreactivity is also greatly enhanced in patients with AD and occurs at reactive astrocytes surrounding cerebral vessels and amyloid plaques. A higher amount of phospho-ERK1,2, likely as result of the ShcA activation, is present in AD brains. In vitro experiments show that the ShcA-CTFs interaction is strictly confined to glial cells when treated with thrombin, which is a well known ShcA and ERK1,2 activator and a regulator of APP cleavage. In untreated cells ShcA does not interact with either APP or CTFs, although they are normally generated. Altogether these data suggest that CTFs are implicated in cell signaling via Shc transduction machinery, likely influencing MAPK activity and glial reaction in AD patients

The WW Domain of Neural Protein FE65 Interacts with Proline-rich Motifs in Mena, the Mammalian Homolog of Drosophila Enabled*

The neural protein FE65 contains two types of protein-protein interaction modules: one WW binding domain and two phosphotyrosine binding domains. The carboxyl-terminal phosphotyrosine binding domain of FE65 interacts in vivo with the beta-amyloid precursor protein, which is implicated in Alzheimer disease. To understand the function of this adapter protein, we identified binding partners for the FE65 WW domain. Proline-rich sequences sharing a proline-proline-leucine-proline core motif were recovered by screening expression libraries for ligands of the FE65 WW domain. Five proteins of molecular masses 60, 75, 80, 140, and 200 kDa could be purified from mouse brain lysates by affinity to the FE65 WW domain. We identified two of these five proteins as the 80- and 140-kDa isoforms encoded by Mena, the mammalian homolog of the Drosophila Enabled gene. Using the SPOTs technique of peptide synthesis, we identified the sequences in Mena that interact with the FE65 WW domain and found that they contain the signature proline-proline-leucine-proline motif. Finally, we demonstrated that Mena binds to FE65 in vivo by coimmunoprecipitation assay from COS cell extracts. The specificity of the Mena-FE65 WW domain association was confirmed by competition assays. Further characterization of the FE65-Mena complex may identify a physiological role for these proteins in beta-amyloid precursor protein biogenesis and may help in understanding the mechanism of molecular changes that underlie Alzheimer disease

Platelet-derived Growth Factor Induces the β-γ-Secretase-mediated Cleavage of Alzheimer's Amyloid Precursor Protein through a Src-Rac-dependent Pathway

The beta-amyloid peptide (Abeta) present in the senile plaques of Alzheimer's disease derives from the cleavage of a membrane protein, named APP, driven by two enzymes, known as beta- and gamma-secretases. The mechanisms regulating this cleavage are not understood. We have developed an experimental system to identify possible extracellular signals able to trigger the cleavage of an APP-Gal4 fusion protein, which is detected by measuring the expression of the CAT gene transcribed under the control of the Gal4 transcription factor, which is released from the membrane upon the cleavage of APP-Gal4. By using this assay, we purified a protein contained in the C6 cell-conditioned medium, which activates the cleavage of APP-Gal4 and which we demonstrated to be PDGF-BB. The APP-Gal4 processing induced by PDGF is dependent on the gamma-secretase activity, being abolished by an inhibitor of this enzyme, and is the consequence of the activation of a pathway downstream of the PDGF-receptor, which includes the non-receptor tyrosine kinase Src and the small G-protein Rac1. These findings are confirmed by the observation that a constitutively active form of Src increases Abeta generation and that, in cells stably expressing APP, the generation of A is strongly decreased by the Src tyrosine kinase inhibitor PP2

Типологія синтаксичних конструкцій в німецькій та українській мовах

Німецька та українська мови є односистемними мовами: обидві належать до індоєвропейської мовної сім’ї. Спільні корені та тривалий період ізольованого розвитку, вказують на те, що вказані мови мають характеристики подібності та відмінності в своій внутрішній будові. Німецька та українська належать до синтетичного типу флективних мов. Це означає, що граматичне значення слів у них виражається, здебільшого, за допомогою системи флексій і реалізується в межах одного графічного слова. Але флективна система німецької мови бідніша, ніж у слов’янських мовах.Немецкий и украинский языки являются односистемными языками: оба принадлежат к индоевропейской языковой семье. Общие корни и длительный период изолированного развития, указывают на то, что указанные языки имеют характеристики сходства и различия в своем внутреннем строении. Немецкий и украинский принадлежат к синтетическому типу флективных языков. Это означает, что грамматическое значение слов в них выражается, в основном, с помощью системы флексий и реализуется в пределах одного графического слова. Но флективная система немецкого языка беднее, чем в славянских языках.German and Ukrainian are single-system languages: both belong to the Indo-European language family. Common roots and a long period of isolated development, indicate that these languages ​​have characteristics of similarity and differences in their internal structure. German and Ukrainian belong to the synthetic type of inflectional languages. This means that the grammatical meaning of words in them is expressed, mainly, with the help of a system of inflexions and is realized within a single graphic word. But the inflectional system of the German language is poorer than in the Slavic languages

Proteomic Analysis of Sera from Common Variable Immunodeficiency Patients Undergoing Replacement Intravenous Immunoglobulin Therapy

Common variable immunodeficiency is the most common form of symptomatic primary antibody failure in adults and children. Replacement immunoglobulin is the standard treatment of these patients. By using a differential proteomic approach based on 2D-DIGE, we examined serum samples from normal donors and from matched, naive, and immunoglobulin-treated patients. The results highlighted regulated expression of serum proteins in naive patients. Among the identified proteins, clusterin/ApoJ serum levels were lower in naive patients, compared to normal subjects. This finding was validated in a wider collection of samples from newly enrolled patients. The establishment of a cellular system, based on a human hepatocyte cell line HuH7, allowed to ascertain a potential role in the regulation of CLU gene expression by immunoglobulins

Interaction of the Phosphotyrosine Interaction/Phosphotyrosine Binding-related Domains of Fe65 with Wild-type and Mutant Alzheimer's β-Amyloid Precursor Proteins

The two tandem phosphotyrosine interaction/phosphotyrosine binding (PID/PTB) domains of the Fe65 protein interact with the intracellular region of the Alzheimer's beta-amyloid precursor protein (APP). This interaction, previously demonstrated in vitro and in the yeast two hybrid system, also takes place in vivo in mammalian cells, as demonstrated here by anti-Fe65 co-immunoprecipitation experiments. This interaction differs from that occurring between other PID/PTB domain-containing proteins, such as Shc and insulin receptor substrate 1, and activated growth factor receptors as follows: (i) the Fe65-APP interaction is phosphorylation-independent; (ii) the region of the APP intracellular domain involved in the binding is larger than that of the growth factor receptor necessary for the formation of the complex with Shc; and (iii) despite a significant similarity the carboxyl-terminal regions of PID/PTB of Fe65 and of Shc are not functionally interchangeable in terms of binding cognate ligands. A role for Fe65 in the pathogenesis of familial Alzheimer's disease is suggested by the finding that mutant APP, responsible for some cases of familial Alzheimer's disease, shows an altered in vivo interaction with Fe65

Novel human anti-claudin 1 mAbs inhibit hepatitis C virus infection and may synergize with anti-SRB1 mAb

Hepatitis C virus (HCV) is a major cause of chronic hepatitis and liver carcinoma and new therapies based on novel targets are needed. The tight junction protein claudin 1 (CLDN-1) is essential for HCV cell entry and spread, and anti-CLDN-1 rat and mouse mAbs are safe and effective in preventing and treating HCV infection in a human liver chimeric mouse model. To accelerate translation of these observations into a novel approach to treat HCV infection and disease in humans, we screened a phage display library of human single-chain antibody fragments by using a panel of CLDN-1-positive and -negative cell lines and identified phage specifically binding to CLDN-1. The 12 clones showing the highest levels of binding were converted into human IgG4. Some of these mAbs displayed low-nanomolar affinity, and inhibited infection of human hepatoma Huh7.5 cells by different HCV isolates in a dose-dependent manner. Cross-competition experiments identified six inhibitory mAbs that recognized distinct epitopes. Combination of the human anti-SRB1 mAb C-1671 with these anti-CLDN-1 mAbs could either increase or reduce inhibition of cell culture-derived HCV infection in vitro. These novel human anti-CLDN-1 mAbs are potentially useful to develop a new strategy for anti-HCV therapy and lend support to the combined use of antibodies targeting the HCV receptors CLDN-1 and SRB1, but indicate that care must be taken in selecting the proper combination