Structural Basis for Inhibition of the MDM2:p53 Interaction by an Optimized MDM2-Binding Peptide Selected with mRNA Display

The oncoprotein MDM2 binds to tumor suppressor protein p53 and inhibits its anticancer activity, which leads to promotion of tumor cell growth and tumor survival. Abrogation of the p53:MDM2 interaction reportedly results in reactivation of the p53 pathway and inhibition of tumor cell proliferation. We recently performed rigorous selection of MDM2-binding peptides by means of mRNA display and identified an optimal 12-mer peptide (PRFWEYWLRLME), named MDM2 Inhibitory Peptide (MIP), which shows higher affinity for MDM2 (and also its homolog, MDMX) and higher tumor cell proliferation suppression activity than known peptides. Here we determined the NMR solution structure of a MIP-MDM2 fusion protein to elucidate the structural basis of the tight binding of MIP to MDM2. A region spanning from Phe³ to Met¹¹ of MIP forms a single α-helix, which is longer than those of the other MDM2-binding peptides. MIP shares a conserved Phe³-Trp⁷-Leu¹⁰ triad, whose side chains are oriented towards and fit into the hydrophobic pockets of MDM2. Additionally, hydrophobic surface patches that surround the hydrophobic pockets of MDM2 are covered by solvent-exposed MIP residues, Trp⁴, Tyr⁶, and Met¹¹. Their hydrophobic interactions extend the interface of the two molecules and contribute to the strong binding. The potential MDM2 inhibition activity observed for MIP turned out to originate from its enlarged binding interface. The structural information obtained in the present study provides a road map for the rational design of strong inhibitors of MDM2:p53 binding.     

Effects of pH and Lactate on Hydrogen Sulfide Production by Oral Veillonella spp.

Indigenous oral bacteria in the tongue coating such as Veillonella have been identified as the main producers of hydrogen sulfide (H₂S), one of the major components of oral malodor. However, there is little information on the physiological properties of H₂S production by oral Veillonella such as metabolic activity and oral environmental factors which may affect H₂S production. Thus, in the present study, the H₂S-producing activity of growing cells, resting cells, and cell extracts of oral Veillonella species and the effects of oral environmental factors, including pH and lactate, were investigated. Type strains of Veillonella atypica, Veillonella dispar, and Veillonella parvula were used. These Veillonella species produced H₂S during growth in the presence of l-cysteine. Resting cells of these bacteria produced H₂S from l-cysteine, and the cell extracts showed enzymatic activity to convert l-cysteine to H₂S. H₂S production by resting cells was higher at pH 6 to 7 and lower at pH 5. The presence of lactate markedly increased H₂S production by resting cells (4.5- to 23.7-fold), while lactate had no effect on enzymatic activity in cell extracts. In addition to H₂S, ammonia was produced in cell extracts of all the strains, indicating that H₂S was produced by the catalysis of cystathionine γ-lyase (EC 4.4.1.1). Serine was also produced in cell extracts of V. atypica and V. parvula, suggesting the involvement of cystathionine β-synthase lyase (EC 4.2.1.22) in these strains. This study indicates that Veillonella produce H₂S from l-cysteine and that their H₂S production can be regulated by oral environmental factors, namely, pH and lactate.     

SEC14 Phospholipid Transfer Protein Is Involved in Lipid Signaling-Mediated Plant Immune Responses in Nicotiana benthamiana

We previously identified a gene related to the SEC14-gene phospholipid transfer protein superfamily that is induced in Nicotiana benthamiana (NbSEC14) in response to infection with Ralstonia solanacearum. We here report that NbSEC14 plays a role in plant immune responses via phospholipid-turnover. NbSEC14-silencing compromised expression of defense–related PR-4 and accumulation of jasmonic acid (JA) and its derivative JA-Ile. Transient expression of NbSEC14 induced PR-4 gene expression. Activities of diacylglycerol kinase, phospholipase C and D, and the synthesis of diacylglycerol and phosphatidic acid elicited by avirulent R. solanacearum were reduced in NbSEC14-silenced plants. Accumulation of signaling lipids and activation of diacylglycerol kinase and phospholipases were enhanced by transient expression of NbSEC14. These results suggest that the NbSEC14 protein plays a role at the interface between lipid signaling-metabolism and plant innate immune responses.     

Mutations that alter the allosteric nature of cAMP receptor protein of Escherichia coli.

Mutations which permit cAMP binding protein (CRP) to act in the absence of cAMP have been isolated by in vitro mutagenesis of a plasmid containing the cloned crp gene. Adenylate cyclase deficient cells harbouring the mutant (crp*) plasmids exhibited a variety of fermentation profiles on MacConkey indicator plates containing various sugars. beta-galactosidase synthesis in cells carrying the crp* plasmids was activated most by the addition of cGMP as well as cAMP. The sites of mutations which are responsible for the cAMP independent phenotype were determined by in vitro recombination and DNA sequencing. The amino acid substitutions in the mutant proteins were found in two specific regions of the crp gene encoding residues 53-62 and 141-148 of CRP polypeptide. The first region may participate in cAMP binding, while the second appears to be the inter-domain region of the N-terminal cAMP-binding and C-terminal DNA-binding domains.     

Effect of antithrombin III on glycoprotein Ib/IX/V activation in human platelets: Suppression of thromboxane A(2) generation

We have previously shown that ristocetin, an activator of glycoprotein Ib/IX/V, induces release of soluble CD40 (sCD40) ligand via thromboxane (TX) A(2) production from human platelets. In the present study, we investigated the effect of antithrombin-III (AT-III), an anticoagulant, on the ristocetin-induced glycoprotein Ib/IX/V activation in human platelets. AT-III inhibited ristocetin-stimulated platelet aggregation. The ristocetin-induced production of 11-dehydro-TXB(2), a stable metabolite of TXA(2), and the release of sCD40 ligand were suppressed by AT-III. AT-III also reduced the ristocetin-stimulated secretion of platelet-derived growth factor (PDGF)-AB. AT-III failed to affect U46619-, a TXA(2) receptor agonist, induced levels of p38 mitogen-activated protein kinase phosphorylation or sCD40 ligand release. AT-III reduced the binding of SZ2, a monoclonal antibody to the sulfated sequence in the α-chain of glycoprotein Ib, to the ristocetin-stimulated platelets. These results strongly suggest that AT-III reduced ristocetin-stimulated release of sCD40 ligand due to inhibiting TXA(2) production in human platelets.     

Structural Analysis of a Trimer of β2-Microgloblin Fragment by Molecular Dynamics Simulations

A peptide ${\beta }_2$-${\text{m}}_{21?31}$, which is a fragment from residue 21 to residue 31 of ${\beta }_2$-microgloblin, is experimentally known to self-assemble and form amyloid fibrils. In order to understand the mechanism of amyloid fibril formations, we applied the replica-exchange molecular dynamics method to the system consisting of three fragments of ${\beta }_2$-${\text{m}}_{21?31}$. From the analyses on the temperature dependence, we found that there is a clear phase transition temperature in which the peptides aggregate with each other. Moreover, we found by the free energy analyses that there are two major stable states: One of them is like amyloid fibrils and the other is amorphous aggregates.     

A Tecpr1-dependent selective autophagy pathway targets bacterial pathogens

Selective autophagy of bacterial pathogens represents a host innate immune mechanism. Selective autophagy has been characterized on the basis of distinct cargo receptors but the mechanisms by which different cargo receptors are targeted for autophagic degradation remain unclear. In this study we identified a highly conserved Tectonin domain-containing protein, Tecpr1, as an Atg5 binding partner that colocalized with Atg5 at Shigella-containing phagophores. Tecpr1 activity is necessary for efficient autophagic targeting of bacteria, but has no effect on rapamycin- or starvation-induced canonical autophagy. Tecpr1 interacts with WIPI-2, a yeast Atg18 homolog and PI(3)P-interacting protein required for phagophore formation, and they colocalize to phagophores. Although Tecpr1-deficient mice appear normal, Tecpr1-deficient MEFs were defective for selective autophagy and supported increased intracellular multiplication of Shigella. Further, depolarized mitochondria and misfolded protein aggregates accumulated in the Tecpr1-knockout MEFs. Thus, we identify a Tecpr1-dependent pathway as important in targeting bacterial pathogens for selective autophagy.     

Endocrine disrupter bisphenol A increases in situ estrogen production in the mouse urogenital sinus

The balance between androgens and estrogens is very important in the development of the prostate, and even small changes in estrogen levels, including those of estrogen-mimicking chemicals, can lead to serious changes. Bisphenol A (BPA), an endocrine-disrupting chemical, is a well-known, ubiquitous, estrogenic chemical. To investigate the effects of fetal exposure to low-dose BPA on the development of the prostate, we examined alterations of the in situ sex steroid hormonal environment in the mouse urogenital sinus (UGS). In the BPA-treated UGS, estradiol (E(2)) levels and CYP19A1 (cytochrome P450 aromatase) activity were significantly increased compared with those of the untreated and diethylstilbestrol (DES)-treated UGS. The mRNAs of steroidogenic enzymes, Cyp19a1 and Cyp11a1, and the sex-determining gene, Nr5a1, were up-regulated specifically in the BPA-treated group. The up-regulation of mRNAs was observed in the mesenchymal component of the UGS as well as in the cerebellum, heart, kidney, and ovary but not in the testis. The number of aromatase-expressing mesenchymal cells in the BPA-treated UGS was approximately twice that in the untreated and DES-treated UGS. The up-regulation of Esrrg mRNA was observed in organs for which mRNAs of steroidogenic enzymes were also up-regulated. We demonstrate here that fetal exposure to low-dose BPA has the unique action of increasing in situ E(2) levels and CYP19A1 (aromatase) activity in the mouse UGS. Our data suggest that BPA might interact with in situ steroidogenesis by altering tissue components, such as the accumulation of aromatase-expressing mesenchymal cells, in particular organs.     

Anti-citrullinated collagen type I antibody is a target of autoimmunity in rheumatoid arthritis

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, but its autoimmune mechanisms are not clearly understood. Recently, anti-citrullinated peptide antibodies have been specifically observed in sera of RA patients. Furthermore, we identified RA-susceptible variant in a gene encoding citrullinating enzyme, peptidylarginine deiminase type 4 (PADI4). Therefore, we hypothesized that proteins which are modified in RA synovium by PADI4 act as autoantigens. Subsequently, we obtained human collagen type I (huCI) as one of the autoantigens using a RA synoviocyte cDNA library by immunoscreening. We also investigated that the levels of anti-citrullinated huCI were significantly higher in RA patient sera than in normal control sera with high specificity (99%) and positively correlated with the levels of anti-cyclic citrullinated peptide (anti-CCP) antibodies. We concluded that huCI is a novel substrate protein of PADIs and that citrullinated huCI is a candidate autoantigen of RA.     

Gadd45a, the gene induced by the mood stabilizer valproic acid, regulates neurite outgrowth through JNK and the substrate paxillin in N1E-115 neuroblastoma cells

Valproic acid (VPA), a mood stabilizer and anticonvulsant, has a variety of neurotrophic functions; however, less is known about how VPA regulates neurite outgrowth. Here, using N1E-115 neuroblastoma cells as the model, we show that VPA upregulates Gadd45a to trigger activation of the downstream JNK cascade controlling neurite outgrowth. VPA induces the phosphorylation of c-Jun N-terminal kinase (JNK) and the substrate paxillin, while VPA induction of neurite outgrowth is inhibited by JNK inhibitors (SP600125 and the small JNK-binding peptide) or a paxillin construct harboring a Ser 178-to-Ala mutation at the JNK phosphorylation. Transfection of Gadd45a, acting through the effector MEKK4, leads to the phosphorylation of the JNK cascade. Conversely, knockdown of Gadd45a with siRNA reduces the effect of VPA. Taken together, these results suggest that upregulation of Gadd45a explains one of the mechanisms whereby VPA induces the neurotrophic effect, providing a new role of Gadd45a in neurite outgrowth.