Naive T cells can be induced to differentiate into Foxp3+ regulatory T cells (iTregs) upon suboptimal T cell receptor (TCR) stimulus, or TCR stimulus in conjunction with TGF-β signaling; however, we do not fully understand how these signals combine to control foxp3 expression. Here, we show that strong TCR activation-in terms of both duration and ligand affinity-causes the accumulation of DNA (cytosine-5)-methyltransferase 1 (DNMT1) and DNMT3b, and their specific enrichment at the foxp3 locus, which leads to increased CpG methylation and inhibited foxp3 transcription. During this process, the augmentation of DNMT1 is regulated through at least two post-transcriptional mechanisms: strong TCR signal inactivates GSK3β to rescue DNMT1 protein from proteasomal degradation; and, strong TCR signal suppresses miR-148a to derepress DNMT1 mRNA translation. Meanwhile, TGF-β signaling antagonizes DNMT1 accumulation via activation of p38 MAP kinase. Thus, independent of transcription factor activation, TCR and TGF-β signals converge on DNMT1 to modulate the expression of foxp3 epigenetically, which marks mother cells' iTreg lineage choice within the genome of differentiating daughter cells.

Sugar beet α-glucosidase (SBG), a member of glycoside hydrolase family 31, shows exceptional long-chain specificity, exhibiting higher kcat/Kms for longer maltooligosaccharides. However, its amino acid sequence is similar to other short-chain-specific α-glucosidases. To gain structural insights into the long-chain-substrate recognition of SBG, a crystal structure complex with the pseudotetrasaccharide, acarbose, was determined at 1.7 Å resolution. The active site pocket of SBG was formed by a (β/α)8 barrel domain and a long loop (the N-loop) bulging from the N-terminal domain similar to other related enzymes. Two residues, Phe236 and Asn237 in the N-loop were important for the long-chain specificity. Kinetic analysis of an Asn237 mutant enzyme and a previous study of a Phe236 mutant enzyme demonstrated that these residues create subsites +2 and +3. The structure also indicated that Phe236 and Asn237 guided the reducing end of long substrates to subdomain b2, which is an additional element inserted into the (β/α)8 barrel domain. Subdomain b2 of SBG included Ser497, which was identified as the residue at subsite +4 by site-directed mutagenesis.

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is a key component in synaptic plasticity and neuronal survival. The aim of this work was to investigate the participation of the PI3K/Akt pathway and its outcome on different molecular targets such as glycogen synthase kinase 3β (GSK3β) and Forkhead box-O (FoxO) transcription factors during mild oxidative stress triggered by iron overload. The exposure of mouse hippocampal neurons (HT22) to different concentrations of Fe(2+) (25-200 μM) for 24 h led us to define a mild oxidative injury status (50 μM Fe(2+)) in which cell morphology showed changes typical of neuronal damage, with increased lipid peroxidation and cellular oxidant levels but no alteration of cellular viability. There was a simultaneous increase in both Akt and GSK3β phosphorylation. Levels of phospho-FoxO3a (inactive form) increased in the cytosolic fraction of cells treated with iron in a PI3K-dependent manner. Moreover, PI3K and Akt translocated to the nucleus in response to oxidative stress. Iron-overloaded cells harboring a constitutively active form of Akt showed decreased oxidants levels. Indeed, glutathione (GSH) synthesis under oxidative stress conditions was regulated by activated Akt. Our results show that activation of the PI3K/Akt pathway during iron-induced neurotoxicity regulates multiple targets such as GSK3β, FoxO transcriptional activity and glutathione metabolism thus modulating neuronal response to oxidative stress.

Presynaptic nerve terminals must maintain stable neurotransmission via synaptic vesicle membrane recycling despite encountering wide fluctuations in the number and frequency of incoming action potentials (AP). However, the molecular mechanism linking variation in neuronal activity to vesicle trafficking is unknown. Here, we combined genetic knockdown and direct physiological measurements of synaptic transmission from paired neurons to show that three isoforms of dynamin, an essential endocytic protein, work individually to match vesicle reuse pathways having distinct rate and time constants with physiological AP frequencies. Dynamin-3 resupplied the readily releasable pool (RRP) with slow kinetics independent of AP frequency, but acted quickly, within 20 ms of the incoming AP. Under high frequency firing, dynamin-1 regulated recycling to the RRP with fast kinetics in a slower time window of greater than 50 ms. Dynamin-2 displayed a hybrid response between the other isoforms. Collectively, our findings show how dynamin isoforms select appropriate vesicle reuse pathways associated with specific neuronal firing patterns.

Protein palmitoylation, a common post-translational lipid modification, plays an important role in protein trafficking and functions. Recently developed palmitoyl-proteomic methods identified many novel substrates. However, the whole picture of palmitoyl substrates has not been clarified. Here, we performed global in silico screening using the CSS-Palm 2.0 program, free software for prediction of palmitoylation sites, and selected 17 candidates as novel palmitoyl substrates. Of the 17 candidates, 10 proteins, including six synaptic proteins (Syd-1, TARP γ-2, TARP γ-8, cornichon-2, CaMKIIα and neurochondrin [Ncdn]/norbin), one focal adhesion protein (zyxin), two ion channels (TRPM8 and TRPC1), and one G-protein coupled receptor (orexin 2 receptor), were palmitoylated. Using the DHHC palmitoylating enzyme library, we found that all tested substrates were palmitoylated by the Golgi-localized DHHC3/7 subfamily. Ncdn, a regulator for neurite outgrowth and synaptic plasticity, was robustly palmitoylated by the DHHC1/10 (zDHHC1/11; z1/11) subfamily, whose substrate has not yet been reported. As predicted by CSS-Palm 2.0, Cys-3 and Cys-4 are the palmitoylation sites for Ncdn. Ncdn was specifically localized in somato-dendritic regions, not in the axon of rat cultured neurons. Stimulated emission depletion (STED) microscopy revealed that Ncdn was localized to Rab5-positive early endosomes in a palmitoylation-dependent manner, where DHHC1/10 (z1/11) were also distributed. Knockdown of DHHC1, 3, or 10 (z11) resulted in the loss of Ncdn from Rab5-positive endosomes. Thus, through in silico screening, we demonstrate that Ncdn and the DHHC1/10 (z1/11) and DHHC3/7 subfamilies are novel palmitoyl substrate-enzyme pairs and that Ncdn palmitoylation plays an essential role in its specific endosomal targeting.

Prostaglandin E2 (PGE2) is a potent lipid mediator that plays a key role in inflammation and carcinogenesis. NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the oxidation of the 15(S)-hydroxyl group of PGE2, which leads to PGE2 biotransformation. In this study, we showed that the 15-PGDH-derived 15-keto-PGE2 is an endogenous peroxisome proliferator activated receptor-γ (PPAR-γ) ligand that causes PPAR-γ dissociation from Smad2/3, allowing Smad2/3 association with TGFBRI and SARA and subsequent Smad2/3 phosphorylation and transcription activation in human cholangiocarcinoma cells. The 15-PGDH/15-keto-PGE2-induced Smad2/3 phosphorylation resulted in the formation of pSmad2/3, TAp63, p53 ternary complex and their binding to TAp63 promoter, inducing TAp63 auto-transcription. The role of TAp63 in 15-PGDH/15-keto-PGE2-induced inhibition of tumor growth was further supported by the observation that knockdown of TAp63 prevented 15-PGDH-induced inhibition of tumor cell proliferation, colony formation and migration. These findings disclose a novel 15-PGDH-mediated 15-keto-PGE2 signaling cascade that interacts with PPAR-γ, Smad2/3 and TAp63.

Oligomerization of the 42-residue peptide Aβ42 plays a key role in the pathogenesis of Alzheimer's disease. Despite great academic and medical interest, the structures of these oligomers have not been well characterized. Site-directed spin labeling combined with electron paramagnetic resonance spectroscopy is a powerful approach for studying structurally ill-defined systems, but its application in amyloid oligomer structure study has not been systematically explored. Here we report a comprehensive structural study on a toxic Aβ42 oligomer, called globulomer, using site-directed spin labeling complemented by other techniques. Transmission electron microscopy shows that these oligomers are globular structures with diameters of ~7-8 nm. Circular dichroism shows primarily β structures. X-ray powder diffraction suggests a highly ordered intra-sheet hydrogen-bonding network and a heterogeneous inter-sheet packing. Residue-level mobility analysis on spin labels introduced at 14 different positions shows a structured state and a disordered state at all labeling sites. Side chain mobility increases from N- to C-terminal regions. Inter-molecular distance measurements at 14 residue positions suggest that C-terminal residues G29-V40 form a tightly packed core with inter-molecular distances in a narrow range of 11.5-12.5 Å. These inter-molecular distances rule out the existence of fibril-like parallel in-register β structures, and strongly suggest an antiparallel β sheet arrangement in Aβ42 globulomers.

Despite the fact that androgen deprivation therapy (ADT) can effectively reduce prostate tumor size, its effect on prostate cancer (PCa) metastasis remains unclear. We examined the existing data of the ADT treated PCa patients to analyze therapy effects on primary tumors size, prostate specific antigen (PSA) values, and metastatic incidence. We found the current ADT might lead to primary tumors reduction with PSA decreases, yet metastases increase in some PCa patients. Using in vitro and in vivo metastasis models with human PCa cell lines, we also evaluated the effects of the currently used anti-andrgoens, Casodex and MDV3100, and the newly developed anti-AR compounds, ASC-J9 and cryptotanshinone (CTS), on PCa cell growth and invasion. In vitro results showing that 10 μM Casodex or MDV3100 treatments suppressed PCa cell growth and reduce PSA level, yet significantly enhanced PCa cells invasion. In vivo mice studies using orthotopic xenograft mouse model also confirmed these results. Mechanism dissection further indicated these Casodex/MDV3100 treatments enhanced the TGF-β1→Smad3→MMP9 pathway, but ASC-J9 and CTS showed promising anti-invasion effects via down-regulation of MMP9 expression. Our findings points out potential risks of the currently used anti-androgens, and provide a potential for the development of a novel strategy targeting both PCa growth and metastasis.

The chemotherapeutic drug cisplatin (CDDP) is widely used in treatment of human cancers. However, the mechanism underlying intrinsic tumor resistance to CDDP remains elusive. Here we demonstrate that treatment with CDDP results in downregulation of cJun expression via caspase9 dependent cleavage of cJun at D65 and MEKK1 mediated ubiquitylation and degradation of cJun in CDDP sensitive cancer cells. In contrast, activation of JNK2 but not JNK1 phosphorylates and upregulates the expression of cJun in CDDP resistant cells. Activated cJun binds to the promoter regions of the MDR1 gene and promotes the expression of MDR1. Expression of a D65A cleavage-resistant cJun mutant suppresses CDDP induced apoptosis of CDDP sensitive cells, whereas depletion of JNK2, cJun, or MDR1 in CDDP resistant cancer cells promotes apoptosis upon CDDP treatment. In addition, mammary gland tumors induced by polyoma virus middle T antigen in jnk2-/- mice are more sensitive to CDDP than are those in jnk2+/+ mice. These findings highlight the instrumental role of c-Jun in the resistance of tumors to treatment with CDDP and indicate that cJun is a molecular target for improving cancer therapy.

Extra cellular Tat is suspected to protect HIV-1 infected cells from cellular immunity. Seropositive patients are unable to produce neutralizing antibodies against Tat and Tat is still secreted under antiviral treatment. In mice, the Tat OYI vaccine candidate generates neutralizing antibodies such as the monoclonal antibody (mAb) 7G12. A peptide called MIMOOX was designed from fragments of Tat OYI identified as the possible binding site for mAb 7G12. MIMOOX was chemically synthesized and its structure was stabilized with a disulfide bridge. Circular Dichroism spectra showed that MIMOOX had mainly beta turns but no alpha helix as Tat OYI. MIMOOX was recognized by mAb 7G12 in ELISA only in reduced condition. Moreover, a competitive recognition assay with mAb 7G12 between MIMOOX and Tat variants showed that MIMOOX mimics a highly conserved surface in Tat variants. Rat immunizations with MIMOOX induce antibodies recognizing Tat variants from the main HIV-1 subtypes and confirm the Tat OYI vaccine rational.