Organelle acidification is important for localisation of vacuolar proteins in Saccharomyces cerevisiae

The acidic environments in the vacuole and other acidic organelles are important for many cellular processes in eukaryotic cells. In this study, we comprehensively investigated the roles of organelle acidification in vacuolar protein localisation in Saccharomyces cerevisiae. After repressing the acidification of acidic compartments by treatment with concanamycin A, a specific inhibitor of vacuolar H⁺-ATPase (V-ATPase), we examined the localisation of GFP-fused proteins that were predicted to localise in the vacuolar lumen or on the vacuolar membrane. Of the 73 proteins examined, 19 changed their localisation to the cytoplasmic region. Localisation changes were evaluated quantitatively using the image processing programme CalMorph. The delocalised proteins included vacuolar hydrolases, V-ATPase subunits, transporters and enzymes for membrane biogenesis, as well as proteins required for protein transport. These results suggest that many alterations in the localisation of vacuolar proteins occur after loss of the acidification of acidic compartments.     

Dual effects of HTLV-1 bZIP factor in suppression of interferon regulatory factor 1

Malignant pleural mesothelioma is known to be widely resistant to therapy and new treatment strategies are needed. Both statins and valproic acid are known to suppress the growth of multiple cancer lines, but the effects on mesothelioma cells are not well defined. In the present study we examined the effects of lovastatin and valproic acid on ACC-MESO-1, which is a human derived mesothelioma cell line. We found that lovastatin (2 μM) and/or valproic acid (5 mM) did not reduce cell viability nor induce apoptosis, but reduced cell invasion. The effect was additive when combined. Furthermore it was speculated that induction of autophagic changes was at least in part involved in this process.     

The ATP-Mediated Regulation of KaiB-KaiC Interaction in the Cyanobacterial Circadian Clock

The cyanobacterial circadian clock oscillator is composed of three clock proteins—KaiA, KaiB, and KaiC, and interactions among the three Kai proteins generate clock oscillation in vitro. However, the regulation of these interactions remains to be solved. Here, we demonstrated that ATP regulates formation of the KaiB-KaiC complex. In the absence of ATP, KaiC was monomeric (KaiC^1mer) and formed a complex with KaiB. The addition of ATP plus Mg²⁺ (Mg-ATP), but not that of ATP only, to the KaiB-KaiC^1mer complex induced the hexamerization of KaiC and the concomitant release of KaiB from the KaiB-KaiC^1mer complex, indicating that Mg-ATP and KaiB compete each other for KaiC. In the presence of ATP and Mg²⁺ (Mg-ATP), KaiC became a homohexameric ATPase (KaiC^6mer) with bound Mg-ATP and formed a complex with KaiB, but KaiC hexamerized by unhydrolyzable substrates such as ATP and Mg-ATP analogs, did not. A KaiC N-terminal domain protein, but not its C-terminal one, formed a complex with KaiB, indicating that KaiC associates with KaiB via its N-terminal domain. A mutant KaiC^6mer lacking N-terminal ATPase activity did not form a complex with KaiB whereas a mutant lacking C-terminal ATPase activity did. Thus, the N-terminal domain of KaiC is responsible for formation of the KaiB-KaiC complex, and the hydrolysis of the ATP bound to N-terminal ATPase motifs on KaiC^6mer is required for formation of the KaiB-KaiC^6mer complex. KaiC^6mer that had been hexamerized with ADP plus aluminum fluoride, which are considered to mimic ADP-Pi state, formed a complex with KaiB, suggesting that KaiB is able to associate with KaiC^6mer with bound ADP-Pi.     

A peptidoglycan monomer with the glutamine to serine change and basic peptides bind in silico to TLR-2 (403–455)

Bacterial cell wall polysaccharides, such as PGN, bind and activate TLR-2, NOD2 and PGRP on monocytes/macrophages and activate inflammation. We found that the peptides containing basic amino acids (cations) at N -terminus and tyrosine at C-terminus interfered with activating ability of PGN. This finding is significant because the ECD of TLR-2 in vivo encounters a large number of proteins or peptides. Some should bind ECD and “pre-form” TLR-2 to respond or not to its activators, although they cannot activate TLR-2 alone. TLR-2 is receptor for a large number of ligands, including lipopeptides and bacterial cell wall glycoproteins. A binding site for lipopeptides has been identified; however, a binding site for soluble or multimeric PGN has not been proposed. To identify the candidate binding sites of peptides and PGN on TLR-2, we modeled docking of peptides and of the PGN monomer (PGN-S-monomer) to extracellular domain (ECD-TLR-2) of the unbound TLR-2. Quantification, in silico, of free energy of binding (DG) identified 2 close sites for peptides and PGN. The PGN-S-monomer binding site is between amino acids TLR-2, 404–430 or more closely TLR-2, 417–428. The peptide-binding site is between amino acids TLR-2, 434–455. Molecular models show PGN-S-monomer inserts its N -acetyl-glucosamine (NAG) deep in the TLR-2 coil, while its terminal lysine interacts with inside (Glu⁴⁰³) and outside pocket (Tyr³⁷⁸). Peptides insert their two N -terminal arginines or their C-terminal tyrosines in the TLR-2 coil. PGN did not bind the lipopeptide-binding site in the TLR-2. It can bind the C-terminus, 572–586 (DG = 0.026 kcal), of “lipopeptide-bound” TLR-2. An additional, low-affinity PGN-binding site is TLR-2 (227–237). MTP, MDP, and lysine-less PGN bind to TLR-2, 87–113. This is the first report identifying candidate binding sites of monomer PGN and peptides on TLR-2. Experimental verification of our findings is needed to create synthetic adjuvant for vaccines. Such synthetic PGN can direct both adjuvant and cancer antigen to TLR-2.     

Distinct steps of cross-linking, self-association, and maturation of tropoelastin are necessary for elastic fiber formation

Elastic fibers play an important role in the characteristic resilience of many tissues. The assembly of tropoelastin into a fibrillar matrix is a complex stepwise process and the deposition and cross-linking of tropoelastin are believed to be key steps of elastic fiber formation. However, the detailed mechanisms of elastic fiber assembly have not been defined yet. Here, we demonstrate the relationship between deposition and the cross-linking/maturation of tropoelastin. Our data show that a C-terminal half-fragment of tropoelastin encoded by exons 16-36 (BH) is deposited onto microfibrils, yet we detect very limited amounts of the cross-linking amino acid, desmosine, an indicator of maturation, whereas the N-terminal half-fragment encoded by exons 2-15 (FH) was deficient for both deposition and cross-linking, suggesting that elastic fiber formation requires full-length tropoelastin molecules. A series of experiments using mutant BH fragments, lacking either exon 16 or 30, or a deletion of both exons showed that self-association of tropoelastin polypeptides was an early step in elastic fiber assembly. Immunofluorescence and Western blot assay showed that the treatment of cell culture medium or conditioned medium with beta-aminopropionitrile to inhibit cross-linking, prevented both the deposition and polymerization of tropoelastin. In conclusion, our present results support the view that self-association and oxidation by lysyl oxidase precedes tropoelastin deposition onto microfibrils and the entire molecule of tropoelastin is required for this following maturation process.     

Analysis of naïve and memory CD4 and CD8 T cell populations in breast cancer patients receiving a HER2/<Emphasis Type="Italic">neu</Emphasis> peptide (E75) and GM-CSF vaccine

We are conducting clinical trials of the E75 peptide as a vaccine in breast cancer (BrCa) patients. We assessed T cell subpopulations in BrCa patients before and after E75 vaccination and compared them to healthy controls. We obtained 17 samples of blood from ten healthy individuals and samples from 22 BrCa patients prior to vaccination. We also obtained pre- and post-vaccination samples of blood from seven BrCa patients who received the E75/GM-CSF vaccine. CD4, CD8, CD45RA, CD45RO, and CCR7 antibodies were used to analyze the CD4+ and CD8+ T cells by four-color flow cytometry. Compared to healthy individuals, BrCa patients have significantly more memory and less naïve T cells and more effector-memory CD8+ and less effector CD4+ T cells. Phenotypic differences in defined circulating CD4+ and CD8+ T cell subpopulations suggest remnants of an active immune response to tumor distinguished by a predominant memory T cell response and by untapped recruitment of naïve helper and cytotoxic T cells. E75 vaccination induced recruitment of both CD4+ and CD8+ naïve T cells while memory response remained stable. Additionally, vaccination induced global activation of all T cells, with specific enhancement of effector CD4+ T cells. E75 vaccination causes activation of both memory and naïve CD4+ and CD8+ T cells, while recruiting additional naïve CD4+ and CD8+ T cells to the overall immune response.     

Positive co‐occurrence between feeding‐associative savannah fishes depends on species and habitat

相似文献