Solution structure of atypical protein kinase C PB1 domain and its mode of interaction with ZIP/p62 and MEK5

Atypical protein kinase C (aPKC) has been implicated in several signaling pathways such as cell polarity, cell survival, and cell differentiation. In contrast to other PKCs, aPKC is unique in having the PB1 (Phox and Bem 1) domain in the N terminus. The aPKC PB1 domain binds with ZIP/p62, Par6, or MEK5 through a PB1-PB1 domain interaction that controls the localization of aPKC. Here, we determined the three-dimensional structure of the PB1 domain of PKCiota by NMR and found that the PB1 domain adopts a ubiquitin fold. The OPCA (OPR, PC, and AID) motif inserted into the ubiquitin fold was presented as a betabetaalpha fold in which the side chains of conserved Asp residues were oriented to the same direction to form an acidic surface. This structural feature suggested that the acidic surface of the PKCiota PB1 domain interacted with the basic surface of the target PB1 domains, and this was confirmed in the case of the PKCiota-ZIP/p62 complex by mutational analysis. Interestingly, in the PKCiota PB1 domain a conserved lysine residue was located on the side opposite to the OPCA motif-presenting surface, suggesting dual roles for the PKCiota PB1 domain in that it could interact with either the conserved lysine residue or the acidic residues on the OPCA motif of the target PB1 domains.     

Structure of POIA1, a homologous protein to the propeptide of subtilisin: implication for protein foldability and the function as an intramolecular chaperone

Solution structure of POIA1 (Pleurotus ostreatus proteinase A inhibitor 1), which functions as an intramolecular chaperone and as an inhibitor to subtilisin, was determined. By making use of the fact that POIA1 is the only structured protein that shows homology to the propeptide of subtilisin, which is unstructured by itself, foldability of this protein was elucidated. It became clear that the evolutionarily conserved residues play two important roles, one for the maintenance of its own structure, and the other for the interaction with subtilisin. Structural softness and mutational tolerance contained in the POIA1 structure makes it an ideal material for designing a foldable protein.     

Postnatal changes in the overall postsynaptic currents evoked in CA1 pyramidal neurons of the rat hippocampus

Evoked fast postsynaptic currents (fPSCs) during the postnatal development of rats (postnatal day 6-70, P6-P70) were systematically examined in hippocampal CA1 pyramidal neurons using whole-cell recordings with biocytin-filled electrodes. Focal stimulation of the stratum radiatum in the CA1 region elicited fPSCs in 80% of the neurons P6-7, 90% of P9-10, and 100% of > or =P11. In neurons P6-7, the fPSCs were exclusively inward and had multiple (on average 5.6) peaks. The fPSCs increased in amplitude with the growth of dendritic arborization, but decreased in the number of peaks. A distinct outward fPSC following the inward fPSC emerged in neurons > or =P11 and was abolished by bicuculline (50 microM). Bicuculline increased the amplitude and duration of the initial inward fPSC (fEPSC) in all age groups and characteristically recruited the polysynaptic second component of fEPSCs in neurons P11-P21. No spontaneous periodic inward current was detected in any age group after blocking GABAA receptors. The coapplication of DL-2-amino-5-phosphonopentanoic acid (AP5, 100 microM) with bicuculline did not eliminate the polysynaptic second component, but the second component was only elicited in slices in which the CA3 region was kept intact. Moreover, the bicuculline- and AP5-resistant second component was due to the burst activity of CA3 pyramidal neurons, which were excited through excitatory recurrents of the Schaffer collaterals. Plausible physiological functions of the generation of the second component in vivo were discussed.     

The spindle pole body duplicates in early G1 phase in the pathogenic yeast Exophiala dermatitidis: an ultrastructural study

The spindle pole body of the pathogenic yeast Exophiala dermatitidis was observed during the cell cycle using freeze-substitution and serial ultrathin sectioning electron microscopy. The spindle pole body was located on the outer membrane of the nuclear envelope and consisted of two disk elements connected by an intervening midpiece in G1 through G2 phases. Each disk element was composed of filamentous materials and measured 150 nm in diameter and 100 nm in thickness. The midpiece had higher electron density and measured 60 nm in length and 40 nm in thickness. At the beginning of prophase, each disk element of the spindle pole body enlarged to more than double in size. They were separated on the nuclear envelope, and associated with numerous cytoplasmic microtubules. At mitosis, the spindle pole body entered the nuclear envelope, associated with numerous nuclear microtubules, and was located at the spindle poles. At the end of telophase, it was extruded back into the cytoplasm from the nuclear envelope. Three-dimensional analysis of cells in different cell cycles suggested that duplication of the spindle pole body took place in early G1 phase. Thus, the location, structure, and duplication cycle of the E. dermatitidis spindle pole body were different from those of Saccharomyces cerevisiae.     

Distinct structural elements that direct solution aggregation and membrane assembly in the channel-forming peptide M2GlyR

Restoration of chloride conductance via the introduction of an anion selective pore, formed by a channel-forming peptide, has been hypothesized as a novel treatment modality for patients with cystic fibrosis (CF). Delivery of these peptide sequences to airway cells from an aqueous environment in the absence of organic solvents is paramount. New highly soluble COOH- and NH(2)-terminal truncated peptides, derived from the second transmembrane segment of the glycine receptor alpha-subunit (M2GlyR), were generated, with decreasing numbers of amino acid residues. NH(2)-terminal lysyl-adducted truncated peptides with lengths of 22, 25, and 27 amino acid residues are equally able to stimulate short circuit current (I(SC)). Peptides with as few as 16 amino acid residues are able to stimulate I(SC), although to a lesser degree. In contrast, COOH-terminal truncated peptides show greatly reduced induced I(SC) values for all peptides fewer than 27 residues in length and show no measurable activity for peptides fewer than 21 residues in length. CD spectra for both the NH(2)- and COOH-truncated peptides have random structure in aqueous solution, and those sequences that stimulated the highest maximal I(SC) are predominantly helical in 40% trifluoroethanol. Peptides with a decreased propensity to form helical structures in TFE also failed to stimulate I(SC). Palindromic peptide sequences based on both the NH(2)- and COOH-terminal halves of M2GlyR were synthesized to test roles of the COOH- and NH(2)-terminal halves of the molecule in solution aggregation and channel forming ability. On the basis of the study presented here, there are distinct, nonoverlapping regions of the M2GlyR sequence that define solution aggregation and membrane channel assembly. Peptides that eliminate solution aggregation with complete retention of channel forming activity were generated.     

Effect of different K+ concentrations on Cryptococcus neoformans phenoloxidase activity

Melanin synthesis in Cryptococcus neoformans, catalyzed by phenoloxidase activity, is one of the oldest virulence factors known. However, until now, the relationship between melanin production in C. neoformans and its virulence has been poorly understood. Among different chemical compounds only Fe3+ and Cu2+ cations enhance the phenoloxidase activity in C. neoformans. A few reports in the literature describe the influence of different cations on C. neoformans phenoloxidase activity, excluding iron. In this study, 13 C. neoformans strains isolated from AIDS patients and 7 from bird droppings (B.D.), were examined in order to clarify the effect of different K+ concentrations on phenoloxidase activity. A new solid and liquid caffeic acid minimal synthetic medium (MSM-CAF) containing only caffeic acid and ferric citrate with different potassium concentrations was used to evaluate C. neoformans phenoloxidase activity. In the MSM-CAF solid medium the degree of brown pigmentation on the agar plates was read on days 1, 2 and 3 of incubation, and the pigmentation of the C. neoformans strains was classed into 5 categories. The brown pigment of the liquid MSM-CAF test tubes were checked after 24 hours of incubation by measuring the optical density (O.D.) at 480 nm. Three C. neoformans AIDS and B.D. strains, randomly chosen, were tested for phenoloxidase activity, according to the modified protocols of Polacheck et al., Torres-Guerrero et al. and Rhodes. According to the results obtained, it has been observed that K+ does not activate the phenoloxidase activity in the C. neoformans AIDS and B.D. strains. In particular, with an increase in potassium concentrations in the MSM-CAF solid and liquid medium, there was a corresponding inhibition of the phenoloxidase activity on both the C. neoformans AIDS and B.D. strains.     

Identification of neuron-specific promoters in Ciona intestinalis

We isolated 5' flanking regions of four genes, Ci-Galphai1, Ci-arr, Ci-vAChTP, and Ci-vGAT, each of which is expressed in distinct sets of neurons in the central nervous system of the ascidian Ciona intestinalis, and we examined their function by introducing green fluorescent protein (GFP)-fusion constructs into Ciona embryos. The reporter gene driven by the 5' flanking region of Ci-Galphai1, Ci-arr, and Ci-vAChTP recapitulated the endogenous gene expression patterns, while that of Ci-vGAT can drive GFP expression in particular subsets of neurons expressing the endogenous gene. Deletion analysis revealed that the Ci-Galphai1 promoter consists of multiple regulatory modules controlling the expression in different types of cells. The GFP fluorescence enabled visualization of cell bodies and axons of different sets of neurons in ascidian larvae. These promoters can be a powerful tool for studying molecular mechanisms of neuronal development as well as neuron networks and functions in ascidians.     

Chemical modification of reveromycin A and its biological activities

Various derivatives of reveromycin A, a novel inhibitor of eukaryotic cell growth, were prepared and their inhibitory effects on both isoleucyl-tRNA synthetase activity and in vitro protein synthesis, and activities on the morphological reversion of src(ts)-NRK cells were assayed. The C5 hydroxyl group and C24 carboxyl group are particularly important for these activities.     

Effective cellulose production by a coculture of Gluconacetobacter xylinus and Lactobacillus mali

A microbial colony that contained a marked amount of cellulose was isolated from vineyard soil. The colony was formed by the associated growth of two bacterial strains: a cellulose-producing acetic acid bacterium (st-60-12) and a lactic acid bacterium (st-20). The 16S rDNA-based taxonomy indicated that st-60-12 belonged to Gluconacetobacter xylinus and st-20 was closely related to Lactobacillus mali. Cocultivation of the two organisms in corn steep liquor/sucrose liquid medium resulted in a threefold higher cellulose yield when compared to the st-60-12 monoculture. A similar enhancement was observed in a coculture with various L. mali strains but not with other Lactobacillus spp. The enhancement of cellulose production was most remarkable when sucrose was supplied as the substrate. L. mali mutants for exocellular polysaccharide (EPS) production were defective in promoting cellulose production, but the addition of EPS to the monoculture of st-60-12 did not affect cellulose productivity. Scanning electron microscopic observation of the coculture revealed frequent association between the st-60-12 and L. mali cells. These results indicate that cell–cell interaction assisted by the EPS-producing L. mali promotes cellulose production in st-60-12.The nucleotide sequences of 16S rDNA that are reported in this paper were submitted to GenBank/EMBL/DDBJ under the accession numbers AB016864 (st-20) and AB016865 (st-60-12).     

Rational design of a mononuclear metal site into the archaeal Rieske-type protein scaffold

Proteins containing Rieske-type [2Fe-2S] clusters play essential functions in all three domains of life. We engineered the two histidine ligands to the Rieske-type [2Fe-2S] cluster in the hyperthermophilic archaeal Rieske-type ferredoxin from Sulfolobus solfataricus to modify types and spacing of ligands and successfully converted the metal and cluster type at the redox-active site with a minimal structural change to a native Rieske-type protein scaffold. Spectroscopic analyses unambiguously established a rubredoxin-type mononuclear Fe3+/2+ center at the engineered local metal-binding site (Zn2+ occupies the iron site depending on the expression conditions). These results show the importance of types and spacing of ligands in the in vivo cluster recognition/insertion/assembly in biological metallosulfur protein scaffolds. We suggest that early ligand substitution and displacement events at the local metal-binding site(s) might have primarily allowed the metal and cluster type conversion in ancestral redox protein modules, which greatly enhanced their capabilities of conducting a wide range of unique redox chemistry in biological electron transfer conduits, using a limited number of basic protein scaffolds.