High-level secretory production of phospholipase A1 by Saccharomyces cerevisiae and Aspergillus oryzae

Phospholipase A1 (PLA1) is a hydrolytic enzyme that catalyzes the removal of the acyl group from position 1 of lecithin to form lysolecithin. The PLA1 gene, which had been cloned from Aspergillus oryzae, was expressed in Saccharomyces cerevisiae and A. oryzae. Through the modification of the medium composition and the feeding conditions of substrate, the production level of PLA1 by S. cerevisiae was increased to a level fivefold higher than that indicated in a previous report. In the case of A. oryzae, introduction of multicopies of PLA1 expression units, and the morphological change from the pellet form to the filamentous form were effective for the enhancement of PLA1 production. We succeeded in producing 3,500 U/ml of PLA1 using an industrial-scale fermentor.     

Purification and properties of a phospholipase A2/lipase preferring phosphatidic acid,bis(monoacylglycerol) phosphate,and monoacylglycerol from rat testis

Phospholipase A(2) (PLA(2)) was purified to homogeneity from the supernatant fraction of rat testis homogenate. The purified 63-kDa enzyme did not require Ca(2+) ions for activity and exhibited both phosphatidic acid-preferring PLA(2) and monoacylglycerol lipase activities with a modest specificity toward unsaturated acyl chains. Anionic detergents enhanced these activities. Serine-modifying irreversible inhibitors, (p-amidinophenyl) methanesulfonyl fluoride and methylarachidonyl fluorophosphonate, inhibited both activities to a similar extent, indicating a single active site is involved in PLA(2) and lipase activities. The sequence of NH(2)-terminal 12 amino acids of purified enzyme was identical to that of a carboxylesterase from rat liver. The optimal pH for PLA(2) activity (around 5.5) differed from that for lipase activity (around 8.0). At pH 5.5 the enzyme also hydrolyzed bis(monoacylglycerol) phosphate, or lysobisphosphatidic acid (LBPA), that has been hitherto known as a secretory PLA(2)-resistant phospholipid and a late endosome marker. LBPA-enriched fractions were prepared from liver lysosome fractions of chloroquine-treated rats, treated with excess of pancreatic PLA(2), and then used for assaying LBPA-hydrolyzing activity. LBPA and the reaction products were identified by microbore normal phase high performance liquid chromatography/electrospray ionization ion-trap mass spectrometry. These enzymatic properties suggest that the enzyme can metabolize phosphatidic and lysobisphosphatidic acids in cellular acidic compartments.     

The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy

Muscle cells respond to mechanical stretch stimuli by triggering downstream signals for myocyte growth and survival. The molecular components of the muscle stretch sensor are unknown, and their role in muscle disease is unclear. Here, we present biophysical/biochemical studies in muscle LIM protein (MLP) deficient cardiac muscle that support a selective role for this Z disc protein in mechanical stretch sensing. MLP interacts with and colocalizes with telethonin (T-cap), a titin interacting protein. Further, a human MLP mutation (W4R) associated with dilated cardiomyopathy (DCM) results in a marked defect in T-cap interaction/localization. We propose that a Z disc MLP/T-cap complex is a key component of the in vivo cardiomyocyte stretch sensor machinery, and that defects in the complex can lead to human DCM and associated heart failure.     

Dissection of upstream regulatory components of the Rho1p effector, 1,3-beta-glucan synthase,in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, one of the main structural components of the cell wall is 1,3-beta-glucan produced by 1,3-beta-glucan synthase (GS). Yeast GS is composed of a putative catalytic subunit encoded by FKS1 and FKS2 and a regulatory subunit encoded by RHO1. A combination of amino acid alterations in the putative catalytic domain of Fks1p was found to result in a loss of the catalytic activity. To identify upstream regulators of 1,3-beta-glucan synthesis, we isolated multicopy suppressors of the GS mutation. We demonstrate that all of the multicopy suppressors obtained (WSC1, WSC3, MTL1, ROM2, LRE1, ZDS1, and MSB1) and the constitutively active RHO1 mutations tested restore 1,3-beta-glucan synthesis in the GS mutant. A deletion of either ROM2 or WSC1 leads to a significant defect of 1,3-beta-glucan synthesis. Analyses of the degree of Mpk1p phosphorylation revealed that among the multicopy suppressors, WSC1, ROM2, LRE1, MSB1, and MTL1 act positively on the Pkc1p-MAPK pathway, another signaling pathway regulated by Rho1p, while WSC3 and ZDS1 do not. We have also found that MID2 acts positively on Pkc1p without affecting 1,3-beta-glucan synthesis. These results suggest that distinct networks regulate the two effector proteins of Rho1p, Fks1p and Pkc1p.     

Dual roles of photosynthetic electron transport in photosystem I biogenesis: light induction of mRNAs and chromatic regulation at post-mRNA level

Light regulation of photosystem I (PSI) biogenesis was studied in a unicellular green alga, Chlamydomonas reinhardtii. When Chlamydomonas cells were transferred from darkness to the light, mRNAs for both nuclear- and chloroplast-encoded PSI subunits were induced in concert. This light induction was inhibited by photosynthetic electron transport (PET) inhibitors, 3-(3,4 dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6 isopropyl-p-benzoquinone, but not by an uncoupler, carbonyl cyanide m-chlorophenylhydrazone. This indicated that PET plays a pivotal role in the light induction of PSI subunit mRNAs, but that photophosphorylation is not necessary. When we irradiated the Chlamydomonas cells with PSI-light (695 nm) or PSII-light (644 nm), which makes the plastoquinone pool oxidative and reductive, respectively, PSII-light caused the accumulation of PSI proteins more abundantly than did PSI-light. However, there was no difference for the PSI subunit mRNA levels between these light sources. From these results, we conclude that PET plays dual roles in the regulation of PSI biogenesis in Chlamydomonas: when cells are illuminated, PET first induces the PSI subunit mRNAs irrespective of the redox state of the intersystem electron carriers, and then their redox state fine-tunes PSI biogenesis at translational and/or post-translational steps to fulfil the chromatic adaptation.     

Molecular isolation and characterization of novel four subisoforms of ECE-2

Although the four polypeptides of blasticidin S (BS) deaminase (BSD) are packed rather tightly coordinated to the "structural and catalytic" zinc atom of each subunit, the C-terminal region of the enzyme was suggested to be somewhat molten and flexible [M. Kimura, S. Sekido, Y. Isogai, and I. Yamaguchi (2000) J. Biochem. 127, 955-963]. To understand roles of this flexible region, we constructed five C-terminal deletion variants of BSD (each successively deleted from the C-terminal end up to five residues) and analyzed their biochemical properties focusing on the structure and activity of the enzyme. BSD and all of the deletion mutants showed the unique rigid conformation (e.g., characterized by their stabilities in SDS solution) and high levels of resistance against protease digestions. Furthermore, both the wild-type and deletion apoenzymes exhibited similar physical properties in thermodynamic refolding into the stable tetramer conformation. However, these small C-terminal deletions exerted deleterious effects on the catalytic efficiency of the enzyme as indicated by their strongly reduced k(cat)/K(m) value. Judging from the altered kinetic parameters and unaltered structural properties of the deletion variants, these C-terminal residues appear to be directly involved in enzyme-substrate interaction. In this short flexible region, Tyr-126, Trp-128, and Gly-130 were the key residues. Most notably, removal of Gly-130 markedly increased K(m) for BS without affecting its k(cat) value. These results indicate that the flexible C-terminal region is important for catalytic function and that a single Gly residue at the C-terminal end of BSD contributes significantly in facilitating access of a substrate to the active site.     

Functional vanilloid receptors in cultured normal human epidermal keratinocytes

Vanilloid receptor subtype 1, VR1, is an ion channel that serves as a polymodal detector of pain-producing chemicals such as capsaicin and protons in primary afferent neurons. Here we showed that both capsaicin and acidification produced elevations in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in cultured human epidermal keratinocytes. The capsaicin- and acidification-evoked increases in [Ca(2+)](i) were inhibited by capsazepine, an antagonist to VR1. VR1-like immunoreactivity was observed in the cells. These findings suggest that functional VR1-like protein is present and functions as a sensor against noxious chemical stimuli, such as capsaicin or acidification, in epidermal keratinocytes.     

Class VI myosin moves processively along actin filaments backward with large steps.

Among a superfamily of myosin, class VI myosin moves actin filaments backwards. Here we show that myosin VI moves processively on actin filaments backwards with large ( approximately 36 nm) steps, nevertheless it has an extremely short neck domain. Myosin V also moves processively with large ( approximately 36 nm) steps and it is believed that myosin V strides along the actin helical repeat with its elongated neck domain that is critical for its processive movement with large steps. Myosin VI having a short neck cannot take this scenario. We found by electron microscopy that myosin VI cooperatively binds to an actin filament at approximately 36 nm intervals in the presence of ATP, raising a hypothesis that the binding of myosin VI evokes "hot spots" on actin filaments that attract myosin heads. Myosin VI may step on these "hot spots" on actin filaments in every helical pitch, thus producing processive movement with 36 nm steps.