Improved metabolic action of a bacterial lysine decarboxylase gene in tobacco hairy root cultures by its fusion to arbcS transit peptide coding sequence

The gene of a bacterial lysine decarboxylase (ldc) fused to arbcS transit peptide coding sequence (tp), and under the control of the CaMV 35S promoter, was expressed in hairy root cultures ofNicotiana tabacum. The fusion of theldc to the targeting signal sequence improved the performance of the bacterial gene in the plant cells in many respects. Nearly all transgenic hairy root cultures harbouring the35S-tp-ldc gene contained distinctly higher lysine decarboxylase activity (from 1.5 to 30 pkat LDC per mg protein) than those which had been transformed with constructs in which the gene had been directly cloned behind the CaMV 35S promoter. The higher enzyme activity led to the accumulation of up to 0.7% cadaverine on a dry mass basis. In addition, part of the cadaverine pool was used for increased biosynthesis of anabasine, an alkaloid which was hardly detectable in control cultures. The best line contained anabasine levels of 0.5% dry mass, which could further be enhanced by feeding of lysine.     

Phosphorylation of elF-4E and initiation of protein synthesis in P19 embryonal carcinoma cells

Mitogenic stimulation of protein synthesis is accompanied by an increase in elF-4E phosphorylation. The effect on protein synthesis by induction of differentiation is less well known. We treated P19 embryonal carcinoma cells with the differentiating agent retinoic acid and found that protein synthesis increased during the first hour of addition. However, the phosphorylation state, as well as the turnover of phosphate on elF-4E, remained unchanged. Apparently, the change in protein synthesis after RA addition is regulated by another mechanism than elF-4E phosphorylation. By using P19 cells overexpressing the EGF receptor, we show that the signal transduction pathway that leads to phosphorylation of elF-4E is present in P19 cells; the EGF-induced change in phosphorylation of elF-4E in these cells is likely to be regulated by a change in elF-4E phosphatase activity. These results suggest that the onset of retinoic acid-induced differentiation is triggered by a signal transduction pathway which involves changes in protein synthesis, but not elF-4E phosphorylation. © 1995 Wiley-Liss, Inc.     

Metallocenter assembly in nickel-containing enzymes

 The five known nickel-dependent enzymes include urease, hydrogenase, carbon monoxide dehydrogenase (and CO dehydrogenase/acetyl-coenzyme A synthase), methyl-S–coenzyme M reductase, and one class of superoxide dismutase. Consistent with their disparate functions, these Ni enzymes have distinct metallocenter structures that vary in Ni coordination geometry, number and types of metals, and the presence of additional components. Sophisticated cellular Ni processing systems have been devised to allow for specific and functional incorporation of Ni into these proteins. This review highlights several themes that are common to the enzyme activation processes and summarizes current concepts related to the enzyme-specific Ni assembly pathways. Received, accepted: 3 April 1997     

The application of strand invasion phenomenon,directed by peptide nucleic acid (PNA) and single‐stranded DNA binding protein (SSB) for the recognition of specific sequences of human endogenous retroviral HERV‐W family

The HERV‐W family of human endogenous retroviruses represents a group of numerous sequences that show close similarity in genetic composition. It has been documented that some members of HERV‐W–derived expression products are supposed to play significant role in humans' pathology, such as multiple sclerosis or schizophrenia. Other members of the family are necessary to orchestrate physiological processes (eg, ERVWE1 coding syncytin‐1 that is engaged in syncytiotrophoblast formation). Therefore, an assay that would allow the recognition of particular form of HERV‐W members is highly desirable. A peptide nucleic acid (PNA)–mediated technique for the discrimination between multiple sclerosis‐associated retrovirus and ERVWE1 sequence has been developed. The assay uses a PNA probe that, being fully complementary to the ERVWE1 but not to multiple sclerosis‐associated retrovirus (MSRV) template, shows high selective potential. Single‐stranded DNA binding protein facilitates the PNA‐mediated, sequence‐specific formation of strand invasion complex and, consequently, local DNA unwinding. The target DNA may be then excluded from further analysis in any downstream process such as single‐stranded DNA‐specific exonuclease action. Finally, the reaction conditions have been optimized, and several PNA probes that are targeted toward distinct loci along whole HERV‐W env sequences have been evaluated. We believe that PNA/single‐stranded DNA binding protein–based application has the potential to selectively discriminate particular HERV‐W molecules as they are at least suspected to play pathogenic role in a broad range of medical conditions, from psycho‐neurologic disorders (multiple sclerosis and schizophrenia) and cancers (breast cancer) to that of an auto‐immunologic background (psoriasis and lupus erythematosus).     

Crystal structure of a novel ATPase RadD from Escherichia coli

The helicase superfamily 2 (SF2) proteins are involved in essentially every step in DNA and RNA metabolism. The radD (yejH) gene, which belongs to SF2, plays an important role in DNA repair. The RadD protein includes all seven conserved SF2 motifs and has shown ATPase activity. Here, we first reported the structure of RadD from Escherichia coli containing two RecA-like domains, a zinc finger motif, and a C-terminal domain. Based on the structure of RadD and other SF2 proteins, we then built a model of the RedD-ATP complex.     

Structure of the Human TELO2-TTI1-TTI2 Complex

Phosphatidylinositol 3-kinase-related protein kinases (PIKKs) play critical roles in various metabolic pathways related to cell proliferation and survival. The TELO2-TTI1-TTI2 (TTT) complex has been proposed to recognize newly synthesized PIKKs and to deliver them to the R2TP complex (RUVBL1-RUVBL2-RPAP3-PIH1D1) and the heat shock protein 90 chaperone, thereby supporting their folding and assembly. Here, we determined the cryo-EM structure of the TTT complex at an average resolution of 4.2 Å. We describe the full-length structures of TTI1 and TELO2, and a partial structure of TTI2. All three proteins form elongated helical repeat structures. TTI1 provides a platform on which TELO2 and TTI2 bind to its central region and C-terminal end, respectively. The TELO2 C-terminal domain (CTD) is required for the interaction with TTI1 and recruitment of Ataxia-telangiectasia mutated (ATM). The N- and C-terminal segments of TTI1 recognize the FRAP-ATM-TRRAP (FAT) domain and the N-terminal HEAT repeats of ATM, respectively. The TELO2 CTD and TTI1 N- and C-terminal segments are required for cell survival in response to ionizing radiation.     

The role of lipid second messengers in aldosterone synthesis and secretion

Second messengers are small rapidly diffusing molecules or ions that relay signals between receptors and effector proteins to produce a physiological effect. Lipid messengers constitute one of the four major classes of second messengers. The hydrolysis of two main classes of lipids, glycerophospholipids and sphingolipids, generate parallel profiles of lipid second messengers: phosphatidic acid (PA), diacylglycerol (DAG), and lysophosphatidic acid versus ceramide, ceramide-1-phosphate, sphingosine, and sphingosine-1-phosphate, respectively. In this review, we examine the mechanisms by which these lipid second messengers modulate aldosterone production at multiple levels. Aldosterone is a mineralocorticoid hormone responsible for maintaining fluid volume, electrolyte balance, and blood pressure homeostasis. Primary aldosteronism is a frequent endocrine cause of secondary hypertension. A thorough understanding of the signaling events regulating aldosterone biosynthesis may lead to the identification of novel therapeutic targets. The cumulative evidence in this literature emphasizes the critical roles of PA, DAG, and sphingolipid metabolites in aldosterone synthesis and secretion. However, it also highlights the gaps in our knowledge, such as the preference for phospholipase D-generated PA or DAG, as well as the need for further investigation to elucidate the precise mechanisms by which these lipid second messengers regulate optimal aldosterone production.