Evidence that the human gene for prostate short-chain dehydrogenase/reductase (PSDR1) encodes a novel retinal reductase (RalR1)

All-trans-retinoic acid is a metabolite of vitamin A (all-trans-retinol) that functions as an activating ligand for a family of nuclear retinoic acid receptors. The intracellular levels of retinoic acid in tissues are tightly regulated, although the mechanisms underlying the control of retinoid metabolism at the level of specific enzymes are not completely understood. In this report we present the first characterization of the retinoid substrate specificity of a novel short-chain dehydrogenase/reductase (SDR) encoded by RalR1/PSDR1, a cDNA recently isolated from the human prostate (Lin, B., White, J. T., Ferguson, C., Wang, S., Vessella, R., Bumgarner, R., True, L. D., Hood, L., and Nelson, P. S. (2001) Cancer Res. 61, 1611-1618). We demonstrate that RalR1 exhibits an oxidoreductive catalytic activity toward retinoids, but not steroids, with at least an 800-fold lower apparent K(m) values for NADP+ and NADPH versus NAD+ and NADH as cofactors. The enzyme is approximately 50-fold more efficient for the reduction of all-trans-retinal than for the oxidation of all-trans-retinol. Importantly, RalR1 reduces all-trans-retinal in the presence of a 10-fold molar excess of cellular retinol-binding protein type I, which is believed to sequester all-trans-retinal from nonspecific enzymes. As shown by immunostaining of human prostate and LNCaP cells with monoclonal anti-RalR1 antibodies, the enzyme is highly expressed in the epithelial cell layer of human prostate and localizes to the endoplasmic reticulum. The enzymatic properties and expression pattern of RalR1 in prostate epithelium suggest that it might play a role in the regulation of retinoid homeostasis in human prostate.     

On the mechanism of interaction of organic solvents with the active site of alpha-chymotrypsin

Kinetic behavior of -chymotrypsin in the reaction of hydrolysis of the N-acetyl-L-tyrosine derivatives was investigated in non-denaturing water–dimethylsulfoxide and water–ethanol mixtures. Similar specific interactions between the two solvents and the active site of -chymotrypsin were shown to result in similar kinetic effects. It is proposed that the changes in the active site structure of the enzyme caused by the interaction with the organic solvents (conformational isomer formation) resulted in two parallel processes—acceleration of the acyl-enzyme formation step and a decrease in the deacylation rate. The possible molecular mechanism of this phenomenon and an adequate kinetic model describing the data are discussed.     

Overproduction of bioactive retinoic acid in cells expressing disease-associated mutants of retinol dehydrogenase 12

Retinol dehydrogenase 12 (RDH12) is an NADP(+)-dependent oxidoreductase that in vitro catalyzes the reduction of all-trans-retinaldehyde to all-trans-retinol or the oxidation of retinol to retinaldehyde depending on substrate and cofactor availability. Recent studies have linked the mutations in RDH12 to severe early-onset autosomal recessive retinal dystrophy. The biochemical basis of photoreceptor cell death caused by mutations in RDH12 is not clear because the physiological role of RDH12 is not yet fully understood. Here we demonstrate that, although bi-directional in vitro, in living cells, RDH12 acts exclusively as a retinaldehyde reductase, shifting the retinoid homeostasis toward the increased levels of retinol and decreased levels of bioactive retinoic acid. The retinaldehyde reductase activity of RDH12 protects the cells from retinaldehyde-induced cell death, especially at high retinaldehyde concentrations, and this protective effect correlates with the lower levels of retinoic acid in RDH12-expressing cells. Disease-associated mutants of RDH12, T49M and I51N, exhibit significant residual activity in vitro, but are unable to control retinoic acid levels in the cells because of their dramatically reduced affinity for NADPH and much lower protein expression levels. These results suggest that RDH12 acts as a regulator of retinoic acid biosynthesis and protects photoreceptors against overproduction of retinoic acid from all-trans-retinaldehyde, which diffuses into the inner segments of photoreceptors from illuminated rhodopsin. These results provide a novel insight into the mechanism of retinal degeneration associated with mutations in RDH12 and are consistent with the observation that RDH12-null mice are highly susceptible to light-induced retinal apoptosis in cone and rod photoreceptors.     

Modeling of the redox state dynamics in photosystem II of Chlorella pyrenoidosa Chick cells and leaves of spinach and Arabidopsis thaliana from single flash-induced fluorescence quantum yield changes on the 100 ns–10 s time scale

Photosynthesis Research - The time courses of the photosystem II (PSII) redox states were analyzed with a model scheme supposing a fraction of 11–25 % semiquinone (with reduced...     

Changes in localization of cellular vesicular apparatus during differentiation of myoblasts into myotubules in cell culture

A study of changes in intracellular processes in the course of the differentiation of myoblasts into myotubules is of great importance for understanding fundamental problems of cell biology. This primarily concerns the change in the spatial organization of the vacuolar apparatus, which reflects changes in the properties of the membranes, cytoskeleton, and processes of vesicular transport in the course of differentiation. In the present work, the distribution of acid organelles (lysosomes, late endosomes, Golgi cisternae) accumulating acridine orange during the process of the formation of myotubules in the L6J1 cell culture is analyzed. The perinuclear localization of acid cell organelles in myoblasts is shown to be replaced by the diffuse distribution of these organelles throughout the entire volume of the myotubule cytoplasm. The use of lipophilic dyes RH 414 or di-8-ANEPPS enables one to compare the process of formation and dynamics of endocytic vesicles in myoblasts and myotubules. For the analysis of nonspecific endocytosis, we also used the semiconductor nanocrystals, i.e., quantum dots (QDs) conjugated with TAT-peptide, a cell-penetrating peptide. We have shown that the QD-TAT complexes enter into myoblasts via endocytosis. It has been established that QD-TAT complexes penetrate myoblasts, but do not penetrate myotubules, even after incubation for 24 h, which can indicate changes in the properties of the plasma membrane in the process of differentiation of skeletal muscle fibers.     

All Three Domains of the Hepatitis C Virus Nonstructural NS5A Protein Contribute to RNA Binding

The hepatitis C virus (HCV) nonstructural protein NS5A is critical for viral genome replication and is thought to interact directly with both the RNA-dependent RNA polymerase, NS5B, and viral RNA. NS5A consists of three domains which have, as yet, undefined roles in viral replication and assembly. In order to define the regions that mediate the interaction with RNA, specifically the HCV 3′ untranslated region (UTR) positive-strand RNA, constructs of different domain combinations were cloned, bacterially expressed, and purified to homogeneity. Each of these purified proteins was probed for its ability to interact with the 3′ UTR RNA using filter binding and gel electrophoretic mobility shift assays, revealing differences in their RNA binding efficiencies and affinities. A specific interaction between domains I and II of NS5A and the 3′ UTR RNA was identified, suggesting that these are the RNA binding domains of NS5A. Domain III showed low in vitro RNA binding capacity. Filter binding and competition analyses identified differences between NS5A and NS5B in their specificities for defined regions of the 3′ UTR. The preference of NS5A, in contrast to NS5B, for the polypyrimidine tract highlights an aspect of 3′ UTR RNA recognition by NS5A which may play a role in the control or enhancement of HCV genome replication.Hepatitis C virus (HCV) is a human pathogen which chronically infects nearly 3% of the world''s population (36, 37). Persistent infection, in 80% of cases, leads to chronic hepatitis which can progress to liver cirrhosis and, in the worst cases, hepatocellular carcinoma (37). Current therapies lack specificity and efficacy due largely to an incomplete understanding of the complex molecular mechanisms of virus infectivity, RNA replication, and assembly (4, 36). HCV is a member of the Flaviviridae family of enveloped viruses (30), with a positive-sense RNA genome of ∼9.6 kb consisting of a single open reading frame (ORF) that encodes 10 structural and nonstructural viral proteins (3, 16, 25). Cap-independent translation of the ORF (29) yields a large polyprotein of approximately 3,000 amino acid residues that is cleaved co- and posttranslationally by host and viral proteases into 10 mature virus proteins; these cleavage products are ordered from the amino to the carboxy terminus as follows: core (C), envelope proteins 1 and 2 (E1 and E2), p7, nonstructural protein 2 (NS2), NS3, NS4A, NS4B, NS5A, and NS5B (3, 16, 25). At the flanking ends of the genome are two highly conserved untranslated regions (UTRs). The 5′ UTR is highly structured and consists of the internal ribosome entry site (IRES), which is important for the initiation of cap-independent translation of the polyprotein (29). The 3′ UTR consists of a short genotype-specific variable region, a tract of variable length comprising solely pyrimidine residues (predominantly U), and a conserved 98-nucleotide sequence, known as the X region, containing three stem-loops (13, 23) (Fig. ​(Fig.1A).1A). The 3′ UTR is the initiation site for the synthesis of the negative-strand RNA during viral replication (13) and is involved in translational regulation.Open in a separate windowFIG. 1.The HCV 3′ UTR RNA. (A) The positive-strand 3′ UTR consists of three distinct regions, i.e., a short genotype-specific variable region, a polypyrimidine tract [poly(U/UC)] of variable length, and a conserved 98-nucleotide sequence known as the X region containing three stable stem-loops. The predicted structure of the genotype 1b 3′ UTR is shown. (B) Left panel, the integrities of in vitro-transcribed radiolabeled full-length 3′ UTR RNAs of genotypes 1b (nucleotides 9375 to 9595) and 2a (nucleotides 9443 to 9678) and the poly(U/UC) (nucleotides 9406 to 9497) and X region (nucleotides 9498 to 9595) of genotype 1b are shown on denaturing polyacrylamide gels. Right panel, the integrities of in vitro-transcribed radiolabeled RNAs comprising the 3′-terminal NS5B-coding region plus the 3′ UTR RNAs of genotypes 1b (nucleotides 9136 to 9595) and 2a (nucleotides 9204 to 9678) (KL-3′ UTR) are shown on denaturing polyacrylamide gels.HCV RNA replication occurs on membranous structures derived from the endoplasmic reticulum (ER) in a complex that includes host cell factors as well as viral nonstructural proteins, including NS5B, the RNA-dependent RNA polymerase (RdRp) which replicates the viral genome in vivo and in vitro (2, 25, 30). Initiation of the synthesis of the negative-strand RNA is thought to occur upon recognition and specific binding of the NS5B polymerase to the 3′ UTR of the genomic RNA (2, 16, 26). This replication activity and template specificity of NS5B in vivo are dependent, however, on the presence of the other nonstructural proteins, such as the proteases NS2 and NS3, which are required for polyprotein processing and helicase activity, and the multifunctional protein NS5A (16).NS5A is a proline-rich phosphoprotein that is absolutely required for viral replication and is also involved in virus particle assembly (9, 10, 20, 22, 35). Its specific function in the latter process is, however, still unknown. NS5A is membrane associated due to the presence of an N-terminal amphipathic helix that serves as a membrane anchor allowing association with ER-derived membranes (Fig. ​(Fig.2)2) (24, 27). The cytoplasmic portion of NS5A is organized into three domains that are separated by low-complexity sequences (Fig. ​(Fig.2A)2A) (20). The X-ray crystal structure of domain I has revealed that it is a zinc binding domain which forms a homodimer with contacts at the N-terminal ends of the molecules; the resultant large, basic groove at the dimeric interface has been proposed to be involved in RNA binding during viral replication (17, 33). NS5A has also been shown to interact with uridylate and guanylate-rich RNA and to bind to the 3′ ends of the HCV positive- and negative-strand RNAs (8). These observations suggest that NS5A may specifically interact with the large U/G stretches in the IRES of the 5′ UTR, implying a role in HCV translation and genome multiplication, while its interactions with the polypyrimidine tract of the 3′ UTR suggest that NS5A may affect the efficiency of RNA synthesis by NS5B (8, 28, 32). The reported interactions with both flanking regions of the HCV genome imply that NS5A may play a role in the switch between translation and replication that must occur during the viral life cycle (8).Open in a separate windowFIG. 2.Domain structure and expression of HCV NS5A. (A) Schematic diagram of the functional domains of NS5A and design of the constructs used in the study (genotype 1b NS5A protein numbering). The N-terminal amphipathic helix of NS5A (black box) is responsible for the interaction of NS5A with membranes. NS5A is organized into three domains that are separated by low-complexity sequences, indicated by black boxes. The NS5A constructs used all lacked the N-terminal amphipathic helix and were designed to include an N-terminal Strep tag and a C-terminal hexahistidine tag. (B and C) SDS-PAGE and Western blot analysis of the NS5A(ΔAH) and NS5A domain constructs purified by nickel affinity and Streptactin tag affinity chromatography. Coomassie brilliant blue-stained gels and Western blots (WB) using anti-NS5A antibodies for NS5A proteins of genotype 1b strain J4 (B) and genotype 2a strain JFH-1 (C) are shown.Among HCV genotypes, domains II and III are less well conserved than domain I (34). By mutational analysis, domain II, along with domain I, has been attributed to the replicase activity of NS5A (12). Contrastingly, domain III has been shown to be dispensable for RNA replication, and large heterologous insertions and deletions in this region can be tolerated, maintaining RNA replication (34). It has been shown, however, that these insertions and deletions within domain III do have an impact on virus particle assembly, highlighting the critical role of domain III NS5A in the viral life cycle (1, 10). Recent nuclear magnetic resonance (NMR) studies of domains II and III of NS5A revealed that they both adopt a natively unfolded state (6, 14, 15). The high degree of disorder and flexibility observed in these domains may contribute to the promiscuity of NS5A, which has been shown to interact with a variety of biological partners essential for NS5A function and virus persistence (11, 18, 19, 21, 31). In addition, regions within domains I and II of NS5A interact with NS5B, stimulating the in vitro activity of the polymerase and supporting the hypothesis that NS5A has a role in the modulation of RNA replication (28, 32).In this study, we have investigated in detail the RNA binding properties of NS5A. We have mapped the RNA binding regions of NS5A using bacterially expressed deletion constructs of NS5A and have assayed their binding affinity for HCV positive-strand 3′ UTR RNA. In addition, we provide evidence that the RNA binding activity of NS5A is specific and that NS5A interacts preferentially with the polypyrimidine region of the 3′ UTR.     

Cytoskeleton-induced alterations of the lectin activity in winter wheat under cold hardening and abscisic acid (ABA)

The roots and leaves of 7-day seedlings of three winter wheat cultivars differing in frost resistant were used to study changes in lectin activity under cytoskeleton modifiers (DMSO-7%; colchicine-1 m m; oryzalin-15 microm; cytochalasin B-15 microm) of non-hardened (23 degrees C) and hardened (2-3 degrees C, 3-7 day) plants. Plants were grown with ABA (30 microm) or without ABA. Pretreatment with colchicine, oryzalin [inhibitors of microtubules (MT) polymerization], cytochalasin B [inhibitor of microfilament (MF) polymerization] increased the activity of cell wall lectins, although pretreatment with DMSO (stabilizer of microtubules) decreased the activity. Both hardening and ABA decreased the effect of the cytoskeletal modifiers. These results could be explained by the appearance of tolerant MTs with less affinity. It is probable that increase in the activity of cell wall lectins may be the compensatory mechanism which stabilizes the cytoskeleton structure in conditions tending to disrupt it. The genotype with low resistance had higher sensitivity of lectin activity to cytoskeleton modifiers than the frost resistant genotype. The results suggest that leaves have more stable MTs and MFs and stronger MT-MF binding than roots.