Real-time visualization of processive myosin 5a-mediated vesicle movement in living astrocytes.

Recycling endosomes in astrocytes show hormone-regulated, actin fiber-dependent delivery to the endosomal sorting pool. Recycling vesicle trafficking was followed in real time using a fusion protein composed of green fluorescent protein coupled to the 29-kDa subunit of the short-lived, membrane-bound enzyme type 2 deiodinase. Primary endosomes budded from the plasma membrane and oscillated near the cell periphery for 1-4 min. The addition of thyroid hormone triggered the processive, centripetal movement of the recycling vesicle in linear bursts at velocities of up to 200 nm/s. Vesicle migration was hormone-specific and blocked by inhibitors of actin polymerization and myosin ATPase. Domain mapping confirmed that the hormone-dependent vesicle-binding domain was located at the C terminus of the motor. In addition, the interruption of normal dimerization of native myosin 5a monomers inactivated vesicle transport, indicating that single-headed myosin 5a motors do not transport cargo in situ. This is the first demonstration of processive hormone-dependent myosin 5a movement in living cells.

Characterization of the subunit structure of the catalytically active type I iodothyronine deiodinase.

Type I iodothyronine deiodinase is a approximately 50-kDa, integral membrane protein that catalyzes the outer ring deiodination of thyroxine. Despite the identification and cloning of a 27-kDa selenoprotein with the catalytic properties of the type I enzyme, the composition and the physical nature of the active deiodinase are unknown. In this report, we use a molecular approach to determine holoenzyme composition, the role of the membrane anchor on enzyme assembly, and the contribution of individual 27-kDa subunits to catalysis. Overexpression of an immunologically unique rat 27-kDa protein in LLC-PK1 cells that contain abundant catalytically active 27-kDa selenoprotein decreased deiodination by approximately 50%, and > 95% of the LLC-PK1 derived 27-kDa selenoprotein was specifically immune precipitated by the anti-rat enzyme antibody. The hybrid enzyme had a molecular mass of 54 kDa and an s(20,w) of approximately 3.5 S indicating that every native 27-kDa selenoprotein partnered with an inert rat 27-kDa subunit in a homodimer. Enzyme assembly did not depend on the presence of the N-terminal membrane anchor of the 27-kDa subunit. Direct visualization of the deiodinase dimer showed that the holoenzyme was sorted to the basolateral plasma membrane of the renal epithelial cell.

Cloning, expression, and functional characterization of the substrate binding subunit of rat type II iodothyronine 5'-deiodinase.

Type II iodothyronine 5'-deiodinase catalyzes the bioactivation of thyroid hormone in the brain. In astrocytes, this approximately 200-kDa, membrane-bound enzyme is composed of at least one p29 subunit, an approximately 60-kDa, cAMP-induced activation protein, and one or more unidentified catalytic subunit(s). Recently, an artificial type II-like selenodeiodinase was engineered by fusing two independent cDNAs together; however, no native type II selenodeiodinase polypeptide is translated in the brain or brown adipose tissue of rats. These data suggest that the native type II 5'-deiodinase in rat brain is unrelated to this artificial selenoprotein. In this report, we describe the cloning of the 29-kDa subunit (p29) of type II 5'-deiodinase from a lambdazapII cDNA library prepared from cAMP-induced astrocytes. The 3.3-kilobase (kb) cDNA encodes an approximately 30-kDa, 277-amino acid long, hydrophobic protein lacking selenocysteine. Northern blot analysis showed that a 3.5-kb p29 mRNA was present in tissues showing type II 5'-deiodinase activity such as brain and cAMP-stimulated astrocytes. Domain-specific, anti-p29 antibodies specifically immunoprecipitated enzyme activity. Overexpression of exogenous p29 or a green fluorescence protein (GFP)-tagged p29 fusion protein led to a >100-fold increase in deiodinating activity in cAMP-stimulated astrocytes, and the increased activity was specifically immunoprecipitated by anti-GFP antibodies. Steady-state reaction kinetics of the enzyme in GFP-tagged p29-expressing astrocytes are identical to those of the native enzyme in brain. Direct injection of replication-deficient Ad5-p29(GFP) virus particles into the cerebral cortex of neonatal rats leads to a approximately 2-fold increase in brain type II 5'-deiodinating activity. These data show 1) that the 3.3-kb p29 cDNA encodes an essential subunit of rat type II iodothyronine 5'-deiodinase and 2) identify the first non-selenocysteine containing subunit of the deiodinase family of enzymes.

The mammalian homolog of the frog type II selenodeiodinase does not encode a functional enzyme in the rat.

Type II iodothyronine deiodinase is a short-lived, membrane-bound enzyme found in rat brain, brown adipose tissue, and cAMP-stimulated astrocytes. Recently, a full-length complementary DNA (cDNA) encoding a 30-kDa, type II-like selenodeiodinase was cloned from frog, and a homologous partial cDNA (rBAT 1.1), containing two in-frame selenocysteine codons (UGA), was isolated from rat brown adipose tissue. Importantly, the rBAT 1.1 cDNA was derived from a 7.5-kb messenger RNA (mRNA) and did not encode a functional selenoenzyne unless an enabling selenocysteine insertion sequence was appended to the presumed coding region and this cDNA. In this study we determined whether the native 7.5-kb SeD2 mRNA in rat tissues programmed the synthesis of the native type II deiodinase using specific antibodies that were raised against the C-terminus of full-length, 30-kDa SeD2 protein and against the catalytic core of SeD2. Direct analysis of the translation products programmed by the native SeD2 mRNA in cAMP-stimulated astrocytes was performed using antisense deoxynucleotides and hybrid selection strategies. (Bu)2cAMP-stimulated rat astrocytes expressed both type II deiodinase activity (approximately 2500 U/mg protein) and contained abundant levels of the 7.5-kb SeD2 mRNA. However, no immunoreactive 30-kDa SeD2 protein was identified by Western analysis, immunoprecipitation, or immunocytochemistry, and the specific C-terminus antiserum failed to immunoprecipitate deiodinase activity from (Bu)2cAMP-stimulated astrocytes, brown adipose tissue or brain. Instead, the native 7.5-kb SeD2 mRNA encoded a 15-kDa protein that terminated at the first UGA codon and contained the catalytically inactive, N-terminal 129 amino acids of SeD2. These data show that the native 7.5-kb SeD2 mRNA in stimulated astrocytes does not encode D2.

Structure-activity relationships of cysteine-lacking pentapeptide derivatives that inhibit ras farnesyltransferase.

Mutational activation of ras has been found in many types of human cancers, including a greater than 50% incidence in colon and about 90% in pancreatic carcinomas. The activity of both native and oncogenic ras proteins requires a series of post-translational processing steps. The first event in this process is the farnesylation of a cysteine residue located in the fourth position from the carboxyl terminus of the ras protein, catalyzed by the enzyme farnesyltransferase (FTase). Inhibitors of FTase are potential candidates for development as antitumor agents. Through a high-volume screening program, the pentapeptide derivative PD083176 (1), Cbz-His-Tyr(OBn)-Ser(OBn)-Trp-DAla-NH2, was identified as an inhibitor of rat brain FTase, with an IC50 of 20 nM. Structure-activity relationships were carried out to determine the importance of the side chain and chirality of each residue. This investigation led to a series of potent FTase inhibitors which lack a cysteine residue as found in the ras peptide substrate. The parent compound (1) inhibited the insulin-induced maturation of Xenopus oocytes (concentration: 5 pmol/oocyte), a process which is dependent on the activation of the ras pathway.

Selenium-regulated translation control of heterologous gene expression: normal function of selenocysteine-substituted gene products.

In eukaryotes, the synthesis of selenoproteins depends on an exogenous supply of selenium, required for synthesis of the novel amino acid, selenocysteine, and on the presence of a "selenium translation element" in the 3' untranslated region of mRNA. The selenium translation element is required to re-interpret the stop codon, UGA, as coding for selenocysteine incorporation and chain elongation. Messenger RNA lacking the selenium translation element and/or an inadequate selenium supply lead to chain termination at the UGA codon. We exploited these properties to provide direct translational control of protein(s) encoded by transfected cDNAs. Selenium-dependent translation of mRNA transcribed from target cDNA was conferred by mutation of an in-frame UGU, coding for cysteine, to UGA, coding for either selenocysteine or termination, then fusing the mutated coding region to a 3' untranslated region containing the selenium translation element of the human cellular glutathione peroxidase gene. In this study, the biological consequences of placing this novel amino acid in the polypeptide chain was examined with two proteins of known function: the rat growth hormone receptor and human thyroid hormone receptor beta 1. UGA (opal) mutant-STE fusion constructs of the cDNAs encoding these two polypeptides showed selenium-dependent expression and their selenoprotein products maintained normal ligand binding and signal transduction. Thus, integration of selenocysteine had little or no consequence on the functional activity of the opal mutants; however, opal mutants were expressed at lower levels than their wild-type counterparts in transient expression assays. The ability to integrate this novel amino acid at predetermined positions in a polypeptide chain provides selenium-dependent translational control to the expression of a wide variety of target genes, allows facile 75Se radioisotopic labeling of the heterologous proteins, and permits site-specific heavy atom substitution.

Structure-activity relationships of the potent combined endothelin-A/endothelin-B receptor antagonist Ac-DDip16-Leu-Asp-Ile-Ile-Trp21: development of endothelin-B receptor selective antagonists.

The endothelins (ETs) are a family of bicyclic 21-amino acid-containing peptides that are highly potent and prolonged vasoconstrictors. The discovery of potent ET antagonists will facilitate the understanding of the physiological and/or pathophysiological role of ET. Structure-activity studies have revealed the importance of the C-terminal hexapeptide (residues 16-21) of ET (His16-Leu17-Asp18-Ile19-Ile20-Trp21) to the development of potent antagonists at both receptor subtypes (ETA and ETB). In particular, it has been shown that Ac-DDip16-Leu-Asp-Ile-Ile-Trp21 (Dip = 3,3-diphenylalanine) has low nanomolar affinity for the two endothelin receptor subtypes and is a functional antagonist of ET activity, both in vitro and in vivo at both receptors. Herein, we will describe the structure-activity relationships of Ac-DDip16-Leu-Asp-Ile-Ile-Trp21 (PD 142893) with a particular emphasis on modifications that lead to enhanced receptor affinity and/or individual receptor subtype selectivity. In particular, we will demonstrate how we utilized PD 142893 to develop ETB receptor selective ligands and the pharmacological differences that exist between species ETB receptors with respect to their affinity for C-terminal hexapeptide antagonists.