Molecular dynamics simulations of human rhinovirus and an antiviral compound.

The human rhinovirus 14 (HRV14) protomer, with or without the antiviral compound WIN 52084s, was simulated using molecular dynamics and rotational symmetry boundary conditions to model the effect of the entire icosahedral capsid. The protein asymmetrical unit, comprising four capsid proteins (VP1, VP2, VP3, and VP4) and two calcium ions, was solvated both on the exterior and the interior to fill the inside of the capsid. The stability of the simulations of this large system (~800 residues and 6,650 water molecules) is comparable to more conventional globular protein simulations. The influence of the antiviral compound on compressibility and positional fluctuations is reported. The compressibility, estimated from the density fluctuations in the region of the binding pocket, was found to be greater with WIN 52084s bound than without the drug, substantiating previous computations on reduced viral systems. An increase in compressibility correlates with an entropically more favorable system. In contrast to the increase in density fluctuations and compressibility, the positional fluctuations decreased dramatically for the external loops of VP1 and the N-terminus of VP3 when WIN 52084s is bound. Most of these VP1 and VP3 loops are found near the fivefold axis, a region whose mobility was not considered in reduced systems, but can be observed with this simulation of the full viral protomer. Altered loop flexibility is consistent with changes in proteolytic sensitivity observed experimentally. Moreover, decreased flexibility in these intraprotomeric loops is noteworthy since the externalization of VP4, part of VP1, and RNA during the uncoating process is thought to involve areas near the fivefold axis. Both the decrease in positional fluctuations at the fivefold axis and the increase in compressibility near the WIN pocket are discussed in relationship to the antiviral activity of stabilizing the virus against uncoating.

Theoretical studies of viral capsid proteins.

Recent results in structural biology and increases in computer power have prompted initial theoretical studies on capsids of nonenveloped icosahedral viruses. The macromolecular assembly of 60 to 180 protein copies into a protein shell results in a structure of considerable size for molecular dynamics simulations. Nonetheless, progress has been made in examining these capsid assemblies from molecular dynamics calculations and kinetic models. The goals of these studies are to understand capsid function and structural properties, including quarternary structural stability, effects of antiviral compounds that bind the capsid and the self-assembly process. The insight that can be gained from the detailed information provided by simulations is demonstrated in studies of human rhinovirus; an entropic basis for the antiviral activity of hydrophobic compounds, predicted from calculated compressibility values, has been corroborated by experimental measurements on poliovirus.

Biosynthesis of endotubin: an apical early endosomal glycoprotein from developing rat intestinal epithelial cells.

Endosomes are the site of sorting of internalized receptors and ligands in all cell types and, in polarized cells, the apical endosomal compartment is involved in the selective transepithelial transport of immunoglobulins and growth factors. The biochemical composition of this specialized compartment remains largely unresolved. We have characterized a glycoprotein, called endotubin, that is located in the apical endosomal tubules of developing rat intestinal epithelial cells. A monoclonal antibody against endotubin recognizes a broad band of 55-60kDa, which upon isoelectric focusing can be resolved into two bands, and a faint band of 140kDa. Metabolic labelling followed by immunoprecipitation indicates that endotubin is synthesized as a 140kDa precursor that is cleaved to the 55-60kDa forms. High pH washing of endosomal membranes removes the 55-60kDa forms from the membrane, whereas the high-molecular-mass form remains membrane associated and appears to be an integral membrane protein. Immunoblotting with a polyclonal antibody against the putative cytoplasmic tail of the protein identifies a 140kDa band and a band of 74kDa, presumably the cleavage product. Immunoprecipitation with antibodies against the 55-60kDa form results in coprecipitation of a 74kDa protein, and immunoprecipitation with antibody against the 74kDa protein results in coprecipitation of the 55-60kDa form. Epitope mapping of the monoclonal antibody binding site supports a proposed type I membrane protein orientation. We propose that endotubin is proteolytically processed into a heterodimer with the 55-60kDa fragment remaining membrane-associated through a non-covalent association with the membrane-bound 74kDa portion of the molecule.

Molecular characterization of an apical early endosomal glycoprotein from developing rat intestinal epithelial cells.

The apical endosomal compartment is thought to be involved in the sorting and selective transport of receptors and ligands across polarized epithelia. To learn about the protein components of this compartment, we have isolated and sequenced a cDNA that encodes a glycoprotein that is located in the apical endosomal tubules of developing rat intestinal epithelial cells. The deduced amino acid sequence predicts a protein of 1216 amino acids with a molecular mass of 133,769 Da. The deduced amino acid sequence together with amino-terminal amino acid sequencing indicate that there is a cleaved 21-amino acid signal sequence at the NH2-terminal portion of the molecule. There is a single hydrophobic region near the carboxyl terminus that has the characteristics of a membrane-spanning domain and a 36-amino acid cytoplasmic tail. We have found that the major form of this protein in intestinal epithelial cells has a molecular mass of 55-60 kDa, which is significantly smaller than the size predicted from the cDNA sequence, suggesting that the protein is synthesized as a large precursor and processed to the smaller form. The smaller form remains associated with the membrane, however, possibly through noncovalent association with the transmembrane portion of the molecule or with another membrane protein. The extracytoplasmic domain is cysteine-rich, with three cysteine-rich repeats that are similar to cysteine repeats present in several receptor proteins. However, there is no other significant similarity to other proteins in the GenBank. The cytoplasmic tail contains a possible internalization motif and several consensus motifs for serine/threonine kinases. Northern blot analysis suggests a single abundant message, and Southern blot analysis is consistent with a single gene and the absence of pseudogenes for this unique endosomal protein.

High-affinity androgen binding and androgenic regulation of alpha 1(I)-procollagen and transforming growth factor-beta steady state messenger ribonucleic acid levels in human osteoblast-like osteosarcoma cells.

Clinical observations have demonstrated a positive effect of estrogens and androgens on the maintenance of structural bone integrity. This study examines the direct effects of androgenic hormones on the osteoblast-like human osteosarcoma cell line, HOS TE85. Employing radiolabeled dihydrotestosterone (DHT), 2800 saturable, high-affinity (dissociation constant = 0.66 nM) androgen binding sites were detected per HOS TE85 cell. Androgen binding was specific in that DHT and testosterone (T) displayed significantly greater competition than the progestins, progesterone and medroxyprogesterone. The expression of androgen receptors in HOS TE85 cells was further substantiated by Northern analysis. A human androgen receptor complementary DNA probe revealed a 9.5 kilobase transcript which corresponds to the predominant human androgen receptor transcript detected in human male reproductive tissues. Androgens were also found to elicit biological responses in HOS TE85 cells. Physiological concentrations of DHT and T decreased HOS TE85 cell proliferation as assessed by cell count. This finding suggests that DHT may also play a role in osteoblast differentiation. In support of this hypothesis, treatment with T (24 h, 10 nM) enhanced the abundance of both alpha 1(I)-procollagen messenger RNA (mRNA) (5-fold) and transforming growth factor-beta mRNA (2.2 fold). The nonaromatizable androgen DHT (24 h, 10 nM) elicited an increase in the steady state concentration of alpha 1(I)-procollagen mRNA similar to the increase observed with T treatment. Thus, in addition to the recent discovery of estradiol receptors and estrogenic regulation of HOS TE85 cells, it is now evident that these osteoblast-like osteosarcoma cells also express high affinity androgen binding sites and can respond biologically to androgens.