Biomedical subjects

J Chroboczek

Publications and source records attributed to J Chroboczek.

At least 37 records · Page 2Linked to original sources

PubMed1996

Adenovirus 3 penton dodecahedron exhibits structural changes of the base on fibre binding.

It was recently shown that co-expression of adenovirus type 3 (Ad3) penton base and fibre in the baculovirus system produces dodecahedral particles, as does the expression of the penton base alone. The structure of both of these dodecahedral particles, with and without fibre, has been determined by cryoelectron microscopy and 3-dimensional reconstruction techniques to a resolution of 25 and 20 A, respectively. The general form of the penton base resembles that of the base protein in the recent reconstruction of adenovirus type 2. There is a remarkable difference in the penton base structure with and without the fibre. The five small protuberances on the outer surface of each base move away from the 5-fold axis by approximately 15 A when the fibre is present. These protuberances are of relatively low density and most probably represent a flexible loop possibly containing the RGD site involved in integrin binding. The fibre is apparently bound to the outer surface of the penton base, rather than inserted into it. The fibre is flexible and the shaft contains two distinct globular regions 26 A in diameter. The volume of the inner cavity of the dodecahedron is 350 +/- 100 nm3. This small volume precludes the use of the inner cavity to house genetic information for gene therapy; however, the possibility remains of linking the gene to the dodecahedron surface in the hope that it will be internalized with the dodecahedron.

Adenoviridae↗

PubMed1995

Antigenic sites on the receptor-binding domain of human adenovirus type 2 fiber.

The trimeric fiber of adenovirus type 2 (Ad2) mediates the first stage of virus-cell attachment, and the distal head region of the fiber has been implicated as the receptor-binding domain. To locate regions on the primary polypeptide sequence of the fiber which may be involved in virus-cell interaction, peptide-based epitope mapping was performed using (1) polyclonal antibodies prepared against both native Ad2 fiber and Ad2 head protein expressed in Escherichia coli and (2) 18 monoclonal antibodies prepared against trimeric Ad2 head protein expressed in baculovirus. The approach using polyclonal antibodies revealed eight domains on the primary sequence of the head which contain one or more continuous epitopes. At least two of these regions were also recognized by monoclonal antibodies reacting against both monomeric and trimeric fiber head protein. The majority of monoclonal antibodies which did not recognize Ad2 head-specific peptides in ELISA were also nonreactive against the monomeric form of protein in Western blot, suggesting that their recognition of trimer is due to the existence of as yet undefined discontinuous epitopes or to alterations in monomer configuration. Our results correspond well with the recently published X-ray crystallographic model of Ad5 fiber head (D. Xia, L.J. Henry, R.D. Gerard, and J. Deisenhofer, Structure 2, 1259-1270, 1994), since most antigenic determinants containing linear epitopes mapped to the outer loops or uppermost beta-sheets in this structure. Four of five neutralizing monoclonal antibodies recognized trimer only and none recognized linear peptides. This might suggest that the trimeric form of fiber is necessary for making contact with the receptor(s) and that discontinuous epitopes on the head domain may be involved in fiber-cell interaction.

Adenoviruses, Human↗

PubMed1995

The avian adenovirus penton: two fibres and one base.

The penton capsomer of mammalian adenoviruses consists of a trimeric, long and thin fibre inserted into a pentameric base. The avian adenoviruses possess a penton which presents another symmetry mismatch: each pentameric base is associated with two fibres. Here we have studied the morphology of the penton of CELO virus, an avian adenovirus, and we have determined the sequence of both fibres, one long and one short. The short fibre is probably associated with the base in the same way as the mammalian viral fibres and we will discuss how the long fibre could be attached. The shafts of all known adenovirus fibres consist of a series of 15-residue repeats. The avian virus fibres show a more complicated and less regular shaft repeat structure with single, double and triple repeats. The sequences of the receptor binding (head) domains of both fibres are very different from all other known fibre head domains and very different from each other, suggesting that the two fibres might bind to different receptors. The genome organization of the sequenced region is rather different from that in human adenoviruses. In particular, a region homologous to the human virus E3 region was not found at the position where it normally occurs in the human virus genome.

Amino Acid Sequence↗

PubMed1995

Adenovirus fiber.

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Adenoviridae↗

PubMed1995

Complete amino acid sequence of Proteus mirabilis PR catalase. Occurrence of a methionine sulfone in the close proximity of the active site.

The catalase of Proteus mirabilis PR, a peroxide-resistant (PR) mutant of Proteus mirabilis, binds strongly NADPH, which is a unique property among known bacterial catalases. The enzyme subunit consists of 484 amino acid residues for a mass of 55,647 daltons. The complete amino acid sequence was resolved through the combination of protein sequencing, mass spectrometry, and nucleotide sequencing of a PCR fragment. The sequence obtained was compared with that of other known catalases. Amino acids of the active site are all conserved as well as essential residues involved in NADPH binding. Among the amino acids interacting with the heme, a methionine sulfone was found at position 53, in place of a valine in most other catalases. The origin of oxidation of this methionine is unknown, but the presence of this modification could change iron accessibility by large substrates or inhibitors. This posttranslational modification was also demonstrated in the wild-type P. mirabilis catalase.

Amino Acid Sequence↗

PubMed1995

Human adenovirus serotype 3 (Ad3) and the Ad3 fiber protein bind to a 130-kDa membrane protein on HeLa cells.

The fiber protein of adenovirus mediates the interaction of adenovirus with cell membrane receptors. We have produced the Ad3 fiber protein in the baculovirus expression system. Biochemical, morphological and functional analyses showed that the recombinant fiber was properly folded and functionally competent. The specific binding of Ad3 virus to two HeLa membrane proteins of 130 and 100 kDa was demonstrated with an overlay protein binding assay. In the same assay, Ad3 fiber only recognized the 130-kDa protein. Divalent cations seemed to be important for the interaction of both virus and fiber with these proteins.

Adenoviruses, Human↗

PubMed1994

The penton base of human adenovirus type 3 has the RGD motif.

The gene encoding the penton base of human adenovirus (Ad) type 3 has been sequenced. The resulting amino-acid sequence has an Arg-Gly-Asp (RGD) motif located near its middle in a hydrophilic region. The same motif is found in serotypes 2, 5 and 12. This sequence was found [Wickham et al., Cell 73 (1993) 309-319] to be involved in the internalisation of Ad2 through an interaction with some specific integrins.

Adenoviruses, Human↗

PubMed1994

Cell-binding domain of adenovirus serotype 2 fiber.

The adenovirus fiber appears as a long, thin projection terminated by a knob (head). The fiber consists of a trimeric protein whose head domain is thought to interact with cell receptors. The head part (amino acids 388 to 582) of adenovirus type 2 fiber was produced in a baculovirus expression system. The purified protein was shown to cross-link into trimers. It was very resistant to proteolytic attack and seemed to attain a high degree of compactness. The head domain efficiently inhibited attachment of adenovirus to receptors on the surface of HeLa cells, thereby confirming the hypothesis that the head domain interacts with viral receptors.

Adenoviruses, Human↗

PubMed1993

Cloning, sequencing, and expression in Escherichia coli of the gene coding for malate dehydrogenase of the extremely halophilic archaebacterium Haloarcula marismortui.

The gene coding for the enzyme malate dehydrogenase (MDH) of the extremely halophilic archaebacterium Haloarcula marismortui was isolated and sequenced. The enzyme is composed of 303 amino acids, and its molecular mass is 32,638 Da. The deduced amino acid sequence of the enzyme was found to be more similar to the sequence of L-lactate dehydrogenase (L-LDH) from various sources than to the sequence of other MDHs. The structural gene was cloned in the Escherichia coli expression vector pET11a, and large amounts of a soluble but inactive form of the enzyme were produced upon its induction. Activation of the enzyme was obtained by increasing the salt concentration to 3 M NaCl. The recombinant protein was purified to homogeneity and shown to be indistinguishable from the native enzyme isolated from halobacteria. These findings present the first example of the successful expression of a halobacterial gene coding for a soluble protein in Escherichia coli and its recovery as a functional enzyme. Site-directed mutagenesis was employed to modify Arg100 on the enzyme to Gln. This modification produced an enzyme that has considerably higher specificity for pyruvate (the substrate of L-LDH) than for oxaloacetate (the substrate of MDH). The mutation also caused a modification in the relative activities of the enzyme at different salt concentrations. The greater similarity of the amino acid sequence of the halobacterial MDH to that of L-LDHs than to that of MDHs sheds light on the molecular evolution of these enzymes.

Amino Acid Sequence↗

PubMed1993

Adenovirus type 40 virions contain two distinct fibers.

Human subgroup F adenoviruses, types 40 and 41, have two genes that could specify different fiber proteins, a long fiber with 21 or 22 pseudorepeat motifs in the shaft, and a short fiber with 12 motifs. We show that for adenovirus type 40, both proteins are expressed from separate late regions of the genome. They are incorporated into the virion with only one fiber per penton base, unlike avian adenoviruses which have two fibers extending from each penton. Comparison of known adenovirus fiber sequences suggests that the two fibers in adenovirus 40 and 41 arose through an external recombination event rather than having evolved from each other directly. Two alternative fibers on the virion may imply the existence of two distinct receptors.

Adenoviruses, Human↗

PubMed1992

The sequence of the genome of adenovirus type 5 and its comparison with the genome of adenovirus type 2.

We report the sequence of 7558 nucleotides of the adenovirus type 5 genome. With this sequence and previously published data, the complete sequence of this genome is now available and can be compared with the already known sequence of the adenovirus type 2 genome. These two serotypes belong to the same subgroup and sequence comparison shows 94.7% homology between the two genomes. The differences are not at all randomly distributed. Transitions between C and T and between A and G account in total for 58.3% of the differences and even for 68.6% for the genome devoid of the fiber and the hexon genes (instead of 33% expected for an equal probability of changes). In the fiber gene the transitions account for 47% of the differences. The detailed analysis of the nucleotide substitution between the two genomes suggests that the Ad2 genome could derive from that of Ad5 one, with the exception of the fiber gene which is likely to be present in Ad2 genome as a result of genetic recombination. The homology between the amino acids sequences of the structural proteins varies from 100% (proteins pVII and IX) to only 69.2% for the fiber.

Adenoviruses, Human↗

PubMed1991

Human adenovirus serotype 3 fiber protein. Comparison of native and recombinant proteins.

We were able to isolate viral fiber and penton from Ad3-infected KB cells using for their detection antibodies obtained against recombinant Ad3 fiber. The native material was examined by electron microscopy and the characteristic fiber shape of a shaft terminated by a globular head was observed. The native fiber was compared with two recombinant fibers synthesized in Escherichia coli cells. One, the Ad3 fiber protein expressed in E. coli with a 14-amino acid NH2-terminal fusion peptide, under the control of the T7 promoter has been described previously. The second is a recombinant Ad3 fiber without the fusion peptide (recAd3fib), expressed in the same system. As with the fusion protein recAd3fib was found to be insoluble upon expression. It was solubilized in 6 M urea and the gradual removal of urea during the purification cycle led to a soluble preparation. Biochemical and biophysical studies show that, similarly to fusion fiber, recAd3fib self-assembles as trimers in prokaryotic cells. Electron microscopy shows that, whereas the fusion fiber consists of a population of heterogeneous particles, recAd3fib has the characteristic morphology and size of the Ad3 trimeric native fiber. Small angle neutron scattering gives a molecular weight consistent with a trimeric fiber and a radius of gyration consistent with the dimensions derived from electron microscopy. These results suggest that the fusion peptide at the NH2 terminus prevents correct protein folding. They also indicate that after solubilization with urea and subsequent renaturation a correctly folded eukaryotic oligomeric protein can be produced in E. coli.

Adenoviridae↗

PubMed1991

The interaction of wheat germ tyrosyl-tRNA synthetase and the tRNA-like end of brome mosaic virus RNA has no effect on in vitro viral protein synthesis and on in vitro encapsidation.

The effect of aminoacylation of the tRNA-like end of brome mosaic virus RNA during in vitro protein synthesis and in vitro viral encapsidation was investigated. The components of the homologous system were: BMV RNA, wheat germ cell-free protein synthesizing system and pure tyrosyl-tRNA synthetase from wheat germ. During in vitro protein synthesis directed with tyrosylated as well as non-tyrosylated BMV RNA, no differences were observed in the amount and in the class of polypeptides formed neither in the velocity of the translation reaction. Excess active TyrRS was added during in vitro translation, without modifying the translation efficiency. BMV RNA and active TyrRS were preincubated prior to translation in order to interact without the translation system components and then subjected to translation in vitro. Similar results were obtained when BMV RNA was preincubated with inactive TyrRS or BSA. These results indicate that the aminoacylation of BMV RNA has no pronounced effect on viral protein synthesis in vitro. During BMV RNA encapsidation either tyrosylated or non-tyrosylated BMV RNA 4 could be encapsidated in a similar way.

Capsid↗

PubMed1990

Adenovirus serotype 3 fibre protein is expressed as a trimer in Escherichia coli.

The adenovirus serotype 3 (Ad3) fibre has been expressed in Escherichia coli as an insoluble protein. The protein was solubilized by extraction with urea. Slow removal of urea during the purification procedure resulted in a soluble Ad3 fibre preparation. Polyacrylamide gel analysis of the purified fibre protein, as well as cross-linking experiments performed on cellular debris of expressing cells, suggest that the recombinant Ad3 fibre self-assembles as a trimer from identical polypeptide chains. Gel filtration gave the same exclusion volume for the purified recombinant fibre and for the native fibre in the protein mixture extracted from the Ad3-infected cells. The recombinant fibre was partially resistant to proteolytic degradation, suggesting a folded structure.

Adenoviridae↗

PubMed1990

Purification and characterization of wild-type and ts 112 mutant protein IIIa of human adenovirus 2 expressed in Escherichia coli.

The expression of the protein IIIa gene from human adenovirus type 2 (Ad2) in Escherichia coli has been described previously (M. Cuillel, M. Milleville, and J. C. D'Halluin, 1987, Gene 55, 295-301). The same construct has now been used to express a protein IIIa gene from an Ad2 mutant ts 112 whose functional mutation occurs in this gene. The mutant virus is defective at nonpermissive temperatures in the latest stage of virus maturation. Both the wild-type and ts 112 recombinant proteins are produced in E. coli in an insoluble form, but are readily solubilized in urea. They have the same molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), they sediment as a monomeric species in sucrose gradient centrifugation, and proteolytic digestion reveals a similar pattern for both proteins. Hydrodynamic studies and electron microscopy show that both proteins have an elongated shape, which can be approximated to a cylinder of 20 nm in length and 2.8 nm in diameter. The only well-established difference between the mutant and the wild-type recombinant protein is the higher solubility of the mutant.

Adenoviruses, Human↗

PubMed1990

Structural proteins of adenovirus. Expression in Escherichia coli.

The fiber proteins of adenovirus serotype 2 Ad2 and serotype 3 Ad3 and structural protein IIIa of wild type Ad2 and Ad2 ts 112 mutant were cloned and expressed in E. coli. For the expression of both fiber proteins a gene expression system based on bacteriophage T7 RNA polymerase was used. The expressed proteins constituted 1-3% of total host cell protein. Both proteins were insoluble and inclusion bodies were observed. The proteins could be purified from cellular debris by extraction with 6 M urea followed by chromatography in the presence of diminishing concentration of urea. The folding of recombinant fiber proteins was assessed by sensitivity to proteases and gel filtration. Both proteins were synthetized as trimers. Ad2 recombinant fiber has a much less compact structure than native Ad2 fiber, since on gel filtration it is excluded before the native fiber. It is also much more sensitive to chymotrypsin digestion than the native protein. Contrary to that, Ad3 recombinant fiber is much less sensitive to proteolytic cleavage and on gel filtration has the same exclusion volume as the trimeric native fiber of Ad3.

Adenoviruses, Human↗

PubMed1989

Synthesis of human adenovirus type 2 fiber protein in Escherichia coli cells.

We have cloned and expressed in Escherichia coli the gene encoding the trimeric fiber protein of human adenovirus type 2. A gene expression system based on bacteriophage T7 RNA polymerase was used. Optimal gene expression was obtained with 1-h induction, at a temperature of 30 degrees C. The synthesized protein constituted about 1% of total host-cell protein. During induction, the growth of bacteria carrying the plasmid containing the fiber gene, was retarded compared with that of bacteria carrying the plasmid without the fiber gene. This toxic effect of fiber protein on bacterial hosts could be diminished by addition of glucose to the medium and by maintaining the pH above 7, thus improving the yield of recombinant fiber protein. The fiber protein produced in E. coli is stable during the course of induction. It is insoluble in buffers at physiological pH, in various salt solutions, and in the presence of nonionic detergents. It can be solubilized in 1% sodium dodecyl sulfate or in urea solutions above 2 M. There are indications that recombinant fiber trimerizes spontaneously, since after the removal of urea by dialysis at pH 8, recombinant fibers runs similarly to native trimeric fiber, on nondenaturing polyacrylamide gels. This trimer has, however, a less compact structure than native Ad2 fiber, since during gel filtration recombinant protein is excluded before native protein. It is also more sensitive to chymotrypsin digestion than native fiber.

Adenoviruses, Human↗

PubMed1988

Tyrosyl-tRNA synthetase from wheat germ.

Tyrosyl-tRNA synthetase (TyrRS) was purified 5,000-fold from wheat germ extract by ultracentrifugation, precipitation with ammonium acetate, and column chromatography. Under denaturing conditions the enzyme ran as a single band on SDS-polyacrylamide electrophoresis with an apparent Mr of 55,000. The native molecular weight determined by gel filtration was 110,000, suggesting a quaternary structure of an alpha 2 type for native TyrRS. Purified enzyme activity, based on the aminoacylation reaction, was studied in terms of Mg2+, ATP, pH, and KCl dependence. Optimum concentrations were 6 mM Mg2+, 4 mM ATP, and 200 mM KCl at pH 8. The Km values for ATP, tyrosine, and tRNA were 40, 3.3, and 1.5 microM, respectively. The instability of the TyrRS activity and the methods used for stabilizing it are discussed. In wheat germ extract we found a second tyrosylating activity that works with Escherichia coli tRNA, but not with wheat germ tRNA. We believe that this enzyme is the mitochondrial tyrosyl-tRNA synthetase of wheat germ.

Adenosine Triphosphate↗