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‣ Deficiency of a protein-repair enzyme results in the accumulation of altered proteins, retardation of growth, and fatal seizures in mice

Kim, Edward; Lowenson, Jonathan D.; MacLaren, Duncan C.; Clarke, Steven; Young, Stephen G.
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 10/06/1997 Português
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447.82504%
l-Asparaginyl and l-aspartyl residues in proteins are subject to spontaneous degradation reactions that generate isomerized and racemized aspartyl derivatives. Proteins containing l-isoaspartyl and d-aspartyl residues can have altered structures and diminished biological activity. These residues are recognized by a highly conserved cytosolic enzyme, the protein l-isoaspartate(d-aspartate) O-methyltransferase (EC 2.1.1.77). The enzymatic methyl esterification of these abnormal residues in vitro can lead to their conversion (i.e., repair) to normal l-aspartyl residues and should therefore prevent the accumulation of potentially dysfunctional proteins in vivo as cells and tissues age. Particularly high levels of the repair methyltransferase are present in the brain, although enyzme activity is present in all vertebrate tissues. To define the physiological relevance of this protein-repair pathway and to determine whether deficient protein repair would cause central nervous system dysfunction, we used gene targeting in mouse embryonic stem cells to generate protein l-isoaspartate(d-aspartate) O-methyltransferase-deficient mice. Analyses of tissues from methyltransferase knockout mice revealed a striking accumulation of protein substrates for this enzyme in the cytosolic fraction of brain...

‣ The epitopes for natural polyreactive antibodies are rich in proline

Tchernychev, Boris; Cabilly, Shmuel; Wilchek, Meir
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 10/06/1997 Português
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“Natural” polyreactive antibodies, which bind in a nonspecific manner to a range of biological molecules both of self- and nonself- origin, are normal constituents of serum and are a significant part of the immune repertoire in many species, including humans. Autoantibodies to sTNF-R (the 55-kDa extracellular domain of the human receptor to tumor necrosis factor α) were affinity purified from normal human sera using immobilized sTNF-R. The isolated anti-sTNF-R IgG bound both native and denatured forms of the receptor with low affinity. These antibodies also bound to different proteins and therefore are considered to be polyreactive. We used the anti-sTNF-R antibodies and purified polyreactive antibodies to mannose-specific lectin from garlic (Allium sativum) for screening a peptide library displayed on filamentous M13 phage. After the biopanning procedure, we failed to find epitopes with a consensus sequence; however, we found that proline is the most frequent amino acid in the selected phagotopes. Proline is commonly present at solvent-exposed sites in proteins, such as loops, turns, N-terminal first turn of helix, and random coils. Thus, structures containing proline can serve as conformation-dependent common “public” epitopes for polyreactive natural antibodies. Our findings may be important for understanding polyreactivity in general and for the significance of polyreactive natural antibodies in immunological homeostasis.

‣ Group I allergens of grass pollen as cell wall-loosening agents

Cosgrove, Daniel J.; Bedinger, Patricia; Durachko, Daniel M.
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 10/06/1997 Português
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447.82504%
Group I allergens are the major allergens of grass pollen, but their biological function is unknown. These proteins are shown here to be structurally related to expansins, which are able to induce extension (creep) of plant cell walls. Extracts of maize pollen possess potent expansin-like activity, as measured in wall extension and wall stress-relaxation assays. This activity is selective for grass cell walls and is, at least partly, due to the action of maize group I allergens. We propose that group I allergens facilitate invasion of the pollen tube into the maternal tissues by loosening the cell walls of the grass stigma and style. Additionally, the presence of related mRNAs in vegetative tissues of rice, Arabidopsis, and soybean implies that allergen homologs may function to loosen walls in growing vegetative tissues as well.

‣ The rational design and construction of a cuboidal iron–sulfur protein

Coldren, Christopher D.; Hellinga, Homme W.; Caradonna, John P.
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 24/06/1997 Português
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Rational protein design is an emerging approach for testing general theories of protein chemistry through the creation of new structures and functions. Here we present the first successful introduction by rational design of a [Fe4S4] cuboidal cluster into the hydrophobic core of Escherichia coli thioredoxin, a protein normally devoid of metal centers. Cuboidal [Fe4S4] is one of the stable forms of self-assembled iron–sulfur clusters that are thought to represent some of the earliest evolved biological redox centers. [Fe4S4] clusters have been recruited for use in a variety of proteins whose functions are central to many of the major biochemical processes ranging from simple soluble electron-transfer agents, to membrane-bound components of electron-transfer chains, to electron reservoirs in complex metalloenzymes such as nitrogenase. By situating an [Fe4S4] cluster into a protein environment not previously adapted by evolution we can explore the factors by which their activity is modulated by the protein matrix.

‣ In vitro and in vivo characterization of novel mRNA motifs that bind special elongation factor SelB

Klug, Stefanie J.; Hüttenhofer, Alexander; Kromayer, Matthias; Famulok, Michael
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 24/06/1997 Português
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447.82504%
The special elongation factor SelB of Escherichia coli promotes selenocysteine incorporation into formate dehydrogenases. This is thought to be achieved through simultaneous binding to selenocysteyl-tRNASec and, in the case of formate dehydrogenase H, to an fdhF mRNA hairpin structure 3′ adjacent to the UGA selenocysteine codon. By in vitro selection, novel RNA sequences (“aptamers”), which can interact tightly and specifically with SelB, were isolated from an RNA library. The library was comprised of mutagenized variants of the wild-type fdhF mRNA hairpin. One-half of the selected sequences contained the apical stem–loop of the fdhF mRNA hairpin highly conserved. Some of the aptamers showed deviations in the primary sequence within this region of the wild-type fdhF hairpin motif while still binding with high affinity to SelB. Binding studies performed with truncated versions of SelB revealed that aptamers binding to different sites on the protein have been selected. To dissect SelB binding to the fdhF hairpin from the overall biological function of this complex, four selected aptamers were analyzed in vivo for UGA readthrough in a lacZ fusion construct. Among these, one promoted UGA readthrough in vivo. Three of the aptamers...

‣ Evolution subverting essentiality: Dispensability of the cell attachment Arg-Gly-Asp motif in multiply passaged foot-and-mouth disease virus

Martínez, Miguel A.; Verdaguer, Nuria; Mateu, Mauricio G.; Domingo, Esteban
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 24/06/1997 Português
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Aphthoviruses use a conserved Arg-Gly-Asp triplet for attachment to host cells and this motif is believed to be essential for virus viability. Here we report that this triplet—which is also a widespread motif involved in cell-to-cell adhesion—can become dispensable upon short-term evolution of the virus harboring it. Foot-and-mouth disease virus (FMDV), which was multiply passaged in cell culture, showed an altered repertoire of antigenic variants resistant to a neutralizing monoclonal antibody. The altered repertoire includes variants with substitutions at the Arg-Gly-Asp motif. Mutants lacking this sequence replicated normally in cell culture and were indistinguishable from the parental virus. Studies with individual FMDV clones indicate that amino acid replacements on the capsid surface located around the loop harboring the Arg-Gly-Asp triplet may mediate in the dispensability of this motif. The results show that FMDV quasispecies evolving in a constant biological environment have the capability of rendering totally dispensable a receptor recognition motif previously invariant, and to ensure an alternative pathway for normal viral replication. Thus, variability of highly conserved motifs, even those that viruses have adapted from functional cellular motifs...

‣ The three-dimensional structure of an H-2Ld-peptide complex explains the unique interaction of Ld with beta-2 microglobulin and peptide

Balendiran, Ganesaratnam K.; Solheim, Joyce C.; Young, Aideen C. M.; Hansen, Ted H.; Nathenson, Stanley G.; Sacchettini, James C.
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 24/06/1997 Português
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Solution at 2.5-Å resolution of the three-dimensional structure of H-2Ld with a single nine-residue peptide provides a structural basis for understanding its unique interaction with beta-2 microglobulin (β2m) and peptide. Consistent with the biological data that show an unusually weak association of Ld with β2m, a novel orientation of the α1/α2 domains of Ld relative to β2m results in a dearth of productive contacts compared with other class I proteins. Characteristics of the Ld antigen-binding cleft determine the unique motif of peptides that it binds. Ld has no central anchor residue due to the presence of several bulky side chains in its mid-cleft region. Also, its cleft is significantly more hydrophobic than that of the other class I molecules for which structures are known, resulting in many fewer H-bonds between peptide and cleft residues. The choice of Pro as a consensus anchor at peptide position 2 appears to be related to the hydrophobicity of the B pocket, and to the unique occurrence of Ile (which mirrors Pro in its inability to form H-bonds) at position 63 on the edge of this pocket. Thus, the paucity of stabilizing H-bonds combined with poor complementarity between peptide postion 2 Pro and the B pocket contribute to the weak association between Ld and its peptide antigen. The unique structural interactions of Ld with β2m and peptide could make Ld more suited than other classical class I molecules to play a role in alternative pathways of antigen presentation.

‣ α-Tocopheryl hydroquinone is an efficient multifunctional inhibitor of radical-initiated oxidation of low density lipoprotein lipids

Neužil, Jiří; Witting, Paul K.; Stocker, Roland
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 22/07/1997 Português
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447.82504%
As the oxidation of low density lipoprotein (LDL) lipids may be a key event in atherogenesis, there is interest in antioxidants as potential anti-atherogenic compounds. Here we report that α-tocopheryl hydroquinone (α-TQH2) strongly inhibited or completely prevented the (per)oxidation of ubiquinol-10 (CoQ10H2), α-tocopherol (α-TOH), and both surface and core lipids in LDL exposed to either aqueous or lipophilic peroxyl radicals, Cu2+, soybean lipoxygenase, or the transition metal-containing Ham’s F-10 medium in the absence or presence of human monocyte-derived macrophages. The antioxidant activity of α-TQH2 was superior to that of several other lipophilic hydroquinones, including endogenous CoQ10H2, which is regarded as LDL’s first line of antioxidant defence. At least three independent activities contributed to the antioxidant action of α-TQH2. First, α-TQH2 readily associated with LDL and instantaneously reduced the lipoprotein’s ubiquinone-10 to CoQ10H2, thereby maintaining this antioxidant in its active form. Second, α-TQH2 directly intercepted aqueous peroxyl radicals, as indicated by the increased rate of its consumption with increasing rates of radical production, independent of LDL’s content of CoQ10H2 and α-TOH. Third...

‣ Detection of single-molecule interactions using correlated thermal diffusion

Mehta, A. D.; Finer, J. T.; Spudich, J. A.
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 22/07/1997 Português
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447.82504%
Observation of discrete, single-molecule binding events allows one to bypass assumptions required to infer single-molecule properties from studies of ensembles of molecules. Optically trapped beads and glass microneedles have been applied to detect single-molecule binding events, but it remains difficult to identify signs of binding events given the large displacements induced by thermal forces. Here, we exploit thermal diffusion by using correlation between motion of optically trapped beads attached to both ends of a single actin filament to track binding events of individual myosin molecules. We use correlated diffusion to measure the stiffness of a single myosin molecule and estimate its thermal fluctuation in a poststroke state as comparable in amplitude to the measured stroke distance. The use of correlated diffusion to measure kinetics of single-molecule interactions and the stiffness of the interacting moieties should be applicable to any pair of interacting molecules, and not limited to biological motors.