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1.
The vacuolating toxin VacA, released by Helicobacter pylori, is an important virulence factor in the pathogenesis of gastritis and gastroduodenal ulcers. VacA contains two subunits: The p58 subunit mediates entry into target cells, and the p34 subunit mediates targeting to mitochondria and is essential for toxicity. In this study we found that targeting to mitochondria is dependent on a unique signal sequence of 32 uncharged amino acid residues at the p34 N-terminus. Mitochondrial import of p34 is mediated by the import receptor Tom20 and the import channel of the outer membrane TOM complex, leading to insertion of p34 into the mitochondrial inner membrane. p34 assembles in homo-hexamers of extraordinary high stability. CD spectra of the purified protein indicate a content of >40% β-strands, similar to pore-forming β-barrel proteins. p34 forms an anion channel with a conductivity of about 12 pS in 1.5 M KCl buffer. Oligomerization and channel formation are independent both of the 32 uncharged N-terminal residues and of the p58 subunit of the toxin. The conductivity is efficiently blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a reagent known to inhibit VacA-mediated apoptosis. We conclude that p34 essentially acts as a small pore-forming toxin, targeted to the mitochondrial inner membrane by a special hydrophobic N-terminal signal.  相似文献   
2.
Motz M  Pääbo S  Kilger C 《BioTechniques》2000,29(2):268-270
A common problem in automated DNA sequencing when applying the Sanger chain termination method is ambiguous base calling caused by band compressions. Band compressions are caused by anomalies in the migration behavior of certain DNA fragments in the polyacrylamide gel because of intramolecular base pairing between guanine and cytosine residues. To reduce such undesired secondary structures, several modifications of the sequencing reaction parameters have been performed previously. Here, we have applied mixtures of the nucleotide analogs 7-deaza-dGTP and dITP instead of dGTP in the cycle sequencing reaction and in combination with varying buffer conditions. Band compressions were particularly well resolved, and reading length was optimal when a ratio of 7-deaza-dGTP:dITP of 4:1 was used in the in vitro DNA synthesis with AmpliTaq FS DNA polymerase. We conclude that the incorporation of both nucleotide analogs at these particular ratios leads to heterogeneous DNA chains that result in a reduction or elimination of intramolecular base pairing and thus a higher accuracy in the base assignment.  相似文献   
3.
The γ-subunit is required for the assembly of ATP synthases and plays a crucial role in their catalytic activity. We stepwise shortened the N-terminus and the C-terminus of the γ-subunit in the mitochondrial ATP synthase of yeast and investigated the relevance of these segments in the assembly of the enzyme and in the growth of the cells. We found that a deletion of 9 residues at the N-terminus or 20 residues at the C-terminus still allowed efficient import of the subunit into mitochondria; however, the assembly of both monomeric and dimeric holoenzymes was partially impaired. γ-Subunits lacking 13 N-terminal residues or 30 C-terminal residues were not assembled. Yeast strains expressing either of the truncated γ-subunits did not grow on non-fermentable carbon sources, indicating that non-assembled parts of the ATP synthase accumulated and impaired essential mitochondrial functions.  相似文献   
4.
Thermostable DNA polymerases are an important tool in molecular biology. To exploit the archaeal repertoire of proteins involved in DNA replication for use in PCR, we elucidated the network of proteins implicated in this process in Archaeoglobus fulgidus. To this end, we performed extensive yeast two-hybrid screens using putative archaeal replication factors as starting points. This approach yielded a protein network involving 30 proteins potentially implicated in archaeal DNA replication including several novel factors. Based on these results, we were able to improve PCR reactions catalyzed by archaeal DNA polymerases by supplementing the reaction with predicted polymerase co-factors. In this approach we concentrated on the archaeal proliferating cell nuclear antigen (PCNA) homologue. This protein is known to encircle DNA as a ring in eukaryotes, tethering other proteins to DNA. Indeed, addition of A. fulgidus PCNA resulted in marked stimulation of PCR product generation. The PCNA-binding domain was determined, and a hybrid DNA polymerase was constructed by grafting this domain onto the classical PCR enzyme from Thermus aquaticus, Taq DNA polymerase. Addition of PCNA to PCR reactions catalyzed by the fusion protein greatly stimulated product generation, most likely by tethering the enzyme to DNA. This sliding clamp-induced increase of PCR performance implies a promising novel micromechanical principle for the development of PCR enzymes with enhanced processivity.  相似文献   
5.
6.
Essential hypertension, with pressure overload leading to left ventricular hypertrophy, often results in coronary artery disease and congestive heart failure. The spontaneously hypertensive rat (SHR) is an attractive model for studying the effects of long-term antihypertensive therapy on the contractile properties of the myocardium. In this study we investigated differences in mechanical and biochemical characteristics of papillary muscles from SHR and normal (Wistar-Kyoto [WKY]) rats as a function of age and treatment. We found that the rate of delayed force redevelopment after rapid stretch was less in SHR than in WKY in every age group studied, even at 2 wk of age, before hypertension was evident in the SHR. In the treated SHR, blood pressure was lower, hypertrophy was reduced and the rate of delayed force redevelopment was increased compared with the untreated SHR. Finally, the pattern of myosin isoenzymes was different in treated than in untreated SHR, being shifted to more of the fast V1 and less of the slow V3 isomyosin. We conclude that long-term antihypertensive therapy not only prevents the development of left ventricular hypertrophy, but may do so by preventing the shift in myosin isoenzyme pattern normally found in hearts subjected to a long-term pressure overload.  相似文献   
7.
Mitochondrial dysfunction and oxidative damage in parkin-deficient mice   总被引:18,自引:0,他引:18  
Loss-of-function mutations in parkin are the predominant cause of familial Parkinson's disease. We previously reported that parkin-/- mice exhibit nigrostriatal deficits in the absence of nigral degeneration. Parkin has been shown to function as an E3 ubiquitin ligase. Loss of parkin function, therefore, has been hypothesized to cause nigral degeneration via an aberrant accumulation of its substrates. Here we employed a proteomic approach to determine whether loss of parkin function results in alterations in abundance and/or modification of proteins in the ventral midbrain of parkin-/- mice. Two-dimensional gel electrophoresis followed by mass spectrometry revealed decreased abundance of a number of proteins involved in mitochondrial function or oxidative stress. Consistent with reductions in several subunits of complexes I and IV, functional assays showed reductions in respiratory capacity of striatal mitochondria isolated from parkin-/- mice. Electron microscopic analysis revealed no gross morphological abnormalities in striatal mitochondria of parkin-/- mice. In addition, parkin-/- mice showed a delayed rate of weight gain, suggesting broader metabolic abnormalities. Accompanying these deficits in mitochondrial function, parkin-/- mice also exhibited decreased levels of proteins involved in protection from oxidative stress. Consistent with these findings, parkin-/- mice showed decreased serum antioxidant capacity and increased protein and lipid peroxidation. The combination of proteomic, genetic, and physiological analyses reveal an essential role for parkin in the regulation of mitochondrial function and provide the first direct evidence of mitochondrial dysfunction and oxidative damage in the absence of nigral degeneration in a genetic mouse model of Parkinson's disease.  相似文献   
8.
Despite a profusion of popular misinformation about the left brain and right brain, there are functional differences between the left and right cerebral hemispheres in humans. Evidence from split-brain patients, individuals with unilateral brain damage, and neuroimaging studies suggest that each hemisphere may be specialized for certain cognitive processes. One way to easily explore these hemispheric asymmetries is with the divided visual field technique, where visual stimuli are presented on either the left or right side of the visual field and task performance is compared between these two conditions; any behavioral differences between the left and right visual fields may be interpreted as evidence for functional asymmetries between the left and right cerebral hemispheres. We developed a simple software package that implements the divided visual field technique, called the Lateralizer, and introduced this experimental approach as a problem-based learning module in a lower-division research methods course. Second-year undergraduate students used the Lateralizer to experimentally challenge and explore theories of the differences between the left and right cerebral hemispheres. Measured learning outcomes after active exploration with the Lateralizer, including new knowledge of brain anatomy and connectivity, were on par with those observed in an upper-division lecture course. Moreover, the project added to the students' research skill sets and seemed to foster an appreciation of the link between brain anatomy and function.  相似文献   
9.
The bcl-2 gene encodes a 26kDa protein which functions as a central regulator of apoptosis. Here we investigated the pathway of Bcl-2alpha into the mitochondrial outer membrane using the yeast Saccharomyces cerevisiae as a model organism. We found that interactions of Bcl-2alpha with the mitochondrial import receptor Tom20 are dependent on two positively charged lysine residues in the immediate vicinity of the carboxy-terminal hydrophobic membrane anchor. The targeting function of these residues is independent of Tom22. Subsequent insertion of Bcl-2alpha into the mitochondrial outer membrane does not require Tom5 or Tom40, indicating that Bcl-2alpha bypasses the general import pore (GIP). Bcl-2alpha shows a unique pattern of interactions with the components of the mitochondrial TOM complex, demonstrating that at least two different pathways lead from the import receptor Tom20 into the mitochondrial outer membrane.  相似文献   
10.
Berberine bridge enzyme (BBE) is a member of the recently discovered family of bicovalently flavinylated proteins. In this group of enzymes, the FAD cofactor is linked via its 8α-methyl group and the C-6 atom to conserved histidine and cysteine residues, His-104 and Cys-166 for BBE, respectively. 6-S-Cysteinylation has recently been shown to have a significant influence on the redox potential of the flavin cofactor; however, 8α-histidylation evaded a closer characterization due to extremely low expression levels upon substitution. Co-overexpression of protein disulfide isomerase improved expression levels and allowed isolation and purification of the H104A protein variant. To gain more insight into the functional role of the unusual dual mode of cofactor attachment, we solved the x-ray crystal structures of two mutant proteins, H104A and C166A BBE, each lacking one of the covalent linkages. Information from a structure of wild type enzyme in complex with the product of the catalyzed reaction is combined with the kinetic and structural characterization of the protein variants to demonstrate the importance of the bicovalent linkage for substrate binding and efficient oxidation. In addition, the redox potential of the flavin cofactor is enhanced additively by the dual mode of cofactor attachment. The reduced level of expression for the H104A mutant protein and the difficulty of isolating even small amounts of the protein variant with both linkages removed (H104A-C166A) also points toward a possible role of covalent flavinylation during protein folding.Since the discovery of the first known example of a covalent bond between a flavin cofactor and an amino acid side chain occurring in enzymes in the 1950s (1), a number of different types of linkages have been identified: 8α-histidylation (either to N1 or to N3), 8α-O-tyrosylation, 8α-S-cysteinylation, and 6-S-cysteinylation. For current reviews relating to these modes of flavin attachment, see Refs. 2 and 3. Recently, another way of covalent tethering of FAD to proteins was discovered in x-ray crystallographic studies on glucooligosaccharide oxidase (GOOX)4 from Acremonium strictum (4). The mode of flavin linkage observed in this case employs both 8α-histidylation and 6-S-cysteinylation to form a bicovalently attached cofactor. Representative members of all these groups have been studied in detail, and several explanations for the role of the covalent flavinylation have been put forward. Some of the suggestions tend to be rather specific for the system being studied, e.g. prevention of cofactor inactivation at the C-6 position for trimethylamine dehydrogenase (5) or facilitation of electron transfer from the flavin to the cytochrome subunit for p-cresol methylhydroxylase (6). Other explanations including the increase of the flavin redox potential due to the covalent linkage (79) and the prevention of cofactor dissociation (10, 11) were found for several enzymes also harboring different types of cofactor attachments. Taking into account that protein stability (12) and optimal binding of substrate molecules (11, 13) are also positively influenced by covalent tethering of the flavin, one might speculate that no generally applicable explanation for the covalent attachment of flavins to proteins exists. Therefore, it seems likely that the large variety of systems operating with one of the above mentioned modes of cofactor tethering might have evolved to also adapt to a diversity of enzymatic challenges.Berberine bridge enzyme (BBE) from Eschscholzia californica is a plant enzyme involved in alkaloid biosynthesis, catalyzing the challenging oxidative cyclization of (S)-reticuline to (S)-scoulerine (Scheme 1). This enzyme was recently shown to belong to the group of flavoenzymes with a bicovalently attached FAD (14). After the discovery of this unusual mode of linkage in the crystal structure of GOOX (4), several members of this group, all belonging to the vanillyl-alcohol oxidase family (15), were identified by biochemical methods (1618) and also structural studies (19). Because some of the suggested benefits of a covalent cofactor attachment can easily be brought about by a single linkage, e.g. prevention of cofactor dissociation or stabilization of the tertiary structure, the two amino acids attached to FAD might have different and individual functions as well as an additive effect on physicochemical properties such as redox potentials or substrate binding and oxidation. To elucidate the relative importance for the overall enzymatic functioning of members of this group, more detailed studies have been performed on GOOX (11), chito-oligosaccharide oxidase (ChitO) from Fusarium graminearum (17), and BBE (20). Common results of these analyses show that the bicovalent FAD has a redox potential of about +130 mV, which is among the highest potentials reported for flavoenzymes. Replacement of one of the amino acids involved in anchoring of the cofactor generally reduces the rate of cofactor reduction and the steady-state turnover rate, but whether this can be directly linked to reduced redox potentials of these mutant proteins has been under debate (11).Open in a separate windowSCHEME 1.Overall reaction catalyzed by BBE.To address these issues further, we report the expression of the H104A mutant protein of BBE. A biochemical characterization of this protein variant with respect to the redox potential, transient kinetics, and steady-state analysis is combined with the structural analysis of both the H104A and the C166A mutant proteins. In addition, a structure of wild type (WT) BBE in complex with the product of the enzyme-catalyzed reaction is presented, which provides further insights toward the involvement of active site amino acids during the course of the reaction. Together with the recently reported x-ray crystal structure of WT BBE with and without substrate bound (21) and the biochemical characterization of the C166A mutant protein (20), these results provide interesting insights into the role of bicovalent FAD attachment in enzymes.  相似文献   
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