Disulfide bond catalysts in Escherichia coli.
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Alkaline Phosphatase↗
Biomedical subjects
T Zander
Publications and source records attributed to T Zander.
Detection of identical Hodgkin-Reed Sternberg cell specific immunoglobulin gene rearrangements in a patient with Hodgkin's disease of mixed cellularity subtype at primary diagnosis and in relapse two and a half years later.
BACKGROUND: The malignant nature of Hodgkin-Reed Sternberg (H-RS) cells has been questioned due to their scarcity in lymphoma tissues. Recently, using micromanipulation of H-RS cells and single cell PCR evidence was obtained that H-RS cells represent a clonal B-cell population. In these studies H-RS cells were isolated from each one lymph node for a given case. In classical Hodgkin's disease (HD) it thus could not be ruled out that H-RS cell clonality reflected a locally restricted clonal proliferation. We analysed biopsy specimens from a patient suffering from HD for the presence of clonally related H-RS cells at primary diagnosis and during relapse of the disease. MATERIALS AND METHODS: In 1994 the H-RS cell line L1236 was generated from the peripheral blood of a patient suffering from a disseminating relapse of HD of mixed cellularity subtype. The patient had relapsed despite intensive treatment including high dose chemotherapy and autologous bone marrow transplantation. The clonal identity of this cell line with H-RS cells in situ was proven by amplifying identical Ig gene rearrangements of the cell line as well as of single H-RS cells picked from the patients bone marrow. Primers covering the CDR3 region were chosen from the H-RS cell specific VH1 gene rearrangement to detect H-RS cells of the identical clone by amplifying the rearranged VH1 genes in tissue samples obtained during disseminating relapsing disease and at primary diagnosis of HD in 1991. RESULTS: The H-RS cell specific DNA sequence was detected in all affected tissues analysed including the cervical lymph node which has been exstirpated at primary diagnosis. CONCLUSION: This finding indicates the existence of a clonal H-RS cell population during the first manifestation of HD and persistence and dissemination of this clone despite aggressive treatment. Thus, in the described case the malignant nature of H-RS cells defined by dissemination and recurrence of the identical H-RS cell clone in relapsing disease is proven.
Adult↗
Clonal relapse in Hodgkin's disease.
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B-Lymphocytes↗
The uncharged surface features surrounding the active site of Escherichia coli DsbA are conserved and are implicated in peptide binding.
DsbA is a protein-folding catalyst from the periplasm of Escherichia coli that interacts with newly translocated polypeptide substrate and catalyzes the formation of disulfide bonds in these secreted proteins. The precise nature of the interaction between DsbA and unfolded substrate is not known. Here, we give a detailed analysis of the DsbA crystal structure, now refined to 1.7 A, and present a proposal for its interaction with peptide. The crystal structure of DsbA implies flexibility between the thioredoxin and helical domains that may be an important feature for the disulfide transfer reaction. A hinge point for domain motion is identified-the type IV beta-turn Phe 63-Met 64-Gly 65-Gly 66, which connects the two domains. Three unique features on the active site surface of the DsbA molecule-a groove, hydrophobic pocket, and hydrophobic patch-form an extensive uncharged surface surrounding the active-site disulfide. Residues that contribute to these surface features are shown to be generally conserved in eight DsbA homologues. Furthermore, the residues immediately surrounding the active-site disulfide are uncharged in all nine DsbA proteins. A model for DsbA-peptide interaction has been derived from the structure of a human thioredoxin:peptide complex. This shows that peptide could interact with DsbA in a manner similar to that with thioredoxin. The active-site disulfide and all three surrounding uncharged surface features of DsbA could, in principle, participate in the binding or stabilization of peptide.
Amino Acid Sequence↗
Structural analysis of three His32 mutants of DsbA: support for an electrostatic role of His32 in DsbA stability.
DsbA, a 21-kDa protein from Escherichia coli, is a potent oxidizing disulfide catalyst required for disulfide bond formation in secreted proteins. The active site of DsbA is similar to that of mammalian protein disulfide isomerases, and includes a reversible disulfide bond formed from cysteines separated by two residues (Cys30-Pro31-His32-Cys33). Unlike most protein disulfides, the active-site disulfide of DsbA is highly reactive and the oxidized form of DsbA is much less stable than the reduced form at physiological pH. His32, one of the two residues between the active-site cysteines, is critical to the oxidizing power of DsbA and to the relative instability of the protein in the oxidized form. Mutation of this single residue to tyrosine, serine, or leucine results in a significant increase in stability (of approximately 5-7 kcal/mol) of the oxidized His32 variants relative to the oxidized wild-type protein. Despite the dramatic changes in stability, the structures of all three oxidized DsbA His32 variants are very similar to the wild-type oxidized structure, including conservation of solvent atoms near the active-site residue, Cys30. These results show that the His32 residue does not exert a conformational effect on the structure of DsbA. The destabilizing effect of His32 on oxidized DsbA is therefore most likely electrostatic in nature.
Binding Sites↗
Why is DsbA such an oxidizing disulfide catalyst?
DsbA, a member of the thioredoxin family of disulfide oxidoreductases, acts in catalyzing disulfide bond formation by donating its disulfide to newly translocated proteins. We have found that the two central residues within the active site Cys-30-Pro-31-His-32-Cys-33 motif are critical in determining the exceptional oxidizing power of DsbA. Mutations that change these two residues can alter the equilibrium oxidation potential of DsbA by more than 1000-fold. A quantitative explanation for the very high redox potential of DsbA was found by measuring the pKa of a single residue, Cys-30. The pKa of Cys-30 varied dramatically from mutant to mutant and could accurately predict the oxidizing power of each DsbA mutant protein.
Amino Acid Sequence↗
Synthetic competition between cytoplasmic folding and translocation of a soluble membrane protein domain.
In wild-type strains of Escherichia coli, alkaline phosphatase (AP), either when present as a soluble protein or when fused to a membrane protein, is only active after translocation to the periplasm. In thioredoxin reductase (trxB) mutants, however, cytoplasmically localized AP can form disulphide bonds and can reach an active conformation. Once it has folded in the cytoplasm, it can no longer be translocated. On the other hand, when AP is fused to periplasmic domains of a membrane protein, translocation can be more rapid than folding. Thus, expressing hybrids of AP and integral membrane proteins in a trxB mutant generates competition between folding of AP in the cytoplasm and its translocation to the periplasm. The cellular localization of AP can be monitored in phosphoserine phosphatase (serB) mutants causing auxotrophy for L-serine. Cytoplasmically but not periplasmically localized AP can compensate for the lack of SerB, leading to growth on indicator plates. As expected, when AP was fused to cytoplasmic domains of membrane proteins, serB-mediated auxotrophy was abolished. Surprisingly, AP fusions to periplasmic domains exhibited a non-uniform response pattern. Fusions that translocate AP rapidly did not complement the SerB defect, while those that export AP only slowly could do so. The usefulness of these strains for studying a variety of aspects related to membrane protein biogenesis is discussed.
Alkaline Phosphatase↗
Sec-independent translocation of a 100-residue periplasmic N-terminal tail in the E. coli inner membrane protein proW.
The ProW protein, located in the inner membrane of Escherichia coli, has a very unusual topology with a 100-residue-long N-terminal tail protruding into the periplasmic space. We have studied the mechanism of membrane translocation of the periplasmic tail by analysing ProW-PhoA and ProW-Lep fusion proteins, both in wild-type cells and in cells with an impaired sec machinery. Our results show that the translocation efficiency is not affected by treatments that compromise the SecA and SecY functions, but that translocation is completely blocked by dissipation of the proton motive force or by the introduction of extra positively charged residues into the N-terminal tail. This suggests that the sec machinery can act properly only on domains located on the C-terminal side of a translocation signal, and that the N-terminal tail is driven through the membrane by a mechanism that involves the proton motive force.
ATP-Binding Cassette Transporters↗
Requirements for translocation of periplasmic domains in polytopic membrane proteins.
Periplasmic domains of cytoplasmic membrane proteins require export signals for proper translocation. These signals were studied by using a MalF-alkaline phosphatase fusion in a genetic selection that allowed the isolation of mislocalization mutants. In the original construct, alkaline phosphatase is fused to the second periplasmic domain of the membrane protein, and its activity is thus confined exclusively to the periplasm. Mutants that no longer translocated alkaline phosphatase were selected by complementation of a serB mutation. A total of 11 deletions in the amino terminus were isolated, all of which spanned at least the third transmembrane segment. This domain immediately precedes the periplasmic domain to which alkaline phosphatase was fused. Our results obtained in vivo support the model that amino-terminal membrane-spanning segments are required for translocation of large periplasmic domains. In addition, we found that the inability to export the alkaline phosphatase domain could be suppressed by a mutation, prlA4, in the secretion apparatus.
ATP-Binding Cassette Transporters↗
Analysis of simulated single ligament transection on the mechanical behaviour of a lumbar functional spinal unit.
The influence of the different lumbar spinal ligaments on intersegmental rotation is not fully understood. In order to explore this effect, a finite element model of the functional spinal unit L3/L4 was loaded with pure moments in the three main anatomic planes. The two extremes--minimum and maximum--ligament stiffness values reported in the literature were applied. After virtual transection of each of the spinal ligaments in turn, the intersegmental rotation and forces in the remaining ligaments were calculated. On flexion, the highest force was found for the posterior longitudinal ligament; on extension and lateral bending for the anterior longitudinal ligament; and on axial rotation for the facet capsular ligament. The strongest influence on intersegmental rotation is exerted by the interspinous ligament on flexion, by the anterior longitudinal ligament on extension and lateral bending, and by the facet capsular ligaments on axial rotation. Ligament stiffness has a strong influence on intersegmental rotation and forces in the ligaments, so that finite element models of spinal segments must be validated by experimental data. This study should help to elucidate the role of the various ligaments.
Biomechanical Phenomena↗