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Biomedical subjects

M Götte

Publications and source records attributed to M Götte.

At least 19 recordsLinked to original sources
PubMed2006

Expression and prognostic impact of the protein tyrosine phosphatases PRL-1, PRL-2, and PRL-3 in breast cancer.

The aim of this study was to investigate the expression of the protein tyrosine phosphatases (PTP) PRL-1, PRL-2, and PRL-3 in human breast cancer and to evaluate its clinical and prognostic significance. PRL-PTP mRNA expression was examined in malignant (n = 7) and nonmalignant (n = 7) cryoconserved breast tissue samples as well as in eight breast cancer cell lines by RT-PCR. Furthermore, protein expression of PRL-3 was analysed semiquantitatively by immunohistochemistry in ductal breast carcinoma in situ (n = 135) and invasive breast cancer (n = 147) by use of tissue microarray technology (TMA). In 24 lymph node-positive patients we selected the corresponding lymph node metastases for analysis of PRL-3 expression, and a validation set (n = 99) of invasive breast cancer samples was examined. Staining results were correlated with clinicopathological parameters and long-term follow-up. PRL-3 mRNA expression was significantly higher in malignant compared to benign breast tissue. For PRL-1 and PRL-2 expression no significant differences were observed. Staining of TMAs showed PRL-3 expression in 85.9% ductal carcinoma in situ and 75.5% invasive breast carcinomas. Analysis of survival parameters revealed a shorter disease-free survival (DFS) in patients with PRL-3-positive carcinomas, and in particular a significantly shorter DFS in nodal-positive patients with PRL-3 overexpressing tumours as compared to PRL-3-negative breast carcinomas (66+/-7 months (95% CI, 52-80) vs 97+/-9 months (95% CI, 79-115); P = 0.032). Moreover, we found a more frequent expression of PRL-3 in lymph node metastases as compared to the primary tumours (91.7 vs 66.7%; P = 0.033). Our results suggest that PRL-3 might serve as a novel prognostic factor in breast cancer, which may help to predict an adverse disease outcome.

Adult↗
PubMed2002

Nonradioactive detection of retroviral-associated RNase H activity in a microplate-based, high-throughput format.

None of the available antiretroviral drugs that are currently used in the clinic to treat infection with HIV-1 is directed against the RNase H active site of the reverse transcriptase. Here we developed a nonradioactive, 96-well plate assay designed to be used for high-throughput screening of compounds capable of inhibiting the RNase H activity of HIV-1 reverse transcriptase. We employed a tRNA as substrate that was labeled with digoxygenin-modified reporter residues. The labeled tRNA was prehybridized with a DNA oligonucleotide that contained a single biotinylated residue at its 5'-terminus to ensure its attachment to streptavidin-coated microplates. The uncleaved, immobilized DNA/tRNA substrate was detected through the use of established ELISA protocols. Incubation with purified HIV-1 reverse transcriptase initiated RNase H degradation and caused a signal reduction to negligible background levels. In contrast, the signal intensity remained unaffected when using an RNase H deficient mutant enzyme. The assay was validated using the hydrazone derivative BBNH that was previously shown to inhibit RNase H degradation below concentrations of 10 microM.

Bacteriological Techniques↗
PubMed2001

Role for human immunodeficiency virus type 1 Tat protein in suppression of viral reverse transcriptase activity during late stages of viral replication.

We have examined the role of the human immunodeficiency virus type 1 (HIV-1) Tat protein in the regulation of reverse transcription. We show that a two-exon but not a one-exon form of Tat markedly suppressed cell-free reverse transcriptase (RT) activity. Conversely, viruses expressing two-exon Tat (pNL43 and pNL101) showed rapid replication kinetics and more efficient endogenous RT activity compared with viruses expressing one-exon Tat (pM1ex). The pM1ex virions, as well as pM1ex-infected cells, also contained higher levels of viral DNA than did either the pNL43 or pNL101 viruses, indicating that reverse transcription might have continued during later stages of viral replication in the absence of the second Tat exon. Moreover, degradation of viral genomic RNA was more apparent in the pM1ex virions. Accordingly, we propose that the two-exon Tat may help augment viral infectivity by suppressing the reverse transcription reaction during late stages of viral synthesis and by preventing the synthesis of potentially deleterious viral DNA products.

Cell Line↗
PubMed2000

Analysis of efficiency and fidelity of HIV-1 (+)-strand DNA synthesis reveals a novel rate-limiting step during retroviral reverse transcription.

We have analyzed the efficiency and accuracy of polymerization at several different stages during the initiation of human immunodeficiency virus type 1 (HIV-1) (+)-strand DNA synthesis. This reaction is of particular interest, as it involves the recruitment by reverse transcriptase of an RNA primer that serves as substrate for both the polymerase and RNase H activities of the enzyme. We found that the correct incorporation of the first two nucleotides was severely compromised and that formation of mismatches was completely absent at this stage of initiation. Although the fidelity of incorporations decreased concomitantly with ensuing polymerization, the elongation of mispaired primers was literally blocked. Instead, mispaired primer strands initiated a switch from active synthesis of DNA to premature RNase H-mediated primer removal. These findings suggest the existence of a fragile equilibrium between these two enzymatic activities that is shifted toward RNase H cleavage once the polymerization process is aggravated. Our data show that the initiation of HIV-1 (+)-strand DNA synthesis differs significantly from reactions involving other primer/template combinations, including tRNA-primed (-)-strand DNA synthesis.

Base Pair Mismatch↗
PubMed2000

Human immunodeficiency virus type-1 reverse transcription can be inhibited in vitro by oligonucleotides that target both natural and synthetic tRNA primers.

Reverse transcription of human immunodeficiency virus type-1 is primed by cellular tRNA(Lys3), which is selectively packaged into viral particles where it is bound at its 3' terminus to a complementary sequence of viral RNA termed the primer binding site (PBS). Since cellular tRNA(Lys3) is highly conserved, it might conceivably serve as a good target for novel antagonists to block reverse transcriptase (RT) activity. In this study, we have examined a number of antisense oligodeoxyribonucleotides (ODNs) that are complementary to different parts of the tRNA primer and, therefore, may interfere with the initiation of RT-mediated DNA synthesis. We found that the stability of complexes between synthetic tRNA(Lys3 )and ODNs was significantly increased when binding occurred via sequences involved in tertiary interactions of the tRNA. In particular, ODNs with complementarity to both the variable and TPsiC stem-loop of tRNA(Lys3 )bound with high affinity to both free tRNA(Lys3 )as well as to the binary tRNA(Lys3)/RNA complex. As a result, the initiation of DNA synthesis was severely compromised under these conditions. Moreover, RT-associated RNase H activity recognized the tRNA within this ternary tRNA(Lys3)/RNA/ODN complex as an RNA template and initiated its degradation. Both this RNase H degradation of tRNA(Lys3 )as well as the altered structure of the tRNA/RNA complex, due to the binding of the ODN, contributed to the inhibition of synthesis of viral DNA. The initiation of RT activity was almost completely blocked when using ODNs that interfered with intermolecular tRNA/RNA interactions that involved both the PBS and sequences outside the PBS. Similar findings were obtained with natural preparation of tRNA(Lys3).

Base Sequence↗
PubMed2000

The M184V mutation in the reverse transcriptase of human immunodeficiency virus type 1 impairs rescue of chain-terminated DNA synthesis.

Nucleoside analog chain terminators such as 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxy-3'-thiacytidine (3TC) represent an important class of drugs that are used in the clinic to inhibit the reverse transcriptase (RT) of human immunodeficiency virus type 1. Recent data have suggested that mutant enzymes associated with AZT resistance are capable of removing the chain-terminating residue with much greater efficiency than wild-type RT and this may, in turn, facilitate rescue of DNA synthesis; these experiments were performed using physiological concentrations of pyrophosphate or nucleoside triphosphates, respectively. The present study demonstrates that the M184V mutation, which confers high-level resistance to 3TC, can severely compromise the removal of chain-terminating nucleotides. Pyrophosphorolysis on 3TC-terminated primer strands was not detectable with M184V-containing, as opposed to wild-type, RT, and rescue of AZT-terminated DNA synthesis was significantly decreased with the former enzyme. Thus, mutated RTs associated with resistance to AZT and 3TC possess opposing, and therefore incompatible, phenotypes in this regard. These results are consistent with tissue culture and clinical data showing sustained antiviral effects of AZT in the context of viruses that contain the M184V mutation in the RT-encoding gene.

Adenosine Triphosphate↗
PubMed1999

HIV-1 reverse transcription: a brief overview focused on structure-function relationships among molecules involved in initiation of the reaction.

An early step in the life cycle of the human immunodeficiency virus type 1 (HIV-1) is reverse transcription of viral RNA into proviral DNA, which can then be integrated into the host cell genome. Reverse transcription is a discontinuous process carried out by the viral encoded reverse transcriptase that displays DNA polymerase activities on RNA and DNA templates as well as an RNase H activity that degrades transcribed RNA. DNA synthesis is initiated by cellular tRNALys3 that binds at its 3'-terminus to the complementary primer binding site of the genomic RNA. The initiation of reverse transcription is itself a complex reaction that requires tRNA placement onto viral RNA and the formation of a specific primer/template complex that is recognized by reverse transcriptase. After initiation takes place, the enzyme translocates from the initially bound RNA/RNA duplex into chimeric replication intermediates and finally accommodates newly synthesized DNA/RNA hybrids. This review focuses on structure-function relationships among these various molecules that are involved in the initiation of HIV-1 reverse transcription.

DNA, Viral↗
PubMed1999

Temporal coordination between initiation of HIV (+)-strand DNA synthesis and primer removal.

In this study, we have analyzed the interdependence between the polymerase and RNase H active sites of human immunodeficiency virus-1 reverse transcriptase (RT) using an in vitro system that closely mimics the initiation of (+)-strand DNA synthesis. Time course experiments show that RT pauses after addition of the 12th DNA residue, and at this stage the RNase H activity starts to cleave the RNA primer from newly synthesized DNA. Comparison of cleavage profiles obtained with 3'- and 5'-end-labeled primer strands indicates that RT now translocates in the opposite direction, i.e. in the 5' direction of the RNA strand. DNA synthesis resumes again in the 3' direction, after the RNA-DNA junction was efficiently cleaved. Moreover, we further characterized complexes generated before, during, and after position +12, by treating these with Fe2+ to localize the RNase H active site on the DNA template. Initially, when RT binds the RNA/DNA substrate, oxidative strand breaks were seen at a distance of 18 base pairs upstream from the primer terminus, whereas 17 base pairs were observed at later stages when the enzyme binds more and more DNA/DNA. These data show that the initiation of (+)-strand synthesis is accompanied by a conformational change of the polymerase-competent complex.

Base Pairing↗
PubMed1999

NMR structure of the chimeric hybrid duplex r(gcaguggc).r(gcca)d(CTGC) comprising the tRNA-DNA junction formed during initiation of HIV-1 reverse transcription.

A high-quality NMR solution structure of the chimeric hybrid duplex r(gcaguggc).r(gcca)d(CTGC) was determined using the program DYANA with its recently implemented new module FOUND, which performs exhaustive conformational grid searches for dinucleotides. To ensure conservative data interpretation, the use of 1H-1H lower distance limit constraints was avoided. The duplex comprises the tRNA-DNA junction formed during the initiation of HIV-1 reverse transcription. It forms an A-type double helix that exhibits distinct structural deviations from a standard A-conformation. In particular, the minor groove is remarkably narrow, and its width decreases from about 7.5 A in the RNA/RNA stem to about 4.5 A in the RNA/DNA segment. This is unexpected, since minor groove widths for A-RNA and RNA/DNA hybrid duplexes of approximately 11 A and approximately 8.5 A, respectively, were previously reported. The present, new structure supports that reverse transcriptase-associated RNaseH specificity is related primarily to conformational adaptability of the nucleic acid in 'induced-fit'-type interactions, rather than the minor groove width of a predominantly static nucleic acid duplex.

Base Sequence↗
PubMed1999

Functions of cell surface heparan sulfate proteoglycans.

The heparan sulfate on the surface of all adherent cells modulates the actions of a large number of extracellular ligands. Members of both cell surface heparan sulfate proteoglycan families, the transmembrane syndecans and the glycosylphosphoinositide-linked glypicans, bind these ligands and enhance formation of their receptor-signaling complexes. These heparan sulfate proteoglycans also immobilize and regulate the turnover of ligands that act at the cell surface. The extracellular domains of these proteoglycans can be shed from the cell surface, generating soluble heparan sulfate proteoglycans that can inhibit interactions at the cell surface. Recent analyses of genetic defects in Drosophila melanogaster, mice, and humans confirm most of these activities in vivo and identify additional processes that involve cell surface heparan sulfate proteoglycans. This chapter focuses on the mechanisms underlying these activities and on the cellular functions that they regulate.

Animals↗
PubMed1998

Dominance of the E89G substitution in HIV-1 reverse transcriptase in regard to increased polymerase processivity and patterns of pausing.

The substitution of a glycine for glutamic acid at position 89 in human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT) (E89G) confers resistance to several nucleoside and non-nucleoside inhibitors of RT. As residue 89 contacts the template strand, it has been suggested that this mutation may modulate the conformation of the RT.template/primer complex. In addition, certain mutations in RT that confer resistance to nucleoside analogs, such as M184V, are located near the polymerase active site. To characterize further these substitutions, we performed processivity assays alongside an analysis of pausing profiles with wild-type (wt) RT and recombinant RTs containing substitutions at E89G, M184V, or both. We now show that E89G RT has higher processivity than wt enzyme as well as a different pattern of pausing sites. Similar findings were obtained with the doubly mutated RT, although enzyme containing only the M184V mutation had lower processivity than wt. Consistent with these observations, and from a mechanistic standpoint, both E89G-containing as well as doubly mutated RT had decreased dissociation constants from a complex consisting of RT and template-primer, in comparison with either wt RT or M184V-containing RT. No significant differences were observed among the various enzymes in regard to Km values for the heteropolymeric RNA template used in these studies. Viruses containing the E89G mutation synthesized longer strand DNA products than either wt viruses or viruses containing only the M184V mutation in endogenous RT assays. Thus, the E89G substitution is a dominant determinant in regard to each of the koff values from an RT.template/primer complex, RT processivity, and specific patterns of pausing during DNA polymerization.

Amino Acid Substitution↗
PubMed1998

Mechanistic studies of early pausing events during initiation of HIV-1 reverse transcription.

We have investigated the role of sequences that surround the primer binding site (PBS) in the reverse transcriptase-mediated initiation of (-) strand DNA synthesis in human immunodeficiency virus type 1. In comparisons of reverse transcription initiated from either the cognate primer tRNALys.3 or a DNA primer D-Lys.3, bound to PBS sequences, we observed that a +3 pausing site occurred in both circumstances. However, the initiation reaction with tRNALys.3 was also characterized by a pausing event after incorporation of the first nucleotide. Alteration of sequences at the 5'-end instead of the 3'-end of the PBS resulted in elimination of the +3 pausing site, suggesting that this site was template sequence-dependent. In contrast, the pausing event at the +1 nucleotide position was still present in experiments that employed either of these mutated RNA templates. The mutations at the 5'-end of the PBS also caused a severely diminished rate of initiation and the strong arrest of reactions at the +1 stage when tRNALys.3 was used as primer. Therefore, we propose that the +1 pausing event is an initiation-specific event in regard to reactions primed by tRNALys.3 and that sequences at the 5'-end of the PBS may facilitate the release of reverse transcription from initiation to elongation.

Base Sequence↗
PubMed1998

Localization of the active site of HIV-1 reverse transcriptase-associated RNase H domain on a DNA template using site-specific generated hydroxyl radicals.

Reverse transcriptase (RT)-associated ribonuclease H (RNase H) can cleave both the RNA template of DNA/RNA hybrids as well as double-stranded (ds) RNA. This report shows that human immunodeficiency virus (HIV)-RT can also cleave the template strand of dsDNA when Mg2+ is replaced by Fe2+ in the RNase H active site of HIV-RT. The cleavage mechanisms as well as the positions of the cut vary depending on whether RNA or DNA is used. While DNA is cleaved 17 base positions upstream of the primer 3'-end, RNA is cleaved 18 base positions upstream. Competition experiments show that Fe2+ replaces the catalytically active Mg2+ of RT-associated RNase H. The bound Fe2+ is the source of locally generated OH-radicals that cleave the most proximate base in the DNA. Electrophoretic mobility studies of the cleaved fragments suggest that DNA is cleaved by an oxidative mechanism, while RNA is cleaved by an enzymatic mechanism which is indistinguishable from the Mg2+-dependent cleavage. The Fe2+-dependent cuts can be used to trace the active site of RT-associated RNase H on dsDNA as well as on dsRNA and DNA/RNA hybrids. The observed 1 base difference in the cleavage positions on DNA and RNA templates can be attributed to conformational differences of the bound nucleic acids. We suggest that the lower pitch of dsRNA and DNA/RNA hybrids compared with dsDNA permits accommodation of an additional base pair in the region between the primer 3'-end and the Fe2+-dependent cleavage position at the RNase H active site.

Binding Sites↗
PubMed1998

A new beat for the SNARE drum.

Eukaryotic cells contain membrane-bound compartments that are connected by trafficking of vesicular intermediates. To maintain compartmental organization, proper targeting of transport vesicles is achieved by specific evolutionarily conserved transmembrane proteins that reside on vesicles and target membranes. According to the original SNARE hypothesis, the formation of a complex of an NEM-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs) and membrane-bound SNAP receptor proteins (SNAREs) ensures docking specificity and leads to membrane fusion driven by the ATPase activity of NSF. Recent results have challenged some aspects of this hypothesis and led to a reassessment of models of SNARE interactions and the events leading to vesicle docking and fusion.

Biological Transport↗
PubMed1997

High expression of the yeast syntaxin-related Vam3 protein suppresses the protein transport defects of a pep12 null mutant.

The Pep12 protein of Saccharomyces cerevisiae is a member of the syntaxin family thought to function as target membrane receptor (t-SNARE) for vesicular intermediates travelling between the Golgi apparatus and the vacuole. Exploiting the temperature-sensitive growth phenotype of pep12 deletion strains, we identified VAM3 as a multicopy suppressor. Vam3p is another syntaxin-related protein which on high expression restored vacuole acidification of pep12 null mutants and effectively suppressed their sorting and maturation defects of vacuolar hydrolases. We conclude that Vam3p acts either as a bypass suppressor or by functionally replacing Pep12p at an endosomal, prevacuolar compartment.

Alkaline Phosphatase↗
PubMed1997

Influence of Mg2+ and temperature on formation of the transcription bubble.

The transcription bubble formed in the binding complex of T7A1 promoter upon Escherichia coli RNA polymerase was analyzed by chemical probes, namely by single-strand specific reagents, to map the unpaired bases in the bubble, and by FeEDTA, to analyze the accessibility of the DNA backbone. The latter probe could also be used as a local hydroxyl radical probe placed close to the Mg2+-binding site in the active center. The data show that the transcription bubble consists of two parts, an Mg2+-dependent part and an Mg2+-independent part, both having individual transition temperatures. The data further suggest that formation of a transcription active open complex is preceded by a transition state complex having enhanced affinity for those Mg2+ ions presumably participating in the formation of the catalytic site. Our data also suggests that the three catalytically active Mg2+ ions in RNA polymerase are functionally not equivalent. One/two of the three Mg2+ ions are responsible for the polymerization, the other two/one for enlargement of the transcription bubble.

Amino Acid Sequence↗
PubMed1997

Vesicular transport: how many Ypt/Rab-GTPases make a eukaryotic cell?

In eukaryotic cells, protein transport through the secretory and endocytic pathways is mediated by vesicular intermediates. Individual transport steps are regulated by Ras-like guanine nucleotide-binding proteins, termed Ypt in yeast or Rab in mammals. The complete sequencing of the Saccharomyces cerevisiae genome has revealed the total number of Ypt GTPases in this organism. There is some redundancy among the 11 Ypt proteins, and only those involved in the biosynthetic pathway are essential for cell viability.

Amino Acid Sequence↗