Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Based on presently available information on the structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, peptides have been synthesized which correspond to the sequence of a particular region of the protein involved in formation of the active heterodimeric form of the enzyme. Several peptides that are 15-19 amino acids long and that are derived from the so-called connection domain of the reverse transcriptase are able to inhibit dimerization of the enzyme and thus inhibit development of its enzymatic activities. In particular, a tryptophan-rich 19-mer corresponding to residues 389-407 was relatively efficient, showing an apparent dissociation constant in the micromolar range for one or both of the subunits. The sequence of this region is identical for both subunits, since one (molecular mass of 51 kDa) is the proteolytic product of the other (molecular mass of 66 kDa). Dissociation of the preformed heterodimer could not be induced by the peptides, but increasing concentrations reduced the rate of dimerization in a concentration-dependent manner until it became immeasurable at high concentrations. The results suggest that inhibition of dimerization of reverse transcriptase is an attractive approach to chemotherapeutic intervention in HIV infection and that further development of peptide-based inhibition strategies is worth pursuing.
 ...
PMID:Inhibition of human immunodeficiency virus type 1 reverse transcriptase dimerization using synthetic peptides derived from the connection domain. 751 98

The nuclear factor kappaB (NF-kappaB) regulates the transcription of genes bearing the kappaB consensus motif. Transmigration of NF-kappaB from the cytoplasm to the nucleus is regulated by the IkappaB family of inhibitory NF-kappaB-binding proteins. Dissociation of the NF-kappaB-IkappaB complex requires both phosphorylation and degradation of IkappaBs. We demonstrate that IL-1beta induces complete IkappaB alpha degradation in Caco-2 cell lines but not in HT-29, T84, SW-480 transformed cell lines, or native colonic epithelial cells. A similar lack of IkappaB alpha degradation in HT-29 cells followed TNF-alpha and bacterial polymer stimulation. IL-1beta stimulated NF-kappaB nuclear translocation and NF-kappaB-dependent IL-1beta and IL-8 expression in both Caco-2 and HT-29 cells as assayed by electrophoretic mobility shift assay, immunofluorescence, kappaB-luciferase transfection, reverse transcriptase-PCR analysis and ELISA. In HT-29 cells stimulated with IL-1beta, IkappaB alpha was phosphorylated and when cycloheximide blocked new protein synthesis, IkappaB alpha was partially degraded. NF-kappaB cytoplasmic to nuclear transmigration was incomplete and preceded IkappaB alpha degradation in 9T-29 cells, in contrast to complete coordinated NF-kappaB nuclear translocation and IkappaB alpha degradation in Caco-2 cells. Greater sensitivity of HT-29 cells to a calpain inhibitor, as measured by IL-8 secretion, suggested enhanced resistance to IkappaB alpha proteolysis. These data show that IL-1beta induces NF-kappaB activity and expression of NF-kappaB-dependent genes in colonic epithelial cells and suggest altered regulation of IkappaB alpha degradation compared with other cell lineages, which may result in their increased responsiveness to therapeutic blockade.
 ...
PMID:Evidence for altered regulation of I kappa B alpha degradation in human colonic epithelial cells. 897 94

We have recently shown that an MCF-7 tumor can be imaged in a mouse by PET with 64Cu-labeled Peptide nucleic acids (PNAs) tethered to the permeation peptide Lys4 that recognize the uniquely overexpressed and very abundant upstream of N-ras or N-ras related gene (unr mRNA) expressed in these cells. Herein we describe how the high affinity antisense PNAs to the unr mRNA were identified and characterized. First, antisense binding sites on the unr mRNA were mapped by an reverse transcriptase random oligonucleotide library (RT-ROL) method that we have improved, and by a serial analysis of antisense binding sites (SAABS) method that we have developed which is similar to another recently described method. The relative binding affinities of oligodeoxynucleotides (ODNs) complementary to the antisense binding sites were then qualitatively ranked by a new Dynabead-based dot blot assay. Dissociation constants for a subset of the ODNs were determined by a new Dynabead-based solution assay and were found to be 300 pM for the best binders in 1 M salt. PNAs corresponding to the ODNs with the highest affinities were synthesized with an N-terminal CysTyr and C-terminal Lys4 sequence. Dissociation constants of these hybrid PNAs were determined by the Dynabead-based solution assay to be about 10 pM for the highest affinity binders.
 ...
PMID:Identification and characterization of high affinity antisense PNAs for the human unr (upstream of N-ras) mRNA which is uniquely overexpressed in MCF-7 breast cancer cells. 1631 3

Binding of HIV reverse transcriptase (RT) to unique substrates that positioned RNA-DNA or DNA-DNA near the polymerase or RNase H domains was measured. The substrates consisted of a 50 nucleotide template and DNA primers ranging from 23 to 43 nucleotides. Five different types of template strands were used: homogeneous (1) RNA or (2) DNA, (3) the first 20 5' nucleotides of DNA and the last 30 RNA, (4) the first 20 RNA and the last 30 DNA, and (5) 15 nucleotides of DNA followed by 5 RNA and then 30 DNA. The different length primers were designed to position RT over various regions of the template. Dissociation rate constants were determined for each of the substrates. Results showed that the severalfold tighter binding to RNA-DNA vs DNA-DNA was determined by binding in the polymerase domain and required only a short 5 base pair RNA-DNA hybrid region. Chimeric substrates with RNA-DNA positioned near the polymerase domain and DNA-DNA near the RNase H domain showed binding comparable to a complete RNA-DNA substrate, while those with the reverse orientation were comparable to DNA-DNA. Interestingly, the first configuration, though binding as tightly as RNA-DNA, could not be cleaved by RT RNase H activity, a finding that could perhaps be exploited in the development of nucleic acid-based inhibitors.
 ...
PMID:Tighter binding of HIV reverse transcriptase to RNA-DNA versus DNA-DNA results mostly from interactions in the polymerase domain and requires just a small stretch of RNA-DNA. 1676 58