Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

PKR (double-stranded RNA-dependent protein kinase) is an important component of host defense to virus infection. Binding of dsRNA to two dsRBDs (double-stranded RNA binding domains) of PKR modulates its own kinase activation. How structural features of natural target RNAs, such as bulges and loops, have an effect on the binding to two dsRBDs of PKR still remains unclear. By using ITC and NMR, we show here that both the bulge and loop of TAR RNA are necessary for the high affinity binding to dsRBD1-dsRBD2 of PKR with 1:1 stoichiometry. The binding site for the dsRBD1-dsRBD2 spans from upper bulge to lower stem of the TAR RNA, based on chemical shift mapping. The backbone resonances in the 40 kDa TAR.dsRBD1-dsRBD2 were assigned. NMR chemical shift perturbation data suggest that the beta1-beta2 loop of the dsRBD1 interacts with the TAR RNA, whereas that of the dsRBD2 is less involved in the TAR RNA recognition. In addition, the residues of the interdomain linker between the dsRBD1 and the dsRBD2 also show large chemical perturbations indicating that the linker is involved in the recognition of TAR RNA. The results presented here provide the biophysical and spectroscopic basis for high-resolution structural studies, and show how local RNA structural features modulate recognition by dsRBDs.
J Mol Biol 2006 Apr 28
PMID:Specific recognition of HIV TAR RNA by the dsRNA binding domains (dsRBD1-dsRBD2) of PKR. 1651 25

Argonaute proteins are the core components of effector complexes that facilitate RNA interference (RNAi). Small interfering RNAs (siRNAs) targeted to promoter regions mediate transcriptional gene silencing (TGS) in human cells through heterochromatin formation. RNAi effector complexes have yet to be implicated in the mechanism of mammalian TGS. Here we describe the role of the human Argonaute-1 homolog (AGO1) in directing TGS at the promoters for human immunodeficiency virus-1 coreceptor CCR5 and tumor suppressor RASSF1A. AGO1 associates with RNA polymerase II (RNAPII) and is required for histone H3 Lys9 dimethylation and TGS. AGO1, TAR RNA-binding protein-2 (7TRBP2) and Polycomb protein EZH2 colocalize to the siRNA-targeted RASSF1A promoter, implicating Polycomb silencing in the mechanism of mammalian TGS. These results establish a connection between RNAi components AGO1 and TRBP2, RNAPII transcription and Polycomb-regulated control of gene expression.
Nat Struct Mol Biol 2006 Sep
PMID:Argonaute-1 directs siRNA-mediated transcriptional gene silencing in human cells. 1693 26

Towards the development of oligonucleotide analogues and siRNA as drugs, one potential alternative to the use of liposomal transfection agents is the covalent conjugation of a cell-penetrating peptide (CPP), with the intention of imparting on the oligonucleotide or siRNA an enhanced ability to enter mammalian cells and reach the appropriate RNA target. We have developed robust methods for the chemical synthesis of disulfide-linked conjugates of oligonucleotide analogues, siRNA and peptide nucleic acids (PNA) with a range of cationic and other CPPs. In a HeLa cell assay with integrated plasmid reporters of Tat-dependent trans-activation at the TAR RNA target in the cell nucleus, we were unable to obtain steric block inhibition of gene expression for conjugates of CPPs with a 12-mer oligonucleotide mixmer of 2'-O-methyl and locked nucleic acids units. By contrast, we were able to obtain some reductions in expression of P38alpha MAP kinase mRNA in HeLa cells using microM concentrations of Penetratin or Tat peptides conjugated to the 3'-end of the sense strand of siRNA. However, the most promising results to date have been with a 16-mer PNA conjugated to the CPP Transportan or a double CPP R(6)-Penetratin, where we have demonstrated Tat-dependent trans-activation inhibition in HeLa cells. Results to date suggest the possibility of development of CPP-PNA conjugates as anti-HIV agents as well as other potential applications involving nuclear cell delivery, such as the redirection of splicing.
Blood Cells Mol Dis
PMID:RNA targeting with peptide conjugates of oligonucleotides, siRNA and PNA. 1711 27

Positive transcription elongation factor b (P-TEFb) complexes, composed of cyclin-dependent kinase 9 (CDK9) and cyclin T1 or T2, are engaged by many cellular transcription regulators that activate or inhibit transcription from specific promoters. The related I-mfa (inhibitor of MyoD family a) and HIC (human I-mfa-domain-containing) proteins function in myogenic differentiation and embryonic development by participating in the Wnt signaling pathway. We report that I-mfa is a novel regulator of P-TEFb. Both HIC and I-mfa interact through their homologous I-mfa domains with cyclin T1 and T2 at two binding sites. One site is the regulatory histidine-rich domain that interacts with CDK9 substrates including RNA polymerase II. The second site contains a lysine and arginine-rich motif that is highly conserved between the two T cyclins. This site overlaps and includes the previously identified Tat/TAR recognition motif of cyclin T1 required for activation of human immunodeficiency virus type 1 (HIV-1) transcription. HIC and I-mfa can serve as substrates for P-TEFb. Their I-mfa domains also bind the activation domain of HIV-1 Tat and inhibit Tat- and P-TEFb-dependent transcription from the HIV-1 promoter. This transcriptional repression is cell-type specific and can operate via Tat and cyclin T1. Genomic and sequence comparisons indicate that the I-mf and HIC genes, as well as flanking genes, diverged from a duplicated chromosomal region. Our findings link I-mfa and HIC to viral replication, and suggest that P-TEFb is modulated in the Wnt signaling pathway.
J Mol Biol 2007 Mar 30
PMID:Developmental regulators containing the I-mfa domain interact with T cyclins and Tat and modulate transcription. 1728 77

The human TAR DNA-binding protein (TDP43) colocalizes with ubiquitinated inclusions in motor neurons in amyotrophic lateral sclerosis (ALS). TDP43 is both a DNA-binding protein with a nuclear export sequence that interacts with (TG)nTm elements in DNA and an RNA-binding protein that interacts with (UG)(6-12) motifs in single-stranded RNA. In control motor neurons, TDP43 was almost exclusively nuclear, whereas in ALS spinal motor neurons, TDP43 was predominantly localized to the cytosol and not the nucleus. TDP43 was observed as punctuate immunoreactivity and as dense skeins, with and without ubiquitinization. We observed that TDP43 stabilizes the human low molecular weight (hNFL) mRNA through a direct interaction with the 3'UTR. TDP43 is a unique hNFL mRNA-binding protein that is altered in its somatotopic localization in ALS spinal motor neurons and potentially contributes to the formation of NF aggregates in ALS through alterations in NF stoichiometry.
Mol Cell Neurosci 2007 Jun
PMID:TDP43 is a human low molecular weight neurofilament (hNFL) mRNA-binding protein. 1748 16

Principles of fragment-based molecular design are presented and discussed in the context of de novo drug design. The underlying idea is to dissect known drug molecules in fragments by straightforward pseudo-retro-synthesis. The resulting building blocks are then used for automated assembly of new molecules. A particular question has been whether this approach is actually able to perform scaffold-hopping. A prospective case study illustrates the usefulness of fragment-based de novo design for finding new scaffolds. We were able to identify a novel ligand disrupting the interaction between the Tat peptide and TAR RNA, which is part of the human immunodeficiency virus (HIV-1) mRNA. Using a single template structure (acetylpromazine) as reference molecule and a topological pharmacophore descriptor (CATS), new chemotypes were automatically generated by our de novo design software Flux. Flux features an evolutionary algorithm for fragment-based compound assembly and optimization. Pharmacophore superimposition and docking into the target RNA suggest perfect matching between the template molecule and the designed compound. Chemical synthesis was straightforward, and bioactivity of the designed molecule was confirmed in a FRET assay. This study demonstrates the practicability of de novo design to generating RNA ligands containing novel molecular scaffolds.
J Comput Aided Mol Des 2008 Feb
PMID:The concept of template-based de novo design from drug-derived molecular fragments and its application to TAR RNA. 1806 2

Dicer, a member of the ribonuclease III family of enzymes, processes double-stranded RNA substrates into approximately 21- to 27-nt products that trigger sequence-directed gene silencing by RNA interference. Although the mechanism of RNA recognition and length-specific cleavage by Dicer has been established, the way in which dicing activity is regulated is unclear. Here, we show that the N-terminal domain of human Dicer, which is homologous to DExD/H-box helicases, substantially attenuates the rate of substrate cleavage. Deletion or mutation of this domain activates human Dicer in both single- and multiple-turnover assays. The catalytic efficiency (k(cat)/K(m)) of the deletion construct is increased by 65-fold over that exhibited by the intact enzyme. Kinetic analysis shows that this activation is almost entirely due to an enhancement in k(cat). Modest stimulation of catalysis by the full-length Dicer enzyme was observed in the presence of the TAR-RNA binding protein, which physically interacts with the DExD/H-box domain. These results suggest that the DExD/H-box domain likely disrupts the functionality of the Dicer active site until a structural rearrangement occurs, perhaps upon assembly with its molecular partners.
J Mol Biol 2008 Jun 27
PMID:Autoinhibition of human dicer by its internal helicase domain. 1850 75

Until a couple of years ago, TAR-DNA-binding protein-43 (TDP-43) was a relatively unknown nuclear protein implicated in transcriptional repression and splicing. Since 2006, when the protein was reported to be present in inclusions in the neurons and/or glial cells of a range of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration with ubiquitin-positive, tau- and alpha-synuclein-negative inclusions (FTLD-U) and Alzheimer's disease (AD), many reports on the medical aspects of TDP-43 have been published. Here, we summarize the current literature on TDP-43, focusing on recent studies that provide clues to the function of TDP-43. Using this information and database analysis, we also suggest a molecular and cellular model for possible events in normal and diseased neurons in relation to the emerging importance of the function and dysfunction of this protein as a target for basic as well as translational research.
Trends Mol Med 2008 Nov
PMID:TDP-43: an emerging new player in neurodegenerative diseases. 1892 8

The TAR RNA binding Protein, TRBP, inhibits the activity of the interferon-induced protein kinase R (PKR), whereas the PKR activator, PACT, activates its function. TRBP and PACT also bind to each other through their double-stranded RNA binding domains (dsRBDs) and their Medipal domains, which may influence their activity on PKR. In a human immunodeficiency virus (HIV) long terminal repeat-luciferase assay, PACT unexpectedly reversed PKR-mediated inhibition of gene expression. In a translation inhibition assay in HeLa cells, PACT lacking the 13 C-terminal amino acids (PACTDelta13), but not full-length PACT, activated PKR and enhanced interferon-mediated repression. In contrast, in the astrocytic U251MG cells that express low TRBP levels, both proteins activate PKR, but PACTDelta13 is stronger. Immunoprecipitation assays and yeast two-hybrid assays show that TRBP and PACTDelta13 interact very weakly due to a loss of binding in the Medipal domain. PACT-induced PKR phosphorylation was restored in Tarbp2(-/-) murine tail fibroblasts and in HEK293T or HeLa cells when TRBP expression was reduced by RNA interference. In HEK293T and HeLa cells, arsenite, peroxide, and serum starvation-mediated stresses dissociated the TRBP-PACT interaction and increased PACT-induced PKR activation, demonstrating the relevance of this control in a physiological context. Our results demonstrate that in cells, TRBP controls PACT activation of PKR, an activity that is reversed by stress.
Mol Cell Biol 2009 Jan
PMID:TRBP control of PACT-induced phosphorylation of protein kinase R is reversed by stress. 1893 60

Tartrate-resistant acid phosphatase (TRAP) is a well-known marker of osteoclasts and bone resorption. Here we have investigated whether osteoblast-like cells (hFOB 1.19) present TRAP activity and how would be its pattern of expression during osteoblastic differentiation. We also observed how the osteoblastic differentiation affected the reduced glutathione levels. TRAP activity was measured using the p-nitrophenylphosphate substrate. The osteogenic potential of hFOB 1.19 cells was studied by measuring alkaline phosphatase activity and mineralized nodule formation. Oxidative stress was determined by HPLC and DNTB assays. TRAP activity and the reduced glutathione-dependent microenvironment were modulated during osteoblastic differentiation. During this phase, TRAP activity, as well as alkaline phosphatase and glutathione increased progressively up to the 21st day, decreasing thereafter. We demonstrate that TRAP activity is modulated during osteoblastic differentiation, possibly in response to the redox state of the cell, since it seemed to depend on suitable levels of reduced glutathione.
J Mol Histol 2008 Dec
PMID:Tartrate-resistant acid phosphatase activity and glutathione levels are modulated during hFOB 1.19 osteoblastic differentiation. 1897 72


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>