Showing papers by "Howard Hughes Medical Institute published in 1993"
TL;DR: P16 seems to act in a regulatory feedback circuit with CDK4, D-type cyclins and retinoblastoma protein, and inhibits the catalytic activity of theCDK4/cyclin D enzymes.
Abstract: The division cycle of eukaryotic cells is regulated by a family of protein kinases known as the cyclin-dependent kinases (CDKs). The sequential activation of individual members of this family and their consequent phosphorylation of critical substrates promotes orderly progression through the cell cycle. The complexes formed by CDK4 and the D-type cyclins have been strongly implicated in the control of cell proliferation during the G1 phase. CDK4 exists, in part, as a multi-protein complex with a D-type cyclin, proliferating cell nuclear antigen and a protein, p21 (refs 7-9). CDK4 associates separately with a protein of M(r) 16K, particularly in cells lacking a functional retinoblastoma protein. Here we report the isolation of a human p16 complementary DNA and demonstrate that p16 binds to CDK4 and inhibits the catalytic activity of the CDK4/cyclin D enzymes. p16 seems to act in a regulatory feedback circuit with CDK4, D-type cyclins and retinoblastoma protein.
3,716 citations
TL;DR: It is found that over expression of p21 inhibits the activity of each member of the cyclin/CDK family, and this results indicate that p21 may be a universal inhibitor of cyclin kinases.
Abstract: Deregulation of cell proliferation is a hallmark of neoplastic transformation. Alteration in growth control pathways must translate into changes in the cell-cycle regulatory machinery, but the mechanism by which this occurs is largely unknown. Compared with normal human fibroblasts, cells transformed with a variety of viral oncoproteins show striking changes in the subunit composition of the cyclin-dependent kinases (CDKs). In normal cells, CDKs exist predominantly in multiple quaternary complexes, each containing a CDK, cyclin, proliferating cell nuclear antigen and the p21 protein. However, in many transformed cells, proliferating cell nuclear antigen and p21 are lost from these multiprotein enzymes. Here we have investigated the significance of this phenomenon by molecular cloning of p21 and in vitro reconstitution of the quaternary cell-cycle kinase complexes. We find that p21 inhibits the activity of each member of the cyclin/CDK family. Furthermore, overexpression of p21 inhibits the proliferation of mammalian cells. Our results indicate that p21 may be a universal inhibitor of cyclin kinases.
3,442 citations
TL;DR: Data suggest that bcl-x plays an important role in both positive and negative regulation of programmed cell death, as well as in tissues containing long-lived postmitotic cells, such as adult brain.
3,172 citations
TL;DR: A dimer of the class II αβ heterodimers is seen in the crystal forms of HLA-DR1, suggesting class II HLA dimerization as a mechanism for initiating the cytoplasmic signalling events in T-cell activation.
Abstract: The three-dimensional structure of the class II histocompatibility glycoprotein HLA-DR1 from human B-cell membranes has been determined by X-ray crystallography and is similar to that of class I HLA. Peptides are bound in an extended conformation that projects from both ends of an 'open-ended' antigen-binding groove. A prominent non-polar pocket into which an 'anchoring' peptide side chain fits is near one end of the binding groove. A dimer of the class II alpha beta heterodimers is seen in the crystal forms of HLA-DR1, suggesting class II HLA dimerization as a mechanism for initiating the cytoplasmic signalling events in T-cell activation.
2,313 citations
TL;DR: The Lon Protease, DnaK.T URNOVER of AB ERR ANT PROT EINS in E. COLI, and more.
Abstract: T URNOVER OF AB ERR ANT PROT EINS IN E. COLI . . . . . . . . . . . . . . . . . 445 The Lon Protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446 DnaK. DnaJ. GrpE. GroEL and GroES . . . . . . . . . . . . . . . . . . . . . . . . . . 447 The Clp Protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448 The DegP Protease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
2,255 citations
TL;DR: Cystic fibrosis is a regulated Cl- channel, for which structure-function relationships have begun to be established, and insight into the functions of individual domains has come from a number of studies.
1,446 citations
TL;DR: The results support a role for the striated muscle dystrophin-glycoprotein complex in linking the actin- based cytoskeleton with the extracellular matrix and suggest that dystophin and dystroglycan may play substantially different functional roles in nonmuscle tissues.
Abstract: The dystrophin-glycoprotein complex was tested for interaction with several components of the extracellular matrix as well as actin. The 156-kD dystrophin-associated glycoprotein (156-kD dystroglycan) specifically bound laminin in a calcium-dependent manner and was inhibited by NaCl (IC50 = 250 mM) but was not affected by 1,000-fold (wt/wt) excesses of lactose, IKVAV, or YIGSR peptides. Laminin binding was inhibited by heparin (IC50 = 100 micrograms/ml), suggesting that one of the heparin-binding domains of laminin is involved in binding dystroglycan while negatively charged oligosaccharide moieties on dystroglycan were found to be necessary for its laminin-binding activity. No interaction between any component of the dystrophin-glycoprotein complex and fibronectin, collagen I, collagen IV, entactin, or heparan sulfate proteoglycan was detected by 125I-protein overlay and/or extracellular matrix protein-Sepharose precipitation. In addition, laminin-Sepharose quantitatively precipitated purified dystrophin-glycoprotein complex, demonstrating that the laminin-binding site is accessible when dystroglycan is associated with the complex. Dystroglycan of nonmuscle tissues also bound laminin. However, the other proteins of the striated muscle dystrophin-glycoprotein complex appear to be absent, antigenically dissimilar or less tightly associated with dystroglycan in nonmuscle tissues. Finally, we show that the dystrophin-glycoprotein complex cosediments with F-actin but does not bind calcium or calmodulin. Our results support a role for the striated muscle dystrophin-glycoprotein complex in linking the actin-based cytoskeleton with the extracellular matrix. Furthermore, our results suggest that dystrophin and dystroglycan may play substantially different functional roles in nonmuscle tissues.
1,333 citations
TL;DR: A candidate gene (Mc1) for Menkes disease is isolated and qualitative or quantitative abnormalities in the mRNA in sixteen of twenty–one Menkes patients are found and predicted to be a P–type cation–transporting ATPase.
Abstract: Menkes disease is an X-linked disorder of copper transport characterized by progressive neurological degeneration and death in early childhood. We have isolated a candidate gene (Mc1) for Menkes disease and find qualitative or quantitative abnormalities in the mRNA in sixteen of twenty-one Menkes patients. Four patients lacking Mc1RNA showed rearrangements of the Menkes gene. The gene codes for a 1,500 amino acid protein, predicted to be a P-type cation-transporting ATPase. The gene product is most similar to a bacterial copper-transporting ATPase and additionally contains six putative metal-binding motifs at the N-terminus. The gene is transcribed in all cell types tested except liver, consistent with the expression of the Menkes defect.
1,316 citations
TL;DR: The three-dimensional structure of an HNF-3/fork head DNA-recognition motif complexed with DNA has been determined by X-ray crystallography at 2.5 Å resolution and the transcription factor fold is very similar to the structure of histone H5.
Abstract: The three-dimensional structure of an HNF-3/fork head DNA-recognition motif complexed with DNA has been determined by X-ray crystallography at 2.5 A resolution. This alpha/beta protein binds B-DNA as a monomer, through interactions with the DNA backbone and through both direct and water-mediated major and minor groove base contacts, inducing a 13 degrees bend. The transcription factor fold is very similar to the structure of histone H5. In its amino-terminal half, three alpha-helices adopt a compact structure that presents the third helix to the major groove. The remainder of the protein includes a twisted, antiparallel beta-structure and random coil that interacts with the minor groove.
1,238 citations
TL;DR: Activation of PKA may be a component of the mechanism that generates L-LTP, and analogs of cAMP induced a potentiation that blocked naturally induced L- LTP, which was blocked by inhibitors of protein synthesis.
Abstract: Hippocampal long-term potentiation (LTP) is thought to serve as an elementary mechanism for the establishment of certain forms of explicit memory in the mammalian brain. As is the case with behavioral memory, LTP in the CA1 region has stages: a short-term early potentiation lasting 1 to 3 hours, which is independent of protein synthesis, precedes a later, longer lasting stage (L-LTP), which requires protein synthesis. Inhibitors of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) blocked L-LTP, and analogs of cAMP induced a potentiation that blocked naturally induced L-LTP. The action of the cAMP analog was blocked by inhibitors of protein synthesis. Thus, activation of PKA may be a component of the mechanism that generates L-LTP.
1,200 citations
TL;DR: It is demonstrated that BoNT/A acts as a zinc-dependent protease that selectively cleaves SNAP-25, a second component of the putative fusion complex mediating synaptic vesicle exocytosis is targeted by a clostridial neurotoxin.
Abstract: Neurotransmitter release is potently blocked by a group of structurally related toxin proteins produced by Clostridium botulinum. Botulinum neurotoxin type B (BoNT/B) and tetanus toxin (TeTx) are zinc-dependent proteases that specifically cleave synaptobrevin (VAMP), a membrane protein of synaptic vesicles. Here we report that inhibition of transmitter release from synaptosomes caused by botulinum neurotoxin A (BoNT/A) is associated with the selective proteolysis of the synaptic protein SNAP-25. Furthermore, isolated or recombinant L chain of BoNT/A cleaves SNAP-25 in vitro. Cleavage occurred near the carboxyterminus and was sensitive to divalent cation chelators. In addition, a glutamate residue in the BoNT/A L chain, presumably required to stabilize a water molecule in the zinc-containing catalytic centre, was required for proteolytic activity. These findings demonstrate that BoNT/A acts as a zinc-dependent protease that selectively cleaves SNAP-25. Thus, a second component of the putative fusion complex mediating synaptic vesicle exocytosis is targeted by a clostridial neurotoxin.
TL;DR: The three-dimensional structure of a TATA-box binding polypeptide complexed with the TATA element of the adenovirus major late promoter has been determined by X-ray crystallography at 2.25 Å resolution.
Abstract: The three-dimensional structure of a TATA-box binding polypeptide complexed with the TATA element of the adenovirus major late promoter has been determined by X-ray crystallography at 2.25 A resolution. Binding of the saddle-shaped protein induces a conformational change in the DNA, inducing sharp kinks at either end of the sequence TATAAAAG. Between the kinks, the right-handed double helix is smoothly curved and partially unwound, presenting a widened minor groove to TBP's concave, antiparallel β-sheet. Side-chain/base interactions are restricted to the minor groove, and include hydrogen bonds, van der Waals contacts and phenylalanine–base stacking interactions.
TL;DR: It is demonstrated that mutations in the human Ca(2+)-sensing receptor gene cause familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT), two inherited conditions characterized by altered calcium homeostasis.
TL;DR: The 2.5 Å crystal structure of a TATA-box complex with yeast TBP shows that the eight base pairs of the TATA box bind to the concave surface of TBP by bending towards the major groove with unprecedented severity.
Abstract: The 2.5 A crystal structure of a TATA-box complex with yeast TBP shows that the eight base pairs of the TATA box bind to the concave surface of TBP by bending towards the major groove with unprecedented severity. This produces a wide open, underwound, shallow minor groove which forms a primarily hydrophobic interface with the entire under-surface of the TBP saddle. The severe bend and a positive writhe radically alter the trajectory of the flanking B-form DNA.
TL;DR: The current finding suggests that PLD activity plays a prominent role in the action of ARF and that ARF may be a key component in the generation of second messengers via phospholipase D.
TL;DR: The patterns of odorant receptor expression in the rat olfactory epithelium are examined to determine whether the mammalian olfaction system employs spatial segregation of sensory input to encode the identity of an odorant stimulus.
TL;DR: The new findings suggest an expansion of the classical ternary complex model of receptor action to include an explicit isomerization of the receptors from an inactive to an active state which couples to the G protein ('allosteric ternARY complex model').
TL;DR: Cell permeable, synthetic ligands were devised that can be used to control the intracellular oligomerization of specific proteins to demonstrate their utility and have the potential to be applied wherever precise control of a signal transduction pathway is desired.
Abstract: Dimerization and oligomerization are general biological control mechanisms contributing to the activation of cell membrane receptors, transcription factors, vesicle fusion proteins, and other classes of intra- and extracellular proteins. Cell permeable, synthetic ligands were devised that can be used to control the intracellular oligomerization of specific proteins. To demonstrate their utility, these ligands were used to induce intracellular oligomerization of cell surface receptors that lacked their transmembrane and extracellular regions but contained intracellular signaling domains. Addition of these ligands to cells in culture resulted in signal transmission and specific target gene activation. Monomeric forms of the ligands blocked the pathway. This method of ligand-regulated activation and termination of signaling pathways has the potential to be applied wherever precise control of a signal transduction pathway is desired.
TL;DR: Alternative splicing of the messenger RNA generates an array of putative membrane-attached, intracellular and secreted signalling proteins, at least some of which are expressed in the developing spinal cord and brain.
Abstract: Glial growth factors, proteins that are mitogenic for Schwann cells, and several ligands for the p±85erbB2 receptor, are products of the same gene. Alternative splicing of the messenger RNA generates an array of putative membrane-attached, intracellular and secreted signalling proteins, at least some of which are expressed in the developing spinal cord and brain. These factors are probably important in the development and regeneration of the nervous system.
TL;DR: Dopamine mediates these diverse effects through interactions with specific receptor proteins, and one of the challenges has been to characterizing the structure, function, and elucidate their properties.
Abstract: The catecholamine dopamine plays a role in the functioning of numerous vertebrate and invertebrate organisms. In mammals, dopamine affects a diverse array of processes, such as motor control, cognition, emotion, neuroendocrine regulation, positive reinforcement, and cardiovascular regulation. Moreover, dopamine responsive systems have been implicated in several pathologic conditions, such as schizophrenia, tardive dyskinesia, Parkinson's disease, Tourette syndrome, hyperprolactinemia, and possibly Huntington's chorea. The quest to find new drugs to combat the symptoms of these conditions has led to the discovery of numerous synthetic agonists and antagoni sts for dopamine receptors. At the cellular and biochemical level, dopamine also exhibits a wide range of effects, including stimulation and inhibition of adenylyl cyclase (Stoof & Kebabian 1984), stimulation of potassium channels (Sasaki & Sato 1 987), alterations of phosphotidyl inositol (Mahan et al 1 990) and arachidonic acid metabolism (Kanterman et al 1991; Piomelli et al 1991), changes in .neuronal firing rates (Walters et al 1987), and alterations of gene expression (Gerfen et alI990). Dopamine mediates these diverse effects through interactions with specific receptor proteins, and one of the challenges has been to i dentify these receptors and elucidate their properties. B y characterizing the structure, function,
TL;DR: In this paper, a crystal structure of activated rod transducin, Gtα-GTPγS, was shown to occlude deep in a cleft between a domain structurally homologous to small GTPases and a helical domain unique to heterotrimeric G proteins.
Abstract: The 2.2 A crystal structure of activated rod transducin, Gtα-GTPγS, shows the bound GTPγS molecule occluded deep in a cleft between a domain structurally homologous to small GTPases and a helical domain unique to heterotrimeric G proteins. The structure, when combined with biochemical and genetic studies, suggests: how an activated receptor might open this cleft to allow nucleotide exchange; a mechanism for GTP-induced changes in effector and receptor binding surfaces; and a mechanism for GTPase activity not evident from previous data.
TL;DR: The HNPP locus is assigned to chromosome 17p11.2 and the presence of a large interstitial deletion associated with this disorder is demonstrated in three unrelated pedigrees, suggesting that these genetic disorders may be the result of reciprocal products of unequal crossover.
TL;DR: The experiments reported here document that the tumor suppressor retinoblastoma protein (pRB) plays an important role in the production and maintenance of the terminally differentiated phenotype of muscle cells, and shows that pRB inactivation, through either phosphorylation, binding to T antigen, or genetic alteration, inhibits myogenesis.
TL;DR: The crystal structure of the Src SH2 domain complexed with a high affinity 11-residue phosphopeptide has been determined at 2.7 A resolution by X-ray diffraction, and comparison with the structure with the high affinity complex reveals only localized and relatively small changes.
TL;DR: Differential screening is used to identify five immediate–early genes induced by neuronal activity that play a role in the structural changes that accompany activity-dependent plasticity and tissue-plasminogen activator (tPA) is one of these.
Abstract: THE requirement of protein and messenger RNA synthesis for long-term memory1,2 suggests that neural activity induced by learning initiates a cascade of gene expression3. Here we use differential screening to identify five immediate–early genes induced by neuronal activity. One of these is tissue-plasminogen activator (tPA), an extracellular serine protease, which is induced with different spatial patterns in the brain by three activity-dependent events: (1) convulsive seizure increases expression of tPA in the whole brain; (2) stimulation of the perforant path produces an epileptiform after-discharge that ultimately leads to kindling increases the levels of tPA throughout the hippocampus bilaterally; and (3) brief high-frequency stimulation of the perforant path that produces long-term potentiation (LTP) causes an NMDA (N -methyl-D-aspartate) receptor-mediated increase in the levels of tPA mRNA which is restricted to the granule cells of the ipsilateral dentate gyrus. As release of tPA is correlated with morphological differentiation4–6, the increased expression of tPA may play a role in the structural changes that accompany activity-dependent plasticity7–10.
TL;DR: It is reported that two interacting components are required for full fraction-B activity, purify one of these components to homogeneity, and show that it is the highly abundant GTP-binding protein Ran (Ras-related nuclear protein)/TC4.
Abstract: TWO cytosolic fractions (A and B) fromXenopus oocytes are sufficient to support protein import into the nuclei of digitonin-permeabilized cells1. Fraction A recognizes the nuclear localization sequence (NLS) and binds the import substrate to the nuclear envelope, whereas fraction B mediates the subsequent passage of the bound substrate into the nucleus. Here we report that two interacting components are required for full fraction-B activity, purify one of these components to homogeneity, and show that it is the highly abundant GTP-binding protein Ran (Ras-related nuclear protein)/TC4.
TL;DR: A E1-deficient adenovirus, encoding CFTR, was administered to a defined area of nasal airway epithelium of three individuals with CF, and there was a decrease in the elevated basal transepithelial voltage, and the normal response to a cAMP agonist was restored.
TL;DR: X-ray crystallography results offer a structural framework for understanding the role of nonanchor peptide side chains in both peptide-MHC binding affinity and TCR recognition.
TL;DR: The three-dimensional structure of the basic/helix–loop–helix/leucine zipper domain of the transcription factor Max complexed with DNA has been determined by X-ray crystallography at 2.9 Å resolution.
Abstract: The three-dimensional structure of the basic/helix–loop–helix/leucine zipper domain of the transcription factor Max complexed with DNA has been determined by X-ray crystallography at 2.9 A resolution. Max binds as a dimer to its recognition sequence CACGTG by direct contacts between the α-helical basic region and the major groove. This symmetric homodimer, a new protein fold, is a parallel, left-handed, four-helix bundle, with each monomer containing two α-helical segments separated by a loop. The two α-helical segments are composed of the basic region plus helix 1 and helix 2 plus the leucine repeat, respectively. As in GCN4, the leucine repeat forms a parallel coiled coil.
TL;DR: The results show that the SWI2 family DNA-dependent ATPase domain has functional con-servation between yeast and humans and suggest that a SWI/SNF protein complex is required for the activation of selective mammalian genes.
Abstract: SEQUENCE-SPECIFIC DNA binding activators of gene transcription may be assisted by SWI2(SNF2)1,2, which contains a DNA-depen-dent ATPase domain3. We have isolated a human complementary DNA encoding a 205K nuclear protein, BRG1, that contains extensive homology to SWI2 and Drosophila brahma4,5. We report here that a SWI2/BRG1 chimaera with the DNA-dependent ATPase domain replaced by corresponding human sequence restored normal mitotic growth and capacity for transcriptional activation to swi2& minus; yeast cells. Point mutation of the conserved ATP binding site lysine abolished this complementation. This mutation in SW12 exerted a dominant negative effect on transcription in yeast. A lysine to arginine substitution at the corresponding residue of BRG1 also generated a transcriptional dominant negative in human cells. BRG1 is exclusively nuclear and present in a high Mr complex of about 2 & times; 106. These results show that the SWI2 family DNA-dependent ATPase domain has functional con-servation between yeast and humans and suggest that a SWI/SNF protein complex is required for the activation of selective mammalian genes.