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1.
Physiol Res ; 63(3): 311-9, 2014.
Article in English | MEDLINE | ID: mdl-24564596

ABSTRACT

Proteasomes appear to be involved in the pathophysiology of various acute and chronic lung diseases. Information on the human lung proteasome in health and disease, however, is sparse. Therefore, we studied whether end-stage pulmonary diseases are associated with alterations in lung 20S/26S proteasome content, activity and 20S subunit composition. Biopsies were obtained from donor lungs (n=7) and explanted lungs from patients undergoing lung transplantation because of end stage chronic obstructive pulmonary disease (COPD; n=7), idiopathic pulmonary fibrosis (IPF, n=7) and pulmonary sarcoidosis (n=5). 20S/26S proteasomes in lung extracts were quantified by ELISA, chymotrypsin-like proteasome peptidase activities measured and 20S proteasome beta subunits analyzed by Western blot. As compared with donor lungs, proteasome content was increased in IPF and sarcoidosis, but not in COPD. The relative distribution of free 20S and 26S proteasomes was similar; 20S proteasome was predominant in all extracts. Proteasome peptidase activities in donor and diseased lungs were indistinguishable. All extracts contained a mixed composition of inducible 20S beta immuno-subunits and their constitutive counterparts; a disease associated distribution could not be identified. A higher content of lung proteasomes in IPF and pulmonary sarcoidosis may contribute to the pathophysiology of human fibrotic lung diseases.


Subject(s)
Lung Diseases/metabolism , Lung/metabolism , Proteasome Endopeptidase Complex/metabolism , Aged , Female , Humans , Male , Middle Aged , Pilot Projects , Tissue Donors
2.
Transpl Infect Dis ; 12(1): 87-93, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19735384

ABSTRACT

Multi-drug resistant (MDR) gram-negative infections among solid organ transplant (SOT) recipients have long been associated with high morbidity and mortality. Acinetobacter baumannii has emerged as a potent nosocomial pathogen with the recent acquisition of resistance to broad-spectrum beta-lactams, aminoglycosides, fluoroquinolones, and most notably, carbapenems. Despite a national rise in carbapenem-resistant A. baumannii (CRAB) infections, outcomes among SOT recipients with this emerging MDR pathogen are largely unknown. This single-center cohort is the first to describe the characteristics, complications, and outcomes among abdominal organ transplant recipients with CRAB. The current study suggests that SOT patients with CRAB suffer from prolonged hospitalization, infection with other MDR organisms, allograft dysfunction and loss, and high overall infection-related mortality.


Subject(s)
Acinetobacter Infections/microbiology , Acinetobacter baumannii/drug effects , Anti-Bacterial Agents/pharmacology , Carbapenems/pharmacology , Organ Transplantation/adverse effects , beta-Lactam Resistance , Acinetobacter Infections/diagnosis , Acinetobacter Infections/mortality , Adult , Aged , Female , Humans , Kidney Transplantation/adverse effects , Liver Transplantation/adverse effects , Male , Middle Aged
3.
J Chem Phys ; 129(6): 064702, 2008 Aug 14.
Article in English | MEDLINE | ID: mdl-18715097

ABSTRACT

We present evidence for the formation of transient hydroxyls from the reaction of water with atomic oxygen on Au(111) and investigate the effect of adsorbed oxygen on the hydrogen bonding of water. Water is evolved in peaks at 175 and 195 K in temperature programed reaction experiments following adsorption of water on oxygen-covered Au(111). The peak at 175 K is ascribed to sublimation of multilayers of water, whereas the peak at 195 K is associated with oxygen-stabilized water or a water-hydroxyl surface complex. Infrared reflection absorption spectra are consistent with the presence of molecular water over the entire range of coverages studied, indicating that isolated stable hydroxyls are not formed. Isotopic exchange of adsorbed (16)O with H(2)(18)O following adsorption and subsequent temperature programed reaction, however, indicates that transient OH species are formed. The extent of oxygen exchange was considerable--up to 70%. The degree of oxygen exchange depends on the initial coverage of oxygen, the surface temperature when preparing oxygen adatoms, and the H(2)(18)O coverage. The hydroxyls are short-lived, forming and disproportionating multiple times before water desorption during temperature programed reaction. It was also found that chemisorbed oxygen is critical in the formation of hydroxyls and stabilizing water, whereas gold oxide does not contribute to these effects. These results identify transient hydroxyls as species that could play a critical role in oxidative chemical reactions on gold, especially in ambient water vapor. The crystallinity of adsorbed water also depended on the degree of surface ordering and chemical modification based on scanning tunneling microscopy and infrared spectra. These results demonstrate that oxidation of interfaces has a major impact on their interaction with water.

4.
Ergonomics ; 47(8): 864-75, 2004 Jun 22.
Article in English | MEDLINE | ID: mdl-15204279

ABSTRACT

Employment rights legislative mandates passed in the USA over the last three decades emphasize the importance of validating performance standards for physically strenuous occupations. This study validated minimally acceptable standards for the muscular strength and endurance necessary to perform fire suppression activities. Incumbent firefighters (n=153) selected for key demographic characteristics completed a simulated set of firefighting tasks (Fire Suppression Evolution) and then a Predictor Test Battery of physical abilities tests. Regression analysis revealed that three predictor test items (hose drag/high rise pack carry; arm lift; arm endurance) combined to significantly predict performance time of the Fire Suppression Evolution (p < or =0.01). Firefighters (n=41) rating videotaped performance times of the Fire Suppression Evolution determined that more than one-half a standard deviation slower than the mean time established by the incumbents reflected unacceptable performance. Approximately 80% of incumbent firefighters passed the minimally acceptable performance standard. Use of the 3-predictor test battery would correctly identify 89% of successful performers and 72% of unsuccessful performers. This study demonstrates that validation of minimal physical performance standards will identify a cohort of individuals with a high probability of not being able to perform critical fire suppression activities. This finding has important implications for examining the relationship between physical performance standards and medical and economic outcomes.


Subject(s)
Personnel Selection/standards , Physical Fitness , Task Performance and Analysis , Adult , Analysis of Variance , Female , Fires , Humans , Lifting , Male , Middle Aged , Physical Endurance , Reproducibility of Results , United States
5.
Mol Cell ; 8(2): 449-54, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11545746

ABSTRACT

E. coli ClpX, a member of the Clp/Hsp100 family of ATPases, remodels multicomponent complexes and facilitates ATP-dependent degradation. Here, we analyze the mechanism by which ClpX destabilizes the exceedingly stable Mu transpososome, a natural substrate for remodeling rather than degradation. We find that ClpX has the capacity to globally unfold transposase monomers, the building blocks of the transpososome. A biochemical probe for protein unfolding reveals that ClpX also unfolds MuA subunits during remodeling reactions, but that not all subunits have their structure extensively modified. In fact, direct recognition and unfolding of a single transposase subunit are sufficient for ClpX to destabilize the entire transpososome. Thus, the ability of ClpX to unfold proteins is sufficient to explain its role in both complex destabilization and ATP-dependent proteolysis.


Subject(s)
Adenosine Triphosphatases/metabolism , Enzyme Stability , Transposases/metabolism , ATPases Associated with Diverse Cellular Activities , Adenosine Triphosphatases/genetics , Chaperonin 60/metabolism , Endopeptidase Clp , Escherichia coli Proteins , Immunoblotting , Macromolecular Substances , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Protein Denaturation , Protein Folding , Protein Subunits , Transposases/genetics
6.
Proc Natl Acad Sci U S A ; 98(19): 10584-9, 2001 Sep 11.
Article in English | MEDLINE | ID: mdl-11535833

ABSTRACT

The ssrA tag, an 11-aa peptide added to the C terminus of proteins stalled during translation, targets proteins for degradation by ClpXP and ClpAP. Mutational analysis of the ssrA tag reveals independent, but overlapping determinants for its interactions with ClpX, ClpA, and SspB, a specificity-enhancing factor for ClpX. ClpX interacts with residues 9-11 at the C terminus of the tag, whereas ClpA recognizes positions 8-10 in addition to residues 1-2 at the N terminus. SspB interacts with residues 1-4 and 7, N-terminal to the ClpX-binding determinants, but overlapping the ClpA determinants. As a result, SspB and ClpX work together to recognize ssrA-tagged substrates efficiently, whereas SspB inhibits recognition of these substrates by ClpA. Thus, dissection of the recognition signals within the ssrA tag provides insight into how multiple proteins function in concert to modulate proteolysis.


Subject(s)
Adenosine Triphosphatases/metabolism , Bacterial Proteins/metabolism , RNA, Bacterial/metabolism , Serine Endopeptidases/metabolism , Transcription Factors , ATPases Associated with Diverse Cellular Activities , Amino Acid Sequence , Endopeptidase Clp , Escherichia coli Proteins , Green Fluorescent Proteins , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Molecular Chaperones , Molecular Sequence Data , Mutagenesis , RNA, Bacterial/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
7.
J Am Chem Soc ; 123(21): 5003-13, 2001 May 30.
Article in English | MEDLINE | ID: mdl-11457328

ABSTRACT

Anion [CMo(N[R]Ar)(3)](-) (R = C(CD(3))(2)CH(3) or (t)Bu, Ar = 3,5-C(6)H(3)Me(2)) containing one-coordinate carbon as a terminal substituent and related molecules have been studied by single-crystal X-ray crystallography, solution and solid-state (13)C NMR spectroscopy, and density functional theory (DFT) calculations. Chemical reactivity patterns for [CMo(N[R]Ar)(3)](-) have been investigated, including the kinetics of proton-transfer self-exchange involving HCMo(N[R]Ar)(3), the carbidomolybdenum anion's conjugate acid. While the Mo triple bond C bond lengths in [K(benzo-15-crown-5)(2)][CMo(N[R]Ar)(3)] and the parent methylidyne, HCMo(N[R]Ar)(3), are statistically identical, the carbide chemical shift of delta 501 ppm is much larger than the delta 282 ppm shift for the methylidyne. Solid-state (13)C NMR studies show the carbide to have a much larger chemical shift anisotropy (CSA, 806 ppm) and smaller (95)Mo--(13)C coupling constant (60 Hz) than the methylidyne (CSA = 447 ppm, (1)J(MoC) = 130 Hz). DFT calculations on model compounds indicate also that there is an increasing MoC overlap population on going from the methylidyne to the terminal carbide. The pK(a) of methylidyne HCMo(N[R]Ar)(3) is approximately 30 in THF solution. Methylidyne HCMo(N[R]Ar)(3) and carbide [CMo(N[R]Ar)(3)](-) undergo extremely rapid proton-transfer self-exchange reactions in THF, with k = 7 x 10(6) M(-1) s(-1). Besides being a strong reducing agent, carbide [CMo(N[R]Ar)(3)](-) reacts as a nucleophile with elemental chalcogens to form carbon-chalcogen bonds and likewise reacts with PCl(3) to furnish a carbon-phosphorus bond.

8.
EMBO J ; 20(12): 3092-100, 2001 Jun 15.
Article in English | MEDLINE | ID: mdl-11406586

ABSTRACT

ClpXP is an ATP-dependent protease that denatures native proteins and translocates the denatured polypeptide into an interior peptidase chamber for degradation. To address the mechanism of these processes, Arc repressor variants with dramatically different stabilities and unfolding half-lives varying from months to seconds were targeted to ClpXP by addition of the ssrA degradation tag. Remarkably, ClpXP degraded each variant at a very similar rate and hydrolyzed approximately 150 molecules of ATP for each molecule of substrate degraded. The hyperstable substrates did, however, slow the ClpXP ATPase cycle. These results confirm that ClpXP uses an active mechanism to denature its substrates, probably one that applies mechanical force to the native structure. Furthermore, the data suggest that denaturation is inherently inefficient or that significant levels of ATP hydrolysis are required for other reaction steps. ClpXP degraded disulfide-cross-linked dimers efficiently, even when just one subunit contained an ssrA tag. This result indicates that the pore through which denatured proteins enter the proteolytic chamber must be large enough to accommodate simultaneous passage of two or three polypeptide chains.


Subject(s)
Adenosine Triphosphatases/metabolism , DNA-Binding Proteins/metabolism , Protein Folding , RNA, Bacterial/metabolism , Repressor Proteins/metabolism , Serine Endopeptidases/metabolism , Viral Proteins/metabolism , Adenosine Triphosphatases/physiology , Adenosine Triphosphate/metabolism , DNA-Binding Proteins/genetics , Dimerization , Endopeptidase Clp , RNA, Bacterial/genetics , Repressor Proteins/genetics , Serine Endopeptidases/physiology , Substrate Specificity , Viral Proteins/genetics , Viral Regulatory and Accessory Proteins
9.
Arterioscler Thromb Vasc Biol ; 21(6): 1017-22, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11397713

ABSTRACT

Endothelium-dependent relaxations mediated by NO are impaired in a mouse model of human atherosclerosis. Our objective was to characterize the mechanisms underlying endothelial dysfunction in aortas of apolipoprotein E (apoE)-deficient mice, treated for 26 to 29 weeks with a lipid-rich Western-type diet. Aortic rings from apoE-deficient mice showed impaired endothelium-dependent relaxations to acetylcholine (10(-)(9) to 10(-)(5) mol/L) and Ca(2+) ionophore (10(-)(9) to 10(-)(6) mol/L) and endothelium-independent relaxations to diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate, 10(-)(10) to 10(-)(5) mol/L) compared with aortic rings from C57BL/6J mice (P<0.05). By use of confocal microscopy of an oxidative fluorescent probe (dihydroethidium), increased superoxide anion (O(2)(-)) production was demonstrated throughout the aortic wall but mainly in smooth muscle cells of apoE-deficient mice. CuZn-superoxide dismutase (SOD) and Mn-SOD protein expressions were unaltered in the aorta exposed to hypercholesterolemia. A cell-permeable SOD mimetic, Mn(III) tetra(4-benzoic acid) porphyrin chloride (10(-)(5) mol/L), reduced O(2)(-) production and partially normalized relaxations to acetylcholine and DEA-NONOate in apoE-deficient mice (P<0.05). [(14)C]L-Citrulline assay showed a decrease of Ca(2+)-dependent NOS activity in aortas from apoE-deficient mice compared with C57BL/6J mice (P<0.05), whereas NO synthase protein expression was unchanged. In addition, cGMP levels were significantly reduced in the aortas of apoE-deficient mice (P<0.05). Our results demonstrate that in apoE-deficient mice on a Western-type fat diet, impairment of endothelial function is caused by increased production of O(2)(-) and reduced endothelial NO synthase enzyme activity. Thus, chemical inactivation of NO with O(2)(-) and reduced biosynthesis of NO are key mechanisms responsible for endothelial dysfunction in aortas of atherosclerotic apoE-deficient mice.


Subject(s)
Apolipoproteins E/genetics , Arteriosclerosis/physiopathology , Endothelium, Vascular/metabolism , Animals , Aorta/drug effects , Aorta/metabolism , Aorta/physiopathology , Arteriosclerosis/metabolism , Blotting, Western , Calcium/metabolism , Culture Techniques , Cyclic AMP/biosynthesis , Cyclic GMP/biosynthesis , Male , Metalloporphyrins/pharmacology , Mice , Mice, Inbred C57BL , Mice, Knockout , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II , Nitric Oxide Synthase Type III , Superoxide Dismutase/immunology , Superoxide Dismutase/metabolism , Superoxides/metabolism , Vasoconstriction , Vasodilation
10.
Protein Sci ; 10(3): 551-9, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11344323

ABSTRACT

The ClpA, ClpB, and ClpC subfamilies of the Clp/HSP100 ATPases contain a conserved N-terminal region of approximately 150 residues that consists of two approximate sequence repeats. This sequence from the Escherichia coli ClpA enzyme is shown to encode an independent structural domain (the R domain) that is monomeric and approximately 40% alpha-helical. A ClpA fragment lacking the R domain showed ATP-dependent oligomerization, protein-stimulated ATPase activity, and the ability to complex with the ClpP peptidase and mediate degradation of peptide and protein substrates, including casein and ssrA-tagged proteins. Compared with the activities of the wild-type ClpA, however, those of the ClpA fragment missing the R domain were reduced. These results indicate that the R domain is not required for the basic recognition, unfolding, and translocation functions that allow ClpA-ClpP to degrade some protein substrates, but they suggest that it may play a role in modulating these activities.


Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Escherichia coli Proteins , Escherichia coli/enzymology , Mutagenesis/genetics , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Terminal Repeat Sequences , Amino Acid Sequence , Biophysical Phenomena , Biophysics , Endopeptidase Clp , Enzyme Activation , Escherichia coli/classification , Escherichia coli/genetics , Hydrolysis , Protein Structure, Tertiary , Sequence Deletion/genetics , Sequence Homology
11.
Mol Microbiol ; 40(1): 141-55, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11298282

ABSTRACT

The Mu B protein is an ATP-dependent DNA-binding protein and an allosteric activator of the Mu transposase. As a result of these activities, Mu B is instrumental in efficient transposition and target-site choice. We analysed in vivo the role of Mu B in the two different recombination reactions performed by phage Mu: non-replicative transposition, the pathway used during integration, and replicative transposition, the pathway used during lytic growth. Utilizing a sensitive PCR-based assay for Mu transposition, we found that Mu B is not required for integration, but enhances the rate and extent of the process. Furthermore, three different mutant versions of Mu B, Mu BC99Y, Mu BK106A, and Mu B1-294, stimulate integration to a similar level as the wild-type protein. In contrast, these mutant proteins fail to support Mu growth. This deficiency is attributable to a defect in formation of an essential intermediate for replicative transposition. Biochemical analysis of the Mu B mutant proteins reveals common features: the mutants retain the ability to stimulate transposase, but are defective in DNA binding and target DNA delivery. These data indicate that activation of transposase by Mu B is sufficient for robust non-replicative transposition. Efficient replicative transposition, however, demands that the Mu B protein not only activate transposase, but also bind and deliver the target DNA.


Subject(s)
Bacteriophage mu/physiology , DNA-Binding Proteins/physiology , Viral Proteins , Virus Integration/physiology , Virus Replication/physiology , Base Sequence , DNA Primers
12.
Nat Struct Biol ; 8(4): 302-7, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11276247

ABSTRACT

Transposases and retroviral integrases promote the movement of DNA segments to new locations within and between genomes. These recombinases function as multimeric protein-DNA complexes. Recent success in solving the crystal structure of a Tn5 transposase--DNA complex provides the first detailed structural information about a member of the transposase/integrase superfamily in its active, DNA-bound state. Here, we summarize the reactions catalyzed by transposases and integrases and review the Tn5 transposase-DNA co-crystal structure. The insights gained from the Tn5 structure and other available structures are considered together with biochemical and genetic data to discuss features that are likely to prove common to the catalytic complexes used by members of this important protein family.


Subject(s)
DNA Transposable Elements , Integrases/chemistry , Retroviridae/enzymology , Transposases/chemistry , Binding Sites , Crystallography, X-Ray , DNA/genetics , DNA/metabolism , Integrases/genetics , Integrases/metabolism , Metals/metabolism , Models, Molecular , Protein Conformation , Transposases/genetics , Transposases/metabolism
13.
J Cardiovasc Pharmacol ; 37(3): 333-8, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11243424

ABSTRACT

Vitamin C has long been known for its beneficial vascular effects, but its mechanism of action remains unclear. Recent reports suggest that vitamin C may prevent endothelial dysfunction by scavenging free radicals and increasing the bioavailability of nitric oxide. To investigate this area further, we studied the effect of vitamin C (10(-4) M) and Mn(III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP; 10(-5) M), a scavenger of superoxide, hydrogen peroxide, and peroxynitrite, on endothelial nitric oxide synthase (eNOS) enzymatic activity in cultured human umbilical vein endothelial cells. L-Citrulline formation (a measure of eNOS enzymatic activity) was significantly increased in cells treated for 24 h with vitamin C. No effect was observed after MnTBAP treatment. Chronic administration of vitamin C also had no effect on eNOS protein expression. Treatment with vitamin C for 24 h significantly increased levels of the eNOS co-factor tetrahydrobiopterin (BH4), whereas MnTBAP did not affect its levels. Sepiapterin (10(-4) M), a precursor of BH4, significantly increased eNOS activity, whereas addition of vitamin C to cells treated with sepiapterin did not cause any further increase in eNOS activity. Our results suggest that the beneficial effect of vitamin C on endothelial function is best explained by increased intracellular BH4 content and subsequent enhancement of eNOS activity. This effect appears to be independent of the ability of vitamin C to scavenge superoxide anions.


Subject(s)
Ascorbic Acid/pharmacology , Biopterins/analogs & derivatives , Biopterins/metabolism , Endothelium, Vascular/metabolism , Cells, Cultured , GTP Cyclohydrolase/metabolism , Humans , Immunoblotting , Metalloporphyrins/pharmacology , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type III
14.
Nat Struct Biol ; 8(3): 230-3, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11224567

ABSTRACT

The Clp/Hsp100 ATPases are hexameric protein machines that catalyze the unfolding, disassembly and disaggregation of specific protein substrates in bacteria, plants and animals. Many family members also interact with peptidases to form ATP-dependent proteases. In Escherichia coli, for instance, the ClpXP protease is assembled from the ClpX ATPase and the ClpP peptidase. Here, we have used multiple sequence alignments to identify a tripeptide 'IGF' in E. coli ClpX that is essential for ClpP recognition. Mutations in this IGF sequence, which appears to be part of a surface loop, disrupt ClpXP complex formation and prevent protease function but have no effect on other ClpX activities. Homologous tripeptides are found only in a subset of Clp/Hsp100 ATPases and are a good predictor of family members that have a ClpP partner. Mapping of the IGF loop onto a homolog of known structure suggests a model for ClpX-ClpP docking.


Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Escherichia coli Proteins , Escherichia coli/enzymology , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/metabolism , Protozoan Proteins/chemistry , Protozoan Proteins/metabolism , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , ATP-Dependent Proteases , Adenosine Triphosphatases/genetics , Amino Acid Sequence , Binding Sites , Endopeptidase Clp , Endopeptidases/chemistry , Escherichia coli/genetics , Heat-Shock Proteins/genetics , Macromolecular Substances , Models, Molecular , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Molecular Sequence Data , Protein Binding , Protein Structure, Quaternary , Protozoan Proteins/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Serine Endopeptidases/genetics
15.
Nat Struct Biol ; 8(5): 302-7, 2001 May.
Article in English | MEDLINE | ID: mdl-11774877

ABSTRACT

Transposases and retroviral integrases promote the movement of DNA segments to new locations within and between genomes. These recombinases function as multimeric protein-DNA complexes. Recent success in solving the crystal structure of a Tn5 transposase--DNA complex provides the first detailed structural information about a member of the transposase/integrase superfamily in its active, DNA-bound state. Here, we summarize the reactions catalyzed by transposases and integrases and review the Tn5 transposase-DNA co-crystal structure. The insights gained from the Tn5 structure and other available structures are considered together with biochemical and genetic data to discuss features that are likely to prove common to the catalytic complexes used by members of this important protein family.


Subject(s)
DNA Transposable Elements , Integrases/chemistry , Retroviridae/enzymology , Transposases/chemistry , Binding Sites , Crystallography, X-Ray , DNA/genetics , DNA/metabolism , Integrases/genetics , Integrases/metabolism , Metals/metabolism , Models, Molecular , Protein Conformation , Transposases/genetics , Transposases/metabolism
16.
Science ; 289(5488): 2354-6, 2000 Sep 29.
Article in English | MEDLINE | ID: mdl-11009422

ABSTRACT

Events that stall bacterial protein synthesis activate the ssrA-tagging machinery, resulting in resumption of translation and addition of an 11-residue peptide to the carboxyl terminus of the nascent chain. This ssrA-encoded peptide tag marks the incomplete protein for degradation by the energy-dependent ClpXP protease. Here, a ribosome-associated protein, SspB, was found to bind specifically to ssrA-tagged proteins and to enhance recognition of these proteins by ClpXP. Cells with an sspB mutation are defective in degrading ssrA-tagged proteins, demonstrating that SspB is a specificity-enhancing factor for ClpXP that controls substrate choice.


Subject(s)
Adenosine Triphosphatases/metabolism , Bacterial Proteins/metabolism , Escherichia coli Proteins , Escherichia coli/metabolism , Oligopeptides/metabolism , Serine Endopeptidases/metabolism , Bacterial Proteins/genetics , Endopeptidase Clp , Escherichia coli/enzymology , Green Fluorescent Proteins , Luminescent Proteins/metabolism , Mutation , Oligopeptides/chemistry , Oligopeptides/genetics , Operon , Ribosomes/metabolism , Substrate Specificity
17.
Science ; 289(5476): 73-4, 2000 Jul 07.
Article in English | MEDLINE | ID: mdl-10928934

ABSTRACT

Transposable DNA elements jump from one location in the genome to another. But, the cut-and-paste molecular machinations that support this nomadic lifestyle are still being unraveled. In their Perspective, Williams and Baker at the Massachusetts Institute of Technology discuss new details of transposon relocation revealed through resolution of the structure of a transposase enzyme bound to DNA (Davies et al.).


Subject(s)
DNA Transposable Elements , DNA/chemistry , DNA/metabolism , Transposases/chemistry , Transposases/metabolism , Amino Acid Motifs , Binding Sites , Catalysis , Crystallography, X-Ray , Ligands , Manganese/metabolism , Nucleic Acid Conformation , Protein Conformation
18.
Mol Cell ; 5(4): 639-48, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10882100

ABSTRACT

ClpXP is a protein machine composed of the ClpX ATPase, a member of the Clp/Hsp100 family of remodeling enzymes, and the ClpP peptidase. Here, ClpX and ClpXP are shown to catalyze denaturation of GFP modified with an ssrA degradation tag. ClpX translocates this denatured protein into the proteolytic chamber of ClpP and, when proteolysis is blocked, also catalyzes release of denatured GFP-ssrA from ClpP in a reaction that requires ATP and additional substrate. Kinetic experiments reveal that multiple reaction steps require collaboration between ClpX and ClpP and that denaturation is the rate-determining step in degradation. These insights into the mechanism of ClpXP explain how it executes efficient degradation in a manner that is highly specific for tagged proteins, irrespective of their intrinsic stabilities.


Subject(s)
Adenosine Triphosphatases/metabolism , Escherichia coli Proteins , Molecular Chaperones/metabolism , Protein Processing, Post-Translational , Serine Endopeptidases/metabolism , ATPases Associated with Diverse Cellular Activities , Adenosine Triphosphate/metabolism , Bacterial Proteins/metabolism , Endopeptidase Clp , Escherichia coli , Green Fluorescent Proteins , Luminescent Proteins/metabolism , Models, Theoretical , Oligopeptides/metabolism , Protein Binding , Protein Denaturation , Protein Folding
19.
Genes Dev ; 13(20): 2725-37, 1999 Oct 15.
Article in English | MEDLINE | ID: mdl-10541558

ABSTRACT

Movement of transposable genetic elements requires the cleavage of each end of the element genome and the subsequent joining of these cleaved ends to a new target DNA site. During Mu transposition, these reactions are catalyzed by a tetramer of four identical transposase subunits bound to the paired Mu DNA ends. To elucidate the organization of active sites within this tetramer, the subunit providing the essential active site DDE residues for each cleavage and joining reaction was determined. We demonstrate that recombination of the two Mu DNA ends is catalyzed by two active sites, where one active site promotes both cleavage and joining of one Mu DNA end. This active site uses all three DDE residues from the subunit bound to the transposase binding site proximal to the cleavage site on the other Mu DNA end (catalysis in trans). In addition, we uncover evidence that the catalytic activity of these two active sites is coupled such that the coordinated joining of both Mu DNA ends is favored during recombination. On the basis of these results, we propose that the DNA joining stage requires a cooperative transition within the transposase-DNA complex. The cooperative utilization of active sites supplied in trans by Mu transposase provides an example of how mobile elements can ensure concomitant recombination of distant DNA sites.


Subject(s)
Bacteriophage mu/genetics , DNA Transposable Elements/genetics , Recombination, Genetic , Bacteriophage mu/metabolism , Base Sequence , Binding Sites/genetics , DNA Primers/genetics , DNA, Viral/genetics , DNA, Viral/metabolism , Molecular Sequence Data , Protein Structure, Quaternary , Transposases/chemistry , Transposases/metabolism
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