Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0162871 (abdominal aortic aneurysm)
 8,664 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Like other AAA proteins, Escherichia coli FtsH, a membrane-bound AAA protease, contains highly conserved aromatic and glycine residues (Phe228 and Gly230) that are predicted to lie in the central pore region of the hexamer. The functions of Phe228 and Gly230 were probed by site-directed mutagenesis. The results of both in vivo and in vitro assays indicate that these conserved pore residues are important for FtsH function and that bulkier, uncharged/apolar residues are essential at position 228. None of the point mutants, F228A, F228E, F228K, or G230A, was able to degrade sigma32, a physiological substrate. The F228A mutant was able to degrade casein, an unfolded substrate, although the other three mutants were not. Mutation of these two pore residues also affected the ATPase activity of FtsH. The F228K and G230A mutations markedly reduced ATPase activity, whereas the F228A mutation caused a more modest decrease in this activity. The F228E mutant was actually more active ATPase. The substrates, sigma32 and casein, stimulated the ATPase activity of wild type FtsH. The ATPase activity of the mutants was no longer stimulated by casein, whereas that of the three Phe228 mutants, but not the G230A mutant, remained sigma32-stimulatable. These results suggest that Phe228 and Gly230 in the predicted pore region of the FtsH hexamer have important roles in proteolysis and its coupling to ATP hydrolysis.
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PMID:Conserved pore residues in the AAA protease FtsH are important for proteolysis and its coupling to ATP hydrolysis. 1451 80

The Hsp100 protein ClpB is a member of the AAA+ protein family that mediates the solubilization of aggregated proteins in cooperation with the DnaK chaperone system. Unstructured polypeptides such as casein or poly-L-lysine have been shown to stimulate the ATPase activity of ClpB and thus may both act as substrates. Here we compared the effects of alpha-casein and poly-L-lysine on the ATPase and chaperone activities of ClpB. alpha-Casein stimulated ATP hydrolysis by both AAA domains of ClpB and inhibited the ClpB-dependent solubilization of aggregated proteins if present in excess. In contrast, poly-L-lysine stimulated exclusively the ATPase activity of the second AAA domain and increased the disaggregation activity of ClpB. Thus poly-L-lysine does not act as substrate, but rather represents an effector molecule, which enhances the chaperone activity of ClpB.
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PMID:Poly-L-lysine enhances the protein disaggregation activity of ClpB. 1455 May 59

NSF and p97 are related AAA proteins implicated in membrane trafficking and organelle biogenesis. p97 is also involved in pathways that lead to ubiquitin-dependent proteolysis, including ER-associated degradation (ERAD). In this study, we have used dominant interfering ATP-hydrolysis deficient mutants (NSF(E329Q) and p97(E578Q)) to compare the function of these AAA proteins in the secretory pathway of mammalian cells. Expressing NSF(E329Q) promotes disassembly of Golgi stacks into dispersed vesicular structures. It also rapidly inhibits glycosaminoglycan sulfation, reflecting disruption of intra-Golgi transport. In contrast, expressing p97(E578Q) does not affect Golgi structure or function; glycosaminoglycans are normally sulfated and secreted, as is the VSV-G ts045 protein. Instead, expression of p97(E578Q) causes ubiquitinated proteins to accumulate on ER membranes and slows degradation of the ERAD substrate cystic-fibrosis transmembrane-conductance regulator. In addition, expression of p97(E578Q) eventually causes the ER to swell. More specific assessment of effects of p97(E578Q) on organelle assembly shows that the Golgi apparatus disperses and reassembles normally after treatment with brefeldin A and during mitosis. These findings demonstrate that ATP-hydrolysis-dependent activities of NSF and p97 in the cell are not equivalent and suggest that only NSF is directly involved in regulating membrane fusion.
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PMID:Distinct roles for the AAA ATPases NSF and p97 in the secretory pathway. 1461 20

Early-onset torsion dystonia is an autosomal dominant movement disorder that has been linked to the deletion of one of a pair of glutamic acid residues in the protein torsinA (E(302/303); DeltaE-torsinA). In transfected cells, DeltaE-torsinA exhibits similar biochemical properties to wild type (WT)-torsinA, but displays a distinct subcellular localization. Primary structural analysis of torsinA suggests that this protein is a membrane-associated member of the AAA family of ATP-binding proteins. However, to date, neither WT- nor DeltaE-torsinA has been obtained in sufficient quantity and purity to permit detailed biochemical and biophysical characterization. Here, we report a baculovirus expression system that provides milligram quantities of purified torsin proteins. Recombinant WT- and DeltaE-torsinA were found to be membrane-associated glycoproteins that required detergents for solubilization and purification. Analysis of the biophysical properties of WT- and DeltaE-torsinA indicated that both proteins were folded monomers in solution that exhibited equivalent denaturation behaviors under thermal and chaotropic (guanidinium chloride) stress. Additionally, both forms of torsinA were found to display ATPase activity with similar k(cat) and K(m) values. Collectively, these data reveal that torsinA is a membrane-associated ATPase and indicate that the DeltaE(302/303) dystonia-associated mutation in this protein does not cause gross changes in its catalytic or structural properties. These findings are consistent with a disease mechanism in which DeltaE-torsinA promotes dystonia through a gain rather than loss of function. The recombinant expression system for torsinA proteins described herein should facilitate further biochemical and structural investigations to test this hypothesis.
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PMID:Recombinant expression, purification, and comparative characterization of torsinA and its torsion dystonia-associated variant Delta E-torsinA. 1469 Apr 43

The motor protein cytoplasmic dynein is responsible for most of the minus-end-directed microtubule traffic within cells. Dynein contains four evolutionarily conserved AAA (ATPase associated with various cellular activities) domains that are thought to bind nucleotide; the role of nucleotide binding and hydrolysis in each of these four AAA domains has constituted an important and unresolved question in understanding dynein's mechanism. Using Saccharomyces cerevisiae cytoplasmic dynein as a model system, we mutagenized residues involved in nucleotide binding or hydrolysis in the four AAA domains and examined the ability of the mutant dyneins to mediate nuclear segregation in vivo and to bind microtubules in vitro. Our analysis shows that an AAA1 hydrolysis mutant blocks dynein function, whereas a triple AAA2/3/4 hydrolysis mutant does not, suggesting that nucleotide binding is required at only one site. We also show that nucleotide binding at AAA3, but not hydrolysis, is essential for motor activity in vivo and ATP-induced dissociation of dynein from microtubules, suggesting that this domain acts as a critical allosteric site. In contrast, mutations in AAA2 cause subtle defects in dynein function, whereas mutation in AAA4 produce no obvious defects. These results show that the four conserved dynein AAA domains have distinct functions in dynein's mechanochemical cycle.
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PMID:Molecular dissection of the roles of nucleotide binding and hydrolysis in dynein's AAA domains in Saccharomyces cerevisiae. 1569 49

The yjoB gene of Bacillus subtilis encodes a 48.8-kDa protein belonging to the AAA family. Members of this family contain a 200-250-amino acid residues AAA domain carrying a Walker A and B ATP-binding site assumed to be part of a molecular chaperone. The yjoB gene belongs to the sigmaW regulon, and members of this regulon have been reported to be transiently induced when cells enter the stationary growth phase. This assumption was confirmed here for yjoB by Western blot experiments and by analysis of a transcriptional fusion. Purified YjoB protein exhibited ATPase activity but was unable to prevent aggregation of denatured citrate synthase. An alignment of YjoB with a subgroup of AAA proteins present in Archaea suggests that YjoB might be involved in the modulation of the activity of one or more proteases.
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PMID:The yjoB gene of Bacillus subtilis encodes a protein that is a novel member of the AAA family. 1475 46

The roles of two adjacent genes in the Staphylococcus aureus chromosome with functions in starvation survival and the response to stressful conditions have been characterized. One of these, hprT, encoding a hypoxanthine-guanine phosphoribosyltransferase homologue, was initially identified in a transposon mutagenesis screen. Mutation of hprT affects starvation survival in amino-acid-limiting conditions and the ability of S. aureus to grow in high-salt concentrations. Downstream of hprT is ftsH, which encodes a membrane-bound, ATP- and Zn(2+)-dependent 'AAA'-type protease. Mutation of ftsH in S. aureus leads to pleiotropic defects including slower growth, sensitivity to salt, acid, methyl viologen and potassium tellurite stresses, and reduced survival in amino-acid- or phosphate-limiting conditions. Both hprT-lacZ and ftsH-lacZ gene fusions are expressed maximally in the post-exponential phase of growth. Although secretion of exoproteins is not affected, an ftsH mutant is attenuated in a murine skin lesion model of pathogenicity.
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PMID:Role of the hprT-ftsH locus in Staphylococcus aureus. 1476 15

Initiation of DNA replication in eukaryotes, archea, and eubacteria requires interaction of structurally conserved ATP-binding initiator proteins and origin DNA to mediate assembly of replisomes. However, the specific requirement for ATP in the early steps of initiation remains unclear. This is true even for the well studied Escherichia coli replication origin, oriC, where the ATP form of initiator DnaA is necessary and sufficient for initial DNA strand separation, but the five DnaA-binding sites (R boxes) with consensus sequence 5'TGTGNAT/AAA bind both active ATP-DnaA and inactive ADP-DnaA with equal affinity. By using dimethyl sulfate footprinting, we recently identified two initiator-binding sites, I2 and I3, with sequence 5'TG/TGGATCAG/A. We now show that sites I2 and I3 preferentially bind DnaA-ATP and are required for origin unwinding. Guanine at position 3 determines DnaA-ATP preference, and changing this base to thymine at both I sites allows DnaA-ADP to bind and open oriC, although DNA strand separation is not precisely localized in the AT-rich region. These observations indicate that specific initiator binding sites within a replication origin can be important determinants of an ATP-dependent molecular switch regulating DNA strand separation.
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PMID:Two discriminatory binding sites in the Escherichia coli replication origin are required for DNA strand opening by initiator DnaA-ATP. 1497 87

ClpB is a member of the bacterial protein-disaggregating chaperone machinery and belongs to the AAA(+) superfamily of ATPases associated with various cellular activities. The mechanism of ClpB-assisted reactivation of strongly aggregated proteins is unknown and the oligomeric state of ClpB has been under discussion. Sedimentation equilibrium and sedimentation velocity show that, under physiological ionic strength in the absence of nucleotides, ClpB from Escherichia coli undergoes reversible self-association that involves protein concentration-dependent populations of monomers, heptamers, and intermediate-size oligomers. Under low ionic strength conditions, a heptamer becomes the predominant form of ClpB. In contrast, ATP gamma S, a nonhydrolyzable ATP analog, as well as ADP stabilize hexameric ClpB. Consistently, electron microscopy reveals that ring-type oligomers of ClpB in the absence of nucleotides are larger than those in the presence of ATP gamma S. Thus, the binding of nucleotides without hydrolysis of ATP produces a significant change in the self-association equilibria of ClpB: from reactions supporting formation of a heptamer to those supporting a hexamer. Our results show how ClpB and possibly other related AAA(+) proteins can translate nucleotide binding into a major structural transformation and help explain why previously published electron micrographs of some AAA(+) ATPases detected both six- and sevenfold particle symmetry.
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PMID:Nucleotide-induced switch in oligomerization of the AAA+ ATPase ClpB. 1497 98

Dyneins are highly complex molecular motors that transport their attached cargo towards the minus end of microtubules. These enzymes are required for many essential motile activities within the cytoplasm and also power eukaryotic cilia and flagella. Each dynein contains one or more heavy chain motor units that consist of an N-terminal stem domain that is involved in cargo attachment, and six AAA+ domains (AAA1-6) plus a C-terminal globular segment that are arranged in a heptameric ring. At least one AAA+ domain (AAA1) is capable of ATP binding and hydrolysis, and the available data suggest that one or more additional domains also may bind nucleotide. The ATP-sensitive microtubule binding site is located at the tip of a 10nm coiled coil stalk that emanates from between AAA4 and AAA5. The function of this motor both in the cytoplasm and the flagellum must be tightly regulated in order to result in useful work. Consequently, dyneins also contain a series of additional components that serve to define the cargo-binding properties of the enzyme and which act as sensors to transmit regulatory inputs to the motor units. Here we describe the two basic dynein designs and detail the various regulatory systems that impinge on this motor within the eukaryotic flagellum.
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PMID:Design and regulation of the AAA+ microtubule motor dynein. 1503 37


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