[25] ATP-dependent protease La (Lon) from Escherichia coli

Goldberg, Alfred L.; Moerschell, Richard P.; Chung, Chin Ha; Maurizi, Michael R.

doi:10.1016/0076-6879(94)44027-1

Cited by 180 publications

(229 citation statements)

References 46 publications

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“…This implies there is a difference in the substrate specificity of the two homologs. This is not unexpected, as the two localize differently and would degrade a vastly different protein pool (8,16). We conclude that a peptide-based approach to developing an inhibitor would be more useful as the observed difference in substrate specificity could potentially be exploited to target an inhibitor specifically to bacterial Lon, thereby decreasing the chance for side-effects resulting from cross-reactivity with the human homolog.…”

Section: Discussionmentioning

confidence: 99%

“…Homologs exist ubiquitously in nature, however they localize to the cytosol in prokaryotes and to the mitochondrial matrix in eukaryotes (8,15,16 (11,(18)(19)(20)(21)(22)(23).…”

Section: Introductionmentioning

confidence: 99%

“…In fact, Lon-deficient S. Typhimurium, when administered as an oral vaccine to mice, conferred subsequent protection against infection by virulent S. Typhimurium (4). Taken together, these studies highlight Lon as an important target in the development of novel therapeutic agents.Lon, also known as the protease La, is a homo-oligomeric ATP-dependent serine protease, which functions in the degradation of damaged and certain short-lived regulatory proteins (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Homologs exist ubiquitously in nature, however they localize to the cytosol in prokaryotes and to the mitochondrial matrix in eukaryotes (8,15,16 (11,(18)(19)(20)(21)(22)(23).…”

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confidence: 99%

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Identification of the Proteasome Inhibitor MG262 as a Potent ATP-Dependent Inhibitor of the Salmonella enterica serovar Typhimurium Lon Protease

2006

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Lon is a homo-oligomeric ATP-dependent serine protease which functions in the degradation of damaged and certain regulatory proteins. The importance of Lon activity in bacterial pathogenicity has led to its emergence as a target in the development of novel antibiotics. As no potent inhibitors of Lon activity have been reported to date, we sought to identify an inhibitor which could serve as a lead compound in the development of a potent Lon-specific inhibitor. To determine whether a nucleotide-or peptide-based inhibitor would be more effective, we evaluated the steady-state kinetic parameters associated with both ATP and peptide hydrolysis by human and Salmonella enterica serovar Typhimurium Lon. Although the ATP hydrolysis activities of both homologs are kinetically indistinguishable, they display marked differences in peptide substrate specificity. This suggests that a peptide-based inhibitor could be developed which would target bacterial Lon, thereby decreasing side-effects due to cross-reactivity with human Lon. Using Salmonella enterica serovar Typhimurium Lon as a model, we evaluated the IC 50 values of a series of commercially available peptide-based inhibitors. Those inhibitors which behave as transition state analogs were the most useful in inhibiting Lon activity. The peptidyl boronate, MG262, was the most potent inhibitor tested (IC 50 = 122 ± 9 nM) and required binding, but not hydrolysis of ATP to initiate inhibition. We hope to use MG262 as a lead compound in the development of future Lon specific inhibitors.The number of pathogenic, antibiotic resistant bacteria increases each year, however the development of new antibiotics to treat them lags behind (1). Recent studies aimed at identifying proteins necessary for virulence have implicated the importance of Lon protease (2,3). Pathogenic Salmonella enterica are responsible for causing a range of human diseases from mild gastroenteritis (serovar Typhimurium and serovar Enteritidis) to typhoid fever (serovar Typhi). It has been demonstrated that Salmonella enteria serovar Typhimurium (S. Typhimurium) Lon protease activity is required for systemic infection in mice, a common study model for S. Typhi infection in humans (3). In fact, Lon-deficient S. Typhimurium, when administered as an oral vaccine to mice, conferred subsequent protection against infection by virulent S. Typhimurium (4). Taken together, these studies highlight Lon as an important target in the development of novel therapeutic agents.Lon, also known as the protease La, is a homo-oligomeric ATP-dependent serine protease, which functions in the degradation of damaged and certain short-lived regulatory proteins (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Homologs exist ubiquitously in nature, however they localize to the cytosol in prokaryotes and to the mitochondrial matrix in eukaryotes (8,15,16 (11,(18)(19)(20)(21)(22)(23).Lon protease is a member of the AAA + superfamily (ATPases Associated with different cellular Activities) along with other ATP-dependent proteases such as ClpXP, H...

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Section: Discussionmentioning

confidence: 99%

“…Homologs exist ubiquitously in nature, however they localize to the cytosol in prokaryotes and to the mitochondrial matrix in eukaryotes (8,15,16 (11,(18)(19)(20)(21)(22)(23).…”

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Identification of the Proteasome Inhibitor MG262 as a Potent ATP-Dependent Inhibitor of the Salmonella enterica serovar Typhimurium Lon Protease

2006

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“…(4) where t is time in seconds, Y is [ADP] in μM, A is the amplitude in μM, k obs is the observed rate constant in s −1 , and C is the end point.…”

Section: (3)mentioning

confidence: 99%

Single-Turnover Kinetic Experiments Confirm the Existence of High- and Low-Affinity ATPase Sites in Escherichia coli Lon Protease

2006

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Lon is an ATP-dependent serine protease which degrades damaged and certain regulatory proteins in vivo. Lon exists as a homo-oligomer and represents one of the simplest ATP dependent proteases because both the protease and ATPase domains are located within each monomeric subunit. Previous pre-steady state kinetic studies revealed functional non-equivalency in the ATPase activity of the enzyme [Vineyard, D., et al. (2005) Biochemistry 44, 1671. Both a high and low affinity ATPase site has been previously reported for Lon [Menon, A.S., & Goldberg, A.L. (1987) J Biol Chem 262, 14921-8]. Because of the differing affinities for ATP, we were able to monitor the activities of the sites separately and determine that they were non-interacting. The high affinity sites hydrolyze ATP very slowly (k obs =0.019 ± 0.002s −1 ), while the low affinity sites hydrolyze ATP quickly at a rate of 17.2 ± 0.09s −1 which is comparable to the previously observed burst rate. Although the high affinity sites hydrolyze ATP slowly they support multiple rounds of peptide hydrolysis, indicating that ATP and peptide hydrolysis are not stoichiometrically linked. However, ATP binding and hydrolysis at both the high and low affinity sites is necessary for optimal peptide cleavage and the stabilization of the conformational change associated with nucleotide binding.Lon is an ATP dependent serine protease functioning to degrade damaged and certain regulatory proteins in vivo (1-10). Lon belongs to the ATPases associated with a variety of cellular activities (AAA + ) superfamily whose members include ClpAP, ClpXP, ClpCP, and HslVU (11,12). They share a conserved Walker A (or P-loop) and Walker B motif which is associated with nucleotide binding and hydrolysis (13). Lon represents one of the simplest of the ATP dependent proteases because both the protease and ATPase domains are located within each monomeric subunit (14,15). Crystal structures of portions of the enzyme have been recently reported, and include an inactive mutant of the Lon protease domain (16)(17)(18). This structure shows Lon as a hexamer organized in a ring with a central cavity which is commonly found in other ATP dependent proteases (11,13,17). Although it is known that ATP modulates Lon's protease activity (4,5,7,19), mechanistic details concerning how the binding and hydrolysis of ATP is coordinated with peptide bond cleavage is not known. However, it has been shown that ATP binding and hydrolysis does not affect the oligomeric state of the enzyme (20,21).We have previously developed a continuous fluorescent peptidase assay to monitor the kinetics of peptide cleavage. As the inner filter effect of fluorescence interferes at high concentrations of 100% fluorescent peptide, we used S3, a 10% mixture of fluorescently labeled peptide with its non-fluorescent analog (S2) (22). No optical signal from the peptide is needed when monitoring ATPase activity so only the non-fluorescent analog (S2) is used to account for the *Corresponding author, email: Irene.lee@case.edu, NIH Pub...

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“…ClpA , ClpP (Kim et al 2000), ClpX (Levchenko et al 1997), FtsH (Herman et al 2003), HslU, HslV (Burton et al 2005), Lon (Goldberg et al 1994), RseA 1-108 , and E (Flynn et al 2004) were purified as described.…”

Section: Proteinsmentioning

confidence: 99%

Design principles of the proteolytic cascade governing the σ^E-mediated envelope stress response in Escherichia coli: keys to graded, buffered, and rapid signal transduction

Chaba¹,

Grigorova²,

Flynn³

et al. 2007

Genes Dev.

104

119

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Proteolytic cascades often transduce signals between cellular compartments, but the features of these cascades that permit efficient conversion of a biological signal into a transcriptional output are not well elucidated. E mediates an envelope stress response in Escherichia coli, and its activity is controlled by regulated degradation of RseA, a membrane-spanning anti-factor. Examination of the individual steps in this protease cascade reveals that the initial, signal-sensing cleavage step is rate-limiting; that multiple ATP-dependent proteases degrade the cytoplasmic fragment of RseA and that dissociation of E from RseA is so slow that most free E must be generated by the active degradation of RseA. As a consequence, the degradation rate of RseA is set by the amount of inducing signal, and insulated from the "load" on and activity of the cytoplasmic proteases. Additionally, changes in RseA degradation rate are rapidly reflected in altered E activity. These design features are attractive as general components of signal transduction pathways governed by unstable negative regulators. Proteolytic cascades are widely used to transduce signals across membranes to enable cells to respond to environmental stress and coordinate processes in different cellular compartments. However, the molecular properties of these cascades that facilitate the desired outputs have rarely been examined. In this work, we examine the individual steps in the protease cascade governing the activity of the E -mediated envelope stress response in Escherichia coli to determine the construction features of this cascade that facilitate faithful transmission of signal and a rapid output.E directs RNA polymerase to transcribe genes encoding proteins that ensure the synthesis, assembly, and homeostasis of outer membrane porins and lipopolysaccharide, the two major components of the unique outer membrane of Gram-negative bacteria (Dartigalongue et al. 2001;Rezuchova et al. 2003;Rhodius et al. 2006). Envelope integrity is required under all growth conditions, and E is an essential transcription factor (De Las Penas et al. 1997a). Perturbations in the integrity and protein-folding state of the envelope caused by temperature upshift, chaperone depletion, or accumulation of unassembled porins increase E activity; conversely, temperature downshift and/or depletion of porins decrease E activity (Mecsas et al. 1993;Hiratsu et al. 1995;Raina et al. 1995;Rouviere et al. 1995;Missiakas et al. 1996;Rouviere and Gross 1996;Ades et al. 2003).The components of the signal transduction system that control E activity are shown in Figure 1. RseA, a membrane-spanning anti-factor, inhibits E activity. The cytoplasmic domain of RseA (RseA 1-108 ) binds to E and its periplasmic domain binds to RseB (De Las Penas et al. 1997b;Missiakas et al. 1997

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[25] ATP-dependent protease La (Lon) from Escherichia coli

Cited by 180 publications

References 46 publications

Identification of the Proteasome Inhibitor MG262 as a Potent ATP-Dependent Inhibitor of the Salmonella enterica serovar Typhimurium Lon Protease

Identification of the Proteasome Inhibitor MG262 as a Potent ATP-Dependent Inhibitor of the Salmonella enterica serovar Typhimurium Lon Protease

Single-Turnover Kinetic Experiments Confirm the Existence of High- and Low-Affinity ATPase Sites in Escherichia coli Lon Protease

Design principles of the proteolytic cascade governing the σ^E-mediated envelope stress response in Escherichia coli: keys to graded, buffered, and rapid signal transduction

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[25] ATP-dependent protease La (Lon) from Escherichia coli

Cited by 180 publications

References 46 publications

Identification of the Proteasome Inhibitor MG262 as a Potent ATP-Dependent Inhibitor of the Salmonella enterica serovar Typhimurium Lon Protease

Identification of the Proteasome Inhibitor MG262 as a Potent ATP-Dependent Inhibitor of the Salmonella enterica serovar Typhimurium Lon Protease

Single-Turnover Kinetic Experiments Confirm the Existence of High- and Low-Affinity ATPase Sites in Escherichia coli Lon Protease

Design principles of the proteolytic cascade governing the σE-mediated envelope stress response in Escherichia coli: keys to graded, buffered, and rapid signal transduction

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Design principles of the proteolytic cascade governing the σ^E-mediated envelope stress response in Escherichia coli: keys to graded, buffered, and rapid signal transduction