Protein unfolding and degradation by the AAA+ Lon protease

Gur, Eyal; Vishkautzan, Marina; Sauer, Robert T.

doi:10.1002/pro.2013

Cited by 40 publications

(49 citation statements)

References 102 publications

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“…Consistent with an adaptor function, SmiA facilitates Londependent proteolysis of SwrA both in vivo and in vitro. Unlike known allosteric activators of Lon, such as unfolded protein substrates, SmiA does not simply enhance general proteolytic activity, as the presence of SmiA did not accelerate the LonAdependent degradation of α-casein or titin (13,16,44). Further, SmiA did not function as a generalized unfolding chaperone, as SmiA did not promote SwrA proteolysis by the heterologous Lon protease from E. coli.…”

Section: Discussionmentioning

confidence: 89%

Adaptor-mediated Lon proteolysis restrictsBacillus subtilishyperflagellation

Mukherjee

Bree

Liu

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The Lon AAA+ protease is a highly conserved intracellular protease that is considered an anticancer target in eukaryotic cells and a crucial virulence regulator in bacteria. Lon degrades both damaged, misfolded proteins and specific native regulators, but how Lon discriminates among a large pool of candidate targets remains unclear. Here we report that Bacillus subtilis LonA specifically degrades the master regulator of flagellar biosynthesis SwrA governed by the adaptor protein swarming motility inhibitor A (SmiA). SmiA-dependent LonA proteolysis is abrogated upon microbe-substrate contact causing SwrA protein levels to increase and elevate flagellar density above a critical threshold for swarming motility atop solid surfaces. Surface contact-dependent cellular differentiation in bacteria is rapid, and regulated proteolysis may be a general mechanism of transducing surface stimuli.

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Section: Discussionmentioning

confidence: 89%

Adaptor-mediated Lon proteolysis restrictsBacillus subtilishyperflagellation

Mukherjee

Bree

Liu

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…These proteases mostly recognize their substrates through sequence motifs at the N or C termini of the substrate proteins, and different proteases appear to have overlapping specificities (1)(2)(3). Additionally, these proteases appear to differ in their intrinsic ability to unfold and degrade substrate proteins, which may also be substrate-dependent, perhaps providing an additional contribution to the specificity of protein degradation (3)(4)(5)(6).…”

mentioning

confidence: 99%

Slippery Substrates Impair ATP-dependent Protease Function by Slowing Unfolding

Kraut

2013

Journal of Biological Chemistry

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Background: ATP-dependent proteases cannot fully degrade so-called "slippery" substrates. Results: Reanalysis indicates ClpXP has a reduced unfolding rate for slippery substrates, not a faster substrate release rate. Conclusion: The proteasome and ClpXP share a common mechanism of inhibition by slippery substrates. Significance: Comparison of the mechanisms of ATP-dependent proteases across species help explain how these unfolding machines work and evolve.

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“…For proteotoxic stress assays (28), the chloramphenicol resistance marker of pBAD33 was replaced with an ampicillin resistance marker from pSH21. Degron-tagged variants of the human titin I27 domain (8,14,29,30) were cloned into a pSH21 vector with an N-terminal His 6 tag. ␤20-cp6-SF GFP and cp6-SF GFP-sul20 (31) were cloned into a pCOLADuet1 vector with an N-terminal His 6 tag followed by a PreScission protease site.…”

Section: Methodsmentioning

confidence: 99%

“…Following purification, protein preparations had less than 5% contaminants, determined on the basis of Coomassie blue staining after SDS-PAGE. Assays for degradation and ATP hydrolysis were performed as described previously (8,14,30,31). Binding of a fluorescently labeled sul20 peptide to proteolytically inactive Lon S679A was assayed by determination of changes in fluorescence anisotropy.…”

Section: Methodsmentioning

confidence: 99%

A Mutation in the N Domain of Escherichia coli Lon Stabilizes Dodecamers and Selectively Alters Degradation of Model Substrates

2013

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Escherichia coli Lon, an ATP-dependent AAA ؉ protease, recognizes and degrades many different substrates, including the RcsA and SulA regulatory proteins. More than a decade ago, the E240K mutation in the N domain of Lon was shown to prevent degradation of RcsA but not SulA in vivo. Here, we characterize the biochemical properties of the E240K mutant in vitro and present evidence that the effects of this mutation are complex. For example, Lon E240K exists almost exclusively as a dodecamer, whereas wild-type Lon equilibrates between hexamers and dodecamers. Moreover, Lon E240K displays degradation defects in vitro that do not correlate in any simple fashion with degron identity, substrate stability, or dodecamer formation. The Lon sequence segment near residue 240 is known to undergo nucleotide-dependent conformational changes, and our results suggest that this region may be important for coupling substrate binding with allosteric activation of Lon protease and ATPase activity. A AAϩ proteases play important biological roles in all organisms (1). Inhibition of the AAA ϩ Lon protease reduces or eliminates virulence in several pathogenic bacteria and is toxic to human lymphoma cells, whereas Lon overexpression improves the life span of some fungi but kills Escherichia coli (2-7). Like most AAA ϩ proteases, bacterial Lon recognizes substrates by binding to specific amino acid sequences called degrons or degradation tags (8, 9). In E. coli, Lon degrades native regulatory proteins, including the RcsA transcription factor and the SulA inhibitor of cell division, but also appears to be responsible for degrading the majority of misfolded proteins, including ␤-galactosidase (10-12). The degron that targets RcsA for Lon degradation is unknown, whereas degrons for SulA (called sul20) and ␤-galactosidase (called ␤20) have been indentified (8, 13). The binding of different degrons stabilizes enzyme conformations with high or low protease activities, and the latter state may allow Lon to function as a chaperone (14).E. coli Lon subunits assemble into a homohexamer, which appears to be the minimal unit of proteolytic function (15). Each subunit contains an N-terminal domain, a central AAA ϩ module, and a C-terminal peptidase domain (16,17). The active sites for peptide bond cleavage are sequestered within a bowl-like chamber formed by the peptidase domains of the hexamer (17, 18). The hexameric AAA ϩ ring of Lon appears to regulate access to this chamber by coupling ATP hydrolysis to conformational changes that unfold and translocate substrates through an axial pore and into the chamber (1). The N domain of Lon is required for stable hexamer formation (19,20), and recent studies suggest that it also stabilizes a dodecamer, with hexamers and dodecamers being populated at physiological Lon concentrations (21). The dodecamer degrades certain substrates as well as the hexamer but degrades other substrates, including casein, at substantially reduced rates (21). Although crystal structures are known for most individual domains of Lon...

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Protein unfolding and degradation by the AAA+ Lon protease

Cited by 40 publications

References 102 publications

Adaptor-mediated Lon proteolysis restrictsBacillus subtilishyperflagellation

Adaptor-mediated Lon proteolysis restrictsBacillus subtilishyperflagellation

Slippery Substrates Impair ATP-dependent Protease Function by Slowing Unfolding

A Mutation in the N Domain of Escherichia coli Lon Stabilizes Dodecamers and Selectively Alters Degradation of Model Substrates

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