PinA Inhibits ATP Hydrolysis and Energy-dependent Protein Degradation by Lon Protease

Hilliard, Jamese J.; Simon, Lee D.; Melderen, Laurence Van; Maurizi, Michael R.

doi:10.1074/jbc.273.1.524

Cited by 28 publications

(18 citation statements)

References 20 publications

(18 reference statements)

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“…For example, the PinA protein of bacteriophage T4 binds to the Lon protease and prevents the degradation of some substrates [32]. ClpAPmediated degradation of ssrA-tagged substrates can be inhibited by SspB binding to the ssrA tag, which masks sequences required for ClpA recognition [33].…”

Section: Adaptor-mediated Recognition Of Substratesmentioning

confidence: 99%

ATP-dependent proteases of bacteria: recognition logic and operating principles

Baker

Sauer

2006

Trends in Biochemical Sciences

254

250

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ATP-powered AAA+ proteases degrade specific proteins in intracellular environments occupied by thousands of different proteins. These proteases operate as powerful molecular machines that unfold stable native proteins before degradation. Understanding how these enzymes choose the 'right' protein substrates at the 'right' time is key to understanding their biological function. Recently, proteomic approaches have identified numerous substrates for some bacterial enzymes and the sequence motifs responsible for recognition. Advances have also been made in elucidating the mechanism and impact of adaptor proteins in regulating substrate choice. Finally, recent biochemical dissection of the ATPase cycle and its coupling to protein unfolding has revealed fundamental operating principles of this important, ubiquitous family of molecular machines. The AAA+ proteasesAAA+ proteases (the term AAA comes from 'ATPase associated with cellular activities') are multimeric machines that function with exquisite specificity in all domains of life to recognize, to unfold and to degrade proteins. Energy-dependent proteases differ widely in their complexity. For example, the 26S proteasome is built from >30 different types of protein, whereas a single type of subunit assembles to form the hexameric FtsH and Lon proteases. Nevertheless, ATP-dependent proteases share common architectural features [1][2][3].For example, the active sites that catalyze peptide bond cleavage are sequestered in a hollow interior chamber, typically constructed from rings of six or seven subunits or domains ( Figure 1a). Substrates enter these degradation compartments through axial channels or portals that are too narrow to admit folded native proteins. This restriction prevents the undesirable cleavage of most cellular proteins but requires the coordinated enzymatic recognition, unfolding and translocation of correct substrates before degradation. Hexameric rings of subunits or domains, which belong to the AAA+ ATPase family, carry out these mechanical denaturation and translocation steps. Moreover, these proteins and related AAA+ enzymes often function to disassemble macromolecular complexes and to resolubilize protein aggregates [4,5]. Substrate recognition can be mediated directly by the AAA+ domains of ATP-dependent proteases or indirectly by additional domains or adaptor proteins (Figure 1b) and ClpS). In particular, we highlight recent developments that have advanced our understanding of the mechanisms by which the AAA+ proteases and their adaptors function. Recognition through peptide 'tags'One mechanism that enables ATP-dependent proteases to recognize specific substrates involves binding to unstructured peptide sequences, which are typically located at the Nterminal or C-terminal end of a target protein. Recognition of 'degradation tags' can be the sole determinant of targeted proteolysis. For example, one quality control system in Escherichia coli adds the 'ssrA tag' sequence (AANDENYALAA) to the C terminus of proteins for which biosynthesis...

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Section: Adaptor-mediated Recognition Of Substratesmentioning

confidence: 99%

ATP-dependent proteases of bacteria: recognition logic and operating principles

Baker

Sauer

2006

Trends in Biochemical Sciences

254

250

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“…Prior studies have shown that substrates and other ligands interact with Lon allosterically (7,(14)(15). However, little is known about the importance of these mechanisms in controlling protein degradation.…”

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confidence: 99%

Degrons in protein substrates program the speed and operating efficiency of the AAA+ Lon proteolytic machine

Gur

Sauer

2009

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

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AAA؉ proteases are ATP-fueled machines that bind protein substrates via a degradation tag, unfold the molecule if necessary, and then translocate the polypeptide into a chamber for proteolysis. Tag recognition is normally viewed as a passive reaction. By contrast, for the AAA؉ Lon protease, we show that degron tags are also regulatory elements that determine protease activity levels. Indeed, different tags fused to the same protein change degradation speeds and energetic efficiencies by 10-fold or more. Degron binding to multiple sites in the Lon hexamer appears to differentially stabilize specific enzyme conformations, including one with high protease and low ATPase activity, and results in positively cooperative degradation. These allosteric mechanisms allow Lon to operate in either a fast or slow proteolysis mode, according to specific physiological needs, and may help maximize degradation of misfolded proteins following stress-induced denaturation.AAAϩ protease ͉ allosteric control ͉ degradation tags

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“…In vivo studies suggested that other ATP-dependent E. coli proteases were not affected by PinA (22). As shown in Table I, PinA did not inhibit casein degradation by the ATP-dependent proteases, ClpAP or ClpYQ (HslUV).…”

Section: Resultsmentioning

confidence: 87%

“…PinA, therefore, appears to be a novel protease inhibitor, highly specific for Lon protease. Further characterizations of the effects of PinA on the proteolytic, peptidase and ATPase activities of Lon are described in the accompanying paper (22).…”

Section: Table IV Reversibility Of Lon Inhibition By Pinamentioning

confidence: 99%

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Isolation and Characterization of the Phage T4 PinA Protein, an Inhibitor of the ATP-dependent Lon Protease ofEscherichia coli

Hilliard¹,

Maurizi²,

Simon³

1998

Journal of Biological Chemistry

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The bacteriophage T4 PinA protein, expression of which leads to inhibition of protein degradation in Escherichia coli cells, has been purified from cells carrying multiple copies of the pinA gene. PinA is a heat-stable protein with a subunit M r of 18,800 and an isoelectric point of 4.6. Under nondenaturing conditions on a gel filtration column, PinA migrated in two peaks corresponding to a dimer and a tetramer. Purified PinA inhibited ATP-dependent protein degradation by Lon protease in vitro; it did not inhibit the activity of other E. coli ATP-dependent proteases, ClpAP or ClpYQ. Furthermore, PinA did not inhibit ATP-independent proteolysis in E. coli cell extracts. PinA binds with high affinity to Lon protease (K d ϳ 10 nM for dimer binding), and a complex with ϳ1 dimer of PinA per tetramer of Lon protease could be isolated by gel filtration. Lon activity was partially restored upon dilution of the PinA-Lon complex to subnanomolar concentrations, indicating that inhibition was reversible and that PinA did not covalently modify Lon protease. PinA was not cleaved by Lon protease, and heating the Lon-PinA complex at 65°C denatured Lon protease and released active PinA. The properties of PinA in vitro suggest that PinA inhibits protein degradation in vivo by forming a tight, reversible complex with Lon protease.

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PinA Inhibits ATP Hydrolysis and Energy-dependent Protein Degradation by Lon Protease

Cited by 28 publications

References 20 publications

ATP-dependent proteases of bacteria: recognition logic and operating principles

ATP-dependent proteases of bacteria: recognition logic and operating principles

Degrons in protein substrates program the speed and operating efficiency of the AAA+ Lon proteolytic machine

Isolation and Characterization of the Phage T4 PinA Protein, an Inhibitor of the ATP-dependent Lon Protease ofEscherichia coli

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