From the Waksman Institute, Rutgers, The State
University of New Jersey, Piscataway, New Jersey, 08855-0759 and the
¶ Laboratory of Cell Biology, National Cancer Institute, National
Institutes of Health, Bethesda, Maryland 20892
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 Mr 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 (Kd ~ 10 nM for dimer binding), and a complex with ~1 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.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Lon protease, the product of the lon gene (1), is one
of the major ATP-dependent proteases of Escherichia
coli. In vivo, Lon is responsible for the degradation
of such specific proteins as SulA (2) RcsA (3), and the N protein
(4), as well as unfolded and abnormal proteins (5). Purified Lon,
subunit Mr 87,000, is an oligomeric protein that
has been reported to exist in either tetrameric or octameric form (6,
7). Sequence analysis suggests that each subunit of Lon contains a
proteolytic active site and an ATPase site distinct from the
proteolytic site (8). Cleavage of small peptides and some small
proteins requires nucleotide binding to Lon but does not require ATP
hydrolysis (9-11), but degradation of high molecular weight proteins
requires ATP hydrolysis (12-14). Under optimal conditions with a
variety of protein substrates, two ATP molecules are hydrolyzed per
peptide bond cleaved (15).
Hydrolysis of ATP may provide energy to help unfold protein substrates, giving them greater access to the proteolytic active site and making them more susceptible to cleavage (13, 16, 17). Recent studies of CcdA degradation by Lon in vitro indicated that the absence of stable secondary structure in protein substrates decreased the requirement for ATP hydrolysis (11). Protein substrates bind to two sites on Lon, the proteolytic active site and an allosteric site, which may serve as the site for the protein remodeling function. Occupancy of the allosteric site by substrates such as unfolded polypeptides activates the peptidase activity against small peptides and enhances proteolysis (12).
E. coli cells infected with bacteriophage T4 show reduced
proteolysis of abnormal proteins and protein fragments (18). Inhibition of protein degradation requires synthesis of T4 proteins made during
the first 10 min after infection at 37 °C (18-20). Clones of T4
genes that lead to inhibition of proteolysis in E. coli cells were obtained by Simon and co-workers (19, 20), and one specific
gene, pinA (proteolysis inhibition
A), which resulted in inhibition of abnormal protein
degradation, was cloned by Skorupski et al. (21). The target
of PinA in vivo appears to be the ATP-dependent Lon protease, because E. coli lon+ cells
expressing a single copy of the T4 pinA gene are
phenotypically Lon (21).
We have purified the PinA protein and shown that purified PinA binds to Lon protease and inhibits its ATP-dependent protein degrading activity in vitro. In the accompanying paper (22), we demonstrate that PinA exerts its affect by blocking ATP hydrolysis by Lon.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials-- All chemicals were obtained from commercial sources unless otherwise specified. [3H]Formaldehyde was obtained from NEN Life Science Products. Clp protease was purified as described previously (23-25).
Growth of Bacterial Strains-- Bacterial strains and plasmids used are described below. Bacteria used to make cell extracts were grown in glucose L broth (31), which contained (per liter) 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, and 2 g of glucose. Glucose L agar contained glucose L broth with 1.5% (w/v) agar. Ampicillin was used at 50 µg/ml as needed.
Purification of Lon Protease-- Lon protease was purified from E. coli SG22030 (lon+) carrying the multicopy plasmid plon+500, which contains lon under its own promoter (5). The purification method has been described (6). Lon was estimated by SDS-PAGE1 to be 90-95% pure and was free of other proteases.
Overexpression and Preparation of PinA--
The pinA
gene product was expressed from a plasmid constructed by J. Tomaschewski2 using the
expression vectors of Tabor and Richardson (26). The host strain was
E. coli LS101, a derivative of K38 (27) carrying a
galE mutation to prevent production of excess capsular polysaccharide in the absence of lon function in
vivo. Cells were transformed with plasmid pGP1-2, which has T7
RNA polymerase under a heat-inducible promoter (26), and plasmid
pT7AT/pin, which has the pinA gene under control
of a T7 promoter. To induce expression of the pinA gene,
cells grown at 28 °C were shifted to 42 °C for 30 min and left at
40 °C for 2 h. Harvested cells were stored at 80 °C.
Biochemical Characterization and Physical Properties-- One-dimensional SDS-PAGE gels were run essentially as described by Laemmli (28), using Mini-PROTEAN II Ready Gels (Bio-Rad). Except as noted, 12% gels were used. The molecular weight of PinA under nondenaturing conditions was determined by gel filtration on a Superose-12 column by comparing the elution time of the PinA protein to the elution times of proteins of known molecular weights. The subunit molecular weight was estimated by SDS-PAGE. The isoelectric point of the PinA protein was determined using Pharmacia isoelectric focusing 3-9 Phastgels. Protein concentrations were determined by the dye-binding method of Bradford (29) using the reagent supplied by Bio-Rad, with bovine serum albumin as the standard. PinA concentrations were measured from the absorbance using the extinction coefficient.
Amino Acid Analysis-- Purified PinA was hydrolyzed in 6 N HCl at 155 °C for 30 and 60 min. The hydrolysates were washed, dried, and derivatized with phenylisothiocyanate (PTC). PTC-amino acids were separated on a C18 reverse phase column (4.6 mm × 15 cm) using the solvent system described by Bidlingmeyer et al. (30). The cysteine content of the PinA protein was determined by performic acid oxidation (31), followed by acid hydrolysis and amino acid analysis. Aromatic amino acids were determined spectrophotometrically by second derivative UV spectroscopy in 6 M guanidine hydrochloride, as described by Levine and Federici (32). Using the amino acid content calculated from the DNA-derived sequence of PinA, the extinction coefficient of the protein was determined from the calculated aromatic amino acid content of the protein and the measured absorbance of a standard solution of the protein.
NH2-terminal Sequence Determination-- To remove salts and buffers prior to sequencing, purified PinA was passed through a HR 10/10 Fast desalting column (Pharmacia) in deionized H2O. Sequencing was performed according to the manufacturer's directions on an Applied Biosystems model 470A Protein Sequencer with a model 120A on-line PTH Amino Acid Analyzer (33) and a model 610A Data Analysis Module and the ABI model 475 Report Generator.
[3H]Methyl -Casein Preparation--
-Casein
was radioactively labeled with [3H]formaldehyde by the
method of Jentoft and Dearborn (34). The specific activity of the
[3H]methyl
-casein was approximately 5 µCi/mg.
Assays for ATP-dependent Proteolytic
Activity--
Assays for proteolytic activity were performed as
described previously (9, 23) or as follows. A solution with 9 µg of [3H]methyl -casein in 250 µl of buffer containing 50 mM Tris-HCl, pH 8.0, 25 mM MgCl2, 1 mM DTT, and 4 mM ATP was incubated for 5 min at
37 °C, and the reaction was initiated by the addition of 0.5-2.0
µg of Lon. Incubation at 37 °C was continued for 15-30 min. The
reaction was terminated by the addition of 310 µl of ice-cold 10%
trichloroacetic acid and 40 µl of 10 mg/ml bovine serum albumin.
Precipitated proteins were separated from trichloroacetic acid-soluble
proteins by centrifugation at 4 °C in an Eppendorf centrifuge at
14,000 × g for 6 min. Radioactivity was determined by
liquid scintillation counting using 0.5 ml of the supernatant in 10 ml
of Scintiverse BD (Fisher) or Aquasol (NEN Life Science Products).
Assays were performed in duplicate, and the measured activity had a
variance of
4%.
Inhibition of Lon by PinA-- PinA was added to standard assays solutions 1-5 min prior to addition of Lon to initiate the assays. Mixing PinA and Lon before adding them to the assay mixtures did not affect the results. The effect of pH on the inhibitory activity of PinA was determined by substituting the following buffers in the assay mixture: 50 mM MES, sodium salt, 10 mM MgCl2, pH 6.0 and 7.0; 50 mM Tris-HCl, 10 mM MgCl2, pH 7.0-9.0; and 50 mM 2-amino-2-methyl-1-propanol HCl, 10 mM MgCl2, pH 9.0-10.5.
Stoichiometry of the Lon-PinA Complex-- A 2.5-ml Sephacryl S-200 (Pharmacia) column was equilibrated at room temperature in buffer B. Lon (20 µg) was loaded onto the column, and 250-µl aliquots of buffer were added at 2 min intervals. Aliquots of 250 µl were collected from the column at each step. The same procedure was used for PinA (20 µg) and for a mixture of 20 µg of Lon and 20 µg of PinA. Nucleotide requirement was determined by equilibrating the column in buffer B containing 50 µM AMPPNP, and then chromatographing Lon and PinA. Alternatively, Lon, PinA, or the PinA-Lon complex was centrifuged through a Bio-Spin column packed with Sephacryl Superfine S-200 according the manufacturer's instructions.
Lon (250 µg), PinA (250 µg), and mixtures of the two proteins were also analyzed by gel filtration on a Superose12 column equilibrated in buffer B. Proteins were eluted at a flow rate of 0.4 ml/min in the same buffer. Protein in the fractions was detected by SDS-PAGE, and fractions were assayed for [3H]methyl ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Purification of the PinA Protein--
PinA was purified to near
homogeneity from extracts of cells in which the protein was
overproduced. Previous work had shown that PinA was a component of the
cytosol and was not associated with the cell
membrane.3 Purified PinA in
fractions from the final gel filtration step is shown in Fig.
1A; PinA was estimated to be
95% pure in the best fractions, which were stored separately and
used for the experiments described below. PinA had a maximum absorbance
at 281 nm and an extinction coefficient of 1.95 (mg/ml)
1
determined by analysis of the second derivative of the UV
absorbance spectrum (32).
|
Inhibitory Activity of PinA--
Inhibition of Lon protease
activity was examined using [3H]methyl -casein as the
substrate and by following the ATP-dependent release of
trichloroacetic acid-soluble peptide fragments. Extracts of cells in
which PinA was overproduced contained an inhibitor of Lon protease, and
no such inhibitory activity was seen with extracts of control cells
with the plasmid vector, which did not contain PinA (data not shown).
The inhibitory activity against Lon protease co-eluted with the PinA
protein during purification (Fig. 1B).
|
|
Stability of PinA-- PinA was stable when heated. Table II shows that PinA inhibited Lon to the same extent before and after heating for 10 min at ~100 °C. Inhibition of Lon by PinA required the intact PinA protein, because digestion of PinA by trypsin or chymotrypsin inactivated PinA (data not shown). This experiment also demonstrated that Lon inhibition was not due to nonproteinaceous inhibitors contaminating the PinA.
|
|
Oligomeric Structure of PinA--
Freshly purified PinA migrated
with an apparent molecular weight of 40,000 on a Superose 12 gel
filtration column in the presence of 0.1-0.2 M KCl and
thus appears to be a dimer. After storage at 20 °C for more than 1 year, PinA species that appeared by gel filtration to be tetramers (see
Fig. 4) and octamers (data not shown)
predominated. No noticeable effect on the ability of PinA to inhibit
Lon protease accompanied these changes in oligomeric state, and it is
possible that the aggregated PinA dissociated to dimers and tetramers
at the dilutions used for assays. High salt concentrations (
0.3
M KCl) cause PinA to dissociate into monomers and, when
present in assay solutions, decrease the inhibitory effects of PinA
(data not shown). These last data suggest either that the monomeric
form of PinA does not inhibit Lon or that the interaction between PinA
and Lon is disrupted by high ionic strength.
|
Demonstration of a Lon-PinA Complex-- PinA binding to Lon was shown by isolation of a complex of the two proteins on a Sephacryl S-200 gel filtration column. Fig. 4 shows the fractions containing PinA and Lon protease when the two proteins were run over the column either separately or after mixing together. When the proteins were chromatographed separately, the elution position of PinA, which was a mixture of dimers and tetramers, was much later than that of Lon. When the proteins were run together, PinA and Lon were found together in fractions eluting slightly ahead of the elution position of Lon alone, and there was a decrease in the protein peak at the elution position of PinA alone (Fig. 4A). There was considerable overlap in the positions of the Lon-PinA complex and Lon alone, partly due to the tendency of Lon to trail severely in these columns. The complex could be isolated in the presence or absence of nucleotide (data not shown).
The proteolytic activity of Lon in fractions containing the isolated complex was assayed and compared with that present in similar fractions when Lon protease was run alone. As shown in Fig. 4B, the casein degrading activity of the PinA-Lon complex was only 5-10% of the activity of Lon protease alone. The amount of PinA bound to Lon was determined in a separate experiment by mixing the two proteins in a ratio of five PinA dimers per subunit of Lon and isolating the complex by gel filtration on a Sephacryl S-200 column. Protein in the fractions corresponding to the complex was quantitated by comparing the intensity of the Coomassie-stained bands to known amounts of Lon or PinA that had been run and stained in parallel. Approximately 1-2 dimers of PinA were bound to 1 tetramer of Lon. Increasing the amount of PinA added to Lon did not increase the PinA found complexed to Lon (data not shown).Release of Active PinA from the Lon-PinA Complex-- The release of active PinA from the PinA-Lon complex was demonstrated by taking advantage of the thermal stability of PinA. Aliquots of Lon and the PinA-Lon complex from the Sephacryl spin-column eluates were incubated at 65 °C for 10 min, after which the aliquots were immediately added to reaction mixtures with and without fresh Lon. Table III shows that, after 10 min at 65 °C, Lon alone or in the complex with PinA had no proteolytic activity. Addition of the heated PinA-Lon complex to assay solutions with fresh Lon resulted in inhibition. The inhibition was not due to interference from the heated and presumably denatured Lon, since the addition of heated Lon alone to the reaction mixture had no effect on casein degradation by fresh nondenatured Lon. Thus, no irreversible change in PinA accompanies binding to and inhibition of Lon protease.
|
Reversibility of Lon Inhibition by PinA-- The PinA-Lon complex was isolated by gel filtration on Sephacryl S-200. Eluates were collected and were assayed for ATP-dependent casein degradation in reaction solutions of different volumes. Table IV shows that Lon in the complex isolated from the column was inhibited >90% when assayed at high concentrations of the complex, but dilution of the complex resulted in a progressive increase in the Lon activity. Similar results were obtained without isolation of the complex by gel filtration. PinA and Lon were premixed in a ratio sufficient to cause >90% inhibition of Lon activity when assayed in 100-µl reaction solutions. Dilution of the same mixture into larger volumes for assay resulted in dissociation of the complex and a 40% gain in Lon activity (Table IV). In other experiments, 40-85% of Lon activity was recovered at 8-16-fold dilution of the PinA-Lon complex into assay solutions (data not shown). These results suggest that, although the PinA-Lon complex is quite stable, complex formation is reversible.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The bacteriophage T4 PinA protein has been purified from E. coli cells carrying a multicopy plasmid with the pinA gene under control of a T7 promoter. Purified PinA inhibits ATP-dependent casein degradation by Lon protease by more than 90%. PinA shares several properties with other polypeptide protease inhibitors. It is relatively small (Mr 20,000), heat-stable, and acidic (pI = 4.6); however, PinA does not inhibit proteases such as trypsin, chymotrypsin, subtilisin, or pepsin but is degraded and inactivated by treatment with these proteases. PinA appears to target Lon protease specifically, and elsewhere (22) we demonstrate that PinA inhibits ATP-dependent protein degradation by Lon protease by blocking the coupling between ATP hydrolysis and peptide bond cleavage.
Inhibition of [3H]methyl -casein degradation by Lon
protease occurs at very low concentrations of PinA, and an apparent
Ki of about 5 nM was calculated under
standard assay conditions. The low Ki implies that
PinA binds tightly to Lon, which was confirmed by showing that it was
possible to isolate a complex of Lon and PinA after gel filtration
chromatography. The complex was formed in the absence of nucleotide as
well as in the presence of the nonhydrolyzable ATP analog, AMPPNP,
indicating that tight binding of PinA to Lon does not require any of
the energy-driven steps involved in protein degradation.
As expected, when used at concentrations comparable to those in
standard assays, the PinA-Lon complex isolated by gel filtration showed
little proteolytic activity against [3H]methyl
-casein. However, dilution of the complex resulted in a small
increase in proteolytic activity, suggesting that inhibition of Lon by
PinA is at least partially reversible. Heating the PinA-Lon complex
released PinA from denatured Lon protease. The free PinA was able to
bind to Lon and inhibit proteolytic activity. SDS gel analysis of PinA
after forming a complex with Lon or released from the complex by
heating showed no evidence of cleavage of the PinA.
The classical protease inhibitors from plants and microbial organisms are characterized by either reversible or irreversible mechanisms (37). Reversible inhibitors have a specific peptide bond, which combines with the active site of the target protease and is then cleaved by the protease. Hydrolysis does not proceed to completion, but an equilibrium between intact and cleaved peptide bonds is established. Irreversible inhibitors combine with the protease and are cleaved at a specific peptide bond. However, the acyl intermediate between the inhibitor and the protease is not hydrolyzed and the inhibitor remains covalently bound to the protease. The latter inhibitor-enzyme complex resists dissociation by urea and SDS. The results of the thermal inactivation and dilution studies of the PinA-Lon complex suggest that PinA may differ from other reversible inhibitors in that it is not cleaved by the protease.
Skorupski et al. (21) have shown that
lon+ cells lysogenic for in which the
pinA gene has been cloned behave phenotypi-cally like
lon mutants. These lysogens produce mucoid colonies,
filament in response to DNA damage, permit efficient plaque formation
by
Ots phage at 40 °C, and exhibit reduced levels of
abnormal protein degradation, all typical of E. coli cells
lacking functional Lon protease (17, 38). Expression of pinA
has no detectable effect on abnormal protein degradation in E. coli null lon strains (21). Our finding that PinA does
not inhibit ClpAP or ClpYQ activity in vitro is consistent
with the idea that PinA displays specificity for Lon protease
alone.
ATP-dependent proteases tend to be high molecular weight,
multimeric enzymes with potential binding sites for regulatory
components. Several endogenous inhibitors that bind reversibly to the
eukaryotic 20 S and 26 S proteasomes have been described, each of which
appears to have a unique mode of action. For example, an inhibitor
described by Chu-Ping et al. (39) apparently acts
allosterically since it inhibits three distinct peptidase activities of
the 20 S proteasome, whereas a ubiquitinated inhibitor from rabbit
reticulocytes blocks ATP-dependent degradation of
ubiquitinated proteins by the 26 S proteasome but has only partial
activity against peptidase activities (40). A multimeric factor, CF2,
combines with the 20 S proteasome and appears to inhibit peptidase
activity (41). Since CF2 and the 20 S proteasome are both components of
the 26 S proteasome, this inhibition probably reflects changes in
accessibility of the proteolytic active sites during assembly of the 26 S proteasome, which has ATP-dependent proteolytic activity
and has a more stringent specificity, preferentially degrading
ubiquitinated proteins. No inhibitors of the E. coli ClpAP
or ClpXP proteases have yet been described. The CIII protein of inhibits the ATP-dependent FtsH (HflB) protease; however,
in vivo data suggest that CIII simply acts as a competitive
substrate for the protease, and CIII does not inhibit Lon protease
in vitro.4 A
search of the GenBank and Swiss-Prot data bases (42) revealed no
homologies to pinA at either the DNA or amino acid sequence level. 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).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Jorg Tomaschewski and Wolfgang Rüger for the pinA expression vector. We also thank Helen Kroh and Jane Duncan for assistance and Clement Woghiren for the NH2-terminal sequencing.
![]() |
FOOTNOTES |
---|
* This work was supported by National Science Foundation Grant DMB-8818950 (to L. D. S.) and by a Robert Wood Johnson predoctoral fellowship (to J. J. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Current address: Robert Wood Johnson Pharmaceutical Research Institute, Raritan, NJ 08869-0602.
To whom all correspondence should be addressed: Waksman
Institute, Rutgers, The State University of New Jersey, Piscataway, NJ
08855-0759. Tel.: 732-445-2912; Fax: 732-445-5735.
1
The abbreviations used are: PAGE, polyacrylamide
gel electrophoresis; AMPPNP, adenyl-5-yl imidodiphosphate; DTT,
dithiothreitol; PIPES, 1,4-piperazinediethanesulfonic acid; MES,
2-(N-morpholino)ethanesulfonic acid; PTC,
phenylisothiocyanate.
2 J. Tomaschewski, unpublished data.
3 H. J. Kim, unpublished observations.
4 M. R. Maurizi, unpublished data.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|