Tài liệu Báo cáo khoa học: The unique sites in SulA protein preferentially cleaved by ATP-dependent

The unique sites in SulA protein preferentially cleaved

by ATP-dependent Lon protease from Escherichia coli

Wataru Nishii1

, Takafumi Maruyama1

, Rieko Matsuoka1

, Tomonari Muramatsu2

and Kenji Takahashi1

1

School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji, Japan; 2

Biophysics Division, National Cancer

Center Research Institute, Chuo-ku, Tokyo, Japan

SulA protein is known to be one of the physiological

substrates of Lon protease, an ATP-dependent protease

from Escherichia coli. In this study, we investigated the

cleavage speci®city of Lon protease toward SulA protein.

The enzyme was shown to cleave  27 peptide bonds in

the presence of ATP. Among them, six peptide bonds

were cleaved preferentially in the early stage of digestion,

which represented an apparently unique cleavage sites

with mainly Leu and Ser residues at the P1, and P1¢

1positions, respectively, and one or two Gln residues in

positions P2±P5. They were located in the central region

and partly in the C-terminal region, both of which are

known to be important for the function of SulA, such as

inhibition of cell growth and interaction with Lon prote￾ase, respectively. The other cleavage sites did not represent

such consensus sequences, though hydrophobic or non￾charged residues appeared to be relatively preferred at the

P1 sites. On the other hand, the cleavage in the absence of

ATP was very much slower, especially in the central

region, than in the presence of ATP. The central region

was predicted to be rich in a helix and b sheet structures,

suggesting that the enzyme required ATP for disrupting

such structures prior to cleavage. Taken together, SulA is

thought to contain such unique cleavage sites in its

functionally and structurally important regions whose

preferential cleavage accelerates the ATP-dependent

degradation of the protein by Lon protease.

Keywords: ATP-dependent protease; Lon protease;

proteolysis; substrate speci®city; SulA.

Lon protease coded by the lon gene of Escherichia coli is an

ATP-dependent cytosolic protease [1]. The enzyme degrades

two types of substrates in vivo. One type of the substrates

includes abnormal proteins such as those with improper

polypeptide length or tertiary structure. Their degradation

should contribute to the quality control of intracellular

proteins. Another involves physiological substrates, such as

SulA, kN, RcsA, CcdA and Pem1, which are short-lived

regulatory proteins, whose speci®c and rapid degradation is

crucial for normal cell growth [2±7]. SulA is one of the most

physiologically important substrates among the second

type. The protein is transcriptionally induced by environ￾mental stresses, such as UV irradiation, and prevents

premature segregation of damaged DNA into daughter cells

during DNA repair processes [8,9]. Induced SulA prevents

the self-assembly of FtsZ protein, leading to the inhibition

of cell division (®lamentation) [10].

The substrate recognition mechanism of Lon protease

has not yet been well clari®ed. The cleavage sites by the

enzyme in vitro have been reported for kN [5] and CcdA [3]

proteins, oxidized insulin B chain and glucagon [5], and

several ¯uorogenic substrates [11]. In these proteins and

peptides, the cleavages occurred mainly after hydrophobic

residues, in spite that not all such sites were cleaved. So far,

however, no more consensus features have been reported in

the primary or higher-order structures of the substrates.

There has been little study on the cleavage sites, particularly

for SulA, by Lon protease. This is presumably because

recombinant SulA was reportedly rather insoluble and/or

unstable [12,13].

In the present study, we were able to prepare SulA in

a soluble form and investigated its cleavage sites by Lon

protease in vitro in the presence and absence of ATP.

The results indicated that Lon protease preferentially

cleaves certain unique sites, mainly in the central region

of SulA, which are functionally and structurally impor￾tant for the protein, thus triggering further rapid and

extensive degradation of SulA in an ATP-dependent

manner.

EXPERIMENTAL PROCEDURES

Preparation of E. coli Lon protease

Recombinant Lon protease was expressed in E. coli

harboring an expression plasmid for the enzyme using T7

promotor (manuscript in preparation). The expressed

enzyme was puri®ed by successive steps of column chro￾matography on phosphocellulose, DEAE-cellulose and

Sephacryl S-300 as described previously [1].

Correspondence to K. Takahashi, School of Life Science, Tokyo

University of Pharmacy and Life Science, 1432-1 Horinouchi,

Hachioji, Tokyo 192-0392. Fax: + 81 426 76 7149,

Tel.: + 81 426 76 7146, E-mail: [email protected]

Abbreviations: LC-MS, liquid chromatography-mass spectrometer;

MBP, maltose binding protein; 4MbNA, 4-methoxy-b-naphthyla￾mide; suc, succinyl; SulA3±169, SulA residues 3±169; SulA23±169,

SulA residues 23±169.

(Received 5 July 2001, revised 8 November 2001, accepted 13

November 2001)

Eur. J. Biochem. 269, 451±457 (2002) Ó FEBS 2002