Bacterial Leader Peptidase Research Articles

64 - Inverse Regulatory Effects of Mitochondrial Superoxide in Resting and Activated T-Lymphocytes

The best-characterized member of eukaryotic membrane-bound translocation proteases is the microsomal signal peptidase. Cytochrome b2(cytb2) and cytochrome cI(cytc1) are cleaved in two successive steps during their import into mitochondria. As the enzyme is inactive toward in vitro-synthesized pre-cytb2 or the cytb2 intermediate generated from pre-cytb2 by addition of purified matrix protease, the substrate is the cytb2 intermediate in a detergent extract of mitochondria from mutant pet ts2858. Conversion of this intermediate to the mature form is analyzed by immunoblotting with antiserum against cytb2. Various properties of inner membrane protease I are discussed in the chapter. It is the only protein that shows sequence homology to bacterial leader peptidase and the only defined protein-mediating intramitochondrial sorting of proteins. Overexpression of the 21.4-kDa protein does not increase the enzyme activity, thereby suggesting that inner membrane protease I is a heterooligomer. The 21.4-kDa subunit is an integral inner membrane protein whose carboxy terminus protrudes into the intermembrane space.

Use of Site-directed Chemical Modification to Study an Essential Lysine in Escherichia coli Leader Peptidase

Escherichia coli leader peptidase, which catalyzes the cleavage of signal peptides from pre-proteins, is an essential, integral membrane serine peptidase that has its active site residing in the periplasmic space. It contains a conserved lysine residue that has been proposed to act as the general base, abstracting the proton from the side chain hydroxyl group of the nucleophilic serine 90. To help elucidate the role of the essential lysine 145 in the activity of E. coli leader peptidase, we have combined site-directed mutagenesis and chemical modification methods to introduce unnatural amino acid side chains at the 145-position. We show that partial activity can be restored to an inactive K145C leader peptidase mutant by reacting it with 2-bromoethylamine.HBr to produce a lysine analog (gamma-thia-lysine) at the 145-position. Modification with the reagents 3-bromopropylamine.HBr and 2-mercaptoethylamine also allowed for partial restoration of activity showing that there is some flexibility in the length requirements of this essential residue. Modification with (2-bromoethyl)trimethylammonium.Br to form a positively charged, nontitratable side chain at the 145-position failed to restore activity to the inactive K145C leader peptidase mutant. This result, along with an inactive K145R mutant result, supports the claim that the lysine side chain at the 145-position is essential due to its ability to form a hydrogen bond(s) or to act as a general base rather than because of an ability to form a critical salt bridge. We find that leader peptidase processes the pre-protein substrate, pro-OmpA nuclease A, with maximum efficiency at pH 9.0, and apparent pKa values for titratable groups at approximately 8.7 and 9.3 are revealed. We show that the lysine modifier maleic anhydride inhibits leader peptidase by reacting with lysine 145. The results of this study are consistent with the hypothesis that the lysine at the 145-position of leader peptidase functions as the active site general base. A model of the active site region of leader peptidase is presented based on the structure of the E. coli UmuD', and a mechanism for bacterial leader peptidase is proposed.

Expression and export in Escherichia coli of fusion proteins containing carboxy-terminally located honeybee prepromelittin.

The aim of this work was to express a eukaryotic pre-protein in Escherichia coli so that it could be obtained intact, without cleavage, by bacterial leader peptidase. To this end, cDNA coding for honeybee prepromelittin was ligated to the 3' end of genes coding for truncated forms of either Protein A or beta-galactosidase (beta-Gal) under the control of inducible promoters, with an oligonucleotide coding for the Factor Xa cleavage site at the junction between the two proteins. The Protein A fusion was expressed in good yield, and about 80% of it formed inclusion bodies. The prepromelittin section of the Protein A fusion caused some export of the intact fusion protein to the growth medium. The prepromelittin beta-Gal fusion was expressed in low yield and became associated with the E. coli cytoplasmic membrane. Its expression was toxic to E. coli. Thus, the synthesis of a full-length eukaryotic pre-protein in E. coli is best achieved when the fusion protein forms inclusion bodies.

Determination of the kinetic parameters of Escherichia coli leader peptidase activity using a continuous assay: the pH dependence and time-dependent inhibition by beta-lactams are consistent with a novel serine protease mechanism.

Bacterial leader peptidase (LPase) is a potential target for the development of novel anti-infective agents, but to data only peptides based upon natural macromolecular substrates have been reported as inhibitors. In this work is described a continuous assay for Escherichia coli LPase activity, based upon Ac-WSASALAKI-AMC (I) as the substrate, that can be monitored either spectrophotometrically or spectrofluorometrically. The LPase reaction is coupled to the liberation of AMC (aminomethylcoumarin) via a nonspecific leucine aminopeptidase. LPase and a short form of the enzyme (LPase-sf) lacking the membrane spanning domains displayed saturable kinetics toward I. The second-order rate constants were approximately 2 x 10(5) M-1 h-1 at pH 7.5 and were comparable to those reported in the literature for peptide substrates based upon natural cleavage sites in preproteins. LPase was inhibited by beta-lactams. [S-(R*,S*)]-4-[(1-(((1-(5-toluoyl)butyl)amino)carbonyl)-3,3-dimethyl-4- oxo-2-azetidinyl)oxyl]benzoic acid (L-684,-248, 588 microM) inhibited the LPase-catalyzed hydrolysis of 50 microM I and 125 microM Ac-WLVP-Nleu-LSFAAEGDDPA-NH2 by 30% and 88% over 1 and 4 h, respectively. The inhibition of LPase by L-684,248 and its C-4 diasteromer was time dependent and yielded second-order rate constants (kinact/Ki) of 12 and 7.7 M-1 min-1, respectively. The process was structurally specific as the C-3 diethyl substituted beta-lactam (C-4 S-isomer) was inactive. The latter data correlate with the LPase preference for alanine at the P1 position of peptide substrates [Kuo et al. (1993) Arch. Biochem. Biophys. 303, 274-280].(ABSTRACT TRUNCATED AT 250 WORDS)

21] Bacterial leader peptidase 1

Publisher Summary This chapter highlights bacterial leader peptidase 1. It describes the purification of leader peptidase, its substrate specificity, sequence homology of the leader peptidase 1 genes, and techniques to probe the structure and function of this protein. Proteins that are secreted out of the cell or translocated across lipid bilayers are usually made with N-terminal extension peptides, termed “leader sequences.” In bacteria, two separate leader peptidases process proteins. Leader peptidase 1 cleaves the majority of the preproteins destined to the cell surface, whereas leader peptidase 2 only processes preproteins that have been first modified with a lipid molecule at the +1 position. The Escherichia coli leader peptidase 1 is an integral membrane protein of 323 amino acid residues. Its physiological role is to release exported proteins from the membrane by removing the leader sequence. Leader peptidase can be assayed using a radiolabeled preprotein substrate or a peptide substrate. It is purified by membrane isolation, Triton X-100 extraction, diethylaminoethyl (DEAE) chromatography, and chromatofocusing.

Mitochondrial protein import: Identification of processing peptidase and of PEP, a processing enhancing protein

Transport of nuclear-encoded precursor proteins into mitochondria includes proteolytic cleavage of aminoterminal targeting sequences in the mitochondrial matrix. We have isolated the processing activity from Neurospora crassa. The final preparation (enriched ca. 10,000-fold over cell extracts) consists of two proteins, the matrix processing peptidase (MPP, 57 kd) and a processing enhancing protein (PEP, 52 kd). The two components were isolated as monomers. PEP is about 15-fold more abundant in mitochondria than MPP. It is partly associated with the inner membrane, while MPP is soluble in the matrix. MPP alone has a low processing activity whereas PEP alone has no apparent activity. Upon recombining both, full processing activity is restored. Our data indicate that MPP contains the catalytic site and that PEP has an enhancing function. The mitochondrial processing enzyme appears to represent a new type of “signal peptidase,” different from the bacterial leader peptidase and the signal peptidase of the endoplasmic reticulum.

Requirements for substrate recognition by bacterial leader peptidase.

Many secreted and membrane proteins have amino-terminal leader peptides which are essential for their insertion across the membrane bilayer. These precursor proteins, whether from prokaryotic or eukaryotic sources, can be processed to their mature forms in vitro by bacterial leader peptidase. While different leader peptides have shared features, they do not share a unique sequence at the cleavage site. To examine the requirements for substrate recognition by leader peptidase, we have truncated M13 procoat, a membrane protein precursor, from both the amino- and carboxy-terminal ends with specific proteases or chemical cleavage agents. The fragments isolated from these reactions were assayed as substrates for leader peptidase. A 16 amino acid residue peptide which spans the leader peptidase cleavage site is accurately cleaved. Neither the basic amino-terminal region nor most of the hydrophobic central region of the leader peptide are essential for accurate cleavage.

Bacterial leader peptidase, a membrane protein without a leader peptide, uses the same export pathway as pre-secretory proteins.

Leader peptidase typifies a group of proteins of the plasma membrane of E. coli which span the membrane and are synthesized without a cleaved amino-terminal leader (signal) sequence. The membrane assembly properties of these proteins have not been previously reported. We find that the membrane electrochemical potential is necessary for the insertion of a large domain of leader peptidase across the membrane. In the absence of potential, the peptidase accumulates inside the cell in tight association with the plasma membrane. Upon restoration of the potential, accumulated peptidase inserts across the membrane, indicating that this insertion is not mechanistically coupled to polypeptide chain growth. The normal, trans-bilayer peptidase and that which accumulates in the absence of potential have different conformations, as shown by the relative resistance of the trans-bilayer enzyme to digestion by trypsin or chymotrypsin in cell lysates. Membrane insertion is accompanied by this conformational change. This assembly reaction has several features predicted by the hypothesis of membrane-triggered folding.

Energetics and intermediates of the assembly of Protein OmpA into the outer membrane of Escherichia coli.

OmpA is a major protein of the outer membrane of Escherichia coli. It is made as a larger precursor, pro-OmpA, which requires a membrane potential for processing. We now show that pro-OmpA accumulates in the cytoplasm of cells treated with carbonyl cyanide m-chlorophenylhydrazone, an uncouple which lowers the membrane potential. Upon restoration of the potential, this pro-OmpA is secreted, processed, and assembled into the outer membrane. Pro-OmpA made in vitro is also recovered with the postribosomal supernatant. It is efficiently processed to OmpA by liposomes which have bacterial leader peptidase that is exclusively internally oriented. These experiments show that: (i) the insertion of pro-OmpA into the plasma membrane is not coupled to its synthesis; (ii) insertion is promoted by the transmembrane electrochemical potential; (iii) pro-OmpA can cross a bilayer spontaneously; and (iv) pro-OmpA is processed by the same leader peptidase which converts M13 procoat to coat.

Reconstitution of rapid and asymmetric assembly of M13 procoat protein into liposomes which have bacterial leader peptidase.

The leader peptidase of Escherichia coli cleaves a 23-residue leader sequence from M13 procoat to yield mature coat protein in virus-infected cells. We have reconstituted pure leader peptidase into vesicles of E. coli lipids and found that these liposomes are active in the conversion of procoat to coat. Trypsin removes all but 10% of the leader peptidase, yet the vesicles retain nearly full capacity to convert procoat to coat, suggesting that only procoat which inserts across the liposomal membrane is a substrate for leader peptidase. This is confirmed by the finding that over 70% of the coat protein produced by these liposomes spans the membrane. The rate at which leader peptidase inside protease-treated liposomes cleaves externally added procoat is comparable to the rate of procoat cleavage by the same amount of leader peptidase in detergent micelles. Thus, procoat can rapidly integrate across a liposomal membrane and be cleaved to coat protein. These findings confirm the central part of the membrane trigger hypothesis that certain proteins (such as procoat) can cross a bilayer without the aid of a proteinaceous pore or transport system.