The HtrA (DegP) protein, essential for Escherichia coli survival at high temperatures, is an endopeptidase

Abstract

As a preliminary step in the understanding of the function of the Escherichia coli HtrA (DegP) protein, which is indispensable for bacterial survival only at elevated temperatures, the protein was purified and partially characterized. The HtrA protein was purified from cells carrying the htrA gene cloned into a multicopy plasmid, resulting in its overproduction. The sequence of the 13 N-terminal amino acids of the purified HtrA protein was determined and was identical to the one predicted for the mature HtrA protein by the DNA sequence of the cloned gene. Moreover, the N-terminal sequence showed that the 48-kilodalton HtrA protein is derived by cleavage of the first 26 amino acids of the pre-HtrA precursor polypeptide and that the point of cleavage follows a typical target sequence recognized by the leader peptidase enzyme. The HtrA protein was shown to be a specific endopeptidase which was inhibited by diisopropylfluorophosphate, suggesting that HtrA is a serine protease.Both procaryotic and eucaryotic cells respond to a variety of stresses by accelerating the rate of synthesis of a group of proteins called heat shock proteins. This response in Escherichia coli is positively regulated by the product of the rpoH (htpR) gene, a32, which enables RNA polymerase to recognize promoters of heat shock genes (21). The heat shock genes all belong to the rpoH (htpR) regulon, are absolutely necessary for bacterial survival at high temperatures, and perform various, although not very well understood, functions in the cell. One of the processes in which the heat shock proteins are involved is cellular proteolysis. For example, the Lon protease belongs to the group of heat shock proteins (7,8). It is known that the accumulation of abnormal proteins leads to the induction of the rpoH (htpR)-dependent heat shock response (1, 12) and that aberrant proteins are frequently subject to proteolysis (13,14,28). It has been shown that, to a large extent, this proteolysis is rpoH (htpR) dependent; i.e., rpoH (htpR) mutant cells are defective in the proteolysis of abnormal proteins such as canavanine-containing proteins, puromycyl peptides, and the X90 fragment of ,B-galactosidase (2,11). This decrease occurs even in rpoH lon mutant bacteria, suggesting that other members of the heat shock regulon are also involved. Indeed, it has been observed that mutations in some of the other heat shock genes result in decreased proteolysis: dnaJ mutants are defective in the degradation of puromycyl peptides and a fragment of 3-galactosidase, groEL mutants are defective in the proteolysis of puromycyl peptides, and dnaK mutants are defective in the degradation of canavanine-containing proteins and puromycyl peptides (16, 25).Recently, the existence of a second heat shock regulon whose transcription is induced at a high temperature independently of rpoH (htpR) by a newly discovered sigma factor, Cr24 ((rE) was demonstrated (9,10,18,27 proteins (23, 24). This finding suggests that the HtrA (DegP) protein may itself be a protease or control the activit...

The HtrA family of serine proteases

“…2). Experimental studies have confirmed that HtrA is a serine protease -purified HtrA shows ATP-independent endopeptidase activity against beta-casein (but not against many other standard protease substrates) that can be inhibited by diisopropyl fluorophosphate, a specific inhibitor of serine proteases (Lipinska et al, 1990). Mutating two of the putative catalytic triad residues, Ser-210 and His-105, leads to a loss of protease activity (Skorko-Glonek et al, 1995a), with no detectable changes in secondary structure (Skorko-Glonek et al, 1995b).…”

“…Lipinska et al (1990) reported that HtrA, when overexpressed in E. coli, was partially degraded within cells to give two 43 kDa proteins that co-purified with the intact 48 kDa protein. Skorko-Glonek et al (1995a) showed that these breakdown products were the result of cutting after Cys-69 and Gln-82 of the mature protein.…”

No death without life: vital functions of apoptotic effectors

“…HtrA2) belongs to a family of serine proteases that is well conserved from bacteria to humans. 88 In bacteria, HtrA2 is localized within the periplasmic space and determines thermotolerance, 89 whereas in healthy eukaryotic cells Omi/HtrA2 is confined to the IMS. After MOMP, the protease is released into the cytosol and promotes apoptosis via caspase-dependent and -independent mechanisms.…”

Identification of the Escherichia coli sohB gene, a multicopy suppressor of the HtrA (DegP) null phenotype

“…The other strategy involved the characterization of a mutant which was defective in proteolysis of certain periplasmic fusion proteins (33). Subsequent studies verified that the product of the htrA gene is indeed an endopeptidase (21). Therefore, the product of the htrA gene is a periplasmic protease that is required only at elevated temperatures.…”