AppppA-binding protein E89 is the Escherichia coli heat shock protein ClpB

Abstract

Dinucleotide AppppA (5',5'''-P1,P4-diadenosine tetraphosphate) is rapidly synthesized in Escherichia coli cells during heat shock. apaH mutants lack AppppN hydrolase activity and, therefore, contain constitutively levels of AppppA, which affect several cellular processes. However, the precise role of AppppA remains undetermined. Photo-crosslinking experiments with radioactively labelled azido-AppppA have shown that a number of proteins, including heat shock proteins DnaK and GroEL, specifically bind to AppppA. Several other unidentified proteins (C40, C45, and E89) also bind strongly to AppppA. In this work, we have identified the AppppA-binding protein E89 as heat shock protein ClpB. In addition, since ClpB belongs to a family of proteins implicated in proteolysis, we have examined the effects of apaH mutants on protein degradation. Constitutively elevated levels of AppppA stimulate lon-independent proteolysis only in heat-shocked cells. We also show that overproduction of ClpB from a plasmid rescues apaH mutants from sensitivity to killing by heat.

Protein Synthesis and the Stress Response

“…apaH mutant cells also showed prolonged synthesis of heat shock protein DnaK after heat shocked cells were returned to 30°C suggesting that the role of dinucleotides would be to modulate the long term response to stress conditions and not to trigger it [76]. The idea that the dinucleotides modulate the stress response is also supported by the fact that these molecules specifically bind several heat shock proteins including DnaK, GroEL [77] and ClpB [78]. Binding to DnaK inhibit its 5'-nucleotidase activity [79], but it is not known if it affects its ability to bind denatured proteins.…”

“…Binding to DnaK inhibit its 5'-nucleotidase activity [79], but it is not known if it affects its ability to bind denatured proteins. Effects of AppppA binding to the other heat shock proteins has not been investigated, although it has been shown that increases in cellular level of Appp(p)N enhance the degradation of abnormal proteins synthesized during incubation with puromycin [78].…”

“…Also, over expression of apaH, the gene coding for AppppN hydrolase that eliminates these dinucleotides in vivo [73], decreased significantly the levels of AppppA, but did not affected protein expression or cell survival under heat shock or H2O2 incubation [75]. Conversely, deletion of apaH, that increases the cellular level of AppppA, does affect cell survival and protein expression on several stress conditions including heat shock [76,77], UV irradiation [76], incubation with N-ethylmaleimide [76] as well during starvation [78]. It was shown that deletion of apaH somehow decreases expression of CAP-cAMP controlled genes which would decrease oxidative phosphorylation and limit further production of oxygen radicals.…”

Null Mutations in a Nudix Gene, ygdP , Implicate an Alarmone Response in a Novel Suppression of Hybrid Jamming

“…If this is the case, presumably the mutant PrlF1 protein has higher-than-wild-type chaperone activity, resulting in the observed posttranslational hyperactivation of Lon. Interestingly, while the mechanisms by which alarmones exert their pleiotropic effects in bacteria are unclear, it has been shown that the alarmone AppppA (diadenosine tetraphosphate) binds to a number of proteins, including the cytoplasmic chaperones, DnaK, GroEL, and ClpB (16,19). Thus, it may be that alarmones modulate the activities of protease-associated chaperones, thus increasing the activity of the proteases.…”

The pnhA Gene of Pasteurella multocida Encodes a Dinucleoside Oligophosphate Pyrophosphatase Member of the Nudix Hydrolase Superfamily

“…The role of the Nudix hydrolase is to remove the excess amounts of these molecules to restore cellular balance. In Escherichia coli, an increased concentration of dinucleoside oligophosphates, such as adenosine (5Ј)-tetraphospho-(5Ј)-adenosine (Ap 4 A), is proposed to be a signal for cell division (38,39) and to be involved in the stress responses by modulating protein refolding by chaperone proteins (21,30).…”