Dataset Information

A mutant HemA protein with positive charge close to the N terminus is stabilized against heme-regulated proteolysis in Salmonella typhimurium.

ABSTRACT: The HemA enzyme (glutamyl-tRNA reductase) catalyzes the first committed step in heme biosynthesis in the enteric bacteria. HemA is mainly regulated by conditional protein stability; it is stable and, consequently, more abundant in heme-limited cells but unstable and less abundant in normally growing cells. Both the Lon and ClpAP energy-dependent proteases contribute to HemA turnover in vivo. Here we report that the addition of two positively charged lysine residues to the third and fourth positions at the HemA N terminus resulted in complete stabilization of the protein. By contrast, the addition of an N-terminal myc epitope tag did not affect turnover. This result confirms the importance of the N-terminal sequence for proteolysis of HemA. This region of the protein also contains a proline flanked by hydrophobic residues, a motif that has been suggested to be important for Lon-mediated proteolysis of UmuD. However, mutation of this motif did not affect the turnover of HemA protein. Cells expressing the stabilized HemA[KK] mutant protein display substantial defects in heme regulation.

SUBMITTER: Wang L

PROVIDER: S-EPMC103631 | biostudies-literature | 1999 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A mutant HemA protein with positive charge close to the N terminus is stabilized against heme-regulated proteolysis in Salmonella typhimurium.

Wang L L Wilson S S Elliott T T

Journal of bacteriology 19991001 19

The HemA enzyme (glutamyl-tRNA reductase) catalyzes the first committed step in heme biosynthesis in the enteric bacteria. HemA is mainly regulated by conditional protein stability; it is stable and, consequently, more abundant in heme-limited cells but unstable and less abundant in normally growing cells. Both the Lon and ClpAP energy-dependent proteases contribute to HemA turnover in vivo. Here we report that the addition of two positively charged lysine residues to the third and fourth positi ...[more]

PMID: 10498716

Similar Datasets

Project description:Macrophages are central for the immune control of intracellular microbes. Heme oxygenase 1 (HO-1, hmox) is the first and rate limiting enzyme in the breakdown of heme originating from degraded senescent erythrocytes and heme-proteins, yielding equal amounts of iron, carbon monoxide and biliverdin. HO-1 is strongly up-regulated in macrophages in response to inflammatory signals, including bacterial endotoxin. In view of the essential role of iron for the growth and proliferation of intracellular bacteria along with known effects of the metal on innate immune function, we examined whether HO-1 plays a role in the control of infection with the intracellular bacterium Salmonella Typhimurium. We studied the course of infection in stably-transfected murine macrophages (RAW264.7) bearing a tetracycline-inducible plasmid producing hmox shRNA and in primary HO-1 knockout macrophages. While uptake of bacteria into macrophages was not affected, a significantly reduced survival of intracellular Salmonella was observed upon hmox knockdown or pharmacological hmox inhibition, which was independent of Nramp1 functionality. This could be traced to limitation of iron availability for intramacrophage bacteria along with enhanced stimulation of innate immune effector pathways, including the formation of reactive oxygen and nitrogen species and increased TNF-α expression. Mechanistically, these latter effects result from intracellular iron limitation with subsequent activation of NF-κB and further inos, tnfa and p47phox transcription along with reduced formation of the anti-inflammatory and radical scavenging molecules, CO and biliverdin as a consequence of HO-1 silencing. Taken together our data provide novel evidence that the infection-driven induction of HO-1 exerts detrimental effects in the early control of Salmonella infection, whereas hmox inhibition can favourably modulate anti-bacterial immune effector pathways of macrophages and promote bacterial elimination.

Dataset Information

A mutant HemA protein with positive charge close to the N terminus is stabilized against heme-regulated proteolysis in Salmonella typhimurium.

Publications

A mutant HemA protein with positive charge close to the N terminus is stabilized against heme-regulated proteolysis in Salmonella typhimurium.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets