Sina Langklotz

Structure and function of the bacterial AAA protease FtsH

Proteolysis of regulatory proteins or key enzymes of biosynthetic pathways is a universal mechanism to rapidly adjust the cellular proteome to particular environmental needs. Among the five energy-dependent AAA(+) proteases in Escherichia coli, FtsH is the only essential protease. Moreover, FtsH is unique owing to its anchoring to the inner membrane. This review describes the structural and functional properties of FtsH. With regard to its role in cellular quality control and regulatory circuits, cytoplasmic and membrane substrates of the FtsH protease are depicted and mechanisms of FtsH-dependent proteolysis are discussed.

The C-terminal end of LpxC is required for degradation by the FtsH protease

Lipopolysaccharide (LPS) biosynthesis is essential in Gram negative bacteria. LpxC, the key enzyme in LPS formation, catalyses the limiting reaction and controls the ratio between LPS and phospholipids. As overproduction of LPS is toxic, the cellular amount of LpxC must be regulated carefully. The membrane‐bound protease FtsH controls the level of LpxC via proteolysis making FtsH the only essential protease of Escherichia coli. We found that the chaperones DnaK and DnaJ co‐purified with LpxC. However, degradation of LpxC was DnaK/J‐independent in contrast to turnover of the heat shock sigma factor σ32 (RpoH). The stability of LpxC in a bacterial one‐hybrid system suggested that a terminus of LpxC might be important for degradation. Different LpxC truncations and extensions were constructed. Removal of at least five amino acids from the C‐terminus abolished degradation by FtsH in vivo. While addition of two aspartic acids to LpxC did not alter its half‐life, the exchange of the last two residues against aspartic acids resulted in stabilization. All stable LpxC enzymes were active in vivo as assayed by their high toxicity. Our data demonstrate that the C‐terminus of LpxC contains a signal sequence necessary for FtsH‐dependent degradation.

Analysis of the Mechanism of Action of Potent Antibacterial Hetero-tri-organometallic Compounds: A Structurally New Class of Antibiotics

Two hetero-tri-organometallic compounds with potent activity against Gram-positive bacteria including multi-resistant Staphylococcus aureus (MRSA) were identified. The compounds consist of a peptide nucleic acid backbone with an alkyne side chain, substituted with a cymantrene, a (dipicolyl)Re(CO)3 moiety, and either a ferrocene (FcPNA) or a ruthenocene (RcPNA). Comparative proteomic analysis indicates the bacterial membrane as antibiotic target structure. FcPNA accumulation in the membrane was confirmed by manganese tracing with atomic absorption spectroscopy. Both organometallics disturbed several essential cellular processes taking place at the membrane such as respiration and cell wall biosynthesis, suggesting that the compounds affect membrane architecture. Correlating with enhanced antibacterial activity, oxidative stress was induced only by the ferrocene-substituted compound. The organometallics described here target the cytoplasmic membrane, a clinically proven antibacterial target structure, feature a bactericidal but non-bacteriolytic mode of action and limited cytotoxicity within the limits of solubility. Thus, FcPNA represents a promising lead structure for the development of a new synthetic class of antibiotics.

Degradation of cytoplasmic substrates by FtsH, a membrane-anchored protease with many talents.

Control of cellular processes by regulated proteolysis is conserved among all organisms. FtsH, the only membrane-anchored AAA protease in bacteria, fulfills a variety of regulatory functions. This review focuses on soluble FtsH substrates in Escherichia coli and in other bacteria and outlines emerging substrate recognition principles.

A trapping approach reveals novel substrates and physiological functions of the essential protease FtsH in Escherichia coli

Background: Only few substrates of the essential membrane-anchored protease FtsH are known. Results: New cytoplasmic and membrane-bound substrates of FtsH were trapped in vivo. Conclusion: FtsH is involved in the sulfatation of molecules, d-amino acid metabolism, and adaptation to anaerobiosis and stress conditions. Significance: The novel FtsH substrates significantly expand our knowledge on the biological functions of this fundamentally important protease. Proteolysis is a universal strategy to rapidly adjust the amount of regulatory and metabolic proteins to cellular demand. FtsH is the only membrane-anchored and essential ATP-dependent protease in Escherichia coli. Among the known functions of FtsH are the control of the heat shock response by proteolysis of the transcription factor RpoH (σ32) and its essential role in lipopolysaccharide biosynthesis by degradation of the two key enzymes LpxC and KdtA. Here, we identified new FtsH substrates by using a proteomic-based substrate trapping approach. An FtsH variant (FtsHtrap) carrying a single amino acid exchange in the proteolytic center was expressed and purified in E. coli. FtsHtrap is devoid of its proteolytic activity but fully retains ATPase activity allowing for unfolding and translocation of substrates into the inactivated proteolytic chamber. Proteins associated with FtsHtrap and wild-type FtsH (FtsHWT) were purified, separated by two-dimensional PAGE, and subjected to mass spectrometry. Over-representation of LpxC in the FtsHtrap preparation validated the trapping strategy. Four novel FtsH substrates were identified. The sulfur delivery protein IscS and the d-amino acid dehydrogenase DadA were degraded under all tested conditions. The formate dehydrogenase subunit FdoH and the yet uncharacterized YfgM protein were subject to growth condition-dependent regulated proteolysis. Several lines of evidence suggest that YfgM serves as negative regulator of the RcsB-dependent stress response pathway, which must be degraded under stress conditions. The proteins captured by FtsHtrap revealed previously unknown biological functions of the physiologically most important AAA+ protease in E. coli.

Control of Lipopolysaccharide Biosynthesis by FtsH-Mediated Proteolysis of LpxC Is Conserved in Enterobacteria but Not in All Gram-Negative Bacteria

Despite the essential function of lipopolysaccharides (LPS) in Gram-negative bacteria, it is largely unknown how the exact amount of this molecule in the outer membrane is controlled. The first committed step in LPS biosynthesis is catalyzed by the LpxC enzyme. In Escherichia coli, the cellular concentration of LpxC is adjusted by the only essential protease in this organism, the membrane-anchored metalloprotease FtsH. Turnover of E. coli LpxC requires a length- and sequence-specific C-terminal degradation signal. LpxC proteins from Salmonella, Yersinia, and Vibrio species carry similar C-terminal ends and, like the E. coli enzyme, were degraded by FtsH. Although LpxC proteins are highly conserved in Gram-negative bacteria, there are striking differences in their C termini. The Aquifex aeolicus enzyme, which is devoid of the C-terminal extension, was stable in E. coli, whereas LpxC from the alphaproteobacteria Agrobacterium tumefaciens and Rhodobacter capsulatus was degraded by the Lon protease. Proteolysis of the A. tumefaciens protein required the C-terminal end of LpxC. High stability of Pseudomonas aeruginosa LpxC in E. coli and P. aeruginosa suggested that Pseudomonas uses a proteolysis-independent strategy to control its LPS content. The differences in LpxC turnover along with previously reported differences in susceptibility against antimicrobial compounds have important implications for the potential of LpxC as a drug target.

Short Antibacterial Peptides with Significantly Reduced Hemolytic Activity can be Identified by a Systematic l-to-d Exchange Scan of their Amino Acid Residues

High systemic toxicity of antimicrobial peptides (AMPs) limits their clinical application to the treatment of topical infections; in parenteral systemic application of AMPs the problem of hemolysis is one of the first to be tackled. We now show that the selectivity of lipidated short synthetic AMPs can be optimized substantially by reducing their hemolytic activity without affecting their activity against methicillin resistant Staphylococcus aureus (MRSA). In order to identify the optimized peptides, two sets of 32 diastereomeric H-(D)Arg-WRWRW-(L)Lys(C(O)CnH2n+1)-NH2 (n = 7 or 9) peptides were prepared using a split-split procedure to perform a systematic L-to-D exchange scan on the central WRWRW-fragment. Compared to the all-L C8-lipidated lead sequence, diastereomeric peptides had very similar antibacterial properties, but were over 30 times less hemolytic. We show that the observed hemolysis and antibacterial activity is affected by both differences in lipophilicity of the different peptides and specific combinations of L- and D-amino acid residues. This study identified several peptides that can be used as tools to precisely unravel the origin of hemolysis and thus help to design even further optimized nontoxic very active short antibacterial peptides.

The Escherichia coli replication inhibitor CspD is subject to growth-regulated degradation by the Lon protease

Post‐translational proteolysis‐dependent regulation of critical cellular processes is a common feature in bacteria. The Escherichia coli Lon protease is involved in the control of the SOS response, acid tolerance and nutritional deprivation. Moreover, Lon plays a role in the regulation of toxin–antitoxin (TA) systems and thereby is linked to persister cell induction. Persister cells represent a small subpopulation that has reversibly switched to a dormant and non‐dividing state without genomic alterations. Formation of persister cells permits viability upon nutritional depletion and severe environmental stresses. CspD is a replication inhibitor, which is induced in stationary phase or upon carbon starvation and increases the production of persister cells. It has remained unknown how CspD activity is counteracted when growth is resumed. Here we report that CspD is subject to proteolysis by the Lon protease both in vivo and in vitro. Turnover of CspD by Lon is strictly adjusted to the growth rate and growth phase of E. coli, reflecting the necessity to control CspD levels according to the physiological conditions.

Tuning the Activity of a Short Arg-Trp Antimicrobial Peptide by Lipidation of a C- or N-Terminal Lysine Side-Chain

The attachment of lipids to C- or N-terminally positioned lysine side-chain amino groups increases the activity of a short synthetic (Arg-Trp)3 antimicrobial peptide significantly, making these peptides even active against pathogenic Gram-negative bacteria. Thus, a peptide with strong activity against S. aureus (1.1-2 μM) and good activity against A. baumannii and P. aeruginosa (9-18 μM) was identified. The most promising peptide causes 50% hemolysis at 285 μM and shows some selectivity against human cancer cell lines. Interestingly, the increased activity of ferrocenoylated peptides is mostly due to the lipophilicity of the organometallic fragment.

Activation of RidA chaperone function by N-chlorination

Escherichia coli RidA is a member of a structurally conserved, yet functionally highly diverse protein family involved in translation inhibition (human), Hsp90-like chaperone activity (fruit fly) and enamine/imine deamination (Salmonella enterica). Here, we show that E. coli RidA modified with HOCl acts as a highly effective chaperone. Although activation of RidA is reversed by treatment with DTT, ascorbic acid, the thioredoxin system and glutathione, it is independent of cysteine modification. Instead, treatment with HOCl or chloramines decreases the amino group content of RidA by reversibly N-chlorinating positively charged residues. N-chlorination increases hydrophobicity of RidA and promotes binding to a wide spectrum of unfolded cytosolic proteins. Deletion of ridA results in an HOCl-sensitive phenotype. HOCl-mediated N-chlorination thus is a cysteine-independent post-translational modification that reversibly turns RidA into an effective chaperone holdase, which plays a crucial role in the protection of cytosolic proteins during oxidative stress.