Next-Generation Trapping of Protease Substrates by Label-Free Proteomics.

AAA+ proteases (ATPases associated with various cellular activities) shape the cellular protein pool in response to environmental conditions. A prerequisite for understanding the underlying recognition and degradation principles is the identification of as many protease substrates as possible. Most previous studies made use of inactive protease variants to trap substrates, which were identified by 2D-gel based proteomics. Since this method is known for limitations in the identification of low-abundant proteins or proteins with many transmembrane domains, we established a trapping approach that overcomes these limitations. We used a proteolytically inactive FtsH variant (FtsHtrap) of Escherichia coli (E. coli) that is still able to bind and translocate substrates into the proteolytic chamber but no longer able to degrade proteins. Proteins associated with FtsHtrap or FtsHwt (proteolytically active FtsH) were purified, concentrated by an 1D-short gel, and identified by LC-coupled mass spectrometry (LC-MS) followed by label-free quantification. The identification of four known FtsH substrates validated this approach and suggests that it is generally applicable to AAA+ proteases.

An Integrated Proteomic Approach Uncovers Novel Substrates and Functions of the Lon Protease in Escherichia coli.

Controlling the cellular abundance and proper function of proteins by proteolysis is a universal process in all living organisms. In Escherichia coli, the ATP-dependent Lon protease is crucial for protein quality control and regulatory processes. To understand how diverse substrates are selected and degraded, unbiased global approaches are needed. We employed a quantitative Super-SILAC (stable isotope labeling with amino acids in cell culture) mass spectrometry approach and compared the proteomes of a lon mutant and a strain producing the protease to discover Lon-dependent physiological functions. To identify Lon substrates, we took advantage of a Lon trapping variant, which is able to translocate substrates but unable to degrade them. Lon-associated proteins were identified by label-free LC-MS/MS. The combination of both approaches revealed a total of 14 novel Lon substrates. Besides the identification of known pathways affected by Lon, for example, the superoxide stress response, our cumulative data suggests previously unrecognized fundamental functions of Lon in sulfur assimilation, nucleotide biosynthesis, amino acid and central energy metabolism.

Intricate Crosstalk Between Lipopolysaccharide, Phospholipid and Fatty Acid Metabolism in Escherichia coli Modulates Proteolysis of LpxC.

Lipopolysaccharides (LPS) in the outer membrane of Gram-negative bacteria provide the first line of defense against antibiotics and other harmful compounds. LPS biosynthesis critically depends on LpxC catalyzing the first committed enzyme in this process. In Escherichia coli, the cellular concentration of LpxC is adjusted in a growth rate-dependent manner by the FtsH protease making sure that LPS biosynthesis is coordinated with the cellular demand. As a result, LpxC is stable in fast-growing cells and prone to degradation in slow-growing cells. One of the factors involved in this process is the alarmone guanosine tetraphosphate (ppGpp) but previous studies suggested the involvement of yet unknown factors in LpxC degradation. We established a quantitative proteomics approach aiming at the identification of proteins that are associated with LpxC and/or FtsH at high or low growth rates. The identification of known LpxC and FtsH interactors validated our approach. A number of proteins involved in fatty acid biosynthesis and degradation, including the central regulator FadR, were found in the LpxC and/or FtsH interactomes. Another protein associated with LpxC and FtsH was WaaH, a LPS-modifying enzyme. When overproduced, several members of the LpxC/FtsH interactomes were able to modulate LpxC proteolysis. Our results go beyond the previously established link between LPS and phospholipid biosynthesis and uncover a far-reaching network that controls LPS production by involving multiple enzymes in fatty acid metabolism, phospholipid biosynthesis and LPS modification.

Strategies in relative and absolute quantitative mass spectrometry based proteomics.

Quantitative mass spectrometry approaches are used for absolute and relative quantification in global proteome studies. To date, relative and absolute quantification techniques are available that differ in quantification accuracy, proteome coverage, complexity and robustness. This review focuses on most common relative or absolute quantification strategies exemplified by three experimental studies. A label-free relative quantification approach was performed for the investigation of the membrane proteome of sensory cilia to the depth of olfactory receptors in Mus musculus. A SILAC-based relative quantification approach was successfully applied for the identification of core components and transient interactors of the peroxisomal importomer in Saccharomyces cerevisiae. Furthermore, AQUA using stable isotopes was exemplified to unraveling the prenylome influenced by novel prenyltransferase inhibitors. Characteristic enrichment and fragmentation strategies for a robust quantification of the prenylome are also summarized.

In vivo trapping of FtsH substrates by label-free quantitative proteomics.

FtsH is the only membrane-bound and essential protease in Escherichia coli. It is responsible for the degradation of regulatory proteins and enzymes such as the heat-shock sigma factor RpoH or LpxC, the key enzyme of lipopolysaccharide biosynthesis. To find new FtsH targets, we trapped substrates in E. coli cells from exponential and stationary growth phase by using a proteolytically inactive FtsH variant. Subsequent analysis of the isolated FtsH-substrate complexes by label-free nanoLC-MS/MS revealed more than 50 putative FtsH substrates, among them five already known substrates. Four out of thirty-seven tested candidates were found to be novel FtsH substrates as shown by in vivo degradation experiments. Six other candidates were degraded by one or more other protease(s). The FtsH substrates SecD and ExbD are involved in transport processes across the membrane, whereas the physiological roles of YlaC and YhbT are yet unknown. The presence of the previously identified YfgM degron in two of the novel substrates suggests general rules for substrate recognition of this unique protease.

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

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 (σ(32)) 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 (FtsH(trap)) carrying a single amino acid exchange in the proteolytic center was expressed and purified in E. coli. FtsH(trap) 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 FtsH(trap) and wild-type FtsH (FtsH(WT)) were purified, separated by two-dimensional PAGE, and subjected to mass spectrometry. Over-representation of LpxC in the FtsH(trap) 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 FtsH(trap) revealed previously unknown biological functions of the physiologically most important AAA(+) protease in E. coli.