Proteolysis to Identify Protease Substrates: Cleave to Decipher

Bhagwat, Sonali R.; Hajela, Krishnan; Kumar, Amit

doi:10.1002/pmic.201800011

Cited by 9 publications

(6 citation statements)

References 145 publications

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“…It is possible that we overlooked other RseP substrates by similar and/or other reasons. For the systematic identification of RseP substrates, it is necessary to improve the methods of sample preparations in mass analyses ( 31 , 78 , 79 ) and also use additional approaches such as the co-isolation of possible substrates with a proteolytically inactive RseP and systematic screening of type II membrane proteins in combination with the mass analysis-based proteomic approach. Such approaches would also be useful to search for the unknown substrate of other IMPs.…”

Section: Discussionmentioning

confidence: 99%

The Escherichia coli S2P intramembrane protease RseP regulates ferric citrate uptake by cleaving the sigma factor regulator FecR

Yokoyama

Niinae

Tsumagari

et al. 2021

Journal of Biological Chemistry

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Escherichia coli RseP, a member of the site-2 protease family of intramembrane proteases, is involved in the activation of the σ E extracytoplasmic stress response and elimination of signal peptides from the cytoplasmic membrane. However, whether RseP has additional cellular functions is unclear. In this study, we used mass spectrometry–based quantitative proteomic analysis to search for new substrates that might reveal unknown physiological roles for RseP. Our data showed that the levels of several Fec system proteins encoded by the fecABCDE operon ( fec operon) were significantly decreased in an RseP-deficient strain. The Fec system is responsible for the uptake of ferric citrate, and the transcription of the fec operon is controlled by FecI, an alternative sigma factor, and its regulator FecR, a single-pass transmembrane protein. Assays with a fec operon expression reporter demonstrated that the proteolytic activity of RseP is essential for the ferric citrate–dependent upregulation of the fec operon. Analysis using the FecR protein and FecR-derived model proteins showed that FecR undergoes sequential processing at the membrane and that RseP participates in the last step of this sequential processing to generate the N-terminal cytoplasmic fragment of FecR that participates in the transcription of the fec operon with FecI. A shortened FecR construct was not dependent on RseP for activation, confirming this cleavage step is the essential and sufficient role of RseP. Our study unveiled that E. coli RseP performs the intramembrane proteolysis of FecR, a novel physiological role that is essential for regulating iron uptake by the ferric citrate transport system.

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Section: Discussionmentioning

confidence: 99%

The Escherichia coli S2P intramembrane protease RseP regulates ferric citrate uptake by cleaving the sigma factor regulator FecR

Yokoyama

Niinae

Tsumagari

et al. 2021

Journal of Biological Chemistry

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show abstract

“…It is possible that we overlooked other RseP substrates by similar and/or other reasons. For the systematic identification of RseP substrates, it is necessary to improve the methods of sample preparations in mass analyses (31, 79, 80) and also use additional approaches such as the co-isolation of possible substrates with a proteolytically inactive RseP and systematic screening of type II membrane proteins in combination with the mass analysis-based proteomic approach. Such approaches would also be useful to search for the unknown substrate of other IMPs.…”

Section: Discussionmentioning

confidence: 99%

Escherichia coliS2P family intramembrane protease RseP is engaged in the regulated sequential cleavages of FecR in the ferric citrate signaling

Yokoyama

Niinae

Tsumagari

et al. 2021

Preprint

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Escherichia coli RseP, a member of the S2P family of intramembrane proteases, is involved in the activation of the σE extracytoplasmic stress response and elimination of remnant signal peptides. However, whether RseP has additional cellular functions is unclear. In this study, we attempted to identify new RseP substrates to explore still unknown physiological roles of this protease. Our mass spectrometry-based quantitative proteomic analysis revealed that the levels of several Fec system proteins encoded by the fecABCDE operon (fec operon) were significantly decreased in an RseP-deficient strain. The Fec system is responsible for the uptake of ferric citrate, and the transcription of the fec operon is controlled by FecI, an alternative sigma factor, and its regulator FecR, a single-pass transmembrane protein. Assays with the fec operon expression reporter demonstrated that the proteolytic activity of RseP is essential for the ferric citrate-dependent upregulation of the fec operon. Analysis using the FecR protein and FecR-derived model proteins showed that FecR undergoes sequential processing at the membrane and that RseP participates in the last step of this sequential processing to generate the N-terminal cytoplasmic fragment of FecR that participates in the transcription of the fec operon with FecI. Ferric citrate signal-dependent generation of this cleavage product is the essential and sufficient role of RseP in the transcriptional activation of the fec operon. Our study unveiled that E. coli RseP performs the intramembrane proteolysis of FecR, a novel physiological role that is essential for regulating iron uptake by the ferric citrate transport system.

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“…Combinations of different experimental strategies are necessary to reveal the physiological substrates and hence, the molecular functions of plant proteases. Quantitative mass spectrometry-based proteomics enables large-scale interrogation of plant proteomes and allows the identification of protease cleavage sites and determination of protease sequence specificity [39,40]. Biochemical profiling of active sites using proteome-derived peptide libraries in combination with quantitative proteomics is useful to simultaneously identify N-terminal and C-terminal cleavage motifs [41].…”

Section: Future Perspectivesmentioning

confidence: 99%

Proteases of Nicotiana benthamiana: an emerging battle for molecular farming

Jutras

Dodds

Hoorn

2020

Current Opinion in Biotechnology

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Molecular farming increasingly uses the tobacco relative Nicotiana benthamiana for production of recombinant proteins through transient expression. Several proteins are produced efficiently with this expression platform, but yields for other proteins are often very low. These low yields are frequently due to endogenous proteases. The latest genome annotations indicate that N. benthamiana encodes for at least 1243 putative proteases that probably act redundantly and consecutively on substrates in different subcellular compartments. Here, we discuss the N. benthamiana protease repertoire that may affect recombinant protein production and recent advances in protease depletion strategies to increase recombinant protein production in N. benthamiana.

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Proteolysis to Identify Protease Substrates: Cleave to Decipher

Cited by 9 publications

References 145 publications

The Escherichia coli S2P intramembrane protease RseP regulates ferric citrate uptake by cleaving the sigma factor regulator FecR

The Escherichia coli S2P intramembrane protease RseP regulates ferric citrate uptake by cleaving the sigma factor regulator FecR

Escherichia coliS2P family intramembrane protease RseP is engaged in the regulated sequential cleavages of FecR in the ferric citrate signaling

Proteases of Nicotiana benthamiana: an emerging battle for molecular farming

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