Selektive Aktivierung der humanen caseinolytischen Protease P (ClpP)

Approaches for profiling protease substrates are critical for defining protease functions, but remain challenging tasks. We combine genetic code expansion, photocrosslinking and proteomics to identify substrates of the mitochondrial (mt) human caseinolytic protease P (hClpP). Sitespecific incorporation of the diazirine-bearing amino acid DiazK into the inner proteolytic chamber of hClpP, followed by UV-irradiation of cells, allows to covalently trap substrate proteins of hClpP and to substantiate hClpP's major involvement in maintaining overall mt homeostasis. In addition to confirming many of the previously annotated hClpP substrates, our approach adds a diverse set of new proteins to the hClpP interactome. Importantly, our workflow allows identifying substrate dynamics upon application of external cues in an unbiased manner. Identification of unique hClpP-substrate proteins upon induction of mt oxidative stress, suggests that hClpP counteracts oxidative stress by processing of proteins that are involved in respiratory chain complex synthesis and maturation as well as in catabolic pathways.

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Substrate Profiling of Mitochondrial Caseinolytic Protease P via a Site‐Specific Photocrosslinking Approach

Nguyen

Gronauer

Nast-Kolb

et al. 2022

Angewandte Chemie

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Substrate Profiling of Mitochondrial Caseinolytic Protease P via a Site‐Specific Photocrosslinking Approach

et al. 2022

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Acyldepsipeptide Probes Facilitate Specific Detection of Caseinolytic Protease P Independent of Its Oligomeric and Activity State

et al. 2020

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Caseinolytic protease P (ClpP) is a tetradecameric peptidase that assembles with chaperones such as ClpX to gain proteolytic activity. Acyldepsipeptides (ADEPs) are small‐molecule mimics of ClpX that bind into hydrophobic pockets on the apical site of the complex, thereby activating ClpP. Detection of ClpP has so far been facilitated with active‐site‐directed probes which depend on the activity and oligomeric state of the complex. To expand the scope of ClpP labeling, we took a stepwise synthetic approach toward customized ADEP photoprobes. Structure–activity relationship studies with small fragments and ADEP derivatives paired with modeling studies revealed the design principles for suitable probe molecules. The derivatives were tested for activation of ClpP and subsequently applied in labeling studies of the wild‐type peptidase as well as enzymes bearing mutations at the active site and an oligomerization sensor. Satisfyingly, the ADEP photoprobes provided a labeling readout of ClpP independent of its activity and oligomeric state.

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Selektive Aktivierung der humanen caseinolytischen Protease P (ClpP)

Cited by 3 publications

References 30 publications

Substrate Profiling of Mitochondrial Caseinolytic Protease P via a Site‐Specific Photocrosslinking Approach

Substrate Profiling of Mitochondrial Caseinolytic Protease P via a Site‐Specific Photocrosslinking Approach

Substrate Profiling of Mitochondrial Caseinolytic Protease P via a Site‐Specific Photocrosslinking Approach

Acyldepsipeptide Probes Facilitate Specific Detection of Caseinolytic Protease P Independent of Its Oligomeric and Activity State

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