Dataset Information

Substrate profiling of mitochondrial caseinolytic protease P via a site-specific photocrosslinking approach

ABSTRACT: The function of proteases is mainly determined by the substrates they are processing. However, substrate identification remains a challenging task. Here, we used genetic code expansion to incorporate a diazirine-lysine amino acid (DiazK) at selected positions of mitochondrial caseinolytic protease P (ClpP) in mammalian cells and determined its substrate scope by proteomic analyses after UV-light dependent photocrosslink. The results confirm its major involvement in maintaining the overall mitochondrial protein homeostasis as evident from alternative approaches. Additionally, novel putative substrates were identified revealing the highly complementary nature of this approach. Furthermore, this allows for the identification of changing substrate dynamics in an unbiased manner. Induction of oxidative stress with rotenone followed by identification of unique proteins corroborate its role in mitochondrial stress response. We hypothesize, that the oxidative stress is counteracted by processing proteins, which are involved in respiratory chain complex proteins synthesis and maturation as well as catabolic enzymes respectively.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Thomas Gronauer

LAB HEAD: Stephan Axel Sieber

PROVIDER: PXD027954 | Pride | 2023-08-22

REPOSITORIES: Pride

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

Nguyen Tuan-Anh TA Gronauer Thomas F TF Nast-Kolb Timon T Sieber Stephan A SA Lang Kathrin K

Angewandte Chemie (International ed. in English) 20220114 10

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). Site-specific 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 substantia ...[more]

PMID: 34847623

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