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In vivo proteolytic profiling of the type I and type II metacaspases in Chlamydomonas reinhardtii exposed to salt stress

ABSTRACT: Metacaspases are cysteine proteases present in plants, fungi and protists. While the association of metacaspases with cell death is studied in a range of organisms, their native substrates are largely unknown. Here, we explored the in vivo proteolytic landscape of the two metacaspases, CrMCA-I and CrMCA-II, present in the green freshwater alga Chlamydomonas reinhardtii, using N-terminal peptide labelling and enrichment during standard conditions and salt stress. Comparison between the cleavage events of CrMCA-I and CrMCA-II in metacaspase mutants revealed unique cleavage preferences and substrate specificity. Degradome analysis demonstrated relevance of the predicted metacaspase substrates to the physiology of Chlamydomonas reinhardtii cells and its adaptation during salt stress. Functional enrichment analysis indicated an involvement of CrMCA-I in photosynthesis and translation, while CrMCA-II seems to be important during the catabolism of carboxylic acids. Altogether, our findings suggest distinct cellular functions of the two metacaspases in C. reinhardtii during salt stress response.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Chlamydomonas Reinhardtii

SUBMITTER: Evy Timmerman

LAB HEAD: Christiane Funk

PROVIDER: PXD051835 | Pride | 2024-06-11

REPOSITORIES: Pride

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			Action	DRS
	F004515.dat	Other
	F004515.mgf	Mgf
	F004516.dat	Other
	F004516.mgf	Mgf
	F004524.dat	Other

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Project description:Metacaspases are part of an evolutionarily broad family of multifunctional cysteine proteases, involved in disease and normal development. Despite the extensive study of metacaspases in the two decades since their discovery, the structure-function relationship of metacaspases remains poorly understood. Furthermore, previous studies on their function have been thwarted by the redundancy in gene copy number and potential phenotypic suppression of genetic mutations, especially in plants. Here, we have solved the X-ray crystal structure of an Arabidopsis thaliana type II metacaspase (AtMCA-IIf) that belongs to a particular sub-group that does not require calcium ions for activation. Compared to crystal structures of other metacaspases and caspases, the AtMCA-IIf active site is structurally similar and poses a conundrum for the catalytic mechanism of the cysteine-histidine dyad. To study metacaspase activity in plants, we developed an in vitro chemical screen to identify small molecule metacaspase inhibitors. Several hits with a minimal thioxodihydropyrimidine-dione (TDP) structure were identified, some being specific inhibitors of AtMCA-IIf. We provide a mechanistic basis for inhibition by the TDP-containing compounds through molecular docking onto the AtMCA-IIf crystal structure. Finally, a TDP-containing compound (TDP6) was effective at inhibiting lateral root emergence in vivo, likely through the inhibition of metacaspases that are specifically expressed in the endodermal cells overlaying developing lateral root primordia. In the future, the small compound inhibitors and crystal structure of AtMCA-IIf can be used to study metacaspases in various other species, such as important human pathogens including those causing neglected diseases.

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Proteomic comparison of Chlamydomonas wildtype and calredoxin knock-down mutants via 14N/15N metabolic labeling

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Identification of heat stress-targets of translational control by large scale analysis of Arabidopsis trancriptome and translatome.

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Protein expression and identification of Tyrp1 Mutant Variants Associated with OCA3 form of albinism.

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