Dataset Information

Cell-based characterization of caspase-3 cleavage sites by LATE and HYATNE

ABSTRACT: Proteins' N-termini contain information about their biochemical properties and functions, and they can undergo co- or post-translational modifications, as well as be processed by proteases. To expand the coverage of the N-terminome, we have developed LATE (LysN Amino Terminal Enrichment), a method that uses selective chemical derivatization of α-amines to isolate the N-terminal peptides. We applied LATE in combination with other N-terminomic method to study caspase-3 mediated proteolysis both in vitro and during apoptosis in cells. This enable us to identify many unreported caspase-3 cleavages, including some that cannot be identified by other methods. Furthermore, we find direct evidence that some of the caspase-3 generated neo-N-termini can be further modified by Nt-acetylation. This provides a global picture of the caspase-3 degradome and uncovers previously unrecognised functional crosstalk between post-translational Nt-acetylation and caspase proteolysis pathways.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Oded Kleifeld

LAB HEAD: Oded Kleifeld

PROVIDER: PXD036648 | Pride | 2023-06-27

REPOSITORIES: Pride

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Publications

In-Depth Characterization of Apoptosis N-Terminome Reveals a Link Between Caspase-3 Cleavage and Posttranslational N-Terminal Acetylation.

Hanna Rawad R Rozenberg Andrey A Saied Layla L Ben-Yosef Daniel D Lavy Tali T Kleifeld Oded O

Molecular & cellular proteomics : MCP 20230524 7

The N termini of proteins contain information about their biochemical properties and functions. These N termini can be processed by proteases and can undergo other co- or posttranslational modifications. We have developed LATE (LysN Amino Terminal Enrichment), a method that uses selective chemical derivatization of α-amines to isolate the N-terminal peptides, in order to improve N-terminome identification in conjunction with other enrichment strategies. We applied LATE alongside another N-termin ...[more]

PMID: 37236440

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Project description:Background and Aims: Inflammasome-mediated caspase-1 activity regulates the maturation and release of the pro-inflammatory cytokines interleukin (IL)-1M-CM-^_ and IL-18. Recently, we showed that caspase-1 deficiency strongly reduces high fat diet-induced adiposity although the mechanism is still unclear. We now aimed to elucidate the mechanism by which caspase-1 deficiency reduces modulates resistance to high fat diet-feeding fat accumulation in adipose tissue by focusing on the role of caspase-1 in the regulation of triglyceride (TG)-rich lipoprotein metabolism. Methods: Caspase-1 deficient and wild-type mice (both C57Bl/6 background) were used to determine postprandial TG kinetics, intestinal TG absorption, VLDL-TG production as well as TG clearance, all of which strongly contribute to the supply of TG for storage in adipose tissue. Micro-array and qPCR analysis were used to unravel intestinal and hepatic metabolic pathways involved. Results: Caspase-1 deficiency reduced the postprandial response to an oral lipid load, while tissue specific clearance of TG-rich lipoproteins was not changed. Indeed, an oral olive oil gavage containing [3H]TG revealed that caspase-1 deficiency significantly decreased intestinal chylomicron-TG production and reduced the uptake of [3H]TG-derived FA by liver, muscle, and adipose tissue. Similarly, caspase-1 deficiency reduced the hepatic VLDL-TG production without reducing VLDL-apoB production, despite an elevated hepatic TG content. Pathway analysis revealed that caspase-1 deficiency reduces intestinal and hepatic expression of genes involved in lipogenesis. Conclusions: Absence of caspase-1 reduces assembly and secretion of TG-rich lipoproteins, thereby reducing the availability of TG-derived FA for uptake by peripheral organs including adipose tissue. We anticipate that caspase-1 represents a novel link between innate immunity and lipid metabolism. Keywords: Expression profiling by array Wild-type (WT) and Casp1-null mice were maintained at lab chow. Animals, aged between 14 and 16 weeks (n=3 per genotype), were killed and liver and intestinal segments were removed. Livers were isolated from mice that were fasted over night, whereas intesines were removed from mice 2 hrs after they received an oral lipid load.Total RNA was isolated and subjected to gene expression profiling.

Project description:Murine BV2 microglia cells were transfected either with siRNA negative control or siRNA against caspase-3 for 48h. Later on some the of the BV2 transfected cells were co-cultured with C6 glioma cells during 6h. We used the SA Biosciences Mouse Wound Healing PCR Array (PAMM-121Z) to quantitate gene expression of relevant genes related to the wound healing process Glioma cells recruit and exploit microglia, resident immune cells of the brain, for their proliferation and invasion capability. The underlying molecular mechanism used by glioma cells to transform microglia into a tumor-supporting phenotype remains elusive. Here, we report that glioma-induced microglia conversion is coupled to a reduction of basal microglial caspase-3 activity, increased S-nitrosylation of mitochondria-associated caspase-3 through inhibition of thioredoxin-2 activity, and demonstrate that caspase-3 inhibition regulates microglial tumor-supporting function. Further, we identified nitric oxide synthase-2 (NOS2) activity originating from the glioma cells as a driving stimulus in the control of microglial caspase-3 activity. Repression of glioma NOS2 expression in vivo led to reduction in both microglia recruitment and tumor expansion, whereas depletion of the microglial caspase-3 gene promoted tumor growth. This study provides evidence that the inhibition of Trx2-mediated denitrosylation of SNO-procaspase-3 is part of the microglial pro-tumoral activation pathway initiated by glioma cancer cells. qPCR gene expression profiling. Three independent experiments of siControl BV2 monoculture, siCaspase3 BV2 monoculture, siControl BV2 cocultured 6h with C6 glioma cells and siCaspase3 BV2 cocultured 6h with C6 glioma cells. Equal amount total RNA from each culture was used for the gene expression analysis. Please note that the raw data for three independent experiments (prior to averaging the data) is provided in the 'Raw_Data_File_with_the_Ct_values_for_3_indep_experiments.txt'.

Dataset Information

Cell-based characterization of caspase-3 cleavage sites by LATE and HYATNE

Publications

In-Depth Characterization of Apoptosis N-Terminome Reveals a Link Between Caspase-3 Cleavage and Posttranslational N-Terminal Acetylation.

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