Summary of Study ST003602

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002230. The data can be accessed directly via it's Project DOI: 10.21228/M8CJ97 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST003602
Study Title	Metabolic effects of cellular necrosis caused by exfoliative toxin C (ExhC) from Mammaliccocus sciuri
Study Summary	Exfoliative toxins (ETs) are glutamyl endopeptidases (GEPs) of the chymotrypsin-like serine protease family (CLSPs) that play crucial roles in diverse skin diseases. Specifically, exfoliative toxin C (ExhC), expressed by Mammaliicoccus sciuri, is an atypical CLSP and is been classified as a moonlighting protein due to its ability to induce necrosis in specific cell lines, inhibits the phagocytic activity of macrophages, and causes skin exfoliation in pigs and mice. The latter function is due to the high specificity of ExhC for porcine and murine desmoglein-1, a cadherin that contributes to cell-cell adhesion within the epidermis. While the amino acid residues responsible for ExhC-induced necrosis have been identified, the specific cellular metabolic pathways it affects to induce cell death remain unclear. Herein, we employed Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS) to explore the metabolic pathways affected by the necrotic activity of ExhC in the BHK-21 cell line. The metabolic profile of cells exposed to sub-toxic doses of ExhC revealed significant alterations in oxidative stress protection, energy production, and gene expression pathways. The data demonstrate the potential mechanisms of action of ExhC and highlight that this toxin causes cellular damage, even at low concentrations.
Institute	São Paulo State University - UNESP
Department	Physics Department (Rio Preto)
Laboratory	NMR Laboratory
Last Name	de Moraes
First Name	Fábio
Address	Cristóvão Colombo Street, 2265
Email	fabio.moraes@unesp.br
Phone	1732212454
Submit Date	2024-11-28
Num Groups	2
Total Subjects	16
Raw Data Available	Yes
Raw Data File Type(s)	fid
Analysis Type Detail	NMR
Release Date	2025-06-21
Release Version	1

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Project:

Project ID:	PR002230
Project DOI:	doi: 10.21228/M8CJ97
Project Title:	Metabolic effects of cellular necrosis caused by exfoliative toxin C (ExhC) from Mammaliccocus sciuri
Project Type:	Untargetd NMR-based metabolomics
Project Summary:	Exfoliative toxins (ETs) are glutamyl endopeptidases (GEPs) of the chymotrypsin-like serine protease family (CLSPs) that play crucial roles in diverse skin diseases. Specifically, exfoliative toxin C (ExhC), expressed by Mammaliicoccus sciuri, is an atypical CLSP and is been classified as a moonlighting protein due to its ability to induce necrosis in specific cell lines, inhibits the phagocytic activity of macrophages, and causes skin exfoliation in pigs and mice. The latter function is due to the high specificity of ExhC for porcine and murine desmoglein-1, a cadherin that contributes to cell-cell adhesion within the epidermis. While the amino acid residues responsible for ExhC-induced necrosis have been identified, the specific cellular metabolic pathways it affects to induce cell death remain unclear. Herein, we employed Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS) to explore the metabolic pathways affected by the necrotic activity of ExhC in the BHK-21 cell line. The metabolic profile of cells exposed to sub-toxic doses of ExhC revealed significant alterations in oxidative stress protection, energy production, and gene expression pathways. The data demonstrate the potential mechanisms of action of ExhC and highlight that this toxin causes cellular damage, even at low concentrations.
Institute:	São Paulo State University - UNESP
Department:	Physics Department (Rio Preto)
Laboratory:	NMR Laboratory
Last Name:	de Moraes
First Name:	Fábio
Address:	Cristóvão Colombo Street, 2265
Email:	fabio.moraes@unesp.br
Phone:	1732212454
Funding Source:	Fapesp and CNPq
Project Comments:	FAPESP, #2020/08615-8 and CNPq, #309940/2019-2
Publications:	to be submitted
Contributors:	Carolina Gismene, Fábio Rogério de Moraes, Anelize Bauermeister, Thyerre Santana Da Costa, Marília Freitas Calmon, Paula Rahal, Rejane Maira Góes, Luiz Alberto Beraldo de Moraes, Ljubica Tasic, Raghuvir Krishnaswamy Arni

Subject:

Subject ID:	SU003731
Subject Type:	Cultured cells
Subject Species:	Mesocricetus Auratus

Factors:

Subject type: Cultured cells; Subject species: Mesocricetus Auratus (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment
SA392541	C1	Control
SA392542	C2	Control
SA392543	C3	Control
SA392544	C4	Control
SA392545	C5	Control
SA392546	C6	Control
SA392547	C7	Control
SA392548	C8	Control
SA392549	C9	Control
SA392550	C10	Control
SA392551	T1	Treatment with Toxin
SA392552	T2	Treatment with Toxin
SA392553	T3	Treatment with Toxin
SA392554	T4	Treatment with Toxin
SA392555	T5	Treatment with Toxin

Showing results 1 to 15 of 15

Showing results 1 to 15 of 15

Collection:

Collection ID:	CO003724
Collection Summary:	Around 1.2 x 107 BHK-21 cells were seeded in 60 mm plates for 24 h and, then, treated with 7.5 µmol.L-1 of ExhC for 48 h. After treatment, the cells were detached with 0.25% trypsin-EDTA (0.25%) (Gibco - Thermo Fisher Scientific, Waltham, MA, USA), followed by two washes with PBS 1x, and the addition of 2 mL of 80% methanol (Sigma-Aldrich). The cell solution was vortexed for one minute followed by centrifugation at 4000 rpm for 20 min at 4 °C. The aqueous extract obtained was lyophilized in a vacuum centrifuge (Concentrator 5301, Eppendorf, Hamburg, Germany) and stored in the freezer at -80 °C. The extraction of intracellular metabolites was also performed with 1.2 x 107 of BHK-21 cells after 48 h of growth in DMEM medium, representing the control experiment. Overall, five samples of intracellular metabolites were obtained in the presence of ExhC (i) and ten samples of intracellular metabolites were obtained in the absence of ExhC (ii). The intracellular samples were diluted in 700 µL of deuterium oxide with 3-(trimethylsilyl)propionic-2,2,3,3-d₄ acid sodium salt - TSP (D2O 99.9%, Sigma Aldrich) and transferred to 5 mm tubes for data acquisition by Nuclear Magnetic Resonance (NMR).
Collection Protocol Filename:	ExhC_protocol.pdf
Sample Type:	Kidney

Treatment:

Treatment ID:	TR003740
Treatment Summary:	Around 1.2 x 107 BHK-21 cells were seeded in 60 mm plates for 24 h and, then, treated with 7.5 µmol.L-1 of ExhC for 48 h.

Sample Preparation:

Sampleprep ID:	SP003738
Sampleprep Summary:	After treatment, the cells were detached with 0.25% trypsin-EDTA (0.25%) (Gibco - Thermo Fisher Scientific, Waltham, MA, USA), followed by two washes with PBS 1x, and the addition of 2 mL of 80% methanol (Sigma-Aldrich). The cell solution was vortexed for one minute followed by centrifugation at 4000 rpm for 20 min at 4 °C. The aqueous extract obtained was lyophilized in a vacuum centrifuge (Concentrator 5301, Eppendorf, Hamburg, Germany) and stored in the freezer at -80 °C. The extraction of intracellular metabolites was also performed with 1.2 x 107 of BHK-21 cells after 48 h of growth in DMEM medium, representing the control experiment. Overall, five samples of intracellular metabolites were obtained in the presence of ExhC (i) and ten samples of intracellular metabolites were obtained in the absence of ExhC (ii). The intracellular samples were diluted in 700 µL of deuterium oxide with 3-(trimethylsilyl)propionic-2,2,3,3-d₄ acid sodium salt - TSP (D2O 99.9%, Sigma Aldrich) and transferred to 5 mm tubes for data acquisition by Nuclear Magnetic Resonance (NMR).

Sampleprep ID:

SP003738

Sampleprep Summary:

After treatment, the cells were detached with 0.25% trypsin-EDTA (0.25%) (Gibco - Thermo Fisher Scientific, Waltham, MA, USA), followed by two washes with PBS 1x, and the addition of 2 mL of 80% methanol (Sigma-Aldrich). The cell solution was vortexed for one minute followed by centrifugation at 4000 rpm for 20 min at 4 °C. The aqueous extract obtained was lyophilized in a vacuum centrifuge (Concentrator 5301, Eppendorf, Hamburg, Germany) and stored in the freezer at -80 °C. The extraction of intracellular metabolites was also performed with 1.2 x 107 of BHK-21 cells after 48 h of growth in DMEM medium, representing the control experiment. Overall, five samples of intracellular metabolites were obtained in the presence of ExhC (i) and ten samples of intracellular metabolites were obtained in the absence of ExhC (ii). The intracellular samples were diluted in 700 µL of deuterium oxide with 3-(trimethylsilyl)propionic-2,2,3,3-d₄ acid sodium salt - TSP (D2O 99.9%, Sigma Aldrich) and transferred to 5 mm tubes for data acquisition by Nuclear Magnetic Resonance (NMR).

Analysis:

Analysis ID:	AN005918
Laboratory Name:	NMR Laboratory
Analysis Type:	NMR
Software Version:	TopSpin 4.4.0
Operator Name:	Fábio
Num Factors:	2
Num Metabolites:	28
Units:	TSP_normalized

NMR:

NMR ID:	NM000296
Analysis ID:	AN005918
Instrument Name:	Bruker
Instrument Type:	FT-NMR
NMR Experiment Type:	1D-1H
Spectrometer Frequency:	600 MHz
NMR Probe:	triple resonance broadband inverse probe (PH TBI 600S3)
NMR Solvent:	90% H2O + 10% D2O
NMR Tube Size:	5mm
Pulse Sequence:	1D Carr-Purcell-Meiboom-Gill
Water Suppression:	pre saturation
Pulse Width:	9.2 us
Power Level:	17.378 W
Receiver Gain:	203
Offset Frequency:	0.5
Presaturation Power Level:	-42.30 dBW
Chemical Shift Ref Cpd:	TSP
Temperature:	25
Number Of Scans:	128
Acquisition Time:	3.89
Relaxation Delay:	4s
Spectral Width:	14 ppm