#METABOLOMICS WORKBENCH fabio_moraes_20241128_110632 DATATRACK_ID:5411 STUDY_ID:ST003602 ANALYSIS_ID:AN005918 PROJECT_ID:PR002230
VERSION             	1
CREATED_ON             	December 5, 2024, 10:20 am
#PROJECT
PR:PROJECT_TITLE                 	Metabolic effects of cellular necrosis caused by exfoliative toxin C (ExhC) from
PR:PROJECT_TITLE                 	Mammaliccocus sciuri
PR:PROJECT_TYPE                  	Untargetd NMR-based metabolomics
PR:PROJECT_SUMMARY               	Exfoliative toxins (ETs) are glutamyl endopeptidases (GEPs) of the
PR:PROJECT_SUMMARY               	chymotrypsin-like serine protease family (CLSPs) that play crucial roles in
PR:PROJECT_SUMMARY               	diverse skin diseases. Specifically, exfoliative toxin C (ExhC), expressed by
PR:PROJECT_SUMMARY               	Mammaliicoccus sciuri, is an atypical CLSP and is been classified as a
PR:PROJECT_SUMMARY               	moonlighting protein due to its ability to induce necrosis in specific cell
PR:PROJECT_SUMMARY               	lines, inhibits the phagocytic activity of macrophages, and causes skin
PR:PROJECT_SUMMARY               	exfoliation in pigs and mice. The latter function is due to the high specificity
PR:PROJECT_SUMMARY               	of ExhC for porcine and murine desmoglein-1, a cadherin that contributes to
PR:PROJECT_SUMMARY               	cell-cell adhesion within the epidermis. While the amino acid residues
PR:PROJECT_SUMMARY               	responsible for ExhC-induced necrosis have been identified, the specific
PR:PROJECT_SUMMARY               	cellular metabolic pathways it affects to induce cell death remain unclear.
PR:PROJECT_SUMMARY               	Herein, we employed Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS)
PR:PROJECT_SUMMARY               	to explore the metabolic pathways affected by the necrotic activity of ExhC in
PR:PROJECT_SUMMARY               	the BHK-21 cell line. The metabolic profile of cells exposed to sub-toxic doses
PR:PROJECT_SUMMARY               	of ExhC revealed significant alterations in oxidative stress protection, energy
PR:PROJECT_SUMMARY               	production, and gene expression pathways. The data demonstrate the potential
PR:PROJECT_SUMMARY               	mechanisms of action of ExhC and highlight that this toxin causes cellular
PR:PROJECT_SUMMARY               	damage, even at low concentrations.
PR:INSTITUTE                     	São Paulo State University - UNESP
PR:DEPARTMENT                    	Physics Department (Rio Preto)
PR:LABORATORY                    	NMR Laboratory
PR:LAST_NAME                     	de Moraes
PR:FIRST_NAME                    	Fábio
PR:ADDRESS                       	Cristóvão Colombo Street, 2265
PR:EMAIL                         	fabio.moraes@unesp.br
PR:PHONE                         	1732212454
PR:FUNDING_SOURCE                	Fapesp and CNPq
PR:PROJECT_COMMENTS              	FAPESP, #2020/08615-8 and CNPq, #309940/2019-2
PR:PUBLICATIONS                  	to be submitted
PR:CONTRIBUTORS                  	Carolina Gismene, Fábio Rogério de Moraes, Anelize Bauermeister, Thyerre
PR:CONTRIBUTORS                  	Santana Da Costa, Marília Freitas Calmon, Paula Rahal, Rejane Maira Góes, Luiz
PR:CONTRIBUTORS                  	Alberto Beraldo de Moraes, Ljubica Tasic, Raghuvir Krishnaswamy Arni
#STUDY
ST:STUDY_TITLE                   	Metabolic effects of cellular necrosis caused by exfoliative toxin C (ExhC) from
ST:STUDY_TITLE                   	Mammaliccocus sciuri
ST:STUDY_SUMMARY                 	Exfoliative toxins (ETs) are glutamyl endopeptidases (GEPs) of the
ST:STUDY_SUMMARY                 	chymotrypsin-like serine protease family (CLSPs) that play crucial roles in
ST:STUDY_SUMMARY                 	diverse skin diseases. Specifically, exfoliative toxin C (ExhC), expressed by
ST:STUDY_SUMMARY                 	Mammaliicoccus sciuri, is an atypical CLSP and is been classified as a
ST:STUDY_SUMMARY                 	moonlighting protein due to its ability to induce necrosis in specific cell
ST:STUDY_SUMMARY                 	lines, inhibits the phagocytic activity of macrophages, and causes skin
ST:STUDY_SUMMARY                 	exfoliation in pigs and mice. The latter function is due to the high specificity
ST:STUDY_SUMMARY                 	of ExhC for porcine and murine desmoglein-1, a cadherin that contributes to
ST:STUDY_SUMMARY                 	cell-cell adhesion within the epidermis. While the amino acid residues
ST:STUDY_SUMMARY                 	responsible for ExhC-induced necrosis have been identified, the specific
ST:STUDY_SUMMARY                 	cellular metabolic pathways it affects to induce cell death remain unclear.
ST:STUDY_SUMMARY                 	Herein, we employed Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS)
ST:STUDY_SUMMARY                 	to explore the metabolic pathways affected by the necrotic activity of ExhC in
ST:STUDY_SUMMARY                 	the BHK-21 cell line. The metabolic profile of cells exposed to sub-toxic doses
ST:STUDY_SUMMARY                 	of ExhC revealed significant alterations in oxidative stress protection, energy
ST:STUDY_SUMMARY                 	production, and gene expression pathways. The data demonstrate the potential
ST:STUDY_SUMMARY                 	mechanisms of action of ExhC and highlight that this toxin causes cellular
ST:STUDY_SUMMARY                 	damage, even at low concentrations.
ST:INSTITUTE                     	São Paulo State University - UNESP
ST:DEPARTMENT                    	Physics Department (Rio Preto)
ST:LABORATORY                    	NMR Laboratory
ST:LAST_NAME                     	de Moraes
ST:FIRST_NAME                    	Fábio
ST:ADDRESS                       	Cristóvão Colombo Street, 2265
ST:EMAIL                         	fabio.moraes@unesp.br
ST:PHONE                         	1732212454
ST:NUM_GROUPS                    	2
ST:TOTAL_SUBJECTS                	16
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Mesocricetus Auratus
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	C1	Sample source:BHK-21 | Treatment:Control	RAW_FILE_NAME(Raw file name)=Files under the folder C1
SUBJECT_SAMPLE_FACTORS           	-	C2	Sample source:BHK-21 | Treatment:Control	RAW_FILE_NAME(Raw file name)=Files under the folder C2
SUBJECT_SAMPLE_FACTORS           	-	C3	Sample source:BHK-21 | Treatment:Control	RAW_FILE_NAME(Raw file name)=Files under the folder C3
SUBJECT_SAMPLE_FACTORS           	-	C4	Sample source:BHK-21 | Treatment:Control	RAW_FILE_NAME(Raw file name)=Files under the folder C4
SUBJECT_SAMPLE_FACTORS           	-	C5	Sample source:BHK-21 | Treatment:Control	RAW_FILE_NAME(Raw file name)=Files under the folder C5
SUBJECT_SAMPLE_FACTORS           	-	C6	Sample source:BHK-21 | Treatment:Control	RAW_FILE_NAME(Raw file name)=Files under the folder C6
SUBJECT_SAMPLE_FACTORS           	-	C7	Sample source:BHK-21 | Treatment:Control	RAW_FILE_NAME(Raw file name)=Files under the folder C7
SUBJECT_SAMPLE_FACTORS           	-	C8	Sample source:BHK-21 | Treatment:Control	RAW_FILE_NAME(Raw file name)=Files under the folder C8
SUBJECT_SAMPLE_FACTORS           	-	C9	Sample source:BHK-21 | Treatment:Control	RAW_FILE_NAME(Raw file name)=Files under the folder C9
SUBJECT_SAMPLE_FACTORS           	-	C10	Sample source:BHK-21 | Treatment:Control	RAW_FILE_NAME(Raw file name)=Files under the folder C10
SUBJECT_SAMPLE_FACTORS           	-	T1	Sample source:BHK-21 | Treatment:Treatment with Toxin	RAW_FILE_NAME(Raw file name)=Files under the folder T1
SUBJECT_SAMPLE_FACTORS           	-	T2	Sample source:BHK-21 | Treatment:Treatment with Toxin	RAW_FILE_NAME(Raw file name)=Files under the folder T2
SUBJECT_SAMPLE_FACTORS           	-	T3	Sample source:BHK-21 | Treatment:Treatment with Toxin	RAW_FILE_NAME(Raw file name)=Files under the folder T3
SUBJECT_SAMPLE_FACTORS           	-	T4	Sample source:BHK-21 | Treatment:Treatment with Toxin	RAW_FILE_NAME(Raw file name)=Files under the folder T4
SUBJECT_SAMPLE_FACTORS           	-	T5	Sample source:BHK-21 | Treatment:Treatment with Toxin	RAW_FILE_NAME(Raw file name)=Files under the folder T5
#COLLECTION
CO:COLLECTION_SUMMARY            	Around 1.2 x 107 BHK-21 cells were seeded in 60 mm plates for 24 h and, then,
CO:COLLECTION_SUMMARY            	treated with 7.5 µmol.L-1 of ExhC for 48 h. After treatment, the cells were
CO:COLLECTION_SUMMARY            	detached with 0.25% trypsin-EDTA (0.25%) (Gibco - Thermo Fisher Scientific,
CO:COLLECTION_SUMMARY            	Waltham, MA, USA), followed by two washes with PBS 1x, and the addition of 2 mL
CO:COLLECTION_SUMMARY            	of 80% methanol (Sigma-Aldrich). The cell solution was vortexed for one minute
CO:COLLECTION_SUMMARY            	followed by centrifugation at 4000 rpm for 20 min at 4 °C. The aqueous extract
CO:COLLECTION_SUMMARY            	obtained was lyophilized in a vacuum centrifuge (Concentrator 5301, Eppendorf,
CO:COLLECTION_SUMMARY            	Hamburg, Germany) and stored in the freezer at -80 °C. The extraction of
CO:COLLECTION_SUMMARY            	intracellular metabolites was also performed with 1.2 x 107 of BHK-21 cells
CO:COLLECTION_SUMMARY            	after 48 h of growth in DMEM medium, representing the control experiment.
CO:COLLECTION_SUMMARY            	Overall, five samples of intracellular metabolites were obtained in the presence
CO:COLLECTION_SUMMARY            	of ExhC (i) and ten samples of intracellular metabolites were obtained in the
CO:COLLECTION_SUMMARY            	absence of ExhC (ii). The intracellular samples were diluted in 700 µL of
CO:COLLECTION_SUMMARY            	deuterium oxide with 3-(trimethylsilyl)propionic-2,2,3,3-d₄ acid sodium salt -
CO:COLLECTION_SUMMARY            	TSP (D2O 99.9%, Sigma Aldrich) and transferred to 5 mm tubes for data
CO:COLLECTION_SUMMARY            	acquisition by Nuclear Magnetic Resonance (NMR).
CO:COLLECTION_PROTOCOL_FILENAME  	ExhC_protocol.pdf
CO:SAMPLE_TYPE                   	Kidney
#TREATMENT
TR:TREATMENT_SUMMARY             	Around 1.2 x 107 BHK-21 cells were seeded in 60 mm plates for 24 h and, then,
TR:TREATMENT_SUMMARY             	treated with 7.5 µmol.L-1 of ExhC for 48 h.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	After treatment, the cells were detached with 0.25% trypsin-EDTA (0.25%) (Gibco
SP:SAMPLEPREP_SUMMARY            	- Thermo Fisher Scientific, Waltham, MA, USA), followed by two washes with PBS
SP:SAMPLEPREP_SUMMARY            	1x, and the addition of 2 mL of 80% methanol (Sigma-Aldrich). The cell solution
SP:SAMPLEPREP_SUMMARY            	was vortexed for one minute followed by centrifugation at 4000 rpm for 20 min at
SP:SAMPLEPREP_SUMMARY            	4 °C. The aqueous extract obtained was lyophilized in a vacuum centrifuge
SP:SAMPLEPREP_SUMMARY            	(Concentrator 5301, Eppendorf, Hamburg, Germany) and stored in the freezer at
SP:SAMPLEPREP_SUMMARY            	-80 °C. The extraction of intracellular metabolites was also performed with 1.2
SP:SAMPLEPREP_SUMMARY            	x 107 of BHK-21 cells after 48 h of growth in DMEM medium, representing the
SP:SAMPLEPREP_SUMMARY            	control experiment. Overall, five samples of intracellular metabolites were
SP:SAMPLEPREP_SUMMARY            	obtained in the presence of ExhC (i) and ten samples of intracellular
SP:SAMPLEPREP_SUMMARY            	metabolites were obtained in the absence of ExhC (ii). The intracellular samples
SP:SAMPLEPREP_SUMMARY            	were diluted in 700 µL of deuterium oxide with
SP:SAMPLEPREP_SUMMARY            	3-(trimethylsilyl)propionic-2,2,3,3-d₄ acid sodium salt - TSP (D2O 99.9%,
SP:SAMPLEPREP_SUMMARY            	Sigma Aldrich) and transferred to 5 mm tubes for data acquisition by Nuclear
SP:SAMPLEPREP_SUMMARY            	Magnetic Resonance (NMR).
#ANALYSIS
AN:SOFTWARE_VERSION              	TopSpin 4.4.0
#NMR
NM:INSTRUMENT_NAME               	Bruker
NM:INSTRUMENT_TYPE               	FT-NMR
NM:NMR_EXPERIMENT_TYPE           	1D-1H
NM:SPECTROMETER_FREQUENCY        	600 MHz
NM:NMR_PROBE                     	triple resonance broadband inverse probe (PH TBI 600S3)
NM:NMR_SOLVENT                   	90% H2O + 10% D2O
NM:NMR_TUBE_SIZE                 	5mm
NM:PULSE_SEQUENCE                	1D Carr-Purcell-Meiboom-Gill
NM:WATER_SUPPRESSION             	pre saturation
NM:PULSE_WIDTH                   	9.2 us
NM:POWER_LEVEL                   	17.378 W
NM:RECEIVER_GAIN                 	203
NM:OFFSET_FREQUENCY              	0.5
NM:PRESATURATION_POWER_LEVEL     	-42.30 dBW
NM:CHEMICAL_SHIFT_REF_CPD        	TSP
NM:TEMPERATURE                   	25
NM:NUMBER_OF_SCANS               	128
NM:ACQUISITION_TIME              	3.89
NM:RELAXATION_DELAY              	4s
NM:SPECTRAL_WIDTH                	14 ppm
#NMR_METABOLITE_DATA
NMR_METABOLITE_DATA:UNITS	TSP_normalized
NMR_METABOLITE_DATA_START
Samples	C1	C2	C3	C4	C5	C6	C7	C8	C9	C10	T1	T2	T3	T4	T5
Factors	Sample source:BHK-21 | Treatment:Control	Sample source:BHK-21 | Treatment:Control	Sample source:BHK-21 | Treatment:Control	Sample source:BHK-21 | Treatment:Control	Sample source:BHK-21 | Treatment:Control	Sample source:BHK-21 | Treatment:Control	Sample source:BHK-21 | Treatment:Control	Sample source:BHK-21 | Treatment:Control	Sample source:BHK-21 | Treatment:Control	Sample source:BHK-21 | Treatment:Control	Sample source:BHK-21 | Treatment:Treatment with Toxin	Sample source:BHK-21 | Treatment:Treatment with Toxin	Sample source:BHK-21 | Treatment:Treatment with Toxin	Sample source:BHK-21 | Treatment:Treatment with Toxin	Sample source:BHK-21 | Treatment:Treatment with Toxin
AMP	0.0017	0.0018	0.0016	0.0017	0.0017	0.0016	0.0016	0.0016	0.0015	0.0016	0.0011	0.001	0.0009	0.0008	0.0008
IMP	0.0016	0.0016	0.0017	0.0015	0.0015	0.0014	0.0018	0.0017	0.0015	0.0015	0.0032	0.0033	0.0037	0.0023	0.0022
Formate	0.0373	0.0378	0.038	0.0369	0.0362	0.0362	0.036	0.0358	0.0356	0.0357	0.0029	0.0014	0.002	0.0014	0.0005
NADP+	0.0003	0.0004	0.0004	0.0002	0.0002	0.0002	0.0002	0.0002	0.0002	0.0003	0.0006	0.0009	0.0008	0.0009	0.0004
NAD+	0.0013	0.0013	0.0013	0.0012	0.0012	0.0012	0.0012	0.0012	0.0012	0.0012	0.0013	0.0013	0.0015	0.0014	0.001
ATP	0.0012	0.0011	0.0012	0.0012	0.0011	0.0011	0.0012	0.0011	0.0012	0.0011	0.0009	0.0007	0.0007	0.0007	0.0006
GTP	0.0001	0.0002	0.0002	0.0001	0.0001	0.0002	0.0001	0.0001	0.0001	0.0001	0.0007	0.0007	0.0007	0.0007	0.0003
UMP	0.0014	0.0015	0.0016	0.0015	0.0015	0.0014	0.0015	0.0015	0.0014	0.0017	0.0016	0.0008	0.0009	0.0008	0.0005
UDP_variants	0.0075	0.0078	0.008	0.0077	0.0076	0.0078	0.0075	0.0076	0.0075	0.0076	0.004	0.0049	0.0071	0.0056	0.0039
Tau-Methylhistidine	0	0.0001	0.0001	0.0001	0.0001	0.0001	0	0.0001	0	0	0.001	0.0007	0.0013	0.0007	0.0009
Tryptophan	0.0011	0.0013	0.0012	0.0011	0.0012	0.001	0.0012	0.0011	0.0011	0.0012	0.0008	0.0007	0.0009	0.0007	0.0007
Phenylalanine	0.0092	0.0096	0.0101	0.0096	0.0094	0.0094	0.0093	0.0095	0.0096	0.0091	0.0071	0.0052	0.0076	0.0059	0.0061
Tyrosine	0.0126	0.0127	0.0133	0.0128	0.0122	0.0123	0.0123	0.0123	0.0123	0.0124	0.0086	0.0067	0.0091	0.0072	0.0081
Fumarate	0.0001	0.0001	0.0002	0.0001	0.0001	0.0001	0.0001	0.0001	0.0001	0.0001	0.0002	0.0001	0.0002	0.0002	0.0001
Glucose-Galactose	0.0006	0.0015	0.0023	0.0009	0.0002	0.0002	0.0003	0.0003	0.0003	0.0002	0.004	0.002	0.0015	0.0005	0.0005
Proline	0.004	0.0043	0.0047	0.0039	0.0036	0.0037	0.0037	0.0038	0.0037	0.0036	0.003	0.0024	0.0032	0.0028	0.0023
Lactate	0.0059	0.0062	0.0063	0.0059	0.0056	0.0056	0.0055	0.0056	0.0055	0.0055	0.006	0.0042	0.005	0.0031	0.004
Creatine	0.0486	0.0491	0.0494	0.0486	0.0484	0.0485	0.0483	0.0484	0.0482	0.0484	0.0537	0.042	0.0549	0.0608	0.0444
Methylamine	0.0022	0.0026	0.0024	0.0024	0.0023	0.0023	0.0023	0.0023	0.0023	0.0022	0.0018	0.0014	0.0017	0.0017	0.0013
Beta-Alanine	0.0187	0.0194	0.0197	0.0192	0.0188	0.0192	0.0194	0.0193	0.0197	0.0197	0.0189	0.0156	0.0182	0.0197	0.0133
Succinate	0.0048	0.0048	0.0049	0.0048	0.0046	0.0047	0.0046	0.0047	0.0047	0.0046	0.0065	0.0032	0.0029	0.0032	0.0033
Acetate	0.0691	0.0697	0.0701	0.0691	0.0685	0.0689	0.0687	0.0689	0.0687	0.0691	0.0282	0.011	0.0205	0.0103	0.0038
Butyrate	0.0021	0.0024	0.0024	0.0023	0.0023	0.0022	0.0021	0.0022	0.0023	0.0022	0.002	0.0019	0.0021	0.0021	0.002
Alanine	0.0486	0.0499	0.0503	0.0484	0.0475	0.0478	0.0476	0.0475	0.0475	0.0477	0.0524	0.031	0.0408	0.0321	0.0371
Valine	0.0286	0.0293	0.03	0.0288	0.0281	0.0281	0.0283	0.0284	0.0282	0.0282	0.0214	0.0158	0.0218	0.0171	0.016
Isoleucine	0.0242	0.0248	0.0255	0.0244	0.0237	0.024	0.0239	0.024	0.0239	0.0242	0.0181	0.0138	0.0194	0.0148	0.0151
Leucine	0.025	0.0244	0.0253	0.0249	0.0252	0.0248	0.0252	0.0253	0.0247	0.0247	0.0184	0.0144	0.02	0.0149	0.0158
Valerate	0.0022	0.0023	0.0023	0.0022	0.0022	0.0023	0.0023	0.0023	0.0022	0.0021	0.0021	0.0024	0.0026	0.0028	0.0025
NMR_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name
AMP
IMP
Formate
NADP+
NAD+
ATP
GTP
UMP
UDP_variants
Tau-Methylhistidine
Tryptophan
Phenylalanine
Tyrosine
Fumarate
Glucose-Galactose
Proline
Lactate
Creatine
Methylamine
Beta-Alanine
Succinate
Acetate
Butyrate
Alanine
Valine
Isoleucine
Leucine
Valerate
METABOLITES_END
#END