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long-term complications, including severe neuronal impairment, developmental
delay, kidney and liver disease, coma, and death. Also, acute metabolic
decompensation and cardiac crises are frequent in these patients, so that early
intervention is required.
No therapeutic treatment actually exists for MMA, so that the only recommended
guideline subscribes limitation in the dietary propiogenic amino acids assumption
and, in the case of vitamin B12-responsive patients, supplementation with vitamin
B12 [1-3].
In order to study altered molecular mechanisms representative of the damage
induced by the disease in patients, two HEK293 cell models were developed. The
first one is a CRISPR/CAS9-based MUT gene knock out (MUT-KO). The knockingout process was monitored by fluorescence and, after collection of the positive
clones, protein signals were (not) detected by western blot. Then, the second cell
model derived from a MUT-KO cell line, engineered to rescue the stable expression
of MUT protein (MUT-RES). As control, a wild type HEK293 cell line (MUT-WT)
was used for the following experiments.
To track the quantitative changes in the global proteome of MUT-KO and MUTRES cells, a Data Independent Acquisition mass spectrometry-based proteomics
experiment was performed.
A total number of 5278 proteins were accurately identified (≥2 unique peptides,
1% False Discovery Rate) and quantified by LC-MS/MS, throughout the abovementioned cell conditions. Data analysis was performed using Spectronaut Pulsar
software and the KO/WT and KO/RES ratios were analyzed to retrieve the relative
quantitative abundances of proteins in KO samples. As expected, MUT protein was
not detected in the KO cell line. Then, the list of both KO/WT and KO/RES
differentially regulated proteins was managed in order to visualize the trend of
abundances in protein levels through the three experimental conditions. Thus,
proteins with a similar trend of abundance within the three conditions were
extrapolated from the list and analyzed by gene ontology enrichment.
The high confidence of these results, obtained with the latest high resolutionaccurate mass proteomic technologies, can be helpful in determining novel and
unknown molecular pathways affected in cellular models recapitulating the general
features of isolated methylmalonic acidemia. The perturbed biological processes
highlighted in this dataset are related to cell survival and organization, supposing an
unbalance between cell division and apoptosis.
The alterations described could be the trigger for the damage in patients and,
thus, this represent the starting point for the identification of potential targets of
therapy to be translated in the clinical practice.
M. Costanzo et al.
long-term complications, including severe neuronal impairment, developmental
delay, kidney and liver disease, coma, and death. Also, acute metabolic
decompensation and cardiac crises are frequent in these patients, so that early
intervention is required.
No therapeutic treatment actually exists for MMA, so that the only recommended
guideline subscribes limitation in the dietary propiogenic amino acids assumption
and, in the case of vitamin B12-responsive patients, supplementation with vitamin
B12 [1-3].
In order to study altered molecular mechanisms representative of the damage
induced by the disease in patients, two HEK293 cell models were developed. The
first one is a CRISPR/CAS9-based MUT gene knock out (MUT-KO). The knockingout process was monitored by fluorescence and, after collection of the positive
clones, protein signals were (not) detected by western blot. Then, the second cell
model derived from a MUT-KO cell line, engineered to rescue the stable expression
of MUT protein (MUT-RES). As control, a wild type HEK293 cell line (MUT-WT)
was used for the following experiments.
To track the quantitative changes in the global proteome of MUT-KO and MUTRES cells, a Data Independent Acquisition mass spectrometry-based proteomics
experiment was performed.
A total number of 5278 proteins were accurately identified (≥2 unique peptides,
1% False Discovery Rate) and quantified by LC-MS/MS, throughout the abovementioned cell conditions. Data analysis was performed using Spectronaut Pulsar
software and the KO/WT and KO/RES ratios were analyzed to retrieve the relative
quantitative abundances of proteins in KO samples. As expected, MUT protein was
not detected in the KO cell line. Then, the list of both KO/WT and KO/RES
differentially regulated proteins was managed in order to visualize the trend of
abundances in protein levels through the three experimental conditions. Thus,
proteins with a similar trend of abundance within the three conditions were
extrapolated from the list and analyzed by gene ontology enrichment.
The high confidence of these results, obtained with the latest high resolutionaccurate mass proteomic technologies, can be helpful in determining novel and
unknown molecular pathways affected in cellular models recapitulating the general
features of isolated methylmalonic acidemia. The perturbed biological processes
highlighted in this dataset are related to cell survival and organization, supposing an
unbalance between cell division and apoptosis.
The alterations described could be the trigger for the damage in patients and,
thus, this represent the starting point for the identification of potential targets of
therapy to be translated in the clinical practice.
M. Costanzo et al.