Costanzo, Michele
(2020)
The proteomic landscape of cellular models for methylmalonic acidemia.
[Tesi di dottorato]
| Tipologia del documento: |
Tesi di dottorato
|
| Lingua: |
English |
| Titolo: |
The proteomic landscape of cellular models for methylmalonic acidemia |
| Autori: |
| Autore | Email |
|---|
| Costanzo, Michele | michele.costanzo@unina.it |
|
| Data: |
13 Marzo 2020 |
| Numero di pagine: |
109 |
| Istituzione: |
Università degli Studi di Napoli Federico II |
| Dipartimento: |
Medicina Molecolare e Biotecnologie Mediche |
| Dottorato: |
Medicina molecolare e biotecnologie mediche |
| Ciclo di dottorato: |
32 |
| Coordinatore del Corso di dottorato: |
| nome | email |
|---|
| Avvedimento, Vittorio Enrico | avvedim@unina.it |
|
| Tutor: |
| nome | email |
|---|
| Ruoppolo, Margherita | [non definito] |
|
| Data: |
13 Marzo 2020 |
| Numero di pagine: |
109 |
| Parole chiave: |
Proteomics; boinformatics; methylmalonic acidemia; metabolic disease |
| Settori scientifico-disciplinari del MIUR: |
Area 05 - Scienze biologiche > BIO/10 - Biochimica |
[error in script]
[error in script]
| Depositato il: |
25 Mar 2020 11:53 |
| Ultima modifica: |
08 Nov 2021 11:52 |
| URI: |
http://www.fedoa.unina.it/id/eprint/13178 |
Abstract
The present PhD thesis project concerned the development of novel cellular models to be used for the study and the advancing knowledge about methylmalonic acidemia (MMA), a rare inborn error of metabolism that occurs as consequence of specific defects in propionyl-CoA and cobalamin (vitamin B12) metabolism. Isolated MMA is caused by specific mutations in methylmalonyl-CoA mutase (MUT) gene, whose protein product converts methylmalonyl-CoA into succinyl-CoA, an intermediate of the Krebs cycle. Mutations in one of the genes involved in the intracellular metabolism of cobalamin, used for the synthesis of MUT cofactor, the adenosylcobalamin, are causative of secondary forms of MMA with or without homocystinuria. Methylmalonic acid is the metabolite that accumulates downstream MUT reaction impairment and, therefore, is used as diagnostic biomarker of MMA. Metabolic alterations of this disease include tissue (mostly brain, liver, kidney) damage and necessity of transplantation to ensure a long-term survival. The causes of metabolic instability are often addressed to the action of secondary metabolites of propionyl-CoA pathway, ammonium accumulation, reactive oxygen species production and mitochondrial impairment with global energy production imbalance and irreversible damages. For all these altered processes, the acting mechanisms are not known. Here, the generation of different cell models that may recapitulate MMA physiopathology was accompanied by the application of different mass spectrometry-based proteomic strategies, employing different instrumentations and techniques, with the aim to describe and elucidate perturbed pathways and unravel possible pathogenetic mechanisms that are still unknown.
The first cellular model for isolated MMA was created by silencing MUT gene in human neuroblastoma SH-SY5Y cell line via siRNA-mediated RNA interference. The proteome of these cells was resolved by monodimensional SDS-PAGE and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a LTQ-Orbitrap XL mass spectrometer. Label-free proteomic analysis by means of the spectral counts approach highlighted common dysregulated signatures related to mitochondrial energy production and oxidation-reduction processes unbalances.
Subsequently, a second cell model for isolated MMA was generated knocking out (KO) MUT gene in human embryonic kidney (HEK) 293 cells using CRISPR/Cas9 genome editing technology. MUT-KO proteome was analyzed by shotgun LC-MS/MS using an Orbitrap-Q Exactive Plus mass spectrometer. Label-free quantitative proteomics based on LFQ method and biochemical assays suggested that MUT-KO cells are adapting to the changes triggered upon MUT knock out by altering their structure with modifications in extracellular matrix, cell adhesion and cytoskeletal components. MUT KO induced metabolic alterations related to toxic compounds accumulations, such as ammonia, and production of ROS that are known damaging cells by interfering in the oxidation-reduction homeostasis. The MUT-deficient cells modify their proteome by a modulation of gene expression that still permits to survive, even in conditions of stress and mitochondrial impairment. On the other hand, an additional stress induced by propionate overload on a system already stressed became fatal. All the alterations found were monitored in an additional cell model of MUT rescuing that was generated from MUT-KO cells in order to control the specificity of the effects of MUT knockout.
In addition to the previous investigation, proteome alterations were deeper investigated in MUT-KO and MUT-Rescue through the employment of a sophisticated high resolution Data-Independent Acquisition-based proteomic analysis carried out with an Orbitrap-Q Exactive HF mass spectrometer.
Finally, to complete the picture of the proteomic landscape of MMA, the possible role of MMACHC protein in the cell was investigated. Interactomics experiments provided the identification of many putative interactors of MMACHC, which may take part in the formation of MUT cofactor. Alternatively, new roles for MMACHC protein in the cell may be discovered making a valuable contribution to the knowledge of vitamin B12 metabolism.
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