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Tsansizi, Lorenza Iolanda (2018) PPM1K, a novel regulator of metabolism and autophagy in the heart. [Ph.D. thesis]

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Abstract (italian or english)

The general aim of this work was to understand the relationship between mitochondria and autophagy, with a focus on heart biology. Autophagy is of extreme importance for cardiac function. Animal models of impaired autophagy display cardiac phenotypes at basal levels as well as when stressed. In this work we dissected molecularly two different faces of mitochondrial biology related to autophagy: BCAA-catabolism mediated regulation of mTORC1, and the role of the deubiquitinating enzyme USP8, involved in EGFR signaling and mitophagy, in heart mitochondrial function and more generally in heart function. Tissues can adapt to availability of different substrates by activating specific catabolic pathways that in most instances converge on mitochondria. Yet, how fluxes of metabolites through these organelles affect cellular processes is unclear. Here we show that PPM1K, a mitochondrial matrix protein phosphatase that controls the rate limiting step of branched chain amino acid (BCAA) catabolism, modulates mTORC1 activation and autophagy. PPM1K levels directly correlated with increased autophagy and reciprocally, PPM1K was induced upon starvation in vitro and in vivo, including in tissues where BCAA catabolism is considered marginal, like heart. Steady state metabolomics of labeled Leucine metabolites revealed that in the absence of PPM1K, TCA cycle intermediates were as expected decreased, whereas Leucine, its ketoisocaproic ketoacid and surprisingly methionine were increased, potentially explaining the mTORC1 dependent autophagy inhibition. Our data suggest how mitochondrial BCAA catabolism can be sensed by mTORC1 to modulate autophagy.
Activating mutations in the USP8 gene, coding for ubiquitin-specific protease 8, a deubiquitinase involved in endocytic trafficking and mitophagy, can cause Cushing’s syndrome. Usp8 inhibitors are therefore scrutinized to treat Cushing’s pituitary adenomas. However, because heart function requires mitophagy, it is unclear if Usp8 inhibitors could be detrimental for the already failing hearts of Cushing’s patients. Here we show that acute Usp8 genetic ablation in the mouse heart impairs mitochondrial function and autophagic clearance. Myocardial Usp8 deletion in adult mice resulted in cardiomyopathy associated with the accumulation of damaged and dysfunctional mitochondria. Mechanistically, we found that USP8 interacted with, and stabilized PINK1 that senses dysfunctional mitochondria and activates Parkin dependent mitophagy. Consequently, in cardiomyocytes and cells lacking USP8, PINK1 was not stabilized upon mitochondrial dysfunction, mitophagy was not activated in response to mitochondrial depolarization and chemical mitochondrial uncouplers led to cell death. Our data not only shed light on the mechanisms of mitophagy regulation, but also recommend caution in investigative anti Usp8 therapy for Cushing’s syndrome.

Abstract (a different language)

The general aim of this work was to understand the relationship between mitochondria and autophagy, with a focus on heart biology. Autophagy is of extreme importance for cardiac function. Animal models of impaired autophagy display cardiac phenotypes at basal levels as well as when stressed. In this work we dissected molecularly two different faces of mitochondrial biology related to autophagy: BCAA-catabolism mediated regulation of mTORC1, and the role of the deubiquitinating enzyme USP8, involved in EGFR signaling and mitophagy, in heart mitochondrial function and more generally in heart function. Tissues can adapt to availability of different substrates by activating specific catabolic pathways that in most instances converge on mitochondria. Yet, how fluxes of metabolites through these organelles affect cellular processes is unclear. Here we show that PPM1K, a mitochondrial matrix protein phosphatase that controls the rate limiting step of branched chain amino acid (BCAA) catabolism, modulates mTORC1 activation and autophagy. PPM1K levels directly correlated with increased autophagy and reciprocally, PPM1K was induced upon starvation in vitro and in vivo, including in tissues where BCAA catabolism is considered marginal, like heart. Steady state metabolomics of labeled Leucine metabolites revealed that in the absence of PPM1K, TCA cycle intermediates were as expected decreased, whereas Leucine, its ketoisocaproic ketoacid and surprisingly methionine were increased, potentially explaining the mTORC1 dependent autophagy inhibition. Our data suggest how mitochondrial BCAA catabolism can be sensed by mTORC1 to modulate autophagy.
Activating mutations in the USP8 gene, coding for ubiquitin-specific protease 8, a deubiquitinase involved in endocytic trafficking and mitophagy, can cause Cushing’s syndrome. Usp8 inhibitors are therefore scrutinized to treat Cushing’s pituitary adenomas. However, because heart function requires mitophagy, it is unclear if Usp8 inhibitors could be detrimental for the already failing hearts of Cushing’s patients. Here we show that acute Usp8 genetic ablation in the mouse heart impairs mitochondrial function and autophagic clearance. Myocardial Usp8 deletion in adult mice resulted in cardiomyopathy associated with the accumulation of damaged and dysfunctional mitochondria. Mechanistically, we found that USP8 interacted with, and stabilized PINK1 that senses dysfunctional mitochondria and activates Parkin dependent mitophagy. Consequently, in cardiomyocytes and cells lacking USP8, PINK1 was not stabilized upon mitochondrial dysfunction, mitophagy was not activated in response to mitochondrial depolarization and chemical mitochondrial uncouplers led to cell death. Our data not only shed light on the mechanisms of mitophagy regulation, but also recommend caution in investigative anti Usp8 therapy for Cushing’s syndrome.

EPrint type:Ph.D. thesis
Tutor:Scorrano, Luca
Ph.D. course:Ciclo 31 > Corsi 31 > BIOSCIENZE
Data di deposito della tesi:30 November 2018
Anno di Pubblicazione:30 November 2018
Key Words:autophagy mitophagy
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/10 Biochimica
Struttura di riferimento:Dipartimenti > Dipartimento di Biologia
Codice ID:11521
Depositato il:06 Nov 2019 12:00
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