Mitochondria are relevant in cellular physiology, but also in promoting apoptotic cell-death and autophagy in response to specific signals. Mitochondrial dysfunctions are involved in a variety of diseases, including cancer, neurodegeneration, chronic infections, inflammation, atherosclerosis and aging. This projects joins four research groups with a common interest in understanding the mitochondrial pathways leading to such crucial cellular responses, focusing on specific changes in mitochondrial physiology, dissecting the mitochondrial functions of specific genes of interest (p53, Ras, RET, VDAC, Parkin, PINK1, DJ-1 and huntingtin) and analyzing in some detail the changes associated to models of neurodegeneration and cancer. Special emphasis will be given to the study of their role in the mechanisms of regulation of apoptosis and autophagy as the unifying theme of this application. We will: 1) dissect the role of p53 and p53-related proteins in mitochondrial apoptosis and autophagy, with a detailed investigation of the effects that p53 can exert on Ca2+ homeostasis and other parameters of mitochondrial physiology. While the mitochondrial function of p53 is established, it is not known if this function is shared by the other p53-related proteins. 2) investigate the mitochondrial effects of Ras signalling in the regulation of apoptosis and autophagy, given its relevance for cell proliferation and survival. Both wild-type Ras, as well as a series of tumor-associated oncogenic mutants will be used. This analysis promises to contribute to our knowledge of the broad cellular consequences of Ras signalling in health and disease. 3) study the role of Voltage Dependent Anion Channels (VDACs) in regulating mitochondrial apoptosis and autophagy, since there are evidences of a functional interaction between VDACs and the Ras-MEK signaling pathway. To gain insight into the molecular function and regulation of these channels, a proteomic approach to map the protein interaction profile of selected VDACs will be implemented. The possible connection between mitochondrial outer membrane proteins, Ras and p53 will be studied, analysing whether VDACs can behave as receptors for these molecules, thus contributing to their recruitment to mitochondria, or if VDAC functions are involved as downstream events in the mitochondrial responses induced by Ras and p53. 4) elucidate the mechanisms of apoptosis deregulation in cancer, focusing on oncocytic tumors, characterized by an abnormal amount of mitochondria due to mtDNA mutations in complex I. It will be defined whether predisposition to tumorigenesis by a severe mitochondrial dysfunction may be related to activation of signalling mechanisms leading to inhibition of cell death. In particular the involvement of mitochondrial biogenesis, autophagy and calcium homeostasis disturbance will be determined. The function of the proto-oncogene receptor RET and its pro-apoptotic fragment, responsible for triggering cell death and promoting increased p53 levels, will also be studied, analysing in particular whether mitochondria are involved. Our preliminary studies have identified several proteins interacting with RET pro-apoptotic fragment, including VDAC2 and AIP, which in turns binds Tom20: the functional consequences of this interaction will be further investigated. 5) investigate the role on mitochondrial Ca2+ and ATP of mitochondrial related proteins parkin, PINK1, DJ-1 and huntingtin (Htt), whose mutations are responsible for Parkinson's and Huntington's disease. Special emphasis will be on their interaction with mitochondrial components, and on their modulation of apoptosis and autophagy, two mechanisms of cell death probably involved in the selective loss of neuronal populations. In particular, for the PD-related proteins a possible connection with the mitochondrial p53 pathway will be investigated. As for Htt, since emerging evidences suggest its interaction with the outer mitochondrial membrane, probably through the PTP (which is still molecularly unknown) and/or its regulating proteins (VDAC, hexokinases, Cyclophilin etc.) efforts will be put on identifying the nature of these interactions and their role on mitochondrial physiology. In summary, this project addresses a number of relevant questions on the mitochondrial functions of crucial regulators of cell growth and proliferation (Ras, p53, VDAC, RET), and aims to better define the molecular mechanisms coupling specific changes in mitochondrial physiology to fundamental cellular responses such as apoptosis and autophagy. A detailed understanding of how these pathways are regulated could allow translation of this information into new approaches for the treatment of a wide variety of diseases, in particular neurodegeneration and cancer.

Apoptosis and autophagy regulation at mitochondrial level: molecular mechanisms and effect of pathologic mutations

PINTON, Paolo
2010

Abstract

Mitochondria are relevant in cellular physiology, but also in promoting apoptotic cell-death and autophagy in response to specific signals. Mitochondrial dysfunctions are involved in a variety of diseases, including cancer, neurodegeneration, chronic infections, inflammation, atherosclerosis and aging. This projects joins four research groups with a common interest in understanding the mitochondrial pathways leading to such crucial cellular responses, focusing on specific changes in mitochondrial physiology, dissecting the mitochondrial functions of specific genes of interest (p53, Ras, RET, VDAC, Parkin, PINK1, DJ-1 and huntingtin) and analyzing in some detail the changes associated to models of neurodegeneration and cancer. Special emphasis will be given to the study of their role in the mechanisms of regulation of apoptosis and autophagy as the unifying theme of this application. We will: 1) dissect the role of p53 and p53-related proteins in mitochondrial apoptosis and autophagy, with a detailed investigation of the effects that p53 can exert on Ca2+ homeostasis and other parameters of mitochondrial physiology. While the mitochondrial function of p53 is established, it is not known if this function is shared by the other p53-related proteins. 2) investigate the mitochondrial effects of Ras signalling in the regulation of apoptosis and autophagy, given its relevance for cell proliferation and survival. Both wild-type Ras, as well as a series of tumor-associated oncogenic mutants will be used. This analysis promises to contribute to our knowledge of the broad cellular consequences of Ras signalling in health and disease. 3) study the role of Voltage Dependent Anion Channels (VDACs) in regulating mitochondrial apoptosis and autophagy, since there are evidences of a functional interaction between VDACs and the Ras-MEK signaling pathway. To gain insight into the molecular function and regulation of these channels, a proteomic approach to map the protein interaction profile of selected VDACs will be implemented. The possible connection between mitochondrial outer membrane proteins, Ras and p53 will be studied, analysing whether VDACs can behave as receptors for these molecules, thus contributing to their recruitment to mitochondria, or if VDAC functions are involved as downstream events in the mitochondrial responses induced by Ras and p53. 4) elucidate the mechanisms of apoptosis deregulation in cancer, focusing on oncocytic tumors, characterized by an abnormal amount of mitochondria due to mtDNA mutations in complex I. It will be defined whether predisposition to tumorigenesis by a severe mitochondrial dysfunction may be related to activation of signalling mechanisms leading to inhibition of cell death. In particular the involvement of mitochondrial biogenesis, autophagy and calcium homeostasis disturbance will be determined. The function of the proto-oncogene receptor RET and its pro-apoptotic fragment, responsible for triggering cell death and promoting increased p53 levels, will also be studied, analysing in particular whether mitochondria are involved. Our preliminary studies have identified several proteins interacting with RET pro-apoptotic fragment, including VDAC2 and AIP, which in turns binds Tom20: the functional consequences of this interaction will be further investigated. 5) investigate the role on mitochondrial Ca2+ and ATP of mitochondrial related proteins parkin, PINK1, DJ-1 and huntingtin (Htt), whose mutations are responsible for Parkinson's and Huntington's disease. Special emphasis will be on their interaction with mitochondrial components, and on their modulation of apoptosis and autophagy, two mechanisms of cell death probably involved in the selective loss of neuronal populations. In particular, for the PD-related proteins a possible connection with the mitochondrial p53 pathway will be investigated. As for Htt, since emerging evidences suggest its interaction with the outer mitochondrial membrane, probably through the PTP (which is still molecularly unknown) and/or its regulating proteins (VDAC, hexokinases, Cyclophilin etc.) efforts will be put on identifying the nature of these interactions and their role on mitochondrial physiology. In summary, this project addresses a number of relevant questions on the mitochondrial functions of crucial regulators of cell growth and proliferation (Ras, p53, VDAC, RET), and aims to better define the molecular mechanisms coupling specific changes in mitochondrial physiology to fundamental cellular responses such as apoptosis and autophagy. A detailed understanding of how these pathways are regulated could allow translation of this information into new approaches for the treatment of a wide variety of diseases, in particular neurodegeneration and cancer.
2010
Pinton, Paolo
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/1399598
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