From nucleus to mitochondria : a ubiquitination study
Author: Richard, Thibaud JC
Date: 2019-02-08
Location: rum C0335, Biomedicum
Time: 09:00
Department: Inst för cell- och molekylärbiologi / Dept of Cell and Molecular Biology
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Licentiate thesis (1.193Mb)
Abstract
Ubiquitination is a post-translational modification achieved by an enzymatic cascade. This
post-translational modification is involved in many crucial cellular processes across different
cellular compartments such as protein turnover via the ubiquitin proteasome system and
various signaling pathways from the DNA damage response to the immune response.
Ubiquitin has seven lysine residues (Lys 6, 11, 27, 29, 33, 48 and 63) onto which other
ubiquitin moieties can be conjugated, forming ubiquitin chains of different types. These
different ubiquitin chains can have different functions and the relationship between both is
often referred to as “the ubiquitin code”. Although the enzymatic cascade leading to
ubiquitination of proteins is well described, the ubiquitin code remains largely unresolved.
Accumulation of insoluble ubiquitinated proteins is a hallmark of neurodegenerative
disorders such as Parkinson’s and Alzheimer’s disease, making the study of these cellular
processes relevant to human health. Specific proteins can also impair the function of the
proteasome such as progerin in the Hutchinson-Gilford Progeria Syndrome (HGPS).
The importance of ubiquitination in many cellular processes and its involvement in many human pathologies inspired us to develop an inducible ubiquitination system that could be used as a tool to better understand the ubiquitin code and its role in different cellular compartments. In paper I, we have engineered a ubiquitin ligase, ProxE3, which assembles specific ubiquitin chain (lysine 63) onto a fluorescent substrate. We have used this tool to generate ubiquitin chains on the surface of mitochondria and investigate mitophagy, more precisely if lysine 63 (K63) ubiquitin chains are sufficient to trigger aggregation of mitochondria or mitophagy. Upon successful ubiquitination of the surface of mitochondria by ProxE3 and depolarization of mitochondria by carbonyl cyanide mchlorophenyl hydrazone (CCCP), peri-nuclear clustering of mitochondria was observed but not mitophagy. The lack of mitophagy indicates that either the amount of K63 ubiquitination is insufficient in our system, that other types of ubiquitin chains are required, that a specific substrate need to be ubiquitinated or that the PINK1 feedforward loop is essential for mitophagy. Nonetheless, this work presents a valid tool for studies of ubiquitination in living cells, while reaffirming the complexity of the regulation of mitophagy.
In paper II, we were interested in ubiquitination in a different cellular compartment: the nucleus. In this paper, we used a human cell-line expressing a fluorescent proteasomal substrate to investigate if the ubiquitin-proteasome system was impaired upon overexpression of progerin. Progerin is a mutated form of lamin A which is the cause of HGPS. It has been suspected that progerin might inhibit the catalytic activity of the proteasome, which could lead to neuronal dysfunction. However, we did not detect proteasomal impairment in human cells overexpressing progerin compared to wild-type lamin A. This observation is further supported by the lack of progerin/lamin A inclusions in hippocampal neurons of HGPS mice, implying that the ubiquitin/proteasome system is not sensitive to the expression of progerin in neurons of mice.
The importance of ubiquitination in many cellular processes and its involvement in many human pathologies inspired us to develop an inducible ubiquitination system that could be used as a tool to better understand the ubiquitin code and its role in different cellular compartments. In paper I, we have engineered a ubiquitin ligase, ProxE3, which assembles specific ubiquitin chain (lysine 63) onto a fluorescent substrate. We have used this tool to generate ubiquitin chains on the surface of mitochondria and investigate mitophagy, more precisely if lysine 63 (K63) ubiquitin chains are sufficient to trigger aggregation of mitochondria or mitophagy. Upon successful ubiquitination of the surface of mitochondria by ProxE3 and depolarization of mitochondria by carbonyl cyanide mchlorophenyl hydrazone (CCCP), peri-nuclear clustering of mitochondria was observed but not mitophagy. The lack of mitophagy indicates that either the amount of K63 ubiquitination is insufficient in our system, that other types of ubiquitin chains are required, that a specific substrate need to be ubiquitinated or that the PINK1 feedforward loop is essential for mitophagy. Nonetheless, this work presents a valid tool for studies of ubiquitination in living cells, while reaffirming the complexity of the regulation of mitophagy.
In paper II, we were interested in ubiquitination in a different cellular compartment: the nucleus. In this paper, we used a human cell-line expressing a fluorescent proteasomal substrate to investigate if the ubiquitin-proteasome system was impaired upon overexpression of progerin. Progerin is a mutated form of lamin A which is the cause of HGPS. It has been suspected that progerin might inhibit the catalytic activity of the proteasome, which could lead to neuronal dysfunction. However, we did not detect proteasomal impairment in human cells overexpressing progerin compared to wild-type lamin A. This observation is further supported by the lack of progerin/lamin A inclusions in hippocampal neurons of HGPS mice, implying that the ubiquitin/proteasome system is not sensitive to the expression of progerin in neurons of mice.
List of papers:
I. Thibaud J.C. Richard, Aldwin Suryo Rahmanto, Olle Sangfelt, Florian A. Salomons and Nico P. Dantuma. An engineered ubiquitin ligase for inducible K63-linked ubiquitylation. [Manuscript]
II. Jean-Ha Baek, Eva Schmidt, Nikenza Viceconte, Charlotte Strandgren, Karin Pernold, Thibaud J.C. Richard, Fred W. van Leeuwen, Nico P. Dantuma, Peter Damberg, Kjell Hultenby, Brun Ulfhake, Enrico Mugnaini, Björn Rozell and Maria Eriksson. Expression of progerin in aging mouse brains reveals structural nuclear abnormalities without detectible significant alterations in gene expression, hippocampal stem cells or behavior. Hum. Mol. Genet. 2015 Mar;24(5):1305-21.
Fulltext (DOI)
Pubmed
View record in Web of Science®
I. Thibaud J.C. Richard, Aldwin Suryo Rahmanto, Olle Sangfelt, Florian A. Salomons and Nico P. Dantuma. An engineered ubiquitin ligase for inducible K63-linked ubiquitylation. [Manuscript]
II. Jean-Ha Baek, Eva Schmidt, Nikenza Viceconte, Charlotte Strandgren, Karin Pernold, Thibaud J.C. Richard, Fred W. van Leeuwen, Nico P. Dantuma, Peter Damberg, Kjell Hultenby, Brun Ulfhake, Enrico Mugnaini, Björn Rozell and Maria Eriksson. Expression of progerin in aging mouse brains reveals structural nuclear abnormalities without detectible significant alterations in gene expression, hippocampal stem cells or behavior. Hum. Mol. Genet. 2015 Mar;24(5):1305-21.
Fulltext (DOI)
Pubmed
View record in Web of Science®
Institution: Karolinska Institutet
Supervisor: Dantuma, Nico
Co-supervisor: Sangfelt, Olle
Issue date: 2019-01-18
Rights:
Publication year: 2019
ISBN: 978-91-7831-306-8
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