scholarly journals Functional Characterisation of the Autophagy ATG12~5/16 Complex in Dictyostelium discoideum

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1179 ◽  
Author(s):  
Malte Karow ◽  
Sarah Fischer ◽  
Susanne Meßling ◽  
Roman Konertz ◽  
Jana Riehl ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Ubiquitin Proteasome System ◽  
Strong Increase ◽  
Knock Out ◽  
E Coli ◽  
Atg Proteins ◽  
Functional Characterisation ◽  
Independent Functions ◽  
Ubiquitin Proteasome ◽  
Essential Function

Macroautophagy, a highly conserved and complex intracellular degradative pathway, involves more than 20 core autophagy (ATG) proteins, among them the hexameric ATG12~5/16 complex, which is part of the essential ubiquitin-like conjugation systems in autophagy. Dictyostelium discoideum atg5 single, atg5/12 double, and atg5/12/16 triple gene knock-out mutant strains displayed similar defects in the conjugation of ATG8 to phosphatidylethanolamine, development, and cell viability upon nitrogen starvation. This implies that ATG5, 12 and 16 act as a functional unit in canonical autophagy. Macropinocytosis of TRITC dextran and phagocytosis of yeast were significantly decreased in ATG5¯ and ATG5¯/12¯ and even further in ATG5¯/12¯/16¯ cells. In contrast, plaque growth on Klebsiella aerogenes was about twice as fast for ATG5¯ and ATG5¯/12¯/16¯ cells in comparison to AX2, but strongly decreased for ATG5¯/12¯ cells. Along this line, phagocytic uptake of Escherichia coli was significantly reduced in ATG5¯/12¯ cells, while no difference in uptake, but a strong increase in membrane association of E. coli, was seen for ATG5¯ and ATG5¯/12¯/16¯ cells. Proteasomal activity was also disturbed in a complex fashion, consistent with an inhibitory activity of ATG16 in the absence of ATG5 and/or ATG12. Our results confirm the essential function of the ATG12~5/16 complex in canonical autophagy, and furthermore are consistent with autophagy-independent functions of the complex and its individual components. They also strongly support the placement of autophagy upstream of the ubiquitin-proteasome system (UPS), as a fully functional UPS depends on autophagy.

scholarly journals ATP13A2 Regulates Cellular α-Synuclein Multimerization, Membrane Association, and Externalization

2021 ◽  
Vol 22 (5) ◽  
pp. 2689
Author(s):  
Jianmin Si ◽  
Chris Van den Haute ◽  
Evy Lobbestael ◽  
Shaun Martin ◽  
Sarah van Veen ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Ubiquitin Proteasome System ◽  
Regulatory Function ◽  
Membrane Association ◽  
Loss Of Function ◽  
Atypical Form ◽  
Polyamine Transport ◽  
Independent Functions ◽  
Ubiquitin Proteasome ◽  
The Impact

ATP13A2, a late endo-/lysosomal polyamine transporter, is implicated in a variety of neurodegenerative diseases, including Parkinson’s disease and Kufor–Rakeb syndrome, an early-onset atypical form of parkinsonism. Loss-of-function mutations in ATP13A2 result in lysosomal deficiency as a consequence of impaired lysosomal export of the polyamines spermine/spermidine. Furthermore, accumulating evidence suggests the involvement of ATP13A2 in regulating the fate of α-synuclein, such as cytoplasmic accumulation and external release. However, no consensus has yet been reached on the mechanisms underlying these effects. Here, we aimed to gain more insight into how ATP13A2 is linked to α-synuclein biology in cell models with modified ATP13A2 activity. We found that loss of ATP13A2 impairs lysosomal membrane integrity and induces α-synuclein multimerization at the membrane, which is enhanced in conditions of oxidative stress or exposure to spermine. In contrast, overexpression of ATP13A2 wildtype (WT) had a protective effect on α-synuclein multimerization, which corresponded with reduced αsyn membrane association and stimulation of the ubiquitin-proteasome system. We also found that ATP13A2 promoted the secretion of α-synuclein through nanovesicles. Interestingly, the catalytically inactive ATP13A2 D508N mutant also affected polyubiquitination and externalization of α-synuclein multimers, suggesting a regulatory function independent of the ATPase and transport activity. In conclusion, our study demonstrates the impact of ATP13A2 on α-synuclein multimerization via polyamine transport dependent and independent functions.

scholarly journals The phenotypes of ATG9, ATG16 and ATG9/16 knock-out mutants imply autophagy-dependent and -independent functions

Open Biology ◽  
2015 ◽  
Vol 5 (4) ◽  
pp. 150008 ◽  
Author(s):  
Qiuhong Xiong ◽  
Can Ünal ◽  
Jan Matthias ◽  
Michael Steinert ◽  
Ludwig Eichinger
Keyword(s):  
Dictyostelium Discoideum ◽  
Legionella Pneumophila ◽  
Double Mutant ◽  
Eukaryotic Cells ◽  
Knock Out ◽  
Cellular Processes ◽  
Ubiquitinated Proteins ◽  
Complex Phenotypes ◽  
Independent Functions ◽  
Mutant Cells

Macroautophagy is a highly conserved intracellular bulk degradation system of all eukaryotic cells. It is governed by a large number of autophagy proteins (ATGs) and is crucial for many cellular processes. Here, we describe the phenotypes of Dictyostelium discoideum ATG16 − and ATG9 − /16 − cells and compare them to the previously reported ATG9 − mutant. ATG16 deficiency caused an increase in the expression of several core autophagy genes, among them atg9 and the two atg8 paralogues. The single and double ATG9 and ATG16 knock-out mutants had complex phenotypes and displayed severe and comparable defects in pinocytosis and phagocytosis. Uptake of Legionella pneumophila was reduced. In addition, ATG9 − and ATG16 − cells had dramatic defects in autophagy, development and proteasomal activity which were much more severe in the ATG9 − /16 − double mutant. Mutant cells showed an increase in poly-ubiquitinated proteins and contained large ubiquitin-positive protein aggregates which partially co-localized with ATG16-GFP in ATG9 − /16 − cells. The more severe autophagic, developmental and proteasomal phenotypes of ATG9 − /16 − cells imply that ATG9 and ATG16 probably function in parallel in autophagy and have in addition autophagy-independent functions in further cellular processes.

scholarly journals The Role of ATG16 in Autophagy and The Ubiquitin Proteasome System

Cells ◽  
2018 ◽  
Vol 8 (1) ◽  
pp. 2 ◽  
Author(s):  
Qiuhong Xiong ◽  
Wenjing Li ◽  
Ping Li ◽  
Min Yang ◽  
Changxin Wu ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Current Knowledge ◽  
Ubiquitin Proteasome System ◽  
Degradation Pathways ◽  
Autophagosome Formation ◽  
Cell Homeostasis ◽  
Independent Functions ◽  
Ubiquitin Proteasome

Autophagy and the ubiquitin proteasome system (UPS) are the two major cellular degradation pathways, which are critical for the maintenance of cell homeostasis. The two pathways differ in their mechanisms and clients. The evolutionary conserved ATG16 plays a key role in autophagy and appears to link autophagy with the UPS. Here, we review the role of ATG16 in different species. We summarize the current knowledge of its functions in autophagosome membrane expansion and autophagosome formation, in Crohn’s disease, and in bacterial sequestration. In addition, we provide information on its autophagy-independent functions and its role in the crosstalk between autophagy and the UPS.

scholarly journals Dictyostelium discoideum has a highly Q/N-rich proteome and shows an unusual resilience to protein aggregation

2015 ◽  
Vol 112 (20) ◽  
pp. E2620-E2629 ◽  
Author(s):  
Liliana Malinovska ◽  
Sandra Palm ◽  
Kimberley Gibson ◽  
Jean-Marc Verbavatz ◽  
Simon Alberti
Keyword(s):  
Dictyostelium Discoideum ◽  
Protein Misfolding ◽  
Prion Proteins ◽  
Sequence Similarity ◽  
Ubiquitin Proteasome System ◽  
Yeast Prion ◽  
Intrinsically Disordered ◽  
Exon 1 ◽  
Ubiquitin Proteasome ◽  
Misfolding Diseases

Many protein-misfolding diseases are caused by proteins carrying prion-like domains. These proteins show sequence similarity to yeast prion proteins, which can interconvert between an intrinsically disordered and an aggregated prion state. The natural presence of prions in yeast has provided important insight into disease mechanisms and cellular proteostasis. However, little is known about prions in other organisms, and it is not yet clear whether the findings in yeast can be generalized. Using bioinformatics tools, we show that Dictyostelium discoideum has the highest content of prion-like proteins of all organisms investigated to date, suggesting that its proteome has a high overall aggregation propensity. To study mechanisms regulating these proteins, we analyze the behavior of several well-characterized prion-like proteins, such as an expanded version of human huntingtin exon 1 (Q103) and the prion domain of the yeast prion protein Sup35 (NM), in D. discoideum. We find that these proteins remain soluble and are innocuous to D. discoideum, in contrast to other organisms, where they form cytotoxic cytosolic aggregates. However, when exposed to conditions that compromise molecular chaperones, these proteins aggregate and become cytotoxic. We show that the disaggregase Hsp101, a molecular chaperone of the Hsp100 family, dissolves heat-induced aggregates and promotes thermotolerance. Furthermore, prion-like proteins accumulate in the nucleus, where they are targeted by the ubiquitin–proteasome system. Our data suggest that D. discoideum has undergone specific adaptations that increase the proteostatic capacity of this organism and allow for an efficient regulation of its prion-like proteome.

scholarly journals HIV-1 antisense protein of different clades induces autophagy and associates to the autophagy factor p62

2018 ◽  
Author(s):  
Zhenlong Liu ◽  
Cynthia Torresilla ◽  
Yong Xiao ◽  
Clément Caté ◽  
Karina Barbosa ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Strong Support ◽  
Ubiquitin Proteasome System ◽  
Expression Vectors ◽  
Autophagosome Formation ◽  
Knock Out ◽  
Mammalian Cell Lines ◽  
Autophagy Induction ◽  
Ubiquitin Proteasome ◽  
Hiv 1

AbstractOver recent years, strong support argues for the existence of an HIV-1 protein encoded by antisense transcripts and termed Antisense Protein (ASP). Furthermore, a recentin silicoanalysis has provided evidence for its recent appearance in the genome of HIV-1. We have previously detected ASP in various mammalian cell lines by Western blot (WB), flow cytometry and confocal microscopy analyses and reported that it induced autophagy, potentially through multimer formation. The aim of the current study was to examine autophagy induction by testing ASP from different clades, and to identify the implicated autophagy factors. We firstly confirmed that NL4.3-derived ASP was interacting with itself and that multimer formation was dependent on its amino region. Removal of this region was associated with reduced level of induced autophagy, as assessed by autophagosome formation but deletion of the most amino cysteine triplet did not totally abrogate multimer and autophagosome formation. Expression vectors of ASP from different clades were next tested and led to detection of monomers and varying levels of multimers with concomitant induced autophagy, as determined by increased LC3-II and decreased p62 (SQSTM1) levels. Through confocal microscopy, ASP was noted to co-localize with p62 and LC3-II in autophagosome-like cellular structures. CRISPR-based knock-out of ATG5, ATG7 and p62 genes led to increased stability in the levels of ASP. Furthermore, co-immunoprecipitation experiments demonstrated the interaction between p62 and ASP, which was dependent on the PB1 domain of p62. Interestingly, immunoprecipitation experiments further supported that ASP is ubiquitinated and that ubiquitination was also responsible for the modulation of its stability. We are thus suggesting that ASP induces autophagy through p62 interaction and that its abundance is controlled by autophagy- and Ubiquitin/Proteasome System (UPS)-mediated degradation in which ubiquitin is playing an important role. Understanding the mechanisms underlying the degradation of ASP is essential to better assess its function.Author SummaryIn the present study, we provide the first evidence that a new HIV-1 protein termed ASP when derived from different clades act similarly in inducing autophagy, an important cellular process implicated in the degradation of excess or defective material. We have gained further knowledge on the mechanism mediate the activation of autophagy and have identified an important interacting partner. Our studies have important ramification in the understanding of viral replication and the pathogenesis associated with HIV-1 in infected individuals. Indeed, autophagy is implicated in antigen presentation during immune response and could thus be rendered inefficient in infected cells, such as dendritic cells. Furthermore, a possible link with HIV-1-associated Neurological Disorder (HAND) might also be a possible association with the capacity of ASP to induce autophagy. Our studies are thus important and demonstrate the importance in conducting further studies on this protein, as it could represent a new interesting target for antiretroviral therapies and vaccine design.

The ubiquitin–proteasome system and skeletal muscle wasting

2005 ◽  
Vol 41 ◽  
pp. 173-186 ◽  
Author(s):  
Didier Attaix ◽  
Sophie Ventadour ◽  
Audrey Codran ◽  
Daniel Béchet ◽  
Daniel Taillandier ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Wasting ◽  
Proteolytic Enzymes ◽  
Cathepsin L ◽  
Ubiquitin Proteasome System ◽  
Complete Breakdown ◽  
Skeletal Muscle Proteins ◽  
Ubiquitin Proteasome ◽  
Tripeptidyl Peptidase Ii ◽  
F Box Protein

The ubiquitin–proteasome system (UPS) is believed to degrade the major contractile skeletal muscle proteins and plays a major role in muscle wasting. Different and multiple events in the ubiquitination, deubiquitination and proteolytic machineries are responsible for the activation of the system and subsequent muscle wasting. However, other proteolytic enzymes act upstream (possibly m-calpain, cathepsin L, and/or caspase 3) and downstream (tripeptidyl-peptidase II and aminopeptidases) of the UPS, for the complete breakdown of the myofibrillar proteins into free amino acids. Recent studies have identified a few critical proteins that seem necessary for muscle wasting {i.e. the MAFbx (muscle atrophy F-box protein, also called atrogin-1) and MuRF-1 [muscle-specific RING (really interesting new gene) finger 1] ubiquitin–protein ligases}. The characterization of their signalling pathways is leading to new pharmacological approaches that can be useful to block or partially prevent muscle wasting in human patients.

The ubiquitin–proteasome system and skeletal muscle wasting

2005 ◽  
Vol 41 (1) ◽  
pp. 173 ◽  
Author(s):  
Didier Attaix ◽  
Sophie Ventadour ◽  
Audrey Codran ◽  
Daniel Béchet ◽  
Daniel Taillandier ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Wasting ◽  
Ubiquitin Proteasome System ◽  
Skeletal Muscle Wasting ◽  
Ubiquitin Proteasome

Theoretical Studies of the Acid-Base Equilibria in a Model Active Site of the Human 20S Proteasome

2020 ◽  
Author(s):  
Jon Uranga ◽  
Lukas Hasecke ◽  
Jonny Proppe ◽  
Jan Fingerhut ◽  
Ricardo A. Mata
Keyword(s):  
Active Site ◽  
Boronic Acid ◽  
Computational Study ◽  
Ubiquitin Proteasome System ◽  
Bayesian Optimization ◽  
Inhibition Mechanism ◽  
20S Proteasome ◽  
Acid Base ◽  
Ubiquitin Proteasome ◽  
Infection Cycles

The 20S Proteasome is a macromolecule responsible for the chemical step in the ubiquitin-proteasome system of degrading unnecessary and unused proteins of the cell. It plays a central role both in the rapid growth of cancer cells as well as in viral infection cycles. Herein, we present a computational study of the acid-base equilibria in an active site of the human proteasome, an aspect which is often neglected despite the crucial role protons play in the catalysis. As example substrates, we take the inhibition by epoxy and boronic acid containing warheads. We have combined cluster quantum mechanical calculations, replica exchange molecular dynamics and Bayesian optimization of non-bonded potential terms in the inhibitors. In relation to the latter, we propose an easily scalable approach to the reevaluation of non-bonded potentials making use of QM/MM dynamics information. Our results show that coupled acid-base equilibria need to be considered when modeling the inhibition mechanism. The coupling between a neighboring lysine and the reacting threonine is not affected by the presence of the inhibitor.

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