Autophagy and ubiquitin–proteasome system contribute to sperm mitophagy after mammalian fertilization

Maternal inheritance of mitochondria and mtDNA is a universal principle in human and animal development, guided by selective ubiquitin-dependent degradation of the sperm-borne mitochondria after fertilization. However, it is not clear how the 26S proteasome, the ubiquitin-dependent protease that is only capable of degrading one protein molecule at a time, can dispose of a whole sperm mitochondrial sheath. We hypothesized that the canonical ubiquitin-like autophagy receptors [sequestosome 1 (SQSTM1), microtubule-associated protein 1 light chain 3 (LC3), gamma-aminobutyric acid receptor-associated protein (GABARAP)] and the nontraditional mitophagy pathways involving ubiquitin-proteasome system and the ubiquitin-binding protein dislocase, valosin-containing protein (VCP), may act in concert during mammalian sperm mitophagy. We found that the SQSTM1, but not GABARAP or LC3, associated with sperm mitochondria after fertilization in pig and rhesus monkey zygotes. Three sperm mitochondrial proteins copurified with the recombinant, ubiquitin-associated domain of SQSTM1. The accumulation of GABARAP-containing protein aggregates was observed in the vicinity of sperm mitochondrial sheaths in the zygotes and increased in the embryos treated with proteasomal inhibitor MG132, in which intact sperm mitochondrial sheaths were observed. Pharmacological inhibition of VCP significantly delayed the process of sperm mitophagy and completely prevented it when combined with microinjection of autophagy-targeting antibodies specific to SQSTM1 and/or GABARAP. Sperm mitophagy in higher mammals thus relies on a combined action of SQSTM1-dependent autophagy and VCP-mediated dislocation and presentation of ubiquitinated sperm mitochondrial proteins to the 26S proteasome, explaining how the whole sperm mitochondria are degraded inside the fertilized mammalian oocytes by a protein recycling system involved in degradation of single protein molecules.

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Post-fertilisation sperm mitophagy: the tale of Mitochondrial Eve and Steve

Reproduction Fertility and Development ◽

10.1071/rd17364 ◽

2018 ◽

Vol 30 (1) ◽

pp. 56 ◽

Cited By ~ 7

Author(s):

Peter Sutovsky ◽

Won-Hee Song

Keyword(s):

Ubiquitin Proteasome System ◽

26S Proteasome ◽

Production Traits ◽

Cell Stage ◽

Mt Dna ◽

Receptor Associated Protein ◽

Sequestosome 1 ◽

Genes Encoding ◽

Ubiquitin Proteasome ◽

Mitochondrial Eve

Preformationist William Harvey’s proclamation of everything live coming from an egg still holds true for mammalian mitochondria and mitochondrial genes. At fertilisation, mitochondria carried into the oocyte cytoplasm by the spermatozoon are sought out and destroyed, leaving only oocyte mitochondria to propagate their mitochondrial (mt) DNA to offspring. This clonal inheritance mode, the ‘mitochondrial Eve’ paradigm, is mediated by oocytes’ resident proteolytic, organelle-targeting mechanisms, including the substrate-specific ubiquitin proteasome system and the autophagic machinery for bulk protein and organelle degradation. Ubiquitination of sperm mitochondria within the cytoplasm of the fertilised oocyte was initially discovered in mammals. More recent studies in Drosophila and Caenorhabditis elegans implicated the ubiquitin-binding autophagy protein sequestosome 1 (SQSTM1) as the early adaptor channelling ubiquitinated sperm mitochondria towards the autophagic machinery. Downstream receptors include microtubule-associated protein 1 light chain 3α (LC3) and GABA type A receptor-associated protein (GABARAP). Among mammals, the domestic pig is the ideal mammalian model of mitochondrial inheritance because of rapid sperm mitophagy at the 1-cell stage of embryo development. Primary recognition of sperm mitochondria by SQSTM1 inside the porcine zygote is followed by GABARAP-containing autophagophore formation, and contributed to by valosin-containing protein (VCP), a 26S proteasome-presenting protein dislocase. Consequently, coinhibition of SQSTM1–GABARAP and VCP activities in the porcine zygotes, resulting in 2- to 4-cell embryos carrying intact sperm mitochondrial sheaths, revived the moniker of ‘Mitochondrial Steve’. Further work will identify the determinants of species specificity of sperm mitophagy and explain the interplay and possible consequences of a mismatch between clonal mitochondrial genome and biparentally inherited chromosomal genes encoding for structural mitochondrial proteins and transcription factors. By better understanding sperm mitophagy and its potential failure, we may be able to alleviate mitochondrial disease and early pregnancy loss in livestock and improve their fitness, reproduction and ability to pass favourable production traits to offspring.

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Accumulation of Mutant Huntingtin Fragments in Aggresome-like Inclusion Bodies as a Result of Insufficient Protein Degradation

Molecular Biology of the Cell ◽

10.1091/mbc.12.5.1393 ◽

2001 ◽

Vol 12 (5) ◽

pp. 1393-1407 ◽

Cited By ~ 423

Author(s):

Stephanie Waelter ◽

Annett Boeddrich ◽

Rudi Lurz ◽

Eberhard Scherzinger ◽

Gerhild Lueder ◽

...

Keyword(s):

Inclusion Bodies ◽

Rna Binding ◽

Ubiquitin Proteasome System ◽

26S Proteasome ◽

Therapeutic Interventions ◽

Ultrastructural Changes ◽

Huntingtin Protein ◽

Fibrillar Morphology ◽

Exon 1 ◽

Ubiquitin Proteasome

The huntingtin exon 1 proteins with a polyglutamine repeat in the pathological range (51 or 83 glutamines), but not with a polyglutamine tract in the normal range (20 glutamines), form aggresome-like perinuclear inclusions in human 293 Tet-Off cells. These structures contain aggregated, ubiquitinated huntingtin exon 1 protein with a characteristic fibrillar morphology. Inclusion bodies with truncated huntingtin protein are formed at centrosomes and are surrounded by vimentin filaments. Inhibition of proteasome activity resulted in a twofold increase in the amount of ubiquitinated, SDS-resistant aggregates, indicating that inclusion bodies accumulate when the capacity of the ubiquitin–proteasome system to degrade aggregation-prone huntingtin protein is exhausted. Immunofluorescence and electron microscopy with immunogold labeling revealed that the 20S, 19S, and 11S subunits of the 26S proteasome, the molecular chaperones BiP/GRP78, Hsp70, and Hsp40, as well as the RNA-binding protein TIA-1, the potential chaperone 14–3-3, and α-synuclein colocalize with the perinuclear inclusions. In 293 Tet-Off cells, inclusion body formation also resulted in cell toxicity and dramatic ultrastructural changes such as indentations and disruption of the nuclear envelope. Concentration of mitochondria around the inclusions and cytoplasmic vacuolation were also observed. Together these findings support the hypothesis that the ATP-dependent ubiquitin–proteasome system is a potential target for therapeutic interventions in glutamine repeat disorders.

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Ccr4 is a novel shuttle factor required for ubiquitin-dependent proteolysis by the 26S proteasome

10.1101/2020.03.30.015370 ◽

2020 ◽

Author(s):

Ganapathi Kandasamy ◽

Ashis Kumar Pradhan ◽

R Palanimurugan

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Degradation ◽

Ubiquitin Proteasome System ◽

Proteasomal Degradation ◽

Rna Degradation ◽

26S Proteasome ◽

Cellular Proteins ◽

Cellular Homeostasis ◽

Substrate Degradation ◽

Ubiquitin Proteasome

AbstractDegradation of short-lived and abnormal proteins are essential for normal cellular homeostasis. In eukaryotes, such unstable cellular proteins are selectively degraded by the ubiquitin proteasome system (UPS). Furthermore, abnormalities in protein degradation by the UPS have been linked to several human diseases. Ccr4 protein is a known component of the Ccr4-Not complex, which has established roles in transcription, mRNA de-adenylation and RNA degradation etc. Excitingly in this study, we show that Ccr4 protein has a novel function as a shuttle factor that promotes ubiquitin-dependent degradation of short-lived proteins by the 26S proteasome. Using a substrate of the well-studied ubiquitin fusion degradation (UFD) pathway, we found that its UPS-mediated degradation was severely impaired upon deletion of CCR4 in Saccharomyces cerevisiae. Additionally, we show that Ccr4 binds to cellular ubiquitin conjugates and the proteasome. In contrast to Ccr4, most other subunits of the Ccr4-Not complex proteins are dispensable for UFD substrate degradation. From our findings we conclude that Ccr4 functions in the UPS as a shuttle factor targeting ubiquitylated substrates for proteasomal degradation.

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Changes in the mitochondrial subproteome of mouse brain Rpn13-binding proteins induced by the neurotoxin MPTP and the neuroprotector isatin

Biomeditsinskaya Khimiya ◽

10.18097/pbmc20216701051 ◽

2021 ◽

Vol 67 (1) ◽

pp. 51-65

Author(s):

O.A. Buneeva ◽

A.T. Kopylov ◽

O.V. Gnedenko ◽

M.V. Medvedeva ◽

I.G. Kapitsa ◽

...

Keyword(s):

Mouse Brain ◽

Binding Proteins ◽

Functional Group ◽

Neuroprotective Effect ◽

Ubiquitin Proteasome System ◽

Mitochondrial Proteins ◽

Proteomic Profiling ◽

Metabolism Regulation ◽

Ubiquitin Proteasome ◽

Biomedical Chemistry

Mitochondrial dysfunction and ubiquitin-proteasome system (UPS) failure contribute significantly to the development of Parkinson's disease (PD). The proteasome subunit Rpn13 located on the regulatory (19S) subparticle play an important role in the delivery of proteins, subjected to degradation, to the proteolytic (20S) part of proteasome. We have previously found several brain mitochondrial proteins specifically bound to Rpn13 (Buneeva et al. (2020) Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry, 14, 297-305). In this study we have investigated the effect of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and the neuroprotector isatin on the mitochondrial subproteome of Rpn13-binding proteins of the mouse brain. Administration of MPTP (30 mg/kg) to animals caused movement disorders typical of PD, while pretreatment with isatin (100 mg/kg, 30 min before MPTP) reduced their severity. At the same time, the injection of MPTP, isatin, or their combination (isatin + MPTP) had a significant impact on the total number and the composition of Rpn13-binding proteins. The injection of MPTP decreased the total number of Rpn13-binding proteins in comparison with control, and the injection of isatin prior to MPTP or without MPTP caused an essential increase in the number of Rpn13-binding proteins, mainly of the functional group of proteins participating in the protein metabolism regulation, gene expression, and differentiation. Selected biosensor validation confirmed the interaction of Rpn13 subunit of proteasome with some proteins (glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, histones H2A and H2B) revealed while proteomic profiling. The results obtained testify that under the conditions of experimental MPTP-induced parkinsonism the neuroprotective effect of isatin may be aimed at the interaction of mitochondria with the components of UPS.

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Ubiquitin carboxyl-terminal hydrolases are required for period maintenance of the circadian clock at high temperature in Arabidopsis

Scientific Reports ◽

10.1038/s41598-019-53229-8 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Ryosuke Hayama ◽

Peizhen Yang ◽

Federico Valverde ◽

Tsuyoshi Mizoguchi ◽

Ikuyo Furutani-Hayama ◽

...

Keyword(s):

Circadian Clock ◽

High Temperatures ◽

Elevated Temperatures ◽

Control Period ◽

Ubiquitin Proteasome System ◽

26S Proteasome ◽

Cellular Processes ◽

Control Functions ◽

Ubiquitin Proteasome ◽

Carboxyl Terminal

AbstractProtein ubiquitylation participates in a number of essential cellular processes including signal transduction and transcription, often by initiating the degradation of specific substrates through the 26S proteasome. Within the ubiquitin-proteasome system, deubiquitylating enzymes (DUBs) not only help generate and maintain the supply of free ubiquitin monomers, they also directly control functions and activities of specific target proteins by modulating the pool of ubiquitylated species. Ubiquitin carboxyl-terminal hydrolases (UCHs) belong to an enzymatic subclass of DUBs, and are represented by three members in Arabidopsis, UCH1, UCH2 and UCH3. UCH1 and UCH2 influence auxin-dependent developmental pathways in Arabidopsis through their deubiquitylation activities, whereas biological and enzymatic functions of UCH3 remain unclear. Here, we demonstrate that Arabidopsis UCH3 acts to maintain the period of the circadian clock at high temperatures redundantly with UCH1 and UCH2. Whereas single uch1, uch2 and uch3 mutants have weak circadian phenotypes, the triple uch mutant displays a drastic lengthening of period at high temperatures that is more extreme than the uch1 uch2 double mutant. UCH3 also possesses a broad deubiquitylation activity against a range of substrates that link ubiquitin via peptide and isopeptide linkages. While the protein target(s) of UCH1-3 are not yet known, we propose that these DUBs act on one or more factors that control period length of the circadian clock through removal of their bound ubiquitin moieties, thus ensuring that the clock oscillates with a proper period even at elevated temperatures.

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Interplay between the Ubiquitin Proteasome System and Ubiquitin-Mediated Autophagy in Plants

Cells ◽

10.3390/cells9102219 ◽

2020 ◽

Vol 9 (10) ◽

pp. 2219 ◽

Cited By ~ 1

Author(s):

Tong Su ◽

Mingyue Yang ◽

Pingping Wang ◽

Yanxiu Zhao ◽

Changle Ma

Keyword(s):

Common Factor ◽

Ubiquitin Proteasome System ◽

26S Proteasome ◽

Regulatory Proteins ◽

Selective Autophagy ◽

Ubiquitin Proteasome ◽

Conjugating Enzymes ◽

Ubiquitin Conjugating Enzymes ◽

Cellular Components ◽

Protein Ligases

All eukaryotes rely on the ubiquitin-proteasome system (UPS) and autophagy to control the abundance of key regulatory proteins and maintain a healthy intracellular environment. In the UPS, damaged or superfluous proteins are ubiquitinated and degraded in the proteasome, mediated by three types of ubiquitin enzymes: E1s (ubiquitin activating enzymes), E2s (ubiquitin conjugating enzymes), and E3s (ubiquitin protein ligases). Conversely, in autophagy, a vesicular autophagosome is formed that transfers damaged proteins and organelles to the vacuole, mediated by a series of ATGs (autophagy related genes). Despite the use of two completely different componential systems, the UPS and autophagy are closely interconnected and mutually regulated. During autophagy, ATG8 proteins, which are autophagosome markers, decorate the autophagosome membrane similarly to ubiquitination of damaged proteins. Ubiquitin is also involved in many selective autophagy processes and is thus a common factor of the UPS and autophagy. Additionally, the components of the UPS, such as the 26S proteasome, can be degraded via autophagy, and conversely, ATGs can be degraded by the UPS, indicating cross regulation between the two pathways. The UPS and autophagy cooperate and jointly regulate homeostasis of cellular components during plant development and stress response.

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The Roles of Ubiquitin-Binding Protein Shuttles in the Degradative Fate of Ubiquitinated Proteins in the Ubiquitin-Proteasome System and Autophagy

Cells ◽

10.3390/cells8010040 ◽

2019 ◽

Vol 8 (1) ◽

pp. 40 ◽

Cited By ~ 27

Author(s):

Katarzyna Zientara-Rytter ◽

Suresh Subramani

Keyword(s):

Protein Quality ◽

Current Knowledge ◽

Binding Protein ◽

Ubiquitin Proteasome System ◽

Oligomeric State ◽

Receptor Associated Protein ◽

Ubiquitin Proteasome ◽

Specific Proteins ◽

And Control ◽

Recognition Code

The ubiquitin-proteasome system (UPS) and autophagy are the two major intracellular protein quality control (PQC) pathways that are responsible for cellular proteostasis (homeostasis of the proteome) by ensuring the timely degradation of misfolded, damaged, and unwanted proteins. Ubiquitination serves as the degradation signal in both these systems, but substrates are precisely targeted to one or the other pathway. Determining how and when cells target specific proteins to these two alternative PQC pathways and control the crosstalk between them are topics of considerable interest. The ubiquitin (Ub) recognition code based on the type of Ub-linked chains on substrate proteins was believed to play a pivotal role in this process, but an increasing body of evidence indicates that the PQC pathway choice is also made based on other criteria. These include the oligomeric state of the Ub-binding protein shuttles, their conformation, protein modifications, and the presence of motifs that interact with ATG8/LC3/GABARAP (autophagy-related protein 8/microtubule-associated protein 1A/1B-light chain 3/GABA type A receptor-associated protein) protein family members. In this review, we summarize the current knowledge regarding the Ub recognition code that is bound by Ub-binding proteasomal and autophagic receptors. We also discuss how cells can modify substrate fate by modulating the structure, conformation, and physical properties of these receptors to affect their shuttling between both degradation pathways.

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CCR4-NOT complex nuclease Caf1 is a novel shuttle factor involved in the degradation of ubiquitin-modified proteins by 26S proteasome

10.1101/2020.05.13.093104 ◽

2020 ◽

Author(s):

Ganapathi Kandasamy ◽

Ashis Kumar Pradhan ◽

R Palanimurugan

Keyword(s):

Protein Degradation ◽

Translational Control ◽

Ubiquitin Proteasome System ◽

26S Proteasome ◽

Control Mechanisms ◽

Gene Expressions ◽

Proteolytic Pathway ◽

Ubiquitin Ligase E3 ◽

Ubiquitin Proteasome ◽

Modified Proteins

AbstractProtein degradation by ubiquitin proteasome system (UPS) is the major selective proteolytic pathway responsible for the degradation of short lived proteins ranging from regulatory proteins to abnormal proteins. Many diseases are associated with abnormal protein degradation; occasionally such dysregulated protein degradation is compensated by various transcriptional and translational control mechanisms in the cell. Among those pathways CCR4-NOT protein complex is responsible for transcriptional and transitional control of various gene expressions. Furthermore, CCR4-NOT complex also has a RING type ubiquitin ligase (E3) which is required for the degradation of several proteins. Here we report a novel function that the CCR4-NOT complex 3’-5’ exonuclease Caf1 is involved in ubiquitindependent degradation of short lived proteins by the 26S proteasome in yeast Saccharomyces cerevisiae. caf1 deletion results in stabilization of R-Ura3 (N-end rule) and Ub-V76-Ura3 (Ubiquitin fusion degradation) substrates from proteasomal degradation. Additionally, caf1 deletion accumulates ubiquitin-modified Ub-V76-Ura3 proteins and Caf1 binds to poly-ubiquitin conjugates and linear tetra ubiquitin chains. Surprisingly, Caf1 interacts with 19S regulatory particle complex of the 26S proteasome. Therefore, we conclude that Caf1 has an exciting novel function as an ubiquitin shuttle factor in which Caf1 targets ubiquitin-modified proteins to 26S proteasome for efficient degradation.

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Releasing the Lockdown: An Emerging Role for the Ubiquitin-Proteasome System in the Breakdown of Transient Protein Inclusions

Biomolecules ◽

10.3390/biom10081168 ◽

2020 ◽

Vol 10 (8) ◽

pp. 1168 ◽

Cited By ~ 1

Author(s):

Yuval Reiss ◽

Elisheva Gur ◽

Tommer Ravid

Keyword(s):

Protein Quality ◽

Ubiquitin Proteasome System ◽

Biological Properties ◽

26S Proteasome ◽

Turnover Rates ◽

Functional Link ◽

Protein Deposits ◽

Dead End ◽

Ubiquitin Proteasome ◽

Quality Control Mechanism

Intracellular protein inclusions are diverse cellular entities with distinct biological properties. They vary in their protein content, sequestration sites, physiological function, conditions for their generation, and turnover rates. Major distinctions have been recognized between stationary amyloids and dynamic, misfolded protein deposits. The former being a dead end for irreversibly misfolded proteins, hence, cleared predominantly by autophagy, while the latter consists of a protein-quality control mechanism, important for cell endurance, where proteins are sequestered during proteotoxic stress and resolved upon its relief. Accordingly, the disaggregation of transient inclusions is a regulated process consisting of protein solubilization, followed by a triage step to either refolding or to ubiquitin-mediated degradation. Recent studies have demonstrated an indispensable role in disaggregation for components of the chaperone and the ubiquitin–proteasome systems. These include heat-shock chaperones of the 40/70/100 kDa families, the proteasome, proteasome substrate shuttling factors, and deubiquitylating enzymes. Thus, a functional link has been established between the chaperone machinery that extracts proteins from transient deposits and 26S proteasome-dependent disaggregation, indicative of a coordinated process. In this review, we discuss data emanating from these important studies and subsequently consolidate the information in the form of a working model for the disaggregation mechanism.

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Recent Advances in the Discovery of Novel Peptide Inhibitors Targeting 26S Proteasome

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520618666180813120012 ◽

2019 ◽

Vol 18 (12) ◽

pp. 1656-1673

Author(s):

Xinjie Gu ◽

Shutao Ma

Keyword(s):

Cancer Therapy ◽

Proteasome Inhibitors ◽

Ubiquitin Proteasome System ◽

Peptide Inhibitors ◽

26S Proteasome ◽

Design Strategies ◽

Intracellular Protein ◽

Primary Tumors ◽

Protein Levels ◽

Ubiquitin Proteasome

Background: The 26S proteasome is a proteolytic complex of multimeric protease, which operates at the executive end of the Ubiquitin-Proteasome System (UPS) and degrades the polyubiquitylated proteins. Methods: After a brief introduction of 26S proteasome and Ubiquitin-Proteasome System (UPS), this review focuses on the structure and function of the 26S proteasome in intracellular protein level regulation. Then, physiological regulation mechanisms and processes are elaborated. In addition, the advantages and defects of approved 26S proteasome inhibitors were discussed. Finally, we summarized the novel peptide 26S proteasome inhibitors according to their structural classifications, highlighting their design strategies, inhibitory activity and Structure-Activity Relationships (SARs). Results: Cellular function maintenance relies on the proteasome metabolizing intracellular proteins to control intracellular protein levels, which is especially important for cancer cells to survive and proliferate. In primary tumors, proteasomes had a higher level and more potent activity. Currently, the approved small peptide inhibitors have proved their specific 26S proteasome inhibitory effects and considerable antitumor activities, but with obvious defects. Increasingly, novel peptide inhibitors are emerging and possess promising values in cancer therapy. Conclusion: Overall, the 26S proteasome is an efficient therapeutic target and novel 26S proteasome inhibitors hold potency for cancer therapy.

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