Podocyte Injury Associated with Mutant α-Actinin-4

Focal segmental glomerulosclerosis (FSGS) is an important cause of proteinuria and nephrotic syndrome in humans. The pathogenesis of FSGS may be associated with glomerular visceral epithelial cell (GEC; podocyte) injury, leading to apoptosis, detachment, and “podocytopenia”, followed by glomerulosclerosis. Mutations in α-actinin-4 are associated with FSGS in humans. In cultured GECs, α-actinin-4 mediates adhesion and cytoskeletal dynamics. FSGS-associated α-actinin-4 mutants show increased binding to actin filaments, compared with the wild-type protein. Expression of an α-actinin-4 mutant in mouse podocytes in vivo resulted in proteinuric FSGS. GECs that express mutant α-actinin-4 show defective spreading and motility, and such abnormalities could alter the mechanical properties of the podocyte, contribute to cytoskeletal disruption, and lead to injury. The potential for mutant α-actinin-4 to injure podocytes is also suggested by the characteristics of this mutant protein to form microaggregates, undergo ubiquitination, impair the ubiquitin-proteasome system, enhance endoplasmic reticulum stress, and exacerbate apoptosis.

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Fas expression promotes proteasomal activity in toxin-induced parkinsonism

Acta Neuropsychiatrica ◽

10.1111/j.1601-5215.2011.00615.x ◽

2012 ◽

Vol 24 (3) ◽

pp. 166-171

Author(s):

Anne M. Landau ◽

Rosmarie Siegrist-Johnstone ◽

Julie Desbarats

Keyword(s):

Neuroblastoma Cells ◽

Death Receptor ◽

Protein Aggregates ◽

Ubiquitin Proteasome System ◽

Wild Type ◽

Proteasomal Activity ◽

Ubiquitin Proteasome ◽

Deficient Mice

Objective: Fas (CD95), commonly categorised as a death receptor due to its well-defined role in apoptosis, can paradoxically also promote neuroprotection. We have previously found that defects in Fas signalling render mice highly susceptible to neural degeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease (PD). Decreased activity of the ubiquitin proteasome system and accumulation of protein aggregates are implicated in PD pathogenesis. Here, we investigate the relationship between Fas and ubiquitin proteasomal activity in neuronal cells.Methods: We performed proteasome assays in neuroblastoma cells and in midbrain cultures of wild-type and Fas-deficient mice.Results: Neuroblastoma cells upregulated proteasomal activity in response to an activating Fas antibody in vitro. Furthermore, neural tissue from Fas-deficient mice showed decreased proteasomal activity compared with the tissue from wild-type mice when exposed to a PD-inducing toxin in vivo.Conclusion: These findings suggest that mechanisms for Fas-mediated neuroprotection may include Fas-induced upregulation of proteasomal activity, and consequently less accumulation of toxic protein aggregates.

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Breakdown of Filamentous Myofibrils by the UPS–Step by Step

Biomolecules ◽

10.3390/biom11010110 ◽

2021 ◽

Vol 11 (1) ◽

pp. 110

Author(s):

Dina Aweida ◽

Shenhav Cohen

Keyword(s):

Protein Quality ◽

Protein Structures ◽

Ubiquitin Proteasome System ◽

Surface Proteins ◽

Catalytic Core ◽

Complex Protein ◽

Ubiquitin Proteasome ◽

Aaa Atpases

Protein degradation maintains cellular integrity by regulating virtually all biological processes, whereas impaired proteolysis perturbs protein quality control, and often leads to human disease. Two major proteolytic systems are responsible for protein breakdown in all cells: autophagy, which facilitates the loss of organelles, protein aggregates, and cell surface proteins; and the ubiquitin-proteasome system (UPS), which promotes degradation of mainly soluble proteins. Recent findings indicate that more complex protein structures, such as filamentous assemblies, which are not accessible to the catalytic core of the proteasome in vitro, can be efficiently degraded by this proteolytic machinery in systemic catabolic states in vivo. Mechanisms that loosen the filamentous structure seem to be activated first, hence increasing the accessibility of protein constituents to the UPS. In this review, we will discuss the mechanisms underlying the disassembly and loss of the intricate insoluble filamentous myofibrils, which are responsible for muscle contraction, and whose degradation by the UPS causes weakness and disability in aging and disease. Several lines of evidence indicate that myofibril breakdown occurs in a strictly ordered and controlled manner, and the function of AAA-ATPases is crucial for their disassembly and loss.

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Identification of the MuRF1 skeletal muscle ubiquitylome through quantitative proteomics

Function ◽

10.1093/function/zqab029 ◽

2021 ◽

Author(s):

Leslie M Baehr ◽

David C Hughes ◽

Sarah A Lynch ◽

Delphi Van Haver ◽

Teresa Mendes Maia ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Atrophy ◽

Potential Role ◽

Ubiquitin Proteasome System ◽

In Vivo Electroporation ◽

Adult Mice ◽

Expression Of Genes ◽

Regulation Of Metabolism ◽

Ubiquitin Proteasome

Abstract MuRF1 (TRIM63) is a muscle-specific E3 ubiquitin ligase and component of the ubiquitin proteasome system. MuRF1 is transcriptionally upregulated under conditions that cause muscle loss, in both rodents and humans, and is a recognized marker of muscle atrophy. In this study, we used in vivo electroporation to determine if MuRF1 overexpression alone can cause muscle atrophy and, in combination with ubiquitin proteomics, identify the endogenous MuRF1 substrates in skeletal muscle. Overexpression of MuRF1 in adult mice increases ubiquitination of myofibrillar and sarcoplasmic proteins, increases expression of genes associated with neuromuscular junction instability, and causes muscle atrophy. A total of 169 ubiquitination sites on 56 proteins were found to be regulated by MuRF1. MuRF1-mediated ubiquitination targeted both thick and thin filament contractile proteins, as well as, glycolytic enzymes, deubiquitinases, p62, and VCP. These data reveal a potential role for MuRF1 in not only the breakdown of the sarcomere, but also the regulation of metabolism and other proteolytic pathways in skeletal muscle.

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Effects of ubiquitin C-terminal hydrolase L1 deficiency on mouse ova

Reproduction ◽

10.1530/rep-11-0128 ◽

2012 ◽

Vol 143 (3) ◽

pp. 271-279 ◽

Cited By ~ 8

Author(s):

Sayaka Koyanagi ◽

Hiroko Hamasaki ◽

Satoshi Sekiguchi ◽

Kenshiro Hara ◽

Yoshiyuki Ishii ◽

...

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Endoplasmic Reticulum ◽

Proteomic Analysis ◽

Ubiquitin Proteasome System ◽

Underlying Mechanism ◽

Wild Type ◽

Transmission Electron ◽

Ubiquitin Proteasome

Maternal proteins are rapidly degraded by the ubiquitin–proteasome system during oocyte maturation in mice. Ubiquitin C-terminal hydrolase L1 (UCHL1) is highly and specifically expressed in mouse ova and is involved in the polyspermy block. However, the role of UCHL1 in the underlying mechanism of polyspermy block is poorly understood. To address this issue, we performed a comprehensive proteomic analysis to identify maternal proteins that were relevant to the role of UCHL1 in mouse ova using UCHL1-deficientgad. Furthermore, we assessed morphological features ingadmouse ova using transmission electron microscopy. NACHT, LRR, and PYD domain-containing (NALP) family proteins and endoplasmic reticulum (ER) chaperones were identified by proteomic analysis. We also found that the ‘maternal antigen that embryos require’ (NLRP5 (MATER)) protein level increased significantly ingadmouse ova compared with that in wild-type mice. In an ultrastructural study,gadmouse ova contained less ER in the cortex than in wild-type mice. These results provide new insights into the role of UCHL1 in the mechanism of polyspermy block in mouse ova.

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Degradation of mutant huntingtin via the ubiquitin/proteasome system is modulated by FE65

Biochemical Journal ◽

10.1042/bj20112175 ◽

2012 ◽

Vol 443 (3) ◽

pp. 681-689 ◽

Cited By ~ 15

Author(s):

Wan Ning Vanessa Chow ◽

Hon Wing Luk ◽

Ho Yin Edwin Chan ◽

Kwok-Fai Lau

Keyword(s):

Cell Death ◽

Degradation Mechanism ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

Adaptor Protein ◽

Ubiquitin Proteasome System ◽

Exon 1 ◽

Ubiquitin Proteasome

An unstable expansion of the polyglutamine repeat within exon 1 of the protein Htt (huntingtin) causes HD (Huntington's disease). Mounting evidence shows that accumulation of N-terminal mutant Htt fragments is the source of disruption of normal cellular processes which ultimately leads to neuronal cell death. Understanding the degradation mechanism of mutant Htt and improving its clearance has emerged as a new direction in developing therapeutic approaches to treat HD. In the present study we show that the brain-enriched adaptor protein FE65 is a novel interacting partner of Htt. The binding is mediated through WW–polyproline interaction and is dependent on the length of the polyglutamine tract. Interestingly, a reduction in mutant Htt protein level was observed in FE65-knockdown cells, and the process requires the UPS (ubiquitin/proteasome system). Moreover, the ubiquitination level of mutant Htt was found to be enhanced when FE65 is knocked down. Immunofluroescence staining revealed that FE65 associates with mutant Htt aggregates. Additionally, we demonstrated that overexpression of FE65 increases mutant Htt-induced cell death both in vitro and in vivo. These results suggest that FE65 facilitates the accumulation of mutant Htt in cells by preventing its degradation via the UPS, and thereby enhances the toxicity of mutant Htt.

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Regulation of cell proliferation by ERK and signal-dependent nuclear translocation of ERK is dependent on Tm5NM1-containing actin filaments

Molecular Biology of the Cell ◽

10.1091/mbc.e14-10-1453 ◽

2015 ◽

Vol 26 (13) ◽

pp. 2475-2490 ◽

Cited By ~ 28

Author(s):

Galina Schevzov ◽

Anthony J. Kee ◽

Bin Wang ◽

Vanessa B. Sequeira ◽

Jeff Hook ◽

...

Keyword(s):

Cell Proliferation ◽

Actin Filaments ◽

Nuclear Translocation ◽

Genetic Manipulation ◽

Wild Type ◽

Proximity Ligation ◽

Mouse Embryo Fibroblasts ◽

Wild Type Mefs

ERK-regulated cell proliferation requires multiple phosphorylation events catalyzed first by MEK and then by casein kinase 2 (CK2), followed by interaction with importin7 and subsequent nuclear translocation of pERK. We report that genetic manipulation of a core component of the actin filaments of cancer cells, the tropomyosin Tm5NM1, regulates the proliferation of normal cells both in vitro and in vivo. Mouse embryo fibroblasts (MEFs) lacking Tm5NM1, which have reduced proliferative capacity, are insensitive to inhibition of ERK by peptide and small-molecule inhibitors, indicating that ERK is unable to regulate proliferation of these knockout (KO) cells. Treatment of wild-type MEFs with a CK2 inhibitor to block phosphorylation of the nuclear translocation signal in pERK resulted in greatly decreased cell proliferation and a significant reduction in the nuclear translocation of pERK. In contrast, Tm5NM1 KO MEFs, which show reduced nuclear translocation of pERK, were unaffected by inhibition of CK2. This suggested that it is nuclear translocation of CK2-phosphorylated pERK that regulates cell proliferation and this capacity is absent in Tm5NM1 KO cells. Proximity ligation assays confirmed a growth factor–stimulated interaction of pERK with Tm5NM1 and that the interaction of pERK with importin7 is greatly reduced in the Tm5NM1 KO cells.

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Impaired T-cell priming in vivo resulting from dysfunction of WASp-deficient dendritic cells

Blood ◽

10.1182/blood-2007-06-096875 ◽

2007 ◽

Vol 110 (13) ◽

pp. 4278-4284 ◽

Cited By ~ 56

Author(s):

Gerben Bouma ◽

Siobhan Burns ◽

Adrian J. Thrasher

Keyword(s):

T Cell ◽

T Lymphocytes ◽

Immune Cell ◽

Wild Type ◽

Cytoskeletal Dynamics ◽

T Cell Priming ◽

And Migration ◽

Priming Activity

The Wiskott-Aldrich syndrome (WAS) is characterized by defective cytoskeletal dynamics affecting multiple immune cell lineages, and leading to immunodeficiency and autoimmunity. The contribution of dendritic cell (DC) dysfunction to the immune dysregulation has not been defined, although both immature and mature WAS knockout (KO) DCs exhibit significant abnormalities of chemotaxis and migration. To exclude environmental confounders as a result of WAS protein (WASp) deficiency, we studied migration and priming activity of WAS KO DCs in vivo after adoptive transfer into wild-type recipient mice. Homing to draining lymph nodes was reduced and WAS KO DCs failed to localize efficiently in T-cell areas. Priming of both CD4+ and CD8+ T lymphocytes by WAS KO DCs preloaded with antigen was significantly decreased. At low doses of antigen, activation of preprimed wild-type CD4+ T lymphocytes by WAS KO DCs in vitro was also abrogated, suggesting that there is a threshold-dependent impairment even if successful DC–T cell colocalization is achieved. Our data indicate that intrinsic DC dysfunction due to WASp deficiency directly impairs the T-cell priming response in vivo, most likely as a result of inefficient migration, but also possibly influenced by suboptimal DC-mediated cognate interaction.

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Efficient APC/C substrate degradation in cells undergoing mitotic exit depends on K11 ubiquitin linkages

Molecular Biology of the Cell ◽

10.1091/mbc.e15-02-0102 ◽

2015 ◽

Vol 26 (24) ◽

pp. 4325-4332 ◽

Cited By ~ 33

Author(s):

Mingwei Min ◽

Tycho E. T. Mevissen ◽

Maria De Luca ◽

David Komander ◽

Catherine Lindon

Keyword(s):

Cell Cycle Progression ◽

Degradation Kinetics ◽

Ubiquitin Proteasome System ◽

Mitotic Exit ◽

Anaphase Promoting Complex ◽

Polyubiquitin Chains ◽

Substrate Degradation ◽

Ubiquitin Proteasome ◽

In Cells

The ubiquitin proteasome system (UPS) directs programmed destruction of key cellular regulators via posttranslational modification of its targets with polyubiquitin chains. These commonly contain Lys-48 (K48)–directed ubiquitin linkages, but chains containing atypical Lys-11 (K11) linkages also target substrates to the proteasome—for example, to regulate cell cycle progression. The ubiquitin ligase called the anaphase-promoting complex/cyclosome (APC/C) controls mitotic exit. In higher eukaryotes, the APC/C works with the E2 enzyme UBE2S to assemble K11 linkages in cells released from mitotic arrest, and these are proposed to constitute an improved proteolytic signal during exit from mitosis. We tested this idea by correlating quantitative measures of in vivo K11-specific ubiquitination of individual substrates, including Aurora kinases, with their degradation kinetics tracked at the single-cell level. All anaphase substrates tested by this methodology are stabilized by depletion of K11 linkages via UBE2S knockdown, even if the same substrates are significantly modified with K48-linked polyubiquitin. Specific examination of substrates depending on the APC/C coactivator Cdh1 for their degradation revealed Cdh1-dependent enrichment of K11 chains on these substrates, whereas other ubiquitin linkages on the same substrates added during mitotic exit were Cdh1-independent. Therefore we show that K11 linkages provide the APC/C with a means to regulate the rate of substrate degradation in a coactivator-specified manner.

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The Slow Wallerian Degeneration Protein, WldS, Binds Directly to VCP/p97 and Partially Redistributes It within the Nucleus

Molecular Biology of the Cell ◽

10.1091/mbc.e05-04-0375 ◽

2006 ◽

Vol 17 (3) ◽

pp. 1075-1084 ◽

Cited By ~ 41

Author(s):

Heike Laser ◽

Laura Conforti ◽

Giacomo Morreale ◽

Till G.M. Mack ◽

Molly Heyer ◽

...

Keyword(s):

Amino Acids ◽

Wallerian Degeneration ◽

Nuclear Protein ◽

Ubiquitin Proteasome System ◽

Wild Type ◽

Ubiquitin Proteasome ◽

Direct Binding ◽

Synthesis Activity ◽

Additional Role

Slow Wallerian degeneration (WldS) mutant mice express a chimeric nuclear protein that protects sick or injured axons from degeneration. The C-terminal region, derived from NAD+ synthesizing enzyme Nmnat1, is reported to confer neuroprotection in vitro. However, an additional role for the N-terminal 70 amino acids (N70), derived from multiubiquitination factor Ube4b, has not been excluded. In wild-type Ube4b, N70 is part of a sequence essential for ubiquitination activity but its role is not understood. We report direct binding of N70 to valosin-containing protein (VCP; p97/Cdc48), a protein with diverse cellular roles including a pivotal role in the ubiquitin proteasome system. Interaction with WldS targets VCP to discrete intranuclear foci where ubiquitin epitopes can also accumulate. WldS lacking its N-terminal 16 amino acids (N16) neither binds nor redistributes VCP, but continues to accumulate in intranuclear foci, targeting its intrinsic NAD+ synthesis activity to these same foci. Wild-type Ube4b also requires N16 to bind VCP, despite a more C-terminal binding site in invertebrate orthologues. We conclude that N-terminal sequences of WldS protein influence the intranuclear location of both ubiquitin proteasome and NAD+ synthesis machinery and that an evolutionary recent sequence mediates binding of mammalian Ube4b to VCP.

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Innate immunity to yeast prions: Btn2p and Cur1p curing of the [URE3] prion is prevented by 60S ribosomal protein deficiency or ubiquitin/proteasome system overactivity

Genetics ◽

10.1093/genetics/iyab013 ◽

2021 ◽

Author(s):

Evgeny E Bezsonov ◽

Herman K Edskes ◽

Reed B Wickner

Keyword(s):

Ubiquitin Ligase ◽

Ribosomal Subunit ◽

Ubiquitin Proteasome System ◽

Negative Regulator ◽

Large Ribosomal Subunit ◽

Seed Number ◽

Wild Type ◽

Yeast Prions ◽

In The Wild ◽

Ubiquitin Proteasome

Abstract [URE3] is an amyloid-based prion of Ure2p, a negative regulator of poor nitrogen source catabolism in Saccharomyces cerevisiae. Overproduced Btn2p or its paralog Cur1p, in processes requiring Hsp42, cure the [URE3] prion. Btn2p cures by collecting Ure2p amyloid filaments at one place in the cell. We find that rpl4aΔ, rpl21aΔ, rpl21bΔ, rpl11bΔ and rpl16bΔ (large ribosomal subunit proteins) or ubr2Δ (ubiquitin ligase targeting Rpn4p, an activator of proteasome genes) reduce curing by overproduced Btn2p or Cur1p. Impaired curing in ubr2Δ or rpl21bΔ is restored by an rpn4Δ mutation. No effect of rps14aΔ or rps30bΔ on curing was observed, indicating that 60S subunit deficiency specifically impairs curing. Levels of Hsp42p, Sis1p or Btn3p are unchanged in rpl4aΔ, rpl21bΔ or ubr2Δ mutants. Overproduction of Cur1p or Btn2p was enhanced in rpn4Δ and hsp42Δ mutants, lower in ubr2Δ strains, and restored to above wild type levels in rpn4Δ ubr2Δ strains. As in the wild-type, Ure2N-GFP colocalizes with Btn2-RFP in rpl4aΔ, rpl21bΔ or ubr2Δ strains, but not in hsp42Δ. Btn2p/Cur1p overproduction cures [URE3] variants with low seed number, but seed number is not increased in rpl4aΔ, rpl21bΔ or ubr2Δ mutants. Knockouts of genes required for the protein sorting function of Btn2p did not affect curing of [URE3], nor did inactivation of the Hsp104 prion-curing activity. Overactivity of the ubiquitin/proteasome system, resulting from 60S subunit deficiency or ubr2Δ, may impair Cur1p and Btn2p curing of [URE3] by degrading Cur1p, Btn2p or another component of these curing systems.

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