scholarly journals Chip Protein U-Box Domain Truncation Affects Purkinje Neuron Morphology and Leads to Behavioral Changes in Zebrafish

2021 ◽  
Vol 14 ◽  
Author(s):  
Yasaman Pakdaman ◽  
Elsa Denker ◽  
Eirik Austad ◽  
William H. J. Norton ◽  
Hans O. Rolfsnes ◽  
...  
Keyword(s):  
Purkinje Cell ◽  
Ubiquitin Ligase ◽  
Cerebellar Atrophy ◽  
Behavioral Changes ◽  
26S Proteasome ◽  
Spinocerebellar Ataxias ◽  
Interacting Protein ◽  
Ubiquitin Ligase Activity ◽  
Wide Range ◽  
The Brain

The ubiquitin ligase CHIP (C-terminus of Hsc70-interacting protein) is encoded by STUB1 and promotes ubiquitination of misfolded and damaged proteins. CHIP deficiency has been linked to several diseases, and mutations in the human STUB1 gene are associated with recessive and dominant forms of spinocerebellar ataxias (SCAR16/SCA48). Here, we examine the effects of impaired CHIP ubiquitin ligase activity in zebrafish (Danio rerio). We characterized the zebrafish stub1 gene and Chip protein, and generated and characterized a zebrafish mutant causing truncation of the Chip functional U-box domain. Zebrafish stub1 has a high degree of conservation with mammalian orthologs and was detected in a wide range of tissues in adult stages, with highest expression in brain, eggs, and testes. In the brain, stub1 mRNA was predominantly detected in the cerebellum, including the Purkinje cell layer and granular layer. Recombinant wild-type zebrafish Chip showed ubiquitin ligase activity highly comparable to human CHIP, while the mutant Chip protein showed impaired ubiquitination of the Hsc70 substrate and Chip itself. In contrast to SCAR16/SCA48 patients, no gross cerebellar atrophy was evident in mutant fish, however, these fish displayed reduced numbers and sizes of Purkinje cell bodies and abnormal organization of Purkinje cell dendrites. Mutant fish also had decreased total 26S proteasome activity in the brain and showed behavioral changes. In conclusion, truncation of the Chip U-box domain leads to impaired ubiquitin ligase activity and behavioral and anatomical changes in zebrafish, illustrating the potential of zebrafish to study STUB1-mediated diseases.

scholarly journals Genetic Dominant Variants in STUB1, Segregating in Families with SCA48, Display In Vitro Functional Impairments Indistinctive from Recessive Variants Associated with SCAR16

2021 ◽  
Vol 22 (11) ◽  
pp. 5870
Author(s):  
Yasaman Pakdaman ◽  
Siren Berland ◽  
Helene J. Bustad ◽  
Sigrid Erdal ◽  
Bryony A. Thompson ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cerebellar Atrophy ◽  
Disease Status ◽  
Functional Impairments ◽  
Interacting Protein ◽  
C Terminus ◽  
Ubiquitin Ligase Activity ◽  
Native Polyacrylamide Gel Electrophoresis

Variants in STUB1 cause both autosomal recessive (SCAR16) and dominant (SCA48) spinocerebellar ataxia. Reports from 18 STUB1 variants causing SCA48 show that the clinical picture includes later-onset ataxia with a cerebellar cognitive affective syndrome and varying clinical overlap with SCAR16. However, little is known about the molecular properties of dominant STUB1 variants. Here, we describe three SCA48 families with novel, dominantly inherited STUB1 variants (p.Arg51_Ile53delinsProAla, p.Lys143_Trp147del, and p.Gly249Val). All the patients developed symptoms from 30 years of age or later, all had cerebellar atrophy, and 4 had cognitive/psychiatric phenotypes. Investigation of the structural and functional consequences of the recombinant C-terminus of HSC70-interacting protein (CHIP) variants was performed in vitro using ubiquitin ligase activity assay, circular dichroism assay and native polyacrylamide gel electrophoresis. These studies revealed that dominantly and recessively inherited STUB1 variants showed similar biochemical defects, including impaired ubiquitin ligase activity and altered oligomerization properties of the CHIP. Our findings expand the molecular understanding of SCA48 but also mean that assumptions concerning unaffected carriers of recessive STUB1 variants in SCAR16 families must be re-evaluated. More investigations are needed to verify the disease status of SCAR16 heterozygotes and elucidate the molecular relationship between SCA48 and SCAR16 diseases.

scholarly journals Assay for Ubiquitin Ligase Activity: High-Throughput Screen for Inhibitors of HDM2

2004 ◽  
Vol 9 (8) ◽  
pp. 695-703 ◽  
Author(s):  
I. V. Davydov ◽  
D. Woods ◽  
Y. J. Safiran ◽  
P. Oberoi ◽  
H. O. Fearnhead ◽  
...  
Keyword(s):  
High Throughput ◽  
Ubiquitin Ligase ◽  
Ubiquitin Ligases ◽  
High Throughput Screen ◽  
Ubiquitin Ligase Activity ◽  
Wide Range

An assay for the autoubiquitination activity of the E3 ligaseHDM2 (Mdm2) was developed and adapted to a high-throughput format to identify inhibitors of this activity. The assay can also be used tomeasure the activity of other E3s andmay be useful in finding both inhibitors and activators of a wide range of different ubiquitin ligases.

scholarly journals PARAQUAT TOLERANCE3 is an E3 ligase and acts as a negative regulator of oxidative stress response

2016 ◽  
Author(s):  
Chao Luo ◽  
Xiao-Teng Cai ◽  
Jin Du ◽  
Tao-Lan Zhao ◽  
Peng-Fei Wang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Genetic Locus ◽  
E3 Ligase ◽  
26S Proteasome ◽  
Negative Regulator ◽  
Biotic And Abiotic Stresses ◽  
Interacting Protein ◽  
Ubiquitination Assay ◽  
Ubiquitin Ligase Activity ◽  
Arabidopsis Mutant

AbstractOxidative damage could be caused in plant cells when biotic and abiotic stresses are imposed. While the response to oxidative stress is well studied, little is known about how the activated response is switched off when oxidative stress is diminished. By studying Arabidopsis mutant paraquat tolerance3, we identified the genetic locus PARAQUAT TOLERANCE3 (PQT3) as a major negative regulator of oxidative stress tolerance. PQT3, encoding an E3 ligase, is rapidly down-regulated by oxidative stress. PQT3 has E3 ubiquitin ligase activity in ubiquitination assay. Subsequently, we identified PRMT4b as a PQT3-interacting protein. By histone methylation, PRMT4b may regulate the expression of APX1 and GPX1, encoding two key enzymes against oxidative stress. Moreover, PQT3 is able to recognize PRMT4b for targeted degradation via 26S proteasome. Therefore, we have identified PQT3 as an E3 ligase that acts as a negative regulator of activated response to oxidative stress.

scholarly journals The APC11 RING-H2 Finger Mediates E2-Dependent Ubiquitination

2000 ◽  
Vol 11 (7) ◽  
pp. 2315-2325 ◽  
Author(s):  
Joel D. Leverson ◽  
Claudio A.P. Joazeiro ◽  
Andrew M. Page ◽  
Han-kuei Huang ◽  
Philip Hieter ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
26S Proteasome ◽  
Anaphase Promoting Complex ◽  
Mitotic Progression ◽  
Protein Substrates ◽  
Ubiquitin Ligase Activity ◽  
Ubiquitin Conjugating Enzyme ◽  
Ubiquitin Conjugating Enzymes

Polyubiquitination marks proteins for degradation by the 26S proteasome and is carried out by a cascade of enzymes that includes ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). The anaphase-promoting complex or cyclosome (APC/C) comprises a multisubunit ubiquitin ligase that mediates mitotic progression. Here, we provide evidence that theSaccharomyces cerevisiae RING-H2 finger protein Apc11 defines the minimal ubiquitin ligase activity of the APC. We found that the integrity of the Apc11p RING-H2 finger was essential for budding yeast cell viability, Using purified, recombinant proteins we showed that Apc11p interacted directly with the Ubc4 ubiquitin conjugating enzyme (E2). Furthermore, purified Apc11p was capable of mediating E1- and E2-dependent ubiquitination of protein substrates, including Clb2p, in vitro. The ability of Apc11p to act as an E3 was dependent on the integrity of the RING-H2 finger, but did not require the presence of the cullin-like APC subunit Apc2p. We suggest that Apc11p is responsible for recruiting E2s to the APC and for mediating the subsequent transfer of ubiquitin to APC substrates in vivo.

scholarly journals Regulation of p53 and MDM2 Activity by MTBP

2005 ◽  
Vol 25 (2) ◽  
pp. 545-553 ◽  
Author(s):  
Mark Brady ◽  
Nikolina Vlatković ◽  
Mark T. Boyd
Keyword(s):  
Ubiquitin Ligase ◽  
Stress Responses ◽  
Nuclear Export ◽  
Cellular Response ◽  
E3 Ubiquitin Ligase ◽  
Ring Finger ◽  
Dependent Manner ◽  
Γ Irradiation ◽  
Ubiquitin Ligase Activity ◽  
Wide Range

ABSTRACT p53 is a critical coordinator of a wide range of stress responses. To facilitate a rapid response to stress, p53 is produced constitutively but is negatively regulated by MDM2. MDM2 can inhibit p53 in multiple independent ways: by binding to its transcription activation domain, inhibiting p53 acetylation, promoting nuclear export, and probably most importantly by promoting proteasomal degradation of p53. The latter is achieved via MDM2's E3 ubiquitin ligase activity harbored within the MDM2 RING finger domain. We have discovered that MTBP promotes MDM2-mediated ubiquitination and degradation of p53 and also MDM2 stabilization in an MDM2 RING finger-dependent manner. Moreover, using small interfering RNA to down-regulate endogenous MTBP in unstressed cells, we have found that MTBP significantly contributes to MDM2-mediated regulation of p53 levels and activity. However, following exposure of cells to UV, but not γ-irradiation, MTBP is destabilized as part of the coordinated cellular response. Our findings suggest that MTBP differentially regulates the E3 ubiquitin ligase activity of MDM2 towards two of its most critical targets (itself and p53) and in doing so significantly contributes to MDM2-dependent p53 homeostasis in unstressed cells.

scholarly journals Disrupted Calcium Signaling in Animal Models of Human Spinocerebellar Ataxia (SCA)

2019 ◽  
Vol 21 (1) ◽  
pp. 216 ◽  
Author(s):  
Francesca Prestori ◽  
Francesco Moccia ◽  
Egidio D’Angelo
Keyword(s):  
Animal Models ◽  
Purkinje Cell ◽  
Calcium Signaling ◽  
Purkinje Cells ◽  
Motor Coordination ◽  
Cerebellar Atrophy ◽  
Cerebellar Nuclei ◽  
Spinocerebellar Ataxias ◽  
Cell Degeneration ◽  
Cerebellar Purkinje Cells

Spinocerebellar ataxias (SCAs) constitute a heterogeneous group of more than 40 autosomal-dominant genetic and neurodegenerative diseases characterized by loss of balance and motor coordination due to dysfunction of the cerebellum and its efferent connections. Despite a well-described clinical and pathological phenotype, the molecular and cellular events that underlie neurodegeneration are still poorly undaerstood. Emerging research suggests that mutations in SCA genes cause disruptions in multiple cellular pathways but the characteristic SCA pathogenesis does not begin until calcium signaling pathways are disrupted in cerebellar Purkinje cells. Ca2+ signaling in Purkinje cells is important for normal cellular function as these neurons express a variety of Ca2+ channels, Ca2+-dependent kinases and phosphatases, and Ca2+-binding proteins to tightly maintain Ca2+ homeostasis and regulate physiological Ca2+-dependent processes. Abnormal Ca2+ levels can activate toxic cascades leading to characteristic death of Purkinje cells, cerebellar atrophy, and ataxia that occur in many SCAs. The output of the cerebellar cortex is conveyed to the deep cerebellar nuclei (DCN) by Purkinje cells via inhibitory signals; thus, Purkinje cell dysfunction or degeneration would partially or completely impair the cerebellar output in SCAs. In the absence of the inhibitory signal emanating from Purkinje cells, DCN will become more excitable, thereby affecting the motor areas receiving DCN input and resulting in uncoordinated movements. An outstanding advantage in studying the pathogenesis of SCAs is represented by the availability of a large number of animal models which mimic the phenotype observed in humans. By mainly focusing on mouse models displaying mutations or deletions in genes which encode for Ca2+ signaling-related proteins, in this review we will discuss the several pathogenic mechanisms related to deranged Ca2+ homeostasis that leads to significant Purkinje cell degeneration and dysfunction.

The Ubiquitin-Proteasome Pathway and Its Role in Cancer

2005 ◽  
Vol 23 (21) ◽  
pp. 4776-4789 ◽  
Author(s):  
Aparna Mani ◽  
Edward P. Gelmann
Keyword(s):  
Cell Cycle ◽  
Protein Degradation ◽  
Ubiquitin Ligase ◽  
De Novo ◽  
Cell Cancer ◽  
26S Proteasome ◽  
Great Promise ◽  
Major Pathway ◽  
Cellular Processes ◽  
Wide Range

Critical cellular processes are regulated, in part, by maintaining the appropriate intracellular levels of proteins. Whereas de novo protein synthesis is a comparatively slow process, proteins are rapidly degraded at a rate compatible with the control of cell cycle transitions and cell death induction. A major pathway for protein degradation is initiated by the addition of multiple 76–amino acid ubiquitin monomers via a three-step process of ubiquitin activation and substrate recognition. Polyubiquitination targets proteins for recognition and processing by the 26S proteasome, a cylindrical organelle that recognizes ubiquitinated proteins, degrades the proteins, and recycles ubiquitin. The critical roles played by ubiquitin-mediated protein turnover in cell cycle regulation makes this process a target for oncogenic mutations. Oncogenes of several common malignancies, for example colon and renal cell cancer, code for ubiquitin ligase components. Cervical oncogenesis by human papillomavirus is also mediated by alteration of ubiquitin ligase pathways. Protein degradation pathways are also targets for cancer therapy, as shown by the successful introduction of bortezomib, an inhibitor of the 26S proteasome. Further work in this area holds great promise toward our understanding and treatment of a wide range of cancers.

scholarly journals Angelman syndrome–associated point mutations in the Zn2+-binding N-terminal (AZUL) domain of UBE3A ubiquitin ligase inhibit binding to the proteasome

2018 ◽  
Vol 293 (47) ◽  
pp. 18387-18399 ◽  
Author(s):  
Simone Kühnle ◽  
Gustavo Martínez-Noël ◽  
Flavien Leclere ◽  
Sebastian D. Hayes ◽  
J. Wade Harper ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Angelman Syndrome ◽  
Protein Complexes ◽  
Point Mutations ◽  
Estrogen Receptor Α ◽  
Autism Spectrum ◽  
26S Proteasome ◽  
Interacting Protein ◽  
Functional Defect ◽  
Spectrum Disorders

Deregulation of the HECT ubiquitin ligase UBE3A/E6AP has been implicated in Angelman syndrome as well as autism spectrum disorders. We and others have previously identified the 26S proteasome as one of the major UBE3A-interacting protein complexes. Here, we characterize the interaction of UBE3A and the proteasomal subunit PSMD4 (Rpn10/S5a). We map the interaction to the highly conserved Zn2+-binding N-terminal (AZUL) domain of UBE3A, the integrity of which is crucial for binding to PSMD4. Interestingly, two Angelman syndrome point mutations that affect the AZUL domain show an impaired ability to bind PSMD4. Although not affecting the ubiquitin ligase or the estrogen receptor α–mediated transcriptional regulation activities, these AZUL domain mutations prevent UBE3A from stimulating the Wnt/β-catenin signaling pathway. Taken together, our data indicate that impaired binding to the 26S proteasome and consequential deregulation of Wnt/β-catenin signaling might contribute to the functional defect of these mutants in Angelman syndrome.

scholarly journals TRIM22 E3 ubiquitin ligase activity is required to mediate antiviral activity against encephalomyocarditis virus

2009 ◽  
Vol 90 (3) ◽  
pp. 536-545 ◽  
Author(s):  
Patrick Eldin ◽  
Laura Papon ◽  
Alexandra Oteiza ◽  
Emiliana Brocchi ◽  
T. Glen Lawson ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Adaptive Immune Response ◽  
Antiviral Effect ◽  
Encephalomyocarditis Virus ◽  
Target Cells ◽  
Virus Infections ◽  
Cellular Genes ◽  
Ubiquitin Ligase Activity ◽  
Wide Range

The interferon (IFN) system is a major effector of the innate immunity that allows time for the subsequent establishment of an adaptive immune response against a wide-range of pathogens. Their diverse biological actions are thought to be mediated by the products of specific but usually overlapping sets of cellular genes induced in the target cells. Ubiquitin ligase members of the tripartite motif (TRIM) protein family have emerged as IFN-induced proteins involved in both innate and adaptive immunity. In this report, we provide evidence that TRIM22 is a functional E3 ubiquitin ligase that is also ubiquitinated itself. We demonstrate that TRIM22 expression leads to a viral protection of HeLa cells against encephalomyocarditis virus infections. This effect is dependent upon its E3 ubiquitinating activity, since no antiviral effect was observed in cells expressing a TRIM22-deletion mutant defective in ubiquitinating activity. Consistent with this, TRIM22 interacts with the viral 3C protease (3C PRO ) and mediates its ubiquitination. Altogether, our findings demonstrate that TRIM22 E3 ubiquitin ligase activity represents a new antiviral pathway induced by IFN against picornaviruses.

scholarly journals The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis.

1995 ◽  
Vol 6 (2) ◽  
pp. 185-197 ◽  
Author(s):  
V Sudakin ◽  
D Ganoth ◽  
A Dahan ◽  
H Heller ◽  
J Hershko ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Embryonic Cell ◽  
26S Proteasome ◽  
Protein Activity ◽  
Cell Cycles ◽  
Ubiquitin Ligase Activity ◽  
M Phase ◽  
Ubiquitin Ligase E3 ◽  
Mitotic Cyclin

The ubiquitin-mediated degradation of mitotic cyclins is required for cells to exit from mitosis. Previous work with cell-free systems has revealed four components required for cyclin-ubiquitin ligation and proteolysis: a nonspecific ubiquitin-activating enzyme E1, a soluble fraction containing a ubiquitin carrier protein activity called E2-C, a crude particulate fraction containing a ubiquitin ligase (E3) activity that is activated during M-phase, and a constitutively active 26S proteasome that degrades ubiquitinated proteins. Here, we identify a novel approximately 1500-kDa complex, termed the cyclosome, which contains a cyclin-selective ubiquitin ligase activity, E3-C. E3-C is present but inactive during interphase; it can be activated in vitro by the addition of cdc2, enabling the transfer of ubiquitin from E2-C to cyclin. The kinetics of E3-C activation suggest the existence of one or more intermediates between cdc2 and E3-C. Cyclosome-associated E3-C acts on both cyclin A and B, and requires the presence of wild-type N-terminal destruction box motifs in each cyclin. Ubiquitinated cyclins are then rapidly recognized and degraded by the proteasome. These results identify the cyclosome-associated E3-C as the component of the cyclin destruction machinery whose activity is ultimately regulated by cdc2 and, as such, the element directly responsible for setting mitotic cyclin levels during early embryonic cell cycles.

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