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 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 Mouse Receptor Interacting Protein 3 Does Not Contain a Caspase-Recruiting or a Death Domain but Induces Apoptosis and Activates NF-κB

1999 ◽  
Vol 19 (10) ◽  
pp. 6500-6508 ◽  
Author(s):  
Nanette J. Pazdernik ◽  
David B. Donner ◽  
Mark G. Goebl ◽  
Maureen A. Harrington
Keyword(s):  
Sequence Similarity ◽  
Kinase Domain ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Death Domain ◽  
Interacting Protein ◽  
C Terminus ◽  
Catalytically Active ◽  
Induced Apoptosis

ABSTRACT The death domain-containing receptor superfamily and their respective downstream mediators control whether or not cells initiate apoptosis or activate NF-κB, events critical for proper immune system function. A screen for upstream activators of NF-κB identified a novel serine-threonine kinase capable of activating NF-κB and inducing apoptosis. Based upon domain organization and sequence similarity, this novel kinase, named mRIP3 (mouse receptor interacting protein 3), appears to be a new RIP family member. RIP, RIP2, and mRIP3 contain an N-terminal kinase domain that share 30 to 40% homology. In contrast to the C-terminal death domain found in RIP or the C-terminal caspase-recruiting domain found in RIP2, the C-terminal tail of mRIP3 contains neither motif and is unique. Despite this feature, overexpression of the mRIP3 C terminus is sufficient to induce apoptosis, suggesting that mRIP3 uses a novel mechanism to induce death. mRIP3 also induced NF-κB activity which was inhibited by overexpression of either dominant-negative NIK or dominant-negative TRAF2. In vitro kinase assays demonstrate that mRIP3 is catalytically active and has autophosphorylation site(s) in the C-terminal domain, but the mRIP3 catalytic activity is not required for mRIP3 induced apoptosis and NF-κB activation. Unlike RIP and RIP2, mRIP3 mRNA is expressed in a subset of adult tissues and is thus likely to be a tissue-specific regulator of apoptosis and NF-κB activity. While the lack of a dominant-negative mutant precludes linking mRIP3 to a known upstream regulator, characterizing the expression pattern and the in vitro functions of mRIP3 provides insight into the mechanism(s) by which cells modulate the balance between survival and death in a cell-type-specific manner.

scholarly journals Diverse Effects of Mutations in Exon II of the von Hippel-Lindau (VHL) Tumor Suppressor Gene on the Interaction of pVHL with the Cytosolic Chaperonin and pVHL-Dependent Ubiquitin Ligase Activity

2002 ◽  
Vol 22 (6) ◽  
pp. 1947-1960 ◽  
Author(s):  
William J. Hansen ◽  
Michael Ohh ◽  
Javid Moslehi ◽  
Keiichi Kondo ◽  
William G. Kaelin ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Ubiquitin Ligase ◽  
Hypoxia Inducible Factor ◽  
Detectable Effect ◽  
Missense Mutations ◽  
Subsequent Formation ◽  
Von Hippel Lindau ◽  
Ubiquitin Ligase Activity

ABSTRACT We examined the biogenesis of the von Hippel-Lindau (VHL) tumor suppressor protein (pVHL) in vitro and in vivo. pVHL formed a complex with the cytosolic chaperonin containing TCP-1 (CCT or TRiC) en route to assembly with elongin B/C and the subsequent formation of the VCB-Cul2 ubiquitin ligase. Blocking the interaction of pVHL with elongin B/C resulted in accumulation of pVHL within the CCT complex. pVHL present in purified VHL-CCT complexes, when added to rabbit reticulocyte lysate, proceeded to form VCB and VCB-Cul2. Thus, CCT likely functions, at least in part, by retaining VHL chains pending the availability of elongin B/C for final folding and/or assembly. Tumor-associated mutations within exon II of the VHL syndrome had diverse effects upon the stability and/or function of pVHL-containing complexes. First, a pVHL mutant lacking the entire region encoded by exon II did not bind to CCT and yet could still assemble into complexes with elongin B/C and elongin B/C-Cul2. Second, a number of tumor-derived missense mutations in exon II did not decrease CCT binding, and most had no detectable effect upon VCB-Cul2 assembly. Many exon II mutants, however, were found to be defective in the binding to and subsequent ubiquitination of hypoxia-inducible factor 1α (HIF-1α), a substrate of the VCB-Cul2 ubiquitin ligase. We conclude that the selection pressure to mutate VHL exon II during tumorigenesis does not relate to loss of CCT binding but may reflect quantitative or qualitative defects in HIF binding and/or in pVHL-dependent ubiquitin ligase activity.

scholarly journals CHIP promotes Runx2 degradation and negatively regulates osteoblast differentiation

2008 ◽  
Vol 181 (6) ◽  
pp. 959-972 ◽  
Author(s):  
Xueni Li ◽  
Mei Huang ◽  
Huiling Zheng ◽  
Yinyin Wang ◽  
Fangli Ren ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Osteoblast Differentiation ◽  
Degradation Mechanism ◽  
Interacting Protein ◽  
E3 Ligases ◽  
Protein Levels ◽  
C Terminus ◽  
Osteoblast Lineage

Runx2, an essential transactivator for osteoblast differentiation, is tightly regulated at both the transcriptional and posttranslational levels. In this paper, we report that CHIP (C terminus of Hsc70-interacting protein)/STUB1 regulates Runx2 protein stability via a ubiquitination-degradation mechanism. CHIP interacts with Runx2 in vitro and in vivo. In the presence of increased Runx2 protein levels, CHIP expression decreases, whereas the expression of other E3 ligases involved in Runx2 degradation, such as Smurf1 or WWP1, remains constant or increases during osteoblast differentiation. Depletion of CHIP results in the stabilization of Runx2, enhances Runx2-mediated transcriptional activation, and promotes osteoblast differentiation in primary calvarial cells. In contrast, CHIP overexpression in preosteoblasts causes Runx2 degradation, inhibits osteoblast differentiation, and instead enhances adipogenesis. Our data suggest that negative regulation of the Runx2 protein by CHIP is critical in the commitment of precursor cells to differentiate into the osteoblast lineage.

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 The CUL1 C-Terminal Sequence and ROC1 Are Required for Efficient Nuclear Accumulation, NEDD8 Modification, and Ubiquitin Ligase Activity of CUL1

2000 ◽  
Vol 20 (21) ◽  
pp. 8185-8197 ◽  
Author(s):  
Manabu Furukawa ◽  
Yanping Zhang ◽  
Joseph McCarville ◽  
Tomohiko Ohta ◽  
Yue Xiong
Keyword(s):  
Nuclear Localization ◽  
Ubiquitin Ligase ◽  
Nuclear Import ◽  
Ring Finger ◽  
Nuclear Accumulation ◽  
Protein Families ◽  
Terminal Sequence ◽  
Ubiquitin Ligase Activity

ABSTRACT Members of the cullin and RING finger ROC protein families form heterodimeric complexes to constitute a potentially large number of distinct E3 ubiquitin ligases. We report here that the highly conserved C-terminal sequence in CUL1 is dually required, both for nuclear localization and for modification by NEDD8. Disruption of ROC1 binding impaired nuclear accumulation of CUL1 and decreased NEDD8 modification in vivo but had no effect on NEDD8 modification of CUL1 in vitro, suggesting that ROC1 promotes CUL1 nuclear accumulation to facilitate its NEDD8 modification. Disruption of NEDD8 binding had no effect on ROC1 binding, nor did it affect nuclear localization of CUL1, suggesting that nuclear localization and NEDD8 modification of CUL1 are two separable steps, with nuclear import preceding and required for NEDD8 modification. Disrupting NEDD8 modification diminishes the IκBα ubiquitin ligase activity of CUL1. These results identify a pathway for regulation of CUL1 activity—ROC1 and the CUL1 C-terminal sequence collaboratively mediate nuclear accumulation and NEDD8 modification, facilitating assembly of active CUL1 ubiquitin ligase. This pathway may be commonly utilized for the assembly of other cullin ligases.

scholarly journals Fat-regulated adaptor protein Dlish binds the growth suppressor Expanded and controls its stability and ubiquitination

2019 ◽  
Vol 116 (4) ◽  
pp. 1319-1324 ◽  
Author(s):  
Xing Wang ◽  
Yifei Zhang ◽  
Seth S. Blair
Keyword(s):  
Ubiquitin Ligase ◽  
Hippo Pathway ◽  
E3 Ubiquitin Ligase ◽  
Growth Control ◽  
Adaptor Protein ◽  
Domain Growth ◽  
C Terminus ◽  
The Hippo Pathway

The Drosophila protocadherin Fat controls organ size through the Hippo pathway, but the biochemical links to the Hippo pathway components are still poorly defined. We previously identified Dlish, an SH3 domain protein that physically interacts with Fat and the type XX myosin Dachs, and showed that Fat’s regulation of Dlish levels and activity helps limit Dachs-mediated inhibition of Hippo pathway activity. We here characterize a parallel growth control pathway downstream of Fat and Dlish. Using immunoprecipitation and mass spectrometry to search for Dlish partners, we find that Dlish binds the FERM domain growth repressor Expanded (Ex); Dlish SH3 domains directly bind sites in the Ex C terminus. We further show that, in vivo, Dlish reduces the subapical accumulation of Ex, and that loss of Dlish blocks the destabilization of Ex caused by loss of Fat. Moreover, Dlish can bind the F-box E3 ubiquitin ligase Slimb and promote Slimb-mediated ubiquitination of Expanded in vitro. Both the in vitro and in vivo effects of Dlish on Ex require Slimb, strongly suggesting that Dlish destabilizes Ex by helping recruit Slimb-containing E3 ubiquitin ligase complexes to Ex.

scholarly journals E3 Ubiquitin Ligase TRIP12: Regulation, Structure, and Physiopathological Functions

2020 ◽  
Vol 21 (22) ◽  
pp. 8515
Author(s):  
Manon Brunet ◽  
Claire Vargas ◽  
Dorian Larrieu ◽  
Jérôme Torrisani ◽  
Marlène Dufresne
Keyword(s):  
Thyroid Hormone ◽  
Ubiquitin Ligase ◽  
Hormone Receptor ◽  
Cell Cycle Progression ◽  
Thyroid Hormone Receptor ◽  
E3 Ubiquitin Ligase ◽  
Autism Spectrum ◽  
Causative Gene ◽  
Interacting Protein ◽  
C Terminus

The Thyroid hormone Receptor Interacting Protein 12 (TRIP12) protein belongs to the 28-member Homologous to the E6-AP C-Terminus (HECT) E3 ubiquitin ligase family. First described as an interactor of the thyroid hormone receptor, TRIP12’s biological importance was revealed by the embryonic lethality of a murine model bearing an inactivating mutation in the TRIP12 gene. Further studies showed the participation of TRIP12 in the regulation of major biological processes such as cell cycle progression, DNA damage repair, chromatin remodeling, and cell differentiation by an ubiquitination-mediated degradation of key protein substrates. Moreover, alterations of TRIP12 expression have been reported in cancers that can serve as predictive markers of therapeutic response. The TRIP12 gene is also referenced as a causative gene associated to intellectual disorders such as Clark–Baraitser syndrome and is clearly implicated in Autism Spectrum Disorder. The aim of the review is to provide an exhaustive and integrated overview of the different aspects of TRIP12 ranging from its regulation, molecular functions and physio-pathological implications.

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