Abstract UBR4 is an E3 ligase (E3) of the N-degron pathway and is involved in neurodevelopment, age-associated muscular atrophy and cancer progression. The location and mechanistic classification of the E3 module within the 600 kDa protein UBR4 remains unknown. Herein, we identify and characterize, at a biochemical and structural level, a distinct E3 module within human UBR4 consisting of a novel “hemiRING” zinc finger, a helical-rich UBR Zinc-finger Interacting (UZI) subdomain, and a predicted backside interacting N-terminal helix. A structure of an E2 conjugating enzyme (E2)-E3 complex provides atomic level insight into the exquisite specificity of the hemiRING towards the E2s UBE2A/B. The UZI subdomain can be considered a component of the E3 module as it has a modest activating effect on the ubiquitin loaded E2 (E2∼Ub), which is complemented by the intrinsically high lysine reactivity of UBE2A. These findings reveal the mechanistic underpinnings of a neuronal N-degron E3 ligase, its specific recruitment of UBE2A, and highlight the underappreciated architectural diversity of cross-brace domains associated with ubiquitin E3 activity.
Understanding the regulatory mechanisms mediating PRNP gene expression is highly relevant to elucidating normal cellular prion protein (PrP) function(s) and the transmissibility of prion protein neurodegenerative diseases. Here, luciferase reporter assays showed that an endoplasmic reticulum stress element (ERSE)-like element, CCAAT-N26-CCACG in the human PRNP promoter, is regulated by ER stress and X-box-binding protein 1 (XBP1) but not by activating transcription factor 6 α (ATF6α). Bioinformatics identified the ERSE-26 motif in 37 other human genes in the absence of canonical ERSE sites except for three genes. Several of these genes are associated with a synaptic function or are involved in oxidative stress. Brefeldin A, tunicamycin, and thapsigargin ER stressors induced gene expression of PRNP and four randomly chosen ERSE-26-containing genes, ERLEC1, GADD45B, SESN2, and SLC38A5, in primary human neuron cultures or in the breast carcinoma MCF-7 cell line, although the level of the response depends on the gene analyzed, the genetic background of the cells, the cell type, and the ER stressor. Overexpression of XBP1 increased, whereas siRNA knockdown of XBP1 considerably reduced, PRNP and ERLEC1 mRNA levels in MCF-7 cells. Taken together, these results identify a novel ER stress regulator, which implicates the ER stress response in previously unrecognized cellular functions.
Similar to many proteins trafficking through the secretory pathway, cellular prion protein (PrP) partly retrotranslocates from the endoplasmic reticulum to the cytosol through the endoplasmic reticulum-associated degradation (ERAD) pathway in an attempt to alleviate accumulation of cellular misfolded PrP. Surprisingly, familial PrP mutants fail to retrotranslocate and simultaneously block normal cellular PrP retrotranslocation. That impairments in retrotranslocation of misfolded proteins could lead to global disruptions in cellular homeostasis prompted further investigations into PrP mutant retrotranslocation defects. A gain- and loss-of-function approach identified human E3 ubiquitin ligase, Hrd1, as a critical regulator of PrP retrotranslocation in mammalian cells. Expression of familial human PrP mutants, V210I(129V) and M232R(129V), not only abolished PrP retrotranslocation, but also that of Hrd1-dependent ERAD substrates, transthyretin TTR(D18G) and α1-anti-trypsin A1AT(NHK). Mutant PrP expression decreased binding immunoglobulin protein (BiP) levels by 50% and attenuated ER stress-induced BiP by increasing BiP turnover 6-fold. Overexpression of BiP with PrP mutants rescued retrotranslocation of PrP, TTR(D18G) and A1AT(NHK). PrP mutants-induced cell death was also rescued by co-expression of BiP. These results show that PrP mutants highjack the Hrd1-dependent ERAD pathway, an action that would result in misfolded protein accumulation especially in terminally differentiated neurons. This could explain the age-dependent neuronal degeneration in familial prion diseases.
L’administration systemique d’atorvastatine s’est montree neuroprotective suivant un traumatisme medullaire, en diminuant la reponse inflammatoire au site de la lesion ainsi qu’en reduisant l’apoptose des oligodendrocytes. Ce dernier epargne la matiere blanche au site de l’insulte et ameliore la locomotion. Le but de cette etude etait de confirmer l’efficacite neuroprotective de l’atorvastatine ainsi que son action precoce, lorsqu’administre post-trauma, sur la limitation de l’apoptose. Des rats Sprague-Dawley femelles ont recu une injection intraperitoneale de : (1) statine/saline (5 mg/kg) 2 h apres une lesion contusive; (2) saline physiologique 2 h post-contusion; ou (3) saline physiologique sans lesion. Les rats traites a la statine ont montre une amelioration significative (p<0.05) de leur locomotion apres 4 semaines post-trauma, comparee au groupe « vehicule » lese. Expliquant cette observation, l’activite de la caspase-3 fut diminuee de 50% (p<0.05) et la methode de TUNEL revela une diminution d’approximativement 20% du nombre de cellules apoptotiques au site lesionnel (p<0.01) 4 h apres l’insulte contusive chez le groupe traite en comparaison aux groupes « vehicules ». Ces resultats demontrent que l’atorvastatine est efficace dans la prevention de l’apoptose precoce au site lesionnel dans un modele experimental de traumatisme medullaire apres seulement 2 h post-traumatisme.
Abstract The cellular prion protein (PrP) is essential for transmissible prion diseases, but its exact physiological function remains unclear. Better understanding the regulation of the human prion protein gene ( PRNP ) expression can provide insight into this elusive function. Spliced XBP1 (sXBP1) was recently shown to mediate endoplasmic reticulum (ER) stress-induced PRNP expression. In this manuscript, we identify Luman, a ubiquitous, non-canonical unfolded protein response (UPR), as a novel regulator of ER stress-induced PRNP expression. Luman activity was transcriptionally and proteolytically activated by the ER stressing drug brefeldin A (BFA) in human neurons, astrocytes, and breast cancer MCF-7 cells. Over-expression of active cleaved Luman (ΔLuman) increased PrP levels, while siRNA-mediated Luman silencing decreased BFA-induced PRNP expression. Site-directed mutagenesis and chromatin immunoprecipitation demonstrated that ΔLuman regulates PRNP expression by interacting with the ER stress response element 26 (ERSE26). Co-over-expression and siRNA-mediated silencing experiments showed that sXBP1 and ΔLuman both up-regulate ER stress-induced PRNP expression. Attempts to understand the function of PRNP up-regulation by Luman excluded a role in atorvastatin-induced neuritogenesis, ER-associated degradation, or proteasomal inhibition-induced cell death. Overall, these results refine our understanding of ER stress-induced PRNP expression and function.
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