The inherited blindness protein AIPL1 regulates the ubiquitin-like FAT10 pathway

Mutations in AIPL1 cause the inherited blindness Leber congenital amaurosis (LCA). AIPL1 has previously been shown to interact with NUB1, which facilitates the proteasomal degradation of proteins modified with the ubiquitin-like protein FAT10. Here we report that AIPL1 binds non-covalently to free FAT10 and FAT10ylated proteins and can form a ternary complex with FAT10 and NUB1. In addition, AIPL1 antagonised the NUB1-mediated degradation of the model FAT10 conjugate, FAT10-DHFR, and pathogenic mutations of AIPL1 were defective in inhibiting this degradation. While all AIPL1 mutants tested still bound FAT10-DHFR, there was a close correlation between the ability of the mutants to interact with NUB1 and their ability to prevent NUB1-mediated degradation. Interestingly, AIPL1 also co-immunoprecipitated the E1 activating enzyme for FAT10, UBA6, suggesting AIPL1 may have a role in directly regulating the FAT10 conjugation machinery. These studies are the first to implicate FAT10 in retinal cell biology and LCA pathogenesis, and reveal a new role of AIPL1 in regulating the FAT10 pathway

NF-κB regulates protein quality control after heat stress through modulation of the BAG3-HspB8 complex.

We previously found that the NF-κB transcription factor is activated during the recovery period after heat shock; moreover, we demonstrated that NF-κB is essential for cell survival after heat shock by activating autophagy, a mechanism that probably helps the cell to cope with hyperthermic stress through clearance of damaged proteins. In this study, we analyze the involvement of NF-κB in basal and heat-stress-induced protein quality control, by comparing the level of multiubiquitylated and/or aggregated proteins, and proteasome and autophagic activity in NF-κB-competent and NF-κB-incompetent cells. We show that NF-κB has only a minor role in basal protein quality control, where it modulates autophagosome maturation. By contrast, NF-κB is shown to be a key player in protein quality control after hyperthermia. Indeed, NF-κB-incompetent cells show highly increased levels of multiubiquitylated and/or aggregated proteins and aggresome clearance defects; a phenotype that disappears when NF-κB activity is restored to normal. We demonstrate that during heat shock recovery NF-κB activates selective removal of misfolded or aggregated proteins--a process also called 'aggrephagy'--by controlling the expression of BAG3 and HSPB8 and by modulating the level of the BAG3-HspB8 complex. Thus NF-κB-mediated increase in the level of the BAG3-HspB8 complex leads to upregulation of aggrephagy and clearance of irreversibly damaged proteins and might increase cell survival in conditions of hyperthermia

NUB1 modulation of GSK3β reduces tau aggregation

Abnormal phosphorylation of the microtubule-associated protein tau in neurodegenerative disorders, including Alzheimer’s disease (AD) and frontotemporal lobar degeneration, is associated with disrupted axonal transport and synaptic dysfunction ultimately manifesting as histopathological lesions of protein aggregates. Glycogen synthase kinase 3b (GSK3b) may be critical for the pathological hyperphosphorylation of tau. Here, we examined the role of the proteasome-associated protein Nedd8 ultimate buster 1 (NUB1) in the neuropathogenic phosphorylation and aggregation of tau. We reveal that NUB1 interacted with both tau and GSK3b to disrupt their interaction, and abolished recruitment of GSK3b to tau inclusions. Moreover, NUB1 reduced GSK3b-mediated phosphorylation of tau and aggregation of tau in intracellular inclusions. Strikingly, NUB1 induced GSK3b degradation. Deletion of the NUB1 ubiquitin-like (UBL) domain did not impair the interaction with tau and GSK3b, and the ability to suppress the phosphorylation and aggregation of tau was not affected. However, the UBL motif was necessary for GSK3b degradation. Deletion of the NUB1 ubiquitin-associated (UBA) domain abrogated the ability of NUB1 to interact with and degrade GSK3b. Moreover, the UBA domain was required to suppress the aggregation of tau. Silencing of NUB1 in cells stabilized endogenous GSK3b and exacerbated tau phosphorylation. Thus, we propose that NUB1, by regulating GSK3b levels, modulates tau phosphorylation and aggregation, and is a key player in neurodegeneration associated with tau pathology. Moreover, NUB1 regulation of GSK3b could modulate numerous signalling pathways in which GSK3b is a centrally important effector

NF kappa B is a central regulator of protein quality control in response to protein aggregation stresses via autophagy modulation

During cell life, proteins often misfold, depending on particular mutations or environmental changes, which may lead to protein aggregates that are toxic for the cell. Such protein aggregates are the root cause of numerous diseases called "protein conformational diseases," such as myofibrillar myopathy and familial amyotrophic lateral sclerosis. To fight against aggregates, cells are equipped with protein quality control mechanisms. Here we report that NF kappa B transcription factor is activated by misincorporation of amino acid analogues into proteins, inhibition of proteasomal activity, expression of the R120G mutated form of HspB5 (associated with myofibrillar myopathy), or expression of the G985R and G93A mutated forms of superoxide dismutase 1 (linked to familial amyotrophic lateral sclerosis). This noncanonical stimulation of NF kappa B triggers the up-regulation of BAG3 and HspB8 expression, two activators of selective autophagy, which relocalize to protein aggregates. Then NF kappa B-dependent autophagy allows the clearance of protein aggregates. Thus NF kappa B appears as a central and major regulator of protein aggregate clearance by modulating autophagic activity. In this context, the pharmacological stimulation of this quality control pathway might represent a valuable strategy for therapies against protein conformational diseases.Ligue contre le Cancer, comite du Rhone Ligue contre le Cancer, comite de Savoie Bonus Qualite Recherche from Universite Claude Bernard Lyon 1 Centre National de la Recherche Scientifique Millennium Institute P09-015-F FONDAP Program 15150012 French Department of research Fondation pour la Recherche Medicale Association Francaise contre les Myopathies/Teletho

Bacillus velezensis BE2 controls wheat and barley diseases by direct antagonism and induced systemic resistance

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AIPL1 interacts with NUB1 to block the degradation of FAT10-DHFR.

<p>Cells were transfected with the indicated constructs, then treated 24 hours later with cycloheximide (CHX) to block protein synthesis and assess degradation of FAT10-DHFR over the indicated times. (A) AIPL1 blocked the NUB1-mediated degradation of FAT10-DHFR, and the effect was stronger in the presence of MG132. (B) AIPL1 delayed the degradation of FAT10-DHFR in the presence of NUB1. The percentage of FAT10 remaining was measured from 3 independent experiments (n = 3), and the level of significance calculated using the Wilcoxon signed-rank test. (C) Pathogenic AIPL1 mutants A197P and C239R were defective in blocking FAT10-DHFR degradation, while the G262S mutant was able to block degradation. (D) NUB1 co-precipitated with both WT and G262S AIPL1, but not with the A197P or C239R mutants. (E) FAT10-DHFR co-precipitated with WT AIPL1, and the mutants A197P, C239R and G262S.</p

AIPL1 binds to FAT10 and FAT10-modified proteins.

<p>(A) Monomeric FAT10 co-precipitates with AIPL1 from transfected SK-N-SH cell lysates. Cells were transfected with the indicated constructs and immunoprecipitation was performed followed by immunoblot analysis. (B) Recombinant purified GST-AIPL1 but not GST can pull down free FAT10 and FAT10-conjugated proteins from HA-FAT10-transfected cell lysates. (C) Recombinant purified GST-AIPL1, but not GST, pulls down recombinant purified His6-FAT10. The position of molecular weight markers is indicated in kilodalton (kDa).</p

AIPL1, FAT10 and NUB1 form a ternary complex.

<p>Cells were transfected with constructs as indicated followed by immunoprecipitation and immunoblot analysis. (A) AIPL1 and FAT10 both co-precipitate with NUB1 (top panel); NUB1 and FAT10 both co-precipitate with AIPL1 (bottom panel). MG132 increased FAT10 steady-state levels and the amount of co-precipitated AIPL1 (top panel) or NUB1 (bottom panel). (B) NUB1 and AIPL1 both co-precipitate with FAT10. The AIPL1 C239R pathogenic mutant immunoprecipitated FAT10, but did not affect its NUB1-mediated degradation profile. (C) The AIPL1 C239R mutant did not immunoprecipitate NUB1 but FAT10 expression promoted their interaction. Heavy (h) and light (l) immunoglobulin chains are indicated. The position of molecular weight markers is indicated in kilodalton (kDa).</p

AIPL1 alters the NUB1-mediated degradation of FAT10.

<p>SK-N-SH Cells were transfected with NUB1-FLAG, HA-FAT10 and Myc-AIPL1 vectors in the presence and absence of the proteasome inhibitor MG132, as indicated. Cell lysates were harvested 24 hours post-transfection and immunoprecipitates were analyzed by immunoblotting to detect the protein indicated. (A) NUB1 interacts with FAT10 and accelerates the degradation of free FAT10 and FAT10-modified proteins. The change in levels of FAT10 was measured from 3 independent experiments (n = 3) of duplicate samples. Heavy (h) and light (l) immunoglobulin chains are indicated. (B) NUB1 co-precipitates with AIPL1. (C) AIPL1 enhances the steady-state levels of free FAT10 and FAT10 modified proteins, both alone and in the presence of NUB1. The change in levels of FAT10 was measured from 5 independent experiments (n = 5) of duplicate samples. (D) HA-FAT10 was visualised by immunocytochemical analysis with anti-HA and Cy2-conjugated secondary antibody. NUB1-mediated degradation of FAT10 is altered by the presence of AIPL1. Scale bar is 20 µM. (C) and (E) A small proportion of AIPL1 is itself covalently modified with FAT10, as detected by anti-HA, anti-AIPL1 and anti-FAT10 (rabbit polyclonal) antibodies. The percentage of AIPL1 modified by FAT10 was measured from 3 independent experiments (n = 3). Conjugation of FAT10 to AIPL1 is prevented using a FAT10 diglycine deletion mutant. The position of molecular weight markers is indicated in kilodalton (kDa).</p

The FAT10 E1 activating enzyme interacts with AIPL1.

<p>Cells were transfected as indicated and subject to immunoprecipitation and immunoblot analysis. (A) Expression of the FAT10 E1 activating enzyme UBA6 increases the amount of FAT10-conjugated proteins including the covalent AIPL1-FAT10 conjugate. AIPL1 altered the profile of UBA6-dependent FAT10 conjugation. (B) UBA6 co-precipitated with AIPL1. (C) Co-precipitation of UBA6 with AIPL1 is abrogated in the presence of FAT10. The position of molecular weight markers is indicated in kilodalton (kDa).</p