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1.
Cell ; 156(6): 1167-1178, 2014 Mar 13.
Article in English | MEDLINE | ID: mdl-24630720

ABSTRACT

Aging entails a progressive decline in protein homeostasis, which often leads to age-related diseases. The endoplasmic reticulum (ER) is the site of protein synthesis and maturation for secreted and membrane proteins. Correct folding of ER proteins requires covalent attachment of N-linked glycan oligosaccharides. Here, we report that increased synthesis of N-glycan precursors in the hexosamine pathway improves ER protein homeostasis and extends lifespan in C. elegans. Addition of the N-glycan precursor N-acetylglucosamine to the growth medium slows aging in wild-type animals and alleviates pathology of distinct neurotoxic disease models. Our data suggest that reduced aggregation of metastable proteins and lifespan extension depend on enhanced ER-associated protein degradation, proteasomal activity, and autophagy. Evidently, hexosamine pathway activation or N-acetylglucosamine supplementation induces distinct protein quality control mechanisms, which may allow therapeutic intervention against age-related and proteotoxic diseases.


Subject(s)
Biosynthetic Pathways , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/metabolism , Hexosamines/metabolism , Longevity , Proteins/metabolism , Amino Acid Sequence , Animals , Autophagy , Caenorhabditis elegans/enzymology , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress , Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/genetics , Humans , Molecular Sequence Data , Mutation , Protein Biosynthesis , Sequence Alignment , Tunicamycin/pharmacology
2.
J Cell Sci ; 130(19): 3322-3335, 2017 Oct 01.
Article in English | MEDLINE | ID: mdl-28827405

ABSTRACT

The mammalian ubiquitin ligase Hrd1 is the central component of a complex facilitating degradation of misfolded proteins during the ubiquitin-proteasome-dependent process of ER-associated degradation (ERAD). Hrd1 associates with cofactors to execute ERAD, but their roles and how they assemble with Hrd1 are not well understood. Here, we identify crucial cofactor interaction domains within Hrd1 and report a previously unrecognised evolutionarily conserved segment within the intrinsically disordered cytoplasmic domain of Hrd1 (termed the HAF-H domain), which engages complementary segments in the cofactors FAM8A1 and Herp (also known as HERPUD1). This domain is required by Hrd1 to interact with both FAM8A1 and Herp, as well as to assemble higher-order Hrd1 complexes. FAM8A1 enhances binding of Herp to Hrd1, an interaction that is required for ERAD. Our findings support a model of Hrd1 complex formation, where the Hrd1 cytoplasmic domain and FAM8A1 have a central role in the assembly and activity of this ERAD machinery.


Subject(s)
Endoplasmic Reticulum-Associated Degradation/physiology , Membrane Proteins/metabolism , Models, Biological , Ubiquitin-Protein Ligases/metabolism , HEK293 Cells , Humans , Membrane Proteins/genetics , Ubiquitin-Protein Ligases/genetics
3.
Proc Natl Acad Sci U S A ; 111(22): E2271-80, 2014 Jun 03.
Article in English | MEDLINE | ID: mdl-24843123

ABSTRACT

The Mediator is a conserved transcriptional coregulator complex required for eukaryotic gene expression. In Caenorhabditis elegans, the Mediator subunit mdt-15 is essential for the expression of genes involved in fatty acid metabolism and ingestion-associated stress responses. mdt-15 loss of function causes defects in reproduction and mobility and shortens lifespan. In the present study, we find that worms with mutated or depleted mdt-15 (mdt-15 worms) exhibit decreased membrane phospholipid desaturation, especially in phosphatidylcholine. Accordingly, mdt-15 worms exhibit disturbed endoplasmic reticulum (ER) homeostasis, as indicated by a constitutively activated ER unfolded protein response (UPR(ER)). Activation of this stress response is only partially the consequence of reduced membrane lipid desaturation, implicating other mdt-15-regulated processes in maintaining ER homeostasis. Interestingly, mdt-15 inactivation or depletion of the lipid metabolism enzymes stearoyl-CoA-desaturases (SCD) and S-adenosyl methionine synthetase (sams-1) activates the UPR(ER) without promoting misfolded protein aggregates. Moreover, these worms exhibit wild-type sensitivity to chemically induced protein misfolding, and they do not display synthetic lethality with mutations in UPR(ER) genes, which cause protein misfolding. Therefore, the constitutively activated UPR(ER) in mdt-15, SCD, and sams-1 worms is not the consequence of proteotoxic stress but likely is the direct result of changes in ER membrane fluidity and composition. Together, our data suggest that the UPR(ER) is induced directly upon membrane disequilibrium and thus monitors altered ER homeostasis.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Endoplasmic Reticulum/metabolism , Proteostasis Deficiencies/metabolism , Transcription Factors/metabolism , Unfolded Protein Response/physiology , Acyl Coenzyme A/metabolism , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Cardiolipins/metabolism , Fatty Acids/metabolism , Homeostasis/physiology , Lipids/biosynthesis , Mitochondria/metabolism , Phosphatidylcholines/metabolism , Phosphatidylethanolamines/metabolism , Transcription Factors/genetics
4.
Nat Cell Biol ; 24(12): 1714-1725, 2022 12.
Article in English | MEDLINE | ID: mdl-36471127

ABSTRACT

The endoplasmic reticulum (ER) coordinates mRNA translation and processing of secreted and endomembrane proteins. ER-associated degradation (ERAD) prevents the accumulation of misfolded proteins in the ER, but the physiological regulation of this process remains poorly characterized. Here, in a genetic screen using an ERAD model substrate in Caenorhabditis elegans, we identified an anti-viral RNA interference pathway, referred to as ER-associated RNA silencing (ERAS), which acts together with ERAD to preserve ER homeostasis and function. Induced by ER stress, ERAS is mediated by the Argonaute protein RDE-1/AGO2, is conserved in mammals and promotes ER-associated RNA turnover. ERAS and ERAD are complementary, as simultaneous inactivation of both quality-control pathways leads to increased ER stress, reduced protein quality control and impaired intestinal integrity. Collectively, our findings indicate that ER homeostasis and organismal health are protected by synergistic functions of ERAS and ERAD.


Subject(s)
Endoplasmic Reticulum , RNA Interference , Endoplasmic Reticulum/genetics
6.
Genetics ; 193(3): 865-76, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23335331

ABSTRACT

Intraneuronal deposition of aggregated proteins in tauopathies, Parkinson disease, or familial encephalopathy with neuroserpin inclusion bodies (FENIB) leads to impaired protein homeostasis (proteostasis). FENIB represents a conformational dementia, caused by intraneuronal polymerization of mutant variants of the serine protease inhibitor neuroserpin. In contrast to the aggregation process, the kinetic relationship between neuronal proteostasis and aggregation are poorly understood. To address aggregate formation dynamics, we studied FENIB in Caenorhabditis elegans and mice. Point mutations causing FENIB also result in aggregation of the neuroserpin homolog SRP-2 most likely within the ER lumen in worms, recapitulating morphological and biochemical features of the human disease. Intriguingly, we identified conserved protein quality control pathways to modulate protein aggregation both in worms and mice. Specifically, downregulation of the unfolded protein response (UPR) pathways in the worm favors mutant SRP-2 accumulation, while mice overexpressing a polymerizing mutant of neuroserpin undergo transient induction of the UPR in young but not in aged mice. Thus, we find that perturbations of proteostasis through impairment of the heat shock response or altered UPR signaling enhance neuroserpin accumulation in vivo. Moreover, accumulation of neuroserpin polymers in mice is associated with an age-related induction of the UPR suggesting a novel interaction between aging and ER overload. These data suggest that targets aimed at increasing UPR capacity in neurons are valuable tools for therapeutic intervention.


Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/metabolism , Endoplasmic Reticulum Stress , Endoplasmic Reticulum/metabolism , Inclusion Bodies/metabolism , Neuropeptides/metabolism , Serpins/genetics , Serpins/metabolism , Age Factors , Amino Acid Sequence , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/metabolism , Heat-Shock Response , Hippocampus/metabolism , Hippocampus/pathology , Humans , Mice , Mice, Mutant Strains , Molecular Sequence Data , Neurons/metabolism , Neurons/pathology , Neuropeptides/chemistry , Neuropeptides/genetics , Point Mutation , Polymerization , Protein Conformation , Protein Folding , Serpins/chemistry , Signal Transduction , Unfolded Protein Response , Neuroserpin
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