Reconstitution defines the roles of p62, NBR1 and TAX1BP1 in ubiquitin condensate formation and autophagy initiation.
Ontology highlight
ABSTRACT:
SUBMITTER: Turco E
PROVIDER: S-EPMC8410870 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The striated muscle-specific tripartite motif (TRIM) proteins TRIM63/MURF1, TRIM55/MURF2 and TRIM54/MURF3 can function as ubiquitin E3 ligases in ubiquitin-mediated muscle protein turnover. Despite their well-characterised roles in muscle atrophy, the dynamics of MURF expression in the development and early postnatal adaptation of striated muscle is largely unknown. Here, we show that MURF2 is expressed at the very onset of mouse cardiac differentiation at embryonic day 8.5, and represents a sensitive marker for differentiating myocardium. During cardiac development, expression shifts from the 50 kDa to the 60 kDa A-isoform, which dominates postnatally. In contrast, MURF1 shows strong postnatal upregulation and MURF3 is not significantly expressed before birth. MURF2 expression parallels that of the autophagy-associated proteins LC3, p62/SQSTM1 and nbr1. SiRNA knockdown of MURF2 in neonatal rat cardiomyocytes disrupts posttranslational microtubule modification and myofibril assembly, and is only partly compensated by upregulation of MURF3 but not MURF1. Knockdown of both MURF2 and MURF3 severely disrupts the formation of ordered Z- and M-bands, likely by perturbed tubulin dynamics. These results suggest that ubiquitin-mediated protein turnover and MURF2 in particular play an unrecognised role in the earliest steps of heart muscle differentiation, and that partial complementation of MURF2 deficiency is afforded by MURF3.
| S-EPMC3047806 | biostudies-literature
Project description:(Macro)autophagy encompasses both an unselective, bulk degradation of cytoplasmic contents as well as selective autophagy of damaged organelles, intracellular microbes, protein aggregates, cellular structures and specific soluble proteins. Selective autophagy is mediated by autophagic adapters, like p62/SQSTM1 and NBR1. p62 and NBR1 are themselves selective autophagy substrates, but they also act as cargo receptors for degradation of other substrates. Surprisingly, we found that homologs of NBR1 are distributed throughout the eukaryotic kingdom, while p62 is confined to the metazoans. As a representative of all organisms having only an NBR1 homolog we studied Arabidopsis thaliana NBR1 (AtNBR1) in more detail. AtNBR1 is more similar to mammalian NBR1 than to p62 in domain architecture and amino acid sequence. However, similar to p62, AtNBR1 homo-polymerizes via the PB1 domain. Hence, AtNBR1 has hybrid properties of mammalian NBR1 and p62. AtNBR1 has 2 UBA domains, but only the C-terminal UBA domain bound ubiquitin. AtNBR1 bound AtATG8 through a conserved LIR (LC3-interacting region) motif and required co-expression of AtATG8 or human GABARAPL2 to be recognized as an autophagic substrate in HeLa cells. To monitor the autophagic sequestration of AtNBR1 in Arabidopsis we made transgenic plants expressing AtNBR1 fused to a pH-sensitive fluorescent tag, a tandem fusion of the red, acid-insensitive mCherry and the acid-sensitive yellow fluorescent proteins. This strategy allowed us to show that AtNBR1 is an autophagy substrate degraded in the vacuole dependent on the polymerization property of the PB1 domain and of expression of AtATG7. A functional LIR was required for vacuolar import.
| S-EPMC3210314 | biostudies-literature
Project description:p62/SQSTM1 is a multivalent protein that has the ability to cause liquid-liquid phase separation and serves as a receptor protein that participates in cargo isolation during selective autophagy. This protein is also involved in the non-canonical activation of the Keap1-Nrf2 system, a major oxidative stress response pathway. Here, we show a role of neighbor of BRCA1 gene 1 (NBR1), an autophagy receptor structurally similar to p62/SQSTM1, in p62-liquid droplet formation and Keap1-Nrf2 pathway activation. Overexpression of NBR1 blocks selective degradation of p62/SQSTM1 through autophagy and promotes the accumulation and phosphorylation of p62/SQSTM1 in liquid-like bodies, which is required for the activation of Nrf2. NBR1 is induced in response to oxidative stress, which triggers p62-mediated Nrf2 activation. Conversely, loss of Nbr1 suppresses not only the formation of p62/SQSTM1-liquid droplets, but also of p62-dependent Nrf2 activation during oxidative stress. Taken together, our results show that NBR1 mediates p62/SQSTM1-liquid droplet formation to activate the Keap1-Nrf2 pathway.
| S-EPMC7054683 | biostudies-literature
Project description:NBR1 (neighbor of BRCA1 gene 1) is a protein commonly found in ubiquitin-positive inclusions in neurodegenerative diseases. Due to its high architectural similarity to the well studied autophagy receptor protein p62/SQSTM1, NBR1 has been thought to analogously bind to ubiquitin-marked autophagic substrates via its C-terminal ubiquitin-associated (UBA) domain and deliver them to autophagosomes for degradation. Unexpectedly, we find that NBR1 differs from p62 in its UBA structure and accordingly in its interaction with ubiquitin. Structural differences are observed on helix ?-3, which is tilted farther from helix ?-2 and extended by approximately one turn in NBR1. This results not only in inhibition of a p62-type self-dimerization of NBR1 UBA but also in a significantly higher affinity for monoubiquitin as compared with p62 UBA. Importantly, the NBR1 UBA-ubiquitin complex structure shows that the negative charge of the side chain in front of the conserved MGF motif in the UBA plays an integral role in the recognition of ubiquitin. In addition, NMR and isothermal titration calorimetry experiments show that NBR1 UBA binds to each monomeric unit of polyubiquitin with similar affinity and by the same surface used for binding to monoubiquitin. This indicates that NBR1 lacks polyubiquitin linkage-type specificity, in good agreement with the nonspecific linkages observed in intracellular ubiquitin-positive inclusions. Consequently, our results demonstrate that the structural differences between NBR1 UBA and p62 UBA result in a much higher affinity of NBR1 for ubiquitin, which in turn suggests that NBR1 may form intracellular inclusions with ubiquitylated autophagic substrates more efficiently than p62.
| S-EPMC4022861 | biostudies-literature
Project description:The ubiquitin-proteasome system and autophagy are the two main proteolytic systems involved in, among other functions, the maintenance of cell integrity by eliminating misfolded and damaged proteins and organelles. Both systems remove their targets after their conjugation with ubiquitin. An interesting, yet incompletely understood problem relates to the fate of the components of the two systems. Here we provide evidence that amino acid starvation enhances polyubiquitination on specific sites of the proteasome, a modification essential for its targeting to the autophagic machinery. The uptake of the ubiquitinated proteasome is mediated by its interaction with the ubiquitin-associated domain of p62/SQSTM1, a process that also requires interaction with LC3. Importantly, deletion of the PB1 domain of p62, which is important for the targeting of ubiquitinated substrates to the proteasome, has no effect on stress-induced autophagy of this proteolytic machinery, suggesting that the domain of p62 that binds to the proteasome determines the function of p62 in either targeting substrates to the proteasome or targeting the proteasome to autophagy.
| S-EPMC5127335 | biostudies-literature
Project description:Ubiquitination, the post-translational modification essential for various intracellular processes, is implicated in multiple aspects of autophagy, the major lysosome/vacuole-dependent degradation pathway. The autophagy machinery adopted the structural architecture of ubiquitin and employs two ubiquitin-like protein conjugation systems for autophagosome biogenesis. Ubiquitin chains that are attached as labels to protein aggregates or subcellular organelles confer selectivity, allowing autophagy receptors to simultaneously bind ubiquitinated cargos and autophagy-specific ubiquitin-like modifiers (Atg8-family proteins). Moreover, there is tremendous crosstalk between autophagy and the ubiquitin-proteasome system. Ubiquitination of autophagy-related proteins or regulatory components plays significant roles in the precise control of the autophagy pathway. In this review, we summarize and discuss the molecular mechanisms and functions of ubiquitin and ubiquitination, in the process and regulation of autophagy.
| S-EPMC7564124 | biostudies-literature
Project description:BackgroundIdiopathic pulmonary fibrosis is a persistent disease of the lung interstitium for which there is no efficacious pharmacological therapy. Protodioscin, a steroidal saponin, possesses diverse pharmacological properties; however, its function in pulmonary fibrosis is yet to be established. Hence, in this investigation, it was attempted to figure out the anti-pulmonary fibrosis influences of protodioscin and its pharmacological properties related to oxidative stress.MethodsA mouse lung fibrosis model was generated using tracheal injections of bleomycin, followed by intraperitoneal injection of different concentrations of protodioscin, and the levels of oxidative stress and fibrosis were detected in the lungs. Multiple fibroblasts were treated with TGF-β to induce their transition to myofibroblasts. It was attempted to quantify myofibroblast markers' expression levels and reactive oxygen species levels as well as Nrf2 activation after co-incubation of TGF-β with fibroblasts and different concentrations of protodioscin. The influence of protodioscin on the expression and phosphorylation of p62, which is associated with Nrf2 activation, were detected, and p62 related genes were predicted by STRING database. The effects of Nrf2 inhibitor or silencing of the Nrf2, p62 and NBR1 genes, respectively, on the activation of Nrf2 by protodioscin were examined. The associations between p62, NBR1, and Keap1 in the activation of Nrf2 by protodioscin was demonstrated using a co-IP assay. Nrf2 inhibitor were used when protodioscin was treated in mice with pulmonary fibrosis and lung tissue fibrosis and oxidative stress levels were detected.ResultsIn vivo, protodioscin decreased the levels of fibrosis markers and oxidative stress markers and activated Nrf2 in mice with pulmonary fibrosis, and these effects were inhibited by Nrf2 inhibitor. In vitro, protodioscin decreased the levels of myofibroblast markers and oxidative stress markers during myofibroblast transition and promoted Nrf2 downstream gene expression, with reversal of these effects after Nrf2, p62 and NBR1 genes were silenced or Nrf2 inhibitors were used, respectively. Protodioscin promoted the binding of NBR1 to p62 and Keap1, thereby reducing Keap1-Nrf2 binding.ConclusionThe NBR1-p62-Nrf2 axis is targeted by protodioscin to reduce oxidative stress and inhibit pulmonary fibrosis.
| S-EPMC11003024 | biostudies-literature
Project description:Alterations in cellular ubiquitin (Ub) homeostasis, known as Ub stress, feature and affect cellular responses in multiple conditions, yet the underlying mechanisms are incompletely understood. Here we report that autophagy receptor p62/sequestosome-1 interacts with E2 Ub conjugating enzymes, UBE2D2 and UBE2D3. Endogenous p62 undergoes E2-dependent ubiquitylation during upregulation of Ub homeostasis, a condition termed as Ub+ stress, that is intrinsic to Ub overexpression, heat shock or prolonged proteasomal inhibition by bortezomib, a chemotherapeutic drug. Ubiquitylation of p62 disrupts dimerization of the UBA domain of p62, liberating its ability to recognize polyubiquitylated cargoes for selective autophagy. We further demonstrate that this mechanism might be critical for autophagy activation upon Ub+ stress conditions. Delineation of the mechanism and regulatory roles of p62 in sensing Ub stress and controlling selective autophagy could help to understand and modulate cellular responses to a variety of endogenous and environmental challenges, potentially opening a new avenue for the development of therapeutic strategies against autophagy-related maladies.
| S-EPMC5520855 | biostudies-literature
Project description:NBR1 was discovered as an autophagy receptor not long after the first described vertebrate autophagy receptor p62/SQSTM1. Since then, p62 has currently been mentioned in >10,000 papers on PubMed, while NBR1 is mentioned in <350 papers. Nonetheless, evolutionary analysis reveals that NBR1, and likely also selective autophagy, was present already in the last eukaryotic common ancestor (LECA), while p62 appears first in the early Metazoan lineage. Furthermore, yeast-selective autophagy receptors Atg19 and Atg34 represent NBR1 homologs. NBR1 is the main autophagy receptor in plants that do not contain p62, while most animal taxa contain both NBR1 and p62. Mechanistic studies are starting to shed light on the collaboration between mammalian NBR1 and p62 in the autophagic degradation of protein aggregates (aggrephagy). Several domains of NBR1 are involved in cargo recognition, and the list of known substrates for NBR1-mediated selective autophagy is increasing. Lastly, roles of NBR1 in human diseases such as proteinopathies and cancer are emerging.
| S-EPMC9582228 | biostudies-literature
Project description:Plant stress responses require both protective measures that reduce or restore stress-inflicted damage to cellular structures and mechanisms that efficiently remove damaged and toxic macromolecules, such as misfolded and damaged proteins. We have recently reported that NBR1, the first identified plant autophagy adaptor with a ubiquitin-association domain, plays a critical role in plant stress tolerance by targeting stress-induced, ubiquitinated protein aggregates for degradation by autophagy. Here we report a comprehensive genetic analysis of CHIP, a chaperone-associated E3 ubiquitin ligase from Arabidopsis thaliana implicated in mediating degradation of nonnative proteins by 26S proteasomes. We isolated two chip knockout mutants and discovered that they had the same phenotypes as the nbr1 mutants with compromised tolerance to heat, oxidative and salt stresses and increased accumulation of insoluble proteins under heat stress. To determine their functional interactions, we generated chip nbr1 double mutants and found them to be further compromised in stress tolerance and in clearance of stress-induced protein aggregates, indicating additive roles of CHIP and NBR1. Furthermore, stress-induced protein aggregates were still ubiquitinated in the chip mutants. Through proteomic profiling, we systemically identified heat-induced protein aggregates in the chip and nbr1 single and double mutants. These experiments revealed that highly aggregate-prone proteins such as Rubisco activase and catalases preferentially accumulated in the nbr1 mutant while a number of light-harvesting complex proteins accumulated at high levels in the chip mutant after a relatively short period of heat stress. With extended heat stress, aggregates for a large number of intracellular proteins accumulated in both chip and nbr1 mutants and, to a greater extent, in the chip nbr1 double mutant. Based on these results, we propose that CHIP and NBR1 mediate two distinct but complementary anti-proteotoxic pathways and protein's propensity to aggregate under stress conditions is one of the critical factors for pathway selection of protein degradation.
| S-EPMC3907298 | biostudies-literature