SIRT2 interferes with autophagy-mediated degradation of protein aggregates in neuronal cells under proteasome inhibition

Abnormal protein aggregates have been suggested as a common pathogenesis of many neurodegenerative diseases. Two well-known protein degradation pathways are responsible for protein homeostasis by balancing protein biosynthesis and degradative processes: the ubiquitin–proteasome system (UPS) and autophagy-lysosomal system. UPS serves as the primary route for degradation of short-lived proteins, but large-size protein aggregates cannot be degraded by UPS. Autophagy is a unique cellular process that facilitates degradation of bulky protein aggregates by lysosome. Recent studies have demonstrated that autophagy plays a crucial role in the pathogenesis of neurodegenerative diseases characterized by abnormal protein accumulation, suggesting that regulation of autophagy may be a valuable therapeutic strategy for the treatment of various neurodegenerative diseases. Sirtuin-2 (SIRT2) is a class III histone deacetylase that is expressed abundantly in aging brain tissue. Here, we report that SIRT2 increases protein accumulation in murine cholinergic SN56 cells and human neuroblastoma SH-SY5Y cells under proteasome inhibition. Overexpression of SIRT2 inhibits lysosome-mediated autophagic turnover by interfering with aggresome formation and also makes cells more vulnerable to accumulated protein-mediated cytotoxicity by MG132 and amyloid beta. Moreover, MG132-induced accumulation of ubiquitinated proteins and p62 as well as cytotoxicity are attenuated in siRNA-mediated SIRT2-silencing cells. Taken together, these results suggest that regulation of SIRT2 could be a good therapeutic target for a range of neurodegenerative diseases by regulating autophagic flux.     

DNA aptamers detecting generic amyloid epitopes

Amyloids are fibrillar protein aggregates resulting from non-covalent autocatalytic polymerization of various structurally and functionally unrelated proteins. Previously we have selected DNA aptamers, which bind specifically to the in vitro assembled amyloid fibrils of the yeast prionogenic protein Sup35. Here we show that such DNA aptamers can be used to detect SDS-insoluble amyloid aggregates of the Sup35 protein, and of some other amyloidogenic proteins, including mouse PrP, formed in yeast cells. The obtained data suggest that these aggregates and the Sup35 amyloid fibrils assembled in vitro possess common conformational epitopes recognizable by aptamers. The described DNA aptamers may be used for detection of various amyloid aggregates in yeast and, presumably, other organisms.     

p62 accumulates and enhances aggregate formation in model systems of familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurode-generative disease characterized by motor neuron death. A hallmark of the disease is the appearance of protein aggregates in the affected motor neurons. We have found that p62, a protein implicated in protein aggregate formation, accumulated progressively in the G93A mouse spinal cord. The accumulation of p62 was in parallel to the increase of polyubiquitinated proteins and mutant SOD1 aggregates. Immunostaining studies showed that p62, ubiquitin, and mutant SOD1 co-localized in the protein aggregates in affected cells in G93A mouse spinal cord. The p62 protein selectively interacted with familial ALS mutants, but not WT SOD1. When p62 was co-expressed with SOD1 in NSC34 cells, it greatly enhanced the formation of aggregates of the ALS-linked SOD1 mutants, but not wild-type SOD1. Cell viability was measured in the presence and absence of overexpressed p62, and the results suggest that the large aggregates facilitated by p62 were not directly toxic to cells under the conditions in this study. Deletion of the ubiquitin-association (UBA) domain of p62 significantly decreased the p62-facilitated aggregate formation, but did not completely inhibit it. Further protein interaction experiments also showed that the truncated p62 with the UBA domain deletion remained capable of interacting with mutant SOD1. The findings of this study show that p62 plays a critical role in forming protein aggregates in familial ALS, likely by linking misfolded mutant SOD1 molecules and other cellular proteins together.     

Functional interaction between sequestosome-1/p62 and autophagy-linked FYVE-containing protein WDFY3 in human osteoclasts

Paget’s disease of bone (PDB) is a late-onset disorder characterised by focal areas of increased bone resorption, with osteoclasts that are increased in size, multinuclearity, number and activity. PDB-causing missense and nonsense variants in the gene encoding Sequestosome-1/p62 (SQSTM1) have been identified, all of which cluster in and around the ubiquitin-associated (UBA) domain of the protein. SQSTM1 is ubiquitously expressed and there is, as yet, no clear reason why these mutations only appear to cause an osteoclast-related phenotype.Using co-immunoprecipitation and tandem mass spectrometry, we identified a novel interaction in human osteoclast-like cells between SQSTM1 and Autophagy-Linked FYVE domain-containing protein (ALFY/WDFY3). Endogenous ALFY and SQSTM1 both localised within the nuclei of osteoclasts and their mononuclear precursors. When osteoclasts were starved to induce autophagy, SQSTM1 and ALFY relocated to the cytoplasm where they formed large aggregates, with cytoplasmic relocalisation appearing more rapid in mature osteoclasts than in precursors in the same culture. Overexpression of wild-type SQSTM1 in HEK293 cells also resulted in the formation of cytoplasmic aggregates containing SQSTM1 and endogenous ALFY, as did overexpression of a PDB-causing missense mutant form of SQSTM1, indicating that this mutation does not impair the formation of SQSTM1- and ALFY-containing aggregates.Expression of ALFY in bone cells has not previously been reported, and the process of autophagy has not been studied with respect to osteoclast activity. We have identified a functional interaction between SQSTM1 and ALFY in osteoclasts under conditions of cell stress. The difference in response to starvation between mature osteoclasts and their precursors may begin to explain the cell-specific functional effects of SQSTM1 mutations in PDB.     

Anti-oligomeric single chain variable domain antibody differentially affects huntingtin and alpha-synuclein aggregates

Huntington's and Parkinson's diseases are both neurodegenerative disorders caused at least in part by misfolding and aggregation of huntingtin (htt) and alpha-synuclein, respectively. Here we use a single chain antibody fragment (scFv) isolated against oligomeric alpha-synuclein to probe similarities and differences between the aggregation and toxic mechanisms of htt and alpha-synuclein. When incubated with htt, the scFv both blocks formation of and promotes dissociation of fibrillar aggregates, but stabilizes formation of cytotoxic oligomeric aggregates. Previous studies with monomeric alpha-synuclein showed the scFv prevented fibrillar aggregation, but blocked toxicity of oligomeric aggregates. These divergent effects suggest the toxic mechanisms of oligomeric aggregates differ among amyloidogenic protein species.     

The DUB-ious lack of ALIS in Salmonella infection: A Salmonella deubiquitinase regulates the autophagy of protein aggregates

Ubiquitinated aggregates are formed in eukaryotic cells in response to several external stimuli, including exposure to bacterial lipopolysaccharide (LPS). Although Salmonella enterica serovar Typhimurium (S. Typhimurium) LPS has been shown to induce aggresome-like induced structures (ALIS) in macrophages, these have not been described in S. Typhimurium-infected macrophages. Given that LPS is present in infection, this suggests that S. Typhimurium might suppress the formation of ALIS. We found that S. Typhimurium induces the formation of ubiquitinated aggregates in epithelial cells and macrophages, but that their presence is masked by the deubiquitinase (DUB) activity of the S. Typhimurium virulence protein, SseL. SseL deubiquitinates SQSTM1/p62-bound proteins found in S. Typhimurium-induced aggregates and ALIS, and reduces the recruitment of autophagic components. While the functions of ALIS and other ubiquitinated aggregates remain unclear, they serve to sequester cytosolic proteins under a variety of stress conditions and are suggested to be involved in host immune defense. During infection, the deubiquitinase activity of SseL reduces autophagic flux in infected cells and favors bacterial replication. This is a new example of how a bacterial pathogen counteracts the autophagy pathway through the action of a translocated virulence protein.     

Cytotoxicity of insulin within its self-assembly and amyloidogenic pathways

Solvational perturbations were employed to selectively tune the aggregational preferences of insulin at 60 degrees C in vitro in purely aqueous acidic solution and in the presence of the model co-solvent ethanol (EtOH) (at 40%(w/w)). Dynamic light scattering (DLS), thioflavin T (ThT)-fluorescence, Fourier transform infrared (FTIR) and atomic force microscopy (AFM) techniques were employed to characterize these pathways biophysically with respect to the pre-aggregational assembly of the protein, the aggregation kinetics, and finally the aggregate secondary structure and morphology. Using cell viability assays, the results were subsequently correlated with the cytotoxicity of the insulin species that form in the two distinct aggregation pathways. In the cosolvent-free solution, predominantly dimeric insulin self-assembles via the well-known amyloidogenic pathway, yielding exclusively fibrillar aggregates, whereas in the solution containing EtOH, the aggregation of predominantly monomeric insulin proceeds via a pathway that leads to exclusively non-fibrillar, amorphous aggregates. Initially present native insulin assemblies as well as partially unfolded monomeric species and low molecular mass oligomeric aggregates could be ruled out as direct and major cytotoxic species. Apart from the slower overall aggregation kinetics under amorphous aggregate promoting conditions, which is due to the chaotropic nature of high EtOH concentrations, however, both pathways were unexpectedly found to evoke insulin aggregates that were cytotoxic to cultured rat insulinoma cells. The observed kinetics of the decrease of cell viabilities correlated well with the results of the DLS, ThT, FTIR and AFM studies, revealing that the formation of cytotoxic species correlated well with the formation of large-sized, beta-sheet-rich assemblies (>500 nm) of both fibrillar and amorphous nature. These results suggest that large-sized, beta-sheet-rich insulin assemblies of both fibrillar and amorphous nature are toxic to pancreatic beta-cells. In the light of the ongoing discussion about putative cytotoxic effects of prefibrillar and fibrillar amyloid aggregates, our results support the hypothesis that, in the case of insulin, factors other than the specific secondary or quarternary structural features of the various different aggregates may define their cytotoxic properties. Two such factors might be the aggregate size and the aggregate propensity to expose hydrophobic surfaces to a polar environment.     

Increased expression of p62 in expanded polyglutamine-expressing cells and its association with polyglutamine inclusions

Huntington's disease is a progressive neurodegenerative disorder that is associated with a CAG repeat expansion in the gene encoding huntingtin. We found that a 60-kDa protein was increased in Neuro2a cells expressing the N-terminal portion of huntingtin with expanded polyglutamine. We purified this protein, and, using mass spectrometry, identified it as p62, an ubiquitin-associated domain-containing protein. A specific p62 antibody stained the ubiquitylated polyQ inclusions in expanded polyglutamine-expressing cells, as well as in the brain of the huntingtin exon 1 transgenic mice. Furthermore, the level of p62 protein and mRNA was increased in expanded polyglutamine-expressing cells. We also found that p62 formed aggresome-like inclusions when p62 was increased in normal Neuro2a cells by a proteasome inhibitor. Knock-down of p62 does not affect the formation of aggresomes or polyglutamine inclusions, suggesting that p62 is recruited to the aggresome or inclusions secondary to their formation. These results suggest that p62 may play important roles as a responsive protein to a polyglutamine-induced stress rather than as a cross-linker between ubiquitylated proteins.     

Fibrillar aggregates of the tumor suppressor p53 core domain

Alzheimer's disease, Parkinson's disease, cystic fibrosis, prion diseases, and many types of cancer are considered to be protein conformation diseases. Most of them are also known as amyloidogenic diseases due to the occurrence of pathological accumulation of insoluble aggregates with fibrillar conformation. Some neuroblastomas, carcinomas, and myelomas show an abnormal accumulation of the wild-type tumor suppressor protein p53 either in the cytoplasm or in the nucleus of the cell. Here we show that the wild-type p53 core domain (p53C) can form fibrillar aggregates after mild perturbation. Gentle denaturation of p53C by pressure induces fibrillar aggregates, as shown by electron and atomic force microscopies, by binding of thioflavin T, and by circular dichroism. On the other hand, heat denaturation produced granular-shaped aggregates. Annular aggregates similar to those found in the early aggregation stages of alpha-synuclein and amyloid-beta were also observed by atomic force microscopy immediately after pressure treatment. Annular and fibrillar aggregates of p53C were toxic to cells, as shown by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction assay. Interestingly, the hot-spot mutant R248Q underwent similar aggregation behavior when perturbed by pressure or high temperature. Fibrillar aggregates of p53C contribute to the loss of function of p53 and seed the accumulation of conformationally altered protein in some cancerous cells.     

Tissue transglutaminase does not contribute to the formation of mutant huntingtin aggregates

The cause of Huntington's disease (HD) is a pathological expansion of the polyglutamine domain within the NH(2)-terminal region of huntingtin. Neuronal intranuclear inclusions and cytoplasmic aggregates composed of the mutant huntingtin within certain neuronal populations are a characteristic hallmark of HD. Because in vitro expanded polyglutamine repeats are glutaminyl-donor substrates of tissue transglutaminase (tTG), it has been hypothesized that tTG may contribute to the formation of these aggregates in HD. Therefore, it is of fundamental importance to establish whether tTG plays a significant role in the formation of mutant huntingtin aggregates in the cell. Human neuroblastoma SH-SY5Y cells were stably transfected with truncated NH(2)-terminal huntingtin constructs containing 18 (wild type) or 82 (mutant) glutamines. In the cells expressing the mutant truncated huntingtin construct, numerous SDS-resistant aggregates were present in the cytoplasm and nucleus. Even though numerous aggregates were present in the mutant huntingtin-expressing cells, tTG did not coprecipitate with mutant truncated huntingtin. Further, tTG was totally excluded from the aggregates, and significantly increasing tTG expression had no effect on the number of aggregates or their intracellular localization (cytoplasm or nucleus). When a YFP-tagged mutant truncated huntingtin construct was transiently transfected into cells that express no detectable tTG due to stable transfection with a tTG antisense construct, there was extensive aggregate formation. These findings clearly demonstrate that tTG is not required for aggregate formation, and does not facilitate the process of aggregate formation. Therefore, in HD, as well as in other polyglutamine diseases, tTG is unlikely to play a role in the formation of aggregates.     

Depletion of the Ubiquitin-binding Adaptor Molecule SQSTM1/p62 from Macrophages Harboring cftr ΔF508 Mutation Improves the Delivery of Burkholderia cenocepacia to the Autophagic Machinery

Cystic fibrosis is the most common inherited lethal disease in Caucasians. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), of which the cftr ΔF508 mutation is the most common. ΔF508 macrophages are intrinsically defective in autophagy because of the sequestration of essential autophagy molecules within unprocessed CFTR aggregates. Defective autophagy allows Burkholderia cenocepacia (B. cepacia) to survive and replicate in ΔF508 macrophages. Infection by B. cepacia poses a great risk to cystic fibrosis patients because it causes accelerated lung inflammation and, in some cases, a lethal necrotizing pneumonia. Autophagy is a cell survival mechanism whereby an autophagosome engulfs non-functional organelles and delivers them to the lysosome for degradation. The ubiquitin binding adaptor protein SQSTM1/p62 is required for the delivery of several ubiquitinated cargos to the autophagosome. In WT macrophages, p62 depletion and overexpression lead to increased and decreased bacterial intracellular survival, respectively. In contrast, depletion of p62 in ΔF508 macrophages results in decreased bacterial survival, whereas overexpression of p62 leads to increased B. cepacia intracellular growth. Interestingly, the depletion of p62 from ΔF508 macrophages results in the release of the autophagy molecule beclin1 (BECN1) from the mutant CFTR aggregates and allows its redistribution and recruitment to the B. cepacia vacuole, mediating the acquisition of the autophagy marker LC3 and bacterial clearance via autophagy. These data demonstrate that p62 differentially dictates the fate of B. cepacia infection in WT and ΔF508 macrophages.     

Linker histone H1 binds to disease associated amyloid-like fibrils

Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most prevalent neurodegenerative diseases of the central nervous system. These two diseases share a common feature in that a normally soluble peptide (amyloid-beta) or protein (alpha-synuclein) aggregates into an ordered fibrillar structure. As well as structural similarities observed between fibrillar aggregates related to these diseases, common pathological processes of increased oxidative injury, excitotoxicity and altered cell cycle are also evident. It was the aim of this study to identify novel interacting proteins to the amyloid-like motif and therefore identify common potential pathways between neurodegenerative diseases that share biophysical properties common to classical amyloid fibrils. Optimal ageing of recombinant proteins to form amyloid-like fibrils was determined by electron microscopy, Congo red birefringement and photo-induced cross-linking. Using pull-down assays the strongest detected interacting protein to the amyloid-like motifs of amyloid-beta, alpha-synuclein and lysozyme was identified as histone H1. The interaction with the amyloid-like motif was confirmed by techniques including surface plasmon resonance and immunohistochemistry. Histone H1 is known to be an integral part of chromatin within the nucleus, with a primary role of binding DNA that enters and exits from the nucleosome, and facilitating the shift in equilibrium of chromatin towards a more condensed form. However, phosphorylated histone H1 is predominantly present in the cytoplasm and as yet the functional significance of this translocation is unknown. This study also found that histone H1 is localised within the cytoplasm of neurons and astrocytes from areas affected by disease as well as amyloid plaques, supporting the hypothesis that histone H1 favoured binding to an ordered fibrillar motif. We conclude that the binding of histone H1 to a general amyloid-like motif indicates that histone H1 may play an important common role in diseases associated with amyloid-like fibrils.     

Fighting disease by selective autophagy of aggregate-prone proteins

Ubiquitinated protein aggregates are hallmarks of a range of human diseases, including neurodegenerative, liver and muscle disorders. These protein aggregates are typically positive for the autophagy receptor p62. Whereas the ubiquitin-proteasome system (UPS) degrades shortlived and misfolded ubiquitinated proteins that are small enough to enter the narrow pore of the barrel-shaped proteasome, the lysosomal pathway of autophagy can degrade larger structures including entire organelles or protein aggregates. This degradation requires autophagy receptors that link the cargo with the molecular machinery of autophagy and is enhanced by certain posttranslational modifications of the cargo. In this review we focus on how autophagy clears aggregate-prone proteins and the relevance of this process to protein aggregate associated diseases.     

Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice

Inactivation of constitutive autophagy results in formation of cytoplasmic protein inclusions and leads to liver injury and neurodegeneration, but the details of abnormalities related to impaired autophagy are largely unknown. Here we used mouse genetic analyses to define the roles of autophagy in the aforementioned events. We report that the ubiquitin- and LC3-binding protein "p62" regulates the formation of protein aggregates and is removed by autophagy. Thus, genetic ablation of p62 suppressed the appearance of ubiquitin-positive protein aggregates in hepatocytes and neurons, indicating that p62 plays an important role in inclusion body formation. Moreover, loss of p62 markedly attenuated liver injury caused by autophagy deficiency, whereas it had little effect on neuronal degeneration. Our findings highlight the unexpected role of homeostatic level of p62, which is regulated by autophagy, in controlling intracellular inclusion body formation, and indicate that the pathologic process associated with autophagic deficiency is cell-type specific.     

TRIM16 controls assembly and degradation of protein aggregates by modulating the p62‐NRF2 axis and autophagy

Sequestration of protein aggregates in inclusion bodies and their subsequent degradation prevents proteostasis imbalance, cytotoxicity, and proteinopathies. The underlying molecular mechanisms controlling the turnover of protein aggregates are mostly uncharacterized. Herein, we show that a TRIM family protein, TRIM16, governs the process of stress‐induced biogenesis and degradation of protein aggregates. TRIM16 facilitates protein aggregate formation by positively regulating the p62‐NRF2 axis. We show that TRIM16 is an integral part of the p62‐KEAP1‐NRF2 complex and utilizes multiple mechanisms for stabilizing NRF2. Under oxidative and proteotoxic stress conditions, TRIM16 activates ubiquitin pathway genes and p62 via NRF2, leading to ubiquitination of misfolded proteins and formation of protein aggregates. We further show that TRIM16 acts as a scaffold protein and, by interacting with p62, ULK1, ATG16L1, and LC3B, facilitates autophagic degradation of protein aggregates. Thus, TRIM16 streamlines the process of stress‐induced aggregate clearance and protects cells against oxidative/proteotoxic stress‐induced toxicity in vitro and in vivo. Taken together, this work identifies a new mechanism of protein aggregate turnover, which could be relevant in protein aggregation‐associated diseases such as neurodegeneration.     

The N-terminal domain of the mammalian nucleoporin p62 interacts with other nucleoporins of the FXFG family during interphase

Nuclear pore complexes (NPCs) provide the only sites for macromolecular transport between nucleus and cytoplasm. The nucleoporin p62, a component of higher eukaryotic NPCs, is located at the central gated channel and involved in nuclear trafficking of various cargos. p62 is organized into an N-terminal segment that contains FXFG repeats and binds the soluble transport factor NTF2, whereas the C-terminal portion associates with other nucleoporins and importin-beta1. We have now identified new components that interact specifically with the p62 N-terminal domain. Using the p62 N-terminal segment as bait, we affinity-purified nucleoporins Nup358, Nup214 and Nup153 from crude cell extracts. In ligand binding assays, the N-terminal p62 segment associated with Nup358 and p62, suggesting their direct binding to the p62 N-terminal portion. Furthermore, p62 was isolated in complex with Nup358, Nup214 and Nup153 from growing HeLa cells, indicating that the interactions Nup358/p62, Nup214/p62 and p62/Nup153 also occur in vivo. The formation of Nup358/p62 and p62/Nup153 complexes was restricted to interphase cells, whereas Nup214/p62 binding was detected in interphase as well as during mitosis. Our results support a model of complex interactions between FXFG containing nucleoporins, and we propose that some of these interactions may contribute to the movement of cargo across the NPC.     

p38MAPK-regulated induction of p62 and NBR1 after photodynamic therapy promotes autophagic clearance of ubiquitin aggregates and reduces reactive oxygen species levels by supporting Nrf2–antioxidant signaling

Emerging evidence indicates that oxidative stress instigates the formation of ubiquitin (Ub) aggregates, substrates of autophagy, through a process requiring the ubiquitin binding adaptors p62/SQSTM1 and NBR1. Here, we have investigated the role of p62 and NBR1 in cell survival after hypericin-mediated photodynamic therapy (Hyp-PDT), a procedure known to incite robust reactive oxygen species (ROS)-based endoplasmic reticulum stress and autophagy pathways. We found that Hyp-PDT stimulated the formation of p62- and NBR1-associated Ub aggregates in normal and cancer cells, which were ultimately removed by autophagy, through a mechanism partially regulated by p38^MAPK. In line with this, genetic or pharmacological p38^MAPK inhibition reduced p62 and NBR1 levels and aggregate formation and impaired Nrf2 activation, thus increasing photo-oxidative stress and cell death. p62-deficient cells, or cells lacking p62 and with reduced levels of NBR1 (through siRNA knockdown), also displayed reduced aggregate formation but exhibited attenuated ROS levels, reduced caspase activation, and improved survival after Hyp-PDT. The increased resistance to photo-oxidative stress exhibited by cells lacking p62 and/or NBR1 was overruled by the inhibition of p38^MAPK, which restored cytotoxic ROS levels, thus indicating the relevance of this signal in the control of cell viability. Taken together these findings provide evidence that in photodynamically treated cells a p38^MAPK-regulated pathway coordinates the p62/NBR1-mediated clearance of cytosolic aggregates and mitigates PDT-induced proteotoxicity. They also reveal that a functional p38^MAPK–Nrf2 signal is required to keep ROS levels in check and protect against PDT-induced proteotoxicity, independent of aggregate formation.