Glucose deprivation causes oxidative stress and stimulates aggresome formation and autophagy in cultured cardiac myocytes

Aggresomes are dynamic structures formed when the ubiquitin–proteasome system is overwhelmed with aggregation-prone proteins. In this process, small protein aggregates are actively transported towards the microtubule-organizing center. A functional role for autophagy in the clearance of aggresomes has also been proposed. In the present work we investigated the molecular mechanisms involved on aggresome formation in cultured rat cardiac myocytes exposed to glucose deprivation. Confocal microscopy showed that small aggregates of polyubiquitinated proteins were formed in cells exposed to glucose deprivation for 6 h. However, at longer times (18 h), aggregates formed large perinuclear inclusions (aggresomes) which colocalized with γ-tubulin (a microtubule-organizing center marker) and Hsp70. The microtubule disrupting agent vinblastine prevented the formation of these inclusions. Both small aggregates and aggresomes colocalized with autophagy markers such as GFP-LC3 and Rab24. Glucose deprivation stimulates reactive oxygen species (ROS) production and decreases intracellular glutathione levels. ROS inhibition by N-acetylcysteine or by the adenoviral overexpression of catalase or superoxide dismutase disrupted aggresome formation and autophagy induced by glucose deprivation. In conclusion, glucose deprivation induces oxidative stress which is associated with aggresome formation and activation of autophagy in cultured cardiac myocytes.     

UCH-L1 aggresome formation in response to proteasome impairment indicates a role in inclusion formation in Parkinson's disease

Aggresomes are associated with many neurodegenerative disorders, including Parkinson's disease, and polyglutamine disorders such as Huntington's disease. These inclusions commonly contain ubiquitylated proteins. The stage at which these proteins are ubiquitylated remains unclear. A malfunction of the ubiquitin/proteasome system (UPS) may be associated with their formation. Conversely, it may reflect an unsuccessful attempt by the cell to remove them. Previously, we demonstrated that overexpression of Parkin, a ubiquitin-protein ligase associated with autosomal recessive juvenile Parkinsonism, generates aggresome-like inclusions in UPS compromised cells. Mutations in the de-ubiquitylating enzyme, UCH-L1, cause a rare form of Parkinsonism. We now demonstrate that overexpression of UCH-L1 also forms ribbon-like aggresomes in response to proteasomal inhibition. Disease-associated mutations, which affect enzymatic activities, significantly increased the number of inclusions. UCH-L1 aggresomes co-localized with ubiquitylated proteins, HSP70, gamma-tubulin and, to a lesser extent, the 20S proteasome and the chaperone BiP. Similar to Parkin inclusions, we found UCH-L1 aggresomes to be surrounded by a tubulin rather than a vimentin cage-like structure. Furthermore, UCH-L1 aggregates with Parkin and alpha-synuclein in some, but not all inclusions, suggesting the heterogeneous nature of these inclusion bodies. This study provides additional evidence that aggregation-prone proteins are likely to recruit UPS components in an attempt to clear proteins from failing proteasomes. Furthermore, UCH-L1 accumulation is likely to play a pathological role in inclusion formation in Parkinson's disease.     

Aggresomes formed by alpha-synuclein and synphilin-1 are cytoprotective

Lewy bodies (LBs), which are the hallmark pathologic features of Parkinson's disease and of dementia with LBs, have several morphologic and molecular similarities to aggresomes. Whether such cytoplasmic inclusions contribute to neuronal death or protect cells from the toxic effects of misfolded proteins remains controversial. In this report, the role of aggresomes in cell viability was addressed in the context of over-expressing alpha-synuclein and its interacting partner synphilin-1 using engineered 293T cells. Inhibition of proteasome activity elicited the formation of juxtanuclear aggregates with characteristics of aggresomes including immunoreactivity for vimentin, gamma-tubulin, ubiquitin, proteasome subunit, and hsp70. As expected from the properties of aggresomes, the microtubule disrupting agents, vinblastin and nocodazole, markedly prevented the formation of these inclusions. Similar to LBs, the phosphorylated form of alpha-synuclein co-localized in these synphilin-1-containing aggresomes. Although the caspase inhibitor z-VAD-fmk significantly reduced the number of apoptotic cells, it had no impact on the percentage of aggresome-positive cells. Finally, quantitative analysis revealed aggresomes in 60% of nonapoptotic cells but only in 10% of apoptotic cells. Additionally, alpha-synuclein-induced apoptosis was not coupled with increased prevalence of aggresome-bearing cells. Taken together, these observations indicate a disconnection between aggresome formation and apoptosis, and support a protective role for these inclusions from the toxicity associated with the combined over-expression of alpha-synuclein and synphilin-1.     

Protein Folding, Misfolding, and Aggregation. Formation of Inclusion Bodies and Aggresomes

In this review the mechanisms of protein folding, misfolding, and aggregation as well as the mechanisms of cell defense against toxic protein aggregates are considered. Misfolded and aggregated proteins in cells are exposed to chaperone-mediated refolding and are degraded by proteasomes if refolding is impossible. Proteolysis-stable protein aggregates accumulate, forming inclusion bodies. In eucaryotic cells, protein aggregates form structures in the pericentrosomal area that have been termed "aggresomes". Formation of aggresomes in cells is a general cellular response to the presence of misfolded proteins when the degrading capacity of the cells is exceeded. The role of aggresomes in disturbance of the proteasomal system operation and in cellular death, particularly in the so-called "protein conformational diseases", is discussed.     

Aggresomes do not represent a general cellular response to protein misfolding in mammalian cells

Background

Aggresomes are juxtanuclear inclusion bodies that have been proposed to represent a general cellular response to misfolded proteins in mammalian cells. Yet, why aggresomes are not a pathological characteristic of protein misfolding diseases is unclear. Here, we investigate if a misfolded protein inevitably forms aggresomes in mammalian cells.

Results

We show that a cytoplasmic form of the prion protein may form aggresomes or dispersed aggregates in different cell lines. In contrast to aggresomes, the formation of dispersed aggregates is insensitive to histone deacetylase 6 inhibitors and does not result in cytoskeleton rearrangements. Modulation of expression levels or proteasome inhibitors does not alter the formation of dispersed aggregates.

Conclusion

Our results establish that aggresomes are not obligatory products of protein misfolding in vivo.     

Aggresomes: A Cellular Response to Misfolded Proteins

Intracellular deposition of misfolded protein aggregates into ubiquitin-rich cytoplasmic inclusions is linked to the pathogenesis of many diseases. Why these aggregates form despite the existence of cellular machinery to recognize and degrade misfolded protein and how they are delivered to cytoplasmic inclusions are not known. We have investigated the intracellular fate of cystic fibrosis transmembrane conductance regulator (CFTR), an inefficiently folded integral membrane protein which is degraded by the cytoplasmic ubiquitin-proteasome pathway. Overexpression or inhibition of proteasome activity in transfected human embryonic kidney or Chinese hamster ovary cells led to the accumulation of stable, high molecular weight, detergent-insoluble, multiubiquitinated forms of CFTR. Using immunofluorescence and transmission electron microscopy with immunogold labeling, we demonstrate that undegraded CFTR molecules accumulate at a distinct pericentriolar structure which we have termed the aggresome. Aggresome formation is accompanied by redistribution of the intermediate filament protein vimentin to form a cage surrounding a pericentriolar core of aggregated, ubiquitinated protein. Disruption of microtubules blocks the formation of aggresomes. Similarly, inhibition of proteasome function also prevented the degradation of unassembled presenilin-1 molecules leading to their aggregation and deposition in aggresomes. These data lead us to propose that aggresome formation is a general response of cells which occurs when the capacity of the proteasome is exceeded by the production of aggregation-prone misfolded proteins.     

Triggering aggresome formation. Dissecting aggresome-targeting and aggregation signals in synphilin 1

Abnormal polypeptides that escape proteasome-dependent degradation and aggregate in cytosol can be transported via microtubules to an aggresome, a recently discovered organelle where aggregated proteins are stored or degraded by autophagy. We used synphilin 1, a protein implicated in Parkinson disease, as a model to study mechanisms of aggresome formation. When expressed in na?ve HEK293 cells, synphilin 1 forms multiple small highly mobile aggregates. However, proteasome or Hsp90 inhibition rapidly triggered their translocation into the aggresome, and surprisingly, this response was independent on the expression level of synphilin 1. Therefore, aggresome formation, but not aggregation of synphilin 1, represents a special cellular response to a failure of the proteasome/chaperone machinery. Importantly, translocation to aggresomes required a special aggresome-targeting signal within the sequence of synphilin 1, an ankyrin-like repeat domain. On the other hand, formation of multiple small aggregates required an entirely different segment within synphilin 1, indicating that aggregation and aggresome formation determinants can be separated genetically. Furthermore, substitution of the ankyrin-like repeat in synphilin 1 with an aggresome-targeting signal from huntingtin was sufficient for aggresome formation upon inhibition of the proteasome. Analogously, attachment of the ankyrin-like repeat to a huntingtin fragment lacking its aggresome-targeting signal promoted its transport to aggresomes. These findings indicate the existence of transferable signals that target aggregation-prone polypeptides to aggresomes.     

Hsp25, a member of the Hsp30 family, promotes inclusion formation in response to stress

Protein aggregates are oligomeric complexes of misfolded proteins, and serve as the seeds of inclusion bodies termed aggresomes in the cells. Heat shock proteins (Hsps) prevent misfolding and aggregate formation. Here, we found that only avian Hsp25 dominantly accumulated in the aggresomes induced by proteasome inhibition. Molecular cloning of chicken Hsp25 (cHsp25) revealed that it belongs to the Hsp30 family, which is a subfamily of the alpha-crystallin/small Hsp gene family. Unexpectedly, overexpression of cHsp25 into HeLa cells promoted inclusion formation whereas overexpression of mouse Hsp27 and its chicken homologue did not. These results suggest that cHsp25 acts differently from other small Hsps on protein aggregates.     

ALIS are stress-induced protein storage compartments for substrates of the proteasome and autophagy

Misfolded proteins can be directed into cytoplasmic aggregates such as aggresomes and dendritic cell aggresome-like induced structures (DALIS). DALIS were originally identified in lipopolysaccharide-stimulated dendritic cells and act as storage compartments for polyubiquitinated Defective Ribosomal Products (DRiPs) prior to their clearance by the proteasome. Here we demonstrate that ubiquitinated protein aggregates that are similar to DALIS, and not related to aggresomes, can be observed in several cell types in response to stress, including oxidative stress, transfection, and starvation. Significantly, both immune and nonimmune cells could form these aggresome-like induced structures (ALIS). Protein synthesis was essential for ALIS formation in response to oxidative stress, indicating that DRiP formation was required. Furthermore, puromycin, which increases DRiP formation, was sufficient to induce ALIS formation. Inhibition of either proteasomes or of autophagy interfered with ALIS clearance in puromycin treated cells. Autophagy inhibition enhanced ALIS formation under a variety of stress conditions. During starvation, ALIS formation in autophagy-deficient cells was only partially inhibited by protein synthesis inhibitors, indicating that both long-lived proteins and DRiPs can be targeted to ALIS. Together, these findings demonstrate that ALIS act as generalized stress-induced protein storage compartments for substrates of the proteasome and autophagy.     

Presenilin-binding protein forms aggresomes in monkey kidney COS-7 cells

A novel presenilin-binding protein (PBP) is specifically expressed in the brain, and its level in the soluble fraction of Alzheimer's disease (AD) brains is much less than that in the age-matched controls. Recently, several proteins, including presenilin (PS), have been found to form structures of aggregated proteins, called aggresomes, when the production of the proteins exceeds their rate of degradation by proteasomes. Based on these observations it has been proposed that the aggresome may represent one of the mechanisms forthe formation of cytoplasmic deposits which are linked to the pathogenesis of neurodegenerative disorders including AD. It is shown here that the overexpression of PBP or the suppression of proteasome activity in monkey kidney COS-7 cells leads to the accumulation of detergent-insoluble and multiubiquitinated PBP aggregates. PBP also forms aggregates in primary cultures of neurons in the presence of a proteasome inhibitors. PBP aggregates have the characteristics of aggresomes, including the localization to microtubule organization centers and the disruption of intermediate filaments. These observations suggest that the malfunctioning of the proteasome can cause the formation of PBP aggresomes, which may lead to AD.     

The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress

The efficient clearance of cytotoxic misfolded protein aggregates is critical for cell survival. Misfolded protein aggregates are transported and removed from the cytoplasm by dynein motors via the microtubule network to a novel organelle termed the aggresome where they are processed. However, the means by which dynein motors recognize misfolded protein cargo, and the cellular factors that regulate aggresome formation, remain unknown. We have discovered that HDAC6, a microtubule-associated deacetylase, is a component of the aggresome. We demonstrate that HDAC6 has the capacity to bind both polyubiquitinated misfolded proteins and dynein motors, thereby acting to recruit misfolded protein cargo to dynein motors for transport to aggresomes. Indeed, cells deficient in HDAC6 fail to clear misfolded protein aggregates from the cytoplasm, cannot form aggresomes properly, and are hypersensitive to the accumulation of misfolded proteins. These findings identify HDAC6 as a crucial player in the cellular management of misfolded protein-induced stress.     

Modulation of prion formation, aggregation, and toxicity by the actin cytoskeleton in yeast

Self-perpetuating protein aggregates transmit prion diseases in mammals and heritable traits in yeast. De novo prion formation can be induced by transient overproduction of the corresponding prion-forming protein or its prion domain. Here, we demonstrate that the yeast prion protein Sup35 interacts with various proteins of the actin cortical cytoskeleton that are involved in endocytosis. Sup35-derived aggregates, generated in the process of prion induction, are associated with the components of the endocytic/vacuolar pathway. Mutational alterations of the cortical actin cytoskeleton decrease aggregation of overproduced Sup35 and de novo prion induction and increase prion-related toxicity in yeast. Deletion of the gene coding for the actin assembly protein Sla2 is lethal in cells containing the prion isoforms of both Sup35 and Rnq1 proteins simultaneously. Our data are consistent with a model in which cytoskeletal structures provide a scaffold for generation of large aggregates, resembling mammalian aggresomes. These aggregates promote prion formation. Moreover, it appears that the actin cytoskeleton also plays a certain role in counteracting the toxicity of the overproduced potentially aggregating proteins.     

Aggresomes and Russell bodies. Symptoms of cellular indigestion?

All cells are equipped with a proteolytic apparatus that eliminates damaged, misfolded and incorrectly assembled proteins. The principal engine of cytoplasmic proteolysis, the 26S proteasome, requires that substrates be unfolded to gain access to the active site; consequently, it is relatively ineffective at degrading aggregated proteins. Cellular indigestion occurs when the production of aggregation-prone proteins exceeds the cell’s (or organelle’s) capacity to eliminate them. Cellular pathways that resolve this indigestion exist, but appear to have limited capacities. Russell bodies and aggresomes are manifestations of cellular indigestion in the endoplasmic reticulum and cytoplasmic compartments, respectively, and are often associated with disease.     

Co-chaperone CHIP Stabilizes Aggregate-prone Malin, a Ubiquitin Ligase Mutated in Lafora Disease

Lafora disease (LD) is an autosomal recessive neurodegenerative disorder caused by mutation in either the dual specificity phosphatase laforin or ubiquitin ligase malin. A pathological hallmark of LD is the accumulation of cytoplasmic polyglucosan inclusions commonly known as Lafora bodies in both neuronal and non-neuronal tissues. How mutations in these two proteins cause disease pathogenesis is not well understood. Malin interacts with laforin and recruits to aggresomes upon proteasome inhibition and was shown to degrade misfolded proteins. Here we report that malin is spontaneously misfolded and tends to be aggregated, degraded by proteasomes, and forms not only aggresomes but also other cytoplasmic and nuclear aggregates in all transfected cells upon proteasomal inhibition. Malin also interacts with Hsp70. Several disease-causing mutants of malin are comparatively more unstable than wild type and form aggregates in most transfected cells even without the inhibition of proteasome function. These cytoplasmic and nuclear aggregates are immunoreactive to ubiquitin and 20 S proteasome. Interestingly, progressive proteasomal dysfunction and cell death is also most frequently observed in the mutant malin-overexpressed cells compared with the wild-type counterpart. Finally, we demonstrate that the co-chaperone carboxyl terminus of the Hsc70-interacting protein (CHIP) stabilizes malin by modulating the activity of Hsp70. All together, our results suggest that malin is unstable, and the aggregate-prone protein and co-chaperone CHIP can modulate its stability.     

Polarised asymmetric inheritance of accumulated protein damage in higher eukaryotes

Disease-associated misfolded proteins or proteins damaged due to cellular stress are generally disposed via the cellular protein quality-control system. However, under saturating conditions, misfolded proteins will aggregate. In higher eukaryotes, these aggregates can be transported to accumulate in aggresomes at the microtubule organizing center. The fate of cells that contain aggresomes is currently unknown. Here we report that cells that have formed aggresomes can undergo normal mitosis. As a result, the aggregated proteins are asymmetrically distributed to one of the daughter cells, leaving the other daughter free of accumulated protein damage. Using both epithelial crypts of the small intestine of patients with a protein folding disease and Drosophila melanogaster neural precursor cells as models, we found that the inheritance of protein aggregates during mitosis occurs with a fixed polarity indicative of a mechanism to preserve the long-lived progeny.     

Aggregation-prone Regions in HYPK Help It to Form Sequestration Complex for Toxic Protein Aggregates

Protein aggregates result from altered structural conformations and they can perturb cellular homeostasis. Prevention mechanisms, which function against protein aggregation by modulatory processes, are diverse and redundant. In this study, we have characterized Huntingtin interacting protein K (HYPK) as a global aggregation-regulatory protein. We report the mechanistic details of how HYPK's aggregation-prone regions allow it to sense and prevent other toxic protein's aggregation by forming unique annular-shaped sequestration complexes. Screenings for interacting partners of different aggregation-prone proteins identify HYPK as a global interacting partner/regulator of Huntingtin97Qexon1, α-Synuclein-A53T and Superoxide dismutase1-G93A. C-terminal hydrophobic region in HYPK makes direct contacts with aggregates to initiate the formation of sequestration complexes. HYPK acts as aggregate sensor by existing in a seeded amyloid-like state which also favors its own concentration-dependent self-oligomerization. Oligomerization of HYPK leads to annular and non-fibrillar/amorphous aggregates. Two hydrophobic segments in the C-terminus of HYPK are responsible for its own aggregations. Self-association of HYPK follows seed nucleation, in which oligomeric HYPK seeds nucleate to annular structures. Annular oligomers of HYPK fuse with each other to form amorphous aggregates. HYPK shows differential interactions with aggregation-prone and non-aggregating proteins, as it preferentially binds to aggregation-prone proteins with higher affinity than native/non-aggregating proteins. This favors the formation of HYPK's sequestration complexes both in cytosol and in ribosome. Besides having aggregation-preventive property, HYPK also reduces the cellular level of toxic proteins. In vivo, HYPK sequestration complexes prevent the formation of toxic protein aggregates to physiologically show positive impact on cell survival and restoration of normal cell physiology.     

α-Synuclein modifies huntingtin aggregation in living cells

Several neurodegenerative disorders are characterized by the accumulation of proteinaceous inclusions in the central nervous system. These inclusions are frequently composed of a mixture of aggregation-prone proteins. Here, we used a bimolecular fluorescence complementation assay to study the initial steps of the co-aggregation of huntingtin (Htt) and α-synuclein (α-syn), two aggregation-prone proteins involved in Huntington's disease (HD) and Parkinson's disease (PD), respectively. We found that Htt (exon 1) oligomerized with α-syn and sequestered it in the cytosol. In turn, α-syn increased the number of cells displaying aggregates, decreased the number of aggregates per cell and increased the average size of the aggregates. Our results support the idea that co-aggregation of aggregation-prone proteins can contribute to the histopathology of neurodegenerative disorders.     

Morphological Changes within the Rat Lateral Ventricle after the Administration of Proteasome Inhibitors

The broad variety of substances that inhibit the action of the ubiquitin-proteasome system (UPS)—known as proteasome inhibitors—have been used extensively in previous studies, and they are currently frequently proposed as a novel form of cancer treatment and as a protective factor in intracerebral hemorrhage treatment. The experimental data on the safest route of proteasome inhibitor administration, their associated side effects, and the possible ways of minimizing these effects have recently become a very important topic. The aim of our present study was to determine the effects of administering of MG-132, lactacystin and epoxomicin, compounds belonging to three different classes of proteasome inhibitors, on the ependymal walls of the lateral ventricle. Observations were made 2 and 8 weeks after the intraventricular administration of the studied substances dissolved in dimethyl sulfoxide (DMSO) into the lateral ventricle of adult Wistar rats. Qualitative and quantitative analysis of brain sections stained with histochemical and inmmunofluorescence techniques showed that the administration of proteasome inhibitors caused a partial occlusion of the injected ventricle in all of the studied animals. The occlusion was due to ependymal cells damage and subsequent ependymal discontinuity, which caused direct contact between the striatum and the lateral nuclei of the septum, mononuclear cell infiltration and the formation of a glial scar between these structures (with the activation of astroglia, microglia and oligodendroglia). Morphologically, the ubiquitin-positive aggregates corresponded to aggresomes, indicating impaired activity of the UPS and the accumulation and aggregation of ubiquitinated proteins that coincided with the occurrence of glial scars. The most significant changes were observed in the wall covering the striatum in animals that were administered epoxomicin, and milder changes were observed in animals administered lactacystin and MG-132. Interestingly, DMSO administration also caused damage to some of the ependymal cells, but the aggresome-like structures were not formed. Our results indicate that all of the studied classes of proteasome inhibitors are detrimental to ependymal cells to some extent, and may cause severe changes in the ventricular system. The safety implications of their usage in therapeutic strategies to attenuate intracerebral hemorrhagic injury and in brain cancer treatment will require further studies.     

Amyloidogenic Regions and Interaction Surfaces Overlap in Globular Proteins Related to Conformational Diseases

Protein aggregation underlies a wide range of human disorders. The polypeptides involved in these pathologies might be intrinsically unstructured or display a defined 3D-structure. Little is known about how globular proteins aggregate into toxic assemblies under physiological conditions, where they display an initially folded conformation. Protein aggregation is, however, always initiated by the establishment of anomalous protein-protein interactions. Therefore, in the present work, we have explored the extent to which protein interaction surfaces and aggregation-prone regions overlap in globular proteins associated with conformational diseases. Computational analysis of the native complexes formed by these proteins shows that aggregation-prone regions do frequently overlap with protein interfaces. The spatial coincidence of interaction sites and aggregating regions suggests that the formation of functional complexes and the aggregation of their individual subunits might compete in the cell. Accordingly, single mutations affecting complex interface or stability usually result in the formation of toxic aggregates. It is suggested that the stabilization of existing interfaces in multimeric proteins or the formation of new complexes in monomeric polypeptides might become effective strategies to prevent disease-linked aggregation of globular proteins.     

Mitochondrial enzymes are protected from stress-induced aggregation by mitochondrial chaperones and the Pim1/LON protease

Proteins in a natural environment are constantly challenged by stress conditions, causing their destabilization, unfolding, and, ultimately, aggregation. Protein aggregation has been associated with a wide variety of pathological conditions, especially neurodegenerative disorders, stressing the importance of adequate cellular protein quality control measures to counteract aggregate formation. To secure protein homeostasis, mitochondria contain an elaborate protein quality control system, consisting of chaperones and ATP-dependent proteases. To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins. We could show that major metabolic pathways in mitochondria were affected by the aggregation of key enzyme components, which were largely inactivated after heat stress. Furthermore, treatment with elevated levels of reactive oxygen species strongly influenced the aggregation behavior, in particular in combination with elevated temperatures. Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems. Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON. We therefore propose that the proteolytic breakdown of aggregation-prone polypeptides represents a major protective strategy to prevent the in vivo formation of aggregates in mitochondria.