Gain in toxic function of stefin B EPM1 mutants aggregates: Correlation between cell death,aggregate number/size and oxidative stress

EPM1 is a rare progressive myoclonus epilepsy accompanied by apoptosis in the cerebellum of patients. Mutations in the gene of stefin B (cystatin B) are responsible for the primary defect underlying EPM1. Taking stefin B aggregates as a model we asked what comes first, protein aggregation or oxidative stress, and how these two processes correlate with cell death.We studied the aggregation in cells of the stefin B wild type, G4R mutant, and R68X fragment before (Ceru et al., 2010, Biol. Cell). The present study was performed on two more missense mutants of human stefin B, G50E and Q71P, and they similarly showed numerous aggregates upon overexpression. Mutant- and oligomer-dependent increase in oxidative stress and cell death in cells bearing aggregates was shown. On the other hand, there was no correlation between the size and number of the aggregates and cell death. We suggest that differences in toxicity of the aggregates depend on whether they are in oligomeric/protofibrillar or fibrillar form. This in turn likely depends on the mutant's 3D structure where unfolded proteins show lower toxicity. Imaging by transmission electron microscopy showed that the aggregates in cells are of different types: bigger perinuclear, surrounded by membranes and sometimes showing vesicle-like invaginations, or smaller, punctual and dispersed throughout the cytoplasm. All EPM1 mutants studied were inactive as cysteine proteases inhibitors and in this way contribute to loss of stefin B functions. Relevance to EPM1 disease by gain in toxic function is discussed.     

The dependency of autophagy and ubiquitin proteasome system during skeletal muscle atrophy

Among the four proteolytic systems in the cell, autophagy and the ubiquitin-proteasome system (UPS) are the main proteolytic events that allow for the removal of cell debris and proteins to maintain cellular homeostasis. Previous studies have revealed that these systems perform their functions independently of each other. However, recent studies indicate the existence of regulatory interactions between these proteolytic systems via ubiquitinated tags and a reciprocal regulation mechanism with several crosstalk points. UPS plays an important role in the elimination of short-lived/soluble misfolded proteins, whereas autophagy eliminates defective organelles and persistent insoluble protein aggregates. Both of these systems seem to act independently; however, disruption of one pathway affects the activity of the other pathway and contributes to different pathological conditions. This review summarizes the recent findings on direct and indirect dependencies of autophagy and UPS and their execution at the molecular level along with the important drug targets in skeletal muscle atrophy.     

In vitro study of stability and amyloid-fibril formation of two mutants of human stefin B (cystatin B) occurring in patients with EPM1

Myoclonus epilepsy of type 1 (EPM1) is a rare monogenic progressive and degenerative epilepsy, also known under the name Unverricht-Lundborg disease. With the aim of comparing their behavior in vitro, wild-type (wt) human stefin B (cystatin B) and the G4R and the R68X mutants observed in EPM1 were expressed and isolated from the Escherichia coli lysate. The R68X mutant (Arg68Stop) is a peptide of 67 amino acids from the N terminus of stefin B. CD spectra have shown that the R68X peptide is not folded, in contrast to the G4R mutant, which folds like wild type. The wild type and the G4R mutant were unfolded by urea and by trifluoroethanol (TFE). It has been shown that both proteins have closely similar stability and that at pH 4.8, where a native-like intermediate was demonstrated, TFE induces unfolding intermediates prior to the major transition to the all-alpha-helical state. Kinetics of fibril formation were followed by Thioflavin T fluorescence while the accompanying changes of morphology were followed by the transmission electron microscopy (TEM). For the two folded proteins the optimal concentration of TFE producing extensive lag phases and high fibril yields was predenaturational, 9% (v/v). The unfolded R68X peptide, which is highly prone to aggregate, formed amyloid fibrils in aqueous solution and in predenaturing 3% TFE. The G4R mutant exhibited a much longer lag phase than the wild type, with the accumulation of prefibrillar aggregates. Implications for pathology in view of the higher toxicity of prefibrillar aggregates to cells are discussed.     

E3 Ubiquitin Ligase CHIP and NBR1-Mediated Selective Autophagy Protect Additively against Proteotoxicity in Plant Stress Responses

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.     

Interaction of human stefin B in the prefibrillar oligomeric form with membranes. Correlation with cellular toxicity

Protein aggregation is central to most neurodegenerative diseases, as shown by familial case studies and by animal models. A modified 'amyloid cascade' hypothesis for Alzheimer's disease states that prefibrillar oligomers, also called amyloid-beta-derived diffusible ligands or globular oligomers, are the responsible toxic agent. It has been proposed that these oligomeric species, as shown for amyloid-beta, beta2-microglobulin or prion fragments, exert toxicity by forming pores in membranes, initiating a cascade of detrimental events for the cell. Interaction of granular aggregates and globular oligomers of an amyloidogenic protein, human stefin B, with model lipid membranes and monolayers was studied. Prefibrillar oligomers/aggregates of stefin B are shown to cause concentration-dependent membrane leaking, in contrast to the homologous stefin A. Prefibrillar oligomers/aggregates of stefin B also increase the surface pressure at an air-water interface, i.e. they have amphipathic character and are surface seeking. In addition, they show stronger interaction with 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] monolayers than native stefin A or nonaggregated stefin B. Prefibrillar aggregates interact predominantly with acidic phospholipids, such as dioleoylphosphatidylglycerol or dipalmitoylphosphatidylserine, as shown by calcein release experiments and surface plasmon resonance. The same preparations are toxic to neuroblastoma cells, as determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, again in contrast to the homologue stefin A, which does not aggregate under any of the conditions studied. This study is aimed to contribute to the general model of cellular toxicity induced by prefibrillar oligomers of amyloidogenic proteins, not necessarily involved in pathology.     

Interaction between Oligomers of Stefin B and Amyloid-�� in Vitro and in Cells

To contribute to the question of the putative role of cystatins in Alzheimer disease and in neuroprotection in general, we studied the interaction between human stefin B (cystatin B) and amyloid-β-(1–40) peptide (Aβ). Using surface plasmon resonance and electrospray mass spectrometry we were able to show a direct interaction between the two proteins. As an interesting new fact, we show that stefin B binding to Aβ is oligomer specific. The dimers and tetramers of stefin B, which bind Aβ, are domain-swapped as judged from structural studies. Consistent with the binding results, the same oligomers of stefin B inhibit Aβ fibril formation. When expressed in cultured cells, stefin B co-localizes with Aβ intracellular inclusions. It also co-immunoprecipitates with the APP fragment containing the Aβ epitope. Thus, stefin B is another APP/Aβ-binding protein in vitro and likely in cells.     

The deubiquitinating enzyme USP36 controls selective autophagy activation by ubiquitinated proteins

Initially described as a nonspecific degradation process induced upon starvation, autophagy is now known also to be involved in the degradation of specific ubiquitinated substrates such as mitochondria, bacteria and aggregated proteins, ensuring crucial functions in cell physiology and immunity. We report here that the deubiquitinating enzyme USP36 controls selective autophagy activation in Drosophila and in human cells. We show that dUsp36 loss of function autonomously inhibits cell growth while activating autophagy. Despite the phenotypic similarity, dUSP36 is not part of the TOR signaling pathway. Autophagy induced by dUsp36 loss of function depends on p62/SQSTM1, an adaptor for delivering cargo marked by polyubiquitin to autophagosomes. Consistent with p62 requirement, dUsp36 mutant cells display nuclear aggregates of ubiquitinated proteins, including Histone H2B, and cytoplasmic ubiquitinated proteins; the latter are eliminated by autophagy. Importantly, USP36 function in p62-dependent selective autophagy is conserved in human cells. Our work identifies a novel, crucial role for a deubiquitinating enzyme in selective autophagy.     

Amyloid fibril formation by human stefins: Structure,mechanism & putative functions

Many questions in the field of protein aggregation to amyloid fibrils remain open. In this review we describe predominantly in vitro studies of oligomerization and amyloid fibril formation by human stefins A and B. In human stefin B amyloidogenesis in vitro we have observed some general and many specific properties of its prefibrillar oligomers and amyloid fibrils. One characteristic feature in common to stefins and cystatins (and possibly some other amyloid proteins) is domain-swapping. In addition to solution structure of the domain-swapped dimer of stefin A, we recently have determined 3D structure of stefin B tetramer, which proved to be composed from two domain-swapped dimers, whose interaction occurs by a proline switch in the loop surrounding the conserved Pro 74. Studying the mechanism of fibril formation by stefin B, we found that the nucleation and fibril elongation reactions have energies of activation (E_a’s) in the range of proline isomerisation, strongly indicating importance of the Pro at site 74 and/or other prolines in the sequence. Correlation between toxicity of the prefibrillar oligomers and their interaction with acidic phospholipids was demonstrated. Stefin B was shown to interact with amyloid-beta peptide of Alzheimer’s disease in an oligomer specific manner, both in vitro and in the cells. It also has been shown that endogenous stefin B (with E at site 31) but especially the EPM1 mutant R68X and Y31-stefin B variant, and to a lesser extent EPM1 mutant G4R, are prone to form aggregates in cells.     

Autophagy was activated in injured astrocytes and mildly decreased cell survival following glucose and oxygen deprivation and focal cerebral ischemia

The present study evaluated autophagy activation in astrocytes and its contribution to astrocyte injury induced by cerebral ischemia and hypoxia. Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion (pMCAO) in rats. In vitro hypoxia in cultured primary astrocytes was induced by the oxygen-glucose deprivation (OGD). Alterations of astrocytes were evaluated with astroglia markers glial fibrillary acidic protein (GFAP). The formation of autophagosomes in astrocytes was examined with transmission electron microscopy (TEM). The expression of autophagy-related proteins were examined with immunoblotting. The role of autophagy in OGD or focal cerebral ischemia-induced death of astrocytes was assessed by pharmacological inhibition of autophagy with 3-methyladenine (3-MA) or bafilomycin A1 (Baf). The results showed that GFAP staining was reduced in the infarct brain areas 3-12 h following pMCAO. Cerebral ischemia or OGD induced activation of autophagy in astrocytes as evidenced by the increased formation of autophagosomes and autolysosomes and monodansylcadaverine (MDC)-labeled vesicles; the increased production of microtubule-associated protein 1 light chain 3 (LC3-II); the upregulation of Beclin 1, lysosome-associated membrane protein 2 (LAMP2) and lysosomal cathepsin B expression; and the decreased levels of cytoprotective Bcl-2 protein in primary astrocytes. 3-MA inhibited OGD-induced the increase in LC3-II and the decline in Bcl-2. Furthermore, 3-MA and Baf slightly but significantly attenuated OGD-induced death of astrocytes. 3-MA also significantly increased the number of GFAP-positive cells and the protein levels of GFAP in the ischemic cortex core 12 h following pMCAO. These results suggest that ischemia or hypoxia-induced autophagic/lysosomal pathway activation may at least partly contribute to ischemic injury of astrocytes.     

The deubiquitinating enzyme USP36 controls selective autophagy activation by ubiquitinated proteins

Initially described as a nonspecific degradation process induced upon starvation, autophagy is now known also to be involved in the degradation of specific ubiquitinated substrates such as mitochondria, bacteria and aggregated proteins, ensuring crucial functions in cell physiology and immunity. We report here that the deubiquitinating enzyme USP36 controls selective autophagy activation in Drosophila and in human cells. We show that dUsp36 loss of function autonomously inhibits cell growth while activating autophagy. Despite the phenotypic similarity, dUSP36 is not part of the TOR signaling pathway. Autophagy induced by dUsp36 loss of function depends on p62/SQSTM1, an adaptor for delivering cargo marked by polyubiquitin to autophagosomes. Consistent with p62 requirement, dUsp36 mutant cells display nuclear aggregates of ubiquitinated proteins, including Histone H2B, and cytoplasmic ubiquitinated proteins; the latter are eliminated by autophagy. Importantly, USP36 function in p62-dependent selective autophagy is conserved in human cells. Our work identifies a novel, crucial role for a deubiquitinating enzyme in selective autophagy.     

Autophagy genes protect against disease caused by polyglutamine expansion proteins in Caenorhabditis elegans

Expanded polyglutamine (polyQ) proteins aggregate intracellularly in Huntington's disease and other neurodegenerative disorders. The lysosomal degradation pathway, autophagy, is known to promote clearance of polyQ protein aggregates in cultured cells. Moreover, basal autophagy in neuronal cells in mice prevents neurodegeneration by suppressing the accumulation of abnormal intracellular proteins. However, it is not yet known whether autophagy genes play a role in vivo in protecting against disease caused by mutant aggregate-prone, expanded polyQ proteins. To examine this question, we used two models of polyQ-induced toxicity in C. elegans, including the expression of polyQ40 aggregates in muscle and the expression of a human huntingtin disease fragment containing a polyQ tract of 150 residues (Htn-Q150) in ASH sensory neurons. Here, we show that genetic inactivation of autophagy genes accelerates the accumulation of polyQ40 aggregates in C. elegans muscle cells and exacerbates polyQ40-induced muscle dysfunction. Autophagy gene inactivation also increases the accumulation of Htn-Q150 aggregates in C. elegans ASH sensory neurons and results in enhanced neurodegeneration. These data provide in vivo genetic evidence that autophagy genes suppress the accumulation of polyQ aggregates and protect cells from disease caused by polyQ toxicity.     

Na+/H+ Exchangers Induce Autophagy in Neurons and Inhibit Polyglutamine-Induced Aggregate Formation

In polyglutamine diseases, an abnormally elongated polyglutamine results in protein misfolding and accumulation of intracellular aggregates. Autophagy is a major cellular degradative pathway responsible for eliminating unnecessary proteins, including polyglutamine aggregates. Basal autophagy constitutively occurs at low levels in cells for the performance of homeostatic function, but the regulatory mechanism for basal autophagy remains elusive. Here we show that the Na⁺/H⁺ exchanger (NHE) family of ion transporters affect autophagy in a neuron-like cell line (Neuro-2a cells). We showed that expression of NHE1 and NHE5 is correlated to polyglutamine accumulation levels in a cellular model of Huntington''s disease, a fatal neurodegenerative disorder characterized by accumulation of polyglutamine-containing aggregate formation in the brain. Furthermore, we showed that loss of NHE5 results in increased polyglutamine accumulation in an animal model of Huntington''s disease. Our data suggest that cellular pH regulation by NHE1 and NHE5 plays a role in regulating basal autophagy and thereby promotes autophagy-mediated degradation of proteins including polyglutamine aggregates.     

MD1 deletion exaggerates cardiomyocyte autophagy induced by heart failure with preserved ejection fraction through ROS/MAPK signalling pathway

In our previous studies, we reported that myeloid differentiation protein 1 (MD1) serves as a negative regulator in several cardiovascular diseases. However, the role of MD1 in heart failure with preserved ejection fraction (HFpEF) and the underlying mechanisms of its action remain unclear. Eight‐week‐old MD1‐knockout (MD1‐KO) and wild‐type (WT) mice served as models of HFpEF induced by uninephrectomy, continuous saline or d‐aldosterone infusion and a 1.0% sodium chloride treatment in drinking water for 4 weeks to investigate the effect of MD1 on HFpEF in vivo. H9C2 cells were treated with aldosterone to evaluate the role of MD1 KO in vitro. MD1 expression was down‐regulated in the HFpEF mice; HFpEF significantly increased the levels of intracellular reactive oxygen species (ROS) and promoted autophagy; and in the MD1‐KO mice, the HFpEF‐induced intracellular ROS and autophagy effects were significantly exacerbated. Moreover, MD1 loss activated the p38‐MAPK pathway both in vivo and in vitro. Aldosterone‐mediated cardiomyocyte autophagy was significantly inhibited in cells pre‐treated with the ROS scavenger N‐acetylcysteine (NAC) or p38 inhibitor SB203580. Furthermore, inhibition with the autophagy inhibitor 3‐methyladenine (3‐MA) offset the aggravating effect of aldosterone‐induced autophagy in the MD1‐KO mice and cells both in vivo and in vitro. Our results validate a critical role of MD1 in the pathogenesis of HFpEF. MD1 deletion exaggerates cardiomyocyte autophagy in HFpEF via the activation of the ROS‐mediated MAPK signalling pathway.     

What to Eat: Evidence for Selective Autophagy in Plants

Autophagy is a macromolecular degradation pathway by which cells recycle their contents as a developmental process, housekeeping mechanism, and response to environmental stress. In plants, autophagy involves the sequestration of cargo to be degraded, transport to the cell vacuole in a double-membrane bound autophagosome, and subsequent degradation by lytic enzymes. Autophagy has generally been considered to be a non-selective mechanism of degradation. However, studies in yeast and animals have found numerous examples of selective autophagy, with cargo including proteins, protein aggregates, and organelles. Recent work has also provided evidence for several types of selective autophagy in plants. The degradation of protein aggregates was the first selective autophagy described in plants, and, more recently, a hybrid protein of the mammalian selective autophagy adaptors p62 and NBR1, which interacts with the autophagy machinery and may function in autophagy of protein aggregates, was described in plants. Other intracellular components have been suggested to be selectively targeted by autophagy in plants, but the current evidence is limited. Here, we discuss recent findings regarding the selective targeting of cell components by autophagy in plants.     

Autophagy Genes Protect Against Disease Caused by Polyglutamine Expansion Proteins in Caenorhabditis elegans

Expanded polyglutamine (polyQ) proteins aggregate intracellularly in Huntington’s disease and other neurodegenerative disorders. The lysosomal degradation pathway, autophagy, is known to promote clearance of polyQ protein aggregates in cultured cells. Moreover, basal autophagy in neuronal cells in mice prevents neurodegeneration by suppressing the accumulation of abnormal intracellular proteins. However, it is not yet known whether autophagy genes play a role in vivo in protecting against disease caused by mutant aggregate-prone, expanded polyQ proteins. To examine this question, we used two models of polyQ-induced toxicity in C. elegans, including the expression of polyQ40 aggregates in muscle and the expression of a human huntingtin disease fragment containing a polyQ tract of 150 residues (Htn-Q150) in ASH sensory neurons. Here, we show that genetic inactivation of autophagy genes accelerates the accumulation of polyQ40 aggregates in C. elegans muscle cells and exacerbates polyQ40-induced muscle dysfunction. Autophagy gene inactivation also increases the accumulation of Htn-Q150 aggregates in C. elegans ASH sensory neurons and results in enhanced neurodegeneration. These data provide in vivo genetic evidence that autophagy genes suppress the accumulation of polyQ aggregates and protect cells from disease caused by polyQ toxicity.     

p62, Ref(2)P and ubiquitinated proteins are conserved markers of neuronal aging,aggregate formation and progressive autophagic defects

Suppression of macroautophagy, due to mutations or through processes linked to aging, results in the accumulation of cytoplasmic substrates that are normally eliminated by the pathway. This is a significant problem in long-lived cells like neurons, where pathway defects can result in the accumulation of aggregates containing ubiquitinated proteins. The p62/Ref(2)P family of proteins is involved in the autophagic clearance of cytoplasmic protein bodies or sequestosomes. These unique structures are closely associated with protein inclusions containing ubiquitin as well as key components of the autophagy pathway. In this study we show that detergent fractionation followed by western blot analysis of insoluble ubiquitinated proteins (IUP), mammalian p62 and its Drosophila homologue, Ref(2)P can be used to quantitatively assess the activity level of aggregate clearance (aggrephagy) in complex tissues. Using this technique we show that genetic or age-dependent changes that modify the long-term enhancement or suppression of aggrephagy can be identified. Moreover, using the Drosophila model system this method can be used to establish autophagy-dependent protein clearance profiles that are occurring under a wide range of physiological conditions including developmental, fasting and altered metabolic pathways. This technique can also be used to examine proteopathies that are associated with human disorders such as frontotemporal dementia, Huntington and Alzheimer disease. Our findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells.     

Isobaric Tag‐Based Protein Profiling of a Nicotine‐Treated Alpha7 Nicotinic Receptor‐Null Human Haploid Cell Line

Nicotinic acetylcholine receptors (nAChR), the primary cell surface targets of nicotine, have implications in various neurological disorders. Here we investigate the proteome‐wide effects of nicotine on human haploid cell lines (wildtype HAP1 and α7KO‐HAP1) to address differences in nicotine‐induced protein abundance profiles between these cell lines. We performed an SPS‐MS3‐based TMT10‐plex experiment arranged in a 2‐3‐2‐3 design with two replicates of the untreated samples and three of the treated samples for each cell line. We quantified 8775 proteins across all ten samples, of which several hundred differed significantly in abundance. Comparing α7KO‐HAP1 and HAP1wt cell lines to each other revealed significant protein abundance alterations; however, we also measured differences resulting from nicotine treatment in both cell lines. Among proteins with increased abundance levels due to nicotine treatment included those previously identified: APP, APLP2, and ITM2B. The magnitude of these changes was greater in HAP1wt compared to the α7KO‐HAP1 cell line, implying a potential role for the α7 nAChR in HAP1 cells. Moreover, the data revealed that membrane proteins and proteins commonly associated with neurons were predominant among those with altered abundance. This study, which is the first TMT‐based proteome profiling of HAP1 cells, defines further the effects of nicotine on non‐neuronal cellular proteomes.