Mitochondrial LonP1 protease is implicated in the degradation of unstable Parkinson's disease-associated DJ-1/PARK 7 missense mutants.

DJ-1/PARK7 mutations are linked with familial forms of early-onset Parkinson's disease (PD). We have studied the degradation of untagged DJ-1 wild type (WT) and missense mutants in mouse embryonic fibroblasts obtained from DJ-1-null mice, an approach closer to the situation in patients carrying homozygous mutations. The results showed that the mutants L10P, M26I, A107P, P158Δ, L166P, E163K, and L172Q are unstable proteins, while A39S, E64D, R98Q, A104T, D149A, A171S, K175E, and A179T are as stable as DJ-1 WT. Inhibition of proteasomal and autophagic-lysosomal pathways had little effect on their degradation. Immunofluorescence and biochemical fractionation studies indicated that M26I, A107P, P158Δ, L166P, E163K, and L172Q mutants associate with mitochondria. Silencing of mitochondrial matrix protease LonP1 produced a strong reduction of the degradation of the mitochondrial-associated DJ-1 mutants A107P, P158Δ, L166P, E163K, and L172Q but not of mutant L10P. These results demonstrated a mitochondrial pathway of degradation of those DJ-1 missense mutants implicated in PD pathogenesis.

Correction: Inhibitors of lysosomal function or serum starvation in control or LAMP2 deficient cells do not modify the cellular levels of Parkinson disease-associated DJ-1/PARK 7 protein.

[This corrects the article DOI: 10.1371/journal.pone.0201152.].

Inhibitors of lysosomal function or serum starvation in control or LAMP2 deficient cells do not modify the cellular levels of Parkinson disease-associated DJ-1/PARK 7 protein.

Mutations in PARK7/DJ-1 gene are associated with familial autosomal recessive Parkinson disease. Recently, lysosomes and chaperone mediated autophagy (CMA) has been reported to participate in the degradation of DJ-1/PARK7 protein. Lamp-2A isoform is considered as the lysosomal receptor for the uptake of proteins being degraded by the CMA pathway. We have used several cell lines with disrupted LAMP2 gene expression and their respective control cells to test the possible role of lysosomal degradation and in particular CMA in DJ-1 /PARK7 degradation. Interruption of LAMP-2 expression did not result in an increase of the steady-state protein levels of DJ-1 /PARK7, as it would have been expected. Furthermore, no change in DJ-1 /PARK7 protein levels were observed upon inhibition of lysosomal function with NH4Cl or NH4Cl plus leupeptin, or after activation of CMA by serum starvation for 24h. Accordingly, we have not found any evidence that DJ-1 /PARK7 protein levels are regulated via lysosomal degradation or the CMA pathway.

CCAAT/Enhancer binding protein ß silencing mitigates glial activation and neurodegeneration in a rat model of Parkinson's disease.

The CCAAT/Enhancer binding protein ß (C/EBPß) is a transcription factor involved in numerous physiological as well as pathological conditions in the brain. However, little is known regarding its possible role in neurodegenerative disorders. We have previously shown that C/EBPß regulates the expression of genes involved in inflammatory processes and brain injury. Here, we have analyzed the effects of C/EBPß interference in dopaminergic cell death and glial activation in the 6-hydroxydopamine model of Parkinson's disease. Our results showed that lentivirus-mediated C/EBPß deprivation conferred marked in vitro and in vivo neuroprotection of dopaminergic cells concomitant with a significant attenuation of the level of the inflammatory response and glial activation. Additionally, C/EBPß interference diminished the induction of α-synuclein in the substantia nigra pars compacta of animals injected with 6-hydroxydopamine. Taking together, these results reveal an essential function for C/EBPß in the pathways leading to inflammatory-mediated brain damage and suggest novel roles for C/EBPß in neurodegenerative diseases, specifically in Parkinson's disease, opening the door for new therapeutic interventions.

Lysine-Less Variants of Spinal Muscular Atrophy SMN and SMNΔ7 Proteins Are Degraded by the Proteasome Pathway.

Spinal muscular atrophy is due to mutations affecting the SMN1 gene coding for the full-length protein (survival motor neuron; SMN) and the SMN2 gene that preferentially generates an exon 7-deleted protein (SMNΔ7) by alternative splicing. To study SMN and SMNΔ7 degradation in the cell, we have used tagged versions at the N- (Flag) or C-terminus (V5) of both proteins. Transfection of those constructs into HeLa cells and treatment with cycloheximide showed that those protein constructs were degraded. Proteasomal degradation usually requires prior lysine ubiquitylation. Surprisingly, lysine-less variants of both proteins tagged either at N- (Flag) or C-terminus (V5) were also degraded. The degradation of the endogenous SMN protein, and the protein constructs mentioned above, was mediated by the proteasome, as it was blocked by lactacystin, a specific and irreversible proteasomal inhibitor. The results obtained allowed us to conclude that SMN and SMNΔ7 proteasomal degradation did not absolutely require internal ubiquitylation nor N-terminal ubiquitylation (prevented by N-terminal tagging). While the above conclusions are firmly supported by the experimental data presented, we discuss and justify the need of deep proteomic techniques for the study of SMN complex components (orphan and bound) turn-over to understand the physiological relevant mechanisms of degradation of SMN and SMNΔ7 in the cell.

Protein degradation in a LAMP-2-deficient B-lymphoblastoid cell line from a patient with Danon disease.

Danon disease, a condition characterized by cardiomyopathy, myopathy, and intellectual disability, is caused by mutations in the LAMP-2 gene. Lamp-2A protein, generated by alternative splicing from the Lamp-2 pre-mRNA, is reported to be the lysosomal membrane receptor essential for the chaperone-mediated autophagic pathway (CMA) aimed to selective protein targeting and translocation into the lysosomal lumen for degradation. To study the relevance of Lamp-2 in protein degradation, a lymphoblastoid cell line was obtained by EBV transformation of B-cells from a Danon patient. The derived cell line showed no significant expression of Lamp-2 protein. The steady-state mRNA and protein levels of alpha-synuclein, IΚBα, Rcan1, and glyceraldehyde-3-phosphate dehydrogenase, four proteins reported to be selective substrates of the CMA pathway, were similar in control and Lamp-2-deficient cells. Inhibition of protein synthesis showed that the half-life of alpha-synuclein, IΚBα, and Rcan1 was similar in control and Lamp-2-deficient cells, and its degradation prevented by proteasome inhibitors. Both in control and Lamp-2-deficient cells, induction of CMA and macroautophagy by serum and aminoacid starvation of cells for 8h produced a similar decrease in IΚBα and Rcan1 protein levels and was prevented by the addition of lysosome and autophagy inhibitors. In conclusion, the results presented here showed that Lamp-2 deficiency in human lymphoblastoid cells did not modify the steady-state levels or the degradation of several protein substrates reported as selective substrates of the CMA pathway.

Proteins directly interacting with mammalian 20S proteasomal subunits and ubiquitin-independent proteasomal degradation.

The mammalian 20S proteasome is a heterodimeric cylindrical complex (α7ß7ß7α7), composed of four rings each composed of seven different α or ß subunits with broad proteolytic activity. We review the mammalian proteins shown to directly interact with specific 20S proteasomal subunits and those subjected to ubiquitin-independent proteasomal degradation (UIPD). The published reports of proteins that interact with specific proteasomal subunits, and others found on interactome databases and those that are degraded by a UIPD mechanism, overlap by only a few protein members. Therefore, systematic studies of the specificity of the interactions, the elucidation of the protein regions implicated in the interactions (that may or may not be followed by degradation) and competition experiments between proteins known to interact with the same proteasomal subunit, are needed. Those studies should provide a coherent picture of the molecular mechanisms governing the interactions of cellular proteins with proteasomal subunits, and their relevance to cell proteostasis and cell functioning.

Epitope Mapping of Antibodies to Alpha-Synuclein in LRRK2 Mutation Carriers, Idiopathic Parkinson Disease Patients, and Healthy Controls.

Alpha-synuclein (Snca) plays a major role in Parkinson disease (PD). Circulating anti-Snca antibodies has been described in PD patients and healthy controls, but they have been poorly characterized. This study was designed to assess the prevalence of anti-Snca reactivity in human subjects carrying the LRRK2 mutation, idiopathic PD (iPD) patients, and healthy controls and to map the epitopes of the anti-Snca antibodies. Antibodies to Snca were detected by ELISA and immunoblotting using purified recombinant Snca in plasma from individuals carrying LRRK2 mutations (104), iPD patients (59), and healthy controls (83). Epitopes of antibodies were mapped using recombinant protein constructs comprising different regions of Snca. Clear positive anti-Snca reactivity showed no correlation with age, sex, years of evolution, or the disability scores for PD patients and anti-Snca reactivity was not prevalent in human patients with other neurological or autoimmune diseases. Thirteen of the positive individuals were carriers of LRRK2 mutations either non-manifesting (8 out 49 screened) or manifesting (5 positive out 55), three positive (out of 59) were iPD patients, and five positive (out of 83) were healthy controls. Epitope mapping showed that antibodies against the N-terminal (a.a. 1-60) or C-terminal (a.a. 109-140) regions of Snca predominate in LRRK2 mutation carriers and iPD patients, being N122 a critical amino acid for recognition by the anti-C-terminal directed antibodies. Anti-Snca circulating antibodies seem to cluster within families carrying the LRRK2 mutation indicating possible genetic or common environmental factors in the generation of anti-Snca antibodies. These results suggest that case-controls' studies are insufficient and further studies in family cohorts of patients and healthy controls should be undertaken, to progress in the understanding of the possible relationship of anti-Snca antibodies and PD pathology.

Mechanism of cleavage of alpha-synuclein by the 20S proteasome and modulation of its degradation by the RedOx state of the N-terminal methionines.

Alpha-synuclein is a small protein implicated in the pathophysiology of Parkinson's disease (PD). We have investigated the mechanism of cleavage of alpha-synuclein by the 20S proteasome. Alpha-synuclein interacts with the C8 (α7) subunit of the proteasome. The N-terminal part of alpha-synuclein (amino acids 1-60) is essential for its proteasomal degradation and analysis of peptides released from proteasomal digestion allows concluding that initial cleavages occur within the N-terminal region of the molecule. Aggregated alpha-synucleins are also degraded by the proteasome with a reduced rate, likely due to Met oxidation. In fact, mild oxidation of alpha-synuclein with H2O2 resulted in the inhibition of its degradation by the proteasome, mainly due to oxidation of Met 1 and 5 of alpha-synuclein. The inhibition was reversed by treatment of the oxidized protein with methionine sulfoxide reductases (MsrA plus MsrB). Similarly, treatment with H2O2 of N2A cells transfected with alpha-synuclein resulted in the inhibition of its degradation that was also reverted by co-transfection of MsrA plus MsrB. These results clearly indicate that oxidative stress, a common feature of PD and other synucleinopathies, promotes a RedOx change in the proteostasis of alpha-synuclein due to Met oxidation and reduced proteasomal degradation; compromised reversion of those oxidative changes would result in the accumulation of oxidative damaged alpha-synuclein likely contributing to the pathogenesis of PD.

The N-terminal region of Nurr1 (a.a 1-31) is essential for its efficient degradation by the ubiquitin proteasome pathway.

NURR1/NR4A2 is an orphan nuclear receptor that is critical for the development and maintenance of mesencephalic dopaminergic neurons and regulates transcription of genes involved in the function of dopaminergic neurons directly via specific NGFI-B response elements (NBRE).and substantial data support a possible role of Nurr1 in the pathogenesis of Parkinson's disease (PD). Here we show that Nurr1 is degraded by the ubiquitin-proteasome pathway and determined that N-terminal region (a.a 1-31) of Nurr1 is essential for an efficient targeting of Nurr1 to degradation in the cell. Nurr1 Δ1-31 has a much longer half-life, and as a consequence its steady-state protein levels were higher, than full-length Nurr1 in the cell. Nurr1 Δ1-31 was as potent as Nurr1 full length in transcriptional luciferase reporter assays after normalization with the corresponding steady-state protein expression levels, either in trans-activation of NBRE or trans-repression of iNOS (inducible NO synthase) reporters. These results suggest that Nurr1 Δ1-31, because of longer persistence in the cell, can be a good candidate for gene and cell therapies in the treatment of PD.

A critical appraisal of quantitative studies of protein degradation in the framework of cellular proteostasis.

Protein homeostasis, proteostasis, is essential to understand cell function. Protein degradation is a crucial component of the proteostatic mechanisms of the cell. Experiments on protein degradation are nowadays present in many investigations in the field of molecular and cell biology. In the present paper, we focus on the different experimental approaches to study protein degradation and present a critical appraisal of the results derived from steady-state and kinetic experiments using detection of unlabelled and labelled protein methodologies with a proteostatic perspective. This perspective allows pinpointing the limitations in interpretation of results and the need of further experiments and/or controls to establish "definitive evidence" for the role of protein degradation in the proteostasis of a given protein or the entire proteome. We also provide a spreadsheet for simple calculations of mRNA and protein decays for mimicking different experimental conditions and a checklist for the analysis of experiments dealing with protein degradation studies that may be useful for researchers interested in the area of protein turnover.

Reduced protein stability of human DJ-1/PARK7 L166P, linked to autosomal recessive Parkinson disease, is due to direct endoproteolytic cleavage by the proteasome.

Parkinson's disease (PD) is characterized by dopaminergic dysfunction and degeneration. DJ-1/PARK7 mutations have been linked with a familial form of early onset PD. In this study, we found that human DJ-1 wild type and the missense mutants M26I, R98Q, A104T and D149A were stable proteins in cells, only the L166P mutant was unstable. In parallel, the former were not degraded and the L166P mutant was directly degraded in vitro by proteasome-mediated endoproteolytic cleavage. Furthermore, genetic evidence in fission yeast showed the direct involvement of proteasome in the degradation of human DJ-1 L166P and the corresponding L169P mutant of SPAC22E12.03c, the human orthologue of DJ-1 in Schizosaccharomyces Pombe, as their protein levels were increased at restrictive temperature in fission yeast (mts4 and pts1-732) harboring temperature sensitive mutations in proteasomal subunits. In total, our results provide evidence that direct proteasomal endoproteolytic cleavage of DJ-1 L166P is the mechanism of degradation contributing to the loss-of-function of the mutant protein, a property not shared by other DJ-1 missense mutants associated with PD.

Synphilin-1 inhibits alpha-synuclein degradation by the proteasome.

Intracellular deposits of aggregated alpha-synuclein are a hallmark of Parkinson's disease. Protein-protein interactions are critical in the regulation of cell proteostasis. Synphilin-1 interacts both in vitro and in vivo with alpha-synuclein promoting its aggregation. We report here that synphilin-1 specifically inhibits the degradation of alpha-synuclein wild-type and its missense mutants by the 20S proteasome due at least in part by the interaction of the ankyrin and coiled-coil domains of synphilin-1 (amino acids 331-555) with the N-terminal region (amino acids 1-60) of alpha-synuclein. Co-expression of synphilin-1 and alpha-synuclein wild-type in HeLa and N2A cells produces a specific increase in the half-life of alpha-synuclein, as degradation of unstable fluorescent reporters is not affected. Synphilin-1 inhibition can be relieved by co-expression of Siah-1 that targets synphilin-1 to degradation. Synphilin-1 inhibition of the proteasomal pathway of degradation of alpha-synuclein may help to understand the pathophysiological changes occurring in PD and other synucleinopathies.

Cytomegalovirus promoter up-regulation is the major cause of increased protein levels of unstable reporter proteins after treatment of living cells with proteasome inhibitors.

Fluorescent unstable proteins obtained by the fusion of a fluorescent protein coding sequence with specific amino acid sequences that promote its fast degradation have become popular to gauge the activity of the ubiquitin/proteasome system in living cells. The steady-state levels of expression of these unstable proteins is low in agreement with their short half-lives, and they accumulate in the cell upon treatment with proteasome inhibitors. We show here that this accumulation is mainly due to transcriptional up-regulation of the cytomegalovirus promoter by proteasome inhibitors and mediated, at least in part, by AP1 transactivation. These simple facts put under quarantine conclusions reached about the activity of the ubiquitin/proteasome pathway in animal cells in culture or in transgenic mice, where popular cytomegalovirus-driven constructs are used, as transcriptional regulation of the expression of the reporter protein construct and not degradation of the unstable protein by the ubiquitin/proteasome system may contribute significantly to the interpretation of the results observed.

Parkin deficiency increases the resistance of midbrain neurons and glia to mild proteasome inhibition: the role of autophagy and glutathione homeostasis.

Parkin mutations in humans produce parkinsonism whose pathogenesis is related to impaired protein degradation, increased free radicals and abnormal neurotransmitter release. In this study, we have investigated whether partial proteasomal inhibition by epoxomicin, an ubiquitin proteasomal system (UPS) irreversible inhibitor, further aggravates the cellular effects of parkin suppression in midbrain neurons and glia. We observed that parkin null (PK-KO) midbrain neuronal cultures are resistant to epoxomicin-induced cell death. This resistance is due to increased GSH and DJ-1 protein levels in PK-KO mice. The treatment with epoxomicin increases, in wild type (WT) cultures, the pro-apoptotic Bax/Bcl-2 ratio, the phosphorylation of tau, and the levels of chaperones heat-shock protein 70 and C-terminal Hsc-interacting protein, but none of these effects took place in epoxomicin-treated PK-KO cultures. Poly-ubiquitinated proteins increased more in WT than in PK-KO-treated neuronal cultures. Parkin accumulated in WT neuronal cultures treated with epoxomicin. Markers of autophagy, such as LC3II/I, were increased in naïve PK-KO cultures, and further increased after treatment with epoxomicin, implying that the blockade of the proteasome in PK-KO neurons triggers the enhancement of autophagy. The treatment with l-buthionine-S,R-sulfoximine and the inhibition of autophagy, however, reverted the increase resistance to epoxomicin of the PK-KO cultures. We also found that PK-KO glial cells, stressed by growth in defined medium and depleted of GSH, were more susceptible to epoxomicin induced cell death than WT glia treated similarly. This susceptibility was linked to reduced GSH levels and less heat-shock protein 70 response, and to activation of p-serine/threonine kinase protein signaling pathway as well as to increased poly-ubiquitinated proteins. These data suggest that mild UPS inhibition is compensated by other mechanisms in PK-KO midbrain neurons. However the depletion of GSH, as happens in stressed glia, suppresses the protection against UPS inhibition-induced cell death. Furthermore, GSH inhibition regulated differentially UPS activity and in old PK-KO mice, which have depletion of GSH, UPS activity is decreased in comparison with that of old-WT.

Infection with Salmonella typhimurium has no effect on the composition and cleavage specificity of the 20S proteasome in human lymphoid cells.

Human leucocyte antigen (HLA)-B27 is strongly associated with spondyloarthropathies, including reactive arthritis. Several Gram-negative bacteria, such as Salmonella typhimurium, can trigger this disease. It has been suggested that peptides derived from bacterial proteins and presented by HLA-B27 to cytotoxic T lymphocytes might show molecular mimicry with autologous peptides, leading to T-cell cross-reaction and autoimmunity. Antigen presentation in Salmonella-infected cells could be modulated by changes in the composition of the proteasome, which is the major proteolytic system that generates major histocompatibility complex class I ligands. In this study we analysed whether the composition or activity of the 20S proteasome was altered upon infection of lymphoid cells by S. typhimurium. Two-dimensional gel electrophoresis failed to show any differences between the composition of 20S proteasomes from cells infected with S. typhimurium for 24 hr, relative to non-infected cells. In addition, digestions of oxidized insulin B-chain with purified 20S proteasomes from non-infected and infected cells generated the same products, indicating that the proteasomal cleavage specificity was not altered upon infection. These data indicate that infection of lymphoid cells by S. typhimurium fails to induce formation of immunoproteasomes or otherwise alter the proteolytic specificity of the 20S proteasome.

Constitutive/hypoxic degradation of HIF-alpha proteins by the proteasome is independent of von Hippel Lindau protein ubiquitylation and the transactivation activity of the protein.

The transcriptional activator complex HIF-1 plays a key role in the long term adaptation of cells and tissues to their hypoxic microenvironment by stimulating the expression of genes involved in angiogenesis and glycolysis. The expression of the HIF-1 complex is regulated by the levels of its HIF-alpha subunits that are degraded under normoxic conditions by the ubiquitin-proteasome system. Whereas this pathway of HIF-alpha protein degradation has been well characterized, little is known of their turnover during prolonged hypoxic conditions. Herein, we describe a pathway by which HIF-1alpha and HIF-2alpha proteins are constitutively degraded during hypoxia by the proteasome system, although without requirement of prior ubiquitylation. The constitutive/hypoxic degradation of HIF-alpha proteins is independent of the presence of VHL, binding to DNA, or the formation of a transcriptionally active HIF-1 complex. These results are further strengthened by the demonstration that HIF-alpha proteins are directly degraded in a reconstituted in vitro assay by the proteasome. Finally, we demonstrate that the persistent down-regulation of HIF-1alpha during prolonged hypoxia is mainly caused by a decreased production of the protein without change in its degradation rate. This constitutive, ubiquitin-independent proteasomal degradation pathway of HIF-alpha proteins has to be taken into account in understanding the biology as well as in the development of therapeutic interventions of highly hypoxic tumors.

Inhibition of 26S proteasome activity by huntingtin filaments but not inclusion bodies isolated from mouse and human brain.

In Huntington's disease (HD), as in the rest of CAG triplet-repeat disorders, the expanded polyglutamine (polyQ)-containing proteins form intraneuronal fibrillar aggregates that are gathered into inclusion bodies (IBs). Since IBs contain ubiquitin and proteasome subunits, it was proposed that inhibition of proteasome activity might underlie pathogenesis of polyQ disorders. Recent in vitro enzymatic studies revealed the inability of eukaryotic proteasomes to digest expanded polyQ, thus suggesting that occasional failure of polyQ to exit the proteasome may interfere with its proteolytic function. However, it has also recently been found that in vitro assembled aggregates made of synthetic polyQ fail to inhibit proteasome activity. Because synthetic polyQ aggregates lack the post-translational modifications found inside affected neurons, such as poly ubiquitylation, we decided to study the effect of mutant huntingtin (htt) aggregates isolated from the Tet/HD94 mouse model and from human HD brain tissue. Here, we show that isolated ubiquitylated filamentous htt aggregates, extracted from IBs by a previously reported method, selectively inhibited the in vitro peptidase activity of the 26S but not of the 20S proteasome in a non-competitive manner. In good agreement, immuno-electron microscopy revealed a direct interaction of htt filaments with the 19S ubiquitin-interacting regulatory caps of the 26S proteasome. Here, we also report a new method for isolation of IBs based on magnetic sorting. Interestingly, isolated IBs did not modify proteasome activity. Our results therefore show that mutant htt filamentous aggregates can inhibit proteasome activity, but only when not recruited into IBs, thus strengthening the notion that IB formation is protective by neutralizing toxicity of dispersed filamentous htt aggregates.

Mechanism of direct degradation of IkappaBalpha by 20S proteasome.

IkappaBalpha regulates activation of the transcription factor NF-kappaB. NF-kappaB is activated in response to several stimuli, i.e. proinflamatory cytokines, infections, and physical stress. This signal dependent pathway involves IkappaBalpha phosphorylation, ubiquitylation, and degradation by 26S proteasome. A signal independent (basal) turnover of IkappaBalpha has also been described. Here, we show that IkappaBalpha can be directly degraded by 20S proteasomes. Deletion constructs of IkappaBalpha allow us to the determine that N-terminal (DeltaN 1-70) and C-terminal regions (DeltaC 280-327, removing the PEST region) of IkappaBalpha are not required for IkappaBalpha degradation, while a further C-terminal deletion including part of the arm repeats (DeltaC2 245-327) almost completely suppress the degradation by 20S proteasome. Binding and competition experiments demonstrate that the degradation of IkappaBalpha involves specific interactions with alpha2(C3) subunit of the proteasome. Finally, p65/relA (not itself a substrate for 20S proteasome) inhibits the degradation of IkappaBalpha by the proteasome. These results recapitulate in vitro the main characteristics of signal independent (basal) turnover of IkappaBalpha demonstrated in intact cells.