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.

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.

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.

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 gene coding for the full-length protein (survival motor neuron; SMN) and the 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.

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.

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

The mammalian 20S proteasome is a heterodimeric cylindrical complex (α7β7β7α7), composed of four rings each composed of seven different α or β 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.

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.

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 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 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.

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.

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.

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.

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.

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.

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.

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.

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.

Generation of DNA double-strand breaks by two independent enzymatic activities in nuclear extracts.

We have reported the existence in rat nuclear extracts of a specific cleavage activity on a DNA fragment containing the human minisatellite MsH42 region (minisatellite plus its flanking sequences). Here, we have developed a system to analyse the nature of the cleavage products from the MsH42 region generated by the nuclear extracts. Our results demonstrated the formation of DNA double-strand breaks (DSB) in the MsH42 region by two different enzymatic activities, and that their distribution along this fragment changes depending on the presence of Mg2+.

alpha-Synuclein expression levels do not significantly affect proteasome function and expression in mice and stably transfected PC12 cell lines.

alpha-Synuclein (alpha-syn) is a small protein of unknown function that is found aggregated in Lewy bodies, the histopathological hallmark of sporadic Parkinson disease and other synucleinopathies. Mutations in the alpha-syn gene and a triplication of its gene locus have been identified in early onset familial Parkinson disease. alpha-Syn turnover can be mediated by the proteasome pathway.

Proteasomal expression, induction of immunoproteasome subunits, and local MHC class I presentation in myofibrillar myopathy and inclusion body myositis.

Inclusion body myositis (IBM) and myofibrillar myopathy (MM) are diseases characterized by the abnormal accumulation of proteins in muscle fibers, including desmin, alphaB-crystallin, gelsolin, actin, kinases, and phospho-tau, along with ubiquitin in muscle fibers, suggesting abnormal protein degradation as a possible cause of the surplus myopathy. Since the ubiquitin-proteasome system plays a crucial role in non-lysosomal protein degradation, the present study has examined by immunohistochemistry the expression of components of the catalytic core of 20S proteasomes and its regulators: 19S and PA28alpha/beta, and the expression of immunoproteasome subunits LMP2, LMP7, and MECL1 in 8 patients with MM and 10 patients with IBM. The patients with MM were from 6 unrelated families, 2 sporadic cases, I with autosomal recessive and 5 with autosomal dominant inheritance.

Neuronal induction of the immunoproteasome in Huntington's disease.

Huntington's disease (HD) inclusions are stained with anti-ubiquitin and anti-proteasome antibodies. This, together with proteasome activity studies on transfected cells, suggest that an impairment of the ubiquitin-proteasome system (UPS) may be key in HD pathogenesis. To test whether proteasome activity is impaired in vivo, we performed enzymatic assays for the three peptidase activities of the proteasome in brain extracts from the HD94 conditional mouse model of HD.