Juvenile Parkinsonism Research Articles

Regulation of Parkin E3 ubiquitin ligase activity.

Parkin is an E3 ubiquitin ligase mutated in autosomal recessive juvenile Parkinson's disease. In addition, it is a putative tumour suppressor, and has roles outside its enzymatic activity. It is critical for mitochondrial clearance through mitophagy, and is an essential protein in most eukaryotes. As such, it is a tightly controlled protein, regulated through an array of external interactions with multiple proteins, posttranslational modifications including phosphorylation and S-nitrosylation, and self-regulation through internal associations. In this review, we highlight some of the recent studies into Parkin regulation and discuss future challenges for gaining a full molecular understanding of the regulation of Parkin E3 ligase activity.

Array CGH increasingly used in prenatal and postnatal testing

Array CGH increasingly used in prenatal and postnatal testing

Neddylation positively regulates the ubiquitin E3 ligase activity of parkin

Mutations in the parkin gene underlie a familial form of Parkinson's disease known as autosomal recessive juvenile Parkinsonism (AR-JP). Dysfunction of parkin, a ubiquitin E3 ligase, has been implicated in the accumulation of ubiquitin proteasome system-destined substrates and eventually leads to cell death. However, regulation of parkin enzymatic activity is incompletely understood. Here we investigated whether the ubiquitin E3 ligase activity of parkin could be regulated by neddylation. We found that parkin could be a target of covalent modification with NEDD8, a ubiquitin-like posttranslational modifier. In addition, NEDD8 attachment caused an increase of parkin activity through the increased binding affinity for ubiquitin-conjugating E2 enzyme as well as the enhanced formation of the complex containing parkin and substrates. These findings point to the functional importance of NEDD8 and suggest that neddylation is one to the diverse modes of parkin regulation, potentially linking it to the pathogenesis of AR-JP.

Selective IGT decision-making impairment in a patient with juvenile Parkinson's disease and pathological gambling: A role for dopaminergic therapy?

The orbitofrontal cortex and the dopaminergic system are structures involved in managing impulsivity and sensibility to reinforcements, and both are typically impaired in Parkinson's disease (PD). Also, l-DOPA treatment can contribute to the development of the ‘Dopamine Dysregulation Syndrome’, a syndrome that can influence the patients' personality and lead to risk-taking behaviors. In this study, we describe the case of a 42-year-old woman (LT) affected by juvenile PD, treated with both l-DOPA and dopamine agonists, who showed a sudden onset of pathological gambling (PG), as the only neuropsychiatric symptom. We assessed LT with a full neuropsychological battery and the Iowa Gambling Task (IGT), in order to describe her specific failure in decision making. LT's performance on the IGT is compared with that of 15 non-demented PD patients under therapy with dopamine agonists and no behavioral dysregulations and with that of 16 age- and education-matched healthy subjects. Results showed fully preserved memory, executive functions, and reasoning abilities for LT, but a remarkable and stable impairment in the IGT. Performance of LT on the IGT is significantly lower than that of both control groups. This case shows, for the first time, that high cognitive functioning and preserved executive functions are no guarantee for advantageous decision making, and that the onset of PG is consistent with selective orbitofrontal disruption and side-effects of dopamine agonist therapy. It is also showed that the IGT is a useful neuropsychological device to detect specific risk-taking behaviors, which compromise functioning in real life.

Parkin-induced defects in neurophysiology and locomotion are generated by metabolic dysfunction and not oxidative stress

Parkinson's disease (PD) is characterized by movement disorders, including bradykinesia. Analysis of inherited, juvenile PD, identified several genes linked via a common pathway to mitochondrial dysfunction. In this study, we demonstrate that the larva of the Drosophila parkin mutant faithfully models the locomotory and metabolic defects of PD and is an excellent system for investigating their inter-relationship. parkin larvae displayed a marked bradykinesia that was caused by a reduction in both the frequency of peristalsis and speed of muscle contractions. Rescue experiments confirmed that this phenotype was due to a defect in the nervous system and not in the muscle. Furthermore, recordings of motoneuron activity in parkin larvae revealed reduced bursting and a striking reduction in evoked and miniature excitatory junction potentials, suggesting a neuronal deficit. This was supported by our observations in parkin larvae that the resting potential was depolarized, oxygen consumption and ATP concentration were drastically reduced while lactate was increased. These findings suggest that neuronal mitochondrial respiration is severely compromised and there is a compensatory switch to glycolysis for energy production.parkin mutants also possessed overgrown neuromuscular synapses, indicative of oxidative stress, which could be rescued by overexpression of parkin or scavengers of reactive oxygen species (ROS). Surprisingly, scavengers of ROS did not rescue the resting membrane potential and locomotory phenotypes. We therefore propose that mitochondrial dysfunction in parkin mutants induces Parkinsonian bradykinesia via a neuronal energy deficit and resulting synaptic failure, rather than as a consequence of downstream oxidative stress.

Protective Effects of 4-Phenylbutyrate Derivatives on the Neuronal Cell Death and Endoplasmic Reticulum Stress

Endoplasmic reticulum (ER) stress responses play an important role in neurodegenerative diseases. Sodium 4-phenylbutyrate (4-PBA) is a terminal aromatic substituted fatty acid that has been used for the treatment of urea cycle disorders. 4-PBA possesses in vitro chemical chaperone activity and reduces the accumulation of Parkin-associated endothelin receptor-like receptor (Pael-R), which is involved in autosomal recessive juvenile parkinsonism (AR-JP). In this study, we show that terminal aromatic substituted fatty acids, including 3-phenylpropionate (3-PPA), 4-PBA, 5-phenylvaleric acid, and 6-phenylhexanoic acid, prevented the aggregation of lactalbumin and bovine serum albumin. Aggregation inhibition increased relative to the number of carbons in the fatty acids. Moreover, these compounds protected cells against ER stress-induced neuronal cell death. The cytoprotective effect correlated with the in vitro chemical chaperone activity. Similarly, cell viability decreased on treatment with tunicamycin, an ER stress inducer, and was dependent on the number of carbons in the fatty acids. Moreover, the expression of glucose-regulated proteins 94 and 78 (GRP94, 78) decreased according to the number of carbons in the fatty acids. Furthermore, we investigated the effects of these compounds on the accumulation of Pael-R in neuroblastoma cells. 3-PPA and 4-PBA significantly suppressed neuronal cell death caused by ER stress induced by the overexpression of Pael-R. Overexpressed Pael-R accumulated in the ER of cells. With 3-PPA and 4-PBA treatment, the localization of the overexpressed Pael-R shifted away from the ER to the cytoplasmic membrane. These results suggest that terminal aromatic substituted fatty acids are potential candidates for the treatment of neurodegenerative diseases.

Suppression of Nrdp1 toxicity by Parkin in Drosophila models

Nrdp1 is a RING finger ubiquitin E3 ligase that interacts with Parkin, and promotes the degradation of Parkin, a causative protein for early onset Autosomal Recessive Juvenile Parkinsonism (AR-JP). To investigate if Nrdp1 plays a role in the pathogenesis of Parkinson’s disease, we generated transgenic Drosophila that expressed Drosophila Nrdp1 (dNrdp1) and dNrdp1 D56V, an aspartic acid to valine mutant at residue 56 that disrupts its ring finger domain, resulting in impaired capacity to degrade its substrate ErbB3. Our data show that a pan-neuronal expression of transgenic dNrdp1 but not dNrdp1 D56V mutant leads to the loss of dopaminergic neurons in brains, resulting in reduction of dopamine production. These flies also manifested decreased flight ability. Co-expression of human Parkin (hParkin) provides protection against toxicity induced by over-expression of dNrdp1, reversing the effects of dNrdp1 on death of dopaminergic neurons, reduction of dopamine production, and decreased flight ability. Taken together, we conclude that Nrdp1 plays a role in neurodegeneration and could be potentially targeted as a therapeutic strategy for Parkinson’s disease.

P32 regulates mitochondrial morphology and dynamics through parkin

Mutations in parkin were first identified in a group of Japanese patients who developed autosomal recessive juvenile Parkinsonism with clinical symptoms similar to idiopathic Parkinson's disease (PD). Parkin is an E3 ligase that targets a number of substrates for ubiquitination. Recent studies show that parkin together with PINK1, another familial-linked PD gene product, is involved in the regulation of mitochondrial dynamics in the cell. In this study, we have identified a mitochondrial protein p32 as a novel interactor of parkin in the brain. We found that p32 can regulate mitochondrial morphology and dynamics by promoting parkin degradation through autophagy. These results suggest that parkin might be an important effector in the regulation of morphology and dynamics of mitochondria.

Role of Inflammatory Related Gene Expression in Clear Cell Renal Cell Carcinoma Development and Clinical Outcomes

Renal cell carcinoma is the eighth most common cancer in the United States and clear cell renal carcinoma is the most common type. Many signaling pathways are implicated in clear cell renal carcinoma development, including the inflammation pathway. However, less is known about how gene expression variation in this pathway influences clear cell renal carcinoma development and clinical outcomes. Gene expression in tumor and adjacent normal tissues from 93 patients was detected using a genome-wide expression array. A panel of 661 inflammation related genes was then analyzed. Differential expression patterns between tumor and normal tissues were identified. Association with recurrence or survival was evaluated with genes showing significant association tested further in a validation set of 258 tumors using an independent platform (quantitative real-time polymerase chain reaction). We identified 151 genes with at least a two-fold change in gene expression between adjacent normal tissue and tumor, of which most were up-regulated in tumors. A total of 20 genes significantly associated with recurrence and/or overall survival were selected for further validation. In the replication data set high expression of GADD45G was significantly associated with a 2.09-fold (95% CI 1.08-6.14, p = 0.034) increased risk of recurrence while high CARD9, NCF2 and CIITA expression was significantly associated with a 2.52-fold (95% CI 1.24-5.12, p = 0.010), 2.26-fold (95% CI 1.12-4.58, p = 0.023) and 2.11-fold (95% CI 1.05-4.27, p = 0.037) increased risk of death, respectively. Results suggest that inflammation gene expression may be significant prognostic biomarkers for the risk of recurrence (GADD45G) and death (CARD9, CIITA and NCF2) in patients with clear cell renal carcinoma.

Analysis of PARK2 gene exon rearrangements in Russian patients with sporadic Parkinson's disease

Deletions and duplications of single exons or exon groups account for a large proportion of the PARK2 gene mutations described in juvenile autosomal recessive Parkinson's disease (PD). We analyzed rearrangements in exons 1 to 12 of the PARK2 gene in Russian sporadic patients with early-onset PD (EOPD) and late-onset PD (LOPD). The frequency of EOPD and LOPD patients carrying these mutations was 12.4% and 3.8%, respectively. The most frequent rearrangements were detected in exons 3 and 4. The odds ratio for EOPD in individuals carrying PARK2 exon deletions and duplications was 13.95 (95% confidence interval [CI], 1.846-105.46; P = .0022). In addition, we found a correlation between exon rearrangements in PARK2 and the age at onset of PD, presence of dystonia, and symmetrical course of the disease. Exon rearrangements in the PARK2 gene play a significant role in the pathogenesis of sporadic PD in Russian patients.

Suicide Without Depression After Withdrawal of a Dopamine Agonist in a Patient With Parkinson's Disease

Back to table of contents Previous article Next article LettersFull AccessSuicide Without Depression After Withdrawal of a Dopamine Agonist in a Patient With Parkinson's DiseaseMélanie Flament, M.D., Gwenolé Loas, M.D., Ph.D., Olivier Godefroy, M.D., Ph.D., and Pierre Krystkowiak, M.D., Ph.D.Mélanie FlamentSearch for more papers by this author, M.D., Gwenolé LoasSearch for more papers by this author, M.D., Ph.D., Olivier GodefroySearch for more papers by this author, M.D., Ph.D., and Pierre KrystkowiakSearch for more papers by this author, M.D., Ph.D.Published Online:1 Oct 2011https://doi.org/10.1176/jnp.23.4.jnpe32AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InEmail To the Editor: A recent multicenter study1 reported that the prevalence of impulse-control disorders (ICD) was 2.72-fold higher in Parkinson's disease (PD) patients treated by dopamine agonists than in PD patients not treated by these medications. Several case reports of attempted suicide or suicide in patients treated by dopamine agonists with pathological gambling have been published.2 A recent cross-sectional study3 on 128 PD patients examined associations between current suicidal and/or death ideation and neurological, neuropsychological, and psychiatric variables. Interestingly, a history of ICD disorders was not a statistically significant factor, but was associated with a trend (p<0.07). On multivariate logistic regression, major depression, psychosis, and elevated score on the Beck Hopelessness Scale were independent predictors of current suicidal and/or death ideation.Case ReportWe report the case of a 44-year-old man treated for juvenile idiopathic Parkinson's disease (PD) with pramipexole (0.7 mg three times daily) and l-dopa-benzeraside (62.5 mg three times daily) for 2 years. The dose of pramipexole was then increased to 0.7-mg tablets 4.5 per day. Several days later, he developed severe ICD with hypersexuality: increased libido, visiting of pornographic websites, and adultery. Six months later, pramipexole was decreased, then stopped, and l-dopa was continued alone. Hypersexuality rapidly resolved, but the patient experienced mixed anxiety/depressive disorder, and he was then admitted to the psychiatric department of a general hospital for 3 weeks and was treated with fluoxetine (20 mg/day). At the last psychiatric visit, 4 weeks after discharge from hospital, the patient's main complaint was related to his sexual activity; he said that he had sexual intercourse much less frequently than while he was taking pramipexole. Despite his marked anxiety, he did not present any symptoms of depression. The following day, he experienced a benign fall while jogging and then committed suicide by hanging from a tree, 2 months after withdrawal of pramipexole.DiscussionThis case report illustrates, for the first time, a potential link between suicide and ICD. Suicide due to depression was excluded by the psychiatric assessment on the day before the patient's death. When pramipexole was stopped, compulsive hypersexuality diminished dramatically and then resolved. The patient was then unable to accept his “normal” sexual activity and experienced a kind of pathological bereavement leading to hopelessness. Another hypothesis could be that the suicide was the result of impulsive behavior secondary to dopamine-agonist treatment, but the fall appeared to be the triggering event in a “hopelessness” patient. This potential cause can be excluded because L-dopa treatment was stable at low dose (187.5 mg/day). However, duration of the illness usually facilitates psychological acceptance and elaboration of the illness by the patient, as proposed by several authors using the term “work of disease” or “disease experience.” The patient's paradoxically increased physical or sexual capacities could considerably interfere with this “work of disease” and increase the risk of suicide. This case report suggests the need to identify patients at risk of suicidal behavior before administering dopamine agonists. The presence of impulse-control disorder and not only depression must therefore be investigated in PD patients who attempt suicide.University Department of NeurologyUniversity Department of PsychiatryUniversité de Picardie Amiens, FranceCorrespondence: Prof. Gwenolé Loas e-mail: Loas.[email protected]fr1. Weintraub D , Koester J , Potenza MN , et al.: Impulse-control disorders in Parkinson disease: a cross-sectional study of 3,090 patients. Arch Neurol 2010; 67:589–595Crossref, Medline, Google Scholar2. Kolla BN , Mansukhani MP , Barraza R , et al.: Impact of dopamine agonists on compulsive behaviors: a case series of pramipexole-induced pathological gambling. Psychosomatics 2010; 51:271–273Crossref, Medline, Google Scholar3. Kostic V , Pekmezovic T , Tomic A , et al.: Suicide and suicidal ideation in Parkinson's disease. J Neurol Sci 2010; 289:40–43Crossref, Medline, Google Scholar FiguresReferencesCited byDetailsCited ByHospital care for mental health and substance abuse conditions in Parkinson's disease9 December 2016 | Movement Disorders, Vol. 31, No. 12Subthalamic Nucleus Versus Globus Pallidus Internus Deep Brain Stimulation: Translating the Rematch Into Clinical Practice10 April 2014 | Movement Disorders Clinical Practice, Vol. 1, No. 1 Volume 23Issue 4 Fall 2011Pages E32-E32 Metrics PDF download History Published online 1 October 2011 Published in print 1 October 2011

Regulation of Intracellular Manganese Homeostasis by Kufor-Rakeb Syndrome-associated ATP13A2 Protein

Mutations in the ATP13A2 gene are associated with Kufor-Rakeb syndrome (KRS) and are found also in patients with various other types of parkinsonism. ATP13A2 encodes a predicted lysosomal P5-type ATPase that plays important roles in regulating cation homeostasis. Disturbance of cation homeostasis in brains is indicated in Parkinson disease pathogenesis. In this study, we explored the biological function of ATP13A2 as well as the pathogenic mechanism of KRS pathogenic ATP13A2 mutants. The results revealed that wild-type ATP13A2, but not the KRS pathogenic ATP13A2 mutants, protected cells from Mn(2+)-induced cell death in mammalian cell lines and primary rat neuronal cultures. In addition, wild-type ATP13A2 reduced intracellular manganese concentrations and prevented cytochrome c release from mitochondria compared with the pathogenic mutants. Furthermore, endogenous ATP13A2 was up-regulated upon Mn(2+) treatment. Our results suggest that ATP13A2 plays important roles in protecting cells against manganese cytotoxicity via regulating intracellular manganese homeostasis. The study provides a potential mechanism of KRS and parkinsonism pathogenesis.

Policing Parkin with a UblD

Mutations in the E3 ubiquitin ligase Parkin are linked to Autosomal Recessive Juvenile Parkinsonism, including a cluster of pathogenic mutations in exon 2 that gives rise to Parkin9s N‐terminal ubiquitin‐like domain (UblD). A study in this issue of The EMBO Journal reveals a new role for this domain, showing that the UblD engages in an intramolecular interaction with Parkin9s C‐terminal region to restrict autoubiquitination. Loss of this autoinhibitory mechanism in pathogenic variants renders Parkin constitutively active. Moreover, other proteins that bind to Parkin9s UblD can also relieve autoinhibition, suggesting control by substrate‐mediated activation.

Autoregulation of Parkin activity through its ubiquitin-like domain

Parkin is an E3-ubiquitin ligase belonging to the RBR (RING-InBetweenRING-RING family), and is involved in the neurodegenerative disorder Parkinson's disease. Autosomal recessive juvenile Parkinsonism, which is one of the most common familial forms of the disease, is directly linked to mutations in the parkin gene. However, the molecular mechanisms of Parkin dysfunction in the disease state remain to be established. We now demonstrate that the ubiquitin-like domain of Parkin functions to inhibit its autoubiquitination. Moreover pathogenic Parkin mutations disrupt this autoinhibition, resulting in a constitutively active molecule. In addition, we show that the mechanism of autoregulation involves ubiquitin binding by a C-terminal region of Parkin. Our observations provide important molecular insights into the underlying basis of Parkinson's disease, and in the regulation of RBR E3-ligase activity.

Successful twin pregnancy in a patient with parkin-associated autosomal recessive juvenile parkinsonism

BackgroundPregnancy in patients with Parkinson disease is a rare occurrence. To the best of our knowledge, the effect of pregnancy as well as treatment in genetically confirmed autosomal recessive juvenile parkinsonism (ARJP) has never been reported. Here, we report the first case of pregnancy in a patient with ARJP associated with a parkin gene mutation, ARJP/PARK2.Case presentationA 27-year-old woman with ARJP/PARK2 was diagnosed as having a spontaneous dichorionic/diamniotic twin pregnancy. Exacerbation of motor disability was noted between ovulation and menstruation before pregnancy as well as during late pregnancy, suggesting that her parkinsonism might have been influenced by fluctuations in the levels of endogenous sex hormones. During the organogenesis period, she was only treated with levodopa/carbidopa, although she continued to receive inpatient hospital care for assistance in the activities of daily living. After the organogenesis period, she was administered sufficient amounts of antiparkinsonian drugs. She delivered healthy male twins, and psychomotor development of both the babies was normal at the age of 2 years.ConclusionPregnancy may worsen the symptoms of ARJP/PARK2, although appropriate treatments with antiparkinsonian drugs and adequate assistance in the activities of daily living might enable successful pregnancy and birth of healthy children.

Overexpression of Nrdp1/FLRF sensitizes cells to oxidative stress

Nrdp1 is a RING finger containing ubiquitin E3 ligase that interacts with and modulates activity of multiple proteins, including ErbB3 and Parkin, a causative protein for early onset recessive juvenile parkinsonism (AR-JP). To investigate the functions of Nrdp1, we have generated stable Tet-On inducible HEK293 cells that overexpress Flag-tagged full length Nrdp1, N-terminal Nrdp1 and C-terminal Nrdp1. We demonstrate that overexpression of full-length Nrdp1, not Nrdp1 N-terminus or Nrdp1 C-terminus in cultured HEK293 cells, inhibits cell growth. In addition, we have treated cells with hydroxynonenal (HNE), 6-hydroxydopamine (6-OHDA), and hydrogen peroxide (H2O2) at different concentrations. We have found that Nrdp1 overexpression sensitizes HEK293 cells to oxidative stressors in a dosage-dependent manner. Our data provide insights into understanding the potential role of Nrdp1 in cell growth, apoptosis and oxidative stress, and in the pathogenesis of Parkinson’s disease.

Study of Methylation Levels of Parkin Gene Promoter in Parkinson's Disease Patients

ABSTRACTMutations in the parkin gene have been significantly linked to autosomal recessive juvenile parkinsonism (ARJP). In addition to its association with ARJP, loss of heterozygosity of parkin has been found in several types of malignant tumors, including ovarian, breast, and hepatocellular tumors. Abnormal methylation of parkin promoter was observed in patients with acute lymphoblastic leukemia and chronic myelogenous leukemia in lymphoid blast crisis. We hypothesized that hypermethylation of the parkin promoter might reduce the expression of parkin, which would contribute to the pathogenesis of parkinson's disease (PD) in idiopathic patients and parkin heterozygotes. In this study, we analyzed samples from 17 PD patients with heterozygous parkin mutations, 17 PD patients without parkin mutations, and 10 normal controls. We determined the levels of methylation of the parkin gene promoter in each group using bisulfite genomic sequencing of a region containing 33 CpG sites in the CpG island of the parkin promoter. The methylation levels indicated hypomethylation, and there were no significant differences in the incidence of CpG site methylation among three groups (P > 0.05). These results suggest that the methylation mechanism is unlikely to play a role in the pathogenesis and development of PD.

Mutant Atp13a2 proteins involved in parkinsonism are degraded by ER-associated degradation and sensitize cells to ER-stress induced cell death

Mutations in ATP13A2 (PARK9) have been linked to juvenile parkinsonism with dementia or Kufor-Rakeb syndrome (KRS). The ATP13A2 gene encodes at least three protein isoforms that arise by alternate splicing. A previous study indicated the Atp13a2(Isoform-1) protein is localized to lysosomes, whereas three separate mutations involved in disease cause retention of the protein in the ER. One speculation is that the mutant Atp13a2(Isoform-1) proteins are misfolded and eliminated by the ER-associated degradation pathway (ERAD), which involves the dislocation of proteins from the ER to the cytoplasm for proteasome degradation. We examined whether Atp13a2 proteins are degraded by ERAD and whether the Atp13a2(Isoform-3) protein has similar localization to the Atp13a2(Isoform-1) protein. Through analysis of protein turnover and by disrupting different steps in the ERAD pathway we demonstrate that mutant Atp13a2(Isoform-1) proteins are indeed eliminated by ERAD. Thus, siRNA-mediated knockdown of erasin, a platform for assembly of an ERAD complex, or expression of a dominant negative form of p97/VCP, a protein essential for dislocation of ERAD substrates, or inhibition of the proteasome all slowed degradation of the mutant Atp13a2(Isoform-1) proteins, but not the wild-type Atp13a2(Isoform-1) protein. Immunoprecipitation assays confirmed that the Atp13a2 proteins are ubiquitinated in accord with degradation by ERAD. In contrast to Atp13a2(Isoform-1), we show Atp13a2(Isoform-3) is localized to the ER and rapidly degraded. Lastly, we show Atp13a2 mutants have increased cytotoxicity and predispose cells to ER-stress-induced cell death. These results provide new insight into the properties of wild-type and mutant Atp13a2 proteins involved in KRS.

The Parkin-Like Human Homolog of Drosophila Ariadne-1 (HHARI) Can Induce Aggresome Formation in Mammalian Cells and Is Immunologically Detectable in Lewy Bodies

Loss of functional Parkin is responsible for the death of midbrain dopaminergic neurons in human autosomal recessive juvenile parkinsonism. Since no cells express functional Parkin, it is unclear why other neuronal and non-neuronal populations are not also endangered. One possible explanation is that other neurons express a redundant ubiquitin-protein ligase (E3) that is absent from dopaminergic neurons. In this study, we demonstrate that human homolog of Drosophila ariadne-1 (HHARI) is a candidate for such a redundant function. In in vitro assays, HHARI binds to many of the same proteins as parkin, including CDCrel-1, synphilin-1, and CASK. In cell culture studies, HHARI forms aggresomes that are indistinguishable from those formed by parkin in terms of morphology, subcellular localization, incorporation of ubiquitin-proteasome components, and dependence on microtubules. In addition, endogenous HHARI is found in human Lewy bodies in both Parkinson's disease and diffuse Lewy body disorder. Taken together, these data suggest that HHARI, and perhaps other Parkin-like E3 ligases, may serve redundant roles for parkin in different cell types.

Impact of Autosomal Recessive Juvenile Parkinson’s Disease Mutations on the Structure and Interactions of the Parkin Ubiquitin-like Domain

Autosomal recessive juvenile parkinsonism (ARJP) is an early onset familial form of Parkinson’s disease. Approximately 50% of all ARJP cases are attributed to mutations in the gene park2, coding for the protein parkin. Parkin is a multidomain E3 ubiquitin ligase with six distinct domains including an N-terminal ubiquitin-like (Ubl) domain. In this work we examined the structure, stability, and interactions of the parkin Ubl domain containing most ARJP causative mutations. Using NMR spectroscopy we show that the Ubl domain proteins containing the ARJP substitutions G12R, D18N, K32T, R33Q, P37L, and K48A retained a similar three-dimensional fold as the Ubl domain, while at least one other (V15M) had altered packing. Four substitutions (A31D, R42P, A46P, and V56E) result in poor folding of the domain, while one protein (T55I) showed evidence of heterogeneity and aggregation. Further, of the substitutions that maintained their three-dimensional fold, we found that four of these (V15M, K32T, R33Q, and P37L) lead to impaired function due to decreased ability to interact with the 19S regulatory subunit S5a. Three substitutions (G12R, D18N, and Q34R) with an uncertain role in the disease did not alter the three-dimensional fold or S5a interaction. This work provides the first extensive characterization of the structural effects of causative mutations within the ubiquitin-like domain in ARJP.