Immunodetection of Parkin protein in vertebrate and invertebrate brains: a comparative study using specific antibodies

Horowitz, Judith M.; Vernace, Vita A.; Myers, John M.; Stachowiak, Michal K.; Hanlon, David; Fraley, Gregory S.; Torres, Germán

doi:10.1016/s0891-0618(00)00111-3

Cited by 45 publications

(20 citation statements)

References 22 publications

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“…Although most studies report a band of ϳ52 kDa for parkin, several studies report the detection of additional putative isoforms from rodent tissues ranging from ϳ22 to ϳ65 kDa (8 -16) and from human tissue ranging from ϳ22 to ϳ65 kDa (5,12,13,(17)(18)(19)(20)(21)(22). The specific antibodies described here detect only doublets of ϳ50 and ϳ44 kDa in mouse and ϳ50 and ϳ46 kDa in human brains.…”

Section: Resultsmentioning

confidence: 77%

“…Parkin has also been found in Lewy body-enriched preparations from human brain (20). However, there is a discrepancy among these reports on the number and size of human versus rodent parkin isoforms as well as discrepancies in the subcellular distribution of parkin (12,13,15,21,23). To clarify some of these issues, sensitive and specific antibodies to parkin were generated.…”

Section: Autosomal Recessive Juvenile Parkinsonism (Ar-jp)mentioning

confidence: 99%

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Novel Monoclonal Antibodies Demonstrate Biochemical Variation of Brain Parkin with Age

Pawlyk

Giasson

Sampathu

et al. 2003

Journal of Biological Chemistry

146

117

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Autosomal recessive juvenile parkinsonism is a movement disorder associated with the degeneration of dopaminergic neurons in substantia nigra pars compacta. The loss of functional parkin caused by parkin gene mutations is the most common single cause of juvenile parkinsonism. Parkin has been shown to aid in protecting cells from endoplasmic reticulum and oxidative stressors presumably due to ubiquitin ligase activity of parkin that targets proteins for proteasomal degradation. However, studies on parkin have been impeded because of limited reagents specific for this protein. Here we report the generation and characterization of a panel of parkin-specific monoclonal antibodies. Biochemical analyses indicate that parkin is present only in the high salt-extractable fraction of mouse brain, whereas it is present in both the high salt-extractable and RIPA-resistant, SDS-extractable fraction in young human brain. Parkin is present at decreased levels in the high salt-extractable fraction and at increased levels in the SDS-extractable fraction from aged human brain. This shift in the extractability of parkin upon aging is seen in humans but not in mice, demonstrating species-specific differences in the biochemical characteristics of murine versus human parkin. Finally, by using these highly specific anti-parkin monoclonal antibodies, it was not possible to detect parkin in ␣-synuclein-containing lesions in ␣-synucleinopathies, thereby challenging prior inferences about the role of parkin in movement disorders other than autosomal recessive juvenile parkinsonism. Autosomal recessive juvenile parkinsonism (AR-JP)1 is an especially insidious form of parkinsonism that can strike as early as the 1st decade of life. A major locus for this disease was mapped to chromosome 6q, and the gene was subsequently identified and termed parkin (1). Human parkin is a 465-amino acid protein with a predicted molecular mass of 52 kDa that contains an N-terminal ubiquitin-like domain, a linker region, and a C-terminal TRIAD domain consisting of two RING fingers on either side of an in-between RING (IBR) finger region (2). Deletions and insertions of one or more exons resulting in premature translation termination are some of the most common mutations in parkin, but numerous missense point mutations in parkin have also been shown to be causal of AR-JP (3). Since the identification of parkin, many studies focused on elucidating the function of this protein, and it has been shown it can function as a ubiquitin-protein isopeptide ligase and that its overexpression in vivo enhances the ubiquitinylation of synphilin-1, Pael-R, and CDCRel-1. Immunoprecipitated parkin has been reported to catalyze the ubiquination of these substrates in vitro as well as O-glycosylated ␣-synuclein p22 and cyclin E (4 -8). Furthermore, mutations linked to AR-JP have been reported to reduce the ubiquination of these substrates. Moreover, emerging data suggest that parkin may protect cells from premature death by targeting misfolded or damaged proteins for degradation ...

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Section: Resultsmentioning

confidence: 77%

Section: Autosomal Recessive Juvenile Parkinsonism (Ar-jp)mentioning

confidence: 99%

Novel Monoclonal Antibodies Demonstrate Biochemical Variation of Brain Parkin with Age

Pawlyk

Giasson

Sampathu

et al. 2003

Journal of Biological Chemistry

146

117

View full text Add to dashboard Cite

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“…However, this antibody had never been tested on any tissue other than brain. Horowitz et al (2001) have shown that the size of the Parkin protein can vary between tissue types. Therefore, the antibody was initially tested on protein extracted from normal ovarian surface epithelium (OVSE) to determine the size of the expected protein and to confirm the expression level of Parkin protein in the normal surface epithelium of the ovary.…”

Section: Western Blot Analysismentioning

confidence: 99%

Alterations in the common fragile site gene Parkin in ovarian and other cancers

et al. 2003

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The cloning and characterization of the common fragile site (CFS) FRA6E (6q26) identified Parkin, the gene involved in the pathogenesis of many cases of juvenile, early-onset and, rarely, late-onset Parkinson's disease, as the third large gene to be localized within a large CFS. Initial analyses of Parkin indicated that in addition to playing a role in Parkinson's disease, it might also be involved in the development and/or progression of ovarian cancer. These analyses also indicated striking similarities among the large CFS-locus genes: fragile histidine triad gene (FHIT; 3p14.2), WW domain-containing oxidoreductase gene (WWOX; 16q23), and Parkin (6q26). Analyses of FHIT and WWOX in a variety of different cancer types have identified the presence of alternative transcripts with whole exon deletions. Interestingly, various whole exon duplications and deletions have been identified for Parkin in juvenile and early-onset Parkinson's patients. Therefore, we performed mutational/exon rearrangement analysis of Parkin in ovarian cancer cell lines and primary tumors. Four (66.7%) cell lines and four (18.2%) primary tumors were identified as being heterozygous for the duplication or deletion of a Parkin exon. Additionally, three of 23 (13.0%) nonovarian tumorderived cell lines were also identified as having a duplication or deletion of one or more Parkin exons. Analysis of Parkin protein expression with antibodies revealed that most of the ovarian cancer cell lines and primary tumors had diminished or absent Parkin expression. While functional analyses have not yet been performed for Parkin, these data suggest that like FHIT and WWOX, Parkin may represent a tumor suppressor gene.

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“…But, expression was more concentrated at the central nervous system (brain and nerve cord) in the later stages, which is in line with its function in the central nervous system. And we performed the immunoblot analysis on IPTG-induced Parkin in E. coli using anti-Parkin antibody (Figure 8), which showed one prominent band with a molecular weight of 54 kDa, as described previously (Horowitz et al, 2001). In Horowitz et al (2001), Parkin was detected in a diverse set of organisms including D. melanogaster, frogs, birds and mouse, indicating that Parkin may play a universal role that has been preserved across different species in evolution.…”

Section: Expression Of D M Elanogaster Parkinmentioning

confidence: 63%