The residual minor ubiquitinated fraction is linked via isopeptide bonds, suggesting that a small fraction of Parkin is itself ubiquitinated. Open in a separate window Fig. by MS for Parkin substrate recognition. B) All individual loading settings (actin western blots) for Fig.?8. (PDF 337?kb) 13024_2017_170_MOESM2_ESM.pdf (338K) GUID:?B53A9D22-9F29-44E3-824A-87E328BC14A6 Additional file 3: Number S3: Reproducibility of the system I. A) Venn diagrams display number of proteins recognized in each BioUb pulldown analysed by MS for Parkin substrate recognition. B) LFQ intensity distribution of recognized proteins in all BioUb pulldowns utilized for Parkin substrate recognition with Perseus software. Non-inputated ideals are demonstrated in green and inputated ideals in Tacalcitol reddish. Note that Perseus replaced LFQ intensities that displayed a value of 0 by low value Tacalcitol intensities within the lower detection limit relating to a normal distribution. LFQ intensities are displayed in Log2 level. (PDF 785?kb) 13024_2017_170_MOESM3_ESM.pdf (786K) GUID:?5D90F7A7-A10F-46DE-ABDD-B66052EB6932 Additional file 4: Number S4: Reproducibility of the system II. Multicorrelation graph of LQF intensities of proteins identified in all BioUb pulldowns analysed by MS Tacalcitol for Parkin substrate recognition. LFQ intensities are displayed in Log2 level. (PDF 149?kb) 13024_2017_170_MOESM4_ESM.pdf (149K) GUID:?CDF4ADB2-E655-4D70-BA3E-A27123C4C06E Additional file 5: Figure S5: Peptide validation to identify the most powerful Parkin responders. Relating to Perseus (C), (Ub), (WT) and (LD). (PDF 414?kb) 13024_2017_170_MOESM6_ESM.pdf (414K) GUID:?02A7B676-8B99-4638-8AEA-A419F0BD1790 Additional file 7: Figure S7: Over-expression of Pax1 hParkin (WT) induced an increase of VPS35 ubiquitination compared to control (C) or to over-expression of inactive hParkin (LD) in four self-employed experiments. (A) The complete gels of Fig.?8a showing four indie in vivo ubiquitination assays for VPS35 in SH-SY5Y cells. Ubiquitination of YFP-tagged VPS35 was analysed by Western blot after capture of the YFP-tagged protein. Mouse anti-GFP antibody was utilized for detecting the captured VPS35 (demonstrated in green), and HRP-conjugated anti-FLAG antibody for monitoring its ubiquitinated portion (demonstrated in reddish). Untagged Parkin over-expression levels in the whole cell components are monitored with anti-Parkin antibody. (B) Quantification of the ubiquitination status of VPS35 relative to the non-modified form was performed calculating the percentage FLAG:GFP with Image-J. The storyline shows relative levels of VPS35 ubiquitination normalized to the GFP levels. (PDF 14700?kb) 13024_2017_170_MOESM7_ESM.pdf (15M) GUID:?57DDF3CD-46D7-48E6-93F2-FB928DD08366 Additional file 8: Figure S8: Alignment of Human being and Parkin. (PDF 61?kb) 13024_2017_170_MOESM8_ESM.pdf (62K) GUID:?80803B90-3084-4191-BF7F-79F1F2DD675D Additional file 9: Table S1: All proteins recognized: Details of all proteins recognized in the whole mass spectrometry analysis. Proteins are divided in Background and Hits (Ubiquitinated). (XLS 2430?kb) 13024_2017_170_MOESM9_ESM.xls (2.4M) GUID:?BEFABFBF-03A9-4A7A-BAE4-9D7358F4CAE1 Additional file 10: Table S2: Probably the most powerful Parkin responders recognized in neurons: Details of the most powerful Parkin responders recognized in neurons. (XLS 58?kb) 13024_2017_170_MOESM10_ESM.xls (59K) GUID:?D17BE34C-9438-4B16-8DD5-D81FD23F8C89 Additional file 11: Table S3: Di-gly Sites: Details of all detected Di-gly peptides. (XLS 154?kb) 13024_2017_170_MOESM11_ESM.xls (154K) GUID:?1F1B5C98-935C-48AE-945C-2C220E28ED9B Additional file 12: Video S1: Parkin over-expression in neurodevelopment results in Parkinsonian-like problems: Bare vials containing 5 male and 5 female, 0C3 days after eclosion flies of display Parkinsonian-like phenotypes including reduced life span, climbing and flying disability, sterility, mitochondrial problems and dopaminergic neurodegeneration [16]. Genetic studies in founded that functions upstream of to keep up mitochondrial integrity [17, 18]. Upon mitochondrial depolarization Red1 accumulates in the Outer Mitochondrial Membrane (OMM), where it phosphorylates both ubiquitin and the Ubiquitin-like (UBL) website of Parkin to recruit and activate latent Parkin ubiquitin ligase activity [19C25]. Activated Parkin ubiquitinates several OMM proteins and promotes both proteasome-dependent degradation of specific proteins and mitophagy, a specialised type of autophagy where the whole mitochondrion is definitely engulfed into autophagosomes [26C28]. Red1 and Parkin are widely considered neuroprotective and different studies have shown that Red1/Parkin over-expression can protect against cell death in a number of contexts in vitro and in vivo [29]. Therefore it has been proposed that drugs advertising Red1/Parkin – dependent mitophagy could serve as effective treatments for PD. However, recent evidence demonstrates that excessive Parkin over-expression results in sensitization to cell death using in vitro [30C32] and in vivo models [33]. It is essential to identify physiologically relevant Parkin substrates to understand the pathways leading to PD in order to develop a treatment. A considerable number of proteins have been reported to be Parkin substrates but most of the work offers relied on cultured cells, mainly of epithelial origin, usually.