Journal: NPJ Parkinson's Disease

Article Title: Opportunities and challenges of alpha-synuclein as a potential biomarker for Parkinson’s disease and other synucleinopathies

doi: 10.1038/s41531-022-00357-0

Figure Lengend Snippet: Analysis of aSyn PTMs species in the different biological fluid specimens: overview of the different techniques, antibodies employed and aSyn PTMs concentration range across control and patient groups.

Article Snippet: , Plasma , ELISA , Capture: anti-α-synuclein N-19 (Santa Cruz Biotechnology); Detection: anti-pS129 (Epitomics) , 143.4 ± 531.8 ng/ml , , 756.8 ± 2419.9 ng/ml , , , , Recombinant aSyn was incubated with casein kinase II (New England Biolabs) , Immunoblotting with a phosphorylation-dependent anti-aSyn antibody, pS129 (Epitomics) and mass spectrometry , NA , Foulds et al. .

Techniques: Concentration Assay, Modification, Western Blot, Dot Blot, Enzyme-linked Immunosorbent Assay, Mass Spectrometry, Luminex, Recombinant, Incubation, Mutagenesis, Electrochemiluminescence

Journal: NPJ Parkinson's Disease

Article Title: Opportunities and challenges of alpha-synuclein as a potential biomarker for Parkinson’s disease and other synucleinopathies

doi: 10.1038/s41531-022-00357-0

Figure Lengend Snippet: a The combination of (1) amplification and detection of minute amounts of aggregated aSyn in biological samples (i.e., aSyn SAA) coupled with cryo-EM and (2) identification and quantification of aSyn species by MS/MS can lead to the discovery and validation of novel biomarkers, relying on structure-based classification and disease-specific aSyn PTMs. Together, these approaches can open new avenues to enable differentiating PD patients from controls and from patients with other synucleinopathies. b Schematic depictions illustrating how attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and surface-enhanced infrared absorption spectroscopy (SEIRA) could be used as complementary approaches in this workflow for high-throughput structural analysis of biological samples, allowing molecular-level differentiation of a monolayer of aSyn monomers and fibrils. Created with BioRender.com and the cryo-EM structures of the aSyn fibrils depicted in the figure are derived from different aSyn recombinant proteins.

Article Snippet: , Plasma , ELISA , Capture: anti-α-synuclein N-19 (Santa Cruz Biotechnology); Detection: anti-pS129 (Epitomics) , 143.4 ± 531.8 ng/ml , , 756.8 ± 2419.9 ng/ml , , , , Recombinant aSyn was incubated with casein kinase II (New England Biolabs) , Immunoblotting with a phosphorylation-dependent anti-aSyn antibody, pS129 (Epitomics) and mass spectrometry , NA , Foulds et al. .

Techniques: Amplification, Cryo-EM Sample Prep, Tandem Mass Spectroscopy, Spectroscopy, High Throughput Screening Assay, Derivative Assay, Recombinant

Journal: PLoS Biology

Article Title: The mechanism of sirtuin 2–mediated exacerbation of alpha-synuclein toxicity in models of Parkinson disease

doi: 10.1371/journal.pbio.2000374

Figure Lengend Snippet: (A) AAV6-mediated delivery of EGFP and mutant aSyn (KQ or KR) into the SN of the rat brain. TH and GFP or aSyn expression was examined in brain sections 3 wk after injection by immunohistochemistry (TH, red; GFP or aSyn, green; DAPI, blue). Representative sections are shown. Scale bar for isolated channels 1,000 μm and for merged channels 500 μm. (B) Stereological counting of the number of TH-positive neurons in the SN. The contralateral SN of the different groups of animals was used as a control (intact). Data in panels are average ± SD. (C) Brain sections stained for aSyn (green), pS129 aSyn (red), and DAPI (Blue). Representative sections are shown. Dashed square boxes delineate the magnification presented on the right. Scale bar for isolated channels 1,000 μm and for merged channels 500 μm and 50 μm. *** p < 0.001, **** p < 0.0001, one-way ANOVA with Bonferroni correction used for statistical calculations. In (B), GFP was used as a control; n = 6–7 animals per condition; five sections from a one-in-six series were analyzed per brain. Data in .

Article Snippet: The following antibodies were used to detect specific synucleins: N-terminal rat aSyn (Mouse, Bd Transduction Laboratories, 610787), recognized amino acid 121–125 of human aSyn (Mouse, Santa-Cruz, SC-12767), pS129 aSyn (Rabbit, Abcam, ab51253), and aggregated forms of aSyn (Mouse, Millipore, clone 5G4, MABN389).

Techniques: Mutagenesis, Expressing, Injection, Immunohistochemistry, Isolation, Staining

Journal: Antioxidants

Article Title: Native α-Synuclein, 3-Nitrotyrosine Proteins, and Patterns of Nitro-α-Synuclein-Immunoreactive Inclusions in Saliva and Submandibulary Gland in Parkinson’s Disease

doi: 10.3390/antiox10050715

Figure Lengend Snippet: Individual total αSyn concentration (pg/mL) in ( A ) saliva and ( B ) serum in patients with IPD and control participants, as measured with ELISA. Mean and standard deviation are represented with solid lines. Abbrev.: αSyn, α-synuclein; IPD, idiopathic Parkinson’s disease; ELISA, enzyme-linked immunosorbent assay.

Article Snippet: The α-Synuclein concentration was evaluated with a commercially available Enzyme-linked Immunosorbent Assay kit (Human aSyn ELISA Kit, cat. #E09S0131, Shanghai BlueGene Biotech CO., LTD, Shanghai, China), following manufacturer’s instructions.

Techniques: Concentration Assay, Control, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Antioxidants

Article Title: Native α-Synuclein, 3-Nitrotyrosine Proteins, and Patterns of Nitro-α-Synuclein-Immunoreactive Inclusions in Saliva and Submandibulary Gland in Parkinson’s Disease

doi: 10.3390/antiox10050715

Figure Lengend Snippet: Individual 3-NT-proteins concentration (µg/mL) in ( A ) saliva and ( B ) serum in patients with IPD and control participants, as measured with ELISA. Mean and standard deviation are represented with solid lines. Abbrev.: 3-NT-proteins, 3-nitrotyrosine proteins; IPD, idiopathic Parkinson’s disease; ELISA, enzyme-linked immunosorbent assay.

Article Snippet: The α-Synuclein concentration was evaluated with a commercially available Enzyme-linked Immunosorbent Assay kit (Human aSyn ELISA Kit, cat. #E09S0131, Shanghai BlueGene Biotech CO., LTD, Shanghai, China), following manufacturer’s instructions.

Techniques: Concentration Assay, Control, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: bioRxiv

Article Title: Dissecting the differential role of C-terminal truncations in the regulation of aSyn pathology formation and the biogenesis of Lewy bodies

doi: 10.1101/2024.11.29.625993

Figure Lengend Snippet: a. Seeding model in primary hippocampal neurons. 70 nM of mouse PFFs were added to neurons at DIV 5 (day in vitro ). Control neurons were treated with Tris buffer used to prepare PFFs. After 4 days of treatment, positive pS129-aSyn aggregates were detected in the extension of the neurons. After 7 days of treatment, the aggregates appeared in the cytosol of the neurons. The number of LB-like inclusions increased over time, as shown at 10 days of treatment. Scale bars = 20 μm. b-e. ICC analysis of the LB-like inclusions that formed at 10 days after adding mouse PFFs to aSyn KO neurons (b) or to WT neurons (c-e). Aggregates were detected using pS129 (MJFR13) in combination with total aSyn (SYN-1), p62, or ubiquitin antibodies. Neurons were counterstained with microtubule-associated protein (MAP2) antibody, and the nucleus was counterstained with DAPI staining. Scale bars = 5 μm. f-g. WB analyses of the insoluble fraction of PFFs-treated WT neurons ( f ) or PFFs-treated KO neurons ( g ). Control neurons were treated with Tris buffer (Tris). After sequential extractions of the soluble and insoluble fractions, cell lysates were analyzed by immunoblotting. Total aSyn, pS129 and actin were respectively detected by SYN-1, pS129 (MJFR13), and actin antibodies. Levels of total aSyn (15 kDa, indicated by a double red asterisk; 12 kDa indicated by a single red asterisk or HMW) or pS129-aSyn were estimated by measuring the WB band intensity and normalized to the relative protein levels of actin. Purple arrows indicate the intermediate aSyn-truncated fragments. The graphs represent the mean +/-SD of 3 independent experiments. ( f ) *p<0.01, **p<0.001, ***p<0.0001* (ANOVA followed by Tukey HSD post-hoc test, Tris vs. PFFs-treated neurons) and # p<0.01, ## p<0.001 (ANOVA followed by Tukey HSD post-hoc test, PFFs-treated neurons D10 vs. D7 or D4 or D1). ( g ) *p<0.01, ***p<0.0001 (ANOVA followed by Tukey HSD post-hoc test, level of aSyn 15 kDa at 1 hour vs. other time-points or levels of aSyn 12 kDa at 1 hour vs. other time-points or Tris vs. PFFs-treated neurons). h-i. Insoluble fractions of aSyn KO primary neurons treated with 70 nM of mouse PFFs for 4 or 14 hours were separated on a 16% Tricine gel. After Coomassie staining, two bands at ∼15 (indicated by a black dashed box) and 12 kDa (indicated by a purple dashed box) were extracted from 16% Tricine gels (See Figure S4). Isolated bands were selected based on the size of the proteolytic fragments observed by WB ( h ) and subjected to proteolytic digestion followed by LC-MS/MS analysis. Proteomic analysis showed that aSyn fragments produced in KO neurons transduced with PFFs result from C-terminal truncation but not from N-terminal cleavage of the PFF seeds. The diagram in i shows the different aSyn fragments generated upon C-terminal truncation and their relative position in a WB. Three fragments (1-135, 1-129, and 1-119) were detected in the upper band sliced, and one main fragment (1-114) was found in the lower band. j. Epitope mapping of antibodies raised against the NAC, N-terminal, or C-terminal domains of aSyn. k. N-terminal antibodies raised against residues 1-5 or residues 1-20 could detect full-length (15 kDa, indicated by a double red asterisk) or truncated (∼12 kDa indicated by a single red asterisk) aSyn in the insoluble fraction of KO neurons treated for 14 hours, confirming that the N-terminal region of aSyn PFF seeds is intact after internalization into the neurons. l. Mapping of the C-terminal cleaved product using antibodies raised against the NAC and the C-terminal domains of aSyn. Immunoblots of insoluble fractions of KO neurons treated with aSyn PFFs showed that the fragment 1-114 generated in these neurons was well recognized by the NAC antibodies [(FL-140; 61-95) and (SYN-1; 91-99)] and a C-terminal antibody raised against the residues 108-120. However, it was not recognized by antibodies raised against peptides bearing residues after 116 in the C-terminal domain [(ab6162; 116-131); (ab131508; 134-138) and (ab52168; 131-135)]. Altogether, our data demonstrate that after internalization, aSyn PFF seeds are efficiently C-terminally truncated before the initiation of the intracellular seeding mechanisms.

Article Snippet: Primary antibodies were directed to human aSyn epitope 103-108 (4B12, 1:1,000, Thermo FisherScientific, USA), mouse aSyn (D37A6, 1:1,000, Cell Signaling Technology, USA), aSyn epitope 1-20 (1:750, homemade), aSyn epitope 91-99 (clone 42, SYN-1, 1:1,000, Becton Dickinson, USA), aSynuclein epitope 134-138 (1:1,500, Abcam, UK), phospho-serine 129 aSyn (1:1,500, Abcam, UK), actin (1:3,000, Cell Signaling Technologies, USA).

Techniques: In Vitro, Control, Staining, Western Blot, Isolation, Liquid Chromatography with Mass Spectroscopy, Produced, Transduction, Generated

Journal: bioRxiv

Article Title: Dissecting the differential role of C-terminal truncations in the regulation of aSyn pathology formation and the biogenesis of Lewy bodies

doi: 10.1101/2024.11.29.625993

Figure Lengend Snippet: a-b. aSyn KO neurons were treated for up to 72 hours with WT fluorescently labelled PFFs 488 . The internalization and the truncation of the seeds were evaluated by confocal imaging. a. One hour after addition to the KO neurons, we observed that most of the intracellular PFFs 488 were co-stained by an antibody raised against the extremity of aSyn C-terminal domain (epitope: 134-138, yellow arrows). C-terminal truncation of the seeds over time was confirmed by the loss of detection of the seeds by the C-terminal aSyn antibody (134-138, red; green arrows). b. The internalization of the seeds via the endo-lysosomal pathway was confirmed by the detection of the fluorescently labelled PFF 488 seeds in LAMP1-positive (late endosome, red) compartments overtime. a-b. Neurons were counterstained with MAP2 antibody and the nucleus with DAPI stain. Scale bars = 10 μm. c. Cathepsin B activity was measured in KO neurons treated with 70 nM of WT PFFs seeds for up to 48 hours. Control neurons were treated with Tris buffer. The graphs represent the mean +/-SD of 3 independent experiments. *p<0.01, **p<0.001, ***p<0.0001 (ANOVA followed by Tukey HSD post-hoc test, Tris vs. PFFs-treated neurons). d. Truncation of aSyn PFFs in the cytosol is confirmed by microinjection. The diagram on the left-hand side shows the experimental approach used to microinject WT PFFs 488 in KO neurons. Cells were fixed after 24 hours and immunostained using the N-terminus antibody (aSyn 1-20) or C-terminus antibody (aSyn 134-138). Confocal imaging showed that WT PFFs 488 were detected by the N-terminus antibody (yellow arrows, merge), but not the C-terminus antibody (green arrows, merge). Neurons were counterstained with MAP2 antibody and the nucleus with DAPI stain. Scale bars = 40 μm.

Article Snippet: Primary antibodies were directed to human aSyn epitope 103-108 (4B12, 1:1,000, Thermo FisherScientific, USA), mouse aSyn (D37A6, 1:1,000, Cell Signaling Technology, USA), aSyn epitope 1-20 (1:750, homemade), aSyn epitope 91-99 (clone 42, SYN-1, 1:1,000, Becton Dickinson, USA), aSynuclein epitope 134-138 (1:1,500, Abcam, UK), phospho-serine 129 aSyn (1:1,500, Abcam, UK), actin (1:3,000, Cell Signaling Technologies, USA).

Techniques: Imaging, Staining, Activity Assay, Control, Microinjection

Journal: bioRxiv

Article Title: Dissecting the differential role of C-terminal truncations in the regulation of aSyn pathology formation and the biogenesis of Lewy bodies

doi: 10.1101/2024.11.29.625993

Figure Lengend Snippet: a-g. Identification of C-terminal truncated fragments by proteomic analysis ( a-b ), WB ( c and ) or confocal imaging ( d-g ). WT neurons were treated with 70 nM of mouse PFFs for 10 days. a-b. The insoluble fractions of PFFs-treated neurons were separated on a 16% Tricine gel. After Coomassie staining, 8 bands were extracted from the Tricine gel ( a ). Isolated bands were subjected to proteolytic digestion using trypsin for C-terminal truncation identification , followed by LC-MS/MS analysis. b. Proteomic analyses showed the presence of the 1-114 and 1-119 C-terminal truncated fragments in the HMW species. c. Table summarizing the capacity of NAC domain, N-terminal, and C-terminal antibodies to detect full-length aSyn (15 kDa), the C-terminally cleaved fragment of aSyn (∼12 kDa), and the HMW formed in WT neurons after 10 days of treatment with PFFs (see WB in ). d-g . Antibody mapping of the newly formed inclusions by confocal imaging using pS129 antibody (81a clone) in combination with N-terminal ( d , epitope 1-20) or C-terminal ( e-g , respective epitopes 108-120, 116-131, or 134-138) antibodies revealed the presence of aSyn-positive aggregates that were not pS129 positive or only partially phosphorylated at S129 residue ( e-f ). Neurons were counterstained with MAP2 antibody and the nucleus with DAPI stain. The white arrows indicate the sub-populations of aggregates localized near the pS129-positive inclusions, and the white asterisk those inside the pS129-positive filamentous structures. Scale bars = 10 μm.

Article Snippet: Primary antibodies were directed to human aSyn epitope 103-108 (4B12, 1:1,000, Thermo FisherScientific, USA), mouse aSyn (D37A6, 1:1,000, Cell Signaling Technology, USA), aSyn epitope 1-20 (1:750, homemade), aSyn epitope 91-99 (clone 42, SYN-1, 1:1,000, Becton Dickinson, USA), aSynuclein epitope 134-138 (1:1,500, Abcam, UK), phospho-serine 129 aSyn (1:1,500, Abcam, UK), actin (1:3,000, Cell Signaling Technologies, USA).

Techniques: Imaging, Staining, Isolation, Liquid Chromatography with Mass Spectroscopy, Residue

Journal: bioRxiv

Article Title: Dissecting the differential role of C-terminal truncations in the regulation of aSyn pathology formation and the biogenesis of Lewy bodies

doi: 10.1101/2024.11.29.625993

Figure Lengend Snippet: a. WB analyses of the truncation pattern of aSyn in human brain tissue from MSA patients and healthy controls. After sequential extractions of the soluble and insoluble fractions, cell lysates were analyzed by immunoblotting. The levels of total aSyn (1-20, SYN-1 or 134-138 antibodies) or pS129 aSyn were estimated by measuring the WB band intensity and normalized to the relative protein levels of actin. The 15 kDa band is indicated by a double red asterisk, the 12 kDa band by a single red asterisk, and the purple arrows indicate the intermediate aSyn-truncated fragments. b-c. Serial sections from the midbrains of PDD (pars compacta) and SNCA duplication (tegmentum) cases were stained with aSyn antibodies raised specifically against the N-terminal (epitope: 1-20), the NAC (91-99), the C-terminal (epitopes: 110-115 and 134-138) or pS129 (EP1536Y) regions. Scale bars = 50 μm.

Article Snippet: Primary antibodies were directed to human aSyn epitope 103-108 (4B12, 1:1,000, Thermo FisherScientific, USA), mouse aSyn (D37A6, 1:1,000, Cell Signaling Technology, USA), aSyn epitope 1-20 (1:750, homemade), aSyn epitope 91-99 (clone 42, SYN-1, 1:1,000, Becton Dickinson, USA), aSynuclein epitope 134-138 (1:1,500, Abcam, UK), phospho-serine 129 aSyn (1:1,500, Abcam, UK), actin (1:3,000, Cell Signaling Technologies, USA).

Techniques: Western Blot, Staining

Journal: Scientific Reports

Article Title: A universal approach to investigate circRNA protein coding function

doi: 10.1038/s41598-019-48224-y

Figure Lengend Snippet: Mouse circRtn4 structure and generation in mammalian cells. ( A ) Schematic representation of the circRtn4 localization within the mouse Rtn4 gene environment and the circRNA cassette for pCircRNA-BE and pCircRNA-DMo vectors; mouse circRtn4 consists of Rtn4 gene exons 2 and 3. The 800 nt inverted repeats (purple colour) within the flanking introns were inserted to promote backsplicing through the formation of inter-intronic base-pairing interactions; flanking introns lack 5′ and 3′ splice sites, which lead to abolished canonical splicing of exon 2 and exon 3; the chimeric intron is displayed in green; & and # indicate the circRtn4 RT-PCR oligonucleotide positions; &: Rtn4-c-R and Rtn4-c-F were used for qRT-PCR to determine circRtn4 levels (results shown in B); #, Rtn4-VR, Rtn4-VF were used for the analysis of circRtn4 backsplicing fidelity (results shown in C); *indicates the position of the oligonucleotide probe for Northern blot hybridization (Rtn4-NB-R1) as displayed in D. ( B ) circRtn4 levels in transfected cells (HeLa, N2a, N2a-swe.10, HEK293 cell); pCMV-MIR empty vector as negative control; BE-Rtn4, pCircRNA-BE-Rtn4; DMo-Rtn4, pCircRNA-DMo-Rtn4; all T-tests were performed in comparison to levels of the control sample, ****P ≤ 0 . 0001 , n ≥ 4; β-actin mRNA was used as internal control. ( C ) Northern blot hybridization for detection of circRtn4 in transfected HEK293 cells. Control-1, pCMV-MIR empty vector; Rtn4-Exon2-Exon3, pCMV-Rtn4-Exon2-Exon3; Control-2, the construct devoid of the downstream portion of the inverted repeat in the 3′ flanking intronic region; BE-Rtn4, pCircRNA-BE-Rtn4; DMo-Rtn4, pCircRNA-DMo-Rtn4; −, no RNase R treatment; +, with RNase R treatment; agarose gel ethidium bromide staining of 28S and 18S rRNAs served as loading control; the weak staining of 18S rRNA is due to co-migration and signal quenching with xylene cyanol loading dye. ( D ) Agarose gel electrophoresis of RT-PCR products of circRtn4 to analyse backsplicing fidelity (PCR primers: Rtn4-VR, Rtn4-VF); pCMV-MIR empty vector as negative control; BE-Rtn4, pCircRNA-BE-Rtn4; DMo-Rtn4, pCircRNA-DMo-Rtn4. The entire un-spliced precursor transcript is ~5.3 kb; the spliced circular isoform of exons 2 and exon 3 devoid of the intron corresponds to 2.4 kb. The intron flanked by exons 2 and 3 is 818 nt long. The expected amplicon size is 2.4 kb. Products migrated between 2.0 and 2.5 kb, indicating that the internal intron was spliced during circRtn4 biogenesis. Lane 1–3, HeLa cells; lane 4–6, N2a cells; lane 7–9, N2a-swe.10 cells; lane 10–12, HEK293 cells. PCR products were sequenced and aligned (data not shown). ( E ) Sequencing of the junction site for circRtn4 backsplicing as revealed by assays in N2a and HEK293 cell lines. The RT-PCR products as displayed in C were sequenced and the junction regions were shown; RT-PCR product-4, 5, 6 were from N2a cells; RT-PCR product-11, 12 were from HEK293 cells.

Article Snippet: Linear mRNA generation of Rtn4 exon 2-exon 3 was achieved by inserting mouse Rtn4 exon 2/intron2/exon 3 into the pCMV-MIR vector (OriGene); resulting in construct pCMV-Rtn4-Exon2-Exon3 (oligonucleotides employed are given in Supplementary Table ).

Techniques: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Northern Blot, Hybridization, Transfection, Plasmid Preparation, Negative Control, Construct, Agarose Gel Electrophoresis, Staining, Migration, Amplification, Sequencing

Journal: Scientific Reports

Article Title: A universal approach to investigate circRNA protein coding function

doi: 10.1038/s41598-019-48224-y

Figure Lengend Snippet: Increase of circRtn4 formation via IME in various cell lines.

Article Snippet: Linear mRNA generation of Rtn4 exon 2-exon 3 was achieved by inserting mouse Rtn4 exon 2/intron2/exon 3 into the pCMV-MIR vector (OriGene); resulting in construct pCMV-Rtn4-Exon2-Exon3 (oligonucleotides employed are given in Supplementary Table ).

Techniques:

Journal: Scientific Reports

Article Title: A universal approach to investigate circRNA protein coding function

doi: 10.1038/s41598-019-48224-y

Figure Lengend Snippet: Formation of circRtn4 with IVS1 and PAT1 introns. ( A ) Schematic representation of the circRtn4 cassette for pCircRNA-IVS1-Rtn4 and pCircRNA-PAT1-Rtn4 constructs. The IVS1 intron is displayed in red and the PAT1 intron in blue colour. ( B ) CircRtn4 formation in N2a cells after transfection with circRtn4 constructs. Control, the empty vector control; BE-Rtn4, pCircRNA-BE-Rtn4; DMo-Rtn4, pCircRNA-DMo-Rtn4; IVS1-Rtn4, pCircRNA-IVS1-Rtn4; PAT1-Rtn4, pCircRNA-PAT1-Rtn4; all T-tests were performed in comparison to formation levels of the control sample, ****P ≤ 0 . 0001 , ***P ≤ 0 . 001 , n ≥ 4.

Article Snippet: Linear mRNA generation of Rtn4 exon 2-exon 3 was achieved by inserting mouse Rtn4 exon 2/intron2/exon 3 into the pCMV-MIR vector (OriGene); resulting in construct pCMV-Rtn4-Exon2-Exon3 (oligonucleotides employed are given in Supplementary Table ).

Techniques: Construct, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: A universal approach to investigate circRNA protein coding function

doi: 10.1038/s41598-019-48224-y

Figure Lengend Snippet: Translation of circRtn4. ( A ) Schematic illustration for the insertion of a FLAG-tag (magenta) with and without stop codon (red) into exon 2 (blue) of the circRtn4 open reading frame. Exons 2 (blue) and 3 (yellow) constitute the circRNA open reading frame; the green arrow, denotes the presumed AUG start codon, magenta arrow denotes the position of the FLAG-tag in pCircRNA-DMo-Rtn4-FLAG and derivatives; the red rectangle denotes the stop codon (UGA) in the pCircRNA-DMo-Rtn4-Stop construct. ( B ) Representation of the “infinite” circRtn4 open reading frame (ORF). The inner circle denotes the circular RNA with exon 2 and exon 3 in blue and yellow. The presumed AUG translation start codon is indicated by a green arrow on the outer circle showing the presumed circRtn4 translation products(s). ( C ) Western blot of circRtn4 translated polypeptides in HEK293 cells detected with antibodies targeting Nogo-A (α-Nogo-A). Control, i.e., the empty vector; BE-Rtn4, pCircRNA-BE-Rtn4; DMo-Rtn4, pCircRNA-DMo-Rtn4; Rtn4-Exon2-Exon3, pCMV-Rtn4-Exon2-Exon3. On the right, polypeptides of higher molecular weights are presumed products of more than one round of circRtn4 circular translation; RTN4-fl indicates the endogenous RTN4 full length protein (Reticulon 4 or Nogo-A); the “monomer” presumably represents a single round of circRtn4 circular translation. The calculated MW of the theoretical “monomer” is 88.2 kDa. Possibly due to its highly acidic pI (4.3) and/or high proline content, it migrates significantly slower in the gel, thus corresponding to 150 kDa , . For calculating the relative protein level, protein from pCMV-Rtn4-Exon2-Exon3 was set as 1; pCircRNA-DMo-Rtn4 expressed less “monomer” protein by a factor of 0.25. ( D ) The putative open reading frame of Rtn4-FLAG circRNA. Annotation as in B: In addition, the aspartic acid (blue D) is resulting from the junction site of backsplicing; the FLAG-tag peptide is in magenta; the glutamic acid residue (E, highlighted in blue) is resulting from the junction site of exon2-exon3; the green methionine (M) is the presumed start codon and the isoleucine (I, orange) is directly N-terminal to M and supports a further round of translation (see below). ( E ) Western blot hybridisation of pCircRNA-DMo-Rtn4-FLAG expression in N2a cells with the anti-FLAG antibody (α-FLAG). Control, the empty vector; DMo-Rtn4-FLAG, pCircRNA-DMo-Rtn4-FLAG; of note, compared to C, the intensity of repeating peptides is higher than “monomer”; the reason is unknown. ( F ) The putative open reading frame of Rtn4-stop circRNA is delineated. Annotation as in B: In addition, the red bar indicates the in-frame stop codon (UGA). ( G ) Western blot showing expression of pCircRNA-DMo-Rtn4-Stop transfected in HEK293 cells with an anti-Nogo A antibody (α-Nogo-A). Control, the empty vector; DMo-Rtn4-Stop, pCircRNA-DMo-Rtn4-Stop; DMo-Rtn4, pCircRNA-DMo-Rtn4. ( H ) Nucleotide sequence and predicted translation products surrounding the inserted sequences encoding the FLAG peptide (magenta) as part of exon 2, the region where exons 2 and 3 are joined, and the site of circularization for construct circRtn4-FLAG. Exon 2 nucleotide sequence is highlighted in blue, and black upper-case letters indicate Rtn4 exon 3. The nucleotides encoding the FLAG-tag are displayed in black lower-case letters. The last G residue (position 2448) is joined to the first nucleotide by circularization yielding a GAU triplet (highlighted in yellow), encoding aspartic acid (D, bold, blue letter, in brackets). Nucleotides are arranged in blocks of 12. The predicted amino acid sequence is given in the IUPAC one-letter amino acid code. Tryptic peptide 46 (Supplementary Fig. ) beginning with TSDETL, bridging the circularization site and containing the FLAG peptide (bold and magenta lettering) is underlined. The glutamic acid residue bridging exons 2 and 3 is highlighted as bold blue letter. Unless additional start codon(s) are used, tryptic peptide 52 (Supplementary Fig. ) starting with IMDLKEQPG (broken line) must be derived from translation into the second circular round across the presumed AUG start codon (marked by a horizontal green arrow). Amino acids are highlighted as above. ( I ) Mass spectrometry of peptide 46, providing direct evidence of circular translation of the junction site in circRtn4-FLAG. Amino acids are highlighted as above. ( J ) Mass spectrometry of peptide 52, providing evidence of circular translation into a second round of circRtn4-FLAG. Amino acids are highlighted as above. ( K ) Open reading frame (ORF) of circRtn4-FLAG-ac. Annotation as in B: In addition, the AC insertion is marked in red and the junction amino acid glutamine (Q, red) is marked. The different ORF beyond the first round of translation is marked in red and the extension of the first round in red and “69 aa”. The substitution to generate the stop codon (UAA) some 207 nucleotides further downstream is not shown in this C-terminal segment (Supplementary Fig. ). ( L ) Western blot analysis of pCircRNA-DMo-Rtn4-ac transfected in N2a cells using an anti-Nogo A antibody (α-Nogo-A). DMo-Rtn4-FLAG, pCircRNA-DMo-Rtn4-FLAG; DMo-Rtn4-FLAG-ac, pCircRNA-DMo-Rtn4-FLAG-ac.

Article Snippet: Linear mRNA generation of Rtn4 exon 2-exon 3 was achieved by inserting mouse Rtn4 exon 2/intron2/exon 3 into the pCMV-MIR vector (OriGene); resulting in construct pCMV-Rtn4-Exon2-Exon3 (oligonucleotides employed are given in Supplementary Table ).

Techniques: FLAG-tag, Construct, Western Blot, Plasmid Preparation, Hybridization, Expressing, Transfection, Sequencing, Derivative Assay, Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of the aggregate-forming protein alpha-synuclein on serine-129 inhibits its DNA-bending properties

doi: 10.1016/j.jbc.2021.101552

Figure Lengend Snippet: Alpha-synuclein and phospho-synuclein bind DNA in a DNA length-dependent manner. A , top , EMSA with increasing alpha-synuclein (aSyn) and serine-129 phosphorylated alpha-synuclein (pSyn) concentrations with DNA of different lengths shows an upward shift of DNA bands greater than 200 bp at higher synuclein concentrations. Shifts produced by aSyn and pSyn of specific bands (within gray rectangles ) are enlarged and shown below. Bottom , larger image of aSyn and pSyn EMSA shows upward shift of specific DNA bands longer than 200 bp depending on aSyn or pSyn concentration. For example, at 57 μM pSyn the 300 bp band exhibits both shifted ( green arrow ) and unshifted ( blue arrow ) species compared with the 4 μM concentration, where only an unshifted band ( blue arrow ) is present. B , top , shifts produced by aSyn ( left ) and pSyn ( right ) with individual DNA lengths (125, 200, 300, 400, and 500 bp). Bottom , group data show the fraction of different length DNAs shifted by 57 μM aSyn or pSyn. Both aSyn and pSyn binding to DNA depends on DNA length (aSyn EL50 = 153 bp, R 2 = 0.986, deviation from zero slope p < 0.0001, Hill slope = 3.1; pSyn EL50 = 281 bp, R 2 = 0.991, deviation from zero slope p < 0.0001, Hill slope = 6.0; four-parameter dose-response curve; N = 3 gels). aSyn produces more shift than pSyn of 125, 200 and 300 bp DNA (shifted fraction: aSyn-125 bp = 0.327 ± 0.015, pSyn = 125 bp = 0.007 ± 0.012, unpaired t test p < 0.0001; aSyn-200 bp = 0.740 ± 0.010, pSyn = 200 bp = 0.120 ± 0.104, unpaired t test p = 0.0005; aSyn-300 bp = 0.803 ± 0.025, pSyn = 300 bp = 0.617 ± 0.050, unpaired t test p = 0.0045; aSyn-400 bp = 0.947 ± 0.012, pSyn = 400 bp = 0.927 ± 0.015, unpaired t test p = 0.145; aSyn-500 bp = 1.000 ± 0.000, pSyn = 500 bp = 1.000 ± 0.000; N = 3 gels). Points at 0 DNA length set to 0.0 shifted fraction based on model for fitting purposes. EMSA, electrophoretic mobility shift assay.

Article Snippet: Human aSyn and pSyn proteins (Proteos, cat. # RP-003, RP-004) and human aSyn, bSyn, gSyn, A30P, E46K, A53T, deltaNAC aSyn (rPeptide, cat. # S-1001, S-1003, S-1007, S-1005, S-1008, S-1002, S-1015) were stored at −80 °C.

Techniques: Produced, Concentration Assay, Binding Assay, Electrophoretic Mobility Shift Assay

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of the aggregate-forming protein alpha-synuclein on serine-129 inhibits its DNA-bending properties

doi: 10.1016/j.jbc.2021.101552

Figure Lengend Snippet: Alpha-synuclein and phospho-synuclein bind DNA with blunt or 5′ overhanging ends with similar affinity. Alpha-synuclein (aSyn, A ) and serine-129 phosphorylated alpha-synuclein (pSyn, B ) bind 300 bp DNA with blunt or 300 bp DNA with a 4 base 5′ overhanging end on both sides similarly (shifted fraction blunt end at 0–4, 16, 29, and 57 μM aSyn: 0.000 ± 0.000, 0.007 ± 0.012, 0.387 ± 0.110, 0.857 ± 0.006; shifted fraction overhanging ends at 0–14, 29 and 57 μM aSyn: 0.000 ± 0.000, 0.417 ± 0.121, 0.917 ± 0.031; comparison of shifted fraction of blunt and overhanging end DNA for each aSyn concentration by paired t test shows no significant differences, p between 0.059 and 0.423; variable slope dose-normalized response curve; N = 3 gels, x-axis aSyn concentration on log scale; shifted fraction blunt end at 0–14, 29 and 57 μM pSyn: 0.000 ± 0.000, 0.250 ± 0.098, 0.607 ± 0.101; shifted fraction overhanging ends at 0–14, 29 and 57 μM pSyn: 0.000 ± 0.000, 0.217 ± 0.188, 0.573 ± 0.161; comparison shifted fraction of blunt and overhanging end DNA for each pSyn concentration by paired t test shows no significant differences, p between 0.469 and 0.604; variable slope dose-normalized response curve; N = 3 gels, x-axis pSyn concentration on log scale).

Article Snippet: Human aSyn and pSyn proteins (Proteos, cat. # RP-003, RP-004) and human aSyn, bSyn, gSyn, A30P, E46K, A53T, deltaNAC aSyn (rPeptide, cat. # S-1001, S-1003, S-1007, S-1005, S-1008, S-1002, S-1015) were stored at −80 °C.

Techniques: Comparison, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of the aggregate-forming protein alpha-synuclein on serine-129 inhibits its DNA-bending properties

doi: 10.1016/j.jbc.2021.101552

Figure Lengend Snippet: Alpha-synuclein and phospho-synuclein DNA binding is increased by the minor groove-binding agent Hoechst 33258 and competes with the major groove-binding agent methyl green. A , increasing concentrations of the DNA minor groove binder Hoechst 33258 (0–16 μM) does not change the signal generated by DNA ( left side of each gel) but does increase shifted fraction caused by 14 μM alpha-synuclein (aSyn) and serine-129 phosphorylated alpha-synuclein (pSyn; aSyn Hoechst EC50 = 21.29 μM, R 2 = 0.653, deviation from zero slope p = 0.0003, three-parameter dose–response curve; N = 4 gels; pSyn Hoechst EC50 = 12.76 μM, R 2 = 0.395, deviation from zero slope p = 0.0002, x-axis Hoechst concentration on log scale). B , in contrast, high concentrations (at 500 nM and above) of the DNA major groove binder methyl green decrease intercalation and/or fluorescence of the Sybr dye used to image DNA ( left side of aSyn and pSyn gels). Addition of 29 μM aSyn or pSyn competes with methyl green and partially restores Sybr dye fluorescence signal. Methyl green does not affect the shifted fraction of DNA in the presence of 29 μM aSyn or pSyn at values where it does not reduce Sybr dye fluorescence (at 250 nM and less, aSyn: R 2 = 0.205, deviation from zero slope p = 0.5473; pSyn: R 2 = 0.748, deviation from zero slope p = 0.1351; N = 3 gels, x-axis methyl green concentration on log scale). The 14 and 29 μM aSyn and pSyn concentrations were chosen since these produced intermediate levels of shift, allowing for detection of possible changes with small-molecule dye addition.

Article Snippet: Human aSyn and pSyn proteins (Proteos, cat. # RP-003, RP-004) and human aSyn, bSyn, gSyn, A30P, E46K, A53T, deltaNAC aSyn (rPeptide, cat. # S-1001, S-1003, S-1007, S-1005, S-1008, S-1002, S-1015) were stored at −80 °C.

Techniques: Binding Assay, Generated, Concentration Assay, Fluorescence, Produced

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of the aggregate-forming protein alpha-synuclein on serine-129 inhibits its DNA-bending properties

doi: 10.1016/j.jbc.2021.101552

Figure Lengend Snippet: Lowering DNA concentration shifts alpha-synuclein-bound complexes into a different state, but this does not happen with phospho-synuclein. A 1 , decreasing 300 bp DNA concentration from 200 nM to 0.1 nM with fixed alpha-synuclein (aSyn) concentration (57 μM) produces a change in the apparent length of the complex from to a lower value shift (running ∼600 bp, green arrow ) to a higher value shift (running >1517 bp DNA, red arrow ). Unshifted (unbound) DNA marked by the blue arrow . A 2 , Western blot showing aSyn protein loaded into each lane. A 3 , DNA gel ( green ) and aSyn Western blot ( red ) localization from the same experiment. A 4 , left , group data showing total shifted fraction as a function of DNA concentration (DNA IC50 = 72.24 nM, R 2 = 0.919, three-parameter dose–response curve, N = 3 gels). Right , shifted fraction of high (>1517 bp) and low (∼600 bp) apparent length complexes (high length complex DNA IC50 = 16.26 nM, R 2 = 0.961, three-parameter dose–response curve, N = 2 gels; low length complex DNA EC50 = 5.90 nM, R 2 = 0.842, three-parameter dose–response curve, N = 2 gels). x-axis DNA concentrations on a log scale. B 1 , decreasing 300 bp DNA concentration from 200 nM to 0.1 nM with fixed serine-129 phosphorylated alpha-synuclein (pSyn) concentration (57 μM) produces no change in the apparent length of the bound complex (running ∼500 bp, green arrow ). Unshifted (unbound) DNA marked by the blue arrow . B 2 , Western blot showing pSyn protein loaded into each lane. B 3 , DNA gel ( green ) and pSyn Western blot ( red ) localization from the same experiment. B 4 , left , group data showing little change in total shifted fraction as a function of DNA concentration (DNA EC50 = 0.38 nM, R 2 = 0.116, three-parameter dose–response curve, N = 3–4 gels). Right , shifted fraction of each complex shows no detectable high (>1517 bp), and only detectable low (∼500 bp) apparent length complexes. x-axis DNA concentrations on a log scale.

Article Snippet: Human aSyn and pSyn proteins (Proteos, cat. # RP-003, RP-004) and human aSyn, bSyn, gSyn, A30P, E46K, A53T, deltaNAC aSyn (rPeptide, cat. # S-1001, S-1003, S-1007, S-1005, S-1008, S-1002, S-1015) were stored at −80 °C.

Techniques: Concentration Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of the aggregate-forming protein alpha-synuclein on serine-129 inhibits its DNA-bending properties

doi: 10.1016/j.jbc.2021.101552

Figure Lengend Snippet: Increasing alpha-synuclein concentration shifts alpha-synuclein-bound complexes into a different state, but this does not happen with phospho-synuclein. A 1 , decreasing alpha-synuclein (aSyn) protein concentration from 57 to 1 μM with 300 bp DNA concentration fixed at either 0.1 nM ( left ) or 6 nM ( right ) produces a change in the apparent length of the complex from a higher value shift (running >1517 bp DNA, red arrow ) to a lower value shift (running ∼600 bp, green arrow ). Unshifted (unbound) DNA marked by the blue arrow . A 2 , Western blot showing aSyn protein loaded into each lane. A 3 , DNA gel ( green ) and aSyn Western blot protein ( red ) localization from the same experiment. A 4 , group data showing shifted fraction as a function of aSyn concentration for the two different fixed DNA concentrations. The low DNA concentration curve is left -shifted (aSyn EC50: low DNA conc. = 0.273 μM, R 2 = 0.619; high DNA conc.=5.846 μM, R 2 = 0.977, three-parameter dose–response curve; N = 3–4 gels, x-axis aSyn concentration on log scale). B 1 , decreasing serine-129 phosphorylated alpha-synuclein (pSyn) protein concentration from 57 to 1 μM with 300 bp DNA concentration fixed at either 0.1 nM ( left ) or 6 nM ( right ) produces only a complex with an apparent length of ∼500 bp ( green arrow ) at the higher pSyn concentrations. Unshifted (unbound) DNA marked by the blue arrow . B 2 , Western blot showing pSyn protein loaded into each lane. B 3 , DNA gel ( green ) and pSyn Western blot ( red ) localization from the same experiment. B 4 , group data showing a similar shifted fraction as a function of pSyn concentration for the two different fixed DNA concentrations (pSyn EC50: low DNA conc.=27.34 μM, R 2 = 0.779; high DNA conc.=32.49 μM, R 2 = 0.981, four-parameter dose–response curve; N = 3 gels, x-axis pSyn concentration on log scale).

Article Snippet: Human aSyn and pSyn proteins (Proteos, cat. # RP-003, RP-004) and human aSyn, bSyn, gSyn, A30P, E46K, A53T, deltaNAC aSyn (rPeptide, cat. # S-1001, S-1003, S-1007, S-1005, S-1008, S-1002, S-1015) were stored at −80 °C.

Techniques: Concentration Assay, Protein Concentration, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of the aggregate-forming protein alpha-synuclein on serine-129 inhibits its DNA-bending properties

doi: 10.1016/j.jbc.2021.101552

Figure Lengend Snippet: Alpha-synuclein binds 304 bp circular DNA forms and phospho-synuclein does not. A , agarose gel electrophoresis shows 300 bp linear DNA (with additional 4 base 5′ overhanging ends on each side) treated with no T4 ligase, T4 ligase, or T4 ligase and the DNA bending protein HMGB1. The untreated sample only contains 300 bp linear DNA. The T4 ligase treated sample contains 300 bp and 604 bp linear DNA and 304 bp circular forms (which run faster than their linear counterpart in the agarose gel system). The sample treated with T4 ligase and HMGB1 shows increased formation of 304 bp circular DNA forms. B , polyacrylamide (6%) gel electrophoresis shows 300 bp linear DNA treated with no T4 ligase, T4 ligase, or T4 ligase and the DNA bending protein HMGB1. The untreated sample only contains 300 bp linear DNA. The T4 ligase treated sample contains 300 bp & 604 bp linear DNA and 300 bp circular forms (which run slower than their linear counterpart in the polyacrylamide gel system). The sample treated with T4 ligase and HMGB1 shows the formation of a new 304 bp circular DNA topoisomer form (which runs somewhat faster due to its more compact nature). C , 300 bp linear DNA first treated with T4 ligase and HMGB1 (to produce some circular forms, as in B ), then with either T7 or T5 exonuclease to remove linear DNA molecules, shows the expected resistance to exonuclease treatment of the 304 bp circular DNA forms. D , top , negative stain transmission electron microscopy of 300 bp linear DNA without ligase treatment shows linear molecules of the expected length (∼100 nm). Scale bar 25 nm. Bottom , linear DNA treated with T4 ligase shows 304 bp circular molecules with the expected diameter (∼30 nm). Scale bar 25 nm. E , only aSyn, and not pSyn, causes a shift of the 304 bp circular DNA forms created in the presence of HMGB1 ( red arrows : circular form, orange arrows : circular topoisomer) to a higher apparent length ( purple arrow ). F , left , synuclein concentration dependence of shift of circular DNA forms created in the presence of HMGB1 shows that only aSyn causes a shift at these concentrations and pSyn does not. Right , group data showing shifted fraction of 304 bp DNA circles and circular topoisomers as a function of synuclein concentration (aSyn: EC50 circular DNA = 1.608 μM, R 2 = 0.967; EC50 circular DNA topoisomer = 1.637 μM, R 2 = 0.993; pSyn: EC50 circular & circular DNA topoisomer undefined; at 3–57 μM one-way ANOVA for each concentration, p between <0.0001 and 0.0079; post-hoc Tukey tests shows no significant differences between two [circle versus topoisomer] aSyn conditions p between 0.453 and 0.960, and two [circle versus topoisomer] pSyn conditions p = 0.252–0.956; there are significant differences between two [aSyn versus pSyn] circle conditions p = 0.001–0.028, and the two [aSyn versus pSyn] topoisomer conditions p ≤ 0.0001–0.002; three-parameter dose–response curve, N = 3 gels, x-axis synuclein concentrations on log scale).

Article Snippet: Human aSyn and pSyn proteins (Proteos, cat. # RP-003, RP-004) and human aSyn, bSyn, gSyn, A30P, E46K, A53T, deltaNAC aSyn (rPeptide, cat. # S-1001, S-1003, S-1007, S-1005, S-1008, S-1002, S-1015) were stored at −80 °C.

Techniques: Agarose Gel Electrophoresis, Nucleic Acid Electrophoresis, Staining, Transmission Assay, Electron Microscopy, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of the aggregate-forming protein alpha-synuclein on serine-129 inhibits its DNA-bending properties

doi: 10.1016/j.jbc.2021.101552

Figure Lengend Snippet: Mutant alpha-synuclein and beta- and gamma-synuclein do not bind DNA like wild-type alpha-synuclein. A 1 , left , increasing concentrations of wild-type (WT) alpha-synuclein (aSyn) and disease-causing point mutations (A30P, E46K, A53T) shift 300 bp DNA to higher apparent lengths, with A30P and A53T aSyn being less efficient at shifting than WT, and E46K being more efficient and shifting to a higher apparent length than WT. Right , deletion of the central aggregation-prone NAC domain of aSyn (deltaNAC) increases the efficiency of shifting but reduces the apparent length of the shifted species compared to WT. A 2 , Coomassie stain showing aSyn proteins loaded into each lane. A 3 , DNA gel ( green ) and Coomassie stain ( red ) localization from the same experiment. A 4 , group data showing shifted fraction as a function of aSyn concentration (aSyn R2: WT = 0.980, A30P = 0.963, E46K = 0.999, A53T = 0.965; shifted fraction 1.4 μM aSyn: WT = 0.550 ± 0.082, A30P = 0.270 ± 0.047, E46K = 0.975 ± 0.028, A53T = 0.379 ± 0.102; shifted fraction 2.9 μM aSyn: WT = 0.743 ± 0.049, A30P = 0.515 ± 0.074, E46K = 0.996 ± 0.007, A53T = 0.611 ± 0.010; at 1.4 μM one-way ANOVA p < 0.0001; post-hoc Tukey tests WT versus A30P, E46K, A53T all p between 0.0002 and 0.0449; at 2.9 μM one-way ANOVA p < 0.0001; post-hoc Tukey tests WT versus A30P, E46K, A53T all p between 0.0004 and 0.0174; shifted fraction 1.4 μM aSyn: WT = 0.597 ± 0.148, deltaNAC = 1.000 ± 0.000; shifted fraction 2.9 μM aSyn: WT = 0.764 ± 0.113, deltaNAC = 1.000 ± 0.000; at 1.4 μM t test p = 0.0092; at 2.9 μM t test p = 0.0225; three-parameter dose–response curve; N = 3 gels, x-axis aSyn concentrations on log scale). B 1 , increasing aSyn concentration produces a shift of 300 bp DNA to an apparent length of ∼600 bp, while beta-synuclein (bSyn) and gamma-synuclein (gSyn) produce no such shift. B 2 , Coomassie stain showing synuclein proteins loaded into each lane. B 3 , DNA gel ( green ) and Coomassie stain ( red ) localization from the same experiment. B 4 , group data showing shifted fraction as a function of synuclein concentration (aSyn R2 = 0.791; shifted fraction: 29 μM aSyn = 0.039 ± 0.020, 57 μM aSyn = 0.199 ± 0.066, all other values including for all bSyn and gSyn concentrations produced an undetectable shift = 0.0; at 29 μM one-way ANOVA p = 0.0087; post-hoc Tukey test aSyn versus bSyn, and aSyn versus gSyn p = 0.0139; at 57 μM one-way ANOVA p = 0.0010; post-hoc Tukey test aSyn versus bSyn, and aSyn versus gSyn p = 0.0017; three-parameter dose–response curve; N = 3 gels, x-axis Syn concentrations on log scale).

Article Snippet: Human aSyn and pSyn proteins (Proteos, cat. # RP-003, RP-004) and human aSyn, bSyn, gSyn, A30P, E46K, A53T, deltaNAC aSyn (rPeptide, cat. # S-1001, S-1003, S-1007, S-1005, S-1008, S-1002, S-1015) were stored at −80 °C.

Techniques: Mutagenesis, Staining, Concentration Assay, Produced

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of the aggregate-forming protein alpha-synuclein on serine-129 inhibits its DNA-bending properties

doi: 10.1016/j.jbc.2021.101552

Figure Lengend Snippet: Model of alpha-synuclein binding to double-stranded DNA and the potential similarities between this DNA binding and binding to phospholipid vesicles. A , single image from an animation (see Fig. S7 for full animation) showing a model of aSyn bound to DNA. The aSyn coordinates come from previous work showing its structure bound to a phospholipid micelle . The two curved alpha-helices of the N-terminal domain of aSyn fit into consecutive major grooves of B form DNA. Lysine residues within the protein are labeled. The C-terminal domain, containing the serine-129 phosphorylation site, is poorly resolved in previous structural work, so it is not clear how this phosphorylation may modulate DNA binding. B , single image from an animation (see Fig. S8 for full animation) showing a model of aSyn bound to DNA with 12 lysine residues labeled, using the same structural coordinates as in ( A ). In this particular model, potential electrostatic interactions between lysine-21, -23, and -80 with the phosphate backbone of DNA are present. C , left , ∼30 nm (in diameter) phospholipid vesicle shown in the presence of no synuclein, low or high alpha-synuclein (aSyn), and high serine-129 phosphorylated alpha-synuclein (pSyn). At low aSyn concentrations, aSyn binds the vesicle given its relatively high affinity for these structures. At high aSyn concentrations, multiple aSyn molecules are bound. To our knowledge, the effects of serine-129 phosphorylation on the interaction of aSyn with phospholipid bilayer vesicles with a diameter of ∼30 nm have not yet been tested. We speculate that serine-129 phosphorylation may inhibit binding in this context. Middle , 300 bp circular DNA having a diameter of ∼30 nm. At low concentrations, aSyn binds DNA given its high affinity for the curved negatively charged DNA surface (due to the phosphate backbone). At high aSyn concentrations, multiple aSyn molecules are bound, analogous to their binding to phospholipid vesicle membranes of similar geometry ( left ). Serine-129 phosphorylation inhibits the ability of synuclein to bind 300 bp circular DNA. Right , 300 bp linear DNA having a length of ∼100 nm. At low aSyn concentrations, aSyn binds as a monomer and bends linear DNA. At high aSyn concentrations, multiple aSyn molecules bind linear DNA and bend it into a conformation that resembles a DNA circle ( middle ). At high pSyn concentrations, pSyn binds as a monomer and bends linear DNA, but is not able to bend it into a circle-like form.

Article Snippet: Human aSyn and pSyn proteins (Proteos, cat. # RP-003, RP-004) and human aSyn, bSyn, gSyn, A30P, E46K, A53T, deltaNAC aSyn (rPeptide, cat. # S-1001, S-1003, S-1007, S-1005, S-1008, S-1002, S-1015) were stored at −80 °C.

Techniques: Binding Assay, Labeling, Phospho-proteomics