Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Prosaposin in CNS health and disease, metabolic stress and exercise adaptation

doi: 10.1007/s00109-026-02643-3

Figure Lengend Snippet: Overview of prosaptide peptides derived from the saposin C domain of prosaposin (PSAP) and their tested therapeutic applications. The full saposin C sequence is shown at the top, with the neurotrophic region ( LIDNNKTEKEIL ) highlighted in green and underlined. Left panel: Sequences of various bioengineered or truncated prosaptide peptides tested in previous studies. Conservative amino acid substitutions (*) and lysine deletions (-) are indicated. Bioactivity is noted where tested. Right panel: Mapping of each prosaptide or full saposin (A–D) to specific disease-relevant therapeutic conditions, including oxidative stress, trauma/injury, ischemia, synucleinopathies, diabetes, allodynia, drug-induced toxicity, and Alzheimer’s disease-related Aβ/tau pathology. Checkboxes indicate whether the peptide has been evaluated in vitro, in vivo, or both. Peptides derived from the neurotrophic region, such as TX14(A), 769, and PS18, show diverse functional relevance across multiple disease models. Created with BioRender.com

Article Snippet: 39269584 , Taha et al. , 2024 , USA , In vivo: C57/B6L male young (3 months) mice , None , PS18 , In vivo: subcutaneous injection daily for 7 days , 2.0 mg/kg PS18 , PS18 obtained from Phoenix Pharmaceuticals (324–431) , PS18-injected animals showed modest but unpowered and independently unvalidated increases in BDNF, SNAP25, and PSD95 in cortical synapatosome fractions. Additionally, there were nonsignificant trends towards increases for SYP1, SYT1, and SAP97 in the cortical synaptosome fractions.

Techniques: Derivative Assay, Sequencing, In Vitro, In Vivo, Functional Assay

Journal: Nucleic Acids Research

Article Title: Phosphorylation of HP1/Swi6 relieves competition with Suv39/Clr4 on nucleosomes and enables H3K9 trimethyl spreading

doi: 10.1093/nar/gkaf1244

Figure Lengend Snippet: Swi6 phosphorylation increases oligomerization working through, or in parallel to, the ARK loop and NTE acidic stretch. ( A ) Production of phosphorylated Swi6 (pSwi6) in E. coli . CKII is co-expressed with Swi6. After lysis and purification, the 6XHis tag is removed from the pSwi6 or unpSwi6 protein by TEV cleavage. ( B ) Mass Spectrometry on pSwi6. Shown is a domain diagram of Swi6. Phosphorylation sites identified in pSwi6 by 2D-ETD-MS are indicated and grouped by detection prevalence in the sample. For prevalence of pS18 and/or p24 peptides, see . ( C ) SEC-MALS on EDC/NHS crosslinked unpSwi6 (black) and pSwi6 (green). Relative refractive index signals (solid lines, left y -axis) and derived molar masses (lines over particular species, right y -axis) are shown as a function of the elution volume. The Swi6 concentration was 100 µM. ( D ) Overview of NTE and CD residues targeted for mutation in subsequent panels. S18/S24 phosphoserines, the CD ARK loop (R93AK94A; loopX), and the CD-preceding acidic stretch (E74-80A; acidicX). ( E–J ) 2-D Density hexbin plots examining silencing at nucleation “green” and spreading “orange” reporter, wild-type, S18/24A, loopX, and acidicX mutants and combinations thereof. Note this experiment was a fully separate run from Fig. and .

Article Snippet: For primary antibodies, we used either a polyclonal anti-Swi6 antibody [ ] or anti-pS18-pS24 antibody (Rockland Immunochemicals, this study) diluted 1:1000, and as loading control an anti-α-tubulin antibody [T6199 (Millipore)] (diluted 1:2000).

Techniques: Phospho-proteomics, Lysis, Purification, Mass Spectrometry, Refractive Index, Derivative Assay, Concentration Assay, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Phosphorylation of HP1/Swi6 relieves competition with Suv39/Clr4 on nucleosomes and enables H3K9 trimethyl spreading

doi: 10.1093/nar/gkaf1244

Figure Lengend Snippet: Swi6 phosphorylation mitigates inhibition of the Clr4-mediated conversion of H3K9me2 to H3K9me3. ( A ) Most Swi6 molecules in the cell are phosphorylated at S18 and S24. Quantitative western blots against total Swi6 and phosphorylated Swi6 at S18 and/or S24. A standard curve of pSwi6 isolated as in Fig. is included in both blots. Total protein lysates from wild-type swi6 and swi6 S18/24A strains were probed with a polyclonal anti-Swi6 antibody (α-Swi6) or an antibody raised against a phosphorylated S18/S24 peptide (α-pS18-pS24). α-Tubulin was used as a loading control. One of two independent experiments is shown. L; ladder. Total fraction of Swi6 phosphorylated in vivo at S18 and/or S24 is adjusted by the prevalence of phosphorylation in the in vitro produced standard (∼0.76, see ). ( B ) Experimental scheme to probe the impact of Swi6 on H3K9 trimethylation. ( C ) Quantitative western blots on the time-dependent formation of H3K9me3 from H3K9me2 mononucleosomes in the presence of pSwi6 or unpSwi6 under single turnover conditions. The same blots were probed with α-H3K9me3 and α-H4 antibodies as a loading and normalization control. ( D ) Single exponential fits of production of H3K9me3 tails over time for indicated concentrations of unpSwi6 or pSwi6. ( E ) Plot of the observed single turnover rate constant measured from exponential fits ( k obs ) against the Swi6 concentration in µM.

Article Snippet: For primary antibodies, we used either a polyclonal anti-Swi6 antibody [ ] or anti-pS18-pS24 antibody (Rockland Immunochemicals, this study) diluted 1:1000, and as loading control an anti-α-tubulin antibody [T6199 (Millipore)] (diluted 1:2000).

Techniques: Phospho-proteomics, Inhibition, Western Blot, Isolation, Control, In Vivo, In Vitro, Produced, Concentration Assay

Journal: Nucleic Acids Research

Article Title: Phosphorylation of HP1/Swi6 relieves competition with Suv39/Clr4 on nucleosomes and enables H3K9 trimethyl spreading

doi: 10.1093/nar/gkaf1244

Figure Lengend Snippet: Swi6 phosphorylation focuses Swi6 onto heterochromatin nucleation sites and away from euchromatin. ( A ) Schematic of Swi6 ChIP-seq experiments. The anti-pS18-pS24 experiment was carried out with wild-type swi6 (and swi6 S18/24A as a negative control, see ). For the anti-FLAG experiments, the endogenous swi6 locus was 3XFLAG tagged in the context of wild-type swi6 (black), swi6 S18/24A (blue), or swi6 S18/24/117–220A ( 6S/A , red). For quantitative normalization, ChIP reactions were supplemented with Drosophila chromatin spike-in. ( B ) Heat maps of spike-in normalized FLAG ChIP-seq signal (in counts per million, CPM) for swi6, swi6 S18/24A , or swi6 6S/A at features previously classified as nucleators . ( C ) As in (B) but for features classified as regions of H3K9me2 spreading . ( D ) FLAG ChIP-seq signal visualization plot for the MAT ΔREIII HSS mating type locus. The solid ChIP/input line for each genotype represents the mean of three repeats, while the shading represents the 95% confidence interval. ( E ) As in (D) but for cen II with zoom-in of the left and right pericentromeric region. The brown dashed boxes indicate siRNAi-generating centers as mapped in . ( F ) Heat map as in (B) and (C), but for H3K9me2 negative regions. ( G ) ChIP-seq signal visualization plots for H3K9me2 negative regions. The solid ChIP/input line for each genotype represents the mean of two repeats, while the shading represents the 95% confidence interval. A high signal replicate was removed for both genotypes. ChIP-seq signal visualization for all three genotypes with all three repeats in .

Article Snippet: For primary antibodies, we used either a polyclonal anti-Swi6 antibody [ ] or anti-pS18-pS24 antibody (Rockland Immunochemicals, this study) diluted 1:1000, and as loading control an anti-α-tubulin antibody [T6199 (Millipore)] (diluted 1:2000).

Techniques: Phospho-proteomics, ChIP-sequencing, Negative Control

Journal: Nucleic Acids Research

Article Title: Phosphorylation of HP1/Swi6 relieves competition with Suv39/Clr4 on nucleosomes and enables H3K9 trimethyl spreading

doi: 10.1093/nar/gkaf1244

Figure Lengend Snippet: S18 and/or S24 phosphorylation contributes to pSwi6’s biochemical behaviors. ( A ) Schematic of phosphorylated Swi6 molecules used in this figure. ( B ) pSwi6 S18/24A is defective in oligomerization. pSwi6 or pSwi6 S18/24A was crosslinked or not (-) at indicated concentrations, separated on SDS–PAGE, and probed with a polyclonal anti-Swi6 antibody. M, monomer; D, dimer; T, tetramer; O, octamer; O + , higher molecular weight species. Below: Quantification of oligomer signal divided by dimer signal for crosslinked species. ( C ) FP with H3K9me0 (open circles) or H3K c 9me3 (MLA, filled circles) mononucleosomes as in Fig. , with pSwi6 (green) and pSwi6 S18/24A (magenta). Error bars represent the standard deviation of three repeats. Relative dissociation constant ( K d ) values in . ( D ) Quantitative western blots on the time-dependent formation of H3K9me3 from H3K9me2 mononucleosomes in the presence of pSwi6 or pSwi6 S18/24A , as in Fig. . Quantification of the signal below. Note that the reactions were not fast enough in this experiment to derive a single exponential observed rate. ( E ) Model of the impact of pSwi6 on Clr4 activity. Left: pSwi6 does not engage with H3K9me0 nucleosomes, clearing the substrate for Clr4, and has reduced interactions with the nucleosome core. The pS18/pS24 NTE releases the ARK loop (light blue), allowing CD–CD contacts. Right: unpSwi6 binds H3K9me3 and me0 nucleosomes, occluding Clr4 access. The S18/S24 unphosphorylated NTE blocks the ARK from engaging CDs and additionally may contribute to increased nucleosome affinity by contacting DNA or octamer.

Article Snippet: For primary antibodies, we used either a polyclonal anti-Swi6 antibody [ ] or anti-pS18-pS24 antibody (Rockland Immunochemicals, this study) diluted 1:1000, and as loading control an anti-α-tubulin antibody [T6199 (Millipore)] (diluted 1:2000).

Techniques: Phospho-proteomics, SDS Page, Molecular Weight, Standard Deviation, Western Blot, Activity Assay