tri methyl histone h3 lys9 d4w1u  (Cell Signaling Technology Inc)

Journal: Science Advances

Article Title: Mechanistic insights into 50 S precursor recognition and targeting by erythromycin resistance methyltransferase

doi: 10.1126/sciadv.aea1545

Figure Lengend Snippet: ( A ) Sucrose density gradient profile shows the presence of a distinct precursor peak due to the U2552C mutation. WT, wild type. ( B ) In vitro methylation assay depicts that the precursor fractions isolated from the U2552C cells are responsive to Erm-mediated methylation however remain unresponsive to a mature 30 S or 50 S subunit (data from n = 3 independent experiments are shown where the mean has been plotted with ± SD). ( C ) Tabulated values obtained from cryo-OrbiSIMS for unmodified sample, SAM-modified samples, and N6-mustard–modified sample. The difference column shows that the additional mass (dalton) appended on to the fragment which corresponds to the modification it has undergone. ( D ) Cryo-EM reconstruction of the U2552C fraction yields three discreet precursor states referred to as states I, II, and III. Particle distribution has been provided beside each ribosomal assembly state. The 2D map of the 23 S rRNA shown next to each precursor state demonstrates differential domain assembly in each of the states (I to VI in blue denote the domains of the 23 S rRNA).

Article Snippet: Briefly, the assay was performed in a methylation buffer [20 mM tris-HCl (pH 7.5), 80 mM NH 4 Cl, 1 mM Mg(OAc) 2 , and 6 mM 2-mercaptoethanol] containing 0.3 μM mature 30 S , mature 50 S , or ribosomal precursor, 0.3 μM MTase, 0.1 μM (3H)-SAM (100 Ci/mmol, American Radiolabeled Chemicals), and 1 U of ribonuclease inhibitor (Thermo Fisher Scientific) in a total reaction volume of 30 μl.

Techniques: Mutagenesis, In Vitro, Methylation, Isolation, Modification, Cryo-EM Sample Prep

Journal: Science Advances

Article Title: Mechanistic insights into 50 S precursor recognition and targeting by erythromycin resistance methyltransferase

doi: 10.1126/sciadv.aea1545

Figure Lengend Snippet: Erms are able to bind to a specific state of the ribosomal precursor where the target helix, H73, is exposed. Erm’s CTD, responsible for global recognition, docks into a transient cleft formed by H42, H95, and H97. At this stage, the NTD or the catalytic domain is further away from H73 (C-II). Subsequently, conformational swaying-like motion in Erm allows its NTD to locally scan for the target base and come in proximity of H73 to facilitate methylation (C-I). Followed by which, Erm retracts and dissociates from the precursor, and the immature ribosome follows its course of biogenesis.

Article Snippet: Briefly, the assay was performed in a methylation buffer [20 mM tris-HCl (pH 7.5), 80 mM NH 4 Cl, 1 mM Mg(OAc) 2 , and 6 mM 2-mercaptoethanol] containing 0.3 μM mature 30 S , mature 50 S , or ribosomal precursor, 0.3 μM MTase, 0.1 μM (3H)-SAM (100 Ci/mmol, American Radiolabeled Chemicals), and 1 U of ribonuclease inhibitor (Thermo Fisher Scientific) in a total reaction volume of 30 μl.

Techniques: Methylation

Journal: bioRxiv

Article Title: GFAP Degradation in TBI: Linking Novel Modified Products to Astrocyte Pathology and Patient Outcome

doi: 10.1101/2025.08.01.668181

Figure Lengend Snippet: A) TBI patient CSF immunoblots of a 15% gel show GFAP detected by a polyclonal (top) and a monoclonal (clone 3E10, bottom) antibody (postinjury days, i+1 through 1+3) of an 84 year old male sustaining facial fractures, subarachnoid and intraventricular hemorrhage and intraparenchymal hematomas from a fall. Observed GFAP band molecular weights were bands (II.) 36-39kDa, bands (III.) 20.4-23kDa, and bands (IV.) 17-19kDa, missed by clone 3E10. B) MS peptide maps of immunoprecipitated GFAP breakdown products (BDPs) of four cut out gel band sets (I.-IV., Suppl. Fig. 1) from six CSF TBI samples of three patients. Percent of fragment sequence coverage (left) by high confidence (green, FDR≤1%) and repeatedly identified medium confidence (yellow FDR≤5%) peptides. Bands (I.) had 359 amino acids (aa) with calculated molecular weight of 41.7kDa and suggested new N- and C-termini of serine 53 (S53) and lysine 411 (K411). Bands (II.) spanned 349 aa from glycine 56 (G56) to leucine (L404) within which 37/38kDa fragments with variable endings between TBI samples were located (Suppl.Fig.1). Bands (III.) contained two GFAP BDPs, 92% covered coil1-containing product of 185 aa, calculated molecular weight of 21.8kDa; 77% covered coil2-containing product of 151 aa and peptides, estimated molecular weight of 17.5kDa. Bands (IV.) 96% covered 181 aa coil1 product with size between 21-21.6kDa -±PTMs; and 77% covered coil2 product of 130 aa with estimated molecular weight of 15-15.3kDa. C) Predicted endings of the two sets of novel TBI patients’ GFAP-BDPs. Coil1 products N-termini were S53 (III.) and alanine, A55 (IV.) and new C-termini were arginine 239 (R239) C-terminus (III.) and A233 (IV.), both stretching beyond previously described caspase 6 cleavage around VELD225 . Coil2 products had consistent N-terminus phenylalanine 261 (F261), see reported caspase 3 cleavage RSKFA↓DLTS , and variably ended with K411 (III.) and with R390 (IV.).

Article Snippet: Pan caspase inhibitor ZVAD-FMK (Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (Enzo/ThermoFisher) is a cell-permeable, irreversible pan-caspase inhibitor effective against a spectrum of caspases (1-10, except 2; Invitrogen) .

Techniques: Western Blot, Immunoprecipitation, Sequencing, Molecular Weight

Journal: bioRxiv

Article Title: GFAP Degradation in TBI: Linking Novel Modified Products to Astrocyte Pathology and Patient Outcome

doi: 10.1101/2025.08.01.668181

Figure Lengend Snippet: A) Unstretched or stretch-injured human astrocytes were treated with calpain inhibitor calpeptin (C, 13mM), caspase inhibitor Z-VAD-FMK (V, 11mM), both (CV), or no drug (−) for 48 hours. Adherent cells were lysed (Whole Cell lysate, WCL), centrifuged fluid pellet contained lifted material (Lifted Cell Pellet) and fluids were concentrated (Conditioned medium, CM). Top: 2sec exposures for 50kDa GFAP and 42-47kDa BDPs. Mid: 1min exposures for 38-25kDa BDP bands. Bottom: 20min (WCL, CM) and 5min (Lifted cell pellet) exposures for 17-25kDa BDPs (See Suppl.Fig.7 for 5hr blot and 48h densitometry). B+C) Live imaging of distinct cerebral human astrocyte morphotypes and their acute trauma stages defined by membrane leak, calpain and caspase activities during 5-6 hours postinjury . B) . Left : Elongated ( Top ) and bushy ( bottom ) morphotypes and injury shapes on phase contrast. Right : Fluorescence images for plasma membrane permeability (5min uptake assay of propidium iodide, [PI+], red), calpain activity (CMAC-tBOC-leucyl-methionine, [CMAC-tBLM], converted substrate, blue) and caspase activity ([NucView488] converted substrate, green). Top : Control fibrous astrocytes display intact processes, acutely stretched cells had thinned, frequently beaded clasmatodendrotic processes (arrows). [PI−] astrocytes were calpain active. [PI+], leaky cells presented variable caspase activity (yellow/orange), lacking calpain-activity. Bottom : Bushy control and stretched astrocytes were calpain active (blue). The majority of stretched bushy astrocytes were calpain and caspase active (teal). [PI+], leaky bushy cells either retained both activities (yellow/white), or lost these protease activities (red, arrows) displaying necrotic, atrophic morphology. Phase-dense vacuoles were unstained. C) Enlarged from B: Individual fibrous (top) and bushy (bottom) astrocytes showing distinct structural and functional features of intact, injured and necrotic astrocyte phenotypes: Intact [PI−], calpain[+]; Wounded : [PI−/+], cytoplasmic and nuclear caspase[+]; Dying: [PI+] nuclear caspase [−/+], calpain[−]. Suppl.Fig.8 for separate fluorescence channels. Scale bars: 100µm.

Article Snippet: Pan caspase inhibitor ZVAD-FMK (Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (Enzo/ThermoFisher) is a cell-permeable, irreversible pan-caspase inhibitor effective against a spectrum of caspases (1-10, except 2; Invitrogen) .

Techniques: Imaging, Membrane, Fluorescence, Clinical Proteomics, Permeability, Activity Assay, Control, Functional Assay