Binary Diamondoid Building Blocks for Molecular Gels

Adamantane is a type of diamondoid molecules that has a cage or globular shape with a diameter of 6.34 ± 0.04 Å. Anisotropic interactions between these truly nanoscopic particles can be induced through the derivatization of the diamondoid cage. Here we explore the gelation of paired systems of adamantane where attractions are introduced through van der Waals forces and hydrogen bonding. Gels are produced through the mixing of 1-adamantanecarboxylic acid (A1C) and 1-adamantylamine (A1N). Upon mixing dimethyl sulfoxide solutions of these molecules at vanishing concentrations, these diamondoid molecules rapidly precipitate. A space-filling gel of the resulting aggregates is observed at approximately 3% by weight. These resulting gels have elastic moduli of 10²–10⁴ Pa in the 3–7 wt % concentration range. At a 1:1 mol ratio of 1-adamantanecarboxylic acid (A1C) and 1-adamantylamine (A1N), the gel’s elastic modulus and yield stress increase as volume fractions ϕ^<i>x</i> and ϕ^<i>y</i> with <i>x</i> ≈ 4.2 and <i>y</i> ≈ 3.5. The dependencies of moduli and yield stress on the volume fraction display characteristics of colloidal gels. Transmission electron microscope (TEM) images indicate that the gels are formed from a network of interwoven and branched fibers which are composed of ∼30 nm crystallites that have undergone oriented aggregation to form fibers

Additional file 1 of Phosphorylation of EZH2 differs HER2-positive breast cancer invasiveness in a site-specific manner

Supplementary Material 1: Supplementary Fig. 1. EZH2-related genes in tumors provided by bioinformatics analysis based on TCGA-BRCA using STRING, and metascape databas

Additional figures of MBP-OtUBD and immobilized OtUBD-resin pulldowns.

(A, B) Ubiquitin pulldowns with different amounts of MBP-OtUBD. In A, the pulldown was performed by first binding MBP-OtUBD to an amylose resin and then incubating the resin with yeast cell lysate. In B, pulldown was performed by incubating the lysate with MBP-OtUBD and then binding the complexes to amylose resin. U, unbound fraction; E, fraction eluted with maltose. The bands seen at the expected molecular mass of MBP-UBD were likely a result of antibody cross-reactivity. (C) Anti-ubiquitin blot of MBP-OtUBD pulldowns from HEK293T whole cell lysates. U, unbound fraction; B, bound fraction (eluted with SDS sample buffer). (D) SYPRO Ruby protein stain of the eluates from OtUBD-resin in Fig 2B. E1/E2/E3, eluted fractions from serial low pH elutions. MBP, maltose-binding protein. (TIF)</p

OtUBD pulldown under denaturing condition specifically enriches for proteins covalently modified with ubiquitin.

(A) Workflow of OtUBD pulldowns following sample denaturation (red arrows) or under native (blue arrows) conditions. In the first case, cell lysate is treated with 8 M urea to denature and dissociate proteins. The denatured lysate is then diluted 1:1 with native buffer to allow ubiquitin to refold and bind to OtUBD resin. Under such conditions, only ubiquitylated proteins are expected to be enriched. In the second case, cell lysate contains native ubiquitylated proteins as well as proteins that interact with them. OtUBD pulldown under such conditions is expected to yield both ubiquitylated substrates and ubiquitin-binding proteins. (B) Outline for the use of tandem Co2+ resin pulldowns to validate OtUBD pulldown results under different conditions. Eluates from OtUBD after lysates were incubated with denaturant (red arrows) or left untreated (blue arrows) are (re)treated with denaturant (8 M urea or 6 M guanidine•HCl) and then subjected to IMAC with a Co2+ resin in denaturing conditions. Proteins covalently modified by His6-ubiquitin bind to the Co2+ resin while proteins that only interact noncovalently with ubiquitin end up in the flow-through. (C) Anti-ubiquitin blot of OtUBD pulldowns following native and urea denaturing treatments performed as described in Fig 3A. FT, flow-through; E, eluted fractions. The image was spliced to remove irrelevant lanes. (D) Total protein present in eluates of the OtUBD pulldowns in Fig 3C visualized by SYPRO Ruby stain. (E) Total protein present in different fractions of the Co2+ IMAC (see Fig 3B; the results shown here used urea as the denaturant) visualized by SYPRO Ruby stain. IN, input; FT, flow-through; E, fraction eluted with 500 mM imidazole. (F) Anti-ubiquitin blot of fractions from Co2+ IMAC (see Fig 3B; the blot shown here used urea as the denaturant) of eluates from native and denaturing OtUBD resin pulldowns. IN, input; FT, flow-through; E, fraction eluted with 500 mM imidazole. *The identity of the prominent approximately 20 kDa species in the flow-through from the native extract is unknown. (G) Anti-ubiquitin blot of OtUBD pulldowns from HeLa cell lysates performed as described in Fig 3A following native or denaturing treatments. The image was spliced to remove irrelevant lanes. IN, input; FT, flow-through; E, fraction eluted with low pH elution buffer. (H) Total protein present in eluates in Fig 3G visualized by SYPRO Ruby stain. (I) Immunoblot analysis of human proteasomal subunit Rpt6 in OtUBD pulldowns following native and urea denaturing treatments of lysates. Unmodified Rpt6 co-purified with OtUBD resin under native conditions but not following denaturation of extract. N, native condition; D, denaturing condition.</p

OtUBD pulldown-proteomics enables profiling of the ubiquitylome and ubiquitin interactome of yeast and human cells.

(A) Venn diagram of yeast proteins identified by OtUBD pulldown-proteomics with the pulldowns performed following either nondenaturing or urea denaturing treatments. The collection of proteins identified in OtUBD pulldowns under denaturing conditions is defined as the ubiquitylome (blue outline). The collection of proteins identified only in native OtUBD pulldowns is defined as the ubiquitin interactome (purple outline). (B) Venn diagram comparing the yeast ubiquitylome defined by OtUBD pulldown-proteomics with 3 previous studies using the di-Gly antibody IP method [60–62]. The numbers listed in the brackets are the total number of ubiquitylated proteins identified in each study. (C, D) Top biological pathways involved in OtUBD pulldown-defined yeast ubiquitylome (C) and ubiquitin interactome (D) based on GO analysis. The numeric values supporting these panels can be found in S3 Data. (E) Venn diagram comparing the human ubiquitylome defined by OtUBD pulldown and FK2 antibody IP performed in the current study. (F) Venn diagram comparing the published human ubiquitylomes defined by diGly antibody [63] and TUBE-based enrichments [64] and the OtUBD-defined ubiquitylome obtained in this study. The numbers listed in the brackets are the total number of ubiquitylated proteins identified in each study. GO, Gene Ontology; TUBE, tandem ubiquitin-binding entity.</p

Additional figures for OtUBD pulldown proteomics experiment in yeast.

(A) Representative anti-ubiquitin western blot of OtUBD pulldowns (under native conditions) used for proteomics analysis. IN, input; FT, flow-through; E1/E2/E3, eluted fractions from a series of low pH elutions. (B) Representative anti-ubiquitin blot of OtUBD pulldown samples following extract denaturation (urea) and used for proteomics analysis. (C) Representative SYPRO Ruby protein stain of OtUBD eluates resolved by SDS-PAGE. (D) Number of proteins detected in each biological replicate of OtUBD pulldown-MS and negative control. Error bar represents difference among technical replicates. (E) Overlay of TIC chromatographs of representative OtUBD pulldowns and negative control samples. The negative controls overall have much less peptide spectra compared to the OtUBD pulldown samples. This figure is generated with Thermo Xcalibur Qual Browser (v3.0.63) using.raw files from the corresponding runs (QEp21-2054_Zhang_A1_Native_UBD_pos, QEp21-2050_zhang_a2_native_ubd_neg, QEp21-2036_zhang_a3_denatured_ubd_pos, QEp21-2032_zhang_a4_denatured_ubd_neg), which have been deposited to the ProteomeXchange Consortium and made available to the public (see the Methods section for details). (F) Adjusted number of proteins detected in each biological replicate of OtUBD pulldowns. Only proteins whose TIC value are at least 20 times higher in the OtUBD pulldown samples compared to the corresponding negative control samples are included. (TIF)</p

Selective MS/MS spectra from OtUBD pulldown samples.

(A) Representative MS/MS spectrum of the Htb2 K111GG peptide. (B) Representative MS/MS spectrum of the Htb2 K123GG peptide. In addition to a/b/y ions, we identified multiple peaks from internal fragmentation and dehydration, potentially due to the serine/threonine-rich nature of the sequence. (C) Representative MS/MS spectrum of the YMR160W T534GG peptide. (TIF)</p

Numerical values for quantitative analyses in Figs 5C, 5D and S4C.

(XLSX)</p

Quality control data of the quantitative proteomics analysis with OtUBD pulldowns.

(A) Representative anti-ubiquitin blot of OtUBD pulldowns from WT, bre1△ and pib1△ yeast lysates used for proteomics analysis. IN, input; FT, flow-through; E, pooled eluted fractions. (B) Representative SYPRO Ruby gel showing the total proteins in eluates from OtUBD pulldown from WT, bre1△, and pib1△ yeast lysates. (C, D) Pearson correlation coefficients were calculated between each sample in the analyzed groups using normalized total TIC. Because ubiquitin is present in exceptionally high levels compared to all other proteins, it was excluded from the dataset for this analysis. With the exception of one pib1Δ sample, correlations between different samples were generally high, as expected if the majority of the ubiquitylome was not affected by deletion of a single E3. The low correlation in the single pib1Δ sample was likely due to an error during sample preparation, so the results were excluded from the quantitation. WT, wild-type. (TIF)</p

Identification of potential E3 substrates by OtUBD pulldown and label-free quantitation.

(A) Scheme for E3 substrate identification using OtUBD pulldown and quantitative proteomics. WT and E3 deletion (bre1Δ and pib1Δ) yeast strains were subjected to OtUBD pulldowns following extract denaturation. The eluted proteins were then analyzed by label-free quantitation. (B) Volcano plot comparing WT and bre1Δ samples. Orange dots represent proteins that were significantly enriched in WT samples compared to bre1Δ samples. Horizontal dashed line indicates p = 0.05. Vertical dash lines indicate relative change of +/− 1.5-fold. The numeric values supporting this figure can be found in S2 Data. (C) Two different ubiquitylation sites identified on histone H2B (Htb2) in different samples. (D) List of proteins that were significantly enriched in WT samples compared to bre1Δ samples (orange dots in B). Green color indicates proteins that were previously reported to be stabilized in bre1Δ yeast. (E) Ubiquitylated proteins detected exclusively in WT but not bre1Δ samples. Green color indicates proteins previously reported to be stabilized in bre1Δ cells. (F) Volcano plot comparing WT and pib1Δ samples. Orange dots represent proteins that were significantly enriched in WT samples compared to pib1Δ samples. Horizontal dash line indicates p = 0.05. Vertical dash lines indicate relative change of +/− 1.5-fold. The numeric values supporting this figure can be found in S2 Data. (G) List of proteins that were significantly enriched in WT samples compared to pib1Δ samples (orange dots in F). Green color indicates proteins that were previously reported to be stabilized in pib1Δ yeast. (H) Ubiquitylated proteins detected exclusively in WT but not pib1Δ samples. Green color indicates proteins previously reported to be stabilized in pib1Δ yeast. WT, wild-type.</p