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Spindlin1 (SPIN1) is a transcriptional coactivator with critical functions in embryonic development and emerging roles in cancer. SPIN1 harbors three Tudor domains, two of which engage the tail of histone H3 by reading the H3-Lys-4 trimethylation and H3-Arg-8 asymmetric dimethylation marks. To gain mechanistic insight into how SPIN1 functions as a transcriptional coactivator, here we purified its interacting proteins. We identified an uncharacterized protein (C11orf84), which we renamed SPIN1 docking protein (SPIN·DOC), that directly binds SPIN1 and strongly disrupts its histone methylation reading ability, causing it to disassociate from chromatin. The Spindlin family of coactivators has five related members (SPIN1, 2A, 2B, 3, and 4), and we found that all of them bind SPIN·DOC. It has been reported previously that SPIN1 regulates gene expression in the Wnt signaling pathway by directly interacting with transcription factor 4 (TCF4). We observed here that SPIN·DOC associates with TCF4 in a SPIN1-dependent manner and dampens SPIN1 coactivator activity in TOPflash reporter assays. Furthermore, knockdown and overexpression experiments indicated that SPIN·DOC represses the expression of a number of SPIN1-regulated genes, including those encoding ribosomal RNA and the cytokine IL1B. In conclusion, we have identified SPIN·DOC as a transcriptional repressor that binds SPIN1 and masks its ability to engage the H3-Lys-4 trimethylation activation mark.

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The ultraviolet (UV) absorption of various sections of the human lens was studied and compared with protein expression paralleling differential UV absorbance in anterior and posterior lenticular tissue. The UV absorbance of serial lens cryostat sections (60 μm) and that of lens capsules was determined using a Shimadzu scanning spectrophotometer, and the absorption coefficients were calculated. Two-dimensional gel electrophoresis was performed using two pooled lenticular protein extracts (anterior and posterior sections). Protein spots were quantified and significantly different spots were identified by mass spectrometry following in-gel digestion with trypsin and chymotrypsin. The UV-C and UV-B absorption of the human lens increased toward the posterior parts of the lens. The anterior and posterior lens capsules also effectively absorbed UV radiation. Levels of molecular chaperone proteins Beta-crystallin B2 (UniProtKB ID:P43320), A3 (UniProtKB ID:P05813) and of glyceraldehyde 3-phosphate dehydrogenase (UniProtKB ID:P04406) were significantly higher in the anterior part of the lens, whereas lens proteins Beta-crystallin B1 (UniProtKB ID:P53674) and Alpha-crystallin A chain (UniProtKB ID:P02489) were higher in the posterior sections. These results provide evidence that differential UV absorption in the anterior and posterior lens is accompanied by differential protein expression.

Glutaminase is a multifunctional enzyme encoded by gene Gls involved in energy metabolism, ammonia trafficking and regeneration of neurotransmitter glutamate. To address the proteomic basis for the neurophenotypes of glutaminase-deficient mice, brain proteins from late gestation wild type, Gls +/− and Gls −/− male mice were subjected to two-dimensional gel electrophoresis, with subsequent identification by mass spectrometry using nano-LC-ESI–MS/MS. Protein spots that showed differential genotypic variation were quantified by immunoblotting. Differentially expressed proteins unambiguously identified by MS/MS included neurocalcin delta, retinol binding protein-1, reticulocalbin-3, cytoskeleton proteins fascin and tropomyosin alpha-4-chain, dihydropyrimidinase-related protein-5, apolipoprotein IV and proteins from protein metabolism proteasome subunits alpha type 2, type 7, heterogeneous nuclear ribonucleoprotein C1/C2 and H, voltage-gated anion-selective channel proteins 1 and 2, ATP synthase subunit β and transitional endoplasmic reticulum ATPase. An interaction network determined by Ingenuity Pathway Analysis revealed a link between glutaminase and calcium, Akt and retinol signaling, cytoskeletal elements, ATPases, ion channels, protein synthesis and the proteasome system, intermediary, nucleic acid and lipid metabolism, huntingtin, guidance cues, transforming growth factor beta-1 and hepatocyte nuclear factor 4-alpha. The network identified involves (a) cellular assembly and organization and (b) cell signaling and cell cycle, suggesting that Gls is crucial for neuronal maturation.

The discovery of inhibitors of methyl- and acetyl-binding domains has provided evidence for the 'druggability' of epigenetic effector molecules. The small-molecule probe UNC1215 prevents methyl-dependent protein-protein interactions by engaging the aromatic cage of MBT domains and, with lower affinity, Tudor domains. Using a library of tagged UNC1215 analogs, we screened a protein-domain microarray of human methyllysine effector molecules to rapidly detect compounds with new binding profiles with either increased or decreased specificity. Using this approach, we identified a compound (EML405) that acquired a novel interaction with the Tudor-domain-containing protein Spindlin1 (SPIN1). Structural studies facilitated the rational synthesis of SPIN1 inhibitors with increased selectivity (EML631-633), which engage SPIN1 in cells, block its ability to 'read' H3K4me3 marks and inhibit its transcriptional-coactivator activity. Protein microarrays can thus be used as a platform to 'target-hop' and identify small molecules that bind and compete with domain-motif interactions.

Protein profiling has revealed the presence of glacontryphan-M, a peptide toxin identified only in the sea snail genus Conus , in the wings of Hebomoia glaucippe (HG). The wings and body of HG were homogenized and the proteins were extracted and analyzed by 2D gel electrophoresis with subsequent in-gel digestion. Posttranslational protein modifications were detected and analyzed by nano–LC-MS/MS. An antibody was generated against glacontryphan-M, and protein extracts from the wings of HG samples from Malaysia, Indonesia, and the Philippines were tested by immunoblotting. Glacontryphan-M was unambiguously identified in the wings of HG containing the following posttranslational protein modifications: monoglutamylation at E55, methylation at E53, quinone modification at W61, cyanylation at C56, and amidation of the C terminus at G63. Immunoblotting revealed the presence of the toxin in the wings of HG from all origins, showing a single band for glacontryphan-M in HG samples from Malaysia and Philippines and a double band in HG samples from Indonesia. Intriguingly, sequence analysis indicated that the Conus glacontryphan is identical to that of HG. The toxin may function as a defense against diverse predators, including ants, mantes, spiders, lizards, green frogs, and birds.

Bilin-binding protein (BBP) is a member of the lipocalin superfamily and a pigment protein in Lepidoptera. It is binding to a series of lipidic compounds but its functions remain to be elucidated. Working on wing proteins in Hebomoia glaucippe, we observed this protein on gels and decided to characterize BBP. A gel-based mass spectrometrical method using two-dimensional gel electrophoresis followed by in-gel digestion of protein spots followed by nano-LC-ESI-MS/MS (ion trap, HCT) identification and characterization of proteins was applied. An antibody was generated against the protein and immunoblotting in the butterfly and mouse brain was carried out. Two spots were identified from the butterfly wing as BBP (P09464) with high sequence coverage. Nitrotyrosination (Y163; as aminotyrosine) was observed and nitration was verified using immunoblotting. Additional posttranslational modifications (PTMs) as hypusine, carboxylation, kynurenine, aminoadipic acid, were proposed. The presence of BBP-immunoreactive protein was also observed in mouse brain. The characterization of BBP showed high sequence similarity with mouse apolipoprotein D and the findings suggest a tentative function of BBP comparable to apolipoproteins. The role of the PTMs remains elusive but nitration, in analogy to nitration effects reported in literature, proposes a role for mechanoelastic proteins and protein-protein interactions.

Although several insect cuticular genes and proteins are annotated and an arthropod cuticular database is available, mass spectrometrical data on cuticular proteins and their post-translational modifications are limited. Wings from Hebemoia glaucippe were analyzed by scanning electron microscopy or homogenized, proteins were extracted and run on 2DE. In-gel digestion was carried out by using trypsin, chymotrypsin and Asp-N and subsequently the resulting peptides and post-translational modifications were identified by ion trap tandem mass spectrometry (nano-LC-ESI-MS/MS; HCT). A complex wing skeleton and the cuticle of H. glaucippe were demonstrated. Cuticle protein 18.6, isoform A, pupal cuticle protein, cuticular protein CPR59A and two putative proteins, putative cuticular protein B2DBJ and putative cuticle protein CPG31 with two expression forms were identified. Two phosphorylation sites on the same peptide, T213 and S214, were identified on putative cuticle protein CPG31, quinone formation was observed at Y76 on cuticular protein CPR59A probably indicating the presence of post-translational modifications. The results may be relevant for the interpretation of mechanoelastic and physical properties of these proteins. Along with the extraordinary architecture the proteinaceous matrix is probably representing or allowing the unusual aerodynamic function of the butterfly wing. Moreover, the results may be important for mechanisms of insecticide and drought resistance.

Protein deamidation is a posttranslational modification with important implications in physiology and medicine. There is, however, no simple technique for a rapid screening of protein deamidation. The deamidating activity of transglutaminase was applied to establish a simple method for the screen of protein deamidation using recombinant human growth hormone, a rat hippocampal membrane fraction, and a cell homogenate enriched in 5-hydroxytryptamine-1A receptor as model systems. Here we report a simple, economic, and fast approach to assess protein deamidation by two electrophoretic methods: differential cleavage on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) via in situ V8 protease digestion and the principle of spot shifting via blue native (BN)-PAGE/two-dimensional (2D)-SDS-PAGE/immunoblotting.

Background Histone posttranslational modifications (PTMs) function to regulate chromatin structure and function in part through the recruitment of effector proteins that harbor specialized “reader” domains. Despite efforts to elucidate reader domain–PTM interactions, the influence of neighboring PTMs and the target specificity of many reader domains is still unclear. The aim of this study was to use a high-throughput histone peptide microarray platform to interrogate 83 known and putative histone reader domains from the chromo and Tudor domain families to identify their interactions and characterize the influence of neighboring PTMs on these interactions. Results Nearly a quarter of the chromo and Tudor domains screened showed interactions with histone PTMs by peptide microarray, revealing known and several novel methyllysine interactions. Specifically, we found that the CBX/HP1 chromodomains that recognize H3K9me also recognize H3K23me2/3—a poorly understood histone PTM. We also observed that, in addition to their interaction with H3K4me3, Tudor domains of the Spindlin family also recognized H4K20me3—a previously uncharacterized interaction. Several Tudor domains also showed novel interactions with H3K4me as well. Conclusions These results provide an important resource for the epigenetics and chromatin community on the interactions of many human chromo and Tudor domains. They also provide the basis for additional studies into the functional significance of the novel interactions that were discovered. Electronic supplementary material The online version of this article (doi:10.1186/s13072-017-0117-5) contains supplementary material, which is available to authorized users.