Poly(ADP-ribosyl)ation is catalyzed by a family of enzymes known as PARPs. We describe a method to characterize the human aspartic acid- and glutamic acid-ADP-ribosylated proteome. We identified 1,048 ADP-ribosylation sites on 340 proteins involved in a wide array of nuclear functions; among these were many previously unknown PARP downstream targets whose ADP-ribosylation was sensitive to PARP inhibitor treatment. We also confirmed that iniparib had a negligible effect on PARP activity in intact cells.

SUMMARY Many DNA and RNA regulatory proteins contain polypeptide domains that are unstructured when analyzed in cell lysates. These domains are typified by an over-representation of a limited number of amino acids and have been termed prion-like, intrinsically disordered or low complexity (LC) domains. When incubated at high concentration, certain of these LC domains polymerize into labile, amyloid-like fibers. Here we report methods allowing the generation of a molecular footprint of the polymeric state of the LC domain of hnRNPA2. By deploying this footprinting technique to probe the structure of the native hnRNPA2 protein present in isolated nuclei, we offer evidence that its LC domain exists in a similar conformation as that described for recombinant polymers of the protein. These observations favor biologic utility to the polymerization of LC domains in the pathway of information transfer from gene to message to protein.

The toxic proline:arginine (PR n ) poly-dipeptide encoded by the (GGGGCC) n repeat expansion in the C9orf72 form of heritable amyotrophic lateral sclerosis (ALS) binds to the central channel of the nuclear pore and inhibits the movement of macromolecules into and out of the nucleus. The PR n poly-dipeptide binds to polymeric forms of the phenylalanine:glycine (FG) repeat domain, which is shared by several proteins of the nuclear pore complex, including those in the central channel. A method of chemical footprinting was used to characterize labile, cross-β polymers formed from the FG domain of the Nup54 protein. Mutations within the footprinted region of Nup54 polymers blocked both polymerization and binding by the PR n poly-dipeptide. The aliphatic alcohol 1,6-hexanediol melted FG domain polymers in vitro and reversed PR n -mediated enhancement of the nuclear pore permeability barrier. These data suggest that toxicity of the PR n poly-dipeptide results in part from its ability to lock the FG repeats of nuclear pore proteins in the polymerized state. Our study offers a mechanistic interpretation of PR n poly-dipeptide toxicity in the context of a prominent form of ALS.C9orf72 repeat expansion | PR n poly-dipeptide | nuclear pore | FG domain | labile cross-β polymers

This work presents the establishment of novel bright-emission small-molecule NIR-II fluorophores for in vivo tumor imaging and NIR-II image-guided sentinel lymph node surgery.

We have discovered a new high-potency thermostable sweet protein, which we name brazzein, in a wild African plant Pentadiplandra brazzeana Baillon. Brazzein is 2,000 times sweeter than sucrose in comparison to 2% sucrose aqueous solution and 500 times in comparison to 10% of the sugar. Its taste is more similar to sucrose than that of thaumatin. Its sweetness is not destroyed by 80°C for 4 h. Brazzein is comprised of 54 amino acid residues, corresponding to a molecular mass of 6,473 Da. Materials and methods MaterialsFruits of Pentadiplandra brazzeana from West Africa were used. Each fruit has a reddish nutshell-like epicarp, under which three to five reniform seeds are located, surrounded by a thick soft layer of red pulp which is sweet and containing brazzein. Protein isolationWe found that the best method to extract brazzein from the pulp was to use 0.1 M phosphate buffer at pH 7.0 containing 5% glycerol, 0.1 mM DTT, 20 &ml PMSF, 0.1 mM EDTA and 0.5% (w/v) PVP at 4°C. The proteins, which precipitated between 30 and 85% ammonium sulfate saturation, were separated on a Sephacryl S-100 column (JXK 26/100; Pharmacia, Piscataway, NJ) in 50 mM phosphate buffer at pH 7.0. Finally, brazzein was purified on a CM-Sepharose CLdB column (XK 16/70; Pharmacia, Piscataway, NJ) by a NaCl gradient of 0.1 to 0.4 M in 20 mM sodium citrate at pH 3.6. *Corresponding author. Fax: (1) (608) 262-7420.Abbreviations: EDTA, ethylenediamine tetra acetic acid; ES&MS, electrospray ionization mass spectrometry; M,, molecular weight; PMSF, phenyhnethylsulfonyl fluoride; PVDF, polyvinylidene difluoride; PVP, polyvinylpolypyrrolidone; RP-HPLC, reverse-phase high performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Tris, Tris(hydroxymethyl)aminomethane. Protein characterizationA tricine system [13] was used in SDS-PAGE. ESI-MS was carried out at the Analytical Chemistry Center of the Medical School of the University of Texas in Houston. An expert taste panel (NutraSweet R&D, Mt. Prospect, IL) compared brazzein with a series of sucrose concentrations. Thermostability assay was carried out by incubating aqueous solutions of 1 mg/ml brazzein in water bathes at 80°C. Every 15 min an aliquot was analyzed by a lab bench taste panel. Table 1 summarizes the results of brazzein purification. The results of SDS-PAGE (Fig. 1) show the molecular weight of brazzein to be about 6,500 Da. Brazzein is a single chain polypeptide as shown by the result of SDS-PAGE and gel filtration. The molecular mass of brazzein determined by ESI-MS is 6473 Da. Brazzein is 2,000 times sweeter than sucrose in comparison to 2% sucrose solution (w/v), and 500 times to 10% of the sugar (w/v). It has a more sucrose-like temporal profile than other sweet proteins, and it cross-adapts with other sweeteners. The sweetness of brazzein remained after incubation at 80°C for 4 h. Sequence determination Results Protein purification and characterization Amino acid sequenceThe results of tryptic and Staphylococcus aureus V8 prote...

The fruit of Pentadiplandra brazzeana Baillon contains a small, sweet-tasting protein named brazzein. The structure of brazzein in solution was determined by proton nuclear magnetic resonance spectroscopy at pH 5.2 and 22 degrees C. The brazzein fold, which contains one alpha-helix and three strands of antiparallel beta-sheet, does not resemble that of either of the other two sweet-tasting proteins with known structures, monellin and thaumatin. Instead, the structure of brazzein resembles those of plant gamma-thionins and defensins and arthropod toxins. Sequence comparisons predict that members of a newly-identified family of serine proteinase inhibitors share the brazzein fold.

The PI3K-Akt-mTORC1 pathway is a highly dynamic network that is balanced and stabilized by a number of feedback inhibition loops1, 2. Specifically, activation of mTORC1 has been shown to lead to the inhibition of its upstream growth factor signaling. Activation of the growth factor receptors is triggered by the binding of their cognate ligands in the extracellular space. However, whether secreted proteins contribute to the mTORC1-dependent feedback loops remains unclear. We found that cells with hyperactive mTORC1 secrete a protein that potently inhibits the function of IGF-1. Using a large-scale, unbiased quantitative proteomic platform, we comprehensively characterized the rapamycin-sensitive secretome in TSC2−/− MEFs, and identified IGFBP5 as a secreted, mTORC1 downstream effector protein. IGFBP5 is a direct transcriptional target of HIF1, which itself is a known mTORC1 target3. IGFBP5 is a potent inhibitor of both the signaling and functional outputs of IGF-1. Once secreted, IGFBP5 cooperates with intracellular branches of the feedback mechanisms to block the activation of IGF-1 signaling. Finally, IGFBP5 is a potential tumor suppressor, and the proliferation of IGFBP5-mutated cancer cells are selectively blocked by IGF-1R inhibitors.

Inhibitors against poly (ADP-ribose) polymerase (PARP) are promising targeted agents currently used to treat BRCA-mutant ovarian cancer and are in clinical trials for other cancer types, including BRCA-mutant breast cancer. To enhance the clinical response to PARP inhibitors (PARPi), understanding the mechanisms underlying PARP inhibitor sensitivity is urgently needed. Here, we show enhancer of zeste homolog 2 (EZH2), an enzyme which catalyzes H3 lysine trimethylation and associates with oncogenic function, contributes to PARPi sensitivity in breast cancer cells. Mechanistically, upon oxidative stress or alkylating DNA damage, PARP1 interacts with and attaches poly ADP-ribose (PAR) chains to EZH2. PARylation of EZH2 by PARP1 then induces PRC2 complex dissociation and EZH2 downregulation, which in turn reduces EZH2-mediated H3 tri-methylation. In contrast, inhibition of PARP by PARPi attenuates alkylating DNA damage-induced EZH2 downregulation, thereby promoting EZH2-mediated gene silencing and cancer stem cell property compared to PARPi-untreated cells. Moreover, the addition of an EZH2 inhibitor sensitizes the BRCA-mutant breast cells to PARPi. Thus, these results may provide a rationale for combining PARP and EZH2 inhibition as a therapeutic strategy for BRCA-mutated breast and ovarian cancers.