Characterization of a chromatin-associated TCF7L1 complex in human embryonic stem cells.

Human embryonic stem cells (hESCs) resemble the pluripotent epiblast cells found in the early postimplantation human embryo and represent the "primed" state of pluripotency. One factor that helps primed pluripotent cells retain pluripotency and prepare genes for differentiation is the transcription factor TCF7L1, a member of a small family of proteins known as T cell factors/Lymphoid enhancer factors (TCF/LEF) that act as downstream components of the WNT signaling pathway. Transcriptional output of the WNT pathway is regulated, in part, by the activity of TCF/LEFs in conjunction with another component of the WNT pathway, ß-CATENIN. Because TCF7L1 plays an important role in regulating pluripotency, we began to characterize the protein complex associated with TCF7L1 when bound to chromatin in hESCs using rapid immunoprecipitation of endogenous proteins (RIME).  Data are available via ProteomeXchange with identifier PXD047582. These data identify known and new partners of TCF7L1 on chromatin and provide novel insights into how TCF7L1 and pluripotency itself might be regulated.

HNF4α isoforms regulate the circadian balance between carbohydrate and lipid metabolism in the liver.

Hepatocyte Nuclear Factor 4α (HNF4α), a master regulator of hepatocyte differentiation, is regulated by two promoters (P1 and P2) which drive the expression of different isoforms. P1-HNF4α is the major isoform in the adult liver while P2-HNF4α is thought to be expressed only in fetal liver and liver cancer. Here, we show that P2-HNF4α is indeed expressed in the normal adult liver at Zeitgeber time (ZT)9 and ZT21. Using exon swap mice that express only P2-HNF4α we show that this isoform orchestrates a distinct transcriptome and metabolome via unique chromatin and protein-protein interactions, including with different clock proteins at different times of the day leading to subtle differences in circadian gene regulation. Furthermore, deletion of the Clock gene alters the circadian oscillation of P2- (but not P1-)HNF4α RNA, revealing a complex feedback loop between the HNF4α isoforms and the hepatic clock. Finally, we demonstrate that while P1-HNF4α drives gluconeogenesis, P2-HNF4α drives ketogenesis and is required for elevated levels of ketone bodies in female mice. Taken together, we propose that the highly conserved two-promoter structure of the Hnf4a gene is an evolutionarily conserved mechanism to maintain the balance between gluconeogenesis and ketogenesis in the liver in a circadian fashion.

MYC acetylated lysine residues drive oncogenic cell transformation and regulate select genetic programs for cell adhesion-independent growth and survival.

The MYC oncogenic transcription factor is acetylated by the p300 and GCN5 histone acetyltransferases. The significance of MYC acetylation and the functions of specific acetylated lysine (AcK) residues have remained unclear. Here, we show that the major p300-acetylated K148(149) and K157(158) sites in human (or mouse) MYC and the main GCN5-acetylated K323 residue are reversibly acetylated in various malignant and nonmalignant cells. Oncogenic overexpression of MYC enhances its acetylation and alters the regulation of site-specific acetylation by proteasome and deacetylase inhibitors. Acetylation of MYC at different K residues differentially affects its stability in a cell type-dependent manner. Lysine-to-arginine substitutions indicate that although none of the AcK residues is required for MYC stimulation of adherent cell proliferation, individual AcK sites have gene-specific functions controlling select MYC-regulated processes in cell adhesion, contact inhibition, apoptosis, and/or metabolism and are required for the malignant cell transformation activity of MYC. Each AcK site is required for anchorage-independent growth of MYC-overexpressing cells in vitro, and both the AcK148(149) and AcK157(158) residues are also important for the tumorigenic activity of MYC transformed cells in vivo. The MYC AcK site-specific signaling pathways identified may offer new avenues for selective therapeutic targeting of MYC oncogenic activities.

Novel potent azetidine-based compounds irreversibly inhibit Stat3 activation and induce antitumor response against human breast tumor growth in vivo.

Signal transducer and activator of transcription (Stat)3 is a valid anticancer therapeutic target. We have discovered a highly potent chemotype that amplifies the Stat3-inhibitory activity of lead compounds to levels previously unseen. The azetidine-based compounds, including H172 (9f) and H182, irreversibly bind to Stat3 and selectively inhibit Stat3 activity (IC50 0.38-0.98 µM) over Stat1 or Stat5 (IC50 > 15.8 µM) in vitro. Mass spectrometry detected the Stat3 cysteine peptides covalently bound to the azetidine compounds, and the key residues, Cys426 and Cys468, essential for the high potency inhibition, were confirmed by site-directed mutagenesis. In triple-negative breast cancer (TNBC) models, treatment with the azetidine compounds inhibited constitutive and ligand-induced Stat3 signaling, and induced loss of viable cells and tumor cell death, compared to no effect on the induction of Janus kinase (JAK)2, Src, epidermal growth factor receptor (EGFR), and other proteins, or weak effects on cells that do not harbor aberrantly-active Stat3. H120 (8e) and H182 as a single agent inhibited growth of TNBC xenografts, and H278 (hydrochloric acid salt of H182) in combination with radiation completely blocked mouse TNBC growth and improved survival in syngeneic models. We identify potent azetidine-based, selective, irreversible Stat3 inhibitors that inhibit TNBC growth in vivo.

TMK-based cell-surface auxin signalling activates cell-wall acidification.

The phytohormone auxin controls many processes in plants, at least in part through its regulation of cell expansion1. The acid growth hypothesis has been proposed to explain auxin-stimulated cell expansion for five decades, but the mechanism that underlies auxin-induced cell-wall acidification is poorly characterized. Auxin induces the phosphorylation and activation of the plasma membrane H+-ATPase that pumps protons into the apoplast2, yet how auxin activates its phosphorylation remains unclear. Here we show that the transmembrane kinase (TMK) auxin-signalling proteins interact with plasma membrane H+-ATPases, inducing their phosphorylation, and thereby promoting cell-wall acidification and hypocotyl cell elongation in Arabidopsis. Auxin induced interactions between TMKs and H+-ATPases in the plasma membrane within seconds, as well as TMK-dependent phosphorylation of the penultimate threonine residue on the H+-ATPases. Our genetic, biochemical and molecular evidence demonstrates that TMKs directly phosphorylate plasma membrane H+-ATPase and are required for auxin-induced H+-ATPase activation, apoplastic acidification and cell expansion. Thus, our findings reveal a crucial connection between auxin and plasma membrane H+-ATPase activation in regulating apoplastic pH changes and cell expansion through TMK-based cell surface auxin signalling.

The Arabidopsis PHD-finger protein EDM2 has multiple roles in balancing NLR immune receptor gene expression.

Plant NLR-type receptors serve as sensitive triggers of host immunity. Their expression has to be well-balanced, due to their interference with various cellular processes and dose-dependency of their defense-inducing activity. A genetic "arms race" with fast-evolving pathogenic microbes requires plants to constantly innovate their NLR repertoires. We previously showed that insertion of the COPIA-R7 retrotransposon into RPP7 co-opted the epigenetic transposon silencing signal H3K9me2 to a new function promoting expression of this Arabidopsis thaliana NLR gene. Recruitment of the histone binding protein EDM2 to COPIA-R7-associated H3K9me2 is required for optimal expression of RPP7. By profiling of genome-wide effects of EDM2, we now uncovered additional examples illustrating effects of transposons on NLR gene expression, strongly suggesting that these mobile elements can play critical roles in the rapid evolution of plant NLR genes by providing the "raw material" for gene expression mechanisms. We further found EDM2 to have a global role in NLR expression control. Besides serving as a positive regulator of RPP7 and a small number of other NLR genes, EDM2 acts as a suppressor of a multitude of additional NLR genes. We speculate that the dual functionality of EDM2 in NLR expression control arose from the need to compensate for fitness penalties caused by high expression of some NLR genes by suppression of others. Moreover, we are providing new insights into functional relationships of EDM2 with its interaction partner, the RNA binding protein EDM3/AIPP1, and its target gene IBM1, encoding an H3K9-demethylase.

Menthol in electronic cigarettes: A contributor to respiratory disease?

Menthol is widely used in tobacco products. This study compared the effects of menthol on human bronchial epithelium using submerged cultures, a VITROCELL® cloud chamber that provides air liquid interface (ALI) exposure without solvents or heating, and a Cultex ALI system that delivers aerosol equivalent to that inhaled during vaping. In submerged culture, menthol significantly increased calcium influx and mitochondrial reactive oxygen species (ROS) via the TRPM8 receptor, responses that were inhibited by a TRPM8 antagonist. VITROCELL® cloud chamber exposure of BEAS-2B monolayers increased mitochondrial protein oxidation, expression of the antioxidant enzyme SOD2, activation of NF-κB, and secretion of inflammatory cytokines (IL-6 and IL-8). Proteomics data collected following ALI exposure of 3D EpiAirway tissue in the Cultex showed upregulation of NRF-2-mediated oxidative stress, oxidative phosphorylation, and IL-8 signaling. Across the three platforms, menthol adversely effected human bronchial epithelium in a manner that could lead to respiratory disease.

Proteome Profile of Endogenous Retrotransposon-Associated Complexes in Human Embryonic Stem Cells.

Long Interspersed Element-1 (LINE-1 or L1) are transposable elements similar to retroviruses that have existed in the genome of primates for millions of years. They encode two Open Reading Frame (ORF) proteins (ORF1p and ORF2p) that bind L1 RNA to form a ribonucleoprotein (RNP) complex and are required for L1 integration into the host genome. Humans have evolved with L1 and found ways to limit L1 activity. To identify partners of the L1 RNP, previous studies used ectopic expression of L1 ORF1/2p or RNA in various cancer cells, which express low levels of the ORF proteins. Whether naturally occurring L1 RNP interacts with the same proteins in non-cancer cells is unknown. Here, the aim is to examine the natural assembly of endogenous L1 RNPs in normal human cells. L1 elements are expressed in human embryonic stem cells (hESCs), derived from pre-implantation embryos. Therefore, these cells are used to immunoprecipitate ORF1p followed by MS to identify proteins that associate with the naturally-occurring L1 ORF1p. Some of the same proteins as well as unique proteins are found interacting with the endogenous L1 ORF1p complexes. The analysis of ORF1p-associated proteins in hESCs can help address important questions in both retrotransposon biology and the biology of hESCs.

Integrated transcriptomic and proteomic analyses of a molecular mechanism of radular teeth biomineralization in Cryptochiton stelleri.

Many species of chiton are known to deposit magnetite (Fe3O4) within the cusps of their heavily mineralized and ultrahard radular teeth. Recently, much attention has been paid to the ultrastructural design and superior mechanical properties of these radular teeth, providing a promising model for the development of novel abrasion resistant materials. Here, we constructed de novo assembled transcripts from the radular tissue of C. stelleri that were used for transcriptome and proteome analysis. Transcriptomic analysis revealed that the top 20 most highly expressed transcripts in the non-mineralized teeth region include the transcripts encoding ferritin, while those in the mineralized teeth region contain a high proportion of mitochondrial respiratory chain proteins. Proteomic analysis identified 22 proteins that were specifically expressed in the mineralized cusp. These specific proteins include a novel protein that we term radular teeth matrix protein1 (RTMP1), globins, peroxidasins, antioxidant enzymes and a ferroxidase protein. This study reports the first de novo transcriptome assembly from C. stelleri, providing a broad overview of radular teeth mineralization. This new transcriptomic resource and the proteomic profiles of mineralized cusp are valuable for further investigation of the molecular mechanisms of radular teeth mineralization in chitons.

Parasitoid Jewel Wasp Mounts Multipronged Neurochemical Attack to Hijack a Host Brain.

The parasitoid emerald jewel wasp Ampulex compressa induces a compliant state of hypokinesia in its host, the American cockroach Periplaneta americana through direct envenomation of the central nervous system (CNS). To elucidate the biochemical strategy underlying venom-induced hypokinesia, we subjected the venom apparatus and milked venom to RNAseq and proteomics analyses to construct a comprehensive "venome," consisting of 264 proteins. Abundant in the venome are enzymes endogenous to the host brain, including M13 family metalloproteases, phospholipases, adenosine deaminase, hyaluronidase, and neuropeptide precursors. The amphipathic, alpha-helical ampulexins are among the most abundant venom components. Also prominent are members of the Toll/NF-κB signaling pathway, including proteases Persephone, Snake, Easter, and the Toll receptor ligand Spätzle. We find evidence that venom components are processed following envenomation. The acidic (pH∼4) venom contains unprocessed neuropeptide tachykinin and corazonin precursors and is conspicuously devoid of the corresponding processed, biologically active peptides. Neutralization of venom leads to appearance of mature tachykinin and corazonin, suggesting that the wasp employs precursors as a prolonged time-release strategy within the host brain post-envenomation. Injection of fully processed tachykinin into host cephalic ganglia elicits short-term hypokinesia. Ion channel modifiers and cytolytic toxins are absent in A. compressa venom, which appears to hijack control of the host brain by introducing a "storm" of its own neurochemicals. Our findings deepen understanding of the chemical warfare underlying host-parasitoid interactions and in particular neuromodulatory mechanisms that enable manipulation of host behavior to suit the nutritional needs of opportunistic parasitoid progeny.

LRRK1 regulation of actin assembly in osteoclasts involves serine 5 phosphorylation of L-plastin.

Mice with disruption of Lrrk1 and patients with nonfunctional mutant Lrrk1 exhibit severe osteopetrosis phenotypes because of osteoclast cytoskeletal dysfunction. To understand how Lrrk1 regulates osteoclast function by modulating cytoskeleton rearrangement, we examined the proteins that are differentially phosphorylated in wild-type mice and Lrrk1-deficient osteoclasts by metal affinity purification coupled liquid chromatography/mass spectrometry (LC/MS) analyses. One of the candidates that we identified by LC/MS is L-plastin, an actin bundling protein. We found that phosphorylation of L-plastin at serine (Ser) residues 5 was present in wild-type osteoclasts but not in Lrrk1-deficient cells. Western blot analyses with antibodies specific for Ser5 phosphorylated L-plastin confirmed the reduced L-plastin Ser5 phosphorylation in Lrrk1 knockout (KO) osteoclasts. micro computed tomography (Micro-CT) analyses revealed that the trabecular bone volume of the distal femur was increased by 27% in the 16 to 21-week-old L-plastin KO females as compared with the wild-type control mice. The ratio of bone volume to tissue volume and connectivity density were increased by 44% and 47% (both P < 0.05), respectively, in L-plastin KO mice. Our data suggest that targeted disruption of L-plastin increases trabecular bone volume, and phosphorylation of Ser5 in L-plastin in the Lrrk1 signaling pathway may in part contribute to actin assembly in mature osteoclasts.

CSI1, PATROL1, and exocyst complex cooperate in delivery of cellulose synthase complexes to the plasma membrane.

Cellulose synthesis occurs exclusively at the plasma membrane by cellulose synthase complexes (CSCs). Therefore, delivery of CSCs to discrete sites at the plasma membrane is critical for cellulose synthesis. Despite their significance, the delivery of CSCs is poorly understood. Here we used proteomics approaches, functional genetics, and live cell imaging to show that the de novo secretion of CSCs is mediated by cooperation among cellulose synthase interactive 1 (CSI1), the plant-specific protein PATROL1, and exocyst complex in Arabidopsis thaliana We propose that CSI1 plays a role in marking the docking site, which allows CSCs-containing vesicles access to the plasma membrane through its interaction with microtubules. PATROL1 assists in exocytosis by its interaction with multiple components, including CSI1, CSCs, and exocyst subunits. Both PATROL1 and the exocyst complex determine the rate of delivery of CSCs to the plasma membrane. By monitoring the exocyst complex, PATROL1, CSI1, and CSCs dynamics in real time, we present a timeline of events for exocytosis of CSCs. Our findings provide unique insights into the evolution of exocytosis in eukaryotes.

Highly Efficient Exosome Isolation and Protein Analysis by an Integrated Nanomaterial-Based Platform.

Exosomes play important roles in mediating intercellular communication and regulating a variety of biological processes, but clear understanding of their functions and biogenesis has not been achieved, due to the high technical difficulties involved in analysis of small vesicular structures that contain a high proportion of membrane structures. Herein, we designed a novel approach to integrate two nanomaterials carrying varied surface properties, the hydrophilic, macroporous graphene foam (GF) and the amphiphilic periodic mesoporous organosilica (PMO), for efficient exosome isolation from human serum and effective protein profiling. The high specific surface area of GF, after modification with the antibody against the exosomal protein marker, CD63, allowed highly specific isolation of exosomes from complex biological samples with high recovery. Since the organic solvent, methanol, turned out to be the most effective lysis solution for releasing the exosomal proteins, the amphiphilic PMO was employed to rapidly recover the exosomal proteins, including the highly hydrophobic membrane proteins. The fine pores of PMO also acted as the nanoreactors to accelerate protein digestion that produced peptides subject to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. A total of 334 proteins with 111 membrane proteins [31% of these contained >2 transmembrane domains (TMD)] were identified using the integrated GF/PMO platform. In contrast, with the commercial exosome isolation kit and the in-solution protein digestion method, only 151 proteins were found, with 28 being membrane proteins (only one contained three TMDs). Our results support that the integrated GF/PMO platform is of great value to facilitate the comprehensive characterization of exosomal proteins for better understanding of their functions and for identification of more exosome-based disease markers.

Structure of a pathogen effector reveals the enzymatic mechanism of a novel acetyltransferase family.

Effectors secreted by the type III secretion system are essential for bacterial pathogenesis. Members of the Yersinia outer-protein J (YopJ) family of effectors found in diverse plant and animal pathogens depend on a protease-like catalytic triad to acetylate host proteins and produce virulence. However, the structural basis for this noncanonical acetyltransferase activity remains unknown. Here, we report the crystal structures of the YopJ effector HopZ1a, produced by the phytopathogen Pseudomonas syringae, in complex with the eukaryote-specific cofactor inositol hexakisphosphate (IP6) and/or coenzyme A (CoA). Structural, computational and functional characterizations reveal a catalytic core with a fold resembling that of ubiquitin-like cysteine proteases and an acetyl-CoA-binding pocket formed after IP6-induced structural rearrangements. Modeling-guided mutagenesis further identified key IP6-interacting residues of Salmonella effector AvrA that are required for acetylating its substrate. Our study reveals the structural basis of a novel class of acetyltransferases and the conserved allosteric regulation of YopJ effectors by IP6.

Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis.

The exocyst complex regulates the last steps of exocytosis, which is essential to organisms across kingdoms. In humans, its dysfunction is correlated with several significant diseases, such as diabetes and cancer progression. Investigation of the dynamic regulation of the evolutionarily conserved exocyst-related processes using mutants in genetically tractable organisms such as Arabidopsis thaliana is limited by the lethality or the severity of phenotypes. We discovered that the small molecule Endosidin2 (ES2) binds to the EXO70 (exocyst component of 70 kDa) subunit of the exocyst complex, resulting in inhibition of exocytosis and endosomal recycling in both plant and human cells and enhancement of plant vacuolar trafficking. An EXO70 protein with a C-terminal truncation results in dominant ES2 resistance, uncovering possible distinct regulatory roles for the N terminus of the protein. This study not only provides a valuable tool in studying exocytosis regulation but also offers a potentially new target for drugs aimed at addressing human disease.

Two serine residues in Pseudomonas syringae effector HopZ1a are required for acetyltransferase activity and association with the host co-factor.

Gram-negative bacteria inject type III secreted effectors (T3SEs) into host cells to manipulate the immune response. The YopJ family effector HopZ1a produced by the plant pathogen Pseudomonas syringae possesses acetyltransferase activity and acetylates plant proteins to facilitate infection. Using mass spectrometry, we identified a threonine residue, T346, as the main autoacetylation site of HopZ1a. Two neighboring serine residues, S349 and S351, are required for the acetyltransferase activity of HopZ1a in vitro and are indispensable for the virulence function of HopZ1a in Arabidopsis thaliana. Using proton nuclear magnetic resonance (NMR), we observed a conformational change of HopZ1a in the presence of inositol hexakisphosphate (IP6), which acts as a eukaryotic co-factor and significantly enhances the acetyltransferase activity of several YopJ family effectors. S349 and S351 are required for IP6-binding-mediated conformational change of HopZ1a. S349 and S351 are located in a conserved region in the C-terminal domain of YopJ family effectors. Mutations of the corresponding serine(s) in two other effectors, HopZ3 of P. syringae and PopP2 of Ralstonia solanacerum, also abolished their acetyltransferase activity. These results suggest that, in addition to the highly conserved catalytic residues, YopJ family effectors also require conserved serine(s) in the C-terminal domain for their enzymatic activity.

Size and surface functionalization of iron oxide nanoparticles influence the composition and dynamic nature of their protein corona.

Nanoparticles (NPs) adsorb proteins when in the biological matrix, and the resulted protein corona could affect NP-cell interactions. The corona has a dynamic nature with the adsorbed proteins constantly exchanging with the free proteins in the matrix at various rates. The rapidly exchanging proteins compose the soft corona, which responds more dynamically to environment changes than the hard corona established by the ones with slow exchange rates. In the present study, the corona formed on the superparamagnetic iron oxide NPs (SPIONs) in human serum was studied by flow field-flow fractionation and ultracentrifugation, which rapidly differentiated the corona proteins based on their exchange rates. By varying the surface hydrophobicity of the SPIONs with a core size around 10 nm, we found out that, the more hydrophobic surface ligand attracted proteins with higher surface hydrophobicity and formed a more dynamic corona with a larger portion of the involved proteins with fast exchange rates. Increasing the core diameter of the SPIONs but keeping the surface ligand the same could also result in a more dynamic corona. A brief investigation of the effect on the cellular uptake of SPIONs using one selected corona protein, transferrin, was conducted. The result showed that, only the stably bound transferrin could significantly enhance cellular uptake, while transferrin bound in a dynamic nature had negligible impact. Our study has led to a better understanding of the relationship between the particle properties and the dynamic nature of the corona, which can help with design of nanomaterials with higher biocompatibility and higher efficacy in biosystems for biomedical applications.

Novel diagnosis for citrus stubborn disease by detection of a spiroplasma citri-secreted protein.

Citrus stubborn disease (CSD), first identified in California, is a widespread bacterial disease found in most arid citrus-producing regions in the United States and the Mediterranean Region. The disease is caused by Spiroplasma citri, an insect-transmitted and phloem-colonizing bacterium. CSD causes significant tree damage resulting in loss of fruit production and quality. Detection of CSD is challenging due to low and fluctuating titer and sporadic distribution of the pathogen in infected trees. In this study, we report the development of a novel diagnostic method for CSD using an S. citri-secreted protein as the detection marker. Microbial pathogens secrete a variety of proteins during infection that can potentially disperse systemically in infected plants with the vascular flow. Therefore, their distribution may not be restricted to the pathogen infection sites and could be used as a biological marker for infection. Using mass spectrometry analysis, we identified a unique secreted protein from S. citri that is highly expressed in the presence of citrus phloem extract. ScCCPP1, an antibody generated against this protein, was able to distinguish S. citri-infected citrus and periwinkle from healthy plants. In addition, the antiserum could be used to detect CSD using a simple direct tissue print assay without the need for sample processing or specialized lab equipment and may be suitable for field surveys. This study provides proof of a novel concept of using pathogen-secreted protein as a marker for diagnosis of a citrus bacterial disease and can probably be applied to other plant diseases.