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Port wine birthmarks (PWBs) are caused by somatic, mosaic mutations in the G protein Guanine nucleotide binding protein alpha subunit q (GNAQ) and are characterized by the formation of dilated, dysfunctional blood vessels in the dermis, eyes, and/or brain. Cutaneous PWBs can be treated by current dermatologic therapy, like laser intervention, to lighten the lesions and diminish nodules that occur in the lesion. Involvement of the eyes and/or brain can result in serious complications and this variation is termed Sturge-Weber Syndrome (SWS). Some of the biggest hurdles preventing development of new therapeutics are unanswered questions regarding disease biology and lack of models for drug screening. In this review, we discuss the current understanding of GNAQ signaling, the standard of care for patients, overlap with other GNAQ-associated or phenotypically similar diseases, as well as deficiencies in current in vivo and in vitro vascular malformation models.

Histidine triad nucleotide-binding protein (HINT) belongs to the histidine triad protein family. Recent studies have demonstrated that HINT1 and HINT2 both play a pivotal role in cancer growth. However, the functions of HINT3 in various types of cancer, including breast cancer (BRCA), have not yet been fully elucidated. In the present study, the role of HINT3 in BRCA was investigated. Based on The Cancer Genome Atlas and reverse transcription-quantitative PCR analyses, HINT3 was found to be decreased in BRCA tissues. In vitro, HINT3 knockdown promoted the proliferation and colony formation of, and 5-ethynyl-2′-deoxyuridine incorporation in MCF-7 and MDA-MB-231 BRCA cells. By contrast, HINT3 overexpression suppressed DNA synthesis and the proliferation of both cell lines. Apoptosis was also found to be modulated by HINT3. In vivo, HINT3 ectopic expression attenuated the tumorigenesis of MDA-MB-231 and MCF-7 cells in a mouse tumor xenograft model. Furthermore, HINT3 silencing or overexpression also enhanced or inhibited, respectively, the migratory capacity of the MCF-7 and MDA-MB-231 cells. Finally, HINT3 upregulated phosphatase and tensin homolog (PTEN) at the transcriptional level, which resulted in the inactivation of AKT/mammalian target of rapamycin (mTOR) signaling both in vitro and in vivo. Taken together, the present study demonstrates that HINT3 inhibits the activation of the PTEN/AKT/mTOR signaling pathway, and suppresses the proliferation, growth, migration and tumor development of MCF-7 and MDA-MB-231 BRCA cells.

In animals, microtubules and centrosomes direct the migration of gamete pronuclei for fertilization. By contrast, flowering plants have lost essential components of the centrosome, raising the question of how flowering plants control gamete nuclei migration during fertilization. Here, we use Arabidopsis thaliana to document a novel mechanism that regulates F-actin dynamics in the female gametes and is essential for fertilization. Live imaging shows that F-actin structures assist the male nucleus during its migration towards the female nucleus. We identify a female gamete-specific Rho-GTPase that regulates F-actin dynamics and further show that actin–myosin interactions are also involved in male gamete nucleus migration. Genetic analyses and imaging indicate that microtubules are dispensable for migration and fusion of male and female gamete nuclei. The innovation of a novel actin-based mechanism of fertilization during plant evolution might account for the complete loss of the centrosome in flowering plants. Sexual reproduction involves combining the genetic material from two parents to create an offspring. The genetic material in the male sperm cell and the female egg cell is contained in the nucleus of each cell. Once these two cells fuse at fertilization, their nuclei must then navigate towards each other and fuse. When an animal egg cell is fertilized, cable-like protein filaments called microtubules guide the two nuclei into contact. These microtubules are organized by a cellular structure called a centrosome. However, flowering plants do not have centrosomes; as such, it was unclear how genetic material from the sperm and egg cells is brought together after fertilization in flowering plants. To investigate this, Kawashima et al. turned to a flowering plant commonly used in research, called Arabidopsis thaliana, and found that microtubules are not needed to guide the nuclei of the sperm and the egg cell after fertilization. Instead, another cable-forming protein—called F-actin—fulfills a similar role in Arabidopsis cells. F-actin filaments often connect together to form a network; and when Kawashima et al. disrupted the F-actin in Arabidopsis egg cells, the nucleus of the sperm cell failed to fuse with that of the female. Pollen from Arabidopsis plants actually contains two sperm cells. One sperm cell fertilizes the egg cell; the other fertilizes the so-called ‘central cell’, which develops into a tissue that nourishes the plant embryo. Kawashima et al. found that the fertilization of both of these cells requires an intact F-actin network. By looking more closely at F-actin networks in the larger central cell, Kawashima et al. discovered that the sperm nucleus becomes surrounded by a star-shaped structure of F-actin cables and that this F-actin structure migrates together with the sperm nucleus. The F-actin network constantly moves inward, from the edges of the cell towards the nucleus, prior to fertilization. This movement is essential for guiding the sperm nucleus towards the central cell nucleus. Kawashima et al. also found that this continual movement of the F-actin network depends on a small signaling protein found in the central cell, called ROP8. It also involves a motor protein that normally transports “cargo”, such as proteins and other molecules, inside cells by walking along the F-actin networks. However, rather than transporting the sperm nucleus as cargo, Kawashima et al. believe that the motor protein instead helps to maintain the inward movement of the F-actin network. One of the next challenges will be to investigate the molecular mechanism that underlies this motor protein's involvement in this dynamic F-actin network.

Guanine nucleotide-binding protein-like-3-like (GNL3L) is a crucial regulator of NF-κB signaling that is aberrantly activated during diverse chemoresistance-associated cellular processes. However, the molecular mechanisms of GNL3L tumor initiation and resistant state are largely unknown. Moreover, the identification of predictive biomarkers is necessary to effectively generate therapeutic strategies for metastatic human colorectal cancer (CRC). This study aims to identify how cells acquire resistance to anticancer drugs and whether the downregulation of miR-4454 is associated with the progression of CRC. Here, we have shown that the overexpression of miR-4454 in resistant tumors is a crucial precursor for the posttranscriptional repression of GNL3L in human chemoresistant CRC progression, and we used doxycycline induced miR-4454 overexpression that significantly reduced tumor volume in a subcutaneous injection nude mice model. Together, these observations highlight that the downregulation of miR-4454 in resistant clones is prominently responsible for maintaining their resistance against anticancer drug therapy. Our study indicates that the development of miR-4454 as a microRNA-based therapeutic approach to silence GNL3L may remarkably reduce oncogenic cell survival that depends on GNL3L/NF-κB signaling, making miR-4454 a candidate for treating metastatic human CRC.

Background Nitrate (NO 3 − ) is the major source of nitrogen (N) for higher plants aside from its function in transducing the N signaling. Improving N use efficiency of crops has been an effective strategy for promotion of the sustainable agriculture worldwide. The regulatory pathways associating with N uptake and the corresponding biochemical processes impact largely on plant N starvation tolerance. Thus, exploration of the molecular mechanism underlying nitrogen use efficiency (NUE) and the gene wealth will pave a way for molecular breeding of N starvation-tolerant crop cultivars. Results In the current study, we characterized the function of TaNBP1 , a guanine nucleotide-binding protein subunit beta gene of wheat ( T. aestivum ), in mediating the plant N starvation response. TaNBP1 protein harbors a conserved W40 domain and the TaNBP1-GFP (green fluorescence protein) signals concentrate at positions of cytoplasm membrane and cytosol. TaNBP1 transcripts are induced in roots and leaves upon N starvation stress and that this upregulated expression is recovered by N recovery treatment. TaNBP1 overexpression confers improved phenotype, enlarged root system architecture (RSA), and increased biomass for plants upon N deprivation relative to the wild type, associating with its role in enhancing N accumulation and improving reactive oxygen species (ROS) homeostasis. Nitrate transporter (NRT) gene NtNRT2.2 and antioxidant enzyme genes NtSOD1 , NtSOD2 , and NtCAT1 are transcriptionally regulated under TaNBP1 and contribute to the improved N acquisition and the increased AE activities of plants. Conclusions Altogether, TaNBP1 is transcriptional response to N starvation stress. Overexpression of this gene enhances plant N starvation adaptation via improvement of N uptake and cellular ROS homeostasis by modifying transcription of NRT gene NtNRT2.2 and antioxidant enzyme genes NtSOD1 , NtSOD2 , and NtCAT1 , respectively. Our research helps to understand the mechanism underlying plant N starvation response and benefits to genetically engineer crop cultivars with improved NUE under the N-saving cultivation conditions.

Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone which is essential in eukaryotes. It is required for the activation and stabilization of a wide variety of client proteins and many of them are involved in important cellular pathways. Since Hsp90 affects numerous physiological processes such as signal transduction, intracellular transport, and protein degradation, it became an interesting target for cancer therapy. Structurally, Hsp90 is a flexible dimeric protein composed of three different domains which adopt structurally distinct conformations. ATP binding triggers directionality in these conformational changes and leads to a more compact state. To achieve its function, Hsp90 works together with a large group of cofactors, termed co-chaperones. Co-chaperones form defined binary or ternary complexes with Hsp90, which facilitate the maturation of client proteins. In addition, posttranslational modifications of Hsp90, such as phosphorylation and acetylation, provide another level of regulation. They influence the conformational cycle, co-chaperone interaction, and inter-domain communications. In this review, we discuss the recent progress made in understanding the Hsp90 machinery.

Sufficient amino acid (AA) transport is essential to ensure the normal physiological function and growth of growing animals. The processes of AA sensing and transport in humans and murine animals, but rarely in goats, have been arousing great interest recently. This study was conducted to investigate the messenger RNA expression patterns of lysophosphatidic acid receptor 5 (LPAR5), guanine nucleotide-binding protein α-transducing 3 (GNAT3) and important partial AA transporters in digestive tracts, metabolic organs and muscles of growing goats. The results showed that these genes were widely expressed in goats, and had different expression patterns. LPAR5, GNAT3, solute carrier (SLC38A2), SLC7A7, SLC7A1 and SLC3A1 were rarely expressed in the rumen, but were highly expressed in the abomasum and intestine which are the main sites of AA absorption. GNAT3, SLC38A1, SLC38A2, SLC6A19, SLC7A7 and SLC7A1 showed comparatively high expression in the pancreas and the vital digestive glands, and the relatively high expression of these nine genes were noted in the tibialis posterior, the active muscle in energy metabolism. The correlation analysis showed that there were certain positive correlation among most genes. The current results indicate that the AA sensing and transport occur extensively in the abomasum and small intestine, metabolic organs and muscle tissues of ruminants, and that related genes have tissue specificity.

α1,3‐galactosyltransferase (α3GalT, EC 2.4.1.151) is a Golgi‐resident, type II transmembrane protein that transfers galactose from UDP‐α‐galactose to the terminal N ‐acetyllactosamine unit of glycoconjugate glycans, producing the Galα1,3Galβ1,4GlcNAc oligosaccharide structure present in most mammalian glycoproteins. Unlike most other mammals, humans and Old World primates do not possess α3GalT activity, which is relevant for the hyperacute rejection observed in pig‐to‐human xenotransplantation. The crystal structure of the catalytic domain of substrate‐free bovine α3GalT, solved and refined to 2.3 Å resolution, has a globular shape with an α/β fold containing a narrow cleft on one face, and shares a UDP‐binding domain (UBD) with the recently solved inverting glycosyltransferases. The substrate‐bound complex, solved and refined to 2.5 Å, allows the description of residues interacting directly with UDP‐galactose. These structural data suggest that the strictly conserved residue E317 is likely to be the catalytic nucleophile involved in galactose transfer with retention of anomeric configuration as accomplished by this enzyme. Moreover, the α3GalT structure helps to identify amino acid residues that determine the specificities of the highly homologous ABO histo‐blood group and glycosphingolipid glycosyltransferases.

G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the Gα subunit 1 . To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR–G-protein complex. By monitoring the transitions of the stimulatory G s protein in complex with the β 2 -adrenergic receptor at short sequential time points after GTP addition, we identified the conformational trajectory underlying G-protein activation and functional dissociation from the receptor. Twenty structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of main events driving G-protein activation in response to GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα switch regions and the α5 helix that weaken the G-protein–receptor interface. Molecular dynamics simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP on closure of the α-helical domain against the nucleotide-bound Ras-homology domain correlates with α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signalling events. Time-resolved cryo-EM is used to capture structural transitions during G-protein activation stimulated by a G-protein-coupled receptor.

Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, such as osteoporosis, diabetes and obesity. Here we report the structure of a full-length class B receptor, the calcitonin receptor, in complex with peptide ligand and heterotrimeric Gα s βγ protein determined by Volta phase-plate single-particle cryo-electron microscopy. The peptide agonist engages the receptor by binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helices 6 and 7. This conformation is accompanied by a 60° kink in helix 6 and a large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the α5-helix of Gα s . Also observed is an extended intracellular helix 8 that contributes to both receptor stability and functional G-protein coupling via an interaction with the Gβ subunit. This structure provides a new framework for understanding G-protein-coupled receptor function. Volta phase-plate cryo-electron microscopy reveals the structure of the full-length calcitonin receptor in complex with its peptide ligand and Gα s βγ. GPCR structure solved by cryo-electron microscopy The use of cryo-electron microscopy (cryo-EM) in structural biology has exploded in recent years as it provides structural information at near atomic resolution without the need for crystallization. However, cryo-EM has typically been limited to proteins larger than 200 kDa because of issues with low contrast. Patrick Sexton and colleagues report the structure of the full-length calcitonin receptor in complex with its peptide ligand and Gα s βγ protein by Volta phase-plate single-particle cryo-EM. This is the first G-protein-coupled receptor (GPCR) structure to be solved at high resolution by cryo-EM, the first full-length class B GPCR reported and only the second in complex with the full heterotrimeric G protein. The structure shows the GPCR in the active state and reveals key information about the conformational changes associated with peptide agonist binding and G-protein coupling in class B receptors.