(3), O-GlcNAc modification is now appreciated as a nutrient-responsive mechanism for modulating signal transduction (4, 5) and transcriptional regulation (6, 7). In contrast to N-and O-linked glycosylation taking place in the endoplasmic reticulum and Golgi apparatus, reversible O-GlcNAcylation occurs in the cytoplasm and nucleus and is catalyzed by O-GlcNAc transferase (OGT), which transfers GlcNAc from UDP-GlcNAc to Ser/Thr residues, and O-GlcNAcase (OGA), which removes it. These highly conserved enzymes are expressed in all mammalian tissues (8 -11).The OGT gene encodes three splice variants differing in the number of N-terminal tetratricopeptide repeats that mediate protein-protein interactions and subcellular localization (8,12). The full-length nucleocytoplasmic form of OGT is OGlcNAc-modified, tyrosine-phosphorylated, and dynamically redistributed within the cell upon insulin stimulation (2,(13)(14)(15). Ablation of OGT is embryonically lethal or developmentally limiting in animal models (10,16,17) with the noted exception of Caenorhabditis elegans. In C. elegans, the phenotypes of oga-1 and ogt-1 null mutants suggest the enzymes are involved in macronutrient storage and life span (18 -20), and subsequent studies revealed the presence of O-GlcNAc modFrom the

O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) catalyze the dynamic cycling of intracellular, post-translational O-GlcNAc modification on thousands of Ser/Thr residues of cytosolic, nuclear, and mitochondrial signaling proteins. The identification of O-GlcNAc modified substrates has revealed a functionally diverse set of proteins, and the extent of O-GlcNAcylation fluctuates in response to nutrients and cellular stress. As a result, OGT and OGA are implicated in widespread, nutrient-responsive regulation of numerous signaling pathways and transcriptional programs. These enzymes are required for normal embryonic development and are dysregulated in metabolic and age-related disease states. While a recent surge of interest in the field has contributed to understanding the functional impacts of protein O-GlcNAcylation, little is known about the upstream mechanisms which modulate OGT and OGA substrate targeting. This review focuses on elements of enzyme structure among splice variants, post-translational modification, localization, and regulatory protein interactions which drive the specificity of OGT and OGA toward different subsets of the cellular proteome. Ongoing efforts in this rapidly advancing field are aimed at revealing mechanisms of OGT and OGA regulation to harness the potential therapeutic benefit of manipulating these enzymes’ activities.

Runx2 [also known as: core binding factor ␣ 1 (CBFA1); acute myeloid leukemia transcription factor 3 (AML3); polyoma enhancer-binding protein 2␣ (PEPB2␣)] is a member of the runt-domain gene family of DNA binding proteins (Runx1, Runx2, and Runx3), which control the expression of numerous genes involved in cell growth, proliferation, and determination of cell lineage (1). Aberrant expression and activity of Runx proteins are implicated in leukemia, metastatic breast cancer, and defects in skeletal development and bone remodeling (2-4). Runx2 is expressed during early embryonic development in mesenchymal and cartilage condensations of the developing bone anlagen, and its transcription, activity, and stability are tightly regulated (5-7). Considered the master regulator of osteoblast differentiation, Runx2 also contributes to chondrocyte maturation (8,9) and, through these cell-types, controls intramembranous and endochondral ossification culminating in the formation of the mineralized skeleton (5, 6, 10). A gain-of-function mutation of Runx2, resulting from duplication of exons 3 to 5, is linked to dysplastic long bone formation, enlarged clavicles, and thickening of the cranial vault (11). Conversely, the rare autosomal dominant disorder, cleidocranial dysplasia, has been traced to multiple loss-of-function mutations within the Cbfa1 gene, and is characterized by defective formation or absence of the clavicle, enlarged fontanelles, and dental abnormalities (9, 12, 13). Mice nullizygous for Runx2 develop a cartilaginous skeletal framework that fails to undergo mineralization caused by the absence of osteoblasts, and these mice die at birth because of respiratory failure (5, 10).

Natural products remain an important source of drug leads covering unique chemical space and providing significant therapeutic value for the control of cancer and infectious diseases resistant to current drugs. Here, we determined the antiproliferative activity of a natural product manzamine A ( 1 ) from an Indo-Pacific sponge following various in vitro cellular assays targeting cervical cancer (C33A, HeLa, SiHa, and CaSki). Our data demonstrated the antiproliferative effects of 1 at relatively low and non-cytotoxic concentrations (up to 4 μM). Mechanistic investigations confirmed that 1 blocked cell cycle progression in SiHa and CaSki cells at G1/S phase and regulated cell cycle-related genes, including restoration of p21 and p53 expression. In apoptotic assays, HeLa cells showed the highest sensitivity to 1 as compared to other cell types (C33A, SiHa, and CaSki). Interestingly, 1 decreased the levels of the oncoprotein SIX1, which is associated with oncogenesis in cervical cancer. To further investigate the structure–activity relationship among manzamine A ( 1 ) class with potential antiproliferative activity, molecular networking facilitated the efficient identification, dereplication, and assignment of structures from the manzamine class and revealed the significant potential in the design of optimized molecules for the treatment of cervical cancer. These data suggest that this sponge-derived natural product class warrants further attention regarding the design and development of novel manzamine analogues, which may be efficacious for preventive and therapeutic treatment of cancer. Additionally, this study reveals the significance of protecting fragile marine ecosystems from climate change-induced loss of species diversity.

The Gastrodia antifungal protein (GAFP) is a monocot mannose-binding lectin isolated from the Asiatic orchid Gastrodia elata. This lectin has previously been shown to provide increased resistance in transgenic Nicotiana tabacum against taxonomically unrelated root pathogens Phytophthora nicotianae, Rhizoctonia solani, and Meloidogyne incognita, but its potential to confer disease resistance in tree species is not known. Agrobacterium tumefaciens-mediated transformation yielded three gafp-1 expressing plum lines (Prunus domestica) designated 4J, 4I, and 5D. These lines possessed one, two, and four copies of the gafp-1 gene, respectively, as demonstrated by DNA blotting. Lines 4J and 4I were not phenotypically different from the nontransformed control line, but line 5D showed significant divergence in leaf morphology and growth habit. Compared with the inoculated control line, lines 4J and 4I exhibited increased tolerance to Phytophthora root rot (PRR) caused by P. cinnamomi. When inoculated with the root-knot nematode, Meloidogyne incognita, the 4J and 4I lines showed a significantly lower degree of root galling than the inoculated control line. Nematode reproduction, as measured by the presence of egg masses and the number of eggs produced per gram fresh root, was significantly reduced in line 4J compared with the inoculated control line. The results of this study suggest that the expression of gafp-1 in the roots of a woody plant may confer some level of resistance to PRR and root-knot nematode. Long-term field trials will be necessary to confirm this hypothesis.

No abstract

The type 1 parathyroid hormone receptor (PTH1R) is a key regulator of calcium homeostasis and bone turnover. Here, we employed SILAC-based quantitative mass spectrometry combined with bioinformatic pathways analysis to examine global changes in protein phosphorylation following short-term stimulation of endogenously expressed PTH1R in osteoblastic cells in vitro. Following 5 min exposure to the conventional agonist, PTH(1-34), we detected significant changes in the phosphorylation of 224 distinct proteins. Kinase substrate motif enrichment demonstrated that consensus motifs for PKA and CAMK2 were the most heavily upregulated within the phosphoproteome, while consensus motifs for mitogen-activated protein kinases were strongly downregulated. Signaling pathways analysis identified ERK1/2 and AKT as important nodal kinases in the downstream network and revealed strong regulation of small GTPases involved in cytoskeletal rearrangement, cell motility, and focal adhesion complex signaling. Our data illustrate the utility of quantitative mass spectrometry in measuring dynamic changes in protein phosphorylation following GPCR activation.

The Gastrodia antifungal protein (GAFP-1) is a monocot mannose-binding lectin found in the Asiatic orchid Gastrodia elata. Transgenic plum (Prunus domestica var. ‘Stanley’) lines (4J and 4I) expressing GAFP-1 exhibit enhanced disease resistance to the stramenopile pathogen Phytophthora cinnamomi and the root-knot nematode Meloidogyne incognita. Rootstocks created from such transgenic lines might be more readily accepted by consumers if it can be shown that foreign gene products are not migrating into a grafted, nontransgenic scion on which fruit is produced. In this study, wild-type (WT) plum tissue was budded onto transgenic plum lines 4J and 4I to create chimeric-grafted trees. Tissues from chimeric-grafted trees were analyzed for gafp-1 transcripts (leaf and root) and protein (leaf, soft shoot, and root) by reverse transcription–polymerase chain reaction and immunodetection, respectively. Transcripts of gafp-1 were detected consistently in the root tissues but not within the leaves of the grafted, WT scions. Similarly, the GAFP-1 lectin was identified within the roots, but not in the soft shoot or leaf tissues of the grafted, WT scions. These results suggest that gafp-1 mRNA and protein are not moving into the WT scion tissues of chimeric-grafted plum trees.