Precision mapping of the human O‐GalNAc glycoproteome through SimpleCell technology

Glycosylation is the most abundant and diverse posttranslational modification of proteins. While several types of glycosylation can be predicted by the protein sequence context, and substantial knowledge of these glycoproteomes is available, our knowledge of the GalNAc‐type O‐glycosylation is highly limited. This type of glycosylation is unique in being regulated by 20 polypeptide GalNAc‐transferases attaching the initiating GalNAc monosaccharides to Ser and Thr (and likely some Tyr) residues. We have developed a genetic engineering approach using human cell lines to simplify O‐glycosylation (SimpleCells) that enables proteome‐wide discovery of O‐glycan sites using ‘bottom‐up’ ETD‐based mass spectrometric analysis. We implemented this on 12 human cell lines from different organs, and present a first map of the human O‐glycoproteome with almost 3000 glycosites in over 600 O‐glycoproteins as well as an improved NetOGlyc4.0 model for prediction of O‐glycosylation. The finding of unique subsets of O‐glycoproteins in each cell line provides evidence that the O‐glycoproteome is differentially regulated and dynamic. The greatly expanded view of the O‐glycoproteome should facilitate the exploration of how site‐specific O‐glycosylation regulates protein function.     

Decrease of Fibulin-3 in Hepatocellular Carcinoma Indicates Poor Prognosis

Fibulin-3, originally identified in senescent and Werner syndrome fibroblasts, has been implicated in cell morphology, growth, adhesion and motility. Fibulin-3 exhibits both antitumor and oncogenic activities towards human cancers; however, the role of Fibulin-3 in hepatocellular carcinoma (HCC) remains elusive. In this study, we showed that both the mRNA and protein levels of Fibulin-3 were remarkably downregulated in HCC cell lines and fresh tissues. Immunohistochemical data revealed that Fibulin-3 was decreased in tumorous tissues in 67.1% (171/255) of cases compared to the corresponding adjacent nontumorous tissues. The results of statistical analysis indicated that low Fibulin-3 expression, defined by the receiver operating characteristic curve (ROC), was significantly associated with tumor differentiation (P = 0.008), clinical stage (P = 0.014) and serum AFP levels (P<0.01). Furthermore, Kaplan-Meier and multivariate analysis suggested that Fibulin-3 is an independent negative prognostic indicator for both overall (P<0.001) and recurrence-free (P = 0.036) survival. In addition, an in vitro study demonstrated that knockdown of Fibulin-3 by siRNA markedly increased cell viability and promoted cell invasion in HCC cells. Collectively, our data suggest that Fibulin-3 exhibits antitumor effects towards HCC and serves as a biomarker of unfavorable prognosis for this deadly disease.     

Extracellular Norepinephrine Clearance by the Norepinephrine Transporter Is Required for Skeletal Homeostasis

Changes in bone remodeling induced by pharmacological and genetic manipulation of β-adrenergic receptor (βAR) signaling in osteoblasts support a role of sympathetic nerves in the regulation of bone remodeling. However, the contribution of endogenous sympathetic outflow and nerve-derived norepinephrine (NE) to bone remodeling under pathophysiological conditions remains unclear. We show here that differentiated osteoblasts, like neurons, express the norepinephrine transporter (NET), exhibit specific NE uptake activity via NET and can catabolize, but not generate, NE. Pharmacological blockade of NE transport by reboxetine induced bone loss in WT mice. Similarly, lack of NE reuptake in norepinephrine transporter (Net)-deficient mice led to reduced bone formation and increased bone resorption, resulting in suboptimal peak bone mass and mechanical properties associated with low sympathetic outflow and high plasma NE levels. Last, daily sympathetic activation induced by mild chronic stress was unable to induce bone loss, unless NET activity was blocked. These findings indicate that the control of endogenous NE release and reuptake by presynaptic neurons and osteoblasts is an important component of the complex homeostatic machinery by which the sympathetic nervous system controls bone remodeling. These findings also suggest that drugs antagonizing NET activity, used for the treatment of hyperactivity disorders, may have deleterious effects on bone accrual.     

S(yp) Y279和Y304介导了BcrAbl与Grb2等蛋白的结合

利用体外定点突变技术获得Ｓｙｐ Ｙ２７９Ｆ、Ｙ３０４Ｆ和Ｙ５４６Ｆ突变的ｃＤＮＡ, 将这些突变体和野生型Ｓｙｐ 分别构建入ｐＸＭ 真核表达载体, 转入Ｋ５６２ 细胞。经Ｗｅｓｔｅｒｎ 印迹证明, 各转染Ｋ５６２ 细胞中都有Ｓｙｐ 蛋白的表达。免疫沉淀与免疫印迹结果发现ＷＴ、Ｙ２７９Ｆ、Ｙ３０４Ｆ和Ｙ５４６Ｆ等４ 种Ｓｙｐ 在胞内均能直接与ＢｃｒＡｂｌ 结合。体外结合实验结果表明Ｙ３０４Ｆ突变导致了Ｓｙｐ 不能与Ｓｈｃ 结合,Ｙ２７９Ｆ突变则导致了Ｓｙｐ 不能与Ｇｒｂ２ 结合。结论是： 作为“接头蛋白”,Ｓｙｐ 可以介导ＢｃｒＡｂｌ 与Ｓｈｃ 和Ｇｒｂ２ 之间的结合;Ｇｒｂ２ 结合在Ｓｙｐ 的Ｙ２７９ 上,Ｓｈｃ 则结合在Ｓｙｐ的Ｙ３０４ 上     

A cel44C-man26A gene of endophytic Paenibacillus polymyxa GS01 has multi-glycosyl hydrolases in two catalytic domains

A bacterial strain Paenibacillus polymyxa GS01 was isolated from the interior of the roots of Korean cultivars of ginseng (Panax ginseng C. A. Meyer). The cel44C-man26A gene was cloned from this endophytic strain. This 4,056-bp gene encodes for a 1,352-aa protein which, based on BLAST search homologies, contains a glycosyl hydrolase family 44 (GH44) catalytic domain, a fibronectin domain type 3, a glycosyl hydrolase family 26 (GH26) catalytic domain, and a cellulose-binding module type 3. The multifunctional enzyme domain GH44 possesses cellulase, xylanase, and lichenase activities, while the enzyme domain GH26 possesses mannanase activity. The Cel44C enzyme expressed in and purified from Escherichia coli has an optimum pH of 7.0 for cellulase and lichenase activities, but is at an optimum pH of 5.0 for xylanase and mannanase activities. The optimum temperature for enzymatic activity was 50°C for all substrates. No detectable enzymatic activity was detected for the Cel44C-Man26A mutants E91A and E222A. These results suggest that the amino acid residues Glu₉₁ and Glu₂₂₂ may play an important role in the glycosyl hydrolases activity of Cel44C-Man26A.     

Historical Biogeography and Diversification of Truffles in the Tuberaceae and Their Newly Identified Southern Hemisphere Sister Lineage

Truffles have evolved from epigeous (aboveground) ancestors in nearly every major lineage of fleshy fungi. Because accelerated rates of morphological evolution accompany the transition to the truffle form, closely related epigeous ancestors remain unknown for most truffle lineages. This is the case for the quintessential truffle genus Tuber, which includes species with socio-economic importance and esteemed culinary attributes. Ecologically, Tuber spp. form obligate mycorrhizal symbioses with diverse species of plant hosts including pines, oaks, poplars, orchids, and commercially important trees such as hazelnut and pecan. Unfortunately, limited geographic sampling and inconclusive phylogenetic relationships have obscured our understanding of their origin, biogeography, and diversification. To address this problem, we present a global sampling of Tuberaceae based on DNA sequence data from four loci for phylogenetic inference and molecular dating. Our well-resolved Tuberaceae phylogeny shows high levels of regional and continental endemism. We also identify a previously unknown epigeous member of the Tuberaceae – the South American cup-fungus Nothojafnea thaxteri (E.K. Cash) Gamundí. Phylogenetic resolution was further improved through the inclusion of a previously unrecognized Southern hemisphere sister group of the Tuberaceae. This morphologically diverse assemblage of species includes truffle (e.g. Gymnohydnotrya spp.) and non-truffle forms that are endemic to Australia and South America. Southern hemisphere taxa appear to have diverged more recently than the Northern hemisphere lineages. Our analysis of the Tuberaceae suggests that Tuber evolved from an epigeous ancestor. Molecular dating estimates Tuberaceae divergence in the late Jurassic (∼156 million years ago), with subsequent radiations in the Cretaceous and Paleogene. Intra-continental diversification, limited long-distance dispersal, and ecological adaptations help to explain patterns of truffle evolution and biodiversity.     

Nematicidal activity of Lysobacter capsici YS1215 and the role of gelatinolytic proteins against root-knot nematodes

Lysobacter capsici YS1215 is a soil-borne strain that could inhibit the growth of phytopathogenic fungi, including Phytophthora capsici, Rhizoctonia solani and Fusarium oxysporum, as well as root-knot nematodes. The effect of different concentrations of bacterial culture filtrate (BCF) of L. capsici YS1215 on the mortality of second-stage juveniles (J2) of Meloidogyne incognita was studied using 24-well plates. The J2 mortality increased with increasing concentrations of BCF. YS1215 also produces gelatinases in the culture filtrate. To study its role in nematicidal activities, the partial purification and the characterisation of gelatinolytic proteins were done from the culture medium of the YS1215. The partially purified proteins showed three clear bands with molecular weights estimated using zymography to be 255.7, 232.1 and 146.4 kDa. The optimal pH and temperature for the proteins were 8.0 and 40°C, respectively. The activity of the proteins was inhibited by ethylenediaminetetraacetic acid, FeCl₃ and 1,10-phenanthroline, whereas it was activated by MnCl₂. The proteins may belong to the group of metalloproteases. Moreover, the proteins could hydrolyse skimmed milk, collagen, gelatin and bovine serum albumin (BSA) as substrates, but not casein. The proteins could induce 75% J2 mortality in five days and degrade the J2 bodies. The present study demonstrates the role of the gelatinolytic proteins in the nematicidal potential of L. capsici YS1215.     

Crystal structures of human HSP90alpha-complexed with dihydroxyphenylpyrazoles

A series of dihydroxyphenylpyrazole compounds were identified as a unique class of reversible Hsp90 inhibitors. The crystal structures for two of the identified compounds complexed with the N-terminal ATP binding domain of human Hsp90alpha were determined. The dihydroxyphenyl ring of the compounds fits deeply into the adenine binding pocket with the C2 hydroxyl group forming a direct hydrogen bond with the side chain of Asp93. The pyrazole ring forms hydrogen bonds to the backbone carbonyl of Gly97, the hydroxyl group of Thr184 and to a water molecule, which is present in all of the published HSP90 structures. One of the identified compounds (G3130) demonstrated cellular activities (in Her-2 degradation and activation of Hsp70 promoter) consistent with the inhibition of cellular Hsp90 functions.     

Transmembrane domain-induced oligomerization is crucial for the functions of syndecan-2 and syndecan-4

The syndecans are known to form homologous oligomers that may be important for their functions. We have therefore determined the role of oligomerization of syndecan-2 and syndecan-4. A series of glutathione S-transferase-syndecan-2 and syndecan-4 chimeric proteins showed that all syndecan constructs containing the transmembrane domain formed SDS-resistant dimers, but not those lacking it. SDS-resistant dimer formation was hardly seen in the syndecan chimeras where each transmembrane domain was substituted with that of platelet-derived growth factor receptor (PDGFR). Increased MAPK activity was detected in HEK293T cells transfected with syndecan/PDGFR chimeras in a syndecan transmembrane domain-dependent fashion. The chimera-induced MAPK activation was independent of both ligand and extracellular domain, implying that the transmembrane domain is sufficient to induce dimerization/oligomerization in vivo. Furthermore, the syndecan chimeras were defective in syndecan-4-mediated focal adhesion formation and protein kinase Calpha activation or in syndecan-2-mediated cell migration. Taken together, these data suggest that the transmembrane domains are sufficient for inducing dimerization and that transmembrane domain-induced oligomerization is crucial for syndecan-2 and syndecan-4 functions.