Construction of a nasopharyngeal carcinoma 2D/MS repository with Open Source XML Database – Xindice

Background  

Many proteomics initiatives require integration of all information with uniformcriteria from collection of samples and data display to publication of experimental results. The integration and exchanging of these data of different formats and structure imposes a great challenge to us. The XML technology presents a promise in handling this task due to its simplicity and flexibility. Nasopharyngeal carcinoma (NPC) is one of the most common cancers in southern China and Southeast Asia, which has marked geographic and racial differences in incidence. Although there are some cancer proteome databases now, there is still no NPC proteome database.     

Purification and part characterization of a novel antibacterial protein Scygonadin, isolated from the seminal plasma of mud crab, Scylla serrata (Forskål, 1775)

A novel protein was isolated from the seminal plasma of the mud crab, Scylla serrata (Forskål, 1775). It exhibited an antibacterial activity against the Gram-positive bacterium Micrococcus leteus with IC90 of 0.125 mg/ml. The extraction procedure for the protein included techniques of acid extraction, ion-exchange chromatography on SP-Sepharose Fast Flow and reverse-phase liquid chromatography on Source 5R RPC. It showed a molecular mass of 10.8 kDa by SDS-PAGE. A partial 20 residue NH₂-terminal sequence was determined by Edman degradation and MS-fingerprint of the protein was conducted. Similarity search in protein databases (BLAST) revealed that the protein exhibited no significant homology to any other reported antimicrobial peptides. We propose the name Scygonadin (from the gonad of S. serrata) for this antibacterial protein.     

Isolation of a Genomic Region Affecting Most Components of Metabolic Syndrome in a Chromosome-16 Congenic Rat Model

Metabolic syndrome is a highly prevalent human disease with substantial genomic and environmental components. Previous studies indicate the presence of significant genetic determinants of several features of metabolic syndrome on rat chromosome 16 (RNO16) and the syntenic regions of human genome. We derived the SHR.BN16 congenic strain by introgression of a limited RNO16 region from the Brown Norway congenic strain (BN-Lx) into the genomic background of the spontaneously hypertensive rat (SHR) strain. We compared the morphometric, metabolic, and hemodynamic profiles of adult male SHR and SHR.BN16 rats. We also compared in silico the DNA sequences for the differential segment in the BN-Lx and SHR parental strains. SHR.BN16 congenic rats had significantly lower weight, decreased concentrations of total triglycerides and cholesterol, and improved glucose tolerance compared with SHR rats. The concentrations of insulin, free fatty acids, and adiponectin were comparable between the two strains. SHR.BN16 rats had significantly lower systolic (18–28 mmHg difference) and diastolic (10–15 mmHg difference) blood pressure throughout the experiment (repeated-measures ANOVA, P < 0.001). The differential segment spans approximately 22 Mb of the telomeric part of the short arm of RNO16. The in silico analyses revealed over 1200 DNA variants between the BN-Lx and SHR genomes in the SHR.BN16 differential segment, 44 of which lead to missense mutations, and only eight of which (in Asb14, Il17rd, Itih1, Syt15, Ercc6, RGD1564958, Tmem161a, and Gatad2a genes) are predicted to be damaging to the protein product. Furthermore, a number of genes within the RNO16 differential segment associated with metabolic syndrome components in human studies showed polymorphisms between SHR and BN-Lx (including Lpl, Nrg3, Pbx4, Cilp2, and Stab1). Our novel congenic rat model demonstrates that a limited genomic region on RNO16 in the SHR significantly affects many of the features of metabolic syndrome.     

A mechanism of leading-edge protrusion in the absence of Arp2/3 complex

Cells employ protrusive leading edges to navigate and promote their migration in diverse physiological environments. Classical models of leading-edge protrusion rely on a treadmilling dendritic actin network that undergoes continuous assembly nucleated by the Arp2/3 complex, forming ruffling lamellipodia. Recent work demonstrated, however, that, in the absence of the Arp2/3 complex, fibroblast cells adopt a leading edge with filopodia-like protrusions (FLPs) and maintain an ability to move, albeit with altered responses to different environmental signals. We show that formin-family actin nucleators are required for the extension of FLPs but are insufficient to produce a continuous leading edge in fibroblasts lacking Arp2/3 complex. Myosin II is concentrated in arc-like regions of the leading edge in between FLPs, and its activity is required for coordinated advancement of these regions with formin-generated FLPs. We propose that actomyosin contraction acting against membrane tension advances the web of arcs between FLPs. Predictions of this model are verified experimentally. The dependence of myosin II in leading-edge advancement helps explain the previously reported defect in directional movement in the Arpc3-null fibroblasts. We provide further evidence that this defect is cell autonomous during chemotaxis.     

A Ribonucleoprotein Complex Protects the Interleukin-6 mRNA from Degradation by Distinct Herpesviral Endonucleases

During lytic Kaposi’s sarcoma-associated herpesvirus (KSHV) infection, the viral endonuclease SOX promotes widespread degradation of cytoplasmic messenger RNA (mRNA). However, select mRNAs escape SOX-induced cleavage and remain robustly expressed. Prominent among these is interleukin-6 (IL-6), a growth factor important for survival of KSHV infected B cells. IL-6 escape is notable because it contains a sequence within its 3’ untranslated region (UTR) that can confer protection when transferred to a SOX-targeted mRNA, and thus overrides the endonuclease targeting mechanism. Here, we pursued how this protective RNA element functions to maintain mRNA stability. Using affinity purification and mass spectrometry, we identified a set of proteins that associate specifically with the protective element. Although multiple proteins contributed to the escape mechanism, depletion of nucleolin (NCL) most severely impacted protection. NCL was re-localized out of the nucleolus during lytic KSHV infection, and its presence in the cytoplasm was required for protection. After loading onto the IL-6 3’ UTR, NCL differentially bound to the translation initiation factor eIF4H. Disrupting this interaction, or depleting eIF4H, reinstated SOX targeting of the RNA, suggesting that interactions between proteins bound to distant regions of the mRNA are important for escape. Finally, we found that the IL-6 3’ UTR was also protected against mRNA degradation by the vhs endonuclease encoded by herpes simplex virus, despite the fact that its mechanism of mRNA targeting is distinct from SOX. These findings highlight how a multitude of RNA-protein interactions can impact endonuclease targeting, and identify new features underlying the regulation of the IL-6 mRNA.     

Sulfolobus shibatae CCA-adding enzyme forms a tetramer upon binding two tRNA molecules: A scrunching-shuttling model of CCA specificity

The tRNA CCA-adding enzyme adds CCA stepwise to immature transfer RNA molecules untemplated, but with high specificity. We examined the oligomerization state of the enzyme from Sulfolobus shibatae and its binding to transfer RNA molecules, using various biophysical and biochemical methods including size exclusion chromatography, multi-angle laser light scattering, small-angle X-ray scattering, and gel electrophoresis band mobility shift assay. The 48 kDa monomer forms a stable salt- resistant dimer in solution. Further dimerization of the dimeric enzyme to form a tetramer is induced by the binding of two tRNA molecules. The formation of a tetramer with only two bound tRNA molecules leads us to suggest that one pair of active sites may be specific for adding two C bases, which results in scrunching of the primer strand. An adjacent second pair of active sites may be specific for adding A after addition of two C bases which makes the 3' terminus long enough to reach the second pair of active sites.