Enterovirus 71 (EV71) is a picornavirus that can cause severe neurological complications in children. Like other picornaviruses, the genomic RNA of EV71 contains a long 59 untranslated region (UTR). Cellular proteins interact with the EV71 59 UTR, and these interactions are important for virus replication. Using an RNA pull-down assay and proteomics approaches, this study identified the heterogeneous nuclear ribonucleoprotein K (hnRNP K) as one of the EV71 59 UTR-associated proteins. The interaction between hnRNP K and the 59 UTR was further confirmed by mapping the interaction regions to stem-loops I-II and IV in the 59 UTR. During EV71 infection, hnRNP K was enriched in the cytoplasm where virus replication occurs, whereas hnRNP K was localized in the nucleus in mock-infected cells. Viral yields were found to be significantly lower in hnRNP K knockdown cells and viral RNA synthesis was delayed in hnRNP K knockdown cells in comparison with negative-control cells treated with small interfering RNA. These results suggest that hnRNP K interacts with the EV71 59 UTR and participates in virus replication.

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Cardiotoxins are small proteins that are found in the venoms of snakes from the Elapidae family. These toxins are known to bind to and disrupt the organization, integrity, and function of the cell membrane. Most of the well-studied cardiotoxins cause depolarization of membrane potentials and/or lysis of red cells. In contrast, CTX V from Naja naja atra displays poor hemolytic activity but is proficient at inducing aggregation and fusion of sphingomyelin vesicles [Chien et al. (1991) J. Biol. Chem. 266, 3252-3259]. To determine whether the unique activity of this CTX is attributable to its tertiary structure, the solution structure of CTX V was determined by NMR methods. On the basis of these studies, this cardiotoxin has the same general topology as other members of the family, and thus its unusual properties do not arise from any gross structural differences that are detectable by solution NMR methods. Molecular dynamics calculations indicate that residues 36-50 show concerted fluctuations. On the basis of sequence similarity, we postulate that residues 30-34 are important in determining the specificity of cardiotoxins for fusion versus lysis of vesicles.

CTP synthase (CTPS) forms compartmentalized filaments in response to substrate availability and environmental nutrient status. However, the physiological role of filaments and mechanisms for filament assembly are not well understood. Here, we provide evidence that CTPS forms filaments in response to histidine influx during glutamine starvation. Tetramer conformation-based filament formation restricts CTPS enzymatic activity during nutrient deprivation. CTPS protein levels remain stable in the presence of histidine during nutrient deprivation, followed by rapid cell growth after stress relief. We demonstrate that filament formation is controlled by methylation and that histidine promotes re-methylation of homocysteine by donating one-carbon intermediates to the cytosolic folate cycle. Furthermore, we find that starvation stress and glutamine deficiency activate the GCN2/ATF4/MTHFD2 axis, which coordinates CTPS filament formation. CTPS filament formation induced by histidine-mediated methylation may be a strategy used by cancer cells to maintain homeostasis and ensure a growth advantage in adverse environments.

Hsp90 is one of the most abundant and conserved proteins in the cell. Reduced levels or activity of Hsp90 causes defects in many cellular processes and also reveals genetic and nongenetic variation within a population. Despite information about Hsp90 protein–protein interactions, a global view of the Hsp90-regulated proteome in yeast is unavailable. To investigate the degree of dependency of individual yeast proteins on Hsp90, we used the “stable isotope labeling by amino acids in cell culture” method coupled with mass spectrometry to quantify around 4,000 proteins in low-Hsp90 cells. We observed that 904 proteins changed in their abundance by more than 1.5-fold. When compared with the transcriptome of the same population of cells, two-thirds of the misregulated proteins were observed to be affected posttranscriptionally, of which the majority were downregulated. Further analyses indicated that the downregulated proteins are highly conserved and assume central roles in cellular networks with a high number of protein interacting partners, suggesting that Hsp90 buffers genetic and nongenetic variation through regulating protein network hubs. The downregulated proteins were enriched for essential proteins previously not known to be Hsp90-dependent. Finally, we observed that downregulation of transcription factors and mating pathway components by attenuating Hsp90 function led to decreased target gene expression and pheromone response, respectively, providing a direct link between observed proteome regulation and cellular phenotypes.

AbsatractAlong with the constant improvement in high-throughput sequencing technology, an increasing number of transcriptome sequencing projects are carried out in organisms without decoded genome information and even on environmental biological samples. To study the biological functions of novel transcripts, the very first task is to identify their potential functions. We present a web-based annotation tool, FunctionAnnotator, which offers comprehensive annotations, including GO term assignment, enzyme annotation, domain/motif identification and predictions for subcellular localization. To accelerate the annotation process, we have optimized the computation processes and used parallel computing for all annotation steps. Moreover, FunctionAnnotator is designed to be versatile, and it generates a variety of useful outputs for facilitating other analyses. Here, we demonstrate how FunctionAnnotator can be helpful in annotating non-model organisms. We further illustrate that FunctionAnnotator can estimate the taxonomic composition of environmental samples and assist in the identification of novel proteins by combining RNA-Seq data with proteomics technology. In summary, FunctionAnnotator can efficiently annotate transcriptomes and greatly benefits studies focusing on non-model organisms or metatranscriptomes. FunctionAnnotator, a comprehensive annotation web-service tool, is freely available online at: http://fa.cgu.edu.tw/. This new web-based annotator will shed light on field studies involving organisms without a reference genome.

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BACKGROUND A previous comparative tissue proteomics study by the authors of the current study led to the identification of caldesmon (CaD) as one of the proteins associated with cervical metastasis of oral cavity squamous cell carcinoma (OSCC). In the current investigation, the authors focused on the potential functions of CaD in patients with OSCC. METHODS CaD expression was examined in tissue samples from 155 patients using immunohistochemical analysis. The expression of CaD variants was determined by Western blot analysis and reverse transcriptase‐polymerase chain reaction. In addition, the specific effects of CaD gene overexpression and silence were determined in OSCC cell lines. RESULTS CaD expression was found to be significantly higher in tumor cells from metastatic lymph nodes compared with primary tumor cells, and was nearly absent in normal oral epithelia. Higher CaD expression was found to be correlated with positive N classification, poor differentiation, perineural invasion, and tumor depth (P = .001, P = .029, P = .001, and P = .031, respectively). In survival analyses, OSCC patients with higher CaD expression were found to have poorer prognosis with regard to disease‐specific survival and disease‐free survival (P = .003 and P = .014, respectively). Multivariate analyses further indicated that higher CaD expression was an independent predictor of disease‐specific survival (P = .043). Serum CaD levels were found to be significantly higher in patients with OSCC, but this finding was not associated with clinicopathological manifestations. Data obtained from in vitro suppression, rescue, and overexpression of CaD in OEC‐M1 cells indicated that CaD promotes migration and invasive processes in OSCC cells. CONCLUSIONS The findings of the current study collectively suggest that the low‐molecular‐weight CaD expression in OSCC tumors is associated with tumor metastasis and patient survival. Cancer 2013;119:4003–4011. © 2013 American Cancer Society.