The PHCCEx domain of Tiam1/2 is a novel protein‐ and membrane‐binding module

Tiam1 and Tiam2 (Tiam1/2) are guanine nucleotide‐exchange factors that possess the PH–CC–Ex (pleckstrin homology, coiled coil and extra) region that mediates binding to plasma membranes and signalling proteins in the activation of Rac GTPases. Crystal structures of the PH–CC–Ex regions revealed a single globular domain, PHCCEx domain, comprising a conventional PH subdomain associated with an antiparallel coiled coil of CC subdomain and a novel three‐helical globular Ex subdomain. The PH subdomain resembles the β‐spectrin PH domain, suggesting non‐canonical phosphatidylinositol binding. Mutational and binding studies indicated that CC and Ex subdomains form a positively charged surface for protein binding. We identified two unique acidic sequence motifs in Tiam1/2‐interacting proteins for binding to PHCCEx domain, Motif‐I in CD44 and ephrinB's and the NMDA receptor, and Motif‐II in Par3 and JIP2. Our results suggest the molecular basis by which the Tiam1/2 PHCCEx domain facilitates dual binding to membranes and signalling proteins.     

High-pressure SAXS study of folded and unfolded ensembles of proteins

A structural interpretation of the thermodynamic stability of proteins requires an understanding of the structural properties of the unfolded state. High-pressure small-angle x-ray scattering was used to measure the effects of temperature, pressure, denaturants, and stabilizing osmolytes on the radii of gyration of folded and unfolded state ensembles of staphylococcal nuclease. A set of variants with the internal Val-66 replaced with Ala, Tyr, or Arg was used to examine how changes in the volume and polarity of an internal microcavity affect the dimensions of the native state and the pressure sensitivity of the ensemble. The unfolded state ensembles achieved for these proteins with high pressure were more compact than those achieved at high temperature, and were all very sensitive to the presence of urea and glycerol. Substitutions at the hydrophobic core detectably altered the conformation of the protein, even in the folded state. The introduction of a charged residue, such as Arg, inside the hydrophobic interior of a protein could dramatically alter the structural properties, even those of the unfolded state. The data suggest that a charge at an internal position can interfere with the formation of transient hydrophobic clusters in the unfolded state, and ensure that the pressure-unfolded form of a protein occupies the maximum volume possible. Only at high temperatures does the radius of gyration of the unfolded state ensemble approach the value for a statistical random coil.     

Ambient Stable Quantitative PCR Reagents for the Detection of Yersinia pestis

Background

Although assays for detecting Yersinia pestis using TaqMan probe-based real-time PCR have been developed for years, little is reported on room-temperature-stable PCR reagents, which will be invaluable for field epidemic surveillance, immediate response to public health emergencies, counter-bioterrorism investigation, etc. In this work, a set of real-time PCR reagents for rapid detection of Y. pestis was developed with extraordinary stability at 37°C.

Methods/Principal Findings

TaqMan-based real-time PCR assays were developed using the primers and probes targeting the 3a sequence in the chromosome and the F1 antigen gene caf1 in the plasmid pMT1of Y. pestis, respectively. Then, carbohydrate mixtures were added to the PCR reagents, which were later vacuum-dried for stability evaluation. The vacuum-dried reagents were stable at 37°C for at least 49 days for a lower concentration of template DNA (10 copies/µl), and up to 79 days for higher concentrations (≥10² copies/µl). The reagents were used subsequently to detect soil samples spiked with Y. pestis vaccine strain EV76, and 5×10⁴ CFU per gram of soil could be detected by both 3a- and caf1-based PCR reagents. In addition, a simple and efficient method for soil sample processing is presented here.

Conclusions/Significance

The vacuum-dried reagents for real-time PCR maintain accuracy and reproducibility for at least 49 days at 37°C, indicating that they can be easily transported at room temperature for field application if the machine for performing real-time PCR is available. This dry reagent is of great significance for routine plague surveillance.     

Constructing recombinant herpesvirus BAC vectors with mating-assisted genetically integrated clone method

The large capacity of pseudorabies virus (PRV) for foreign DNA and broad host range make it a prospective tool for the preparation of vaccines and agents of gene and tumour therapy. Here we introduced a cloning strategy that facilitates construction of recombinant PRV?CBAC vectors based on mating-assisted genetically integrated clone (MAGIC). The target gene was cloned into a small conditionally replicating donor plasmid, followed by shuffling to a recipient PRV?CBAC plasmid in vivo of Escherichia coli through MAGIC. The average efficiency of successful clones was 89%. Moreover, permanent integration of unwanted sequences was avoided.     

Phage display selection of peptides that inhibit metastasis ability of gastric cancer cells with high liver-metastatic potential

Organ-specific metastasis is an important character of cancer cells. Cancer cells that can metastasize to a special organ were thought to have different proteins in cell membrane, which might have potential utility as diagnostic markers and therapeutic targets. In the present work, based on high liver-metastatic gastric cancer cells, XGC9811-L, a screening approach with phage displayed peptide library, was successfully used to isolate 8-mer peptide ligands binding to the target cells. The phage20 had the highest binding efficiency to XGC9811-L cells, which also displayed remarkable cell specificity. Peptide20 that was displayed on phage20 could suppress the motility and invasion of XGC9811-L significantly. The adhesive ability of XGC9811-L to collagen IV was also inhibited by peptide20. Furthermore, phage20 could significantly reduce the incidence of liver metastasis of gastric cancer transplanted into nude mice and was also beneficial for the reduction the number of metastatic nodules in the liver. In conclusion, the phage display is an effective method to screen for the new molecules associated with organ-specific metastasis. The selected peptide20 can reverse the liver metastasis behavior of the gastric cancer cells.     

Stepwise deamidation of ribonuclease A at five sites determined by top down mass spectrometry

Although deamidation at asparagine and glutamine has been found in numerous studies of a variety of proteins, in almost all cases the analytical methodology that was used could detect only a single site of deamidation. For the extensively studied case of reduced bovine ribonuclease A (13,689 Da), only Asn67 deamidation has been demonstrated previously, although one study found three monodeamidated fractions. Here top down tandem mass spectrometry shows that Asn67 deamidation is extensive before Asn71 and Asn94 react; these are more than half deamidated before Asn34 reacts, and its deamidation is extensive before that at Gln74 is initiated. Except for the initial Asn67 site, these large reactivity differences correlate poorly with neighboring amino acid identities and instead indicate residual conformational effects despite the strongly denaturing media that were used; deamidation at Asn67 could enhance that at Asn71, and these enhance that at Gln74. This success in the site-specific quantitation of deamidation in a 14 kDa protein mixture, despite the minimal 1 Da (-NH2 --> -OH) change in the molecular mass, is further evidence of the broad applicability of the top down MS/MS methodology for characterization of protein posttranslational modifications.     

Role of Bmi1 in H2A ubiquitylation and Hox gene silencing

Posttranslational histone modifications play a crucial role in the regulation of chromatin structure and gene activity. In previous studies, we identified the histone H2A ubiquitin ligase as Ring2, together in a complex with Ring1, Bmi1, and HPH2 (human polyhomeotic 2). We report here that the oncogene Bmi1 stimulates H2A ubiquitylation both in vitro and in vivo and that Bmi1-regulated H2A ubiquitylation is required for Hox gene silencing and normal cell growth. Our studies indicate that Bmi1 maintains the integrity of the complex through simultaneous interactions with the other subunits. We reconstituted the functional human H2A ubiquitin ligase complex and a panel of subcomplexes of different subunits. Comparisons of the H2A ubiquitin ligase activities of these different complexes revealed that Bmi1 stimulates the H2A ubiquitin ligase activity of Ring2 (and Ring1). Additionally, we demonstrated that the HoxC5 gene is regulated by ubiquitylated H2A in HeLa cells and that ubiquitylated H2A is localized on 5' regulatory regions of the HoxC5 gene. The role of Bmi1 in H2A ubiquitylation and HoxC5 gene expression in vivo was analyzed by RNA interference experiments. Knockdown of Bmi1 causes a global and loci-specific loss of H2A ubiquitylation, up-regulation of the HoxC5 gene, and slower cell growth. Intriguingly, Ring2 binds to its target regions in Bmi1 knockdown cells. Therefore, our studies reveal that Bmi1 is required for H2A ubiquitylation and suggest that H2A ubiquitylation regulates Bmi1-mediated gene expression.     

Phosphatidylinositol-3 phosphatase myotubularin-related protein 6 negatively regulates CD4 T cells

Intracellular Ca2+ levels rapidly rise following cross-linking of the T-cell receptor (TCR) and function as a critical intracellular second messenger in T-cell activation. It has been relatively under appreciated that K+ channels play an important role in Ca2+ influx into T lymphocytes by helping to maintain a negative membrane potential which provides an electrochemical gradient to drive Ca2+ influx. Here we show that the Ca2+-activated K+ channel, KCa3.1, which is critical for Ca2+ influx in reactivated naive T cells and central memory T cells, requires phosphatidylinositol-3 phosphatase [PI(3)P] for activation and is inhibited by the PI(3)P phosphatase myotubularin-related protein 6 (MTMR6). Moreover, by inhibiting KCa3.1, MTMR6 functions as a negative regulator of Ca2+ influx and proliferation of reactivated human CD4 T cells. These findings point to a new and unexpected role for PI(3)P and the PI(3)P phosphatase MTMR6 in the regulation of Ca2+ influx in activated CD4 T cells and suggest that MTMR6 plays a critical role in setting a minimum threshold for a stimulus to activate a T cell.