Mechanical Signaling on the Single Protein Level Studied Using Steered Molecular Dynamics

Efficient communication between the cell and its external environment is of the utmost importance to the function of multicellular organisms. While signaling events can be generally characterized as information exchange by means of controlled energy conversion, research efforts have hitherto mainly been concerned with mechanisms involving chemical and electrical energy transfer. Here, we review recent computational efforts addressing the function of mechanical force in signal transduction. Specifically, we focus on the role of steered molecular dynamics (SMD) simulations in providing details at the atomic level on a group of protein domains, which play a fundamental role in signal exchange by responding properly to mechanical strain. We start by giving a brief introduction to the SMD technique and general properties of mechanically stable protein folds, followed by specific examples illustrating three general regimes of signal transfer utilizing mechanical energy: purely mechanical, mechanical to chemical, and chemical to mechanical. Whenever possible the physiological importance of the example at hand is stressed to highlight the diversity of the processes in which mechanical signaling plays a key role. We also provide an overview of future challenges and perspectives for this rapidly developing field.     

Effect of pathogenic cysteine mutations on FGFR3 transmembrane domain dimerization in detergents and lipid bilayers

Mutations in fibroblast growth factor receptors are known as the genetic basis of skeletal growth disorders. The mechanism of pathogenesis, as determined by mutation-induced changes in receptor structure, interactions, and function, is elusive. Here we study three pathogenic Cys mutations, associated with either thanatophoric dysplasia or achondroplasia, in the TM domain of fibroblast growth factor receptors 3 (FGFR3). We characterize the dimerization propensities of the mutant TM domains in detergents and in lipid bilayers, in the presence and absence of reducing agents, and compare them to previous measurements of wild-type. We find that the Cys mutations increase the propensity for dimerization in detergent, with the Cys370 mutant exhibiting the highest propensity for disulfide bond formation, the Cys371 mutant having an intermediate propensity, and Cys375 the lowest. Thus, disulfide bonds readily form in detergents, with efficiency that correlates with the severity of the phenotype. In lipid bilayers, however, the Cys370 mutant, which dimerizes strongly in detergent, behaves as the wild-type, suggesting that Cys370-mediated disulfide bonds do not form between the isolated TM domains in bilayers. Thus, the nature of the hydrophobic environment plays an important role in defining the structure and flexibility of transmembrane dimers. These results and previous findings from cellular studies lead us to propose a conformational flexibility mechanism of receptor stabilization as a basis for disregulated FGFR3 signaling in thanatophoric dysplasia and achondroplasia.     

Mre11 and Ku regulation of double-strand break repair by gene conversion and break-induced replication

The yeast Mre11-Rad50-Xrs2 (MRX) and Ku complexes regulate single-strand resection at DNA double-strand breaks (DSB), a key early step in homologous recombination (HR). A prior plasmid gap repair study showed that mre11 mutations, which slow single-strand resection, reduce gene conversion tract lengths and the frequency of associated crossovers. Here we tested whether mre11Delta or nuclease-defective mre11 mutations reduced gene conversion tract lengths during HR between homologous chromosomes in diploid yeast. We found that mre11 mutations reduced the efficiency of HR but did not reduce tract lengths or crossovers, despite substantially reduced end-resection at the test (ura3) locus. End-resection is increased in yku70Delta, but this change also had no effect on tract lengths. Thus, heteroduplex formation and tract lengths are not regulated by the extent of end-resection during DSB repair in a chromosomal context. In a plasmid-chromosome DSB repair assay, tract lengths were again similar in wild-type and mre11Delta, but they were reduced in mre11Delta in a gap repair assay. These results indicate that tract lengths are not affected by the extent of end processing when broken ends can invade nearby sites, perhaps because MRX coordination of the two broken ends is dispensable when ends invade nearby sites. Although HR outcome was largely unaffected in mre11 mutants, break-induced replication (BIR) and chromosome loss increased, suggesting that Mre11 function in mitotic HR is limited to early HR stages. Interestingly, yku70Delta suppressed BIR in mre11 mutants. BIR is also elevated in rad51 mutants, but yku70Delta did not suppress BIR in a rad51 background. These results indicate that Mre11 functions in Rad51-independent BIR, and that Ku functions in Rad51-dependent BIR.     

Defective hepatic lipoprotein receptor binding of beta-very low density lipoproteins from type III hyperlipoproteinemic patients. Importance of apolipoprotein E

Apolipoprotein (apo-) E2 and beta-migrating very low density lipoproteins (beta-VLDL) (which were isolated from type III hyperlipoproteinemic subjects) both demonstrated defective binding to apo-E and apo-B,E receptors on dog liver membranes and to apo-B,E low density lipoproteins (LDL) receptors on fibroblasts. The defective binding activity of the apo-E2 and beta-VLDL varied from very poor to nearly normal. The ability of the beta-VLDL to interact with hepatic apo-E receptors was enhanced by the addition of normal apo-E3 to the beta-VLDL. Furthermore, cysteamine treatment of the apo-E2 in beta-VLDL enhanced binding of the beta-VLDL to both apo-E and apo-B,E receptors. The importance of apo-E in mediating the receptor binding of beta-VLDL to these receptors was confirmed by using monoclonal antibodies. The residual binding activity of beta-VLDL to apo-E and apo-B,E receptors was inhibited by greater than 90% with anti-apo-E, while the addition of anti-apo-B had little effect. The apo-B in the beta-VLDL was capable of binding to apo-B,E receptors after the hydrolysis of the beta-VLDL triglycerides with milk lipoprotein lipase. Lipase treatment yielded, two subfractions of beta-VLDL. One fraction (d = 1.02 to 1.03 g/ml) was enriched with apo-B100; the other fraction (d less than 1.006 g/ml) was enriched with apo-B48 and apo-E2. Significantly increased amounts of the apo-B100-enriched fraction bound to apo-B,E receptors. Inhibition of this binding caused by the addition of anti-apo-B indicated that the binding activity of this subfraction was mediated by apo-B100. The apo-B48-enriched fraction did not show a significant increase in receptor binding, suggesting that apo-B48 does not bind to these receptors. In a control experiment, it was shown that triglyceride-rich VLDL, which contain normal apo-E3 and apo-B100, bind significantly to both liver apo-E receptors and fibroblast apo-B,E receptors. This binding activity was inhibited by greater than 90% with anti-apo-E. Lipase hydrolysis of the VLDL did not further enhance their receptor-binding activity. These results demonstrate that apo-E, and not apo-B, is the major determinant mediating the receptor-binding activity of cholesterol-rich beta-VLDL and triglyceride-rich VLDL.     

Paenibacillus brassicae sp. nov., isolated from cabbage rhizosphere in Beijing, China

A novel Gram-positive, rod-shaped, motile, spore-forming, nitrogen-fixing bacterium, designated strain 112^T, was isolated from cabbage rhizosphere in Beijing, China. The strain was found to grow at 10–40 °C and pH 4–11, with an optimum of 30 °C and pH 7.0, respectively. Phylogenetic analysis based on a fragment of the full-length 16S rRNA gene sequence revealed that strain 112^T is a member of the genus Paenibacillus. High levels of 16S rRNA gene similarities were found between strain 112^T, Paenibacillus sabinae DSM 17841^T (97.82 %) and Paenibacillus forsythiae DSM 17842^T (97.22 %). However, the DNA–DNA hybridization values between strain 112^T and the type strains of these two species were 10.36 and 6.28 %, respectively. The predominant menaquinone was found to be menaquinone 7 (MK-7). The major fatty acids were determined to be anteiso-C_15:0 and C_16:0. The major polar lipids were found to be diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and unknown aminophospholipids. The cell wall peptidoglycan was found to contain meso-diaminopimelic acid. The DNA G+C content was determined to be 55.4 mol%. On the basis of its phenotypic characteristics, 16S rRNA gene sequence analysis and the value of DNA–DNA hybridization, strain 112^T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus brassicae sp. nov. is proposed. The type strain is 112^T (= ACCC 01125^T = DSM 24983^T).     

The mechanical stability of ubiquitin is linkage dependent

Ubiquitin chains are formed through the action of a set of enzymes that covalently link ubiquitin either through peptide bonds or through isopeptide bonds between their C terminus and any of four lysine residues. These naturally occurring polyproteins allow one to study the mechanical stability of a protein, when force is applied through different linkages. Here we used single-molecule force spectroscopy techniques to examine the mechanical stability of N-C-linked and Lys48-C-linked ubiquitin chains. We combined these experiments with steered molecular dynamics (SMD) simulations and found that the mechanical stability and unfolding pathway of ubiquitin strongly depend on the linkage through which the mechanical force is applied to the protein. Hence, a protein that is otherwise very stable may be easily unfolded by a relatively weak mechanical force applied through the right linkage. This may be a widespread mechanism in biological systems.     

云南省6个松墨天牛种群形态差异分析

对云南省松墨天牛Monochamus alternatus Hope 6个地理种群159个个体体长、前胸背板纵长、前胸背板横宽值及前胸背板斑纹进行了差异系数计算、聚类分析及判别分析.各松墨天牛种群间的差异系数均小于1.28;聚类分析表明,畹町种群形态与其他5个种群差异较大;基于4个形态特征建立的判别函数可以将畹町种群与云南其他种群区别开来.畹町种群与其他松墨天牛种群的形态差异可能由于其遗传差异造成.     

Towards precise classification of cancers based on robust gene functional expression profiles

Background

Despite the continuous production of genome sequence for a number of organisms, reliable, comprehensive, and cost effective gene prediction remains problematic. This is particularly true for genomes for which there is not a large collection of known gene sequences, such as the recently published chicken genome. We used the chicken sequence to test comparative and homology-based gene-finding methods followed by experimental validation as an effective genome annotation method.

Results

We performed experimental evaluation by RT-PCR of three different computational gene finders, Ensembl, SGP2 and TWINSCAN, applied to the chicken genome. A Venn diagram was computed and each component of it was evaluated. The results showed that de novo comparative methods can identify up to about 700 chicken genes with no previous evidence of expression, and can correctly extend about 40% of homology-based predictions at the 5' end.

Conclusions

De novo comparative gene prediction followed by experimental verification is effective at enhancing the annotation of the newly sequenced genomes provided by standard homology-based methods.     

Sarcocystis cruzi: comparative studies confirm natural infections of buffaloes

Controversy exists concerning whether cattle and water buffalo sustain infections with cysts of distinct arrays of species in the genus Sarcocystis. In particular, morphologically similar parasites have been alternately ascribed to Sarcocystis cruzi or to Sarcocystis levinei, depending on their occurrence in cattle or water buffalo. We used light and transmission electron microscopy, genetic analysis, and experimental infections of definitive canine hosts to determine whether consistent differences could be identified from parasites derived from several natural infections of each host, examining several tissue types (esophagus, skeletal muscles, and heart). Cysts derived from cattle and water buffalo shared similar structure; variation among 18S rRNA sequences did not segregate consistently according to intermediate host type; parasites derived from cattle and water buffalo induced similar outcomes in the canine definitive host. One cattle specimen harbored unusually large (macroscopic) sarcocysts which nonetheless conformed to previously reported ultrastructural and genetic features of S. cruzi. Finding no consistent basis to differentiate between them, we conclude that the parasites infecting each host and tissue type correspond to S. cruzi. In our sample, no phylogenetically distinct taxon was sampled which might correspond to a distinct taxon previously described as S. levinei. Either that taxon was missed by our sampling effort, or it may represent a junior synonym to S. cruzi, which would then cycle between dogs and a broader range of intermediate bovine hosts than was previously considered.