Binding of the human homolog of the Drosophila discs large tumor suppressor protein to the mitochondrial ribosomal protein MRP-S34

The human homolog of the Drosophila discs large tumor suppressor protein (hDLG) functions as a scaffolding protein that facilitates the transmission of diverse downstream signals. Here we show that hDLG interacts through its PDZ domains with the carboxy-terminal S/TXV motif of the mitochondrial ribosomal protein S-34 (MRP-S34). Our results suggest that hDLG interacts with MRP-S34 prior to entry of MRP-S34 into the mitochondria and may be involved in the trafficking of MRP-S34.     

Single-molecule detection of phosphorylation-induced plasticity changes during ezrin activation

Ezrin-radixin-moesin protein family provides a regulated link between the cortical actin cytoskeleton and the plasma membrane. Phosphorylation of ezrin has been functionally linked to membrane dynamics and plasticity. Our recent study demonstrated that phosphorylation of the conserved T567 residue of ezrin alters the physiology of gastric parietal cells. However, the molecular mechanism of phosphorylation-induced ezrin activation has remained elusive. Here we use atomic force microscopy (AFM) to probe phosphorylation-mediated activation of ezrin in single molecules. The phospho-mimicking and non-phosphorylatable mutant ezrin proteins were generated and purified to homogeneity. Comparative analyses of two ezrin mutants by AFM demonstrate the unfolding of the N- and C-terminal domains upon the phospho-activation. To measure the physical force underlying the inter-domain contact during mechanical unfolding, we probed the defined region of ezrin using the N-terminal ezrin coated onto the AFM tip. Comparative force measurements indicate that T567 phosphorylation-induced unfolding of ezrin favors the inter-molecular association. Taken together, these results provide molecular illustration of phosphorylation elicited functional activation of ERM proteins and indicate that stimulus-induced protein conformational change can be used as a signaling mechanism orchestrating cellular dynamics.     

Modeling of pathogen survival during simulated gastric digestion

The objective of the present study was to develop a mathematical model of pathogenic bacterial inactivation kinetics in a gastric environment in order to further understand a part of the infectious dose-response mechanism. The major bacterial pathogens Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. were examined by using simulated gastric fluid adjusted to various pH values. To correspond to the various pHs in a stomach during digestion, a modified logistic differential equation model and the Weibull differential equation model were examined. The specific inactivation rate for each pathogen was successfully described by a square-root model as a function of pH. The square-root models were combined with the modified logistic differential equation to obtain a complete inactivation curve. Both the modified logistic and Weibull models provided a highly accurate fitting of the static pH conditions for every pathogen. However, while the residuals plots of the modified logistic model indicated no systematic bias and/or regional prediction problems, the residuals plots of the Weibull model showed a systematic bias. The modified logistic model appropriately predicted the pathogen behavior in the simulated gastric digestion process with actual food, including cut lettuce, minced tuna, hamburger, and scrambled egg. Although the developed model enabled us to predict pathogen inactivation during gastric digestion, its results also suggested that the ingested bacteria in the stomach would barely be inactivated in the real digestion process. The results of this study will provide important information on a part of the dose-response mechanism of bacterial pathogens.     

Spatial Control of Proton Pump H,K-ATPase Docking at the Apical Membrane by Phosphorylation-coupled Ezrin-Syntaxin 3 Interaction

The digestive function of the stomach depends on acidification of the gastric lumen. Acid secretion into the lumen is triggered by activation of a cAMP-dependent protein kinase (PKA) cascade, which ultimately results in the insertion of gastric H,K-ATPases into the apical plasma membranes of parietal cells. A coupling protein is ezrin whose phosphorylation at Ser-66 by PKA is required for parietal cell activation. However, little is known regarding the molecular mechanism(s) by which ezrin operates in gastric acid secretion. Here we show that phosphorylation of Ser-66 induces a conformational change of ezrin that enables its association with syntaxin 3 (Stx3) and provides a spatial cue for H,K-ATPase trafficking. This conformation-dependent association is specific for Stx3, and the binding interface is mapped to the N-terminal region. Biochemical analyses show that inhibition of ezrin phosphorylation at Ser-66 prevents ezrin-Stx3 association and insertion of H,K-ATPase into the apical plasma membrane of parietal cells. Using atomic force microscopic analyses, our study revealed that phosphorylation of Ser-66 induces unfolding of ezrin molecule to allow Stx3 binding to its N terminus. Given the essential role of Stx3 in polarized secretion, our study presents the first evidence in which phosphorylation-induced conformational rearrangement of the ezrin molecule provides a spatial cue for polarized membrane trafficking in epithelial cells.     

Visual analysis of concerted cleavage by type IIF restriction enzyme SfiI in subsecond time region

Many DNA regulatory factors require communication between distantly separated DNA sites for their activity. The type IIF restriction enzyme SfiI is often used as a model system of site communication. Here, we used fast-scanning atomic force microscopy to monitor the DNA cleavage process with SfiI and the changes in the single SfiI-DNA complex in the presence of either Mg²⁺ or Ca²⁺ at a scan rate of 1–2 fps. The increased time resolution allowed us to visualize the concerted cleavage of the protein at two cognate sites. The four termini generated by the cleavage were released in a multistep manner. The high temporal resolution enabled us to visualize the translocation of a DNA strand on a looped complex and intersegmental transfer of the SfiI protein in which swapping of the site is performed without protein dissociation. On the basis of our results, we propose that the SfiI tetramer can remain bound to one of the sites even after cleavage, allowing the other site on the DNA molecule to fill the empty DNA-binding cleft by combining a one-dimensional diffusion-mediated sliding and a segment transfer mechanism.     

Site-specific attachment of a protein to a carbon nanotube end without loss of protein function

Establishing a nanobiohybrid device largely relies on the availability of various bioconjugation procedures which allow coupling of biomolecules and inorganic materials. Especially, site-specific coupling of a protein to nanomaterials is highly useful and significant, since it can avoid adversely affecting the protein's function. In this study, we demonstrated a covalent coupling of a protein of interest to the end of carbon nanotubes without affecting protein's function. A modified Staudinger-Bertozzi ligation was utilized to couple a carbon nanotube end with an azide group which is site-specifically incorporated into a protein of interest. We demonstrated that Ca(2+)-sensor protein, calmodulin, can be attached to the end of the nanotubes without affecting the ability to bind to the substrate in a calcium-dependent manner. This procedure can be applied not only to nanotubes, but also to other nanomaterials, and therefore provides a fundamental technique for well-controlled protein conjugation.     

Ageing,the Urban-Rural Gap and Disability Trends: 19 Years of Experience in China - 1987 to 2006

Background

As the age of a population increases, so too does the rate of disability. In addition, disability is likely to be more common in rural compared with urban areas. The present study aimed to examine the influence of rapid population changes in terms of age and rural/urban residence on the prevalence of disability.

Methods

Data from the 1987 and 2006 China Sampling Surveys on Disability were used to estimate the impacts of rapid ageing and the widening urban-rural gap on the prevalence of disability. Stratum specific rates of disability were estimated by 5-year age-group and type of residence. The decomposition of rates method was used to calculate the rate difference for each stratum between the two surveys.

Results

The crude disability rate increased from 4.89% in 1987 to 6.39% in 2006, a 1.5% increase over the 19 year period. However, after the compositional effects from the overall rates of changing age-structure in 1987 and 2006 were eliminated by standardization, the disability rate in 1987 was 6.13%, which is higher than that in 2006 (5.91%). While in 1987 the excess due to rural residence compared with urban was <1.0%, this difference increased to >1.5% by 2006, suggesting a widening disparity by type of residence. When rates were decomposed, the bulk of the disability could be attributed to ageing, and very little to rural residence. However, a wider gap in prevalence between rural and urban areas could be observed in some age groups by 2006.

Conclusion

The increasing number of elderly disabled persons in China and the widening discrepancy of disability prevalence between urban and rural areas may indicate that the most important priorities for disability prevention in China are to reinforce health promotion in older adults and improve health services in rural communities.     

Condensin architecture and interaction with DNA: regulatory non-SMC subunits bind to the head of SMC heterodimer

Condensin and cohesin are two protein complexes that act as the central mediators of chromosome condensation and sister chromatid cohesion, respectively. The basic underlying mechanism of action of these complexes remained enigmatic. Direct visualization of condensin and cohesin was expected to provide hints to their mechanisms. They are composed of heterodimers of distinct structural maintenance of chromosome (SMC) proteins and other non-SMC subunits. Here, we report the first observation of the architecture of condensin and its interaction with DNA by atomic force microscopy (AFM). The purified condensin SMC heterodimer shows a head-tail structure with a single head composed of globular domains and a tail with the coiled-coil region. Unexpectedly, the condensin non-SMC trimers associate with the head of SMC heterodimers, producing a larger head with the tail. The heteropentamer is bound to DNA in a distributive fashion, whereas condensin SMC heterodimers interact with DNA as aggregates within a large DNA-protein assembly. Thus, non-SMC trimers may regulate the ATPase activity of condensin by directly interacting with the globular domains of SMC heterodimer and alter the mode of DNA interaction. A model for the action of heteropentamer is presented.     

Fast-scanning atomic force microscopy reveals the ATP/ADP-dependent conformational changes of GroEL

In order to fold non-native proteins, chaperonin GroEL undergoes numerous conformational changes and GroES binding in the ATP-dependent reaction cycle. We constructed the real-time three-dimensional-observation system at high resolution using a newly developed fast-scanning atomic force microscope. Using this system, we visualized the GroES binding to and dissociation from individual GroEL with a lifetime of 6 s (k=0.17 s(-1)). We also caught ATP/ADP-induced open-closed conformational changes of individual GroEL in the absence of qGroES and substrate proteins. Namely, the ATP/ADP-bound GroEL can change its conformation 'from closed to open' without additional ATP hydrolysis. Furthermore, the lifetime of open conformation in the presence of ADP ( approximately 1.0 s) was apparently lower than those of ATP and ATP-analogs (2-3 s), meaning that ADP-bound open-form is structurally less stable than ATP-bound open-form. These results indicate that GroEL has at least two distinct open-conformations in the presence of nucleotide; ATP-bound prehydrolysis open-form and ADP-bound open-form, and the ATP hydrolysis in open-form destabilizes its open-conformation and induces the 'from open to closed' conformational change of GroEL.