The dynamic nature of netrin-1 and the structural basis for glycosaminoglycan fragment-induced filament formation.

Netrin-1 is a bifunctional chemotropic guidance cue that plays key roles in diverse cellular processes including axon pathfinding, cell migration, adhesion, differentiation, and survival. Here, we present a molecular understanding of netrin-1 mediated interactions with glycosaminoglycan chains of diverse heparan sulfate proteoglycans (HSPGs) and short heparin oligosaccharides. Whereas interactions with HSPGs act as platform to co-localise netrin-1 close to the cell surface, heparin oligosaccharides have a significant impact on the highly dynamic behaviour of netrin-1. Remarkably, the monomer-dimer equilibrium of netrin-1 in solution is abolished in the presence of heparin oligosaccharides and replaced with highly hierarchical and distinct super assemblies leading to unique, yet unknown netrin-1 filament formation. In our integrated approach we provide a molecular mechanism for the filament assembly which opens fresh paths towards a molecular understanding of netrin-1 functions.

Solution structure of the cytoplasmic domain of NhaP2 a K+/H+ antiporter from Vibrio cholera.

NhaP2 is a K+/H+ antiporter from Vibrio cholerae which consists of a transmembrane domain and a cytoplasmic domain of approximately 200 amino acids, both of which are required for cholera infectivity. Here we present the solution structure for a 165 amino acid minimal cytoplasmic domain (P2MIN) form of the protein. The structure reveals a compact N-terminal domain which resembles a Regulator of Conductance of K+ channels (RCK) domain connected to a more open C-terminal domain via a flexible 20 amino acid linker. NMR titration experiments showed that the protein binds ATP through its N-terminal domain, which was further supported by waterLOGSY and Saturation Transfer Difference NMR experiments. The two-domain organisation of the protein was confirmed by BIOSAXS, which also revealed that there are no detectable-ATP-induced conformational changes in the protein structure. Finally, in contrast to all known RCK domain structures solved to date, the current work shows that the protein is a monomer.

Nanoscale Assembly of High-Mobility Group AT-Hook 2 Protein with DNA Replication Fork.

High mobility group AT-hook 2 (HMGA2) protein is composed of three AT-hook domains. HMGA2 expresses at high levels in both embryonic stem cells and cancer cells, where it interacts with and stabilizes replication forks (RFs), resulting in elevated cell proliferation rates. In this study, we demonstrated that HMGA2 knockdown reduces cell proliferation. To understand the features required for interaction between HMGA2 and RFs, we studied the solution structure of HMGA2, free and in complex with RFs, using an integrated host of biophysical techniques. Circular dichroism and NMR experiments confirmed the disordered state of unbound HMGA2. Dynamic light scattering and sedimentation velocity experiments demonstrated that HMGA2 and RF are monodisperse in solution, and form an equimolar complex. Small-angle x-ray scattering studies revealed that HMGA2 binds in a side-by-side orientation to RF where 3 AT-hooks act as a clamp to wrap around a distorted RF. Thus, our data provide insights into how HMGA2 interacts with stalled RFs and the function of the process.

A review on sludge dewatering indices.

Dewatering of sludge from sewage treatment plants is proving to be a significant challenge due to the large amounts of residual sludges generated annually. In recent years, research and development have focused on improving the dewatering process in order to reduce subsequent costs of sludge management and transport. To achieve this goal, it is necessary to establish reliable indices that reflect the efficiency of sludge dewatering. However, the evaluation of sludge dewaterability is not an easy task due to the highly complex nature of sewage sludge and variations in solid-liquid separation methods. Most traditional dewatering indices fail to predict the maximum cake solids content achievable during full-scale dewatering. This paper reviews the difficulties in assessing sludge dewatering performance, and the main techniques used to evaluate dewatering performance are compared and discussed in detail. Finally, the paper suggests a new dewatering index, namely the modified centrifugal index, which is demonstrated to be an appropriate indicator for estimating the final cake solids content as well as simulating the prototype dewatering process.

The Dynamic Landscape of the Full-Length HIV-1 Transactivator of Transcription.

The type 1 human immunodeficiency virus (HIV-1) transactivator of transcription (Tat) is a small RNA-binding protein essential for viral gene expression and replication. It has also been shown to bind to a large number of human proteins and to modulate many different cellular activities. We have used nuclear magnetic resonance (NMR) spectroscopy and hydrogen exchange chemistry to measure backbone dynamics over the millisecond to picosecond time scales. Sequential backbone assignment was facilitated by several isotope labeling schemes, including uniform labeling, site-specific labeling, and unlabeling. (15)N NMR relaxation parameters were measured and analyzed by reduced spectral density mapping and the Lipari-Szabo Model-Free approach to characterize the backbone dynamics on the picosecond to nanosecond time scale. The results indicate that the protein exists in an extended disordered conformational ensemble. NMR relaxation dispersion profiles show that on the millisecond time scale no conformational exchange is detected for any of the residues, supporting the model of a disordered backbone. NMR chemical shift differences from random coil values suggest that some segments of the protein have a modest propensity to fold; comparison to X-ray diffraction structures of Tat complexes indicates that some segments of the protein function through an induced-fit mechanism whereas other segments likely operate by conformational selection. Surprisingly, measured hydrogen exchange rates are higher than predicted for a disordered polymer, but this is explained as being caused by the high net charge on the protein that enhances base-catalyzed hydrogen exchange. The dynamics results provide a deeper understanding of the protein conformational ensemble and form a foundation for future studies of the conformational changes that accompany the formation of the superelongation complex that activates viral transcription.

Association between nitrotyrosine levels and microvascular density in human breast cancer.

Nitrotyrosine (NO2Y) is a global marker of protein modification by reactive nitrogen species such as peroxynitrite derived from nitric oxide (NO). Because NO and its derivatives are postulated to enhance carcinogenesis, we used stable isotope dilution mass spectrometry to measure the levels of NO2Y in 30 samples of human breast cancer of varying pathologic types. In the samples tested, the NO2Y levels were generally low (average of 14.1 +/- 9.2 micromol NO2Y per mole of tyrosine). Breast cancers with a high microvascular density, however, had a significantly higher average level of NO2Y than tumors with a low microvascular density (20 v.s. 10 micromol NO2Y per mole of tyrosine, p = 0.007 by two-tailed t-test, assuming unequal variances of two samples). There was no apparent association between NO2Y levels and the differentiation of the tumors, tumor aneuploidy, estrogen receptor status, HER-2 expression, lymph node status, or infiltration of the tumors by neutrophils or eosinophils. When the tissues were stained by immunohistochemistry for NO2Y, the NO2Y was localized predominantly within inflammatory cells located immediately adjacent to blood vessels at the edges of the tumors. NO2Y was generally not evident within the tumor cells or inflammatory cells in the stroma. We conclude that low levels of reactive nitrogen species are located predominantly within inflammatory cells near blood vessels of breast cancer and that higher NO2Y levels are associated with an increased density of blood vessels. Our findings, therefore, support a possible association between inflammatory cells and reactive nitrogen species in modulating microvascular density at the edges of breast cancer.