Conjugation to Nedd8 instigates ubiquitylation and down-regulation of activated receptor tyrosine kinases

When appended to the epidermal growth factor receptor (EGFR), ubiquitin serves as a sorting signal for lysosomal degradation. Here we demonstrate that the ubiquitin ligase of EGFR, namely c-Cbl, also mediates receptor modification with the ubiquitin-like molecule Nedd8. EGF stimulates receptor neddylation, which enhances subsequent ubiquitylation, as well as sorting of EGFR for degradation. Multiple lysine residues, located within the tyrosine kinase domain of EGFR, serve as attachment sites for Nedd8. A set of clathrin coat-associated binders of ubiquitin also bind Nedd8, but they undergo ubiquitylation, not neddylation. We discuss the emerging versatility of the concerted action of ubiquitylation and neddylation in the process that desensitizes growth factor-activated receptor tyrosine kinases.     

Geldanamycins trigger a novel Ron degradative pathway, hampering oncogenic signaling

Ron, the tyrosine kinase receptor for macrophage-stimulating protein is responsible for proliferation and migration of cells from different tissues. Ron can acquire oncogenic potential by single point mutations in the kinase domain, and dysregulated Ron signaling has been involved in the development of different human cancers. We have previously shown that ligand-activated Ron recruits the negative regulator c-Cbl, which mediates its ubiquitylation and degradation. Here we report that Ron is ubiquitylated also by the U-box E3 ligase C-terminal Hsc70-interacting protein (CHIP), recruited via chaperone intermediates Hsp90 and Hsc70. Gene silencing shows that CHIP activity is necessary to mediate Ron degradation upon cell treatment with Hsp90 inhibitors geldanamycins. The oncogenic Ron(M1254T) receptor escapes from c-Cbl negative regulation but retains a strong association with CHIP. This constitutively active mutant of Ron displays increased sensitivity to geldanamycins, enhanced physical interaction with Hsp90, and more rapid degradation rate. Cell growth and migration, as well as the transforming potential evoked by Ron(M1254T), are abrogated upon Hsp90 inhibition. These data highlight a novel mechanism for Ron degradation and propose Hsp90 antagonists like geldanamycins as suitable pharmacological agents for therapy of cancers where altered Ron signaling is involved.     

EGF-ERBB signalling: towards the systems level

Signalling through the ERBB/HER receptors is intricately involved in human cancer and already serves as a target for several cancer drugs. Because of its inherent complexity, it is useful to envision ERBB signalling as a bow-tie-configured, evolvable network, which shares modularity, redundancy and control circuits with robust biological and engineered systems. Because network fragility is an inevitable trade-off of robustness, systems-level understanding is expected to generate therapeutic opportunities to intercept aberrant network activation.     

Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli

The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas) constitute a recently identified prokaryotic defense mechanism against invading nucleic acids. Activity of the CRISPR/Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called 'adaptation', (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the alien nucleic acid. Here we describe a robust assay in Escherichia coli to explore the hitherto least-studied process, adaptation. We identify essential genes and DNA elements in the leader sequence and in the array which are essential for the adaptation step. We also provide mechanistic insights on the insertion of the repeat-spacer unit by showing that the first repeat serves as the template for the newly inserted repeat. Taken together, our results elucidate fundamental steps in the adaptation process of the CRISPR/Cas system.     

Protein hot spots: the islands of stability

Understanding the structural basis of protein-protein interactions (PPIs) may shed light on the organization and functioning of signal transduction and metabolic networks and may assist in structure-based design of ligands (drugs) targeting protein-protein interfaces. The residues at the bimolecular interface, designated as the hot spots, contribute most of the free binding energy of PPI. To date, there is no conclusive atomistic explanation for the unique functional properties of the hot spots. We hypothesized that backbone compliance may play a role in protein-protein recognition and in the mechanism of binding of small-molecule compounds to protein surfaces. We used a steered molecular dynamics simulation to explore the compliance properties of the backbone of surface-exposed residues in several model proteins: interleukin-2, mouse double minute protein 2 and proliferating cell nuclear antigen. We demonstrated that protein surfaces exhibit distinct patterns in which highly immobile residues form defined clusters ("stability patches") alternating with areas of moderate to high mobility. These "stability patches" tend to localize in functionally important regions involved in protein-protein recognition. We propose a mechanism by which the distinct structural organization of the hot spots may contribute to their role in mediating PPI and facilitating binding of structurally diverse small-molecule compounds to protein surfaces.     

Microbial Functional Diversity Associated with Plant Litter Decomposition Along a Climatic Gradient

Predicted changes in climate associated with increased greenhouse gas emissions can cause increases in global mean temperature and changes in precipitation regimes. These changes may affect key soil processes, e.g., microbial CO(2) evolution and biomass, mineralization rates, primary productivity, biodiversity, and litter decomposition, which play an important role in carbon and nutrient cycling in terrestrial ecosystems. Our study examined the changes in litter microbial communities and decomposition along a climatic gradient, ranging from arid desert to humid Mediterranean regions in Israel. Wheat straw litter bags were placed in arid, semi-arid, Mediterranean, and humid Mediterranean sites. Samples were collected seasonally over a 2-year period in order to evaluate mass loss, litter moisture, C/N ratio, bacterial colony-forming units (CFUs), microbial CO(2) evolution and biomass, microbial functional diversity, and catabolic profile. Decomposition rate was the highest during the first year of the study at the Mediterranean and arid sites. Community-level physiological profile and microbial biomass were the highest in summer, while bacterial CFUs were the highest in winter. Microbial functional diversity was found to be highest at the humid Mediterranean site, whereas substrate utilization increased at the arid site. Our results support the assumption that climatic factors control litter degradation and regulate microbial activity.     

Effects of forest wildfire on soil microbial-community activity and chemical components on a temporal-seasonal scale

Background

Soil response and rehabilitation after wildfires are affected by natural environmental factors such as seasonality, and other time-dependent changes, such as vegetation recovery (e.g., % soil cover). These changes affect soil microbial-community activity. During summer 2006, almost 1,200 hectares (ha) of coniferous forest in northern Israel, including Byria Forest, burned.

Methods

Soil samples were collected seasonally from severely burned and unburned areas, on a time scale of 7?days to 4?years after wildfire. Chemical and microbial parameters of the forest soil system were examined.

Results

Results obtained show that increase in total soluble nitrogen (TSN) in burned areas may limit microbial activity during the first year after wildfire. Two years after wildfire, soil TSN levels in burned areas decreased to unburned levels after plant growth, allowing the microbial community to proliferate.

Conclusions

Wildfire had a significant impact on TSN, soil moisture (SM), and microbial nitrogen (MBN) compared to seasonality. These parameters are recommended for monitoring post-fire soil state. The direct effect of wildfire on soil constituents at the study site was stronger during the first 2–4?years. Indirect changes due to vegetation cover could have a longer effect on burned soil systems and should be further examined.     

Tree-hole breeding mosquitoes in Israel

A survey was conducted to evaluate the number of tree-hole breeding mosquito species and their distribution in the six principal woodland types in Israel. Out of approximately 3,000 mature trees examined, only 38 contained holes that retained water for extended periods of time, and breeding mosquitoes were observed in 27 of them. Two specialized tree-hole breeders, Aedes pulchritarsis Rondani and Aedes geniculatus Oliver, were found breeding at several sites in northern Israel, always at locations 500 m above sea level (a.s.l) and with high annual precipitation. Aedes albopictus Skuse which, in Israel, is known as an opportunistic container breeder, was found in this study to have adapted remarkably well to breeding in tree holes and was found in most forest types investigated and in most tree species which had adequate tree holes. Two other species, Culiseta annulata Schrank and Culex pipiens Linnaeus instars, were found in one of the tree holes, but did not survive to reach maturity.     

iNKT Cells Suppress the CD8(+) T Cell Response to a Murine Burkitt's-Like B Cell Lymphoma

The T cell response to B cell lymphomas differs from the majority of solid tumors in that the malignant cells themselves are derived from B lymphocytes, key players in immune response. B cell lymphomas are therefore well situated to manipulate their surrounding microenvironment to enhance tumor growth and minimize anti-tumor T cell responses. We analyzed the effect of T cells on the growth of a transplantable B cell lymphoma and found that iNKT cells suppressed the anti-tumor CD8(+) T cell response. Lymphoma cells transplanted into syngeneic wild type (WT) mice or Jalpha18(-/-) mice that specifically lack iNKT cells grew initially at the same rate, but only the mice lacking iNKT cells were able to reject the lymphoma. This effect was due to the enhanced activity of tumor-specific CD8(+) T cells in the absence of iNKT cells, and could be partially reversed by reconstitution of iNKT cells in Jalpha 18(-/-) mice. Treatment of tumor-bearing WT mice with alpha -galactosyl ceramide, an activating ligand for iNKT cells, reduced the number of tumor-specific CD8(+) T cells. In contrast, lymphoma growth in CD1d1(-/-) mice that lack both iNKT and type II NKT cells was similar to that in WT mice, suggesting that type II NKT cells are required for full activation of the anti-tumor immune response. This study reveals a tumor-promoting role for iNKT cells and suggests their capacity to inhibit the CD8(+) T cell response to B cell lymphoma by opposing the effects of type II NKT cells.