Rule-based modeling and simulations of the inner kinetochore structure

Background

Combinatorial complexity is a central problem when modeling biochemical reaction networks, since the association of a few components can give rise to a large variation of protein complexes. Available classical modeling approaches are often insufficient for the analysis of very large and complex networks in detail. Recently, we developed a new rule-based modeling approach that facilitates the analysis of spatial and combinatorially complex problems. Here, we explore for the first time how this approach can be applied to a specific biological system, the human kinetochore, which is a multi-protein complex involving over 100 proteins.

Results

Applying our freely available SRSim software to a large data set on kinetochore proteins in human cells, we construct a spatial rule-based simulation model of the human inner kinetochore. The model generates an estimation of the probability distribution of the inner kinetochore 3D architecture and we show how to analyze this distribution using information theory. In our model, the formation of a bridge between CenpA and an H3 containing nucleosome only occurs efficiently for higher protein concentration realized during S-phase but may be not in G1. Above a certain nucleosome distance the protein bridge barely formed pointing towards the importance of chromatin structure for kinetochore complex formation. We define a metric for the distance between structures that allow us to identify structural clusters. Using this modeling technique, we explore different hypothetical chromatin layouts.

Conclusions

Applying a rule-based network analysis to the spatial kinetochore complex geometry allowed us to integrate experimental data on kinetochore proteins, suggesting a 3D model of the human inner kinetochore architecture that is governed by a combinatorial algebraic reaction network. This reaction network can serve as bridge between multiple scales of modeling. Our approach can be applied to other systems beyond kinetochores.     

Apoplastic and cytosolic expression of full‐size antibodies and antibody fragments in Nicotiana tabacum

We compared the expression of a functional recombinant TMVspecific fullsize antibody (rAb29) in both the apoplast and cytosol of tobacco plants and a single chain antibody fragment (scFv29), derived from rAb29, was expressed in the cytosol. Cloned heavy and light chain cDNAs of fullsize rAb29, which binds to TMV coat protein monomers, were integrated into the plant expression vector pSS. The fullsize rAb29 was expressed in the cytosol and targeted to the apoplast by including the original murine antibody leader sequences. Levels of functional fullsize rAb29 expression were high in the apoplast (up to 8.5g per gram leaf tissue), whereas cytosolic expression was low or at the ELISA detection limit. Sequences of the variable domains of rAb29 light and heavy chain were used to generate the single chain antibody scFv29, which was expressed in the periplasmic space of E.coli and showed the same binding specificity as fullsize rAb29. In addition, scFv29 was functionally expressed in the cytosol of tobacco plants and plant derived scFv29 maintained same binding specificity to TMVcoat protein monomers as rAb29.     

Acute metal stress in Populus tremula×P. alba (717‐1B4 genotype): Leaf and cambial proteome changes induced by cadmium2+

The comprehension of metal homeostasis in plants requires the identification of molecular markers linked to stress tolerance. Proteomic changes in leaves and cambial zone of Populus tremula×P. alba (717‐1B4 genotype) were analyzed after 61 days of exposure to cadmium (Cd) 360 mg/kg soil dry weight in pot‐soil cultures. The treatment led to an acute Cd stress with a reduction of growth and photosynthesis. Cd stress induced changes in the display of 120 spots for leaf tissue and 153 spots for the cambial zone. It involved a reduced photosynthesis, resulting in a profound reorganisation of carbon and carbohydrate metabolisms in both tissues. Cambial cells underwent stress from the Cd actually present inside the tissue but also a deprivation of photosynthates caused by leaf stress. An important tissue specificity of the response was observed, according to the differences in cell structures and functions.     

Surface hydrolysis of sphingomyelin by the outer membrane protein Rv0888 supports replication of Mycobacterium tuberculosis in macrophages

Sphingomyelinases secreted by pathogenic bacteria play important roles in host–pathogen interactions ranging from interfering with phagocytosis and oxidative burst to iron acquisition. This study shows that the Mtb protein Rv0888 possesses potent sphingomyelinase activity cleaving sphingomyelin, a major lipid in eukaryotic cells, into ceramide and phosphocholine, which are then utilized by Mtb as carbon, nitrogen and phosphorus sources, respectively. An Mtb rv0888 deletion mutant did not grow on sphingomyelin as a sole carbon source anymore and replicated poorly in macrophages indicating that Mtb utilizes sphingomyelin during infection. Rv0888 is an unusual membrane protein with a surface‐exposed C‐terminal sphingomyelinase domain and a putative N‐terminal channel domain that mediated glucose and phosphocholine uptake across the outer membrane in an M. smegmatis porin mutant. Hence, we propose to name Rv0888 as SpmT (sp hingomyelinase of M ycobacterium t uberculosis). Erythrocyte membranes contain up to 27% sphingomyelin. The finding that Rv0888 accounts for half of Mtb's hemolytic activity is consistent with its sphingomyelinase activity and the observation that Rv0888 levels are increased in the presence of erythrocytes and sphingomyelin by 5‐ and 100‐fold, respectively. Thus, Rv0888 is a novel outer membrane protein that enables Mtb to utilize sphingomyelin as a source of several essential nutrients during intracellular growth.     

Possible functions of extracellular peroxidases in stress-induced generation and detoxification of active oxygen species

Extracellular peroxidases are classified as free, or ionically or covalently bound to the cell wall. In addition, peroxidase-like activities have often been demonstrated at the outer surface of protoplasts and plasma membrane preparations. Under certain conditions apoplastic peroxidases have been shown to contribute to the formation of superoxide and hydrogen peroxide during the `oxidative burst' through the oxidation of a reductant. However, the identity of this reductant remains unclear. It has been suggested that the production of these active oxygen species may play important roles in plant responses to biotic and abiotic stress. Extracellular release of pre-existing and de novo synthesis of apoplastic peroxidases is regulated by changing environmental conditions. While the oxidative burst could potentially be harmful to a plant's own cells, tissues can rapidly metabolize even high concentrations of hydrogen peroxide. Recent work has shown that when extracellular hydrogen peroxide exceeds the supplies of reductants, class II and class III peroxidases can display catalase-like activity. Under these conditions, hydrogen peroxide is able to act as both oxidizing and reducing substrate. It seems likely therefore, that a further role of extracellular peroxidases is to protect plants from the consequences of the oxidative burst that they themselves are responsible for producing.     

Solid-state NMR [13C,15N] resonance assignments of the nucleotide-binding domain of a bacterial cyclic nucleotide-gated channel

Channels regulated by cyclic nucleotides are key signalling proteins in several biological pathways. The regulatory aspect is conferred by a C-terminal cyclic nucleotide-binding domain (CNBD). We report resonance assignments of the CNBD of a bacterial mlCNG channel obtained using 2D and 3D solid-state NMR under Magic-angle Spinning conditions. A secondary chemical shift analysis of the 141 residue protein suggests a three-dimensional fold seen in earlier X-ray and solution-state NMR work and points to spectroscopic polymorphism for a selected set of resonances.     

Elongated mouse chromosomes suitable for enhanced molecular cytogenetics

Characterization of genetic disorders in humans and animal models requires identification of chromosomal aberrations. However, identifying fine deletions or insertion in metaphase chromosomes has been always a challenge due to limitations of resolution. In this study we developed a rapid method for chromosome elongation using two different intercalating agents: ethidium bromide and 5-bromo-2′-deoxyuridine (BrdU), together with a short-term mitotic block using colcemid. About 70% of the chromosomes from cells that underwent this elongation procedure reached three times longer than those prepared from control cells. FISH experiments using elongated chromosomes revealed a duplicated region of chromosome 11 that was not visible in cells prepared with conventional methods.