A model of restriction endonuclease MvaI in complex with DNA: a template for interpretation of experimental data and a guide for specificity engineering

R.MvaI is a Type II restriction enzyme (REase), which specifically recognizes the pentanucleotide DNA sequence 5'-CCWGG-3' (W indicates A or T). It belongs to a family of enzymes, which recognize related sequences, including 5'-CCSGG-3' (S indicates G or C) in the case of R.BcnI, or 5'-CCNGG-3' (where N indicates any nucleoside) in the case of R.ScrFI. REases from this family hydrolyze the phosphodiester bond in the DNA between the 2nd and 3rd base in both strands, thereby generating a double strand break with 5'-protruding single nucleotides. So far, no crystal structures of REases with similar cleavage patterns have been solved. Characterization of sequence-structure-function relationships in this family would facilitate understanding of evolution of sequence specificity among REases and could aid in engineering of enzymes with new specificities. However, sequences of R.MvaI or its homologs show no significant similarity to any proteins with known structures, thus precluding straightforward comparative modeling. We used a fold recognition approach to identify a remote relationship between R.MvaI and the structure of DNA repair enzyme MutH, which belongs to the PD-(D/E)XK superfamily together with many other REases. We constructed a homology model of R.MvaI and used it to predict functionally important amino acid residues and the mode of interaction with the DNA. In particular, we predict that only one active site of R.MvaI interacts with the DNA target at a time, and the cleavage of both strands (5'-CCAGG-3' and 5'-CCTGG-3') is achieved by two independent catalytic events. The model is in good agreement with the available experimental data and will serve as a template for further analyses of R.MvaI, R.BcnI, R.ScrFI and other related enzymes.     

The Balbiani body in the oocytes of a common cellar spider, Pholcus phalangioides (Araneae: Pholcidae)

Previtellogenic oocytes of a common cellar spider, Pholcus phalangioides, contain a single aggregation of organelles referred here to as the Balbiani body. It is a well defined ooplasmic structure predominantly composed of fine granular nuage, RNA rich material but comprising also mitochondria, vesicles of endoplasmic reticulum and stacks of Golgi cysternae. The Balbiani body originates early during previtellogenesis in the form of a cap-shaped mass in juxtaposition to one pole of the oocyte nucleus. During later stages of previtellogenic growth the Balbiani body translocates as a single body towards the ooplasm periphery. The results presented indicate that Balbiani body translocation is cytoskeleton independent. Balbiani body repositioning does not result in the localization of its components to any distinct, asymmetrically situated region of the ooplasm but, instead, ends up with their even dispersion in the oocyte cortex. The Balbiani body in Pholcus does not seem to be implicated either in germ cell determination or organelle inheritance. Its homology with similar organelle accumulations in the oocytes of other species is discussed.     

Assignment of the absolute configuration of P-chiral 5' mRNA cap analogues containing phosphorothioate moiety

Enzymatic cleavage of the P-chiral diastereoisomers of the 5' mRNA cap analogue bearing phosphorothioate moiety in alfa position of 5',5'-triphosphate bridge (m(7)Gppp(S)G D1 and D2) was performed by human Decapping Scavenger (DcpS) enzyme. Analysis of the degradation products allowed to estimate the absolute configuration at the asymmetric phosphorus atoms in examined compounds via correlation with the R(P) and S(P) diastereoisomers of guanosine 5'-O-(1-thiodiphosphate) (GDPalphaS).     

Affinity of dinucleotide cap analogues for human decapping scavenger (hDcpS)

Eukaryotic cells utilize scavenger decapping enzymes to degrade cap structure following 3'-5' mRNA decay. Human DcpS recently has been described as a highly specific hydrolase (a member of the HIT family) that catalyses the cleavage of m(7)GpppG and short capped oligoribonucleotides. We have demonstrated here that cap-1 (m(7)GpppGm) is a preferred substrate among several investigated dinucleotide cap analogues m(7)Gp(n)N (n = 3-5, N is a purine or pyrimidine base) and m(7)GMP is always one of the reaction product. Cap analogues containing pyrimidine base instead of guanine or diphosphate chain are resistant to hydrolysis catalyzed by human scavenger. Contrary to the other enzymes of HIT family, hDcpS activity is not stimulated by Mg(2+).     

Laccaria bicolor aquaporin LbAQP1 is required for Hartig net development in trembling aspen (Populus tremuloides)

The development of ectomycorrhizal associations is crucial for growth of many forest trees. However, the signals that are exchanged between the fungus and the host plant during the colonization process are still poorly understood. In this study, we have identified the relationship between expression patterns of Laccaria bicolor aquaporin LbAQP1 and the development of ectomycorrhizal structures in trembling aspen (Populus tremuloides) seedlings. The peak expression of LbAQP1 was 700‐fold higher in the hyphae within the root than in the free‐living mycelium after 24 h of direct interaction with the roots. Moreover, in LbAQP1 knock‐down strains, a non‐mycorrhizal phenotype was developed without the Hartig net and the expression of the mycorrhizal effector protein MiSSP7 quickly declined after an initial peak on day 5 of interaction of the fungal hyphae with the roots. The increase in the expression of LbAQP1 required a direct contact of the fungus with the root and it modulated the expression of MiSSP7. We have also determined that LbAQP1 facilitated NO, H₂O₂ and CO₂ transport when heterologously expressed in yeast. The report demonstrates that the L. bicolor aquaporin LbAQP1 acts as a molecular signalling channel, which is fundamental for the development of Hartig net in root tips of P. tremuloides.     

Characterization of N-cadherin unbinding properties in non-malignant (HCV29) and malignant (T24) bladder cells

The expression of N‐cadherin, characteristic of various cancers, very often leads to changes in the cells' adhesive properties. Thus, we sought to find out if N‐cadherin expressed in various, but cancer‐related cells, differs in its functional properties that could contribute to variations in cells' phenotypes. In our work, measurements of an unbinding force of a single N‐cadherin molecule, probed with the same antibody both on a surface of living non‐malignant (HCV29) and malignant cells (T24) of bladder cancer, were carried out with the use of an atomic force microscopy. The results show the enhanced N‐cadherin level in T24 malignant cells (8.7% vs. 3.6% obtained for non‐malignant one), confirmed by the Western blot and the immunohistochemical staining. The effect was accompanied by changes in unbinding properties of an individual N‐cadherin molecule. Lower unbinding force values (26.1 ± 7.1 pN) in non‐malignant cells reveal less stable N‐cadherin complexes, as compared to malignant cells (61.7 ± 14.6 pN). This suggests the cancer‐related changes in a structure of the binding site of the antibody, located at the extracellular domain of N‐cadherin. Copyright © 2011 John Wiley & Sons, Ltd.     

Diurnal and seasonal changes of leaf lamina hydraulic conductance in bur oak (Quercus macrocarpa) and trembling aspen (Populus tremuloides)

The aim of this study was to examine the diurnal and seasonal variations in the sensitivity of leaf lamina (K _lam) hydraulic conductance to irradiance in bur oak (Quercus macrocarpa Michx.) and trembling aspen (Populus tremuloides Michx.), which vary in their responses of K _lam to irradiance. K _lam was determined using the high-pressure method and the measurements were carried out in June, August and September. The irradiance response of K _lam in bur oak was present throughout the day and declined in senescing leaves. In trembling aspen, K _lam declined from morning to late afternoon and drastically decreased before the onset of leaf senescence, but it was not sensitive to irradiance. In both tree species, the capacity of the petioles to supply water to leaf lamina changed during the day in accordance with the ability of the leaf lamina to transport water. Petiole hydraulic conductivity (K _pet) declined during the season in bur oak leaves, while it tended to increase in trembling aspen leaves. There was no correlation between the K _lam values and air temperature or light intensity at the time of leaf collection. For trembling aspen, K _pet was negatively correlated with the air temperature suggesting sensitivity to drought. We conclude that the water transport properties of petioles and leaf lamina in the two studied tree species reflect their ecological adaptations. Trembling aspen leaves have high hydraulic conductivity and high stomatal conductance regardless of the irradiance level, consistent with the rapid growth and high demand for water. In contrast, the increased lamina hydraulic conductivity and stomatal conductance under high irradiance in bur oak trees reflect a water conservation strategy.     

CENP-C is a structural platform for kinetochore assembly

Centromeres provide a region of chromatin upon which kinetochores are assembled in mitosis. Centromeric protein C (CENP-C) is a core component of this centromeric chromatin that, when depleted, prevents the proper formation of both centromeres and kinetochores. CENP-C localizes to centromeres throughout the cell cycle via its C-terminal part, whereas its N-terminal part appears necessary for recruitment of some but not all components of the Mis12 complex of the kinetochore. We now find that all kinetochore proteins belonging to the KMN (KNL1/Spc105, the Mis12 complex, and the Ndc80 complex) network bind to the N-terminal part of Drosophila CENP-C. Moreover, we show that the Mis12 complex component Nnf1 interacts directly with CENP-C in vitro. To test whether CENP-C's N-terminal part was sufficient to recruit KMN proteins, we targeted it to the centrosome by fusing it to a domain of Plk4 kinase. The Mis12 and Ndc80 complexes and Spc105 protein were then all recruited to centrosomes at the expense of centromeres, leading to mitotic abnormalities typical of cells with defective kinetochores. Thus, the N-terminal part of Drosophila CENP-C is sufficient to recruit core kinetochore components and acts as the principal linkage between centromere and kinetochore during mitosis.     

Protein networks as logic functions in development and cancer

Many biological and clinical outcomes are based not on single proteins, but on modules of proteins embedded in protein networks. A fundamental question is how the proteins within each module contribute to the overall module activity. Here, we study the modules underlying three representative biological programs related to tissue development, breast cancer metastasis, or progression of brain cancer, respectively. For each case we apply a new method, called Network-Guided Forests, to identify predictive modules together with logic functions which tie the activity of each module to the activity of its component genes. The resulting modules implement a diverse repertoire of decision logic which cannot be captured using the simple approximations suggested in previous work such as gene summation or subtraction. We show that in cancer, certain combinations of oncogenes and tumor suppressors exert competing forces on the system, suggesting that medical genetics should move beyond cataloguing individual cancer genes to cataloguing their combinatorial logic.