The nuclear transport machinery recognizes nucleoplasmin-histone complexes

The nuclear transport of the chromatin remodeling protein nucleoplasmin and chromatin building histones is mediated by importins. Nucleoplasmin (NP) contains a classical bipartite nuclear localization signal (NLS) that is recognized by the importin α/β heterodimer, while histones present multiple NLS-like motifs that are recognized by importin β family members for nuclear targeting. To explore the possibility of a cotransport of histones and their chaperone NP to the nucleus, we have analyzed the assembly of complexes of NP/histones with importins by means of fluorescence anisotropy, centrifugation in sucrose gradients, and isothermal titration calorimetry. Data show that importin α ΔIBB (a truncated form of importin α lacking the autoinhibitory N-terminal domain) and histones (linker, H5, and nucleosomal core, H2AH2B) can simultaneously bind to NP. Analysis of the binding energetics reveals an enthalpy-driven formation of high affinity ternary, NP/Δα/H5 and NP/Δα/H2AH2B, complexes. We find that different amount of importin α molecules can be loaded on NP/histone complexes dependent on the histone type, linker or core, and the amount of bound histones. We further demonstrate that NP/H5 complexes can also incorporate importin α/β, thus forming quaternary NP/histones/α/β complexes that might represent a putative coimport pathway for nuclear import of histones and their chaperone protein NP, enhancing the histone import efficiency.     

Influence of Cu2+ on the Amino Acids Profile of Saccharomyces cerevisiae RD1 during Growth

The growth and the amino acid composition of the strain Saccharomyces cerevisiae RD1 were studied in the presence of copper ions. The accumulation of biomass was inhibited with the increase of Cu²⁺ concentration. It should be noted that the synthesis of aromatic amino acids was promoted at lower Cu²⁺ concentration (100 mg·L^?1), but at higher concentrations the inhibiting effect was significant. The decreases of the amino acid contents with the increase of Cu²⁺ concentration varied upon their type. The total amount of amino acids was much lower at 300 and 400 mg·L^?1 Cu²⁺.     

A Mechanism for Histone Chaperoning Activity of Nucleoplasmin: Thermodynamic and Structural Models

Nucleoplasmin (NP), a histone chaperone, acts as a reservoir for histones H2A-H2B in Xenopus laevis eggs and can displace sperm nuclear basic proteins and linker histones from the chromatin fiber of sperm and quiescent somatic nuclei. NP has been proposed to mediate the dynamic exchange of histones during the expression of certain genes and assists the assembly of nucleosomes by modulating the interaction between histones and DNA. Here, solution structural models of full-length NP and NP complexes with the functionally distinct nucleosomal core and linker histones are presented for the first time, providing a picture of the physical interactions between the nucleosomal and linker histones with NP core and tail domains. Small-angle X-ray scattering and isothermal titration calorimetry reveal that NP pentamer can accommodate five histones, either H2A-H2B dimers or H5, and that NP core and tail domains are intimately involved in the association with histones. The analysis of the binding events, employing a site-specific cooperative model, reveals a negative cooperativity-based regulatory mechanism for the linker histone/nucleosomal histone exchange. The two histone types bind with drastically different intrinsic affinity, and the strongest affinity is observed for the NP variant that mimicks the hyperphosphorylated active protein. The different “affinity windows” for H5 and H2A-H2B might allow NP to fulfill its histone chaperone role, simultaneously acting as a reservoir for the core histones and a chromatin decondensing factor. Our data are compatible with the previously proposed model where NP facilitates nucleosome assembly by removing the linker histones and depositing H2A-H2B dimers onto DNA.     

Recognition of nucleoplasmin by its nuclear transport receptor importin α/β: insights into a complete import complex

Nuclear import of the pentameric histone chaperone nucleoplasmin (NP) is mediated by importin α, which recognizes its nuclear localization sequence (NLS), and importin β, which interacts with α and is in charge of the translocation of the NP/α/β complex through the nuclear pore. Herein, we characterize the assembly of a functional transport complex formed by full-length NP with importin α/β. Isothermal titration calorimetry (ITC) was used to analyze the thermodynamics of the interactions of importin α with β, α with NP, and the α/β heterodimer with NP. Our data show that binding of both importin α and α/β to NP is governed by a favorable enthalpic contribution and that NP can accommodate up to five importin molecules per NP pentamer. Phosphomimicking mutations of NP, which render the protein active in histone chaperoning, do not modulate the interaction with importin. Using small-angle X-ray scattering, we model the α/β heterodimer, NP/α, and NP/α/β solution structures, which reveal a glimpse of a complete nuclear import complex with an oligomeric cargo protein. The set of alternative models, equally well fitting the scattering data, yields asymmetric elongated particles that might represent consecutive geometries the complex can adopt when stepping through the nuclear pore.     

Sensitivity of the photosynthetic apparatus to UV-A radiation: role of light-harvesting complex II–photosystem II supercomplex organization

In this work we study the effect of UV-A radiation on the function of the photosynthetic apparatus in thylakoid membranes with different organization of the light-harvesting complex II–photosystem II (LHCII–PSII) supercomplex. Leaves and isolated thylakoid membranes from a number of previously characterized pea species with different LHCII size and organization were subjected to UV-A treatment. A relationship was found between the molecular organization of the LHCII (ratio of the oligomeric to monomeric forms of LHCII) and UV-A-induced changes both in the energy transfer from PSII to PSI and between the chlorophyll–protein complexes within the LHCII–PSII supercomplex. Dependence on the organization of the LHCII was also found with regard to the degree of inhibition of the photosynthetic oxygen evolution. The susceptibility of energy transfer and oxygen evolution to UV-A radiation decreased with increasing LHCII oligomerization when the UV-A treatment was performed on isolated thylakoid membranes, in contrast to the effect observed in thylakoid membranes isolated from pre-irradiated pea leaves. The data suggest that UV-A radiation leads mainly to damage of the PSIIα centers. Comparison of membranes with different organization of their LHCII–PSII supercomplex shows that the oligomeric forms of LHCII play a key role for sensitivity to UV-A radiation of the photosynthetic apparatus. S. G. Taneva is Associated member of the Institute of Biophysics, Bulgarian Academy of Sciences.     

DnaJ recruits DnaK to protein aggregates

Thermal stress might lead to protein aggregation in the cell. Reactivation of protein aggregates depends on Hsp100 and Hsp70 chaperones. We focus in this study on the ability of DnaK, the bacterial representative of the Hsp70 family, to interact with different aggregated model substrates. Our data indicate that DnaK binding to large protein aggregates is mediated by DnaJ, and therefore it depends on its affinity for the cochaperone. Mutations in the structural region of DnaK known as the "latch" decrease the affinity of the chaperone for DnaJ, resulting in a defective activity as protein aggregate-removing agent. As expected, the chaperone activity is recovered when DnaJ concentration is raised to overcome the lower affinity of the mutant for the cochaperone, suggesting that a minimum number of aggregate-bound DnaK molecules is necessary for its efficient reactivation. Our results provide the first experimental evidence of DnaJ-mediated recruiting of ATP-DnaK molecules to the aggregate surface.     

Defects in cell growth regulation by C18:0-ceramide and longevity assurance gene 1 in human head and neck squamous cell carcinomas

In this study, endogenous long chain ceramides were measured in 32 human head and neck squamous cell carcinoma (HNSCC) and 10 nonsquamous head and neck carcinoma tumor tissues, as compared with adjacent noncancerous tissues, by liquid chromatography/mass spectroscopy. Interestingly, only one specific ceramide, C(18:0)-ceramide, was selectively down-regulated in the majority of HNSCC tumor tissues. On the other hand, in nonsquamous tumor tissues, this selectivity for C18-ceramide was not detected. These data suggested the hypotheses that decreased levels of C18-ceramide might impart a growth advantage to HNSCC cells and that increased generation of C18-ceramide may be involved in the inhibition of growth. These roles were examined by reconstitution of C18-ceramide at physiologically relevant concentrations in UM-SCC-22A cells (squamous cell carcinoma of hypopharynx) via overexpression of mammalian upstream regulator of growth and differentiation factor 1 (mUOG1), a mouse homologue of longevity assurance gene 1 (mLAG1), which has been shown to specifically induce the generation of C18-ceramide. Liquid chromatography/mass spectroscopy analysis showed that overexpression of the mLAG1/mUOG1 resulted in increased levels of only C(18:0)-ceramide by approximately 2-fold, i.e. concentrations similar to those of normal head and neck tissues. Importantly, increased generation of C18-ceramide by mLAG1/mUOG1 inhibited cell growth (approximately 70-80%), which mechanistically involved the modulation of telomerase activity and induction of apoptotic cell death by mitochondrial dysfunction. In conclusion, this study demonstrates, for the first time, a biological role for LAG1 and C18-ceramide in the regulation of growth of HNSCC.     

Structural rearrangements in chloroplast thylakoid membranes revealed by differential scanning calorimetry and circular dichroism spectroscopy. Thermo-optic effect

The thermo-optic mechanism in thylakoid membranes was earlier identified by measuring the thermal and light stabilities of pigment arrays with different levels of structural complexity [Cseh, Z., et al. (2000) Biochemistry 39, 15250-15257]. (According to the thermo-optic mechanism, fast local thermal transients, arising from the dissipation of excess, photosynthetically not used, excitation energy, induce elementary structural changes due to the "built-in" thermal instabilities of the given structural units.) The same mechanism was found to be responsible for the light-induced trimer-to-monomer transition in LHCII, the main chlorophyll a/b light-harvesting antenna of photosystem II (PSII) [Garab, G., et al. (2002) Biochemistry 41, 15121-15129]. In this paper, differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy on thylakoid membranes of barley and pea are used to correlate the thermo-optically inducible structural changes with well-discernible calorimetric transitions. The thylakoid membranes exhibited six major DSC bands, with maxima between about 43 and 87 degrees C. The heat sorption curves were analyzed both by mathematical deconvolution of the overall endotherm and by a successive annealing procedure; these yielded similar thermodynamic parameters, transition temperature and calorimetric enthalpy. A systematic comparison of the DSC and CD data on samples with different levels of complexity revealed that the heat-induced disassembly of chirally organized macrodomains contributes profoundly to the first endothermic event, a weak and broad DSC band between 43 and 48 degrees C. Similarly to the main macrodomain-associated CD signals, this low enthalpy band could be diminished by prolonged photoinhibitory preillumination, the extent of which depended on the temperature of preillumination. By means of nondenaturing, "green" gel electrophoresis and CD fingerprinting, it is shown that the second main endotherm, around 60 degrees C, originates to a large extent from the monomerization of LHCII trimers. The main DSC band, around 70 degrees C, which exhibits the highest enthalpy change, and another band around 75-77 degrees C relate to the dismantling of LHCII and other pigment-protein complexes, which under physiologically relevant conditions cannot be induced by light. The currently available data suggest the following sequence of events of thermo-optically inducible changes: (i) unstacking of membranes, followed by (ii) lateral disassembly of the chiral macrodomains and (iii) monomerization of LHCII trimers. We propose that thermo-optical structural reorganizations provide a structural flexibility, which is proportional to the intensity of the excess excitation, while for their localized nature, the structural stability of the system can be retained.     

Chemical composition of Corallina mediterranea Areschoug and Corallina granifera Ell. et Soland

The composition of sterols, volatiles and some polar compounds from three Corallina samples (C. granifera and C. mediterranea from the Black Sea and C. mediterranea from the Mediterranean Sea) was established. The sterol composition of the Black Sea samples was similar but it differs from that of the Mediterranean sample. The composition of the volatiles was very complex. The main groups of constituent were hydrocarbons, alcohols, carbonyl compounds, acids and their esters, terpenes. The composition of the polar components, soluble in n-butanol, was also established. There were some differences in the chemical composition of the two Black Sea species, which may be due to the biodiversity between them, while the differences in the composition of the two C. mediterranea samples could be due to the differences in the environment (salinity, temperature, pollution, etc.).     

Chromosomal disorders and nuclear and cell destruction in cleaving human embryos

Three types of defects of preimplantation embryogenesis contribute to the developmental arrest of cleaving human embryos: blastomere fragmentation, abnormal nuclear status and chromosomal disorders. Data concerning the relation and succession of these abnormalities during first mitotic cycles of the human zygote are controversial and mainly empirical at present. In this study we have performed simultaneous evaluation of blastomere fragmentation, nuclear apoptotic changes and the ploidy of four chromosomes (1, 5, 19 and X or 18, 21, X and Y) in 193 human embryos. Another group of 28 embryos was subjected to TUNEL for confirmation of apoptosis in blastomere nuclei. Nuclei with apoptotic chromatin were seen in nearly 1/10 of blastomeres of embryos with good morphology and in more than 1/5 of blastomeres of embryos with more than 20% fragmentation. The correct number of investigated chromosomes was registered in 85.2% of successfully tested embryos. Chromatin apoptotic changes are the only limiting factor for the success of chromosomal FISH tests. Nearly 1/2 of embryos with at least one apoptotic nucleus were chromosomally abnormal. For the embryos that contain only normal nuclei, the rate of ploid normality was more than 89%. The rate of euploidy was higher (66%) in embryos with a significant degree of cell fragmentation. Moderate cell fragmentation was not related to significant increase of chromatin and chromosomal disorders. In a substantial portion of abnormal blastomeres, chromatin damage preceded cell fragmentation. Nuclear destruction in human blastomeres was illustrated by fluorograms of different stages of chromatin lesions.