Cross-metathesis mediated synthesis of new acyclic nucleoside phosphonates

With the commercial availability of well-defined ruthenium metathesis catalysts which combine high stability and broad functional group compatibility, olefin metathesis is now routinely integrated in various syntheses. We will report here the overwhelming power and scope of cross-metathesis in the area of new acyclic nucleoside phosphonates. Scope and limitations of this approach, and especially the E/Z stereocontrol, are discussed on selected examples from our drug discovery group.     

Phylogenetic diversity in sulphate-reducing bacterial communities from oxidised and reduced bottom sediments of the Barents Sea

In the bottom sediments from a number of the Barents Sea sites, including coastal areas of the Novaya Zemlya, Franz Josef Land, and Svalbard archipelagos, sulphate reduction rates were measured and the phylogenetic composition of sulphate-reducing bacterial (SRB) communities was analysed for the first time. Molecular genetic analysis of the sequences of the 16S rRNA and dsrB genes (the latter encodes the β-subunit of dissimilatory (bi)sulphite reductase) revealed significant differences in the composition of bacterial communities in different sampling stations and sediment horizons of the Barents Sea depending on the physicochemical conditions. The major bacteria involved in reduction of sulphur compounds in Arctic marine bottom sediments belonged to Desulfobulbaceae, Desulfobacteraceae, Desulfovibrionaceae, Desulfuromonadaceae, and Desulfarculaceae families, as well as to uncultured clades SAR324 and Sva0485. Desulfobulbaceae and Desulfuromonadaceae predominated in the oxidised (E_h = 154–226 mV) upper layers of the sediments (up to 9% and 5.9% from all reads of the 16S rRNA gene sequences in the sample, correspondingly), while in deeper, more reduced layers (E_h = ?210 to ?105 mV) the share of Desulfobacteraceae in the SRB community was also significant (up to 5%). The highest relative abundance of members of Desulfarculaceae family (3.1%) was revealed in reduced layers of sandy-clayey sediments from the Barents Sea area affected by currents of transformed (mixed, with changed physicochemical characteristics) Atlantic waters.

     

PHOTOSYSTEM II PROTEIN33, a Protein Conserved in the Plastid Lineage,Is Associated with the Chloroplast Thylakoid Membrane and Provides Stability to Photosystem II Supercomplexes in Arabidopsis

Photosystem II (PSII) is a multiprotein complex that catalyzes the light-driven water-splitting reactions of oxygenic photosynthesis. Light absorption by PSII leads to the production of excited states and reactive oxygen species that can cause damage to this complex. Here, we describe Arabidopsis (Arabidopsis thaliana) At1g71500, which encodes a previously uncharacterized protein that is a PSII auxiliary core protein and hence is named PHOTOSYSTEM II PROTEIN33 (PSB33). We present evidence that PSB33 functions in the maintenance of PSII-light-harvesting complex II (LHCII) supercomplex organization. PSB33 encodes a protein with a chloroplast transit peptide and one transmembrane segment. In silico analysis of PSB33 revealed a light-harvesting complex-binding motif within the transmembrane segment and a large surface-exposed head domain. Biochemical analysis of PSII complexes further indicates that PSB33 is an integral membrane protein located in the vicinity of LHCII and the PSII CP43 reaction center protein. Phenotypic characterization of mutants lacking PSB33 revealed reduced amounts of PSII-LHCII supercomplexes, very low state transition, and a lower capacity for nonphotochemical quenching, leading to increased photosensitivity in the mutant plants under light stress. Taken together, these results suggest a role for PSB33 in regulating and optimizing photosynthesis in response to changing light levels.PSII is a multiprotein complex in plants with 31 identified polypeptides (Wegener et al., 2011; Pagliano et al., 2013). It is associated with an extrinsic trimeric light-harvesting complex (LHC), forming the PSII-LHCII supercomplex. The PSII complex performs a remarkable biochemical reaction, the oxidation of water using light energy from the sun, which profoundly contributes to the overall biomass accumulation in the biosphere (Barber et al., 2004). Consequently, the stability and functional integrity of the PSII-LHCII supercomplex is crucially important for photosynthetic function. The energy of a photon, either absorbed directly by PSII or indirectly via energy transfer from adjacent antenna chlorophyll (Chl) molecules, excites the PSII reaction center P680. The excited state, P680*, can transfer an electron to pheophytin, producing the most powerful oxidant known in biology, P680⁺, which can remove electrons from water. Excessive input of excitation energy into PSII saturates the electron transfer system and causes either acceptor or donor site limitation in the complex. This results in increased production of reactive oxygen species (ROS): singlet oxygen at the PSII donor side and superoxide at the acceptor side (Munné-Bosch et al., 2013). Several protective mechanisms have been documented that decrease the production of singlet oxygen at the PSII donor side in photosynthetic eukaryotes. Notably, reducing energy transfer from LHC to PSII via nonphotochemical quenching (NPQ) is a key avoidance mechanism (Ruban and Murchie, 2012).Despite years of intensive study of PSII structure and function, new proteins that are associated with the PSII complex continue to be discovered, including an increasing number involved in the stability and organization of PSII-LHCII supercomplexes (García-Cerdán et al., 2011; Lu et al., 2011a; Wegener et al., 2011). Two complementary approaches (Merchant et al., 2007; Lu et al., 2008, 2011b; Ajjawi et al., 2010) that utilize phylogenomics (GreenCut) and large-scale phenotypic mutant screening (Chloroplast 2010 Project; http://www.plastid.msu.edu/) were employed by our groups to discover novel plant proteins with roles in photosynthesis. GreenCut identifies proteins found only in photosynthetic organisms, and it is likely that many of them are involved in biochemical processes associated with the structure, assembly, or function of the photosynthetic apparatus and the chloroplast that houses it (Merchant et al., 2007; Karpowicz et al., 2011). The Chloroplast 2010 Project was a large-scale reverse-genetic mutant screen in which thousands of homozygous Arabidopsis (Arabidopsis thaliana) transfer DNA (T-DNA) insertion lines were analyzed for defects in the rise and decay kinetics of Chl fluorescence (Lu et al., 2008, 2011a, 2011b; Ajjawi et al., 2010).The GreenCut and Chloroplast 2010 approaches both identified the Arabidopsis At1g71500 locus as encoding a protein of unknown function with potential relevance to photosynthesis. In this work, we demonstrate that plant lines carrying three independent mutations at this locus display severe light-induced photoinhibition due to a less stable supramolecular organization of PSII. Biochemical analyses revealed that this protein is associated with PSII complexes, and since the last described PSII protein was called PHOTOSYSTEM II PROTEIN32 (PSB32), we named the gene PSB33. The nuclear genome-encoded PSB33 is predicted to have a chloroplast transit peptide and a transmembrane domain. The biochemical analyses presented below indicate that PSB33 is required for the proper interaction and stability of PSII-LHCII supercomplexes and, in turn, in regulating photosynthesis in response to fluctuating light levels.     

Orientation of B798 BChl a Q y transition dipoles in Chloroflexus aurantiacus chlorosomes: polarized transient absorption spectroscopy studies

Photosynthesis Research - Isotropic and anisotropic pump-probe spectra of Cfx. aurantiacus chlorosomes were measured on the fs-through ps-time scales for the B798 BChl a Q y band upon direct...     

The interaction of Gα13 with integrin β1 mediates cell migration by dynamic regulation of RhoA

Heterotrimeric G protein Gα₁₃ is known to transmit G protein–coupled receptor (GPCR) signals leading to activation of RhoA and plays a role in cell migration. The mechanism underlying the role of Gα₁₃ in cell migration, however, remains unclear. Recently we found that Gα₁₃ interacts with the cytoplasmic domain of integrin β₃ subunits in platelets via a conserved ExE motif. Here we show that a similar direct interaction between Gα₁₃ and the cytoplasmic domain of the integrin β₁ subunit plays a critical role in β₁-dependent cell migration. Point mutation of either glutamic acid in the Gα₁₃-binding ⁷⁶⁷EKE motif in β₁ or treatment with a peptide derived from the Gα₁₃-binding sequence of β₁ abolished Gα₁₃–β₁ interaction and inhibited β₁ integrin–dependent cell spreading and migration. We further show that the Gα₁₃-β₁ interaction mediates β₁ integrin–dependent Src activation and transient RhoA inhibition during initial cell adhesion, which is in contrast to the role of Gα₁₃ in mediating GPCR-dependent RhoA activation. These data indicate that Gα₁₃ plays dynamic roles in both stimulating RhoA via a GPCR pathway and inhibiting RhoA via an integrin signaling pathway. This dynamic regulation of RhoA activity is critical for cell migration on β₁ integrin ligands.     

Comparative analysis of genome maintenance genes in naked mole rat,mouse, and human

Genome maintenance (GM) is an essential defense system against aging and cancer, as both are characterized by increased genome instability. Here, we compared the copy number variation and mutation rate of 518 GM‐associated genes in the naked mole rat (NMR), mouse, and human genomes. GM genes appeared to be strongly conserved, with copy number variation in only four genes. Interestingly, we found NMR to have a higher copy number of CEBPG, a regulator of DNA repair, and TINF2, a protector of telomere integrity. NMR, as well as human, was also found to have a lower rate of germline nucleotide substitution than the mouse. Together, the data suggest that the long‐lived NMR, as well as human, has more robust GM than mouse and identifies new targets for the analysis of the exceptional longevity of the NMR.     

SIRT6 rescues the age related decline in base excision repair in a PARP1-dependent manner

In principle, a decline in base excision repair (BER) efficiency with age should lead to genomic instability and ultimately contribute to the onset of the aging phenotype. Although multiple studies have indicated a negative link between aging and BER, the change of BER efficiency with age in humans has not been systematically analyzed. Here, with foreskin fibroblasts isolated from 19 donors between 20 and 64 y of age, we report a significant decline of BER efficiency with age using a newly developed GFP reactivation assay. We further observed a very strong negative correlation between age and the expression levels of SIRT6, a factor which is known to maintain genomic integrity by improving DNA double strand break (DSB) repair. Our mechanistic study suggests that, similar to the regulatory role that SIRT6 plays in DNA DSB repair, SIRT6 regulates BER in a PARP1-depdendent manner. Moreover, overexpression of SIRT6 rescues the decline of BER in aged fibroblasts. In summary, our results uncovered the regulatory mechanisms of BER by SIRT6, suggesting that SIRT6 reactivation in aging tissues may help delay the process of aging through improving BER.     

Isotope-reinforced polyunsaturated fatty acids protect yeast cells from oxidative stress

The facile abstraction of bis-allylic hydrogens from polyunsaturated fatty acids (PUFAs) is the hallmark chemistry responsible for initiation and propagation of autoxidation reactions. The products of these autoxidation reactions can form cross-links to other membrane components and damage proteins and nucleic acids. We report that PUFAs deuterated at bis-allylic sites are much more resistant to autoxidation reactions, because of the isotope effect. This is shown using coenzyme Q-deficient Saccharomyces cerevisiae coq mutants with defects in the biosynthesis of coenzyme Q (Q). Q functions in respiratory energy metabolism and also functions as a lipid-soluble antioxidant. Yeast coq mutants incubated in the presence of the PUFA α-linolenic or linoleic acid exhibit 99% loss of colony formation after 4 h, demonstrating a profound loss of viability. In contrast, coq mutants treated with monounsaturated oleic acid or with one of the deuterated PUFAs, 11,11-D₂-linoleic or 11,11,14,14-D₄-α-linolenic acid, retain viability similar to wild-type yeast. Deuterated PUFAs also confer protection to wild-type yeast subjected to heat stress. These results indicate that isotope-reinforced PUFAs are stabilized compared to standard PUFAs, and they protect coq mutants and wild-type yeast cells against the toxic effects of lipid autoxidation products. These findings suggest new approaches to controlling ROS-inflicted cellular damage and oxidative stress.