New Structural Insights into Phosphorylation-free Mechanism for Full Cyclin-dependent Kinase (CDK)-Cyclin Activity and Substrate Recognition

Pho85 is a versatile cyclin-dependent kinase (CDK) found in budding yeast that regulates a myriad of eukaryotic cellular functions in concert with 10 cyclins (called Pcls). Unlike cell cycle CDKs that require phosphorylation of a serine/threonine residue by a CDK-activating kinase (CAK) for full activation, Pho85 requires no phosphorylation despite the presence of an equivalent residue. The Pho85-Pcl10 complex is a key regulator of glycogen metabolism by phosphorylating the substrate Gsy2, the predominant, nutritionally regulated form of glycogen synthase. Here we report the crystal structures of Pho85-Pcl10 and its complex with the ATP analog, ATPγS. The structure solidified the mechanism for bypassing CDK phosphorylation to achieve full catalytic activity. An aspartate residue, invariant in all Pcls, acts as a surrogate for the phosphoryl adduct of the phosphorylated, fully activated CDK2, the prototypic cell cycle CDK, complexed with cyclin A. Unlike the canonical recognition motif, SPX(K/R), of phosphorylation sites of substrates of several cell cycle CDKs, the motif in the Gys2 substrate of Pho85-Pcl10 is SPXX. CDK5, an important signal transducer in neural development and the closest known functional homolog of Pho85, does not require phosphorylation either, and we found that in its crystal structure complexed with p25 cyclin a water/hydroxide molecule remarkably plays a similar role to the phosphoryl or aspartate group. Comparison between Pho85-Pcl10, phosphorylated CDK2-cyclin A, and CDK5-p25 complexes reveals the convergent structural characteristics necessary for full kinase activity and the variations in the substrate recognition mechanism.     

Hydralazine and Organic Nitrates Restore Impaired Excitation-Contraction Coupling by Reducing Calcium Leak Associated with Nitroso-Redox Imbalance

Although the combined use of hydralazine and isosorbide dinitrate confers important clinical benefits in patients with heart failure, the underlying mechanism of action is still controversial. We used two models of nitroso-redox imbalance, neuronal NO synthase-deficient (NOS1^−/−) mice and spontaneously hypertensive heart failure rats, to test the hypothesis that hydralazine (HYD) alone or in combination with nitroglycerin (NTG) or isosorbide dinitrate restores Ca²⁺ cycling and contractile performance and controls superoxide production in isolated cardiomyocytes. The response to increased pacing frequency was depressed in NOS1^−/− compared with wild type myocytes. Both sarcomere length shortening and intracellular Ca²⁺ transient (Δ[Ca²⁺]_i) responses in NOS1^−/− cardiomyocytes were augmented by HYD in a dose-dependent manner. NTG alone did not affect myocyte shortening but reduced Δ[Ca²⁺]_i across the range of pacing frequencies and increased myofilament Ca²⁺ sensitivity thereby enhancing contractile efficiency. Similar results were seen in failing myocytes from the heart failure rat model. HYD alone or in combination with NTG reduced sarcoplasmic reticulum (SR) leak, improved SR Ca²⁺ reuptake, and restored SR Ca²⁺ content. HYD and NTG at low concentrations (1 μm), scavenged superoxide in isolated cardiomyocytes, whereas in cardiac homogenates, NTG inhibited xanthine oxidoreductase activity and scavenged NADPH oxidase-dependent superoxide more efficiently than HYD. Together, these results revealed that by reducing SR Ca²⁺ leak, HYD improves Ca²⁺ cycling and contractility impaired by nitroso-redox imbalance, and NTG enhanced contractile efficiency, restoring cardiac excitation-contraction coupling.     

Lithium Down-regulates Histone Deacetylase 1 (HDAC1) and Induces Degradation of Mutant Huntingtin

Lithium is an effective mood stabilizer that has been clinically used to treat bipolar disorder for several decades. Recent studies have suggested that lithium possesses robust neuroprotective and anti-tumor properties. Thus far, a large number of lithium targets have been discovered. Here, we report for the first time that HDAC1 is a target of lithium. Lithium significantly down-regulated HDAC1 at the translational level by targeting HDAC1 mRNA. We also showed that depletion of HDAC1 is essential for the neuroprotective effects of lithium and for the lithium-mediated degradation of mutant huntingtin through the autophagic pathway. Our studies explain the multiple functions of lithium and reveal a novel mechanism for the function of lithium in neurodegeneration.     

Niche Partition of Bacteriovorax Operational Taxonomic Units Along Salinity and Temporal Gradients in the Chesapeake Bay Reveals Distinct Estuarine Strains

The predatory Bacteriovorax are Gram-negative bacteria ubiquitous in saltwater systems that prey upon other Gram-negative bacteria in a similar manner to the related genus Bdellovibrio. Among the phylogenetically defined clusters of Bacteriovorax, cluster V has only been isolated from estuaries suggesting that it may be a distinct estuarine phylotype. To assess this hypothesis, the spatial and temporal distribution of cluster V and other Bacteriovorax phylogenetic assemblages along the salinity gradient of Chesapeake Bay were determined. Cluster V was expected to be found in significantly greater numbers in low to moderate salinity waters compared to high salinity areas. The analyses of water and sediment samples from sites in the bay revealed cluster V to be present at the lower salinity and not high salinity sites, consistent with it being an estuarine phylotype. Cluster IV had a similar distribution pattern and may also be specifically adapted to estuaries. While the distribution of clusters V and IV were similar for salinity, they were distinct on temperature gradients, being found in cooler and in warmer temperatures, respectively. The differentiation of phylotype populations along the salinity and temporal gradients in Chesapeake Bay revealed distinct niches inhabited by different phylotypes of Bacteriovorax and unique estuarine phylotypes.     

Major QTL for Fusarium crown rot resistance in a barley landrace

Fusarium crown rot (FCR) is a serious cereal disease in semi-arid regions worldwide. In assisting the effort of breeding cultivars with enhanced resistance, we identified several barley genotypes with high levels of FCR resistance. One of these genotypes, AWCS079 which is a barley landrace originating from Japan, was investigated by developing and assessing three populations of recombinant inbred lines. Two QTL, one located on the long arm of chromosome 1H (designated as Qcrs.cpi-1H) and the other on 3HL (designated as Qcrs.cpi-3H), were found to be responsible for the FCR resistance of this genotype. Qcrs.cpi-1H is novel as no other FCR loci have been reported on this chromosome arm. Qcrs.cpi-3H co-located with a reduced height (Rht) locus and the effectiveness of the former was significantly affected by the latter. The total phenotypic variance explained by these two QTL was over 60 %. Significant effects were detected for each of the QTL in each of the three populations assessed. The existence of these loci with major effects should not only facilitate breeding and exploitation of FCR-resistant barley cultivars but also their further characterization based on fine mapping and map-based gene cloning.     

Rapid development of molecular markers by next-generation sequencing linked to a gene conferring phomopsis stem blight disease resistance for marker-assisted selection in lupin (Lupinus angustifolius L.) breeding

Selection for phomopsis stem blight disease (PSB) resistance is one of the key objectives in lupin (Lupinus angustifolius L.) breeding programs. A cross was made between cultivar Tanjil (resistant to PSB) and Unicrop (susceptible). The progeny was advanced into F₈ recombinant inbred lines (RILs). The RIL population was phenotyped for PSB disease resistance. Twenty plants from the RIL population representing disease resistance and susceptibility was subjected to next-generation sequencing (NGS)-based restriction site-associated DNA sequencing on the NGS platform Solexa HiSeq2000, which generated 7,241 single nucleotide polymorphisms (SNPs). Thirty-three SNP markers showed the correlation between the marker genotypes and the PSB disease phenotype on the 20 representative plants, which were considered as candidate markers linked to a putative R gene for PSB resistance. Seven candidate markers were converted into sequence-specific PCR markers, which were designated as PhtjM1, PhtjM2, PhtjM3, PhtjM4, PhtjM5, PhtjM6 and PhtjM7. Linkage analysis of the disease phenotyping data and marker genotyping data on a F₈ population containing 187 RILs confirmed that all the seven converted markers were associated with the putative R gene within the genetic distance of 2.1 CentiMorgan (cM). One of the PCR markers, PhtjM3, co-segregated with the R gene. The seven established PCR markers were tested in the 26 historical and current commercial cultivars released in Australia. The numbers of “false positives” (showing the resistance marker allele band but lack of the putative R gene) for each of the seven PCR markers ranged from nil to eight. Markers PhtjM4 and PhtjM7 are recommended in marker-assisted selection for PSB resistance in the Australian national lupin breeding program due to its wide applicability on breeding germplasm and close linkage to the putative R gene. The results demonstrated that application of NGS technology is a rapid and cost-effective approach in development of markers for molecular plant breeding.     

Development of a novel recombinant strain Zygosacharomyces rouxii JL2011 for 1,3-propanediol production from glucose

1,3-propanediol (1,3-PDO) is one of the most important industrial chemicals due to its highly desired properties and its wide applications as a key component of the emerging polymer industry. Biotechnology route has been one of the most interesting methods for 1,3-PDO production, whereas, the dha genes were essential to 1,3-PDO biosynthesis. In this study, we cloned and placed the dha cassettes under the control of a glyceraldehyde 3-phosphate dehydrogenase gene promoter pGAP and homologous ZrFPS1 gene promoter pZrfps1; these two promoters were further integrated into the chromosome of Z. rouxii JL2011 to generate recombinant strain JL2011-GZ and JL2011-ZZ, respectively. The results showed that the two strains could produce 1,3-PDO from glucose with a final yield of 6.9 and 10.3 g/l, respectively. The engineered strain JL2011-ZZ showed a 2.3- and 1.5-fold increase in the specific activities and final concentration of 1,3-PDO, respectively, with respect to JL2011-GZ. Batch fermentation with aerobic/micro-aerobic combined strategy of JL2011-ZZ resulted a titer of 17.1 g/l and a yield from glucose of 8.6 %. These results demonstrated that JL2011-ZZ would be a potential strain for 1,3-PDO production from glucose.     

Increasing oxidative stress tolerance and subculturing stability of Cordyceps militaris by overexpression of a glutathione peroxidase gene

Like other filamentous fungi, the medicinal ascomycete Cordyceps militaris frequently degenerates during continuous maintenance in culture by showing loss of the ability to reproduce sexually or asexually. Degeneration of fungal cultures has been related with cellular accumulation of reactive oxygen species (ROS). In this study, an antioxidant glutathione peroxidase (Gpx) gene from Aspergillus nidulans was engineered into two C. militaris strains, i.e., the Cm01 strain which can fruit normally and the Cm04 strain which has lost the ability to form fruiting bodies on different media through subculturing. The results showed that the mitotically stable mutants had higher Gpx activities and stronger capacity to scavenge cellular ROS than their parental strains. Most significantly, the fruiting ability of Cm04 strain was restored by overexpression of the antioxidant enzyme. However, after being successively transferred for up to ten generations, two of three Cm04 mutants again lost the ability to fruit on insect pupae while Cm01 transformants remained fertile. This study confirms the relationship between fungal culture degeneration and cellular ROS accumulation. Our results indicate that genetic engineering with an antioxidant gene can be an effective way to reverse fungal degeneration during subculturing.