Cdk Phosphorylation of a Nucleoporin Controls Localization of Active Genes through the Cell Cycle

Many inducible genes in yeast are targeted to the nuclear pore complex when active. We find that the peripheral localization of the INO1 and GAL1 genes is regulated through the cell cycle. Active INO1 and GAL1 localized at the nuclear periphery during G1, became nucleoplasmic during S-phase, and then returned to the nuclear periphery during G2/M. Loss of peripheral targeting followed the initiation of DNA replication and was lost in cells lacking a cyclin-dependent kinase (Cdk) inhibitor. Furthermore, the Cdk1 kinase and two Cdk phosphorylation sites in the nucleoporin Nup1 were required for peripheral targeting of INO1 and GAL1. Introduction of aspartic acid residues in place of either of these two sites in Nup1 bypassed the requirement for Cdk1 and resulted in targeting of INO1 and GAL1 to the nuclear periphery during S-phase. Thus, phosphorylation of a nuclear pore component by cyclin dependent kinase controls the localization of active genes to the nuclear periphery through the cell cycle.     

Monitoring the Interaction between β2-Microglobulin and the Molecular Chaperone αB-crystallin by NMR and Mass Spectrometry: αB-CRYSTALLIN DISSOCIATES β2-MICROGLOBULIN OLIGOMERS*

The interaction at neutral pH between wild-type and a variant form (R3A) of the amyloid fibril-forming protein β₂-microglobulin (β2m) and the molecular chaperone αB-crystallin was investigated by thioflavin T fluorescence, NMR spectroscopy, and mass spectrometry. Fibril formation of R3Aβ2m was potently prevented by αB-crystallin. αB-crystallin also prevented the unfolding and nonfibrillar aggregation of R3Aβ2m. From analysis of the NMR spectra collected at various R3Aβ2m to αB-crystallin molar subunit ratios, it is concluded that the structured β-sheet core and the apical loops of R3Aβ2m interact in a nonspecific manner with the αB-crystallin. Complementary information was derived from NMR diffusion coefficient measurements of wild-type β2m at a 100-fold concentration excess with respect to αB-crystallin. Mass spectrometry acquired in the native state showed that the onset of wild-type β2m oligomerization was effectively reduced by αB-crystallin. Furthermore, and most importantly, αB-crystallin reversibly dissociated β2m oligomers formed spontaneously in aged samples. These results, coupled with our previous studies, highlight the potent effectiveness of αB-crystallin in preventing β2m aggregation at the various stages of its aggregation pathway. Our findings are highly relevant to the emerging view that molecular chaperone action is intimately involved in the prevention of in vivo amyloid fibril formation.     

Gonadal development and sex-specific demographics of the shovelnose sturgeon in the Middle Mississippi River

Harvest of the shovelnose sturgeon, Scaphirhynchus platorynchus for caviar has increased. To determine whether populations can withstand increased harvest, detailed information regarding sexual demographics is needed. We describe gender and reproductive development of 306 shovelnose sturgeon from the Middle Mississippi River (River km 0–322) during September 2001 through December 2003. Using dissection and histology, we identified three of the four gonadal stages described previously for male lake sturgeon and all seven stages for females. Males reached maturity at a smaller size than did females. Gonads can be rapidly inspected for sex and stage of development for the shovelnose sturgeon. The sex ratio was not different from 1 : 1. Seven intersexual fish occurred. Female fecundity was positively related to body weight (number of eggs = 30.24 × body weight − 8392; P = 0.013; r² = 0.45) and weakly related to fork length (number of eggs = 146.37 × fork length − 66 176, P = 0.053, r² = 0.23).     

The three-dimensional crystal structure of the PrpF protein of Shewanella oneidensis complexed with trans-aconitate: insights into its biological function

In bacteria, the dehydration of 2-methylcitrate to yield 2-methylaconitate in the 2-methylcitric acid cycle is catalyzed by a cofactor-less (PrpD) enzyme or by an aconitase-like (AcnD) enzyme. Bacteria that use AcnD also require the function of the PrpF protein, whose function was previously unknown. To gain insights into the function of PrpF, the three-dimensional crystal structure of the PrpF protein from the bacterium Shewanella oneidensis was solved at 2.0 A resolution. The protein fold of PrpF is strikingly similar to those of the non-PLP-dependent diaminopimelate epimerase from Haemophilus influenzae, a putative proline racemase from Brucella melitensis, and to a recently deposited structure of a hypothetical protein from Pseudomonas aeruginosa. Results from in vitro studies show that PrpF isomerizes trans-aconitate to cis-aconitate. It is proposed that PrpF catalysis of the cis-trans isomerization proceeds through a base-catalyzed proton abstraction coupled with a rotation about C2-C3 bond of 2-methylaconitate, and that residue Lys73 is critical for PrpF function. The newly identified function of PrpF as a non-PLP-dependent isomerase, together with the fact that PrpD-containing bacteria do not require PrpF, suggest that the isomer of 2-methylaconitate that serves as a substrate of aconitase must have the same stereochemistry as that synthesized by PrpD. From this, it follows that the 2-methylaconitate isomer generated by AcnD is not a substrate of aconitase, and that PrpF is required to generate the correct isomer. As a consequence, the isomerase activity of PrpF may now be viewed as an integral part of the 2-methylcitric acid cycle.     

KSRP is critical in governing hepatic lipid metabolism through controlling Per2 expression

Hepatic lipid metabolism is controlled by integrated metabolic pathways. Excess accumulation of hepatic TG is a hallmark of nonalcoholic fatty liver disease, which is associated with obesity and insulin resistance. Here, we show that KH-type splicing regulatory protein (KSRP) ablation reduces hepatic TG levels and diet-induced hepatosteatosis. Expression of period 2 (Per2) is increased during the dark period, and circadian oscillations of several core clock genes are altered with a delayed phase in Ksrp^−/− livers. Diurnal expression of some lipid metabolism genes is also disturbed with reduced expression of genes involved in de novo lipogenesis. Using primary hepatocytes, we demonstrate that KSRP promotes decay of Per2 mRNA through an RNA-protein interaction and show that increased Per2 expression is responsible for the phase delay in cycling of several clock genes in the absence of KSRP. Similar to Ksrp^−/− livers, both expression of lipogenic genes and intracellular TG levels are also reduced in Ksrp^−/− hepatocytes due to increased Per2 expression. Using heterologous mRNA reporters, we show that the AU-rich element-containing 3′ untranslated region of Per2 is responsible for KSRP-dependent mRNA decay. These findings implicate that KSRP is an important regulator of circadian expression of lipid metabolism genes in the liver likely through controlling Per2 mRNA stability.     

Nucleotide sequence of Bacillus phage phi 29 genes 14 and 15: homology of gene 15 with other phage lysozymes.

The nucleotide sequence of Bacillus phage phi 29 genes 14 (g14) and 15 (g15) have been determined and shown to encode proteins with molecular weights of 15,014 and 28,022, respectively. The g14 open reading frame (ORF) was confirmed by sequencing a sus14(1241) mutant. Gene product 15 (gp15) has considerable homology with Salmonella phage P22 lysozyme and lesser homology with Escherichia coli phage T4 lysozyme. Putative translation signals are identified. In addition, the role of a previously described promoter, B2, is discussed.     

The origin and evolution of mosquito APE retroposons

The detection of horizontal transfer is important to understanding the origin and spread of transposable elements and in assessing their impact on genetic diversity. The occurrence of the phenomenon is not in doubt for two of the three major groups of elements, but is disputed for retroposons, largely on the grounds of data paucity and overreliance on divergence estimates between host species. We present here the most wide-ranging retroposon data set assembled to date for a species group, the mosquitoes. The results provide no evidence for horizontal transfer events and show conclusively that four previously reported events, involving Juan-A, Juan-C, T1, and Q, did not occur. We propose that the origin of all known mosquito retroposons can be attributed to vertical inheritance and that retroposons have therefore been a persistent source of genetic diversity in mosquito genomes since the emergence of the taxon. Furthermore, the data confirm that the unprecedented levels of retroposon diversity previously reported in Anopheles gambiae extends to at least seven other species representing five genera and all three mosquito subfamilies. Most notably, this included the L1 elements, which are not known in other insects. A number of novel well-defined monophyletic groups were also identified, particularly, JM2 and JM3 within the Jockey clade, which included sequences from seven and five mosquito species, respectively. As JM3 does not contain an Anopheles element, this represents a good example of stochastic loss and the best out of at least four found in this study. This exceptionally diverse data set when compared with the wealth of data available for the many unrelated species with which mosquitoes have intimate contact through blood feeding ought to be fertile ground for the discovery of horizontal transfer events. The absence of positive results therefore supports the view that retroposon horizontal transfer does not occur or is far more exceptional than for other types of transposable elements.     

Rotaviruses Associate with Cellular Lipid Droplet Components To Replicate in Viroplasms,and Compounds Disrupting or Blocking Lipid Droplets Inhibit Viroplasm Formation and Viral Replication

Rotaviruses are a major cause of acute gastroenteritis in children worldwide. Early stages of rotavirus assembly in infected cells occur in viroplasms. Confocal microscopy demonstrated that viroplasms associate with lipids and proteins (perilipin A, ADRP) characteristic of lipid droplets (LDs). LD-associated proteins were also found to colocalize with viroplasms containing a rotaviral NSP5-enhanced green fluorescent protein (EGFP) fusion protein and with viroplasm-like structures in uninfected cells coexpressing viral NSP2 and NSP5. Close spatial proximity of NSP5-EGFP and cellular perilipin A was confirmed by fluorescence resonance energy transfer. Viroplasms appear to recruit LD components during the time course of rotavirus infection. NSP5-specific siRNA blocked association of perilipin A with NSP5 in viroplasms. Viral double-stranded RNA (dsRNA), NSP5, and perilipin A cosedimented in low-density gradient fractions of rotavirus-infected cell extracts. Chemical compounds interfering with LD formation (isoproterenol plus isobutylmethylxanthine; triacsin C) decreased the number of viroplasms and inhibited dsRNA replication and the production of infectious progeny virus; this effect correlated with significant protection of cells from virus-associated cytopathicity. Rotaviruses represent a genus of another virus family utilizing LD components for replication, pointing at novel therapeutic targets for these pathogens.Rotaviruses are a major cause of acute gastroenteritis in infants and young children, producing a high burden of disease worldwide and over 600,000 deaths per annum, mainly in developing countries (43). Recently, two live attenuated rotavirus vaccines (49, 58) have been licensed in various countries, and their widespread use in universal mass vaccination programs is being implemented (55).Rotaviruses form a genus of the Reoviridae family. They contain a genome of 11 segments of double-stranded RNA (dsRNA) encoding six structural proteins (VP1, VP2, VP3, VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). After entry into the host cell the outer layer of the triple-layered particles (TLPs; infectious virions) is removed in endocytic vesicles, and the resulting double-layered particles (DLPs) actively transcribe mRNAs from the 11 RNA segments and release them into the cytoplasm. The mRNAs are translated into proteins but also act as templates for dsRNA synthesis (RNA replication). The early stages of viral morphogenesis and viral RNA replication occur in cytoplasmic inclusion bodies termed “viroplasms.” Partially assembled DLPs are released from viroplasms and receive their outer layer in the rough endoplasmic reticulum (RER), forming TLPs (for details, see Estes and Kapikian [20]).The rotavirus nonstructural proteins NSP2 and NSP5 are major components of viroplasms (20, 47). These two proteins alone are sufficient to induce the formation of viroplasm-like structures (VLS) (21). Blocking of either NSP2 or NSP5 in rotavirus-infected cells significantly reduces viroplasm formation and the production of infectious viral progeny (11, 54, 57). Until now, host cell proteins involved in viroplasm formation have not been identified.Morphological similarities between viroplasms and lipid droplets (LDs) prompted us to investigate their relationship. Both structures have phosphoproteins (NSP5 and perilipin A, respectively) inserted on their surface in ringlike shapes (16, 34). LDs are intracellular organelles involved in lipid and carbohydrate metabolism. They consist of a neutral lipid core surrounded by a phospholipid monolayer containing LD-associated proteins; those include proteins of the PAT family consisting of perilipin, adipophilin (adipose differentiation-related protein [ADRP]), and TIP-47 (9, 37). Lipolysis from LDs is regulated by hormones such as catecholamines, which trigger the phosphorylation of hormone-sensitive lipase (HSL) and perilipin A and induce LD fragmentation. Incubating adipocytes with the β-adrenergic agonist isoproterenol and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) activates this pathway (27, 34). LD formation can also be blocked by triacsin C, a specific inhibitor of long chain acyl coenzyme A synthetases (30, 39).We demonstrate here that rotavirus viroplasms colocalize with the LD-associated proteins perilipin A and ADRP and also with the lipids of LDs. These interactions appear to be required for the formation of functional viroplasms and the production of infectious viral progeny, since compounds dispersing LDs or blocking LD formation significantly decrease the number and size of viroplasms and the amount of infectious progeny. Taken together, these findings strongly suggest a critical role of LDs in rotavirus replication.