THE IDENTIFICATION AND CHARACTERIZATION OF AN INNER ACROSOMAL MEMBRANE ASSOCIATED PROTEIN, IAM38, RESPONSIBLE FOR SECONDARY SPERM-ZONA BINDING DURING FERTILIZATION

During mammalian fertilization, the exposure of the inner acrosomal membrane (IAM) after acrosomal exocytosis is essential for the secondary binding between sperm and zona pellucida (ZP) of the oocyte, a prerequisite for sperm penetration through the ZP. The identification of the sperm protein(s) responsible for secondary binding has posed a challenge for researchers. We were able to isolate a sperm head fraction in which the IAM was exposed. Attached to the IAM was an electon dense layer, which we termed the IAM extracellular coat (IAMC). The IAMC was also observable in acrosome reacted sperm. High salt extraction removed the IAMC including a prominent 38 kDa polypeptide, referred to as IAM38. Antibodies raised against IAM38 confirmed its presence in the IAMC of intact, sonicated, and acrosome-reacted sperm. Sequencing of IAM38 revealed it as the ortholog of porcine SP38, a protein that was found to bind specifically to ZP2 but whose intra-acrosomal location was not known. We showed that IAM38 occupied the leading edge of sperm contact with the zona pellucida during fertilization, and that secondary binding and fertilization were inhibited in vitro by antibodies directed against IAM38. As for the mechanism of secondary sperm-zona binding by IAM38, we provided evidence that the synthetic peptide derived from the ZP2-binding motif of IAM38 had a competitive inhibitory effect on both sperm-zona binding and fertilization while its mutant form was ineffective. In summary, our study provides a novel approach to obtain direct information on the peripheral and integral protein composition of the IAM and consolidates IAM38 as a genuine secondary sperm-zona binding protein. In addition, our investigation also provides an ultrastructural description of the origin, expression and assembly of IAM38 during spermatogenesis. It shows that IAM38 is originally secreted by the Golgi apparatus as part of the dense contents of the proacrosomic granules but later, during acrosome capping phase of spermiogenesis, is redistributed to the inner periphery of the acrosomal membrane. This relocation occurs at the time of acrosomal compaction, an obligatory structural change that fails to occur in Zpbp1-/- knockout mice, which do not express IAM38 and are infertile.

Antioxidant supplementation reduces skeletal muscle mitochondrial biogenesis

Purpose: Exercise increases the production of reactive oxygen species (ROS) in skeletal muscle, and athletes often consume antioxidant supplements in the belief they will attenuate ROS-related muscle damage and fatigue during exercise. However, exercise-induced ROS may regulate beneficial skeletal muscle adaptations, such as increased mitochondrial biogenesis. We therefore investigated the effects of long-term antioxidant supplementation with vitamin E and alpha-lipoic acid on changes in markers of mitochondrial biogenesis in the skeletal muscle of exercise-trained and sedentary rats. Methods: Male Wistar rats were divided into four groups: 1) sedentary control diet, 2) sedentary antioxidant diet, 3) exercise control diet, and 4) exercise antioxidant diet. Animals ran on a treadmill 4 d.wk(-1) at similar to 70% V (over dot)O(2max) for up to 90 min.d(-1) for 14 wk. Results: Consistent with the augmentation of skeletal muscle mitochondrial biogenesis and antioxidant defenses, after training there were significant increases in peroxisome proliferator-activated receptor F coactivator 1 alpha (PGC-1 alpha) messenger RNA (mRNA) and protein, cytochrome C oxidase subunit IV (COX IV) and cytochrome C protein abundance, citrate synthase activity, Nfe2l2, and SOD2 protein (P < 0.05). Antioxidant supplementation reduced PGC-1 alpha mRNA, PGC-1 alpha and COX IV protein, and citrate synthase enzyme activity (P < 0.05) in both sedentary and exercise-trained rats. Conclusions: Vitamin E and alpha-lipoic acid supplementation suppresses skeletal muscle mitochondrial biogenesis, regardless of training status.

DRB2 is required for microRNA biogenesis in Arabidopsis thaliana

Background The Arabidopsis thaliana (Arabidopsis) DOUBLE-STRANDED RNA BINDING (DRB) protein family consists of five members, DRB1 to DRB5. The biogenesis of two developmentally important small RNA (sRNA) species, the microRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) by DICER-LIKE (DCL) endonucleases requires the assistance of DRB1 and DRB4 respectively. The importance of miRNA-directed target gene expression in plant development is exemplified by the phenotypic consequence of loss of DRB1 activity (drb1 plants). Principal Findings Here we report that the developmental phenotype of the drb235 triple mutant plant is the result of deregulated miRNA biogenesis in the shoot apical meristem (SAM) region. The expression of DRB2, DRB3 and DRB5 in wild-type seedlings is restricted to the SAM region. Small RNA sequencing of the corresponding tissue of drb235 plants revealed altered miRNA accumulation. Approximately half of the miRNAs detected remained at levels equivalent to those of wild-type plants. However, the accumulation of the remaining miRNAs was either elevated or reduced in the triple mutant. Examination of different single and multiple drb mutants revealed a clear association between the loss of DRB2 activity and altered accumulation for both the elevated and reduced miRNA classes. Furthermore, we show that the constitutive over-expression of DRB2 outside of its wild-type expression domain can compensate for the loss of DRB1 activity in drb1 plants. Conclusions/Significance Our results suggest that in the SAM region, DRB2 is both antagonistic and synergistic to the role of DRB1 in miRNA biogenesis, adding an additional layer of gene regulatory complexity in this developmentally important tissue.

Characterization of two homologous disulfide bond systems involved in virulence factor Biogenesis in Uropathogenic Escherichia coli CFT073

Disulfide bond (DSB) formation is catalyzed by disulfide bond proteins and is critical for the proper folding and functioning of secreted and membrane-associated bacterial proteins. Uropathogenic Escherichia coli (UPEC) strains possess two paralogous disulfide bond systems: the well-characterized DsbAB system and the recently described DsbLI system. In the DsbAB system, the highly oxidizing DsbA protein introduces disulfide bonds into unfolded polypeptides by donating its redox-active disulfide and is in turn reoxidized by DsbB. DsbA has broad substrate specificity and reacts readily with reduced unfolded proteins entering the periplasm. The DsbLI system also comprises a functional redox pair; however, DsbL catalyzes the specific oxidative folding of the large periplasmic enzyme arylsulfate sulfotransferase (ASST). In this study, we characterized the DsbLI system of the prototypic UPEC strain CFT073 and examined the contributions of the DsbAB and DsbLI systems to the production of functional flagella as well as type 1 and P fimbriae. The DsbLI system was able to catalyze disulfide bond formation in several well-defined DsbA targets when provided in trans on a multicopy plasmid. In a mouse urinary tract infection model, the isogenic dsbAB deletion mutant of CFT073 was severely attenuated, while deletion of dsbLI or assT did not affect colonization.

Bull sperm plasma and acrosomal membranes: Fluorescence studies of lipid phase fluidity

Bull sperm plasma and outer acrosomal membranes have been isolated by discontinuous sucrose density gradient centrifugation and Ca2+-ATPase activity has been determined for both the membranes. Pyrene excimer fluorescence and diphenylhexatriene fluorescence polarization studies show that the lipid phase of the sperm plasma membranes is more fluid than the lipids of the outer acrosomal membranes. Approximately, a three fold increase in the cholesterol content has been found in the outer acrosomal membranes as compared to that in the plasma membranes.

Rhythmic activity of utilization of mevalonate for biogenesis of cholesterol

The utilization of mevalonate for biogenesis of cholesterol shows rhythmic activity with a peak at midnight and the step responsible is likely to be between mevalonate and isopentenyl pyrophosphate.

Nature of the stimulation of biogenesis of cholesterol in the liver by noradrenaline

1.Administration of noradrenaline increased the incorporation of [1-14C]acetate into hepatic sterols and the activity of liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase. 2. The stimulation was observed at short time-intervals with a maximum at 4h and was progressive with increasing concentrations of noradrenaline. 3. Protein synthesis de novo was a necessary factor for the effect. 4. The stimulatory effect was not mediated through the adrenergic receptors, but appears to involve a direct action of the hormone within the hepatocyte.

Impact of resistance exercise on ribosome biogenesis is acutely regulated by post-exercise recovery strategies

Muscle hypertrophy occurs following increased protein synthesis, which requires activation of the ribosomal complex. Additionally, increased translational capacity via elevated ribosomal RNA (rRNA) synthesis has also been implicated in resistance training-induced skeletal muscle hypertrophy. The time course of ribosome biogenesis following resistance exercise (RE) and the impact exerted by differing recovery strategies remains unknown. In the present study, the activation of transcriptional regulators, the expression levels of pre-rRNA, and mature rRNA components were measured through 48 h after a single-bout RE. In addition, the effects of either low-intensity cycling (active recovery, ACT) or a cold-water immersion (CWI) recovery strategy were compared. Nine male subjects performed two bouts of high-load RE randomized to be followed by 10 min of either ACT or CWI. Muscle biopsies were collected before RE and at 2, 24, and 48 h after RE. RE increased the phosphorylation of the p38-MNK1-eIF4E axis, an effect only evident with ACT recovery. Downstream, cyclin D1 protein, total eIF4E, upstream binding factor 1 (UBF1), and c-Myc proteins were all increased only after RE with ACT. This corresponded with elevated abundance of the pre-rRNAs (45S, ITS-28S, ITS-5.8S, and ETS-18S) from 24 h after RE with ACT. In conclusion, coordinated upstream signaling and activation of transcriptional factors stimulated pre-rRNA expression after RE. CWI, as a recovery strategy, markedly blunted these events, suggesting that suppressed ribosome biogenesis may be one factor contributing to the impaired hypertrophic response observed when CWI is used regularly after exercise.

Role of Magmas in protein transport and human mitochondria biogenesis

Magmas, a conserved mammalian protein essential for eukaryotic development, is overexpressed in prostate carcinomas and cells exposed to granulocyte-macrophage colony-stimulating factor (GM-CSF). Reduced Magmas expression resulted in decreased proliferative rates in cultured cells. However, the cellular function of Magmas is still elusive. In this report, we have showed that human Magmas is an ortholog of Saccharomyces cerevisiae Pam16 having similar functions and is critical for protein translocation across mitochondrial inner membrane. Human Magmas shows a complete growth complementation of delta pam16 yeast cells at all temperatures. On the basis of our analysis, we report that Magmas localizes into mitochondria and is peripherally associated with inner mitochondrial membrane in yeast and humans. Magmas forms a stable subcomplex with J-protein Pam18 or DnaJC19 through its C-terminal region and is tethered to TIM23 complex of yeast and humans. Importantly, amino acid alterations in Magmas leads to reduced stability of the subcomplex with Pam18 that results in temperature sensitivity and in vivo protein translocation defects in yeast cells. These observations highlight the central role of Magmas in protein import and mitochondria biogenesis. In humans, absence of a functional DnaJC19 leads to dilated cardiac myophathic syndrome (DCM), a genetic disorder with characteristic features of cardiac myophathy and neurodegeneration. We propose that the mutations resulting in decreased stability of functional Magmas:DnaJC19 subcomplex at human TIM23 channel leads to impaired protein import and cellular respiration in DCM patients. Together, we propose a model showing how Magmas:DnaJC19 subcomplex is associated with TIM23 complex and thus regulates mitochondrial import process.