Glycogen synthase kinase homolog Rim11 regulates lipid synthesis through the phosphorylation of Pah1 phosphatidate phosphatase in yeast

Pah1 phosphatidate (PA) phosphatase plays a major role in triacylglycerol synthesis in Saccharomyces cerevisiae by producing its precursor diacylglycerol and concurrently regulates de novo phospholipid synthesis by consuming its precursor PA. The function of Pah1 requires its membrane localization, which is controlled by its phosphorylation state. Pah1 is dephosphorylated by the Nem1-Spo7 protein phosphatase, whereas its phosphorylation occurs by multiple known and unknown protein kinases. In this work, we show that Rim11, a yeast homolog of mammalian glycogen synthase kinase-3β, is a protein kinase that phosphorylates Pah1 on serine (Ser12, Ser602, and Ser818) and threonine (Thr163, Thr164, Thr522) residues. Enzymological characterization of Rim11 showed that its K_m for Pah1 (0.4 μM) is similar to those of other Pah1-phosphorylating protein kinases, but its K_m for ATP (30 μM) is significantly higher than those of these same kinases. Furthermore, we demonstrate Rim11 phosphorylation of Pah1 does not require substrate prephosphorylation but was increased ∼2-fold upon its prephosphorylation by the Pho85-Pho80 protein kinase. In addition, we show Rim11-phosphorylated Pah1 was a substrate for dephosphorylation by Nem1-Spo7. Finally, we demonstrate the Rim11 phosphorylation of Pah1 exerted an inhibitory effect on its PA phosphatase activity by reduction of its catalytic efficiency. Mutational analysis of the major phosphorylation sites (Thr163, Thr164, and Ser602) indicated that Rim11-mediated phosphorylation at these sites was required to ensure Nem1-Spo7-dependent localization of the enzyme to the membrane. Overall, these findings advance our understanding of the phosphorylation-mediated regulation of Pah1 function in lipid synthesis.     

Phosphatidic Acid Mediates the Nem1-Spo7/Pah1 Phosphatase Cascade in Yeast Lipid Synthesis

In the yeast Saccharomyces cerevisiae, the PAH1-encoded Mg²⁺-dependent phosphatidate (PA) phosphatase Pah1 regulates the bifurcation of PA to diacylglycerol (DAG) for triacylglycerol (TAG) synthesis and to CDP-DAG for phospholipid synthesis. Pah1 function is mainly regulated via control of its cellular location by phosphorylation and dephosphorylation. Pah1 phosphorylated by multiple protein kinases is sequestered in the cytosol apart from its substrate PA in the membrane. The phosphorylated Pah1 is then recruited and dephosphorylated by the protein phosphatase complex Nem1 (catalytic subunit)-Spo7 (regulatory subunit) in the endoplasmic reticulum. The dephosphorylated Pah1 hops onto and scoots along the membrane to recognize PA for its dephosphorylation to DAG. Here, we developed a proteoliposome model system that mimics the Nem1-Spo7/Pah1 phosphatase cascade to provide a tool for studying Pah1 regulation. Purified Nem1-Spo7 was reconstituted into phospholipid vesicles prepared in accordance with the phospholipid composition of the nuclear/endoplasmic reticulum membrane. The Nem1-Spo7 phosphatase reconstituted in the proteoliposomes, which were measured 60 nm in an average diameter, was catalytically active on Pah1 phosphorylated by Pho85-Pho80, and its active site was located at the external side of the phospholipid bilayer. Moreover, we determined that PA stimulated the Nem1-Spo7 activity, and the regulatory effect was governed by the nature of the phosphate headgroup but not by the fatty acyl moiety of PA. The reconstitution system for the Nem1-Spo7/Pah1 phosphatase cascade, which starts with the phosphorylation of Pah1 by Pho85-Pho80 and ends with the production of DAG, is a significant advance to understand a regulatory cascade in yeast lipid synthesis.Supplementary key words: phosphatidate, diacylglycerol, triacylglycerol, phosphatidate phosphatase, Pho85-Pho80, Nem1-Spo7 protein phosphatase, endoplasmic reticulum, phospholipid bilayer, reconstitution, proteoliposome     

Proportional Upregulation of CD97 Isoforms in Glioblastoma and Glioblastoma-Derived Brain Tumor Initiating Cells

CD97 is a novel glioma antigen that confers an invasive phenotype and poor survival in patients with glioblastoma (GBM), the most aggressive primary malignant brain tumor. The short isoform of CD97, known as EGF(1,2,5), has been shown to promote invasion and metastasis, but its role in gliomas and GBM-derived brain tumor initiating cells (BTICs) has not been studied. We sought to characterize CD97 expression among gliomas and identify the specific isoforms expressed. The short isoform of CD97 was identified in GBM and GBM-derived BTICs, but not low grade or anaplastic astrocytomas. All samples expressing the EGF(1,2,5) isoform were also found to express the EGF(1,2,3,5) isoform. These isoforms are believed to possess similar ligand binding patterns and interact with chondroitin sulfate, a component of the extracellular matrix, and the integrin α5β1. Using data acquired from the Cancer Genome Atlas (TCGA), we show that CD97 is upregulated among the classical and mesenchymal subtypes of GBM and significantly decreased among IDH1 mutant GBMs. Given its proven roles in tumor invasion, expression among aggressive genetic subtypes of GBM, and association with overall survival, CD97 is an attractive therapeutic target for patients with GBM.     

Is Aberrant DNA Methylation a Key Factor in Molar Incisor Hypomineralization?

Molar incisor hypomineralization (MIH) is a qualitative disturbance of the enamel of the permanent molars and/or incisors. Its etiology is not clearly defined but is connected with different factors occurring before and after birth. It remains difficult to identify a single factor or group of factors, and the problem is further complicated by various overlapping mechanisms. In this study, we attempted to determine whether DNA methylation—an epigenetic mechanism—plays a key role in the etiology of MIH. We collected the epithelium of the oral mucosa from children with MIH and healthy individuals and analyzed its global DNA methylation level in each child using a 5-mC DNA ELISA kit after DNA isolation. There was no statistically significant difference between the global DNA methylation levels in the study and control groups. Then, we also analyzed the associations of the DNA methylation levels with different prenatal, perinatal, and postnatal factors, using appropriate statistical methods. Factors such as number of pregnancies, number of births, type of delivery, varicella infection (under 3 years old), and high fever (under 3 years old) were significantly important. This work can be seen as the first step towards further studies of the epigenetic background of the MIH etiology.     

The potential function of post-fledging dispersal behavior in first breeding territory selection for males of a migratory bird

One possible hypothesis for the function of post-fledging dispersal is to locate a suitable future breeding area. This post-fledging period may be particularly important in migratory species because they have a limited period to gather information prior to autumn migration, and in protandrous species, males must quickly acquire a territory after returning from spring migration to maximize their fitness. Here we use color-ring resightings to investigate how the post-fledging dispersal movements of the Cyprus wheatear Oenanthe cypriaca, a small migratory passerine, relate to their first breeding territory the following year when they return from migration. We found that males established first breeding territories that were significantly closer to their post-fledging location than to their natal sites or to post-fledging locations of other conspecifics, but these patterns were not apparent in females. Our findings suggest that familiarity with potential breeding sites may be important for juveniles of migratory species, particularly for the sex that acquires and advertises breeding territories. Exploratory dispersal prior to a migrant’s first autumn migration may contribute toward its breeding success the following year, further highlighting the importance of early seasonal breeding on fitness and population dynamics more generally.     

Anti-oxidant activity of superoxide dismutase and glutathione peroxidase enzymes in skeletal muscles from slaughter cattle infected with Taenia saginata

It is known that highly reactive oxygene species produced during normal cellular metabolism represent a powerful effector mechanism against parasites. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) belong to the main defense anti-oxidants that prevent the formation of new free radical species. The aim of this study was to assess the activities of SOD and GPx in cattle tissues infected with Taenia saginata. We observed a statistically significant increase in the SOD and GPx activities (p = 0.00003, 0.00008, respectively, Student’s t-test) in skeletal muscles infected with T. saginata in spectrophotometric analysis. With the use of western blot technique, SOD synthesis stimulation has appeared in the host tissues containing cysticerci in contrast with the control samples. There was no statistically significant increase in the GPx band intensity observed in the studied samples in comparison to controls (Gene Tools Version 4.01 program). These results support the significance of anti-oxidant processes in host defense mechanism during parasitic infections.     

Evaluation of anti-human antibodies for immunohistochemistry on archival nonhuman primate tissues

Abstract: A panel of commercially available antibodies which recognize specific antigens on human tissues was developed for use in immunohistochemistry on tissues from eight species of nonhuman primates. Antibodies were selected for potential usefulness in diagnostic pathology, and for effectiveness in formalin-fixed, paraffin-embedded tissues. Tissues from four species of macaques and four New World monkeys were evaluated. Using these antibodies we were able to identify 17/21 antigens examined in all eight species, and 21/21 antigens in the four species of macaques. Detailed immunohistochemistry protocols are presented, along with a systematic approach to developing a protocol for a new antibody.