Involvement of Protein Tyrosine Phosphatases BcPtpA and BcPtpB in Regulation of Vegetative Development,Virulence and Multi-Stress Tolerance in Botrytis cinerea

Tyrosine phosphorylation and dephosphorylation have emerged as fundamentally important mechanisms of signal transduction and regulation in eukaryotic cells, governing many processes, but little has been known about their functions in filamentous fungi. In this study, we deleted two putative protein tyrosine phosphatase (PTP) genes (BcPTPA and BcPTPB) in Botrytis cinerea, encoding the orthologs of Saccharomyces cerevisiae Ptp2 and Ptp3, respectively. Although BcPtpA and BcPtpB have opposite functions in conidiation, they are essential for sclerotial formation in B. cinerea. BcPTPA and BcPTPB deletion mutants ΔBcPtpA-10 and ΔBcPtpB-4 showed significantly increased sensitivity to osmotic and oxidative stresses, and to cell wall damaging agents. Inoculation tests showed that both mutants exhibited dramatically decreased virulence on tomato leaves, apples and grapes. In S. cerevisiae, it has been shown that Ptp2 and Ptp3 negatively regulate the high-osmolarity glycerol (HOG) pathway and the cell wall integrity (CWI) pathway. Although both BcPtpA and BcPtpB were able to inactive Hog1 and Mpk1 in S. cerevisiae, in contrast to S. cerevisiae, they positively regulate phosphorylation of BcSak1 (the homologue of Hog1) and BcBmp3 (the homologue of Mpk1) in B. cinerea under stress conditions. These results demonstrated that functions of PTPs in B. cinerea are different from those in S. cerevisiae, and BcPtpA and BcPtpB play important roles in regulation of vegetative development, virulence and in adaptation to oxidative, osmotic and cell-wall damage stresses in B. cinerea.     

Activation of AMPK by Bitter Melon Triterpenoids Involves CaMKKβ

We recently showed that bitter melon-derived triterpenoids (BMTs) activate AMPK and increase GLUT4 translocation to the plasma membrane in vitro, and improve glucose disposal in insulin resistant models in vivo. Here we interrogated the mechanism by which these novel compounds activate AMPK, a leading anti-diabetic drug target. BMTs did not activate AMPK directly in an allosteric manner as AMP or the Abbott compound (A-769662) does, nor did they activate AMPK by inhibiting cellular respiration like many commonly used anti-diabetic medications. BMTs increased AMPK activity in both L6 myotubes and LKB1-deficient HeLa cells by 20–35%. Incubation with the CaMKKβ inhibitor, STO-609, completely attenuated this effect suggesting a key role for CaMKKβ in this activation. Incubation of L6 myotubes with the calcium chelator EGTA-AM did not alter this activation suggesting that the BMT-dependent activation was Ca²⁺-independent. We therefore propose that CaMKKβ is a key upstream kinase for BMT-induced activation of AMPK.     

Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein

Hepatitis C virus (HCV) co-opts hepatic lipid pathways to facilitate its pathogenesis. The virus alters cellular lipid biosynthesis and trafficking, and causes an accumulation of lipid droplets (LDs) that gives rise to hepatic steatosis. Little is known about how these changes are controlled at the molecular level, and how they are related to the underlying metabolic states of the infected cell. The HCV core protein has previously been shown to independently induce alterations in hepatic lipid homeostasis. Herein, we demonstrate, using coherent anti-Stokes Raman scattering (CARS) microscopy, that expression of domain 2 of the HCV core protein (D2) fused to GFP is sufficient to induce an accumulation of larger lipid droplets (LDs) in the perinuclear region. Additionally, we performed fluorescence lifetime imaging of endogenous reduced nicotinamide adenine dinucleotides [NAD(P)H], a key coenzyme in cellular metabolic processes, to monitor changes in the cofactor’s abundance and conformational state in D2-GFP transfected cells. When expressed in Huh-7 human hepatoma cells, we observed that the D2-GFP induced accumulation of LDs correlated with an increase in total NAD(P)H fluorescence and an increase in the ratio of free to bound NAD(P)H. This is consistent with an approximate 10 fold increase in cellular NAD(P)H levels. Furthermore, the lifetimes of bound and free NAD(P)H were both significantly reduced – indicating viral protein-induced alterations in the cofactors’ binding and microenvironment. Interestingly, the D2-expressing cells showed a more diffuse localization of NAD(P)H fluorescence signal, consistent with an accumulation of the co-factor outside the mitochondria. These observations suggest that HCV causes a shift of metabolic control away from the use of the coenzyme in mitochondrial electron transport and towards glycolysis, lipid biosynthesis, and building of new biomass. Overall, our findings demonstrate that HCV induced alterations in hepatic metabolism is tightly linked to alterations in NAD(P)H functional states.     

Methylation of KvDMR1 involved in regulating the imprinting of CDKN1C gene in cattle

The CDKN1C gene encodes a cyclin‐dependent kinase inhibitor and is one of the key genes involved in the development of Beckwith–Wiedemann syndrome and cancer. In this study, using a direct sequencing approach based on a single nucleotide polymorphism (SNP) at genomic DNA and cDNA levels, we show that CDKN1C exhibits monoallelic expression in all seven studied organs (heart, liver, spleen, lung, kidney, muscle and subcutaneous fat) in cattle. To investigate how methylation regulates imprinting of CDKN1C in cattle, allele‐specific methylation patterns in two putative differential methylation regions (DMRs), the CDKN1C DMR and KvDMR1, were analyzed in three tissues (liver, spleen and lung) using bisulfite sequencing PCR. Our results show that in the CDKN1C DMR both parental alleles were unmethylated in all three analyzed tissues. In contrast, KvDMR1 was differentially methylated between the two parental alleles in the same tissues. Statistical analysis showed that there is a significant difference in the methylation level between the two parental alleles (P < 0.01), confirming that this region is the DMR of KvDMR1 and that it may be correlated with CDKN1C imprinting.