Elena S. Suvorova

Cytoprotective Nrf2 Pathway Is Induced In Chronically Txnrd 1-Deficient Hepatocytes

Background Metabolically active cells require robust mechanisms to combat oxidative stress. The cytoplasmic thioredoxin reductase/thioredoxin (Txnrd1/Txn1) system maintains reduced protein dithiols and provides electrons to some cellular reductases, including peroxiredoxins. Principal Findings Here we generated mice in which the txnrd1 gene, encoding Txnrd1, was specifically disrupted in all parenchymal hepatocytes. Txnrd1-deficient livers exhibited a transcriptome response in which 56 mRNAs were induced and 12 were repressed. Based on the global hybridization profile, this represented only 0.3% of the liver transcriptome. Since most liver mRNAs were unaffected, compensatory responses were evidently effective. Nuclear pre-mRNA levels indicated the response was transcriptional. Twenty-one of the induced genes contained known antioxidant response elements (AREs), which are binding sites for the oxidative and chemical stress-induced transcription factor Nrf2. Txnrd1-deficient livers showed increased accumulation of nuclear Nrf2 protein and chromatin immunoprecipitation on the endogenous nqo1 and aox1 promoters in fibroblasts indicated that Txnrd1 ablation triggered in vivo assembly of Nrf2 on each. Conclusions Chronic deletion of Txnrd1 results in induction of the Nrf2 pathway, which contributes to an effective compensatory response.

Hepatocytes lacking thioredoxin reductase 1 have normal replicative potential during development and regeneration

Cells require ribonucleotide reductase (RNR) activity for DNA replication. In bacteria, electrons can flow from NADPH to RNR by either a thioredoxin-reductase- or a glutathione-reductase-dependent route. Yeast and plants artificially lacking thioredoxin reductases exhibit a slow-growth phenotype, suggesting glutathione-reductase-dependent routes are poor at supporting DNA replication in these organisms. We have studied proliferation of thioredoxin-reductase-1 (Txnrd1)-deficient hepatocytes in mice. During development and regeneration, normal mice and mice having Txnrd1-deficient hepatocytes exhibited similar liver growth rates. Proportions of hepatocytes that immunostained for PCNA, phosphohistone H3 or incorporated BrdU were also similar, indicating livers of either genotype had similar levels of proliferative, S and M phase hepatocytes, respectively. Replication was blocked by hydroxyurea, confirming that RNR activity was required by Txnrd1-deficient hepatocytes. Regenerative thymidine incorporation was similar in normal and Txnrd1-deficient livers, further indicating that DNA synthesis was unaffected. Using genetic chimeras in which a fluorescently marked subset of hepatocytes was Txnrd1-deficient while others were not, we found that the multigenerational contributions of both hepatocyte types to development and to liver regeneration were indistinguishable. We conclude that, in mouse hepatocytes, a Txnrd1-independent route for the supply of electrons to RNR can fully support DNA replication and normal proliferative growth.

Cre activity in fetal albCre mouse hepatocytes: Utility for developmental studies.

The albCre transgene, having Cre recombinase driven by the serum albumin (alb) gene promoter, is commonly used to generate adult mice having reliable hepatocyte‐specific recombination of loxP‐flanked (“floxed”) alleles. Based on previous studies, it has been unclear whether albCre transgenes are also reliable in fetal and juvenile mice. Perinatal liver undergoes a dynamic transition from being predominantly hematopoietic to predominantly hepatic. We evaluated Cre activity during this transition in albCre mice using a sensitive two‐color fluorescent reporter system. From fetal through adult stages, in situ patterns of Cre‐dependent recombination of the reporter closely matched expression of endogenous Alb mRNA or protein, indicating most or all hepatocytes, including those in fetal and juvenile livers, had expressed Cre and recombined the reporter. Our results indicate the albCre transgene is effective in converting simple floxed alleles in fetal and neonatal mice and is an appropriate tool for studies on hepatocyte development. genesis 47:789–792, 2009.

Different Endocytosis Pathways of the C5a Receptor and the N-formyl Peptide Receptor

Two chemoattractant receptors, C5aR (the complement fragment C5a receptor) and FPR (the N‐formyl peptide receptor), are involved in neutrophil activation at sites of inflammation. In this study, we found major differences in the intracellular trafficking of the receptors in transfected Chinese hamster ovary (CHO) cells. Western blot analysis showed that FPR was stable during a 3 h stimulation with ligand, but C5aR was reduced in quantity by 50%. Not all C5aR was targeted directly for degradation however; a small, but visible fraction of the receptor became re‐phosphorylated upon subsequent addition of ligand, suggesting that some of the receptor had cycled to the cell surface. Light membrane fractions isolated from activated cells showed C5aR distribution at the bottom of a glycerol gradient, colocalizing with the main distribution of the late endosomal/lysosomal marker LAMP2, whereas FPR was found at the bottom of the gradient as well as in the middle of the gradient, where it cofractionated with the early/sorting endosomal marker Rab5. Using fluorescence microscopy, we observed ligand‐dependent redistribution of C5aR‐EGFP from the plasma membrane to LAMP2‐positive compartments, whereas FPR‐EGFP showed significant colocalization with the early/sorting endosomes. Analysis of endogenous C5aR and FPR in neutrophils revealed a pattern similar to the CHO transfectants: C5aR underwent degradation after prolonged ligand stimulation, while FPR did not. Finally, we confirmed the down‐regulation of C5aR in a functional assay by showing reduced chemotaxis toward C5a in both CHO transfectants and neutrophils after preincubation with C5a. A similar decrease in FPR‐mediated chemotaxis was not observed.

Discovery of a splicing regulator required for cell cycle progression.

In the G1 phase of the cell division cycle, eukaryotic cells prepare many of the resources necessary for a new round of growth including renewal of the transcriptional and protein synthetic capacities and building the machinery for chromosome replication. The function of G1 has an early evolutionary origin and is preserved in single and multicellular organisms, although the regulatory mechanisms conducting G1 specific functions are only understood in a few model eukaryotes. Here we describe a new G1 mutant from an ancient family of apicomplexan protozoans. Toxoplasma gondii temperature-sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. Transcriptome studies demonstrate that gene expression is downregulated in the mutant at the restrictive temperature due to a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. This study demonstrates investigations into diverse unicellular eukaryotes, like the Apicomplexa, have the potential to yield new insights into important mechanisms conserved across modern eukaryotic kingdoms.

Nuclear actin-related protein is required for chromosome segregation in Toxoplasma gondii.

Apicomplexa parasites use complex cell cycles to replicate that are not well understood mechanistically. We have established a robust forward genetic strategy to identify the essential components of parasite cell division. Here we describe a novel temperature sensitive Toxoplasma strain, mutant 13-20C2, which growth arrests due to a defect in mitosis. The primary phenotype is the mis-segregation of duplicated chromosomes with chromosome loss during nuclear division. This defect is conditional-lethal with respect to temperature, although relatively mild in regard to the preservation of the major microtubule organizing centers. Despite severe DNA loss many of the physical structures associated with daughter budding and the assembly of invasion structures formed and operated normally at the non-permissive temperature before completely arresting. These results suggest there are coordinating mechanisms that govern the timing of these events in the parasite cell cycle. The defect in mutant 13-20C2 was mapped by genetic complementation to Toxoplasma chromosome III and to a specific mutation in the gene encoding an ortholog of nuclear actin-related protein 4. A change in a conserved isoleucine to threonine in the helical structure of this nuclear actin related protein leads to protein instability and cellular mis-localization at the higher temperature. Given the age of this protist family, the results indicate a key role for nuclear actin-related proteins in chromosome segregation was established very early in the evolution of eukaryotes.

Initial stages of 2,4,6-trinitrotoluene transformation by microorganisms

Screening of a wide range of microorganisms (32 strains) isolated from various anthropogenic and natural environments and of a number of collection strains showed that the early stages of 2,4,6-trinitrotoluene (TNT) transformation by the majority of the strains studied resulted in the formation of hydroxylaminodinitrotoluenes (HADNTs). The levels of HADNTs were in a number of cases comparable to the initial TNT level. The alternative reductive attack on TNT through the reduction of the aromatic ring was not characteristic of most of the prokaryotes studied. The susceptibility to the toxic effect of TNT was different for gram-positive and gram-negative bacteria.

Role of the carboxyl terminal di-leucine in phosphorylation and internalization of C5a receptor.

The carboxyl tail of G protein-coupled receptors contains motifs that regulate receptor interactions with intracellular partners. Activation of the human neutrophil complement fragment C5a receptor (C5aR) is terminated by phosphorylation of the carboxyl tail followed by receptor internalization. In this study, we demonstrated that bulky hydrophobic residues in the membrane-proximal region of the C5aR carboxyl tail play an important role in proper structure and function of the receptor: Substitution of leucine 319 with alanine (L319A) resulted in receptor retention in the endoplasmic reticulum, whereas a L318A substitution allowed receptor transport to the cell surface, but showed slow internalization upon activation, presumably due to a defect in phosphorylation by both PKC and GRK. Normal agonist-induced activation of ERK1/2 and intracellular calcium release suggested that the L318A mutation did not affect receptor signaling. Binding of GRK2 and PKCbetaII to intracellular loop 3 of C5aR in vitro indicated that mutagenesis of L318 did not affect kinase binding. Limited proteolysis with trypsin revealed a conformational difference between wild type and mutant receptor. Our studies support a model in which the L318/L319 stabilizes an amphipathic helix (Q305-R320) in the membrane-proximal region of C5aR.

A nucleolar AAA-NTPase is required for parasite division

Apicomplexa division involves several distinct phases shared with other eukaryote cell cycles including a gap period (G1) prior to chromosome synthesis, although how progression through the parasite cell cycle is controlled is not understood. Here we describe a cell cycle mutant that reversibly arrests in the G1 phase. The defect in this mutant was mapped by genetic complementation to a gene encoding a novel AAA‐ATPase/CDC48 family member called TgNoAP1. TgNoAP1 is tightly regulated and expressed in the nucleolus during the G1/S phases. A tyrosine to a cysteine change upstream of the second AAA+ domain in the temperature sensitive TgNoAP1 allele leads to conditional protein instability, which is responsible for rapid cell cycle arrest and a primary defect in 28S rRNA processing as confirmed by knock‐in of the mutation back into the parent genome. The interaction of TgNoAP1 with factors of the snoRNP and R2TP complexes indicates this protein has a role in pre‐rRNA processing. This is a novel role for a cdc48‐related chaperone protein and indicates that TgNoAP1 may be part of a dynamic mechanism that senses the health of the parasite protein machinery at the initial steps of ribosome biogenesis and conveys that information to the parasite cell cycle checkpoint controls.

Toxoplasma gondii Cyclic AMP-Dependent Protein Kinase Subunit 3 Is Involved in the Switch from Tachyzoite to Bradyzoite Development

ABSTRACT Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects warm-blooded vertebrates, including humans. Asexual reproduction in T. gondii allows it to switch between the rapidly replicating tachyzoite and quiescent bradyzoite life cycle stages. A transient cyclic AMP (cAMP) pulse promotes bradyzoite differentiation, whereas a prolonged elevation of cAMP inhibits this process. We investigated the mechanism(s) by which differential modulation of cAMP exerts a bidirectional effect on parasite differentiation. There are three protein kinase A (PKA) catalytic subunits (TgPKAc1 to -3) expressed in T. gondii. Unlike TgPKAc1 and TgPKAc2, which are conserved in the phylum Apicomplexa, TgPKAc3 appears evolutionarily divergent and specific to coccidian parasites. TgPKAc1 and TgPKAc2 are distributed in the cytomembranes, whereas TgPKAc3 resides in the cytosol. TgPKAc3 was genetically ablated in a type II cyst-forming strain of T. gondii (PruΔku80Δhxgprt) and in a type I strain (RHΔku80Δhxgprt), which typically does not form cysts. The Δpkac3 mutant exhibited slower growth than the parental and complemented strains, which correlated with a higher basal rate of tachyzoite-to-bradyzoite differentiation. 3-Isobutyl-1-methylxanthine (IBMX) treatment, which elevates cAMP levels, maintained wild-type parasites as tachyzoites under bradyzoite induction culture conditions (pH 8.2/low CO2), whereas the Δpkac3 mutant failed to respond to the treatment. This suggests that TgPKAc3 is the factor responsible for the cAMP-dependent tachyzoite maintenance. In addition, the Δpkac3 mutant had a defect in the production of brain cysts in vivo, suggesting that a substrate of TgPKAc3 is probably involved in the persistence of this parasite in the intermediate host animals. IMPORTANCE Toxoplasma gondii is one of the most prevalent eukaryotic parasites in mammals, including humans. Parasites can switch from rapidly replicating tachyzoites responsible for acute infection to slowly replicating bradyzoites that persist as a latent infection. Previous studies have demonstrated that T. gondii cAMP signaling can induce or suppress bradyzoite differentiation, depending on the strength and duration of cAMP signal. Here, we report that TgPKAc3 is responsible for cAMP-dependent tachyzoite maintenance while suppressing differentiation into bradyzoites, revealing one mechanism underlying how this parasite transduces cAMP signals during differentiation. Toxoplasma gondii is one of the most prevalent eukaryotic parasites in mammals, including humans. Parasites can switch from rapidly replicating tachyzoites responsible for acute infection to slowly replicating bradyzoites that persist as a latent infection. Previous studies have demonstrated that T. gondii cAMP signaling can induce or suppress bradyzoite differentiation, depending on the strength and duration of cAMP signal. Here, we report that TgPKAc3 is responsible for cAMP-dependent tachyzoite maintenance while suppressing differentiation into bradyzoites, revealing one mechanism underlying how this parasite transduces cAMP signals during differentiation.