Calcium-release channels in paramecium. Genomic expansion, differential positioning and partial transcriptional elimination

The release of Ca²⁺ from internal stores is a major source of signal Ca²⁺ in almost all cell types. The internal Ca²⁺ pools are activated via two main families of intracellular Ca²⁺-release channels, the ryanodine and the inositol 1,4,5-trisphosphate (InsP₃) receptors. Among multicellular organisms these channel types are ubiquitous, whereas in most unicellular eukaryotes the identification of orthologs is impaired probably due to evolutionary sequence divergence. However, the ciliated protozoan Paramecium allowed us to prognosticate six groups, with a total of 34 genes, encoding proteins with characteristics typical of InsP₃ and ryanodine receptors by BLAST search of the Paramecium database. We here report that these Ca²⁺-release channels may display all or only some of the characteristics of canonical InsP₃ and ryanodine receptors. In all cases, prediction methods indicate the presence of six trans-membrane regions in the C-terminal domains, thus corresponding to canonical InsP₃ receptors, while a sequence homologous to the InsP₃-binding domain is present only in some types. Only two types have been analyzed in detail previously. We now show, by using antibodies and eventually by green fluorescent protein labeling, that the members of all six groups localize to distinct organelles known to participate in vesicle trafficking and, thus, may provide Ca²⁺ for local membrane-membrane interactions. Whole genome duplication can explain radiation within the six groups. Comparative and evolutionary evaluation suggests derivation from a common ancestor of canonical InsP₃ and ryanodine receptors. With one group we could ascertain, to our knowledge for the first time, aberrant splicing in one thoroughly analyzed Paramecium gene. This yields truncated forms and, thus, may indicate a way to pseudogene formation. No comparable analysis is available for any other, free-living or parasitic/pathogenic protozoan.     

Minimizing DNA contamination by using UNG-coupled quantitative real-time PCR on degraded DNA samples: application to ancient DNA studies

PCR analyses of ancient and degraded DNA suffer from their extreme sensitivity to contamination by modern DNA originating, in particular, from carryover contamination with previously amplified or cloned material. Any strategy for limiting carryover contamination would also have to be compatible with the particular requirements of ancient DNA analyses. These include the need (i) to amplify short PCR products due to template fragmentation; (ii) to clone PCR products in order to track possible base misincorporation resulting from damaged templates; and (iii) to avoid incomplete decontamination causing artifactual sequence transformation. Here we show that the enzymatic decontamination procedures based upon dUTP- and uracil-N-glycosylase (UNG) can be adapted to meet the specific requirements of ancient DNA research. Thus, efficiency can be improved to vastly reduce the amplification of fragments < or = 100 bp. Secondly, the use of an Escherichia coli strain deficient in both UNG and dUTPase allows for the cloning of uracil-containing PCR products and offers protection from plasmid DNA contamination, and, lastly, PCR products amplified from UNG-degraded material are free of misleading sequence modifications.     

PAK5 kinase is an inhibitor of MARK/Par-1, which leads to stable microtubules and dynamic actin

MARK/Par-1 is a kinase involved in development of embryonic polarity. In neurons, MARK phosphorylates tau protein and causes its detachment from microtubules, the tracks of axonal transport. Because the target sites of MARK on tau occur at an early stage of Alzheimer neurodegeneration, we searched for interaction partners of MARK. Here we report that MARK2 is negatively regulated by PAK5, a neuronal member of the p21-activated kinase family. PAK5 suppresses the activity of MARK2 toward its target, tau protein. The inhibition requires the binding between the PAK5 and MARK2 catalytic domains, but does not require phosphorylation. In transfected Chinese hamster ovary (CHO) cells both kinases show a vesicular distribution with partial colocalization on endosomes containing AP-1/2. Although MARK2 transfected alone destabilizes microtubules and stabilizes actin stress fibers, PAK5 keeps microtubules stable through the down-regulation of MARK2 but destabilizes the F-actin network so that stress fibers and focal adhesions disappear and cells develop filopodia. The results point to an inverse relationship between actin- and microtubule-related signaling by the PAK5 and MARK2 pathways that affect both cytoskeletal networks.     

Local atrial natriuretic peptide signaling prevents hypertensive cardiac hypertrophy in endothelial nitric-oxide synthase-deficient mice

The crucial functions of atrial natriuretic peptide (ANP) and endothelial nitric oxide/NO in the regulation of arterial blood pressure have been emphasized by the hypertensive phenotype of mice with systemic inactivation of either the guanylyl cyclase-A receptor for ANP (GC-A-/-) or endothelial nitric-oxide synthase (eNOS-/-). Intriguingly, similar levels of arterial hypertension are accompanied by marked cardiac hypertrophy in GC-A-/-, but not in eNOS-/-, mice, suggesting that changes in local pathways regulating cardiac growth accelerate cardiac hypertrophy in the former and protect the heart of the latter. Our recent observations in mice with conditional, cardiomyocyte-restricted GC-A deletion demonstrated that ANP locally inhibits cardiomyocyte growth. Abolition of these local, protective effects may enhance the cardiac hypertrophic response of GC-A-/- mice to persistent increases in hemodynamic load. Notably, eNOS-/- mice exhibit markedly increased cardiac ANP levels, suggesting that increased activation of cardiac GC-A can prevent hypertensive heart disease. To test this hypothesis, we generated mice with systemic inactivation of eNOS and cardiomyocyte-restricted deletion of GC-A by crossing eNOS-/- and cardiomyocyte-restricted GC-A-deficient mice. Cardiac deletion of GC-A did not affect arterial hypertension but significantly exacerbated cardiac hypertrophy and fibrosis in eNOS-/- mice. This was accompanied by marked cardiac activation of both the mitogen-activated protein kinase (MAPK) ERK 1/2 and the phosphatase calcineurin. Our observations suggest that local ANP/GC-A/cyclic GMP signaling counter-regulates MAPK/ERK- and calcineurin/nuclear factor of activated T cells-dependent pathways of cardiac myocyte growth in hypertensive eNOS-/- mice.     

Clathrin- and caveolin-1-independent endocytosis: entry of simian virus 40 into cells devoid of caveolae

Simian Virus 40 (SV40) has been shown to enter host cells by caveolar endocytosis followed by transport via caveosomes to the endoplasmic reticulum (ER). Using a caveolin-1 (cav-1)-deficient cell line (human hepatoma 7) and embryonic fibroblasts from a cav-1 knockout mouse, we found that in the absence of caveolae, but also in wild-type embryonic fibroblasts, the virus exploits an alternative, cav-1-independent pathway. Internalization was rapid (t1/2 = 20 min) and cholesterol and tyrosine kinase dependent but independent of clathrin, dynamin II, and ARF6. The viruses were internalized in small, tight-fitting vesicles and transported to membrane-bounded, pH-neutral organelles similar to caveosomes but devoid of cav-1 and -2. The viruses were next transferred by microtubule-dependent vesicular transport to the ER, a step that was required for infectivity. Our results revealed the existence of a virus-activated endocytic pathway from the plasma membrane to the ER that involves neither clathrin nor caveolae and that can be activated also in the presence of cav-1.     

Hyperphosphorylation and aggregation of tau in experimental autoimmune encephalomyelitis

Axonal damage is a major morphological correlate and cause of permanent neurological deficits in patients with multiple sclerosis (MS), a multifocal, inflammatory and demyelinating disease of the central nervous system. Hyperphosphorylation and pathological aggregation of microtubule-associated protein tau is a common feature of many neurodegenerative diseases with axonal degeneration including Alzheimer's disease. We have therefore analyzed tau phosphorylation, solubility and distribution in the brainstem of rats with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Tau was hyperphosphorylated at several sites also phosphorylated in Alzheimer's disease and became partially detergent-insoluble in EAE brains. Morphological examination demonstrated accumulation of amorphous deposits of abnormally phosphorylated tau in the cell body and axons of neurons within demyelinating plaques. Hyperphosphorylation of tau was accompanied by up-regulation of p25, an activator of cyclin-dependent kinase 5. Phosphorylation of tau, activation of cdk5, and axonal pathology were significantly reduced when diseased rats were treated with prednisolone, a standard therapy of acute relapses in MS. Hyperphosphorylation of tau was not observed in a genetic or nutritional model of axonal degeneration or demyelination, suggesting that inflammation as detected in the brains of rats with EAE is the specific trigger of tau pathology. In summary, our data provide evidence that axonal damage in EAE and possibly MS is linked to tau pathology.     

Human Tau Isoforms Assemble into Ribbon-like Fibrils That Display Polymorphic Structure and Stability

Fibrous aggregates of Tau protein are characteristic features of Alzheimer disease. We applied high resolution atomic force and EM microscopy to study fibrils assembled from different human Tau isoforms and domains. All fibrils reveal structural polymorphism; the “thin twisted” and “thin smooth” fibrils resemble flat ribbons (cross-section ∼10 × 15 nm) with diverse twist periodicities. “Thick fibrils” show periodicities of ∼65–70 nm and thicknesses of ∼9–18 nm such as routinely reported for “paired helical filaments” but structurally resemble heavily twisted ribbons. Therefore, thin and thick fibrils assembled from different human Tau isoforms challenge current structural models of paired helical filaments. Furthermore, all Tau fibrils reveal axial subperiodicities of ∼17–19 nm and, upon exposure to mechanical stress or hydrophobic surfaces, disassemble into uniform fragments that remain connected by thin thread-like structures (∼2 nm). This hydrophobically induced disassembly is inhibited at enhanced electrolyte concentrations, indicating that the fragments resemble structural building blocks and the fibril integrity depends largely on hydrophobic and electrostatic interactions. Because full-length Tau and repeat domain constructs assemble into fibrils of similar thickness, the “fuzzy coat” of Tau protein termini surrounding the fibril axis is nearly invisible for atomic force microscopy and EM, presumably because of its high flexibility.     

Both thioredoxin 2 and glutaredoxin 2 contribute to the reduction of the mitochondrial 2-Cys peroxiredoxin Prx3

The proteins from the thioredoxin family are crucial actors in redox signaling and the cellular response to oxidative stress. The major intracellular source for oxygen radicals are the components of the respiratory chain in mitochondria. Here, we show that the mitochondrial 2-Cys peroxiredoxin (Prx3) is not only substrate for thioredoxin 2 (Trx2), but can also be reduced by glutaredoxin 2 (Grx2) via the dithiol reaction mechanism. Grx2 reduces Prx3 exhibiting catalytic constants (K(m), 23.8 μmol·liter(-1); V(max), 1.2 μmol·(mg·min)(-1)) similar to Trx2 (K(m), 11.2 μmol·liter(-1); V(max), 1.1 μmol·(mg·min)(-1)). The reduction of the catalytic disulfide of the atypical 2-Cys Prx5 is limited to the Trx system. Silencing the expression of either Trx2 or Grx2 in HeLa cells using specific siRNAs did not change the monomer:dimer ratio of Prx3 detected by a specific 2-Cys Prx redox blot. Only combined silencing of the expression of both proteins led to an accumulation of oxidized protein. We further demonstrate that the distribution of Prx3 in different mouse tissues is either linked to the distribution of Trx2 or Grx2. These results introduce Grx2 as a novel electron donor for Prx3, providing further insights into pivotal cellular redox signaling mechanisms.