The DNA damage-inducible UbL-UbA protein Ddi1 participates in Mec1-mediated degradation of Ho endonuclease

Ho endonuclease initiates a mating type switch by making a double-strand break at the mating type locus, MAT. Ho is marked by phosphorylation for rapid destruction by functions of the DNA damage response, MEC1, RAD9, and CHK1. Phosphorylated Ho is recruited for ubiquitylation via the SCF ubiquitin ligase complex by the F-box protein, Ufo1. Here we identify a further DNA damage-inducible protein, the UbL-UbA protein Ddi1, specifically required for Ho degradation. Ho interacts only with Ddi1; it does not interact with the other UbL-UbA proteins, Rad23 or Dsk2. Ho must be ubiquitylated to interact with Ddi1, and there is no interaction when Ho is produced in mec1 or Deltaufo1 mutants that do not support its degradation. Ddi1 binds the proteasome via its N-terminal ubiquitin-like domain (UbL) and interacts with ubiquitylated Ho via its ubiquitin-associated domain (UbA); both domains of Ddi1 are required for association of ubiquitylated Ho with the proteasome. Despite being a nuclear protein, Ho is exported to the cytoplasm for degradation. In the absence of Ddi1, ubiquitylated Ho is stabilized and accumulates in the cytoplasm. These results establish a role for Ddi1 in the degradation of a natural ubiquitylated substrate. The specific interaction between Ho and Ddi1 identifies an additional function associated with DNA damage involved in its degradation.     

Mitochondrial myopathy and sideroblastic anemia (MLASA): missense mutation in the pseudouridine synthase 1 (PUS1) gene is associated with the loss of tRNA pseudouridylation

A missense mutation in the PUS1 gene affecting a highly conserved amino acid has been associated with mitochondrial myopathy and sideroblastic anemia (MLASA), a rare autosomal recessive oxidative phosphorylation disorder. The PUS1 gene encodes the enzyme pseudouridine synthase 1 (Pus1p) that is known to pseudouridylate tRNAs in other species. Total RNA was isolated from lymphoblastoid cell lines established from patients, parents, unaffected siblings, and unrelated controls, and the tRNAs were assayed for the presence of pseudouridine (Psi) at the expected positions. Mitochondrial and cytoplasmic tRNAs from MLASA patients are lacking modification at sites normally modified by Pus1p, whereas tRNAs from controls, unaffected siblings, or parents all have Psi at these positions. In addition, there was no Pus1p activity in an extract made from a cell line derived from a patient with MLASA. Immunohistochemical staining of Pus1p in cell lines showed nuclear, cytoplasmic, and mitochondrial distribution of the protein, and there is no difference in staining between patients and unaffected family members. MLASA is thus associated with absent or greatly reduced tRNA pseudouridylation at specific sites, implicating this pathway in its molecular pathogenesis.     

Molecular systematics,GISH and the origin of hybrid taxa in Nicotiana (Solanaceae)

Phylogenetic relationships in the genus Nicotiana were investigated using parsimony analyses of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (nrDNA). In addition, origins of some amphidiploid taxa in Nicotiana were investigated using the techniques of genomic in situ hybridization (GISH), and the results of both sets of analyses were used to evaluate previous hypotheses about the origins of these taxa. Phylogenetic analyses of the ITS nrDNA data were performed on the entire genus (66 of 77 naturally occurring species, plus three artificial hybrids), comprising both diploid and polyploid taxa, and on the diploid taxa only (35 species) to examine the effects of amphidiploids on estimates of relationships. All taxa, regardless of ploidy, produced clean, single copies of the ITS region, even though some taxa are hybrids. Results are compared with a published plastid (matK) phylogeny using fewer, but many of the same, taxa. The patterns of relationships in Nicotiana, as seen in both analyses, are largely congruent with each other and previous evolutionary ideas based on morphology and cytology, but some important differences are apparent. None of the currently recognized subgenera of Nicotiana is monophyletic and, although most of the currently recognized sections are coherent, others are clearly polyphyletic. Relying solely upon ITS nrDNA analysis to reveal phylogenetic patterns in a complex genus such as Nicotiana is insufficient, and it is clear that conventional analysis of single data sets, such as ITS, is likely to be misleading in at least some respects about evolutionary history. ITS sequences of natural and well-documented amphidiploids are similar or identical to one of their two parents-usually, but not always, the maternal parent-and are not in any sense themselves 'hybrid'. Knowing how ITS evolves in artificial amphidiploids gives insight into what ITS analysis might reveal about naturally occurring amphidiploids of unknown origin, and it is in this perspective that analysis of ITS sequences is highly informative.     

Human genetic disease caused by de novo mitochondrial-nuclear DNA transfer

Transfer of nucleic acid from cytoplasmic organelles to the nuclear genome is a well-established mechanism of evolutionary change in eukaryotes. Such transfers have occurred throughout evolution, but so far, none has been shown unequivocally to occur de novo to cause a heritable human disease. We have characterized a patient with a de novo nucleic acid transfer from the mitochondrial to the nuclear genome, a transfer that is responsible for a sporadic case of Pallister-Hall syndrome, a condition usually inherited in an autosomal dominant fashion. This mutation, a 72-bp insertion into exon 14 of the GLI3 gene, creates a premature stop codon and predicts a truncated protein product. Both the mechanism and the cause of the mitochondrial-nuclear transfer are unknown. Although the conception of this patient was temporally and geographically associated with high-level radioactive contamination following the Chernobyl accident, this case cannot, on its own, be used to establish a causal relationship between radiation exposure and this rare type of mutation. Thus, for the time being, it must be considered as an intriguing coincidence. Nevertheless, these data serve to demonstrate that de novo mitochondrial-nuclear transfer of nucleic acid is a novel mechanism of human inherited disease.     

Functional reentry in cultured monolayers of neonatal rat cardiac cells

Previous studies of reentrant arrhythmias in the heart have been performed in computer models and tissue experiments. We hypothesized that confluent monolayers of cardiac cells can provide a simple, controlled, and reproducible experimental model of reentry. Neonatal rat ventricular cells were cultured on 22-mm-diameter coverslips and stained with the voltage-sensitive dye RH-237. Recordings of transmembrane potentials were obtained from 61 sites with the use of a contact fluorescence imaging system. An electrical field stimulus, followed by a point stimulus, induced 39 episodes of sustained reentry and 21 episodes of nonsustained reentry. Sustained reentry consisted of single-loop (n = 18 monolayers) or figure-of-eight (n = 4) patterns. The cycle length, action potential duration at 80% repolarization, and conduction velocity were (in means +/- SE) 358 +/- 33 ms, 118 +/- 12 ms, and 12.9 +/- 1.0 cm/s for single loop and 311 +/- 78 ms, 137 +/- 18 ms, and 7.8 +/- 1.3 cm/s for figure-of-eight, respectively. Electrical termination by 6- to 13-V/cm field pulses or 15- to 20-V point stimuli was successful in 60% of the attempts. In summary, highly stable reentry can be induced, sustained for extensive periods of time, and electrically terminated in monolayers of cultured neonatal rat cardiac myocytes.     

Mapping the interaction of bradykinin 1-5 with the exodomain of human protease activated receptor 4

The angiotensin converting enzyme breakdown product of bradykinin, bradykinin 1-5 (RPPGF), inhibits thrombin-induced human or mouse platelet aggregation. RPPGF binds to the exodomain of human protease-activated receptor 1 (PAR1). Studies determined if RPPGF also binds to the exodomain of human PAR4. RPPGF binds to a peptide of the thrombin cleavage site on PAR4. Recombinant wild-type and mutated exodomain of human PAR4 was prepared. The N-terminal arginine on RPPGF binds to the P2 position or proline46 on PAR4 to block thrombin cleavage. These data indicate that RPPGF influences thrombin activity by binding to the thrombin cleavage site on both PAR4 and PAR1.     

Missense mutation in pseudouridine synthase 1 (PUS1) causes mitochondrial myopathy and sideroblastic anemia (MLASA)

Mitochondrial myopathy and sideroblastic anemia (MLASA) is a rare, autosomal recessive oxidative phosphorylation disorder specific to skeletal muscle and bone marrow. Linkage analysis and homozygosity testing of two families with MLASA localized the candidate region to 1.2 Mb on 12q24.33. Sequence analysis of each of the six known genes in this region, as well as four putative genes with expression in bone marrow or muscle, identified a homozygous missense mutation in the pseudouridine synthase 1 gene (PUS1) in all patients with MLASA from these families. The mutation is the only amino acid coding change in these 10 genes that is not a known polymorphism, and it is not found in 934 controls. The amino acid change affects a highly conserved amino acid, and appears to be in the catalytic center of the protein, PUS1p. PUS1 is widely expressed, and quantitative expression analysis of RNAs from liver, brain, heart, bone marrow, and skeletal muscle showed elevated levels of expression in skeletal muscle and brain. We propose deficient pseudouridylation of mitochondrial tRNAs as an etiology of MLASA. Identification of the pathophysiologic pathways of the mutation in these families may shed light on the tissue specificity of oxidative phosphorylation disorders.     

Enhanced detection sensitivity using a novel solid-phase incorporated affinity fluorescent protein biosensor

We engineered green fluorescent protein (GFP) into affinity fluorescent proteins (aFPs) biosensors. The aFPs detect protein-protein interactions by enhanced fluorescence intensity. In a proof of principle demonstration, aFPs containing haemagglutinin (HA) tag bind specifically to the anti-HA antibody. The sensitivity and specificity is enhanced 28-fold by incorporation of aFPs into solid-phase surface.     

The nitric oxide donor sodium nitroprusside requires the 18?kDa Translocator Protein to induce cell death

Various studies have shown that several lethal agents induce cell death via the mitochondrial 18 kDa Translocator Protein (TSPO). In this study we tested the possibility that nitric oxide (NO) is the signaling component inducing the TSPO to initiate cell death process. Cell viability assays included Trypan blue uptake, propidium iodide uptake, lactate dehydrogenase release, and DNA fragmentation. These assays showed that application of the specific TSPO ligand PK 11195 reduced these parameters for the lethal effects of the NO donor sodium nitroprusside (SNP) by 41, 27, 40, and 42 %, respectively. TSPO silencing by siRNA also reduced the measured lethal effects of SNP by 50 % for all of these four assays. With 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxyanilide (XTT) changes in metabolic activity were detected. PK 11195 and TSPO knockdown fully prevented the reductions in XTT signal otherwise induced by SNP. Collapse of the mitochondrial membrane potential was studied with the aid of JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine chloride). PK 11195 and TSPO knockdown reduced, respectively by 36 and 100 %, the incidence of collapse of the mitochondrial membrane potential otherwise induced by SNP. 10-N-Nonyl-Acridine Orange (NAO) was used to detect mitochondrial reactive oxygen species generation due to SNP. PK 11195 and TSPO knockdown reduced this effect of SNP by 65 and 100 %, respectively. SNP did not affect TSPO protein expression and binding characteristics, and also did not cause TSPO S-nitrosylation. However, β-actin and various other proteins (not further defined) were S-nitrosylated. In conclusion, TSPO is required for the lethal and metabolic effects of the NO donor SNP, but TSPO itself is not S-nitrosylated.