In order to quantify the curing effects of phenanthridine on yeast prion, we introduced semi-denaturing agarose gel electrophoresis and fluorescence recovery after photobleaching techniques to quantify the curing effects of phenanthridine on yeast prion at the protein and cellular levels with the [PSI+] yeast strain expressing GFP-Sup35p (NGMC). The results showed that these two approaches could precisely quantify the curing effects of phenanthridine on [PSI+] cells. After a treatment for 1 through 5 days with phenanthridine, the curing rates of [PSI+] cells were 0%, 0%, 51.7%, 87.5% and 94.4%, respectively. Meanwhile, we quantified the sizes of Sup35p polymers in phenanthridine induced pink phenotype cells. The aggregation status in 1-2 days phenanthridine treated cells were similar to those in [PSI+] cells, while the aggregation status in 3-5 days phenanthridine treated cells were similar to those in [psi(-)] cells.
Computer Simulation ; Models, Biological ; Peptide Termination Factors ; metabolism ; Phenanthridines ; pharmacology ; Prions ; drug effects ; genetics ; metabolism ; Saccharomyces cerevisiae ; cytology ; drug effects ; metabolism ; Saccharomyces cerevisiae Proteins ; metabolism

Protozoan ciliates are a group of unicellular eukaryotes. The special characteristics of stop codons usage in termination of protein biosynthesis in ciliates cells makes them an ideal model to study the mechanism of stop codon recognition of polypeptides release factors. To localize the functional positions of biomolecules in ciliates cell, we constructed a macronuclear artificial chromosome containing a gene encoding red fluorescence protein (EoMAC_R) based on the structural characteristics of ciliates chromosome. Three factors, L11, eRF1a, and eRF3 that are involved in termination process of protein synthesis were colocalized in Euplotes octocarinatus cells by using novel EoMAC_R and the previously constructed EoMAC_G. The results indicated that protein synthesis mainly occurred inside the "C" shape macronucleus, suggesting that EoMAC could be a useful tool for localizing biomolecules in ciliates cell.
Chromosomes, Artificial ; Codon, Terminator ; metabolism ; Euplotes ; chemistry ; Peptide Termination Factors ; analysis ; genetics ; metabolism ; Peptides ; metabolism ; Protein Biosynthesis ; genetics ; Protozoan Proteins ; analysis ; genetics ; Ribosomal Proteins ; analysis ; genetics

Child ; Female ; Hereditary Sensory and Motor Neuropathy ; genetics ; Heterozygote ; Humans ; Mitochondrial Proteins ; genetics ; Mutation ; genetics ; Optic Atrophy ; genetics ; Peptide Termination Factors ; genetics

Sup35 in its native state is a translation termination factor in Saccharomyces cerevisiae. The prion domain of Sup35p can form amyloid-like proteinaceous fibrils in vitro and in vivo. Furthermore, the in-register cross beta-sheet structure of Sup35p amyloid fibrils is similar to those formed in other species. Therefore, studies on mechanism of Sup35p self-assembly can be an appropriate model to study protein misfolding-related diseases and prion biology. Because of its ability to self-assemble into nanowires, the prion domain of Sup35p has been widely used in biotechnology and nanotechnology.
Amino Acid Sequence ; Amyloid ; chemistry ; metabolism ; Molecular Sequence Data ; Peptide Termination Factors ; chemistry ; Prions ; chemistry ; Protein Conformation ; Saccharomyces cerevisiae ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; chemistry

Bacterial Proteins ; chemistry ; genetics ; metabolism ; DNA, Bacterial ; chemistry ; genetics ; metabolism ; Gene Expression Regulation, Bacterial ; physiology ; Glutathione ; metabolism ; Listeria monocytogenes ; chemistry ; genetics ; metabolism ; Peptide Termination Factors ; chemistry ; genetics ; metabolism

This article reports the clinical features and C12orf65 gene mutations of a girl with autosomal recessive spastic paraplegia-55. The 8-year-old girl experienced disease onset at the age of 5 years and had optic atrophy as the main clinical manifestation, with slow movements in standing up and a slight duck-shaped gait. Peripheral blood DNA samples were collected from this child and her parents and brother to perform high-throughput whole-exome sequencing and high-throughput mitochondrial genome sequencing. Sanger sequencing was performed for verification. The results showed two compound heterozygous mutations, c.394C>T and c.447_449delGGAinsGT, in the C12orf65 gene. The former mutation came from her father and was a known pathogenic mutation, and the latter came from her mother and was a novel mutation which had not been reported in literature. This study expands the mutation spectrum of the C12orf65 gene and thus provides a molecular basis for the etiological diagnosis of the child and the genetic counseling of the family.
Child ; Female ; High-Throughput Nucleotide Sequencing ; Humans ; Male ; Mitochondrial Proteins ; genetics ; Mutation ; Pedigree ; Peptide Termination Factors ; genetics ; Spastic Paraplegia, Hereditary ; genetics

BACKGROUNDTo investigate the dynamics of amyloid fiber formation of yeast (Saccharomyces cerevisiae) prion protein Sup35NM under the native condition to provide materials and clues for the elucidation of amyloid fiber formation.

METHODSThe Sup35NM gene was cloned and expressed in E. coli. The recombinant Sup35NM protein was purified under denaturing conditions through Nickel-Sepharose chromatography. Aliquots were removed at designated time points for transmission electron microscopy (TEM), circular dichroism (CD) spectra, protease K resistance assay, as well as thioflavin T (ThT) binding assay.

RESULTSThe Sup35NM expressed and purified under denaturing conditions. The morphological alteration of the Sup35NM in PBS (pH7.4) during the protein aggregation and amyloid fiber formation was visualized by TEM. The CD assay showed that the course of amyloid fiber formation underwent a conformational shift from alpha-helix to beta-sheet. The fibers had higher capacity of resistance to protease K digestion compared to the monomers. ThT fluorescence assay displayed a rapid growth phase before reaching a final equilibrium phase during the fiber formation, and the higher concentration of Sup35NM could greatly accelerate the fiber formation in vitro.

CONCLUSIONYeast prion protein Sup35NM forms amyloid readily under native conditions in vitro. The dynamics of Sup35NM amyloid formation may provide supporting evidences for the nucleating polymerization models of amyloid fiber formation.

Amyloid beta-Peptides ; genetics ; metabolism ; ultrastructure ; Electrophoresis, Polyacrylamide Gel ; Endopeptidase K ; metabolism ; Kinetics ; Microscopy, Electron ; Peptide Termination Factors ; Prions ; genetics ; metabolism ; ultrastructure ; Protein Binding ; Recombinant Proteins ; metabolism ; ultrastructure ; Saccharomyces cerevisiae ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; genetics ; metabolism ; ultrastructure ; Thiazoles ; metabolism