The role of Sse1 in the de novo formation and variant determination of the [PSI+] prion.

Yeast prions are a group of non-Mendelian genetic elements transmitted as altered and self-propagating conformations. Extensive studies in the last decade have provided valuable information on the mechanisms responsible for yeast prion propagation. How yeast prions are formed de novo and what cellular factors are required for determining prion "strains" or variants--a single polypeptide capable of existing in multiple conformations to result in distinct heritable phenotypes--continue to defy our understanding. We report here that Sse1, the yeast ortholog of the mammalian heat-shock protein 110 (Hsp110) and a nucleotide exchange factor for Hsp70 proteins, plays an important role in regulating [PSI+] de novo formation and variant determination. Overproduction of the Sse1 chaperone dramatically enhanced [PSI+] formation whereas deletion of SSE1 severely inhibited it. Only an unstable weak [PSI+] variant was formed in SSE1 disrupted cells whereas [PSI+] variants ranging from very strong to very weak were formed in isogenic wild-type cells under identical conditions. Thus, Sse1 is essential for the generation of multiple [PSI+] variants. Mutational analysis further demonstrated that the physical association of Sse1 with Hsp70 but not the ATP hydrolysis activity of Sse1 is required for the formation of multiple [PSI+] variants. Our findings establish a novel role for Sse1 in [PSI+] de novo formation and variant determination, implying that the mammalian Hsp110 may likewise be involved in the etiology of protein-folding diseases.

Protein quaternary structure determination using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and chemical crosslinking

A means of analyzing protein quaternary structure using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI MS) and chemical crosslinking was evaluated. Proteins of known oligomeric structure, as well as monomeric proteins, were analyzed to evaluate the method. The quaternary structure of proteins of unknown or uncertain structure was investigated using this technique. The stoichiometry of recombinant E. coli carbamoyl phosphate synthetase and recombinant human farnesyl protein transferase were determined to be heterodimers using glutaraldehyde crosslinking, agreeing with the stoichiometry found for the wild type proteins. The stoichiometry of the gamma subunit of E. coli DNA polymerase III holoenzyme was determined in solution without the presence of other subunits to be a homotetramer using glutaraldehyde crosslinking and MALDI MS analysis. Chi and psi subunits of E. coli DNA polymerase III subunits appeared to form a heterodimer when crosslinked with heterobifunctional photoreactive crosslinkers.^ Comparison of relative % peak areas obtained from MALDI MS analysis of crosslinked proteins and densitometric scanning of silver stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels showed excellent qualitative agreement for the two techniques, but the quantitative analyses differed, sometimes significantly. This difference in quantitation could be due to SDS-PAGE conditions (differential staining, loss of sample) or to MALDI MS conditions (differences in ionization and/or detection). Investigation of pre-purified crosslinked monomers and dimers recombined in a specific ratio revealed the presence of mass discrimination in the MALDI MS process. The calculation of mass discrimination for two different MALDI time-of-flight instruments showed the loss of a factor of approximately 2.6 in relative peak area as the m/z value doubles over the m/z range from 30,000 to 145,000 daltons.^ Indirect symmetry was determined for tetramers using glutaraldehyde crosslinking with MALDI MS analysis. Mathematical modelling and simple graphing allowed the determination of the symmetry for several tetramers known to possess isologous D2 symmetry. These methods also distinguished tetramers that did not fit D2 symmetry such as apo-avidin. The gamma tetramer of E. coli DNA polymerase III appears to have isologous D2 symmetry. ^

Role of cytochrome P450 in DNA damage produced by treatment of colon cells with 1,2-dimethylhydrazine

The study of colon cancer has taken advantage of the development of a model in animals in which tumors in the colon are easily induced by chemical treatment. When 1,2-dimethylhydrazine (DMH) is injected into rats tumor growth is observed in colon in preference to other tissues. This observation led us to investigate the Cytochrome P450 system in colon and its participation in the particular “colon sensitivity” to DMH. It has been established that the Cytochrome P450 system participates in the metabolism of DMH and the methyl carbonium product of Cytochrome P450 activation of DMH is responsible for DNA damage which is considered an initial step to carcinogenesis. The Cytochrome P450 system is a reasonable place to search for an explanation of this organotropic effect of DMH and we feel that the knowledge obtained from this study can take us closer to understanding the development of colonic malignancy. In our study we used a human colon cell line (LS174T) treated with DMH. The Cytochrome P450 system in the cells was manipulated with inducers of different isoforms of Cytochrome P450. The effect of DMH on colon cells was measured by determination of O-6-methylguanine which is a DNA adduct derived from the metabolism of this chemical and is associated with development of tumors. Our results support the hypothesis that Cytochrome P450 plays an important role in the damage to cellular DNA by DMH. This damage is increased after induction of Cytochromes P450 1A1 and 2E1. The effect of inhibition of the methyltransferase and glutathione systems on protection against DMH damage in colon demonstrated the importance of the protective role of the former and the lack of effective protection of the latter system. ^