Neo-epitope Peptides as Biomarkers of Disease Progression for Muscular Dystrophies and Other Myopathies.

For several decades, serological biomarkers of neuromuscular diseases as dystrophies, myopathies and myositis have been limited to routine clinical biochemistry panels. Gauging the pathological progression is a prerequisite for proper treatment and therefore identifying accessible, easy to monitor biomarkers that can predict the disease progression would be an important advancement. Most muscle diseases involve accelerated muscle fiber degradation, inflammation, fatty tissue substitution and/or fibrosis. All these pathological traits have been shown to give rise to serological peptide biomarkers in other tissues, underlining the potential application of existing biomarkers of such traits in muscle disorders. A significant quantity of tissue is involved in these pathological mechanisms alongside with qualitative changes in protein turnover in myofibrillar, extra-cellular matrix and immunological cell protein fractions accompanied by alterations in body fluids. We propose that protein and peptides can leak out of the afflicted muscles and can be of use in diagnosis, prediction of pathology trajectory and treatment efficacy. Proteolytic cleavage systems are especially modulated during a range of muscle pathologies, thereby giving rise to peptides that are differentially released during disease manifestation. Therefore, we believe that pathology-specific post-translational modifications like cleavages can give rise to neoepitope peptides that may represent a promising class of peptides for discovery of biomarkers pertaining to neuromuscular diseases.

Studies on the function of yeast phosphofructokinase subunits by in vitro mutagenesis.

Genetic and biochemical analysis of phosphofructokinase in the yeast Saccharomyces cerevisiae led to contradictory hypotheses about the function of the subunits of this heterooctameric enzyme. To gain further insight, we exchanged four evolutionary conserved amino acid residues in each of the two yeast subunits affecting presumed catalytic and regulatory functions. In conjunction with a complementary wild-type subunit, each of the mutant subunits led to a loss of a maximum of 50% of phosphofructokinase activity as compared to wild-type cells. Km values for fructose 6-phosphate were increased in most of these mutants. None of the mutant subunits lacking catalytical functions was able to complement the glucose-negative phenotype of a yeast pfk1 pfk2 double mutant when expressed from a single-copy vector. For the beta-subunits, the other mutants did complement, whereas for the alpha-subunits they did not. Concentrations of fructose 1,6-bisphosphate did not drastically change in metabolite determinations in strains carrying one mutant allele, suggesting that the effect of the mutations introduced can be largely compensated by in vivo regulatory mechanisms, as long as one functional subunit is present. The data implicate that each of the yeast phosphofructokinase subunits can serve catalytically as well as regulatory functions.

Coregulation of the Kluyveromyces lactis lactose permease and beta-galactosidase genes is achieved by interaction of multiple LAC9 binding sites in a 2.6 kbp divergent promoter.

The coregulated genes LAC4 and LAC12 encoding beta-galactosidase and lactose permease, respectively, are responsible for the ability of the milk yeast Kluyveromyces lactis to utilise lactose. They are divergently transcribed and separated by an unusually large intergenic region of 2.6 kbp. Mapping of the upstream border of the beta-galactosidase gene (LAC4) promoter by introduction of mutations at the chromosomal locus showed that LAC4 and LAC12 share the same upstream activation sites (UAS). The UASs represent binding sites for the trans-activator LAC9, a K. lactis homologue of GAL4, conforming to the consensus sequence 5'-CGG(N5)A/T(N5)CCG-3'. Two binding sites are located in front of each of the genes at almost symmetrical positions. beta-galactosidase activity measurements as well as quantitation of LAC4 and LAC12 mRNA levels demonstrated that all four sites are required for full induction. LAC4 proximal and a LAC12 proximal sites cooperate in activating transcription of both genes. These sites are more than 1.7 kbp apart and the distal site is located more than 2.3 kbp upstream of the respective start of transcription. Thus, the distance between interacting sites is larger than in any of the well characterised yeast promoters. The contribution to gene activation differs for individual binding sites and correlates with the relative affinity of LAC9 for these sites in vitro suggesting that LAC9 binding is a rate limiting step for LAC promoter function.