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The mechanisms leading to disability and the long-term efficacy and safety of disease modifying drugs (DMDs) in multiple sclerosis (MS) are unclear. We aimed at building a prospective cohort of MS patients with standardized collection of demographic, clinical, MRI data and body fluids that can be used to develop prognostic indicators and biomarkers of disease evolution and therapeutic response. The Swiss MS Cohort (SMSC) is a prospective observational study performed across seven Swiss MS centers including patients with MS, clinically isolated syndrome (CIS), radiologically isolated syndrome or neuromyelitis optica. Neurological and radiological assessments and biological samples are collected every 6-12 months. We recruited 872 patients (clinically isolated syndrome [CIS] 5.5%, relapsing-remitting MS [RRMS] 85.8%, primary progressive MS [PPMS] 3.5%, secondary progressive MS [SPMS] 5.2%) between June 2012 and July 2015. We performed 2,286 visits (median follow-up 398 days) and collected 2,274 serum, plasma and blood samples, 152 cerebrospinal fluid samples and 1,276 brain MRI scans. 158 relapses occurred and expanded disability status scale (EDSS) scores increased in PPMS, SPMS and RRMS patients experiencing relapses. Most RRMS patients were treated with fingolimod (33.4%), natalizumab (24.5%) or injectable DMDs (13.6%). The SMSC will provide relevant information regarding DMDs efficacy and safety and will serve as a comprehensive infrastructure available for nested research projects.

The 90-kDa heat shock protein (Hsp90) is a molecular chaperone that is very abundant even at normal temperature. It is highly conserved and essential for viability in yeast. To delineate functional domains of Hsp90, we have performed a deletion analysis of one of the two Hsp90 isoforms from budding yeast, Hsp82. The Hsp82 derivatives were tested for complementation of a Hsp90-deficient yeast strain and for their ability to function in two signal transduction pathways that depend on Hsp90. Surprisingly, we found that two salient features of Hsp90 sequences from eukaryotes, the N-terminal charged domain and the extremely conserved C-terminal pentapeptide MEEVD, are dispensable for viability as well as for proper regulation of vertebrate steroid receptors and for pheromone signaling. Moreover, we describe, to our knowledge, the first dominant negative mutant of Hsp90: A Hsp82 derivative that lacks amino acids 538–552 fails to complement but has a dominant negative effect on viability of wild-type strains at moderately elevated temperatures. This mutant may become a valuable tool to study Hsp90 functions both in yeast and in mammalian cells.

We demonstrate that the hormone-binding domain (HBD) of the human estrogen receptor (ER) can function as an autonomous regulatory domain in the budding yeast, Saccharomyces cerevisiae. As in mammalian cells, the HBD can subject the activity of a heterologous protein, which is fused to it, to hormonal control. Thus, a chimeric transcriptional activator consisting of (i) the DNA-binding domain of GAL4, (ii) the ER HBD, and (iii) the activation domain of viral protein 16 (VP16) stimulates both episomal and integrated reporter genes exclusively in the presence of steroid hormone. Steroids being gratuitous signals for yeast, this fusion protein is a convenient tool for highly regulated production of proteins of interest. Notably, it can be exploited to activate the commonly used galactose-inducible expression vectors without switching the carbon source.

The Dd ras gene produces three transcripts during Dictyostelium development. The largest transcript (L-) can be induced by external addition of cAMP even in cells prevented from aggregating, whereas shorter transcripts (S1- and S2-) expression requires cell aggregate formation. We show the presence of two independent promoters for L- and S-transcripts by deletion analysis of Dd ras fragments fused to CAT reporter genes reintroduced in Dictyostelium. A direct repeat upstream of S-transcript start sites which seems involved in S-promoter function, modulates also L-RNA accumulation. Furthermore removal of sequences between this repeat and the AUG protein start codon reduces the level of L-transcripts in aggregates. This study allowed to uncover the intricate pattern of sequences participating in the regulation of Dd ras expression.

The heat-shock protein 90 (Hsp90) is a cytosolic molecular chaperone that is highly abundant even at normal temperature. Specific functions for Hsp90 have been proposed based on the characterization of its interactions with certain transcription factors and kinases including Raf in vertebrates and flies. We therefore decided to address the role of Hsp90 for MAP kinase pathways in the budding yeast, an organism amenable to both genetic and biochemical analyses. We found that both basal and induced activities of the pheromone-signaling pathway depend on Hsp90. Signaling is defective in strains expressing low levels or point mutants of yeast Hsp90 (Hsp82), or human Hsp90β instead of the wild-type protein. Ste11, a yeast equivalent of Raf, forms complexes with wild-type Hsp90 and depends on Hsp90 function for accumulation. For budding yeast, Ste11 represents the first identified endogenous “substrate” of Hsp90. Moreover, Hsp90 functions in steroid receptor and pheromone signaling can be genetically separated as the Hsp82 point mutant T525I and the human Hsp90β are specifically defective for the former and the latter, respectively. These findings further corroborate the view that molecular chaperones must also be considered as transient or stable components of signal transduction pathways.

Publisher Summary This chapter discusses a novel approach that involves the posttranslational regulation of a constitutively expressed fusion protein. The method is based on the fact that the hormone binding domain (HBD) of steroid receptors can be used as an autonomous regulatory cassette to subject many heterologous protein functions to hormonal control in cis . Fusion proteins are maintained in an inactive state in the absence of hormone and are rapidly activated by addition of the cognate hormone. The hormone-reversible protein inactivation function of the HBD works by a relatively unspecific mechanism involving steric hindrance by the HSP90 complex. This method should be applicable to any protein with at least one essential function that is sensitive to steric hindrance. Hence, this regulatory system should work in the cytosolic and nuclear compartments of any organism that provides the components of the HSP90 complex. The choice of HBD is primarily dictated by the biological system rather than by the heterologous protein itself because the activation of endogenous steroid receptors is usually undesirable.