Defining the Specificity of Cotranslationally Acting Chaperones by Systematic Analysis of mRNAs Associated with Ribosome-Nascent Chain Complexes

Polypeptides exiting the ribosome must fold and assemble in the crowded environment of the cell. Chaperones and other protein homeostasis factors interact with newly translated polypeptides to facilitate their folding and correct localization. Despite the extensive efforts, little is known about the specificity of the chaperones and other factors that bind nascent polypeptides. To address this question we present an approach that systematically identifies cotranslational chaperone substrates through the mRNAs associated with ribosome-nascent chain-chaperone complexes. We here focused on two Saccharomyces cerevisiae chaperones: the Signal Recognition Particle (SRP), which acts cotranslationally to target proteins to the ER, and the Nascent chain Associated Complex (NAC), whose function has been elusive. Our results provide new insights into SRP selectivity and reveal that NAC is a general cotranslational chaperone. We found surprising differential substrate specificity for the three subunits of NAC, which appear to recognize distinct features within nascent chains. Our results also revealed a partial overlap between the sets of nascent polypeptides that interact with NAC and SRP, respectively, and showed that NAC modulates SRP specificity and fidelity in vivo. These findings give us new insight into the dynamic interplay of chaperones acting on nascent chains. The strategy we used should be generally applicable to mapping the specificity, interplay, and dynamics of the cotranslational protein homeostasis network

Multivalent contacts of the Hsp70 Ssb contribute to its architecture on ribosomes and nascent chain interaction

Hsp70 chaperones assist de novo folding of newly synthesized proteins in all cells. In yeast, the specialized Hsp70 Ssb directly binds to ribosomes. The structural basis and functional mode of recruitment of Ssb to ribosomes is not understood. Here, we present the molecular details underlying ribosome binding of Ssb in Saccharomyces cerevisiae. This interaction is multifaceted, involving the co-chaperone RAC and two specific regions within Ssb characterized by positive charges. The C-terminus of Ssb mediates the key contact and a second attachment point is provided by a KRR-motif in the substrate binding domain. Strikingly, ribosome binding of Ssb is not essential. Autonomous ribosome attachment becomes necessary if RAC is absent, suggesting a dual mode of Ssb recruitment to nascent chains. We propose, that the multilayered ribosomal interaction allows positioning of Ssb in an optimal orientation to the tunnel exit guaranteeing an efficient nascent polypeptide interaction.publishe

Clinical trials in pediatric ALS: a TRICALS feasibility study

Background: Pediatric investigation plans (PIPs) describe how adult drugs can be studied in children. In 2015, PIPs for Amyotrophic Lateral Sclerosis (ALS) became mandatory for European marketing-authorization of adult treatments, unless a waiver is granted by the European Medicines Agency (EMA). Objective: To assess the feasibility of clinical studies on the effect of therapy in children (<18 years) with ALS in Europe. Methods: The EMA database was searched for submitted PIPs in ALS. A questionnaire was sent to 58 European ALS centers to collect the prevalence of pediatric ALS during the past ten years, the recruitment potential for future pediatric trials, and opinions of ALS experts concerning a waiver for ALS. Results: Four PIPs were identified; two were waived and two are planned for the future. In total, 49 (84.5%) centers responded to the questionnaire. The diagnosis of 44,858 patients with ALS was reported by 46 sites; 39 of the patients had an onset < 18 years (prevalence of 0.008 cases per 100,000 or 0.087% of all diagnosed patients). The estimated recruitment potential (47 sites) was 26 pediatric patients within five years. A majority of ALS experts (75.5%) recommend a waiver should apply for ALS due to the low prevalence of pediatric ALS. Conclusions: ALS with an onset before 18 years is extremely rare and may be a distinct entity from adult ALS. Conducting studies on the effect of disease-modifying therapy in pediatric ALS may involve lengthy recruitment periods, high costs, ethical/legal implications, challenges in trial design and limited information