Correction: Mpp10 represents a platform for the interaction of multiple factors within the 90S pre-ribosome.

[This corrects the article DOI: 10.1371/journal.pone.0183272.].

Development of a Healthy Lifestyle Assessment Toolkit for the General Public.

The prevalence of age-related non-communicable chronic diseases has increased worldwide, being the leading causes of morbidity and death in many world regions, including in Europe. Innovative models and strategies focused on preventive care, including early identification of risk factors underlying disease onset and progression, and proper modification of lifestyle habits and behaviors, might contribute to promote quality of life, healthy living and active aging. Healthy Lifestyle Innovative Quarters for Cities and Citizens (HeaLIQs4cities) is an EIT Health-funded project aiming to engage, empower and educate citizens toward healthy lifestyles. One of the major objectives of this project was to develop a toolkit for a rapid and informal assessment of healthy lifestyles, to be used at different levels of care pathways, including in informal public environments. In this paper, we describe the methodology underlying the development of the toolkit, which resulted from the collaboration of an interdisciplinary focus group of academic experts, from medicine, sport sciences, psychology, health economics, and innovative technologies applied to health. The following eight components were included in the toolkit: (1) anthropometric assessment and cardiometabolic parameters; (2) physical activity and exercise; (3) well-being, social cohesion, and functional independence; (4) nutrition; (5) mental health; (6) smoking, drinking, and use of illicit substances; (7) sleep habits and quality; and (8) health and disease. A traffic light rating system indicating the risk score was used (low: green; moderate: yellow; and relevant: orange) for each of the 8 components, together with recommendations for the toolkit users. After completing the reduced version of the toolkit, individuals showing moderate or relevant risk in one or more of the 8 dimensions, were invited to participate in a more detailed assessment (toolkit long version), based on deeper and scientifically validated tools. The toolkit was incorporated in eVida, a web-based platform that focuses on delivering services to personalized health and well-being. The validation of the current toolkit has been applied in wide-ranging public events in three different European Regions. Large scale deployment of the toolkit is expected to profit from the Reference Site Collaborative Network of the European Innovation Partnership on Active and Healthy Aging (EIP on AHA).

Mpp10 represents a platform for the interaction of multiple factors within the 90S pre-ribosome.

In eukaryotes, ribosome assembly is a highly complex process that involves more than 200 assembly factors that ensure the folding, modification and processing of the different rRNA species as well as the timely association of ribosomal proteins. One of these factors, Mpp10 associates with Imp3 and Imp4 to form a complex that is essential for the normal production of the 18S rRNA. Here we report the crystal structure of a complex between Imp4 and a short helical element of Mpp10 to a resolution of 1.88 Å. Furthermore, we extend the interaction network of Mpp10 and characterize two novel interactions. Mpp10 is able to bind the ribosome biogenesis factor Utp3/Sas10 through two conserved motifs in its N-terminal region. In addition, Mpp10 interacts with the ribosomal protein S5/uS7 using a short stretch within an acidic loop region. Thus, our findings reveal that Mpp10 provides a platform for the simultaneous interaction with multiple proteins in the 90S pre-ribosome.

A conserved protein with AN1 zinc finger and ubiquitin-like domains modulates Cdc48 (p97) function in the ubiquitin-proteasome pathway.

Regulated protein degradation mediated by the ubiquitin-proteasome system (UPS) is critical to eukaryotic protein homeostasis. Often vital to degradation of protein substrates is their disassembly, unfolding, or extraction from membranes. These processes are catalyzed by the conserved AAA-ATPase Cdc48 (also known as p97). Here we characterize the Cuz1 protein (Cdc48-associated UBL/zinc finger protein-1), encoded by a previously uncharacterized arsenite-inducible gene in budding yeast. Cuz1, like its human ortholog ZFAND1, has both an AN1-type zinc finger (Zf_AN1) and a divergent ubiquitin-like domain (UBL). We show that Cuz1 modulates Cdc48 function in the UPS. The two proteins directly interact, and the Cuz1 UBL, but not Zf_AN1, is necessary for binding to the Cdc48 N-terminal domain. Cuz1 also associates, albeit more weakly, with the proteasome, and the UBL is dispensable for this interaction. Cuz1-proteasome interaction is strongly enhanced by exposure of cells to the environmental toxin arsenite, and in a proteasome mutant, loss of Cuz1 enhances arsenite sensitivity. Whereas loss of Cuz1 alone causes only minor UPS degradation defects, its combination with mutations in the Cdc48(Npl4-Ufd1) complex leads to much greater impairment. Cuz1 helps limit the accumulation of ubiquitin conjugates on both the proteasome and Cdc48, suggesting a possible role in the transfer of ubiquitylated substrates from Cdc48 to the proteasome or in their release from these complexes.

Biochemical and biophysical characterization of recombinant yeast proteasome maturation factor ump1.

Protein degradation is essential for maintaining cellular homeostasis. The proteasome is the central enzyme responsible for non-lysosomal protein degradation in eukaryotic cells. Although proteasome assembly is not yet completely understood, a number of cofactors required for proper assembly and maturation have been identified. Ump is a short-lived maturation factor required for the efficient biogenesis of the 20S proteasome. Upon the association of the two precursor complexes, Ump is encased and is rapidly degraded after the proteolytic sites in the interior of the nascent proteasome are activated. In order to further understand the mechanisms behind proteasomal maturation, we expressed and purified yeast Ump in E. coli for biophysical and structural analysis. We show that recombinant Ump is purified as a mixture of different oligomeric species and that oligomerization is mediated by intermolecular disulfide bond formation involving the only cysteine residue present in the protein. Furthermore, a combination of bioinformatic, biochemical and structural analysis revealed that Ump shows characteristics of an intrinsically disordered protein, which might become structured only upon interaction with the proteasome subunits.

Functional and structural characterization of a novel mannosyl-3-phosphoglycerate synthase from Rubrobacter xylanophilus reveals its dual substrate specificity.

Rubrobacter xylanophilus is the only actinobacterium known to accumulate the organic solute mannosylglycerate (MG); moreover, the accumulation of MG is constitutive. The key enzyme for MG synthesis, catalysing the conversion of GDP-mannose (GDP-Man) and D-3-phosphoglycerate (3-PGA) into the phosphorylated intermediate mannosyl-3-phosphoglycerate and GDP, was purified from R. xylanophilus cell extracts and the corresponding gene was expressed in E. coli. Despite the related solute glucosylglycerate (GG) having never been detected in R. xylanophilus, the cell extracts and the pure recombinant mannosyl-3-phosphoglycerate synthase (MpgS) could also synthesize glucosyl-3-phosphoglycerate (GPG), the precursor of GG, in agreement with the higher homology of the novel MpgS towards GPG-synthesizing mycobacterial glucosyl-3-phosphoglycerate synthases (GpgS) than towards MpgSs from hyper/thermophiles, known to accumulate MG under salt or thermal stress. To understand the specificity and substrate ambiguity of this novel enzyme, we determined the crystal structure of the unliganded MpgS and of its complexes with the nucleotide and sugar donors, at 2.2, 2.8 and 2.5 Å resolution respectively. The first three-dimensional structures of a protein from this extremely gamma-radiation-resistant thermophile here reported show that MpgS (GT81 family) contains a GT-A like fold and clearly explain its nucleotide and sugar-donor specificity. In the GDP-Man complex, a flexible loop ((254) RQNRHQ(259) ), located close to the active site moves towards the incoming sugar moiety, providing the ligands for both magnesium ion co-ordination and sugar binding. A triple mutant of R. xylanophilus MpgS, mimicking the (206) PLAGE(210) loop stabilizing hydrogen bond network observed for mycobacterial GpgSs, reduces significantly the affinity to GDP-Man, implicating this loop in the sugar-donor discrimination.

Mycobacterium tuberculosis glucosyl-3-phosphoglycerate synthase: structure of a key enzyme in methylglucose lipopolysaccharide biosynthesis.

Tuberculosis constitutes today a serious threat to human health worldwide, aggravated by the increasing number of identified multi-resistant strains of Mycobacterium tuberculosis, its causative agent, as well as by the lack of development of novel mycobactericidal compounds for the last few decades. The increased resilience of this pathogen is due, to a great extent, to its complex, polysaccharide-rich, and unusually impermeable cell wall. The synthesis of this essential structure is still poorly understood despite the fact that enzymes involved in glycosidic bond synthesis represent more than 1% of all M. tuberculosis ORFs identified to date. One of them is GpgS, a retaining glycosyltransferase (GT) with low sequence homology to any other GTs of known structure, which has been identified in two species of mycobacteria and shown to be essential for the survival of M. tuberculosis. To further understand the biochemical properties of M. tuberculosis GpgS, we determined the three-dimensional structure of the apo enzyme, as well as of its ternary complex with UDP and 3-phosphoglycerate, by X-ray crystallography, to a resolution of 2.5 and 2.7 A, respectively. GpgS, the first enzyme from the newly established GT-81 family to be structurally characterized, displays a dimeric architecture with an overall fold similar to that of other GT-A-type glycosyltransferases. These three-dimensional structures provide a molecular explanation for the enzyme's preference for UDP-containing donor substrates, as well as for its glucose versus mannose discrimination, and uncover the structural determinants for acceptor substrate selectivity. Glycosyltransferases constitute a growing family of enzymes for which structural and mechanistic data urges. The three-dimensional structures of M. tuberculosis GpgS now determined provide such data for a novel enzyme family, clearly establishing the molecular determinants for substrate recognition and catalysis, while providing an experimental scaffold for the structure-based rational design of specific inhibitors, which lay the foundation for the development of novel anti-tuberculosis therapies.

Crystallization and preliminary crystallographic analysis of mannosyl-3-phosphoglycerate synthase from Rubrobacter xylanophilus.

Rubrobacter xylanophilus is the only Gram-positive bacterium known to synthesize the compatible solute mannosylglycerate (MG), which is commonly found in hyperthermophilic archaea and some thermophilic bacteria. Unlike the salt-dependent pattern of accumulation observed in (hyper)thermophiles, in R. xylanophilus MG accumulates constitutively. The synthesis of MG in R. xylanophilus was tracked from GDP-mannose and 3-phosphoglycerate, but the genome sequence of the organism failed to reveal any of the genes known to be involved in this pathway. The native enzyme was purified and its N-terminal sequence was used to identify the corresponding gene (mpgS) in the genome of R. xylanophilus. The gene encodes a highly divergent mannosyl-3-phosphoglycerate synthase (MpgS) without relevant sequence homology to known mannosylphosphoglycerate synthases. In order to understand the specificity and enzymatic mechanism of this novel enzyme, it was expressed in Escherichia coli, purified and crystallized. The crystals thus obtained belonged to the hexagonal space group P6(5)22 and contained two protein molecules per asymmetric unit. The structure was solved by SIRAS using a mercury derivative.