Introduction The nuclear pore complex (NPC) is the primary gateway for transport of macromolecules between the nucleus and cytoplasm, serving as both a critical mediator and regulator of gene expression. NPCs are enormous ~120 MDa macromolecular machines embedded in the nuclear envelope, each containing ~1000 protein subunits, termed nucleoporins. Despite substantial progress in visualizing the overall shape of the NPC by cryoelectron tomography and in determining atomic resolution crystal structures of nucleoporins, the molecular architecture of the assembled NPC remains poorly understood, hindering the design of mechanistic studies that could investigate its many roles in cell biology. Rationale Existing cryoelectron tomographic reconstructions of the NPC remain too low in resolution to allow for de novo structure determination of the NPC or unbiased docking of nucleoporin fragment crystal structures. We sought to bridge this resolution gap by first defining the interaction network of the NPC, focusing on the evolutionarily conserved symmetric core. We developed protocols to reconstitute NPC protomers from purified, recombinant proteins, which enabled the generation of a high-resolution biochemical interaction map of the NPC symmetric core. We next determined high-resolution crystal structures of key nucleoporin interactions, providing spatial restraints for their relative orientation. Lastly, by superposing crystal structures that overlapped in sequence, we generated accurate full-length structures of the large scaffold nucleoporins. Supported by this biochemical data, we used sequential, unbiased searches to place the nucleoporin crystal structures into a previously determined cryoelectron tomographic reconstruction of the intact human NPC, thus generating a composite structure of the entire NPC symmetric core. Results Our analysis revealed that the inner and outer rings of the NPC utilize disparate mechanisms of interaction. While the structured coat nucleoporins of the outer ring form extensive surface contacts, the scaffold proteins of the inner ring are bridged by flexible sequences in linker nucleoporins. Our composite structure revealed a defined spoke architecture with limited cross-spoke interactions. Most nucleoporins are present in 32 copies, with notable exceptions of Nup170 and Nup188. Lastly, we observed the arrangement of the channel nucleoporins, which orient their N-termini into two sixteen-membered rings, ensuring that their N-terminal FG repeats project evenly into the central transport channel. Conclusion Our composite structure of the NPC symmetric core can be used as a platform for the rational design of experiments to probe NPC structure and function. Each nucleoporin occupies multiple distinct biochemical environments, explaining how such a large macromolecular complex can be assembled from a relatively small number of unique genes. Our integrated, bottom-up approach provides a paradigm for the biochemical and structural characterization of similarly large biological mega-assemb...

Metabolic challenge protocols, such as the oral glucose tolerance test, can uncover early alterations in metabolism preceding chronic diseases. Nevertheless, most metabolomics data accessible today reflect the fasting state. To analyze the dynamics of the human metabolome in response to environmental stimuli, we submitted 15 young healthy male volunteers to a highly controlled 4 d challenge protocol, including 36 h fasting, oral glucose and lipid tests, liquid test meals, physical exercise, and cold stress. Blood, urine, exhaled air, and breath condensate samples were analyzed on up to 56 time points by MS- and NMR-based methods, yielding 275 metabolic traits with a focus on lipids and amino acids. Here, we show that physiological challenges increased interindividual variation even in phenotypically similar volunteers, revealing metabotypes not observable in baseline metabolite profiles; volunteer-specific metabolite concentrations were consistently reflected in various biofluids; and readouts from a systematic model of β-oxidation (e.g., acetylcarnitine/palmitylcarnitine ratio) showed significant and stronger associations with physiological parameters (e.g., fat mass) than absolute metabolite concentrations, indicating that systematic models may aid in understanding individual challenge responses. Due to the multitude of analytical methods, challenges and sample types, our freely available metabolomics data set provides a unique reference for future metabolomics studies and for verification of systems biology models.

The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic transport. We present the reconstitution and interdisciplinary analyses of the ~425-kDa inner ring complex (IRC), which forms the central transport channel and diffusion barrier of the NPC, revealing its interaction network and equimolar stoichiometry. The Nsp1•Nup49•Nup57 channel nucleoporin hetero-trimer (CNT) attaches to the IRC solely through the adaptor nucleoporin Nic96. The CNT•Nic96 structure reveals that Nic96 functions as an assembly sensor that recognizes the three dimensional architecture of the CNT, thereby mediating the incorporation of a defined CNT state into the NPC. We propose that the IRC adopts a relatively rigid scaffold that recruits the CNT to primarily form the diffusion barrier of the NPC, rather than enabling channel dilation.

Overproduced yeast ribosomal protein (RP) Rpl26 fails to assemble into ribosomes and is degraded in the nucleus/nucleolus by a ubiquitin-proteasome system quality control pathway comprising the E2 enzymes Ubc4/Ubc5 and the ubiquitin ligase Tom1. tom1 cells show reduced ubiquitination of multiple RPs, exceptional accumulation of detergent-insoluble proteins including multiple RPs, and hypersensitivity to imbalances in production of RPs and rRNA, indicative of a profound perturbation to proteostasis. Tom1 directly ubiquitinates unassembled RPs primarily via residues that are concealed in mature ribosomes. Together, these data point to an important role for Tom1 in normal physiology and prompt us to refer to this pathway as ERISQ, for excess ribosomal protein quality control. A similar pathway, mediated by the Tom1 homolog Huwe1, restricts accumulation of overexpressed hRpl26 in human cells. We propose that ERISQ is a key element of the quality control machinery that sustains protein homeostasis and cellular fitness in eukaryotes.DOI: http://dx.doi.org/10.7554/eLife.19105.001

Previously, we reported strong influences of genetic variants on metabolic phenotypes, some of them with clinical relevance. Here, we hypothesize that DNA methylation may have an important and potentially independent effect on human metabolism. To test this hypothesis, we conducted what is to the best of our knowledge the first epigenome-wide association study (EWAS) between DNA methylation and metabolic traits (metabotypes) in human blood. We assess 649 blood metabolic traits from 1814 participants of the Kooperative Gesundheitsforschung in der Region Augsburg (KORA) population study for association with methylation of 457 004 CpG sites, determined on the Infinium HumanMethylation450 BeadChip platform. Using the EWAS approach, we identified two types of methylome–metabotype associations. One type is driven by an underlying genetic effect; the other type is independent of genetic variation and potentially driven by common environmental and life-style-dependent factors. We report eight CpG loci at genome-wide significance that have a genetic variant as confounder (P = 3.9 × 10−20 to 2.0 × 10−108, r2 = 0.036 to 0.221). Seven loci display CpG site-specific associations to metabotypes, but do not exhibit any underlying genetic signals (P = 9.2 × 10−14 to 2.7 × 10−27, r2 = 0.008 to 0.107). We further identify several groups of CpG loci that associate with a same metabotype, such as 4-vinylphenol sulfate and 4-androsten-3-beta,17-beta-diol disulfate. In these cases, the association between CpG-methylation and metabotype is likely the result of a common external environmental factor, including smoking. Our study shows that analysis of EWAS with large numbers of metabolic traits in large population cohorts are, in principle, feasible. Taken together, our data suggest that DNA methylation plays an important role in regulating human metabolism.