Discovering the nuclear localization signal of Werner Helicase Interacting Protein 1.

Our study maps the classic nuclear localization signal (cNLS) domain within WRNIP that directs the protein's nuclear positioning.

Global, in situ analysis of the structural proteome in individuals with Parkinson's disease to identify a new class of biomarker.

Parkinson's disease (PD) is a prevalent neurodegenerative disease for which robust biomarkers are needed. Because protein structure reflects function, we tested whether global, in situ analysis of protein structural changes provides insight into PD pathophysiology and could inform a new concept of structural disease biomarkers. Using limited proteolysis-mass spectrometry (LiP-MS), we identified 76 structurally altered proteins in cerebrospinal fluid (CSF) of individuals with PD relative to healthy donors. These proteins were enriched in processes misregulated in PD, and some proteins also showed structural changes in PD brain samples. CSF protein structural information outperformed abundance information in discriminating between healthy participants and those with PD and improved the discriminatory performance of CSF measures of the hallmark PD protein α-synuclein. We also present the first analysis of inter-individual variability of a structural proteome in healthy individuals, identifying biophysical features of variable protein regions. Although independent validation is needed, our data suggest that global analyses of the human structural proteome will guide the development of novel structural biomarkers of disease and enable hypothesis generation about underlying disease processes.

AI-based structure prediction empowers integrative structural analysis of human nuclear pores.

INTRODUCTION The eukaryotic nucleus pro-tects the genome and is enclosed by the two membranes of the nuclear envelope. Nuclear pore complexes (NPCs) perforate the nuclear envelope to facilitate nucleocytoplasmic transport. With a molecular weight of ∼120 MDa, the human NPC is one of the larg-est protein complexes. Its ~1000 proteins are taken in multiple copies from a set of about 30 distinct nucleoporins (NUPs). They can be roughly categorized into two classes. Scaf-fold NUPs contain folded domains and form a cylindrical scaffold architecture around a central channel. Intrinsically disordered NUPs line the scaffold and extend into the central channel, where they interact with cargo complexes. The NPC architecture is highly dynamic. It responds to changes in nuclear envelope tension with conforma-tional breathing that manifests in dilation and constriction movements. Elucidating the scaffold architecture, ultimately at atomic resolution, will be important for gaining a more precise understanding of NPC function and dynamics but imposes a substantial chal-lenge for structural biologists. RATIONALE Considerable progress has been made toward this goal by a joint effort in the field. A synergistic combination of complementary approaches has turned out to be critical. In situ structural biology techniques were used to reveal the overall layout of the NPC scaffold that defines the spatial reference for molecular modeling. High-resolution structures of many NUPs were determined in vitro. Proteomic analysis and extensive biochemical work unraveled the interaction network of NUPs. Integra-tive modeling has been used to combine the different types of data, resulting in a rough outline of the NPC scaffold. Previous struc-tural models of the human NPC, however, were patchy and limited in accuracy owing to several challenges: (i) Many of the high-resolution structures of individual NUPs have been solved from distantly related species and, consequently, do not comprehensively cover their human counterparts. (ii) The scaf-fold is interconnected by a set of intrinsically disordered linker NUPs that are not straight-forwardly accessible to common structural biology techniques. (iii) The NPC scaffold intimately embraces the fused inner and outer nuclear membranes in a distinctive topol-ogy and cannot be studied in isolation. (iv) The conformational dynamics of scaffold NUPs limits the resolution achievable in structure determination. RESULTS In this study, we used artificial intelligence (AI)-based prediction to generate an exten-sive repertoire of structural models of human NUPs and their subcomplexes. The resulting models cover various domains and interfaces that so far remained structurally uncharac-terized. Benchmarking against previous and unpublished x-ray and cryo-electron micros-copy structures revealed unprecedented accu-racy. We obtained well-resolved cryo-electron tomographic maps of both the constricted and dilated conformational states of the hu-man NPC. Using integrative modeling, we fit-ted the structural models of individual NUPs into the cryo-electron microscopy maps. We explicitly included several linker NUPs and traced their trajectory through the NPC scaf-fold. We elucidated in great detail how mem-brane-associated and transmembrane NUPs are distributed across the fusion topology of both nuclear membranes. The resulting architectural model increases the structural coverage of the human NPC scaffold by about twofold. We extensively validated our model against both earlier and new experimental data. The completeness of our model has enabled microsecond-long coarse-grained molecular dynamics simulations of the NPC scaffold within an explicit membrane en-vironment and solvent. These simulations reveal that the NPC scaffold prevents the constriction of the otherwise stable double-membrane fusion pore to small diameters in the absence of membrane tension. CONCLUSION Our 70-MDa atomically re-solved model covers >90% of the human NPC scaffold. It captures conforma-tional changes that occur during dilation and constriction. It also reveals the precise anchoring sites for intrinsically disordered NUPs, the identification of which is a prerequisite for a complete and dy-namic model of the NPC. Our study exempli-fies how AI-based structure prediction may accelerate the elucidation of subcellular ar-chitecture at atomic resolution. [Figure: see text].

Conserved exchange of paralog proteins during neuronal differentiation.

Gene duplication enables the emergence of new functions by lowering the evolutionary pressure that is posed on the ancestral genes. Previous studies have highlighted the role of specific paralog genes during cell differentiation, for example, in chromatin remodeling complexes. It remains unexplored whether similar mechanisms extend to other biological functions and whether the regulation of paralog genes is conserved across species. Here, we analyze the expression of paralogs across human tissues, during development and neuronal differentiation in fish, rodents and humans. Whereas ∼80% of paralog genes are co-regulated, a subset of paralogs shows divergent expression profiles, contributing to variability of protein complexes. We identify 78 substitutions of paralog pairs that occur during neuronal differentiation and are conserved across species. Among these, we highlight a substitution between the paralogs SEC23A and SEC23B members of the COPII complex. Altering the ratio between these two genes via RNAi-mediated knockdown is sufficient to influence neuron differentiation. We propose that remodeling of the vesicular transport system via paralog substitutions is an evolutionary conserved mechanism enabling neuronal differentiation.

Bioaccumulation of therapeutic drugs by human gut bacteria.

Bacteria in the gut can modulate the availability and efficacy of therapeutic drugs. However, the systematic mapping of the interactions between drugs and bacteria has only started recently1 and the main underlying mechanism proposed is the chemical transformation of drugs by microorganisms (biotransformation). Here we investigated the depletion of 15 structurally diverse drugs by 25 representative strains of gut bacteria. This revealed 70 bacteria-drug interactions, 29 of which had not to our knowledge been reported before. Over half of the new interactions can be ascribed to bioaccumulation; that is, bacteria storing the drug intracellularly without chemically modifying it, and in most cases without the growth of the bacteria being affected. As a case in point, we studied the molecular basis of bioaccumulation of the widely used antidepressant duloxetine by using click chemistry, thermal proteome profiling and metabolomics. We find that duloxetine binds to several metabolic enzymes and changes the metabolite secretion of the respective bacteria. When tested in a defined microbial community of accumulators and non-accumulators, duloxetine markedly altered the composition of the community through metabolic cross-feeding. We further validated our findings in an animal model, showing that bioaccumulating bacteria attenuate the behavioural response of Caenorhabditis elegans to duloxetine. Together, our results show that bioaccumulation by gut bacteria may be a common mechanism that alters drug availability and bacterial metabolism, with implications for microbiota composition, pharmacokinetics, side effects and drug responses, probably in an individual manner.

Nucleoporin Nup155 is part of the p53 network in liver cancer.

Cancer-relevant signalling pathways rely on bidirectional nucleocytoplasmic transport events through the nuclear pore complex (NPC). However, mechanisms by which individual NPC components (Nups) participate in the regulation of these pathways remain poorly understood. We discover by integrating large scale proteomics, polysome fractionation and a focused RNAi approach that Nup155 controls mRNA translation of p21 (CDKN1A), a key mediator of the p53 response. The underlying mechanism involves transcriptional regulation of the putative tRNA and rRNA methyltransferase FTSJ1 by Nup155. Furthermore, we observe that Nup155 and FTSJ1 are p53 repression targets and accordingly find a correlation between the p53 status, Nup155 and FTSJ1 expression in murine and human hepatocellular carcinoma. Our data suggest an unanticipated regulatory network linking translational control by and repression of a structural NPC component modulating the p53 pathway through its effectors.

Pervasive Protein Thermal Stability Variation during the Cell Cycle.

Quantitative mass spectrometry has established proteome-wide regulation of protein abundance and post-translational modifications in various biological processes. Here, we used quantitative mass spectrometry to systematically analyze the thermal stability and solubility of proteins on a proteome-wide scale during the eukaryotic cell cycle. We demonstrate pervasive variation of these biophysical parameters with most changes occurring in mitosis and G1. Various cellular pathways and components vary in thermal stability, such as cell-cycle factors, polymerases, and chromatin remodelers. We demonstrate that protein thermal stability serves as a proxy for enzyme activity, DNA binding, and complex formation in situ. Strikingly, a large cohort of intrinsically disordered and mitotically phosphorylated proteins is stabilized and solubilized in mitosis, suggesting a fundamental remodeling of the biophysical environment of the mitotic cell. Our data represent a rich resource for cell, structural, and systems biologists interested in proteome regulation during biological transitions.

Landscape of nuclear transport receptor cargo specificity.

Nuclear transport receptors (NTRs) recognize localization signals of cargos to facilitate their passage across the central channel of nuclear pore complexes (NPCs). About 30 different NTRs constitute different transport pathways in humans and bind to a multitude of different cargos. The exact cargo spectrum of the majority of NTRs, their specificity and even the extent to which active nucleocytoplasmic transport contributes to protein localization remains understudied because of the transient nature of these interactions and the wide dynamic range of cargo concentrations. To systematically map cargo-NTR relationships in situ, we used proximity ligation coupled to mass spectrometry (BioID). We systematically fused the engineered biotin ligase BirA* to 16 NTRs. We estimate that a considerable fraction of the human proteome is subject to active nuclear transport. We quantified the specificity and redundancy in NTR interactions and identified transport pathways for cargos. We extended the BioID method by the direct identification of biotinylation sites. This approach enabled us to identify interaction interfaces and to discriminate direct versus piggyback transport mechanisms. Data are available via ProteomeXchange with identifier PXD007976.

Capturing protein communities by structural proteomics in a thermophilic eukaryote.

The arrangement of proteins into complexes is a key organizational principle for many cellular functions. Although the topology of many complexes has been systematically analyzed in isolation, their molecular sociology in situ remains elusive. Here, we show that crude cellular extracts of a eukaryotic thermophile, Chaetomium thermophilum, retain basic principles of cellular organization. Using a structural proteomics approach, we simultaneously characterized the abundance, interactions, and structure of a third of the C. thermophilum proteome within these extracts. We identified 27 distinct protein communities that include 108 interconnected complexes, which dynamically associate with each other and functionally benefit from being in close proximity in the cell. Furthermore, we investigated the structure of fatty acid synthase within these extracts by cryoEM and this revealed multiple, flexible states of the enzyme in adaptation to its association with other complexes, thus exemplifying the need for in situ studies. As the components of the captured protein communities are known-at both the protein and complex levels-this study constitutes another step forward toward a molecular understanding of subcellular organization.

Pre-assembled Nuclear Pores Insert into the Nuclear Envelope during Early Development.

Nuclear pore complexes (NPCs) span the nuclear envelope (NE) and mediate nucleocytoplasmic transport. In metazoan oocytes and early embryos, NPCs reside not only within the NE, but also at some endoplasmic reticulum (ER) membrane sheets, termed annulate lamellae (AL). Although a role for AL as NPC storage pools has been discussed, it remains controversial whether and how they contribute to the NPC density at the NE. Here, we show that AL insert into the NE as the ER feeds rapid nuclear expansion in Drosophila blastoderm embryos. We demonstrate that NPCs within AL resemble pore scaffolds that mature only upon insertion into the NE. We delineate a topological model in which NE openings are critical for AL uptake that nevertheless occurs without compromising the permeability barrier of the NE. We finally show that this unanticipated mode of pore insertion is developmentally regulated and operates prior to gastrulation.

In situ structural analysis of the human nuclear pore complex.

Nuclear pore complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of approximately 30 nucleoporins (Nups), 15 are structured and form the Y and inner-ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ~60 nm in diameter. The scaffold is decorated with transport-channel Nups that often contain phenylalanine-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here we combine cryo-electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modelling to generate, to our knowledge, the most comprehensive architectural model of the human nuclear pore complex to date. Our data suggest previously unknown protein interfaces across Y complexes and to inner-ring complex members. We show that the transport-channel Nup358 (also known as Ranbp2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport-channel Nups. We conclude that, similar to coated vesicles, several copies of the same structural building block--although compositionally identical--engage in different local sets of interactions and conformations.

Histone Deacetylase Inhibitors (HDACi) Cause the Selective Depletion of Bromodomain Containing Proteins (BCPs).

Histone deacetylases (HDACs) and acetyltransferases control the epigenetic regulation of gene expression through modification of histone marks. Histone deacetylase inhibitors (HDACi) are small molecules that interfere with histone tail modification, thus altering chromatin structure and epigenetically controlled pathways. They promote apoptosis in proliferating cells and are promising anticancer drugs. While some HDACi have already been approved for therapy and others are in different phases of clinical trials, the exact mechanism of action of this drug class remains elusive. Previous studies have shown that HDACis cause massive changes in chromatin structure but only moderate changes in gene expression. To what extent these changes manifest at the protein level has never been investigated on a proteome-wide scale. Here, we have studied HDACi-treated cells by large-scale mass spectrometry based proteomics. We show that HDACi treatment affects primarily the nuclear proteome and induces a selective decrease of bromodomain-containing proteins (BCPs), the main readers of acetylated histone marks. By combining time-resolved proteome and transcriptome profiling, we show that BCPs are affected at the protein level as early as 12 h after HDACi treatment and that their abundance is regulated by a combination of transcriptional and post-transcriptional mechanisms. Using gene silencing, we demonstrate that the decreased abundance of BCPs is sufficient to mediate important transcriptional changes induced by HDACi. Our data reveal a new aspect of the mechanism of action of HDACi that is mediated by an interplay between histone acetylation and the abundance of BCPs. Data are available via ProteomeXchange with identifier PXD001660 and NCBI Gene Expression Omnibus with identifier GSE64689.

Integrated structural analysis of the human nuclear pore complex scaffold.

The nuclear pore complex (NPC) is a fundamental component of all eukaryotic cells that facilitates nucleocytoplasmic exchange of macromolecules. It is assembled from multiple copies of about 30 nucleoporins. Due to its size and complex composition, determining the structure of the NPC is an enormous challenge, and the overall architecture of the NPC scaffold remains elusive. In this study, we have used an integrated approach based on electron tomography, single-particle electron microscopy, and crosslinking mass spectrometry to determine the structure of a major scaffold motif of the human NPC, the Nup107 subcomplex, in both isolation and integrated into the NPC. We show that 32 copies of the Nup107 subcomplex assemble into two reticulated rings, one each at the cytoplasmic and nuclear face of the NPC. This arrangement may explain how changes of the diameter are realized that would accommodate transport of huge cargoes.