Sec61 channel subunit Sbh1/Sec61ß promotes ER translocation of proteins with suboptimal targeting sequences and is fine-tuned by phosphorylation.

The highly conserved endoplasmic reticulum (ER) protein translocation channel contains one nonessential subunit, Sec61ß/Sbh1, whose function is poorly understood so far. Its intrinsically unstructured cytosolic domain makes transient contact with ER-targeting sequences in the cytosolic channel vestibule and contains multiple phosphorylation sites suggesting a potential for regulating ER protein import. In a microscopic screen, we show that 12% of a GFP-tagged secretory protein library depends on Sbh1 for translocation into the ER. Sbh1-dependent proteins had targeting sequences with less pronounced hydrophobicity and often no charge bias or an inverse charge bias which reduces their insertion efficiency into the Sec61 channel. We determined that mutating two N-terminal, proline-flanked phosphorylation sites in the Sbh1 cytosolic domain to alanine phenocopied the temperature-sensitivity of a yeast strain lacking SBH1 and its ortholog SBH2. The phosphorylation site mutations reduced translocation into the ER of a subset of Sbh1-dependent proteins, including enzymes whose concentration in the ER lumen is critical for ER proteostasis. In addition, we found that ER import of these proteins depended on the activity of the phospho-S/T-specific proline isomerase Ess1 (PIN1 in mammals). We conclude that Sbh1 promotes ER translocation of substrates with suboptimal targeting sequences and that its activity can be regulated by a conformational change induced by N-terminal phosphorylation.

Engineering Material Properties of Transcription Factor Condensates to Control Gene Expression in Mammalian Cells and Mice.

Phase separation of biomolecules into condensates is a key mechanism in the spatiotemporal organization of biochemical processes in cells. However, the impact of the material properties of biomolecular condensates on important processes, such as the control of gene expression, remains largely elusive. Here, the material properties of optogenetically induced transcription factor condensates are systematically tuned, and probed for their impact on the activation of target promoters. It is demonstrated that transcription factors in rather liquid condensates correlate with increased gene expression levels, whereas stiffer transcription factor condensates correlate with the opposite effect, reduced activation of gene expression. The broad nature of these findings is demonstrated in mammalian cells and mice, as well as by using different synthetic and natural transcription factors. These effects are observed for both transgenic and cell-endogenous promoters. The findings provide a novel materials-based layer in the control of gene expression, which opens novel opportunities in optogenetic engineering and synthetic biology.

A longer isoform of Stim1 is a negative SOCE regulator but increases cAMP-modulated NFAT signaling.

Alternative splicing is a potent modifier of protein function. Stromal interaction molecule 1 (Stim1) is the essential activator of store-operated Ca2+ entry (SOCE) triggering activation of transcription factors. Here, we characterize Stim1A, a splice variant with an additional 31 amino acid domain inserted in frame within its cytosolic domain. Prominent expression of exon A is found in astrocytes, heart, kidney, and testes. Full-length Stim1A functions as a dominant-negative regulator of SOCE and ICRAC, facilitating sequence-specific fast calcium-dependent inactivation and destabilizing gating of Orai channels. Downregulation or absence of native Stim1A results in increased SOCE. Despite reducing SOCE, Stim1A leads to increased NFAT translocation. Differential proteomics revealed an interference of Stim1A with the cAMP-SOCE crosstalk by altered modulation of phosphodiesterase 8 (PDE8), resulting in reduced cAMP degradation and increased PIP5K activity, facilitating NFAT activation. Our study uncovers a hitherto unknown mechanism regulating NFAT activation and indicates that cell-type-specific splicing of Stim1 is a potent means to regulate the NFAT signalosome and cAMP-SOCE crosstalk.

Redox signals at the ER-mitochondria interface control melanoma progression.

Reactive oxygen species (ROS) are emerging as important regulators of cancer growth and metastatic spread. However, how cells integrate redox signals to affect cancer progression is not fully understood. Mitochondria are cellular redox hubs, which are highly regulated by interactions with neighboring organelles. Here, we investigated how ROS at the endoplasmic reticulum (ER)-mitochondria interface are generated and translated to affect melanoma outcome. We show that TMX1 and TMX3 oxidoreductases, which promote ER-mitochondria communication, are upregulated in melanoma cells and patient samples. TMX knockdown altered mitochondrial organization, enhanced bioenergetics, and elevated mitochondrial- and NOX4-derived ROS. The TMX-knockdown-induced oxidative stress suppressed melanoma proliferation, migration, and xenograft tumor growth by inhibiting NFAT1. Furthermore, we identified NFAT1-positive and NFAT1-negative melanoma subgroups, wherein NFAT1 expression correlates with melanoma stage and metastatic potential. Integrative bioinformatics revealed that genes coding for mitochondrial- and redox-related proteins are under NFAT1 control and indicated that TMX1, TMX3, and NFAT1 are associated with poor disease outcome. Our study unravels a novel redox-controlled ER-mitochondria-NFAT1 signaling loop that regulates melanoma pathobiology and provides biomarkers indicative of aggressive disease.

How phosphorylation of peptides affects their interaction with 14-3-3η domains.

Members of the 14-3-3 domain family have important functions as adapter domains. Via an amphipathic groove on their protein surface they typically bind to disordered C-terminals of other proteins. Importantly, binding partners of 14-3-3 domains usually contain a phosphorylated serine or threonine residue at their binding interface and possess one of three different sequence motifs. Binding of the respective unphosphorylated versions of the peptides is typically strongly disfavored. There is a wealth of structural and thermodynamic data available for the phosphorylated forms but not for the unphosphorylated forms as the binding affinities seem to be too weak to be measurable experimentally. Here, we characterized the mechanistic details that govern the preference for the binding of phosphorylated peptides to 14-3-3η domains by means of molecular dynamics (MD) simulations. We found that the phosphate group is ideally coordinated in the binding pocket whereas the respective unphosphorylated side-chain counterpart is not. Thus, the binding preference results from the tight coordination of the phosphorylated residue at the center of the binding interface. Furthermore, MD simulations of 14-3-3η dimers showed a preference for the simultaneous binding of two phosphorylated peptides in agreement with their experimentally observed cooperativity.

How does Sec63 affect the conformation of Sec61 in yeast?

The Sec complex catalyzes the translocation of proteins of the secretory pathway into the endoplasmic reticulum and the integration of membrane proteins into the endoplasmic reticulum membrane. Some substrate peptides require the presence and involvement of accessory proteins such as Sec63. Recently, a structure of the Sec complex from Saccharomyces cerevisiae, consisting of the Sec61 channel and the Sec62, Sec63, Sec71 and Sec72 proteins was determined by cryo-electron microscopy (cryo-EM). Here, we show by co-precipitation that the Sec61 channel subunit Sbh1 is not required for formation of stable Sec63-Sec61 contacts. Molecular dynamics simulations started from the cryo-EM conformation of Sec61 bound to Sec63 and of unbound Sec61 revealed how Sec63 affects the conformation of Sec61 lateral gate, plug, pore region and pore ring diameter via three intermolecular contact regions. Molecular docking of SRP-dependent vs. SRP-independent signal peptide chains into the Sec61 channel showed that the pore regions affected by presence/absence of Sec63 play a crucial role in positioning the signal anchors of SRP-dependent substrates nearby the lateral gate.

Optimized Data Set and Feature Construction for Substrate Prediction of Membrane Transporters.

α-Helical transmembrane proteins termed membrane transporters mediate the passage of small hydrophilic substrate molecules across biological lipid bilayer membranes. Annotating the specific substrates of the dozens to hundreds of individual transporters of an organism is an important task. In the past, machine learning classifiers have been successfully trained on pan-organism data sets to predict putative substrates of transporters. Here, we critically examine the selection of an optimal data set of protein sequence features for the classification task. We focus on membrane transporters of the three model organisms Escherichia coli, Arabidopsis thaliana, and Saccharomyces cerevisiae, as well as human. We show that organism-specific classifiers can be robustly trained if at least 20 samples are available for each substrate class. If information from position-specific scoring matrices is included, such classifiers have F1 scores between 0.85 and 1.00. For the largest data set (A. thaliana), a 4-class classifier yielded an F-score of 0.97. On a pan-organism data set composed of transporters of all four organisms, amino acid and sugar transporters were predicted with an F1 score of 0.91.

Interorganelle Tethering to Endocytic Organelles Determines Directional Cytokine Transport in CD4+ T Cells.

Delivery of vesicles to their desired destinations plays a central role in maintaining proper cell functionality. In certain scenarios, depending on loaded cargos, the vesicles have spatially distinct destinations. For example, in T cells, some cytokines (e.g., IL-2) are polarized to the T cell-target cell interface, whereas the other cytokines are delivered multidirectionally (e.g., TNF-α). In this study, we show that in primary human CD4+ T cells, both TNF-α+ and IL-2+ vesicles can tether with endocytic organelles (lysosomes/late endosomes) by forming membrane contact sites. Tethered cytokine-containing vesicle (CytV)-endocytic organelle pairs are released sequentially. Only endocytic organelle-tethered CytVs are preferentially transported to their desired destination. Mathematical models suggest that endocytic organelle tethering could regulate the direction of cytokine transport by selectively attaching different microtubule motor proteins (such as kinesin and dynein) to the corresponding CytVs. These findings establish the previously unknown interorganelle tethering to endocytic organelles as a universal solution for directional cytokine transport in CD4+ T cells. Modulating tethering to endocytic organelles can, therefore, coordinately control directionally distinct cytokine transport.

Mycolactone enhances the Ca2+ leak from endoplasmic reticulum by trapping Sec61 translocons in a Ca2+ permeable state.

The Mycobacterium ulcerans exotoxin, mycolactone, is an inhibitor of co-translational translocation via the Sec61 complex. Mycolactone has previously been shown to bind to, and alter the structure of the major translocon subunit Sec61α, and change its interaction with ribosome nascent chain complexes. In addition to its function in protein translocation into the ER, Sec61 also plays a key role in cellular Ca2+ homeostasis, acting as a leak channel between the endoplasmic reticulum (ER) and cytosol. Here, we have analysed the effect of mycolactone on cytosolic and ER Ca2+ levels using compartment-specific sensors. We also used molecular docking analysis to explore potential interaction sites for mycolactone on translocons in various states. These results show that mycolactone enhances the leak of Ca2+ ions via the Sec61 translocon, resulting in a slow but substantial depletion of ER Ca2+. This leak was dependent on mycolactone binding to Sec61α because resistance mutations in this protein completely ablated the increase. Molecular docking supports the existence of a mycolactone-binding transient inhibited state preceding translocation and suggests mycolactone may also bind Sec61α in its idle state. We propose that delayed ribosomal release after translation termination and/or translocon 'breathing' during rapid transitions between the idle and intermediate-inhibited states allow for transient Ca2+ leak, and mycolactone's stabilisation of the latter underpins the phenotype observed.

Synthesis, Biological Evaluation and Docking Studies of Ring-Opened Analogues of Ipomoeassin F.

The plant-derived macrocyclic resin glycoside ipomoeassin F (Ipom-F) binds to Sec61α and significantly disrupts multiple aspects of Sec61-mediated protein biogenesis at the endoplasmic reticulum, ultimately leading to cell death. However, extensive assessment of Ipom-F as a molecular tool and a therapeutic lead is hampered by its limited production scale, largely caused by intramolecular assembly of the macrocyclic ring. Here, using in vitro and/or in cellula biological assays to explore the first series of ring-opened analogues for the ipomoeassins, and indeed all resin glycosides, we provide clear evidence that macrocyclic integrity is not required for the cytotoxic inhibition of Sec61-dependent protein translocation by Ipom-F. Furthermore, our modeling suggests that open-chain analogues of Ipom-F can interact with multiple sites on the Sec61α subunit, most likely located at a previously identified binding site for mycolactone and/or the so-called lateral gate. Subsequent in silico-aided design led to the discovery of the stereochemically simplified analogue 3 as a potent, alternative lead compound that could be synthesized much more efficiently than Ipom-F and will accelerate future ipomoeassin research in chemical biology and drug discovery. Our work may also inspire further exploration of ring-opened analogues of other resin glycosides.

DNA methylation and the core pluripotency network.

From the onset of fertilization, the genome undergoes cell division and differentiation. All of these developmental transitions and differentiation processes include cell-specific signatures and gradual changes of the epigenome. Understanding what keeps stem cells in the pluripotent state and what leads to differentiation are fascinating and biomedically highly important issues. Numerous studies have identified genes, proteins, microRNAs and small molecules that exert essential effects. Notably, there exists a core pluripotency network that consists of several transcription factors and accessory proteins. Three eminent transcription factors, OCT4, SOX2 and NANOG, serve as hubs in this core pluripotency network. They bind to the enhancer regions of their target genes and modulate, among others, the expression levels of genes that are associated with Gene Ontology terms related to differentiation and self-renewal. Also, much has been learned about the epigenetic rewiring processes during these changes of cell fate. For example, DNA methylation dynamics is pivotal during embryonic development. The main goal of this review is to highlight an intricate interplay of (a) DNA methyltransferases controlling the expression levels of core pluripotency factors by modulation of the DNA methylation levels in their enhancer regions, and of (b) the core pluripotency factors controlling the transcriptional regulation of DNA methyltransferases. We discuss these processes both at the global level and in atomistic detail based on information from structural studies and from computer simulations.

TFmiR2: constructing and analyzing disease-, tissue- and process-specific transcription factor and microRNA co-regulatory networks.

SUMMARY: TFmiR2 is a freely available web server for constructing and analyzing integrated transcription factor (TF) and microRNA (miRNA) co-regulatory networks for human and mouse. TFmiR2 generates tissue- and biological process-specific networks for the set of deregulated genes and miRNAs provided by the user. Furthermore, the service can now identify key driver genes and miRNAs in the constructed networks by utilizing the graph theoretical concept of a minimum connected dominating set. These putative key players as well as the newly implemented four-node TF-miRNA motifs yield novel insights that may assist in developing new therapeutic approaches. AVAILABILITY AND IMPLEMENTATION: The TFmiR2 web server is available at http://service.bioinformatik.uni-saarland.de/tfmir2. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

ProPores2: Web Service and Stand-Alone Tool for Identifying, Manipulating, and Visualizing Pores in Protein Structures.

Surface pockets, cavities, and tunnels in the 3D structures of proteins play integral functional roles such as enabling enzymatic catalysis, ligand binding, or transport of ions or small molecules across biomembranes. ProPores2 facilitates understanding and analysis of these processes by identifying pores and lining residues, determining their axes, and opening closed connections via side-chain rotation. The fast stand-alone tool introduces a novel mode for pore identification, improved axis determination, and additional features such as parallel batch processing and a graphical user interface. The new web service features an integrated and customizable protein viewer with an option to analyze and view more than one structure at once. This feature facilitates side-by-side comparisons of pores in different conformations of the same protein or of identified pores before and after opening gates within the same protein. ProPores2 is freely and publicly available at https://service.bioinformatik.uni-saarland.de/propores.

Molecular Modeling of Signal Peptide Recognition by Eukaryotic Sec Complexes.

Here, we review recent molecular modelling and simulation studies of the Sec translocon, the primary component/channel of protein translocation into the endoplasmic reticulum (ER) and bacterial periplasm, respectively. Our focus is placed on the eukaryotic Sec61, but we also mention modelling studies on prokaryotic SecY since both systems operate in related ways. Cryo-EM structures are now available for different conformational states of the Sec61 complex, ranging from the idle or closed state over an inhibited state with the inhibitor mycolactone bound near the lateral gate, up to a translocating state with bound substrate peptide in the translocation pore. For all these states, computational studies have addressed the conformational dynamics of the translocon with respect to the pore ring, the plug region, and the lateral gate. Also, molecular simulations are addressing mechanistic issues of insertion into the ER membrane vs. translocation into the ER, how signal-peptides are recognised at all in the translocation pore, and how accessory proteins affect the Sec61 conformation in the co- and post-translational pathways.

Quantitative Proteomics and Differential Protein Abundance Analysis after the Depletion of PEX3 from Human Cells Identifies Additional Aspects of Protein Targeting to the ER.

Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of around 10,000 different soluble and membrane proteins in humans. It involves the co- or post-translational targeting of precursor polypeptides to the ER, and their subsequent membrane insertion or translocation. So far, three pathways for the ER targeting of precursor polypeptides and four pathways for the ER targeting of mRNAs have been described. Typically, these pathways deliver their substrates to the Sec61 polypeptide-conducting channel in the ER membrane. Next, the precursor polypeptides are inserted into the ER membrane or translocated into the ER lumen, which may involve auxiliary translocation components, such as the TRAP and Sec62/Sec63 complexes, or auxiliary membrane protein insertases, such as EMC and the TMCO1 complex. Recently, the PEX19/PEX3-dependent pathway, which has a well-known function in targeting and inserting various peroxisomal membrane proteins into pre-existent peroxisomal membranes, was also found to act in the targeting and, putatively, insertion of monotopic hairpin proteins into the ER. These either remain in the ER as resident ER membrane proteins, or are pinched off from the ER as components of new lipid droplets. Therefore, the question arose as to whether this pathway may play a more general role in ER protein targeting, i.e., whether it represents a fourth pathway for the ER targeting of precursor polypeptides. Thus, we addressed the client spectrum of the PEX19/PEX3-dependent pathway in both PEX3-depleted HeLa cells and PEX3-deficient Zellweger patient fibroblasts by an established approach which involved the label-free quantitative mass spectrometry of the total proteome of depleted or deficient cells, as well as differential protein abundance analysis. The negatively affected proteins included twelve peroxisomal proteins and two hairpin proteins of the ER, thus confirming two previously identified classes of putative PEX19/PEX3 clients in human cells. Interestingly, fourteen collagen-related proteins with signal peptides or N-terminal transmembrane helices belonging to the secretory pathway were also negatively affected by PEX3 deficiency, which may suggest compromised collagen biogenesis as a hitherto-unknown contributor to organ failures in the respective Zellweger patients.

Quantitative Proteomics and Differential Protein Abundance Analysis after Depletion of Putative mRNA Receptors in the ER Membrane of Human Cells Identifies Novel Aspects of mRNA Targeting to the ER.

In human cells, one-third of all polypeptides enter the secretory pathway at the endoplasmic reticulum (ER). The specificity and efficiency of this process are guaranteed by targeting of mRNAs and/or polypeptides to the ER membrane. Cytosolic SRP and its receptor in the ER membrane facilitate the cotranslational targeting of most ribosome-nascent precursor polypeptide chain (RNC) complexes together with the respective mRNAs to the Sec61 complex in the ER membrane. Alternatively, fully synthesized precursor polypeptides are targeted to the ER membrane post-translationally by either the TRC, SND, or PEX19/3 pathway. Furthermore, there is targeting of mRNAs to the ER membrane, which does not involve SRP but involves mRNA- or RNC-binding proteins on the ER surface, such as RRBP1 or KTN1. Traditionally, the targeting reactions were studied in cell-free or cellular assays, which focus on a single precursor polypeptide and allow the conclusion of whether a certain precursor can use a certain pathway. Recently, cellular approaches such as proximity-based ribosome profiling or quantitative proteomics were employed to address the question of which precursors use certain pathways under physiological conditions. Here, we combined siRNA-mediated depletion of putative mRNA receptors in HeLa cells with label-free quantitative proteomics and differential protein abundance analysis to characterize RRBP1- or KTN1-involving precursors and to identify possible genetic interactions between the various targeting pathways. Furthermore, we discuss the possible implications on the so-called TIGER domains and critically discuss the pros and cons of this experimental approach.

Analysis of the dynamics of Staphylococcus aureus binding to white blood cells using whole blood assay and geno-to-pheno mapping.

Given that binding and internalization of bacteria to host cells promotes infections and invasion, we aimed at characterizing how various S. aureus isolates adhere to and are internalized by different white blood cells. In particular, the role of genetic determinants on the association kinetics should be unveiled. A flow cytometric (FACS) whole blood assay with fluorescently labelled isolates was applied to 56 clinical S. aureus isolates. This phenotypic data was then linked to previously obtained genotyping data (334 genes) with the help of a redescription mining algorithm. Professional phagocytes showed a time-dependent increase of bacterial adhesion and internalization. Isolates showing higher affinity to granulocytes were associated with lower binding to monocytes. In contrast binding activity between S. aureus and lymphocytes could be subdivided into two phases. Preliminary binding (phase 1) was highest directly after co-incubation and was followed by S. aureus detachment or by sustained binding of a small lymphocyte subset (phase 2). Strain-dependent low granulocyte binding was observed for clonal complex 5 (CC5) isolates (MRSA), as compared to CC30 and CC45 (MSSA). S. aureus isolates associated with low granulocyte phagocytosis were characterized by the presence (cap8, can) and the absence (sasG, lukD, isdA, splA, setC) of specific hybridization signals.

Functions and Mechanisms of the Human Ribosome-Translocon Complex.

The membrane of the endoplasmic reticulum (ER) in human cells harbors the protein translocon, which facilitates membrane insertion and translocation of almost every newly synthesized polypeptide targeted to organelles of the secretory pathway. The translocon comprises the polypeptide-conducting Sec61 channel and several additional proteins, which are associated with the heterotrimeric Sec61 complex. This ensemble of proteins facilitates ER targeting of precursor polypeptides, Sec61 channel opening and closing, and modification of precursor polypeptides in transit through the Sec61 complex. Recently, cryoelectron tomography of translocons in native ER membranes has given unprecedented insights into the architecture and dynamics of the native, ribosome-associated translocon and the Sec61 channel. These structural data are discussed in light of different Sec61 channel activities including ribosome receptor function, membrane insertion or translocation of newly synthesized polypeptides as well as the possible roles of the Sec61 channel as a passive ER calcium leak channel and regulator of ATP/ADP exchange between cytosol and ER.

Differential analysis of combinatorial protein complexes with CompleXChange.

BACKGROUND: Although a considerable number of proteins operate as multiprotein complexes and not on their own, organism-wide studies so far are only able to quantify individual proteins or protein-coding genes in a condition-specific manner for a sizeable number of samples, but not their assemblies. Consequently, there exist large amounts of transcriptomic data and an increasing amount of data on proteome abundance, but quantitative knowledge on complexomes is missing. This deficiency impedes the applicability of the powerful tool of differential analysis in the realm of macromolecular complexes. Here, we present a pipeline for differential analysis of protein complexes based on predicted or manually assigned complexes and inferred complex abundances, which can be easily applied on a whole-genome scale. RESULTS: We observed for simulated data that results obtained by our complex abundance estimation algorithm were in better agreement with the ground truth and physicochemically more reasonable compared to previous efforts that used linear programming while running in a fraction of the time. The practical usability of the method was assessed in the context of transcription factor complexes in human monocyte and lymphoblastoid samples. We demonstrated that our new method is robust against false-positive detection and reports deregulated complexomes that can only be partially explained by differential analysis of individual protein-coding genes. Furthermore we showed that deregulated complexes identified by the tool potentially harbor significant yet unused information content. CONCLUSIONS: CompleXChange allows to analyze deregulation of the protein complexome on a whole-genome scale by integrating a plethora of input data that is already available. A platform-independent Java binary, a user guide with example data and the source code are freely available at https://sourceforge.net/projects/complexchange/ .

Topology Consistency of Disease-specific Differential Co-regulatory Networks.

BACKGROUND: Sets of differentially expressed genes often contain driver genes that induce disease processes. However, various methods for identifying differentially expressed genes yield quite different results. Thus, we investigated whether this affects the identification of key players in regulatory networks derived by downstream analysis from lists of differentially expressed genes. RESULTS: While the overlap between the sets of significant differentially expressed genes determined by DESeq, edgeR, voom and VST was only 26% in liver hepatocellular carcinoma and 28% in breast invasive carcinoma, the topologies of the regulatory networks constructed using the TFmiR webserver for the different sets of differentially expressed genes were found to be highly consistent with respect to hub-degree nodes, minimum dominating set and minimum connected dominating set. CONCLUSIONS: The findings suggest that key genes identified in regulatory networks derived by systematic analysis of differentially expressed genes may be a more robust basis for understanding diseases processes than simply inspecting the lists of differentially expressed genes.