The TOR kinase pathway is relevant for nitrogen signaling and antagonism of the mycoparasite Trichoderma atroviride.

Trichoderma atroviride (Ascomycota, Sordariomycetes) is a well-known mycoparasite applied for protecting plants against fungal pathogens. Its mycoparasitic activity involves processes shared with plant and human pathogenic fungi such as the production of cell wall degrading enzymes and secondary metabolites and is tightly regulated by environmental cues. In eukaryotes, the conserved Target of Rapamycin (TOR) kinase serves as a central regulator of cellular growth in response to nutrient availability. Here we describe how alteration of the activity of TOR1, the single and essential TOR kinase of T. atroviride, by treatment with chemical TOR inhibitors or by genetic manipulation of selected TOR pathway components affected various cellular functions. Loss of TSC1 and TSC2, that are negative regulators of TOR complex 1 (TORC1) in mammalian cells, resulted in altered nitrogen source-dependent growth of T. atroviride, reduced mycoparasitic overgrowth and, in the case of Δtsc1, a diminished production of numerous secondary metabolites. Deletion of the gene encoding the GTPase RHE2, whose mammalian orthologue activates mTORC1, led to rapamycin hypersensitivity and altered secondary metabolism, but had an only minor effect on vegetative growth and mycoparasitic overgrowth. The latter also applied to mutants missing the npr1-1 gene that encodes a fungus-specific kinase known as TOR target in yeast. Genome-wide transcriptome analysis confirmed TOR1 as a regulatory hub that governs T. atroviride metabolism and processes associated to ribosome biogenesis, gene expression and translation. In addition, mycoparasitism-relevant genes encoding terpenoid and polyketide synthases, peptidases, glycoside hydrolases, small secreted cysteine-rich proteins, and G protein coupled receptors emerged as TOR1 targets. Our results provide the first in-depth insights into TOR signaling in a fungal mycoparasite and emphasize its importance in the regulation of processes that critically contribute to the antagonistic activity of T. atroviride.

Early response in phosphorylation of ribosomal protein S6 is associated with sensitivity to trametinib in colorectal cancer cells.

Mutations in RAS or BRAF are associated with poor prognosis and resistance to epidermal growth factor receptor (EGFR)-targeted therapy in colorectal cancer (CRC). Despite their common ability to activate downstream genes such as MEK and ERK, the therapeutic benefit of MEK inhibitors for patients with RAS/BRAF mutant CRC is limited, highlighting the need for biomarkers to predict the efficacy of MEK inhibition. Previously, we reported that a change in phosphorylation of ribosomal protein S6 (pS6) after MEK inhibition was significantly associated with sensitivity to MEK inhibition in gastric cancer cells. Here, we investigated the value of the response in pS6 for predicting the efficacy of trametinib, a MEK inhibitor, in patients with RAS/BRAF mutant CRC using patient-derived CRC organoids. We found that a subset of CRC cell lines and organoids were sensitive to trametinib. The change in phosphorylated ERK, a downstream molecule of the RAS/RAF/MEK pathway, was not significantly associated with trametinib sensitivity. On the other hand, only those with sensitivity showed a reduction of pS6 levels in response to trametinib. The change in pS6 after trametinib treatment was detectable by Western blotting, immunohistochemistry or immunocytochemistry. We also demonstrated an impact of MEK inhibition on pS6 in vivo using a xenograft model. Our data suggest that, in combination with patient-derived organoids, immunostaining-based detection of pS6 could be useful for prediction of trametinib sensitivity.

Capturing the Mechanism Underlying TOP mRNA Binding to LARP1.

The RNA-binding protein La-related protein 1 (LARP1) plays a central role in ribosome biosynthesis. Its C-terminal DM15 region binds the 7-methylguanosine (m7G) cap and 5' terminal oligopyrimidine (TOP) motif characteristic of transcripts encoding ribosomal proteins and translation factors. Under the control of mammalian target of rapamycin complex 1 (mTORC1), LARP1 regulates translation of these transcripts. Characterizing the dynamics of DM15-TOP recognition is essential to understanding this fundamental biological process. We use molecular dynamics simulations, biophysical assays, and X-ray crystallography to reveal the mechanism of DM15 binding to TOP transcripts. Residues C-terminal to the m7G-binding site play important roles in cap recognition. Furthermore, we show that the unusually static pocket that recognizes the +1 cytosine characteristic of TOP transcripts drives binding specificity. Finally, we demonstrate that the DM15 pockets involved in TOP-specific m7GpppC-motif recognition are likely druggable. Collectively, these studies suggest unique opportunities for further pharmacological development.

Novel AU-rich proximal UTR sequences (APS) enhance CXCL8 synthesis upon the induction of rpS6 phosphorylation.

The role of ribosomal protein S6 (rpS6) phosphorylation in mRNA translation remains poorly understood. Here, we reveal a potential role in modulating the translation rate of chemokine (C-X-C motif) ligand 8 (CXCL8 or Interleukin 8, IL8). We observed that more CXCL8 protein was being secreted from less CXCL8 mRNA in primary macrophages and macrophage-like HL-60 cells relative to other cell types. This correlated with an increase in CXCL8 polyribosome association, suggesting an increase in the rate of CXCL8 translation in macrophages. The cell type-specific expression levels were replicated by a CXCL8- UTR-reporter (Nanoluc reporter flanked by the 5' and 3' UTR of CXCL8). Mutations of the CXCL8-UTR-reporter revealed that cell type-specific expression required: 1) a 3' UTR of at least three hundred bases; and 2) an AU base content that exceeds fifty percent in the first hundred bases of the 3' UTR immediately after the stop codon, which we dub AU-rich proximal UTR sequences (APS). The 5' UTR of CXCL8 enhanced expression at the protein level and conferred cell type-specific expression when paired with a 3' UTR. A search for other APS-positive mRNAs uncovered TNF alpha induced protein 6 (TNFAIP6), another mRNA that was translationally upregulated in macrophages. The elevated translation of APS-positive mRNAs in macrophages coincided with elevated rpS6 S235/236 phosphorylation. Both were attenuated by the ERK1/2 signaling inhibitors, U0126 and AZD6244. In A549 cells, rpS6 S235/236 phosphorylation was induced by TAK1, Akt or PKA signaling. This enhanced the translation of the CXCL8-UTR-reporters. Thus, we propose that the induction of rpS6 S235/236 phosphorylation enhances the translation of mRNAs that contain APS motifs, such as CXCL8 and TNFAIP6. This may contribute to the role of macrophages as the primary producer of CXCL8, a cytokine that is essential for immune cell recruitment and activation.

Go-Kit: A Tool To Enable Energy Landscape Exploration of Proteins.

Coarse-grained Go̅-like models, based on the principle of minimal frustration, provide valuable insight into fundamental questions in the field of protein folding and dynamics. In conjunction with commonly used molecular dynamics (MD) simulations, energy landscape exploration methods like discrete path sampling (DPS) with Go̅-like models can provide quantitative details of the thermodynamics and kinetics of proteins. Here we present Go-kit, a software that facilitates the setup of MD and DPS simulations of several flavors of Go̅-like models. Go-kit is designed for use with MD (GROMACS) and DPS (PATHSAMPLE) simulation engines that are open source. The Go-kit code is written in python2.7 and is also open source. A case study for the ribosomal protein S6 is discussed to illustrate the utility of the software, which is available at https://github.com/gokit1/gokit .

Energy Landscape of the Designed Protein Top7.

To fold on biologically relevant time scales, proteins have evolved funnelled energy landscapes with minimal energetic trapping. However, the polymeric nature of proteins and the spatial arrangement of secondary structural elements can create topological traps and slow folding. It is challenging to identify, visualize, and quantify such topological trapping. Designed proteins have not had the benefit of evolution, and it has been hypothesized that de novo designed protein topologies may therefore feature more topological trapping. Structure-based models (SBMs) are inherently funnelled, removing most energetic trapping, and can thus be used to isolate the effect of protein topology on the landscape. Here, we compare Top7, a designed protein with a topology unknown in nature, to S6, a naturally occurring ribosomal protein of similar size and topology. Possible kinetic traps and the energetic barriers separating them from the native state are elucidated. We find that, even with an SBM, the potential energy landscape (PEL) of the designed protein is more frustrated than that of the natural protein. We then quantify the effect of adding non-native hydrophobic interactions and coarse-grained side-chains through a frustration density parameter. A clear increase in frustration is observed including side-chains, whereas adding hydrophobic interactions leads to a narrowing of the funnel and a decrease in complexity. The most likely (un)folding routes for all models are derived through the construction of probability contact maps. The ability to quantitatively understand and optimize the organization of the PEL for designed proteins may enable us to design structure-seeking landscapes, mimicking the effect of evolution.

JMJD5 is a human arginyl C-3 hydroxylase.

Oxygenase-catalysed post-translational modifications of basic protein residues, including lysyl hydroxylations and Nε-methyl lysyl demethylations, have important cellular roles. Jumonji-C (JmjC) domain-containing protein 5 (JMJD5), which genetic studies reveal is essential in animal development, is reported as a histone Nε-methyl lysine demethylase (KDM). Here we report how extensive screening with peptides based on JMJD5 interacting proteins led to the finding that JMJD5 catalyses stereoselective C-3 hydroxylation of arginine residues in sequences from human regulator of chromosome condensation domain-containing protein 1 (RCCD1) and ribosomal protein S6 (RPS6). High-resolution crystallographic analyses reveal overall fold, active site and substrate binding/product release features supporting the assignment of JMJD5 as an arginine hydroxylase rather than a KDM. The results will be useful in the development of selective oxygenase inhibitors for the treatment of cancer and genetic diseases.

Ribosomal Protein S6 Phosphorylation: Four Decades of Research.

The phosphorylation of ribosomal protein S6 (rpS6) has been described for the first time about four decades ago. Since then, numerous studies have shown that this modification occurs in response to a wide variety of stimuli on five evolutionarily conserved serine residues. However, despite a large body of information on the respective kinases and the signal transduction pathways, the physiological role of rpS6 phosphorylation remained obscure until genetic manipulations were applied in both yeast and mammals in an attempt to block this modification. Thus, studies based on both mice and cultured cells subjected to disruption of the genes encoding rpS6 and the respective kinases, as well as the substitution of the phosphorylatable serine residues in rpS6, have laid the ground for the elucidation of the multiple roles of this protein and its posttranslational modification. This review focuses primarily on newly identified kinases that phosphorylate rpS6, pathways that transduce various signals into rpS6 phosphorylation, and the recently established physiological functions of this modification. It should be noted, however, that despite the significant progress made in the last decade, the molecular mechanism(s) underlying the diverse effects of rpS6 phosphorylation on cellular and organismal physiology are still poorly understood.

Constructing a folding model for protein S6 guided by native fluctuations deduced from NMR structures.

The diversity in a set of protein nuclear magnetic resonance (NMR) structures provides an estimate of native state fluctuations that can be used to refine and enrich structure-based protein models (SBMs). Dynamics are an essential part of a protein's functional native state. The dynamics in the native state are controlled by the same funneled energy landscape that guides the entire folding process. SBMs apply the principle of minimal frustration, drawn from energy landscape theory, to construct a funneled folding landscape for a given protein using only information from the native structure. On an energy landscape smoothed by evolution towards minimal frustration, geometrical constraints, imposed by the native structure, control the folding mechanism and shape the native dynamics revealed by the model. Native-state fluctuations can alternatively be estimated directly from the diversity in the set of NMR structures for a protein. Based on this information, we identify a highly flexible loop in the ribosomal protein S6 and modify the contact map in a SBM to accommodate the inferred dynamics. By taking into account the probable native state dynamics, the experimental transition state is recovered in the model, and the correct order of folding events is restored. Our study highlights how the shared energy landscape connects folding and function by showing that a better description of the native basin improves the prediction of the folding mechanism.

Analyses of the folding properties of ferredoxin-like fold proteins by means of a coarse-grained Go model: relationship between the free energy profiles and folding cores.

The folding mechanisms of proteins with multi-state transitions, the role of the intermediate states, and the precise mechanism how each transition occurs are significant on-going research issues. In this study, we investigate ferredoxin-like fold proteins which have a simple topology and multi-state transitions. We analyze the folding processes by means of a coarse-grained Go model. We are able to reproduce the differences in the folding mechanisms between U1A, which has a high-free-energy intermediate state, and ADA2h and S6, which fold into the native structure through two-state transitions. The folding pathways of U1A, ADA2h, S6, and the S6 circular permutant, S6_p54-55, are reproduced and compared with experimental observations. We show that the ferredoxin-like fold contains two common regions consisting folding cores as predicted in other studies and that U1A produces an intermediate state due to the distinct cooperative folding of each core. However, because one of the cores of S6 loses its cooperativity and the two cores of ADA2h are tightly coupled, these proteins fold into the native structure through a two-state mechanism.

Bacterial RNA motif in the 5' UTR of rpsF interacts with an S6:S18 complex.

Approximately half the transcripts encoding ribosomal proteins in Escherichia coli include a structured RNA motif that interacts with a specific ribosomal protein to inhibit gene expression, thus allowing stoichiometric production of ribosome components. However, many of these RNA structures are not widely distributed across bacterial phyla. It is increasingly common for RNA motifs associated with ribosomal protein genes to be identified using comparative genomic methods, yet these are rarely experimentally validated. In this work, we characterize one such motif that precedes operons containing rpsF and rpsR, which encode ribosomal proteins S6 and S18. This RNA structure is widely distributed across many phyla of bacteria despite differences within the downstream operon, and examples are present in both E. coli and Bacillus subtilis. We demonstrate a direct interaction between an example of the RNA from B. subtilis and an S6:S18 complex using in vitro binding assays, verify our predicted secondary structure, and identify a putative protein-binding site. The proposed binding site bears a strong resemblance to the S18 binding site within the 16S rRNA, suggesting molecular mimicry. This interaction is a valuable addition to the canon of ribosomal protein mRNA interactions. This work shows how experimental verification translates computational results into concrete knowledge of biological systems.

S6:S18 ribosomal protein complex interacts with a structural motif present in its own mRNA.

Prokaryotic ribosomal protein genes are typically grouped within highly conserved operons. In many cases, one or more of the encoded proteins not only bind to a specific site in the ribosomal RNA, but also to a motif localized within their own mRNA, and thereby regulate expression of the operon. In this study, we computationally predicted an RNA motif present in many bacterial phyla within the 5' untranslated region of operons encoding ribosomal proteins S6 and S18. We demonstrated that the S6:S18 complex binds to this motif, which we hereafter refer to as the S6:S18 complex-binding motif (S6S18CBM). This motif is a conserved CCG sequence presented in a bulge flanked by a stem and a hairpin structure. A similar structure containing a CCG trinucleotide forms the S6:S18 complex binding site in 16S ribosomal RNA. We have constructed a 3D structural model of a S6:S18 complex with S6S18CBM, which suggests that the CCG trinucleotide in a specific structural context may be specifically recognized by the S18 protein. This prediction was supported by site-directed mutagenesis of both RNA and protein components. These results provide a molecular basis for understanding protein-RNA recognition and suggest that the S6S18CBM is involved in an auto-regulatory mechanism.

Folding without charges.

Surface charges of proteins have in several cases been found to function as "structural gatekeepers," which avoid unwanted interactions by negative design, for example, in the control of protein aggregation and binding. The question is then if side-chain charges, due to their desolvation penalties, play a corresponding role in protein folding by avoiding competing, misfolded traps? To find out, we removed all 32 side-chain charges from the 101-residue protein S6 from Thermus thermophilus. The results show that the charge-depleted S6 variant not only retains its native structure and cooperative folding transition, but folds also faster than the wild-type protein. In addition, charge removal unleashes pronounced aggregation on longer timescales. S6 provides thus an example where the bias toward native contacts of a naturally evolved protein sequence is independent of charges, and point at a fundamental difference in the codes for folding and intermolecular interaction: specificity in folding is governed primarily by hydrophobic packing and hydrogen bonding, whereas solubility and binding relies critically on the interplay of side-chain charges.

Trimming down a protein structure to its bare foldons: spatial organization of the cooperative unit.

Folding of the ribosomal protein S6 is a malleable process controlled by two competing, and partly overlapping, folding nuclei. Together, these nuclei extend over most of the S6 structure, except the edge strand ß2, which is consistently missing in the folding transition states; despite being part of the S6 four-stranded sheet, ß2 seems not to be part of the cooperative unit of the protein. The question is then whether ß2 can be removed from the S6 structure without compromising folding cooperativity or native state integrity. To investigate this, we constructed a truncated variant of S6 lacking ß2, reducing the size of the protein from 96 to 76 residues (S6(Δß2)). The new S6 variant expresses well in Escherichia coli and has a well dispersed heteronuclear single quantum correlation spectrum and a perfectly wild-type-like crystal structure, but with a smaller three-stranded ß-sheet. Moreover, S6(Δß2) displays an archetypical v-shaped chevron plot with decreased slope of the unfolding limb, as expected from a protein with maintained folding cooperativity and reduced size. The results support the notion that foldons, as defined by the structural distribution of the folding nuclei, represent a property-based level of hierarchy in the build-up of larger protein structures and suggest that the role of ß2 in S6 is mainly in intermolecular binding, consistent with the position of this strand in the ribosomal assembly.

Ions and the protein surface revisited: extensive molecular dynamics simulations and analysis of protein structures in alkali-chloride solutions.

Proteins interact with ions in various ways. The surface of proteins has an innate capability to bind ions, and it is also influenced by the screening of the electrostatic potential owing to the presence of salts in the bulk solution. Alkali metal ions and chlorides interact with the protein surface, but such interactions are relatively weak and often transient. In this paper, computer simulations and analysis of protein structures are used to characterize the interactions between ions and the protein surface. The results show that the ion-binding properties of protein residues are highly variable. For example, alkali metal ions are more often associated with aspartate residues than with glutamates, whereas chlorides are most likely to be located near arginines. When comparing NaCl and KCl solutions, it was found that certain surface residues attract the anion more strongly in NaCl. This study demonstrates that protein-salt interactions should be accounted for in the planning and execution of experiments and simulations involving proteins, particularly if subtle structural details are sought after.

Mitotic lymphoma cells are characterized by high expression of phosphorylated ribosomal S6 protein.

Growth factors and mitogens influence signaling pathways and often induce the activity of p70S6 kinase (p70S6K), which in turn phosphorylates the ribosomal S6 protein (S6). Although recent data are rather conflicting, the overall view suggests that phosphorylated S6 is a regulator of global protein synthesis, cell proliferation, cell size and glucose homeostasis. In the present work, emphasis was given to cell cycle-dependent activation of S6 focusing mainly on human lymphoid and lymphoma cells. Paraffin-embedded human tissue blocks from lymph node and different tumor biopsies as well as in vitro cell lines were investigated by immunohistochemistry, immunocytochemistry, flow cytometry and Western blotting using antibodies directed against phospho-S6, phospho-mTOR, phospho-p70S6K and phospho-Histone H3. To enrich the cell number in different phases of the cell cycle, nocodazole, staurosporine or rapamycin were used in cell cultures. We observed strong phospho-S6 positivity by immunostainings in the dividing lymphoid cells of reactive lymph nodes and in lymphoma cells cultured in vitro. Phospho-S6 protein levels were shown to be elevated throughout mitosis in lymphoma cells; however, the high expression of phospho-S6 in mitotic cells was not a general hallmark of tumor cell types studied so far: phospho-S6-negative mitotic cells were detected in several carcinoma and sarcoma biopsies. These observations may have practical implications as they raise the possibility to consider p70S6K and/or S6 as a potential therapeutic target-besides mTOR-in certain lymphomas and perhaps in clinical immunosuppression.

Different cytoplasmic/nuclear distribution of S6 protein phosphorylated at S240/244 and S235/236.

Higher eukaryotic ribosome biogenesis takes place in the nucleolus and requires the import of ribosomal proteins from the cytoplasm. The ribosomal protein S6 is essential for the formation of ribosome subunits, and in mice S6 heterozygosity triggers embryonal lethality. Downstream of the mTOR (mammalian target of rapamycin) and MAPK (mitogen-activated protein kinase) signalling pathways S6 protein is phosphorylated at clustered residues S235/236 and S240/244 upon numerous physiological and pathological stimuli. Here, we show that S240/244-phosphorylated S6 is predominantly nuclear but also detectable in the cytoplasm, whereas S235/236-phosphorylated S6 is almost exclusively localized to the nucleus of primary human cells and virtually undetectable in the cytoplasm. However, in transformed cells the latter can also be detected in the cytoplasm. Experiments with the mTOR inhibitor rapamycin revealed that neither blocking the phosphorylation of S6 at S235/236 and S240/244 nor arresting the cell cycle affects the cytoplasmic/nuclear localization of S6 protein. Our findings provide new insights into the regulation of S6 phosphorylation and S6 protein localization in mammalian cells.

Solution structures and backbone dynamics of the ribosomal protein S6 and its permutant P(54-55).

The ribosomal protein S6 from Thermus thermophilus has served as a model system for the study of protein folding, especially for understanding the effects of circular permutations of secondary structure elements. This study presents the structure of a permutant protein, the 96-residue P(54-55), and the structure of its 101-residue parent protein S6(wt) in solution. The data also characterizes the effects of circular permutation on the backbone dynamics of S6. Consistent with crystallographic data on S6(wt), the overall solution structures of both P(54-55) and S6(wt) show a beta-sheet of four antiparallel beta-strands with two alpha-helices packed on one side of the sheet. In clear contrast to the crystal data, however, the solution structure of S6(wt) reveals a disordered loop in the region between beta-strands 2 and 3 (Leu43-Phe60) instead of a well-ordered stretch and associated hydrophobic mini-core observed in the crystal structure. Moreover, the data for P(54-55) show that the joined wild-type N- and C-terminals form a dynamically robust stretch with a hairpin structure that complies with the in silico design. Taken together, the results explain why the loop region of the S6(wt) structure is relatively insensitive to mutational perturbations, and why P(54-55) is more stable than S6(wt): the permutant incision at Lys54-Asp55 is energetically neutral by being located in an already disordered loop whereas the new hairpin between the wild-type N- and C-termini is stabilizing.

The HD-exchange motions of ribosomal protein S6 are insensitive to reversal of the protein-folding pathway.

An increasing number of protein structures are found to encompass multiple folding nuclei, allowing their structures to be formed by several competing pathways. A typical example is the ribosomal protein S6, which comprises two folding nuclei (sigma1 and sigma2) defining two competing pathways in the folding energy landscape: sigma1 --> sigma2 and sigma2 --> sigma1. The balance between the two pathways, and thus the order of folding events, is easily controlled by circular permutation. In this study, we make use of this ability to manipulate the folding pathway to demonstrate that the dynamic motions of the S6 structure are independent of how the protein folds. The HD-exchange protection factors remain the same upon complete reversal of the folding order. The phenomenon arises because the HD-exchange motions and the high-energy excitations controlling the folding pathway occur at separated free-energy levels: the Boltzmann distribution of unproductive unfolding attempts samples all unfolding channels in parallel, even those that end up in excessively high barriers. Accordingly, the findings provide a simple rationale for how to interpret native-state dynamics without the need to invoke fluctuations off the normal unfolding reaction coordinate.

Protein folding simulations: from coarse-grained model to all-atom model.

Protein folding is an important and challenging problem in molecular biology. During the last two decades, molecular dynamics (MD) simulation has proved to be a paramount tool and was widely used to study protein structures, folding kinetics and thermodynamics, and structure-stability-function relationship. It was also used to help engineering and designing new proteins, and to answer even more general questions such as the minimal number of amino acid or the evolution principle of protein families. Nowadays, the MD simulation is still undergoing rapid developments. The first trend is to toward developing new coarse-grained models and studying larger and more complex molecular systems such as protein-protein complex and their assembling process, amyloid related aggregations, and structure and motion of chaperons, motors, channels and virus capsides; the second trend is toward building high resolution models and explore more detailed and accurate pictures of protein folding and the associated processes, such as the coordination bond or disulfide bond involved folding, the polarization, charge transfer and protonate/deprotonate process involved in metal coupled folding, and the ion permeation and its coupling with the kinetics of channels. On these new territories, MD simulations have given many promising results and will continue to offer exciting views. Here, we review several new subjects investigated by using MD simulations as well as the corresponding developments of appropriate protein models. These include but are not limited to the attempt to go beyond the topology based Go-like model and characterize the energetic factors in protein structures and dynamics, the study of the thermodynamics and kinetics of disulfide bond involved protein folding, the modeling of the interactions between chaperonin and the encapsulated protein and the protein folding under this circumstance, the effort to clarify the important yet still elusive folding mechanism of protein BBL, the development of discrete MD and its application in studying the alpha-beta conformational conversion and oligomer assembling process, and the modeling of metal ion involved protein folding.