The MASTL/PP2A cell cycle kinase-phosphatase module restrains PI3K-Akt activity in an mTORC1-dependent manner.

The AKT-mTOR pathway is a central regulator of cell growth and metabolism. Upon sustained mTOR activity, AKT activity is attenuated by a feedback loop that restrains upstream signaling. However, how cells control the signals that limit AKT activity is not fully understood. Here, we show that MASTL/Greatwall, a cell cycle kinase that supports mitosis by phosphorylating the PP2A/B55 inhibitors ENSA/ARPP19, inhibits PI3K-AKT activity by sustaining mTORC1- and S6K1-dependent phosphorylation of IRS1 and GRB10. Genetic depletion of MASTL results in an inefficient feedback loop and AKT hyperactivity. These defects are rescued by the expression of phosphomimetic ENSA/ARPP19 or inhibition of PP2A/B55 phosphatases. MASTL is directly phosphorylated by mTORC1, thereby limiting the PP2A/B55-dependent dephosphorylation of IRS1 and GRB10 downstream of mTORC1. Downregulation of MASTL results in increased glucose uptake in vitro and increased glucose tolerance in adult mice, suggesting the relevance of the MASTL-PP2A/B55 kinase-phosphatase module in controlling AKT and maintaining metabolic homeostasis.

Development of anti-membrane type 1-matrix metalloproteinase nanobodies as immunoPET probes for triple negative breast cancer imaging.

Triple-negative breast cancer (TNBC) is characterized by aggressiveness and high rates of metastasis. The identification of relevant biomarkers is crucial to improve outcomes for TNBC patients. Membrane type 1-matrix metalloproteinase (MT1-MMP) could be a good candidate because its expression has been reported to correlate with tumor malignancy, progression and metastasis. Moreover, single-domain variable regions (VHHs or Nanobodies) derived from camelid heavy-chain-only antibodies have demonstrated improvements in tissue penetration and blood clearance, important characteristics for cancer imaging. Here, we have developed a nanobody-based PET imaging strategy for TNBC detection that targets MT1-MMP. A llama-derived library was screened against the catalytic domain of MT1-MMP and a panel of specific nanobodies were identified. After a deep characterization, two nanobodies were selected to be labeled with gallium-68 (68Ga). ImmunoPET imaging with both ([68Ga]Ga-NOTA-3TPA14 and [68Ga]Ga-NOTA-3CMP75) in a TNBC mouse model showed precise tumor-targeting capacity in vivo with high signal-to-background ratios. (68Ga)Ga-NOTA-3CMP75 exhibited higher tumor uptake compared to (68Ga)Ga-NOTA-3TPA14. Furthermore, imaging data correlated perfectly with the immunohistochemistry staining results. In conclusion, we found a promising candidate for nanobody-based PET imaging to be further investigated as a diagnostic tool in TNBC.

Structure of the RAF1-HSP90-CDC37 complex reveals the basis of RAF1 regulation.

RAF kinases are RAS-activated enzymes that initiate signaling through the MAPK cascade to control cellular proliferation, differentiation, and survival. Here, we describe the structure of the full-length RAF1 protein in complex with HSP90 and CDC37 obtained by cryoelectron microscopy. The reconstruction reveals a RAF1 kinase with an unfolded N-lobe separated from its C-lobe. The hydrophobic core of the N-lobe is trapped in the HSP90 dimer, while CDC37 wraps around the chaperone and interacts with the N- and C-lobes of the kinase. The structure indicates how CDC37 can discriminate between the different members of the RAF family. Our structural analysis also reveals that the folded RAF1 assembles with 14-3-3 dimers, suggesting that after folding RAF1 follows a similar activation as B-RAF. Finally, disruption of the interaction between CDC37 and the DFG segment of RAF1 unveils potential vulnerabilities in attempting the pharmacological degradation of RAF1 for therapeutic purposes.

Structural basis of Nrd1-Nab3 heterodimerization.

Heterodimerization of RNA binding proteins Nrd1 and Nab3 is essential to communicate the RNA recognition in the nascent transcript with the Nrd1 recognition of the Ser5-phosphorylated Rbp1 C-terminal domain in RNA polymerase II. The structure of a Nrd1-Nab3 chimera reveals the basis of heterodimerization, filling a missing gap in knowledge of this system. The free form of the Nrd1 interaction domain of Nab3 (NRID) forms a multi-state three-helix bundle that is clamped in a single conformation upon complex formation with the Nab3 interaction domain of Nrd1 (NAID). The latter domain forms two long helices that wrap around NRID, resulting in an extensive protein-protein interface that would explain the highly favorable free energy of heterodimerization. Mutagenesis of some conserved hydrophobic residues involved in the heterodimerization leads to temperature-sensitive phenotypes, revealing the importance of this interaction in yeast cell fitness. The Nrd1-Nab3 structure resembles the previously reported Rna14/Rna15 heterodimer structure, which is part of the poly(A)-dependent termination pathway, suggesting that both machineries use similar structural solutions despite they share little sequence homology and are potentially evolutionary divergent.

Urine NMR-based TB metabolic fingerprinting for the diagnosis of TB in children.

Tuberculosis (TB) is a major cause of morbidity and mortality in children, and early diagnosis and treatment are crucial to reduce long-term morbidity and mortality. In this study, we explore whether urine nuclear magnetic resonance (NMR)-based metabolomics could be used to identify differences in the metabolic response of children with different diagnostic certainty of TB. We included 62 children with signs and symptoms of TB and 55 apparently healthy children. Six of the children with presumptive TB had bacteriologically confirmed TB, 52 children with unconfirmed TB, and 4 children with unlikely TB. Urine metabolic fingerprints were identified using high- and low-field proton NMR platforms and assessed with pattern recognition techniques such as principal components analysis and partial least squares discriminant analysis. We observed differences in the metabolic fingerprint of children with bacteriologically confirmed and unconfirmed TB compared to children with unlikely TB (p = 0.041 and p = 0.013, respectively). Moreover, children with unconfirmed TB with X-rays compatible with TB showed differences in the metabolic fingerprint compared to children with non-pathological X-rays (p = 0.009). Differences in the metabolic fingerprint in children with different diagnostic certainty of TB could contribute to a more accurate characterisation of TB in the paediatric population. The use of metabolomics could be useful to improve the prediction of TB progression and diagnosis in children.

The structure of transcription termination factor Nrd1 reveals an original mode for GUAA recognition.

Transcription termination of non-coding RNAs is regulated in yeast by a complex of three RNA binding proteins: Nrd1, Nab3 and Sen1. Nrd1 is central in this process by interacting with Rbp1 of RNA polymerase II, Trf4 of TRAMP and GUAA/G terminator sequences. We lack structural data for the last of these binding events. We determined the structures of Nrd1 RNA binding domain and its complexes with three GUAA-containing RNAs, characterized RNA binding energetics and tested rationally designed mutants in vivo. The Nrd1 structure shows an RRM domain fused with a second α/ß domain that we name split domain (SD), because it is formed by two non-consecutive segments at each side of the RRM. The GUAA interacts with both domains and with a pocket of water molecules, trapped between the two stacking adenines and the SD. Comprehensive binding studies demonstrate for the first time that Nrd1 has a slight preference for GUAA over GUAG and genetic and functional studies suggest that Nrd1 RNA binding domain might play further roles in non-coding RNAs transcription termination.

Structural basis for interdomain communication in SHIP2 providing high phosphatase activity.

SH2-containing-inositol-5-phosphatases (SHIPs) dephosphorylate the 5-phosphate of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) and play important roles in regulating the PI3K/Akt pathway in physiology and disease. Aiming to uncover interdomain regulatory mechanisms in SHIP2, we determined crystal structures containing the 5-phosphatase and a proximal region adopting a C2 fold. This reveals an extensive interface between the two domains, which results in significant structural changes in the phosphatase domain. Both the phosphatase and C2 domains bind phosphatidylserine lipids, which likely helps to position the active site towards its substrate. Although located distant to the active site, the C2 domain greatly enhances catalytic turnover. Employing molecular dynamics, mutagenesis and cell biology, we identify two distinct allosteric signaling pathways, emanating from hydrophobic or polar interdomain interactions, differentially affecting lipid chain or headgroup moieties of PI(3,4,5)P3. Together, this study reveals details of multilayered C2-mediated effects important for SHIP2 activity and points towards interesting new possibilities for therapeutic interventions.

Discovery of first-in-class reversible dual small molecule inhibitors against G9a and DNMTs in hematological malignancies.

The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death. CM-272 significantly prolongs survival of AML, ALL and DLBCL xenogeneic models. Our results represent the discovery of first-in-class dual inhibitors of G9a/DNMTs and establish this chemical series as a promising therapeutic tool for unmet needs in haematological tumours.

Alternative Activation Mechanisms of Protein Kinase B Trigger Distinct Downstream Signaling Responses.

Protein kinase B (PKB/Akt) is an important mediator of signals that control various cellular processes including cell survival, growth, proliferation, and metabolism. PKB promotes these processes by phosphorylating many cellular targets, which trigger distinct downstream signaling events. However, how PKB is able to selectively target its substrates to induce specific cellular functions remains elusive. Here we perform a systematic study to dissect mechanisms that regulate intrinsic kinase activity versus mechanisms that specifically regulate activity toward specific substrates. We demonstrate that activation loop phosphorylation and the C-terminal hydrophobic motif are essential for high PKB activity in general. On the other hand, we identify membrane targeting, which for decades has been regarded as an essential step in PKB activation, as a mechanism mainly affecting substrate selectivity. Further, we show that PKB activity in cells can be triggered independently of PI3K by initial hydrophobic motif phosphorylation, presumably through a mechanism analogous to other AGC kinases. Importantly, different modes of PKB activation result in phosphorylation of distinct downstream targets. Our data indicate that specific mechanisms have evolved for signaling nodes, like PKB, to select between various downstream events. Targeting such mechanisms selectively could facilitate the development of therapeutics that might limit toxic side effects.

Interaction Networks in Protein Folding via Atomic-Resolution Experiments and Long-Time-Scale Molecular Dynamics Simulations.

The integration of atomic-resolution experimental and computational methods offers the potential for elucidating key aspects of protein folding that are not revealed by either approach alone. Here, we combine equilibrium NMR measurements of thermal unfolding and long molecular dynamics simulations to investigate the folding of gpW, a protein with two-state-like, fast folding dynamics and cooperative equilibrium unfolding behavior. Experiments and simulations expose a remarkably complex pattern of structural changes that occur at the atomic level and from which the detailed network of residue-residue couplings associated with cooperative folding emerges. Such thermodynamic residue-residue couplings appear to be linked to the order of mechanistically significant events that take place during the folding process. Our results on gpW indicate that the methods employed in this study are likely to prove broadly applicable to the fine analysis of folding mechanisms in fast folding proteins.

Two singular types of CCCH tandem zinc finger in Nab2p contribute to polyadenosine RNA recognition.

The seven C-terminal CCCH-type zinc fingers of Nab2p bind the poly(A) tail of mRNA (∼A25). Using NMR, we demonstrated that the first four (Zf1-Zf4) contain two structurally independent tandems (TZF12 and TZF34) and bind A12 with moderate affinity (KD = 2.3 µM). Nab2p TZF12 contains a long α helix that contacts the zinc fingers Zf1 and Zf2 to arrange them similarly to Zf6-7 in the Nab2p Zf5-7 structure. Nab2p TZF34 exhibits a distinctive two-fold symmetry of the zinc centers with mutual recognition of histidine ligands. Our mutagenesis and NMR data demonstrate that the α helix of TZF12 and Zf3 of TZF34 define the RNA-binding interface, while Zf1, Zf2, and Zf4 seem to be excluded. These results further our understanding of polyadenosine RNA recognition by the CCCH domain of Nab2p. Moreover, we describe a hypothetical mechanism for controlling poly(A) tail length with specific roles for TZF12, TZF34, and Zf5-7 domains.

Helical peptides from VEGF and Vammin hotspots for modulating the VEGF-VEGFR interaction.

The design, synthesis, conformational studies and binding affinity for VEGF receptors of a collection of linear and cyclic peptide analogues of the N-terminal α-helix fragments 13-25 of VEGF and 1-13 of Vammin are described. Linear 13(14)-mer peptides were designed with the help of an AGADIR algorithm and prepared following peptide solid-phase synthetic protocols. Cyclic peptide derivatives were prepared on-resin from linear precursors with conveniently located Glu and Lys residues, by the formation of amide linkages. Conformational analysis, CD and NMR, showed that most synthesized peptides have a clear tendency to be structured as α-helices in solution. Some of the peptides were able to bind a VEGFR-1 receptor with moderate affinity. In addition to the described key residues (Phe17, Tyr21 and Tyr25), Val14 and Val20 seem to be relevant for affinity.

Nuclear magnetic resonance study of protein-protein interactions involving apoptosis regulator Diva (Boo) and the BH3 domain of proapoptotic Bcl-2 members.

According to biochemical assays, the Bcl-2 protein Diva from mouse regulates programmed cell death by heterodimerizing with other members of the family and by interacting with the apoptotic protease-activating factor Apaf-1. In typical Bcl-2 heterodimers, peptide fragments comprising the Bcl-2 homology domain 3 (BH3 domain) of proapoptotic members are capable of forming functional complexes with prosurvival proteins. High-resolution structural studies have revealed that the BH3 peptide forms an α-helix positioned in a canonical hydrophobic cleft of the antiapoptotic protein. Because Diva shows mutations in conserved residues within this area, it has been proposed to have a different interacting surface. However, we showed previously that Diva binds through the canonical groove the BH3 peptide of the human Bcl-2 killing member Harakiri. To further test Diva's binding capabilities, here we show Nuclear Magnetic Resonance (NMR) data, indicating that Diva binds peptides derived from the BH3 domain of several other proapoptotic Bcl-2 proteins, including mouse Harakiri, Bid, Bak and Bmf. We have measured the binding affinities of the heterodimers, which show significant variability. Structural models of the protein-peptide complexes based on NMR chemical shift perturbation data indicate that the binding surface is analogous. These models do not rely on NMR NOE (Nuclear Overhauser Effect) data, and thus our results can only suggest that the complexes share similar intermolecular interactions. However, the observed affinity differences correlate with the α-helical population of the BH3-peptides obtained from circular dichroism experiments, which highlights a role of conformational selection in the binding mechanism. Altogether, our results shed light on important factors governing Diva-BH3 peptide molecular recognition mode.

Cyclic amino acid linkers stabilizing key loops of brain derived neurotrophic factor.

Based on ß-turn-like BDNF loops 2 and 4, involved in receptor interaction, cyclic peptide replicas were designed, synthesized and tested. In addition to the native turn residues, the cyclic peptides include a linker unit between the N- and C-termini, selected by molecular modeling among various non-proteinogenic cyclic amino acids. NMR conformational studies showed that most of the cyclic peptides were able to adopt turn-like structures. Several of the analogues displayed significant inhibition of the BDNF-induced TrkB receptor phosphorylation, and hence could be useful templates for developing improved antagonists for this receptor.

Pub1p C-terminal RRM domain interacts with Tif4631p through a conserved region neighbouring the Pab1p binding site.

Pub1p, a highly abundant poly(A)+ mRNA binding protein in Saccharomyces cerevisiae, influences the stability and translational control of many cellular transcripts, particularly under some types of environmental stresses. We have studied the structure, RNA and protein recognition modes of different Pub1p constructs by NMR spectroscopy. The structure of the C-terminal RRM domain (RRM3) shows a non-canonical N-terminal helix that packs against the canonical RRM fold in an original fashion. This structural trait is conserved in Pub1p metazoan homologues, the TIA-1 family, defining a new class of RRM-type domains that we propose to name TRRM (TIA-1 C-terminal domain-like RRM). Pub1p TRRM and the N-terminal RRM1-RRM2 tandem bind RNA with high selectivity for U-rich sequences, with TRRM showing additional preference for UA-rich ones. RNA-mediated chemical shift changes map to ß-sheet and protein loops in the three RRMs. Additionally, NMR titration and biochemical in vitro cross-linking experiments determined that Pub1p TRRM interacts specifically with the N-terminal region (1-402) of yeast eIF4G1 (Tif4631p), very likely through the conserved Box1, a short sequence motif neighbouring the Pab1p binding site in Tif4631p. The interaction involves conserved residues of Pub1p TRRM, which define a protein interface that mirrors the Pab1p-Tif4631p binding mode. Neither protein nor RNA recognition involves the novel N-terminal helix, whose functional role remains unclear. By integrating these new results with the current knowledge about Pub1p, we proposed different mechanisms of Pub1p recruitment to the mRNPs and Pub1p-mediated mRNA stabilization in which the Pub1p/Tif4631p interaction would play an important role.

Trp-Trp pairs as ß-hairpin stabilisers: hydrogen-bonded versus non-hydrogen-bonded sites.

Trp-Trp pairs have emerged as a successful strategy for ß-hairpin stabilization. Using loop 3 of Vammin as a template, we experimentally demonstrate that the contribution of Trp-Trp pairs to ß-hairpin stability depends on ß-sheet periodicity, that is, they are stabilising at non-hydrogen-bonded sites, but not at hydrogen-bonded positions.

Structural autonomy of a ß-hairpin peptide derived from the pneumococcal choline-binding protein LytA.

The cell wall of Streptococcus pneumoniae and several other micro-organisms is decorated with a number of the so-called choline-binding proteins (CBPs) that recognise the choline residues in the bacterial surface by means of highly conserved, concatenated 20-aa sequences termed choline-binding repeats (CBRs), that are composed of a loop and a ß-hairpin structure. In this work, we have investigated the ability to fold in aqueous solution of a 14-aa peptide (LytA197₋210[wt]) and a single derivative of it, LytA197₋210[ND], corresponding to one of the six ß-hairpins of the LytA pneumococcal amidase. Intrinsic fluorescence and circular dichroism spectroscopical measurements showed that both peptides spontaneously acquire a non-random conformation which is also able to bind the natural ligand choline. Furthermore, nuclear magnetic resonance techniques allowed the calculation of the structure of the LytA197₋210[ND] peptide, which displayed a ß-hairpin conformation highly similar to that found within the full-length C-LytA module. These results provide a structural basis for the modular organisation of CBPs and suggest the use of CBRs as new templates for the design of stable ß-hairpins.

Tryptophan residues: scarce in proteins but strong stabilizers of ß-hairpin peptides.

Tryptophan plays important roles in protein stability and recognition despite its scarcity in proteins. Except as fluorescent groups, they have been used rarely in peptide design. Nevertheless, Trp residues were crucial for the stability of some designed minimal proteins. In 2000, Trp-Trp pairs were shown to contribute more than any other hydrophobic interaction to the stability of ß-hairpin peptides. Since then, Trp-Trp pairs have emerged as a paradigm for the design of stable ß-hairpins, such as the Trpzip peptides. Here, we analyze the nature of the stabilizing capacity of Trp-Trp pairs by reviewing the ß-hairpin peptides containing Trp-Trp pairs described up to now, the spectroscopic features and geometry of the Trp-Trp pairs, and their use as binding sites in ß-hairpin peptides. To complete the overview, we briefly go through the other relevant ß-hairpin stabilizing Trp-non-Trp interactions and illustrate the use of Trp in the design of short peptides adopting α-helical and mixed α/ß motifs. This review is of interest in the field of rational design of proteins, peptides, peptidomimetics, and biomaterials.