PLEKHA7 signaling is necessary for the growth of mutant KRAS driven colorectal cancer.

Plekha7 (Pleckstrin homology [PH] domain containing, family A member 7) regulates the assembly of proteins of the cytoplasmic apical zonula adherens junction (AJ), thus ensuring cell-cell adhesion and tight-junction barrier integrity. Little is known of Plekha7 function in cancer. In colorectal cancer (CRC) Plekha7 expression is elevated compared to adjacent normal tissue levels, increasing with clinical stage. Plekha7 was present at plasma membrane AJ with wild-type KRas (wt-KRas) but was dispersed in cells expressing mutant KRas (mut-KRas). Fluorescence lifetime imaging microscopy (FLIM) indicated a direct Plekha7 interaction with wt-KRas but scantily with mut-KRas. Inhibiting Plekha7 specifically decreased mut-KRas cell signaling, proliferation, attachment, migration, and retarded mut-KRAS CRC tumor growth. Binding of diC8-phosphoinositides (PI) to the PH domain of Plekha7 was relatively low affinity. This may be because a D175 amino acid residue plays a "sentry" role preventing PI(3,4)P2 and PI(3,4,5)P3 binding. Molecular or pharmacological inhibition of the Plekha7 PH domain prevented the growth of mut-KRas but not wt-KRas cells. Taken together the studies suggest that Plekha7, in addition to maintaining AJ structure plays a role in mut-KRas signaling and phenotype through interaction of its PH domain with membrane mut-KRas, but not wt-KRas, to increase the efficiency of mut-KRas downstream signaling.

Structural basis for the association of PLEKHA7 with membrane-embedded phosphatidylinositol lipids.

PLEKHA7 (pleckstrin homology domain containing family A member 7) plays key roles in intracellular signaling, cytoskeletal organization, and cell adhesion, and is associated with multiple human cancers. The interactions of its pleckstrin homology (PH) domain with membrane phosphatidyl-inositol-phosphate (PIP) lipids are critical for proper cellular localization and function, but little is known about how PLEKHA7 and other PH domains interact with membrane-embedded PIPs. Here we describe the structural basis for recognition of membrane-bound PIPs by PLEHA7. Using X-ray crystallography, nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the interaction of PLEKHA7 with PIPs is multivalent, distinct from a discrete one-to-one interaction, and induces PIP clustering. Our findings reveal a central role of the membrane assembly in mediating protein-PIP association and provide a roadmap for understanding how the PH domain contributes to the signaling, adhesion, and nanoclustering functions of PLEKHA7.

An Inhibitor of the Pleckstrin Homology Domain of CNK1 Selectively Blocks the Growth of Mutant KRAS Cells and Tumors.

Cnk1 (connector enhancer of kinase suppressor of Ras 1) is a pleckstrin homology (PH) domain-containing scaffold protein that increases the efficiency of Ras signaling pathways, imparting efficiency and specificity to the response of cell proliferation, survival, and migration. Mutated KRAS (mut-KRAS) is the most common proto-oncogenic event, occurring in approximately 25% of human cancers and has no effective treatment. In this study, we show that selective inhibition of Cnk1 blocks growth and Raf/Mek/Erk, Rho and RalA/B signaling in mut-KRAS lung and colon cancer cells with little effect on wild-type (wt)-KRAS cells. Cnk1 inhibition decreased anchorage-independent mut-KRas cell growth more so than growth on plastic, without the partial "addiction" to mut-KRAS seen on plastic. The PH domain of Cnk1 bound with greater affinity to PtdIns(4,5)P2 than PtdIns(3,4,5)P3, and Cnk1 localized to areas of the plasma membranes rich in PtdIns, suggesting a role for the PH domain in the biological activity of Cnk1. Through molecular modeling and structural modification, we identified a compound PHT-7.3 that bound selectively to the PH domain of Cnk1, preventing plasma membrane colocalization with mut-KRas. PHT-7.3 inhibited mut-KRas, but not wild-type KRas cancer cell and tumor growth and signaling. Thus, the PH domain of Cnk1 is a druggable target whose inhibition selectively blocks mutant KRas activation, making Cnk1 an attractive therapeutic target in patients with mut-KRAS-driven cancer. SIGNIFICANCE: These findings identify a therapeutic strategy to selectively block oncogenic KRas activity through the PH domain of Cnk1, which reduces its cell membrane binding, decreasing the efficiency of Ras signaling and tumor growth.

Identification and characterization of small molecule inhibitors of the ubiquitin ligases Siah1/2 in melanoma and prostate cancer cells.

Inhibition of ubiquitin ligases with small molecule remains a very challenging task, given the lack of catalytic activity of the target and the requirement of disruption of its interactions with other proteins. Siah1/2, which are E3 ubiquitin ligases, are implicated in melanoma and prostate cancer and represent high-value drug targets. We utilized three independent screening approaches in our efforts to identify small-molecule Siah1/2 inhibitors: Affinity Selection-Mass Spectrometry, a protein thermal shift-based assay and an in silico based screen. Inhibitors were assessed for their effect on viability of melanoma and prostate cancer cultures, colony formation, prolyl-hydroxylase-HIF1α signaling, expression of selected Siah2-related transcripts, and Siah2 ubiquitin ligase activity. Several analogs were further characterized, demonstrating improved efficacy. Combination of the top hits identified in the different assays demonstrated an additive effect, pointing to complementing mechanisms that underlie each of these Siah1/2 inhibitors.

Diabetes reversal by inhibition of the low-molecular-weight tyrosine phosphatase.

Obesity-associated insulin resistance plays a central role in type 2 diabetes. As such, tyrosine phosphatases that dephosphorylate the insulin receptor (IR) are potential therapeutic targets. The low-molecular-weight protein tyrosine phosphatase (LMPTP) is a proposed IR phosphatase, yet its role in insulin signaling in vivo has not been defined. Here we show that global and liver-specific LMPTP deletion protects mice from high-fat diet-induced diabetes without affecting body weight. To examine the role of the catalytic activity of LMPTP, we developed a small-molecule inhibitor with a novel uncompetitive mechanism, a unique binding site at the opening of the catalytic pocket, and an exquisite selectivity over other phosphatases. This inhibitor is orally bioavailable, and it increases liver IR phosphorylation in vivo and reverses high-fat diet-induced diabetes. Our findings suggest that LMPTP is a key promoter of insulin resistance and that LMPTP inhibitors would be beneficial for treating type 2 diabetes.

Definition of a Novel Feed-Forward Mechanism for Glycolysis-HIF1α Signaling in Hypoxic Tumors Highlights Aldolase A as a Therapeutic Target.

The hypoxia-inducible transcription factor HIF1α drives expression of many glycolytic enzymes. Here, we show that hypoxic glycolysis, in turn, increases HIF1α transcriptional activity and stimulates tumor growth, revealing a novel feed-forward mechanism of glycolysis-HIF1α signaling. Negative regulation of HIF1α by AMPK1 is bypassed in hypoxic cells, due to ATP elevation by increased glycolysis, thereby preventing phosphorylation and inactivation of the HIF1α transcriptional coactivator p300. Notably, of the HIF1α-activated glycolytic enzymes we evaluated by gene silencing, aldolase A (ALDOA) blockade produced the most robust decrease in glycolysis, HIF-1 activity, and cancer cell proliferation. Furthermore, either RNAi-mediated silencing of ALDOA or systemic treatment with a specific small-molecule inhibitor of aldolase A was sufficient to increase overall survival in a xenograft model of metastatic breast cancer. In establishing a novel glycolysis-HIF-1α feed-forward mechanism in hypoxic tumor cells, our results also provide a preclinical rationale to develop aldolase A inhibitors as a generalized strategy to treat intractable hypoxic cancer cells found widely in most solid tumors. Cancer Res; 76(14); 4259-69. ©2016 AACR.

Molecular signatures of hemagglutinin stem-directed heterosubtypic human neutralizing antibodies against influenza A viruses.

Recent studies have shown high usage of the IGHV1-69 germline immunoglobulin gene for influenza hemagglutinin stem-directed broadly-neutralizing antibodies (HV1-69-sBnAbs). Here we show that a major structural solution for these HV1-69-sBnAbs is achieved through a critical triad comprising two CDR-H2 loop anchor residues (a hydrophobic residue at position 53 (Ile or Met) and Phe54), and CDR-H3-Tyr at positions 98±1; together with distinctive V-segment CDR amino acid substitutions that occur in positions sparse in AID/polymerase-η recognition motifs. A semi-synthetic IGHV1-69 phage-display library screen designed to investigate AID/polη restrictions resulted in the isolation of HV1-69-sBnAbs that featured a distinctive Ile52Ser mutation in the CDR-H2 loop, a universal CDR-H3 Tyr at position 98 or 99, and required as little as two additional substitutions for heterosubtypic neutralizing activity. The functional importance of the Ile52Ser mutation was confirmed by mutagenesis and by BCR studies. Structural modeling suggests that substitution of a small amino acid at position 52 (or 52a) facilitates the insertion of CDR-H2 Phe54 and CDR-H3-Tyr into adjacent pockets on the stem. These results support the concept that activation and expansion of a defined subset of IGHV1-69-encoded B cells to produce potent HV1-69-sBnAbs does not necessarily require a heavily diversified V-segment acquired through recycling/reentry into the germinal center; rather, the incorporation of distinctive amino acid substitutions by Phase 2 long-patch error-prone repair of AID-induced mutations or by random non-AID SHM events may be sufficient. We propose that these routes of B cell maturation should be further investigated and exploited as a pathway for HV1-69-sBnAb elicitation by vaccination.

αE-catenin is an autoinhibited molecule that coactivates vinculin.

αE-catenin, an essential component of the adherens junction, interacts with the classical cadherin-ß-catenin complex and with F-actin, but its precise role is unknown. αE-catenin also binds to the F-actin-binding protein vinculin, which also appears to be important in junction assembly. Vinculin and αE-catenin are homologs that contain a series of helical bundle domains, D1-D5. We mapped the vinculin-binding site to a sequence in D3a comprising the central two helices of a four-helix bundle. The crystal structure of this peptide motif bound to vinculin D1 shows that the two helices adopt a parallel, colinear arrangement suggesting that the αE-catenin D3a bundle must unfold in order to bind vinculin. We show that αE-catenin D3 binds strongly to vinculin, whereas larger fragments and full-length αE-catenin bind approximately 1,000-fold more weakly. Thus, intramolecular interactions within αE-catenin inhibit binding to vinculin. The actin-binding activity of vinculin is inhibited by an intramolecular interaction between the head (D1-D4) and the actin-binding D5 tail. In the absence of F-actin, there is no detectable binding of αE-catenin D3 to full-length vinculin; however, αE-catenin D3 promotes binding of vinculin to F-actin whereas full-length αE-catenin does not. These findings support the combinatorial or "coincidence" model of activation in which binding of high-affinity proteins to the vinculin head and tail is required to shift the conformational equilibrium of vinculin from a closed, autoinhibited state to an open, stable F-actin-binding state. The data also imply that αE-catenin must be activated in order to bind to vinculin.

Structure of complement C6 suggests a mechanism for initiation and unidirectional, sequential assembly of membrane attack complex (MAC).

The complement membrane attack complex (MAC) is formed by the sequential assembly of C5b with four homologous proteins as follows: one copy each of C6, C7, and C8 and 12-14 copies of C9. Together these form a lytic pore in bacterial membranes. C6 through C9 comprise a MAC-perforin domain flanked by 4-9 "auxiliary" domains. Here, we report the crystal structure of C6, the first and longest of the pore proteins to be recruited by C5b. Comparisons with the structures of the C8αßγ heterodimer and perforin show that the central domain of C6 adopts a "closed" (perforin-like) state that is distinct from the "open" conformations in C8. We further show that C6, C8α, and C8ß contain three homologous subdomains ("upper," "lower," and "regulatory") related by rotations about two hinge points. In C6, the regulatory segment includes four auxiliary domains that stabilize the closed conformation, inhibiting release of membrane-inserting elements. In C8ß, rotation of the regulatory segment is linked to an opening of the central ß-sheet of its clockwise partner, C8α. Based on these observations, we propose a model for initiation and unidirectional propagation of the MAC in which the auxiliary domains play key roles: in the assembly of the C5b-8 initiation complex; in driving and regulating the opening of the ß-sheet of the MAC-performin domain of each new recruit as it adds to the growing pore; and in stabilizing the final pore. Our model of the assembled pore resembles those of the cholesterol-dependent cytolysins but is distinct from that recently proposed for perforin.

Wide prevalence of heterosubtypic broadly neutralizing human anti-influenza A antibodies.

BACKGROUND: Lack of life-long immunity against influenza viruses represents a major global health care problem with profound medical and economic consequences. A greater understanding of the broad-spectrum "heterosubtypic" neutralizing human antibody (BnAb) response to influenza should bring us closer toward a universal influenza vaccine. METHODS: Serum samples obtained from 77 volunteers in an H5N1 vaccine study were analyzed for cross-reactive antibodies (Abs) against both subtype hemagglutinins (HAs) and a highly conserved pocket on the HA stem of Group 1 viruses. Cross-reactive Abs in commercial intravenous immunoglobulin were affinity purified using H5-coupled beads followed by step-wise monoclonal antibody competition or acid elution. Enzyme-linked immunosorbent assays were used to quantify cross-binding, and neutralization activity was determined with HA-pseudotyped viruses. RESULTS: Prevaccination serum samples have detectable levels of heterosubtypic HA binding activity to both Group 1 and 2 influenza A viruses, including subtypes H5 and H7, respectively, to which study subjects had not been vaccinated. Two different populations of Broadly neutralizing Abs (BnAbs) were purified from intravenous immunoglobulin by H5 beads: ~0.01% of total immunoglobulin G can bind to HAs from both Group 1 and 2 and neutralize H1N1 and H5N1 viruses; ~0.001% is F10-like Abs directed against the HA stem pocket on Group 1 viruses. CONCLUSION: These data--to our knowledge, for the first time--quantitatively show the presence, albeit at low levels, of two populations of heterosubtypic BnAbs against influenza A in human serum. These observations warrant further investigation to determine their origin, host polymorphism(s) that may affect their expression levels and how to boost these BnAb responses by vaccination to reach sustainable protective levels.

Structural insights into inhibition of Bacillus anthracis sporulation by a novel class of non-heme globin sensor domains.

Pathogenesis by Bacillus anthracis requires coordination between two distinct activities: plasmid-encoded virulence factor expression (which protects vegetative cells from immune surveillance during outgrowth and replication) and chromosomally encoded sporulation (required only during the final stages of infection). Sporulation is regulated by at least five sensor histidine kinases that are activated in response to various environmental cues. One of these kinases, BA2291, harbors a sensor domain that has ∼35% sequence identity with two plasmid proteins, pXO1-118 and pXO2-61. Because overexpression of pXO2-61 (or pXO1-118) inhibits sporulation of B. anthracis in a BA2291-dependent manner, and pXO2-61 expression is strongly up-regulated by the major virulence gene regulator, AtxA, it was suggested that their function is to titrate out an environmental signal that would otherwise promote untimely sporulation. To explore this hypothesis, we determined crystal structures of both plasmid-encoded proteins. We found that they adopt a dimeric globin fold but, most unusually, do not bind heme. Instead, they house a hydrophobic tunnel and hydrophilic chamber that are occupied by fatty acid, which engages a conserved arginine and chloride ion via its carboxyl head group. In vivo, these domains may therefore recognize changes in fatty acid synthesis, chloride ion concentration, and/or pH. Structure-based comparisons with BA2291 suggest that it binds ligand and dimerizes in an analogous fashion, consistent with the titration hypothesis. Analysis of newly sequenced bacterial genomes points to the existence of a much broader family of non-heme, globin-based sensor domains, with related but distinct functionalities, that may have evolved from an ancestral heme-linked globin.

Structural basis of membrane targeting by the Dock180 family of Rho family guanine exchange factors (Rho-GEFs).

The Dock180 family of atypical Rho family guanine nucleotide exchange factors (Rho-GEFs) regulate a variety of processes involving cellular or subcellular polarization, including cell migration and phagocytosis. Each contains a Dock homology region-1 (DHR-1) domain that is required to localize its GEF activity to a specific membrane compartment where levels of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) are up-regulated by the local activity of PtdIns 3-kinase. Here we define the structural and energetic bases of phosphoinositide specificity by the DHR-1 domain of Dock1 (a GEF for Rac1), and show that DHR-1 utilizes a C2 domain scaffold and surface loops to create a basic pocket on its upper surface for recognition of the PtdIns(3,4,5)P(3) head group. The pocket has many of the characteristics of those observed in pleckstrin homology domains. We show that point mutations in the pocket that abolish phospholipid binding in vitro ablate the ability of Dock1 to induce cell polarization, and propose a model that brings together recent mechanistic and structural studies to rationalize the central role of DHR-1 in dynamic membrane targeting of the Rho-GEF activity of Dock180.

Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses.

Influenza virus remains a serious health threat, owing to its ability to evade immune surveillance through rapid genetic drift and reassortment. Here we used a human non-immune antibody phage-display library and the H5 hemagglutinin ectodomain to select ten neutralizing antibodies (nAbs) that were effective against all group 1 influenza viruses tested, including H5N1 'bird flu' and the H1N1 'Spanish flu'. The crystal structure of one such nAb bound to H5 shows that it blocks infection by inserting its heavy chain into a conserved pocket in the stem region, thus preventing membrane fusion. Nine of the nAbs employ the germline gene VH1-69, and all seem to use the same neutralizing mechanism. Our data further suggest that this region is recalcitrant to neutralization escape and that nAb-based immunotherapy is a promising strategy for broad-spectrum protection against seasonal and pandemic influenza viruses.

Selection of anthrax toxin protective antigen variants that discriminate between the cellular receptors TEM8 and CMG2 and achieve targeting of tumor cells.

Anthrax toxin, a three-component protein toxin secreted by Bacillus anthracis, assembles into toxic complexes at the surface of receptor-bearing eukaryotic cells. The protective antigen (PA) protein binds to receptors, either tumor endothelial cell marker 8 (TEM8) or CMG2 (capillary morphogenesis protein 2), and orchestrates the delivery of the lethal and edema factors into the cytosol. TEM8 is reported to be overexpressed during tumor angiogenesis, whereas CMG2 is more widely expressed in normal tissues. To extend prior work on targeting of tumor with modified anthrax toxins, we used phage display to select PA variants that preferentially bind to TEM8 as compared with CMG2. Substitutions were randomly introduced into residues 605-729 of PA, within the C-terminal domain 4 of PA, which is the principal region that contacts receptor. Candidates were characterized in cellular cytotoxicity assays with Chinese hamster ovary (CHO) cells expressing either TEM8 or CMG2. A PA mutant having the substitutions R659S and M662R had enhanced specificity toward TEM8-overexpressing CHO cells. This PA variant also displayed broad and potent tumoricidal activity to various human tumor cells, especially to HeLa and A549/ATCC cells. By contrast, the substitution N657Q significantly reduced toxicity to TEM8 but not CMG2-overexpressing CHO cells. Our results indicate that certain amino acid substitutions within PA domain 4 create anthrax toxins that selectively kill human tumor cells. The PA R659S/M662R protein may be useful as a therapeutic agent for cancer treatment.

Nod2 mutation in Crohn's disease potentiates NF-kappaB activity and IL-1beta processing.

Variants of NOD2, an intracellular sensor of bacteria-derived muramyl dipeptide (MDP), increase susceptibility to Crohn's disease (CD). These variants are thought to be defective in activation of nuclear factor kappaB (NF-kappaB) and antibacterial defenses, but CD clinical specimens display elevated NF-kappaB activity. To illuminate the pathophysiological function of NOD2, we introduced such a variant to the mouse Nod2 locus. Mutant mice exhibited elevated NF-kappaB activation in response to MDP and more efficient processing and secretion of the cytokine interleukin-1beta (IL-1beta). These effects are linked to increased susceptibility to bacterial-induced intestinal inflammation and identify NOD2 as a positive regulator of NF-kappaB activation and IL-1beta secretion.

Crystal structure of a complex between anthrax toxin and its host cell receptor.

Anthrax toxin consists of the proteins protective antigen (PA), lethal factor (LF) and oedema factor (EF). The first step of toxin entry into host cells is the recognition by PA of a receptor on the surface of the target cell. Subsequent cleavage of receptor-bound PA enables EF and LF to bind and form a heptameric PA63 pre-pore, which triggers endocytosis. Upon acidification of the endosome, PA63 forms a pore that inserts into the membrane and translocates EF and LF into the cytosol. Two closely related host cell receptors, TEM8 and CMG2, have been identified. Both bind to PA with high affinity and are capable of mediating toxicity. Here, we report the crystal structure of the PA-CMG2 complex at 2.5 A resolution. The structure reveals an extensive receptor-pathogen interaction surface mimicking the non-pathogenic recognition of the extracellular matrix by integrins. The binding surface is closely conserved in the two receptors and across species, but is quite different in the integrin domains, explaining the specificity of the interaction. CMG2 engages two domains of PA, and modelling of the receptor-bound PA63 heptamer suggests that the receptor acts as a pH-sensitive brace to ensure accurate and timely membrane insertion. The structure provides new leads for the discovery of anthrax anti-toxins, and should aid the design of cancer therapeutics.

Structural basis for vinculin activation at sites of cell adhesion.

Vinculin is a highly conserved intracellular protein with a crucial role in the maintenance and regulation of cell adhesion and migration. In the cytosol, vinculin adopts a default autoinhibited conformation. On recruitment to cell-cell and cell-matrix adherens-type junctions, vinculin becomes activated and mediates various protein-protein interactions that regulate the links between F-actin and the cadherin and integrin families of cell-adhesion molecules. Here we describe the crystal structure of the full-length vinculin molecule (1,066 amino acids), which shows a five-domain autoinhibited conformation in which the carboxy-terminal tail domain is held pincer-like by the vinculin head, and ligand binding is regulated both sterically and allosterically. We show that conformational changes in the head, tail and proline-rich domains are linked structurally and thermodynamically, and propose a combinatorial pathway to activation that ensures that vinculin is activated only at sites of cell adhesion when two or more of its binding partners are brought into apposition.

Cell adhesion: a FERM grasp of membrane dynamics.

Two recent papers provide the first evidence for a common mechanism of targeting and activation of an enzyme that is important in the rapid regulation of both focal adhesion assembly during cell migration and synaptic vesicle recycling at nerve terminals.

Cysteine regulation of protein function--as exemplified by NMDA-receptor modulation.

Until recently cysteine residues, especially those located extracellularly, were thought to be important for metal coordination, catalysis and protein structure by forming disulfide bonds - but they were not thought to regulate protein function. However, this is not the case. Crucial cysteine residues can be involved in modulation of protein activity and signaling events via other reactions of their thiol (sulfhydryl; -SH) groups. These reactions can take several forms, such as redox events (chemical reduction or oxidation), chelation of transition metals (chiefly Zn(2+), Mn(2+) and Cu(2+)) or S-nitrosylation [the catalyzed transfer of a nitric oxide (NO) group to a thiol group]. In several cases, these disparate reactions can compete with one another for the same thiol group on a single cysteine residue, forming a molecular switch composed of a latticework of possible redox, NO or Zn(2+) modifications to control protein function. Thiol-mediated regulation of protein function can also involve reactions of cysteine residues that affect ligand binding allosterically. This article reviews the basis for these molecular cysteine switches, drawing on the NMDA receptor as an exemplary protein, and proposes a molecular model for the action of S-nitrosylation based on recently derived crystal structures.