Allosteric switch regulates protein–protein binding through collective motion

Many biological processes depend on allosteric communication between different parts of a protein, but the role of internal protein motion in propagating signals through the structure remains largely unknown. Through an experimental and computational analysis of the ground state dynamics in ubiquitin, we identify a collective global motion that is specifically linked to a conformational switch distant from the binding interface. This allosteric coupling is also present in crystal structures and is found to facilitate multispecificity, particularly binding to the ubiquitin-specific protease (USP) family of deubiquitinases. The collective motion that enables this allosteric communication does not affect binding through localized changes but, instead, depends on expansion and contraction of the entire protein domain. The characterization of these collective motions represents a promising avenue for finding and manipulating allosteric networks.Intermolecular interactions are one of the key mechanisms by which proteins mediate their biological functions. For many proteins, these interactions are enhanced or suppressed by allosteric networks that couple distant regions together (1). The mechanisms by which these networks function are just starting to be understood (2–4), and many of the important details have yet to be uncovered. In particular, the role of intrinsic protein motion and kinetics remains particularly poorly characterized. A number of structural ensembles representing ubiquitin motion have been recently proposed (5–9). Additionally, it has been suggested that through motion at the binding interface, its free state visits the same conformations found in complex with its many binding partners (5, 10). However, it remains an unanswered question if the dynamics that enable this multispecificity are only clustered around the canonical binding interface or whether this motion is allosterically coupled to the rest of the protein, especially given the presence of motion at distal sites (11).     

Host–rabies virus protein–protein interactions as druggable antiviral targets

We present an unconventional approach to antiviral drug discovery, which is used to identify potent small molecules against rabies virus. First, we conceptualized viral capsid assembly as occurring via a host-catalyzed biochemical pathway, in contrast to the classical view of capsid formation by self-assembly. This suggested opportunities for antiviral intervention by targeting previously unappreciated catalytic host proteins, which were pursued. Second, we hypothesized these host proteins to be components of heterogeneous, labile, and dynamic multi-subunit assembly machines, not easily isolated by specific target protein-focused methods. This suggested the need to identify active compounds before knowing the precise protein target. A cell-free translation-based small molecule screen was established to recreate the hypothesized interactions involving newly synthesized capsid proteins as host assembly machine substrates. Hits from the screen were validated by efficacy against infectious rabies virus in mammalian cell culture. Used as affinity ligands, advanced analogs were shown to bind a set of proteins that effectively reconstituted drug sensitivity in the cell-free screen and included a small but discrete subfraction of cellular ATP-binding cassette family E1 (ABCE1), a host protein previously found essential for HIV capsid formation. Taken together, these studies advance an alternate view of capsid formation (as a host-catalyzed biochemical pathway), a different paradigm for drug discovery (whole pathway screening without knowledge of the target), and suggest the existence of labile assembly machines that can be rendered accessible as next-generation drug targets by the means described.     

H-Ras forms dimers on membrane surfaces via a protein–protein interface

The lipid-anchored small GTPase Ras is an important signaling node in mammalian cells. A number of observations suggest that Ras is laterally organized within the cell membrane, and this may play a regulatory role in its activation. Lipid anchors composed of palmitoyl and farnesyl moieties in H-, N-, and K-Ras are widely suspected to be responsible for guiding protein organization in membranes. Here, we report that H-Ras forms a dimer on membrane surfaces through a protein–protein binding interface. A Y64A point mutation in the switch II region, known to prevent Son of sevenless and PI3K effector interactions, abolishes dimer formation. This suggests that the switch II region, near the nucleotide binding cleft, is either part of, or allosterically coupled to, the dimer interface. By tethering H-Ras to bilayers via a membrane-miscible lipid tail, we show that dimer formation is mediated by protein interactions and does not require lipid anchor clustering. We quantitatively characterize H-Ras dimerization in supported membranes using a combination of fluorescence correlation spectroscopy, photon counting histogram analysis, time-resolved fluorescence anisotropy, single-molecule tracking, and step photobleaching analysis. The 2D dimerization K_d is measured to be ∼1 × 10³ molecules/µm², and no higher-order oligomers were observed. Dimerization only occurs on the membrane surface; H-Ras is strictly monomeric at comparable densities in solution. Analysis of a number of H-Ras constructs, including key changes to the lipidation pattern of the hypervariable region, suggest that dimerization is a general property of native H-Ras on membrane surfaces.In mammalian signal transduction, Ras functions as a binary switch in fundamental processes including proliferation, differentiation, and survival (1). Ras is a network hub; various upstream signaling pathways can activate Ras-GDP to Ras-GTP, which subsequently selects between multiple downstream effectors to elicit a varied but specific biochemical response (2, 3). Signaling specificity is achieved by a combination of conformational plasticity in Ras itself (4, 5) and dynamic control of Ras spatial organization (6, 7). Isoform-specific posttranslational lipidation targets the main H-, N-, and K-Ras isoforms to different subdomains of the plasma membrane (8–10). For example, H-Ras localizes to cholesterol-sensitive membrane domains, whereas K-Ras does not (11). A common C-terminal S-farnesyl moiety operates in concert with one (N-Ras) or two (H-Ras) palmitoyl groups, or with a basic sequence of six lysines in K-Ras4B (12), to provide the primary membrane anchorage. Importantly, the G-domain (residues 1–166) and the hypervariable region (HVR) (residues 167–189) dynamically modulate the lipid anchor localization preference to switch between distinct membrane populations (13). For example, repartitioning of H-Ras away from cholesterol-sensitive membrane domains is necessary for efficient activation of the effector Raf and GTP loading of the G-domain promotes this redistribution by a mechanism that requires the HVR (14). However, the molecular details of the coupling between lipid anchor partitioning and nucleotide-dependent protein–membrane interactions remain unclear.In addition to biochemical evidence for communication between the C-terminal membrane binding region and the nucleotide binding pocket, NMR and IR spectroscopic observations suggest that the HVR and lipid anchor membrane insertion affects Ras structure and orientation (15–17). Molecular dynamics (MD) modeling of bilayer-induced H-Ras conformations has identified two nucleotide-dependent states, which differ in HVR conformation, membrane contacts, and G-domain orientation (18). In vivo FRET measurements are consistent with a reorientation of Ras with respect to the membrane upon GTP binding (19, 20). Further modeling showed that the membrane binding region and the canonical switch I and II regions communicate across the protein via long-range side-chain interactions (21) in a conformational selection mechanism (22). Whereas these allosteric modes likely contribute to Ras partitioning and reorientation in vivo, direct functional consequences on Ras protein–protein interactions are poorly understood.Members of the Ras superfamily of small GTPases are widely considered to be monomeric (23). However, several members across the Ras GTPase subfamilies are now known to dimerize (24–28), and a class of small GTPases that use dimerization instead of GTPase activating proteins (GAPs) for GTPase activity has been identified (29). Recently, semisynthetic natively lipidated N-Ras was shown to cluster on supported membranes in vitro, in a manner broadly consistent with molecular mechanics (MM) modeling of dimers (30). For Ras, dimerization could be important because Raf, which is recruited to the membrane by binding to Ras, requires dimerization for activation. Soluble Ras does not activate Raf in vitro (31), but because artificial dimerization of GST-fused H-Ras leads to Raf activation in solution, it has been hypothesized that Ras dimers exist on membranes (32). However, presumed dimers were only detected after chemical cross-linking (32), and the intrinsic oligomeric properties of Ras remain unknown.Here, we use a combination of time-resolved fluorescence spectroscopy and microscopy to characterize H-Ras(C118S, 1–181) and H-Ras(C118S, 1–184) [referred to as Ras(C181) and Ras(C181,C184) from here on] anchored to supported lipid bilayers. By tethering H-Ras to membranes at cys181 (or both at cys181 and cys184) via a membrane-miscible lipid tail, we eliminate effects of lipid anchor clustering while preserving the HVR region between the G-domain and the N-terminal palmitoylation site at cys181 (or cys184), which is predicted to undergo large conformational changes upon membrane binding and nucleotide exchange (18). Labeling is achieved through a fluorescent Atto488-linked nucleotide. Fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence anisotropy (TRFA) show that H-Ras forms surface density-dependent clusters. Photon counting histogram (PCH) analysis and single-molecule tracking (SMT) reveal that H-Ras clusters are dimers and that no higher-order oligomers are formed. A Y64A point mutation in the loop between beta strand 3 (β3) and alpha helix 2 (α2) abolishes dimer formation, suggesting that the corresponding switch II (SII) region is either part of, or allosterically coupled to, the dimer interface. The 2D dimerization K_d is measured to be on the order of 1 × 10³ molecules/µm², within the broad range of Ras surface densities measured in vivo (10, 33–35). Dimerization only occurs on the membrane surface; H-Ras is strictly monomeric at comparable densities in solution, suggesting that a membrane-induced structural change in H-Ras leads to dimerization. Comparing singly lipidated Ras(C181) and doubly lipidated Ras(C181,C184) reveals that dimer formation is insensitive to the details of HVR lipidation, suggesting that dimerization is a general property of H-Ras on membrane surfaces.     

Phosphorylation of aquaporin-2 regulates its endocytosis and protein–protein interactions

The water channel aquaporin-2 (AQP2) is essential for urine concentration. Vasopressin regulates phosphorylation of AQP2 at four conserved serine residues at the COOH-terminal tail (S256, S261, S264, and S269). We used numerous stably transfected Madin–Darby canine kidney cell models, replacing serine residues with either alanine (A), which prevents phosphorylation, or aspartic acid (D), which mimics the charged state of phosphorylated AQP2, to address whether phosphorylation is involved in regulation of (i) apical plasma membrane abundance of AQP2, (ii) internalization of AQP2, (iii) AQP2 protein–protein interactions, and (iv) degradation of AQP2. Under control conditions, S256D- and 269D-AQP2 mutants had significantly greater apical plasma membrane abundance compared to wild type (WT)-AQP2. Activation of adenylate cyclase significantly increased the apical plasma membrane abundance of all S-A or S-D AQP2 mutants with the exception of 256D-AQP2, although 256A-, 261A-, and 269A-AQP2 mutants increased to a lesser extent than WT-AQP2. Biotin internalization assays and confocal microscopy demonstrated that the internalization of 256D- and 269D-AQP2 from the plasma membrane was slower than WT-AQP2. The slower internalization corresponded with reduced interaction of S256D- and 269D-AQP2 with several proteins involved in endocytosis, including Hsp70, Hsc70, dynamin, and clathrin heavy chain. The mutants with the slowest rate of internalization, 256D- and 269D-AQP2, had a greater protein half-life (t_1/2 = 5.1 h and t_1/2 = 4.4 h, respectively) compared to WT-AQP2 (t_1/2 = 2.9 h). Our results suggest that vasopressin-mediated membrane accumulation of AQP2 can be controlled via regulated exocytosis and endocytosis in a process that is dependent on COOH terminal phosphorylation and subsequent protein–protein interactions.     

A modular toolkit to inhibit proline-rich motif–mediated protein–protein interactions

Small-molecule competitors of protein–protein interactions are urgently needed for functional analysis of large-scale genomics and proteomics data. Particularly abundant, yet so far undruggable, targets include domains specialized in recognizing proline-rich segments, including Src-homology 3 (SH3), WW, GYF, and Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Here, we present a modular strategy to obtain an extendable toolkit of chemical fragments (ProMs) designed to replace pairs of conserved prolines in recognition motifs. As proof-of-principle, we developed a small, selective, peptidomimetic inhibitor of Ena/VASP EVH1 domain interactions. Highly invasive MDA MB 231 breast-cancer cells treated with this ligand showed displacement of VASP from focal adhesions, as well as from the front of lamellipodia, and strongly reduced cell invasion. General applicability of our strategy is illustrated by the design of an ErbB4-derived ligand containing two ProM-1 fragments, targeting the yes-associated protein 1 (YAP1)-WW domain with a fivefold higher affinity.Proline-rich segments (PRSs) belong to the most abundant sequence motifs of the proteome (1), interacting frequently with PRS-recognizing domains (PRDs), such as EVH1, SH3, GYF, and WW. Although exhibiting different tertiary structures, PRDs expose clusters of aromatic residues, forming a shallow, corrugated binding groove with a hydrogen bond-donating residue (W, Y) in the central position. In the bound state, PRSs often show a conformation closely related to the ideal left-handed polyproline II (PPII) helix characterized by backbone angles of Φ = −78° and Ψ = +146° (2). As a consequence of the axial symmetry of PPII helices, two different types of consensus motifs occur: one containing PxxP specifically recognized by the EVH1 and SH3 domains, the other comprising xPPx, typical for motifs binding at WW and GYF domains. The conserved prolines represent the core of the consensus motifs and interact intimately with the exposed aromatic side chains. They cannot be replaced by any other natural amino acid without complete loss of affinity (2, 3). On the other hand, the core motif alone binds only very weakly to its PRD. Further interactions of flanking residues located outside the core motif contribute substantially to both affinity and specificity. Incorporation of nonnatural amino acids in place of such specificity-determining residues is therefore often beneficial for binding (4–9). However, peptide ligands display a number of disadvantages when used as competitors, among them metabolic instability and often low cell permeability. Cell-permeable small molecules that grant the ability to modulate the function of PRDs are still not available.Here, we present a modular concept for the systematic development of such low-molecular weight compounds. It is based on molecular building blocks that can replace the conserved prolines within the core motif without any loss of affinity. Combinations of such building blocks allow complete replacement of the proline-rich core motifs. They may be supplemented with organic scaffolds addressing the flanking epitopes to obtain peptidomimetic inhibitors of PRDs, highly desirable for functional analysis of PRS-mediated protein–protein interactions.As proof of concept, we developed a peptidomimetic inhibitor targeting the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family Ena/VASP homology 1 (EVH1) domains. This protein family is involved in modulation of the actin cytoskeleton, a complex and highly regulated process, which is the driving force of directed cell migration (10, 11) and plays important roles in disease-relevant processes like tumor metastasis (12, 13). The Ena/VASP family proteins [i.e., VASP, enabled homolog (EnaH), and Ena-VASP–like (EVL) (14–16)] are notably localized at focal adhesions and lamellipodia. Single Ena/VASP protein deletions are mostly compensated for the other members of the family (17); however, triple knock-out mice are embryonic lethal (18, 19). The proteins comprise EVH1 and Ena/VASP homology 2 (EVH2) domains, separated by a proline-rich region. Although EVH2 binds to the barbed ends of actin filaments, EVH1 interacts with proteins, like zyxin or lamellipodin (Lpd also called RAPH1), that contain the class 1 EVH1 consensus motif [FYWL]P.ϕP (ϕ is an aliphatic amino acid) (2, 20–22). Using our peptidomimetic inhibitor, we show that inhibition of the Ena/VASP family EVH1 domains strongly influences both cellular localization of VASP as well as cell migration.     

C-reactive protein and cardiovascular diseases

See related artide,pages 44-48.Recently many new disease markers and risk factorshave been proposed,but it is not yet clear how far thenew markers are validated as predictive risk factors enableus to increase accuracy as well as enhancing our ability topredict cardiovascular (CV) events and to plan preventionand therapy.     

Analysing protein–protein interaction networks of human liver cancer cell lines with diverse metastasis potential

Purpose Human hepatocellular carcinoma (HCC) is one of the most mortal tumor. In a previous study, we had constructed glycoprotein expression profiles and glycoprotein databases of three human liver cancer cell lines with diverse metastasis potential. In order to discover vital glycoproteins related to pathogenesis and metastasis of HCC, in this study we analyzed previous data with bioinformatic approach. Methods We took previous data to draw the protein–protein interaction (PPI) networks of liver cell lines by searching IntACT database and then using Pajeck software. Further more, we compared the differences between the three PPI networks by drawing the PPI networks of differential glycoproteins and by naming differential display PPI networks. Results Large numbers of proliferation and apoptosis-relative proteins interact with the differential glycoproteins, and among the differential glycoproteins there are many interactions. Conclusions We conclude that neither single nor several proteins cause malignant proliferation of liver cells. “Molecule groups” concept should be introduced into diagnosis and metastasis prediction of the HCC.     

Highly neurotoxic monomeric α-helical prion protein

Prion diseases are infectious and belong to the group of protein misfolding neurodegenerative diseases. In these diseases, neuronal dysfunction and death are caused by the neuronal toxicity of a particular misfolded form of their cognate protein. The ability to specifically target the toxic protein conformer or the neuronal death pathway would provide powerful therapeutic approaches to these diseases. The neurotoxic forms of the prion protein (PrP) have yet to be defined but there is evidence suggesting that at least some of them differ from infectious PrP (PrP(Sc)). Herein, without making an assumption about size or conformation, we searched for toxic forms of recombinant PrP after dilution refolding, size fractionation, and systematic biological testing of all fractions. We found that the PrP species most neurotoxic in vitro and in vivo (toxic PrP, TPrP) is a monomeric, highly α-helical form of PrP. TPrP caused autophagy, apoptosis, and a molecular signature remarkably similar to that observed in the brains of prion-infected animals. Interestingly, highly α-helical intermediates have been described for other amyloidogenic proteins but their biological significance remains to be established. We provide unique experimental evidence that a monomeric α-helical form of an amyloidogenic protein represents a cytotoxic species. Although toxic PrP has yet to be purified from prion-infected brains, TPrP might be the equivalent of one highly neurotoxic PrP species generated during prion replication. Because TPrP is a misfolded, highly neurotoxic form of PrP reproducing several features of prion-induced neuronal death, it constitutes a useful model to study PrP-induced neurodegenerative mechanisms.     

How fast is protein hydrophobic collapse?

One of the most recurring questions in protein folding refers to the interplay between formation of secondary structure and hydrophobic collapse. In contrast with secondary structure, it is hard to isolate hydrophobic collapse from other folding events. We have directly measured the dynamics of protein hydrophobic collapse in the absence of competing processes. Collapse was triggered with laser-induced temperature jumps in the acid-denatured form of a simple protein and monitored by fluorescence resonance energy transfer between probes placed at the protein ends. The relaxation time for hydrophobic collapse is only approximately equal to 60 ns at 305 K, even faster than secondary structure formation. At higher temperatures, as the protein becomes increasingly compact by a stronger hydrophobic force, we observe a slowdown of the dynamics of collapse. This dynamic hydrophobic effect is a high-temperature analogue of the dynamic glass transition predicted by theory. Our results indicate that in physiological conditions many proteins will initiate folding by collapsing to an unstructured globule. Local motions will presumably drive the following search for native structure in the collapsed globule.     

Targeting diverse protein–protein interaction interfaces with α/β-peptides derived from the Z-domain scaffold

Peptide-based agents derived from well-defined scaffolds offer an alternative to antibodies for selective and high-affinity recognition of large and topologically complex protein surfaces. Here, we describe a strategy for designing oligomers containing both α- and β-amino acid residues (“α/β-peptides”) that mimic several peptides derived from the three-helix bundle “Z-domain” scaffold. We show that α/β-peptides derived from a Z-domain peptide targeting vascular endothelial growth factor (VEGF) can structurally and functionally mimic the binding surface of the parent peptide while exhibiting significantly decreased susceptibility to proteolysis. The tightest VEGF-binding α/β-peptide inhibits the VEGF₁₆₅-induced proliferation of human umbilical vein endothelial cells. We demonstrate the versatility of this strategy by showing how principles underlying VEGF signaling inhibitors can be rapidly extended to produce Z-domain–mimetic α/β-peptides that bind to two other protein partners, IgG and tumor necrosis factor-α. Because well-established selection techniques can identify high-affinity Z-domain derivatives from large DNA-encoded libraries, our findings should enable the design of biostable α/β-peptides that bind tightly and specifically to diverse targets of biomedical interest. Such reagents would be useful for diagnostic and therapeutic applications.Designed molecules that bind selectively to specific sites on proteins may serve as inhibitors of medically important macromolecular interactions or diagnostic tools for biomarker detection. Small molecules often fail for these applications because of the relatively large and irregularly shaped target surfaces (1–3). In contrast, large polypeptides (e.g., antibodies) can frequently be developed to recognize a protein surface with high affinity and selectivity and represent the state of the art for engineering ligands for specific biomacromolecular targets. Large polypeptides, however, suffer several disadvantages for in vivo applications, including costly production, low storage stability, and/or low bioavailability because of rapid proteolytic degradation (4, 5).Backbone-modified peptides, an underexplored class of molecules, are proving to be a fruitful source of tight-binding and specific protein ligands. Peptidic oligomers that contain β-amino acid residues interspersed among α-residues (“α/β-peptides”) can effectively mimic the recognition surface projected by an α-helix and thereby disrupt or augment protein–protein interactions in which one partner contributes a single helix to the interface (6, 7). The unnatural backbone diminishes α/β-peptide susceptibility to proteolytic degradation relative to conventional peptides (α-residues only, “α-peptides”). As a result, α/β-peptides can exhibit improved pharmacokinetic properties in vivo relative to analogous α-peptides (8, 9). To date, however, the α/β-peptide strategy has been restricted to mimicry of isolated α-helices, which is a significant limitation given that most protein–protein interactions are mediated by surfaces that are broader than can be covered by a single, regular helix (1–4, 10).Several small proteins have been explored as scaffolds that can be adapted to interact with structurally diverse protein-binding partners (11–13). The defined tertiary structures of such scaffolds allow them to present large binding surfaces that can engage large and complementary surfaces on target proteins. The “Z-domain” or “affibody” scaffold (14) is a widely studied example that is derived from domain B of staphylococcal protein A (15). The parent Z-domain (Z-IgG) (Fig. 1A) is a 58-residue engineered analog of domain B that retains affinity for the Fc portion of IgG, the natural binding partner of protein A (16). Z-IgG adopts a three-helix bundle tertiary structure, with a large surface (>600 Ų buried in the interface with Fc) formed by helices 1 and 2 contributing most of the Fc-contacting residues. Helix 3 stabilizes the Z-domain fold by packing against the other two helices (15, 17).Open in a separate windowFig. 1.Design of α/β-peptides based on the Z-domain scaffold. (A) Sequences of peptides previously derived from the Z-domain scaffold Z-VEGF, Z-IgG, and Z-TNFα targeting VEGF (19), IgG (16), and TNFα (20), respectively. Helices 1, 2, and 3 are indicated by brackets. For Z-VEGF and Z-TNFα, residues on the protein-binding face of helices 1 and 2 that were identified via randomization and selection (including the unintentionally incorporated Ala¹⁴ in Z-VEGF) are shown in red. For Z-IgG, the parent Z-domain, red positions indicate the corresponding residues that contact IgG. Sequences are arranged based on structural alignment of helical regions. (B) Strategy for the design of α/β-peptide mimics of Z-VEGF (shown in yellow and red). Red residues indicate selected residues that contact VEGF_8–109 (shown in gray) in the cocrystal structure. Sites targeted for nonnatural amino acid substitutions shown in teal. Figure is based on PDB ID code 3S1K.The composite surface displayed by helices 1 and 2 of the Z-domain scaffold can be crafted for specific binding to diverse protein partners because the three-helix bundle tertiary structure tolerates substitutions at solvent-exposed positions (18). Combinatorial randomization of as many as 13 solvent-exposed positions on helices 1 and 2, followed by affinity-based selection, has identified Z-domain derivatives that bind to a variety of targets (12, 14), including vascular endothelial growth factor (VEGF) (peptide Z-VEGF; Fig. 1 A and B) (19), tumor necrosis factor-α (TNFα) (peptide Z-TNFα; Fig. 1A) (20), and human epidermal growth factor receptor 2 (HER2) (21). Such Z-domain analogs might represent alternatives to antibodies for selective detection of disease marker proteins or for blocking deleterious signal transduction (11–14). In many cases, selection from a phage library has identified Z-domain derivatives that exhibit dissociation constants (K_D) in the nanomolar range for a chosen protein target. Affinity maturation can enhance binding to K_D values in the picomolar range (21). Recent clinical evaluations of radiolabeled Z-domain derivatives targeting HER2 revealed that these peptides could be safely used to image HER2-overexpressing lesions in breast cancer patients (22), a result that highlights the medical promise of the Z-domain scaffold.The high α-helix content of the Z-domain scaffold led us to envision that α/β-peptide analogs could be developed as binding partners for target proteins (23). We hypothesized that α→β replacements focused at sites distinct from the positions within helices 1 and 2 that mediate target recognition could reduce susceptibility to proteolytic degradation while maintaining high affinity for the partner. This design hypothesis is encouraged by two reports of Z-domain derivatives lacking helix 3 that retained affinity for their designated targets (24–26). Here, we describe the development of α/β-peptides that structurally and functionally mimic Z-VEGF. We demonstrate the versatility of this α/β-peptide strategy by showing how principles revealed in the VEGF-based effort can be extended to achieve functional mimicry of Z-domain peptides (Z-IgG and Z-TNFα) that bind to two other protein partners, IgG and TNFα.     

Estrogen receptor α inhibitor activates the unfolded protein response,blocks protein synthesis,and induces tumor regression

Recurrent estrogen receptor α (ERα)-positive breast and ovarian cancers are often therapy resistant. Using screening and functional validation, we identified BHPI, a potent noncompetitive small molecule ERα biomodulator that selectively blocks proliferation of drug-resistant ERα-positive breast and ovarian cancer cells. In a mouse xenograft model of breast cancer, BHPI induced rapid and substantial tumor regression. Whereas BHPI potently inhibits nuclear estrogen–ERα-regulated gene expression, BHPI is effective because it elicits sustained ERα-dependent activation of the endoplasmic reticulum (EnR) stress sensor, the unfolded protein response (UPR), and persistent inhibition of protein synthesis. BHPI distorts a newly described action of estrogen–ERα: mild and transient UPR activation. In contrast, BHPI elicits massive and sustained UPR activation, converting the UPR from protective to toxic. In ERα⁺ cancer cells, BHPI rapidly hyperactivates plasma membrane PLCγ, generating inositol 1,4,5-triphosphate (IP₃), which opens EnR IP₃R calcium channels, rapidly depleting EnR Ca²⁺ stores. This leads to activation of all three arms of the UPR. Activation of the PERK arm stimulates phosphorylation of eukaryotic initiation factor 2α (eIF2α), resulting in rapid inhibition of protein synthesis. The cell attempts to restore EnR Ca²⁺ levels, but the open EnR IP₃R calcium channel leads to an ATP-depleting futile cycle, resulting in activation of the energy sensor AMP-activated protein kinase and phosphorylation of eukaryotic elongation factor 2 (eEF2). eEF2 phosphorylation inhibits protein synthesis at a second site. BHPI’s novel mode of action, high potency, and effectiveness in therapy-resistant tumor cells make it an exceptional candidate for further mechanistic and therapeutic exploration.Estrogens, acting via estrogen receptor α (ERα), stimulate tumor growth (1–3). Approximately 70% of breast cancers are ERα-positive and most deaths due to breast cancer are in patients with ERα⁺ tumors (2, 4). Endocrine therapy using aromatase inhibitors to block estrogen production, or tamoxifen and other competitor antiestrogens, often results in selection and outgrowth of resistant tumors. Although 30–70% of epithelial ovarian tumors are ERα-positive (1), endocrine therapy is largely ineffective (5–7). After several cycles of chemotherapy, tumors recur as resistant ovarian cancer (5), and most patients die within 5 years (8).Noncompetitive ERα inhibitors targeting this unmet therapeutic need, including DIBA, TPBM, TPSF, and LRH-1 inhibitors that reduce ERα levels, show limited specificity, require high concentrations (>5 μM), and usually have not advanced through preclinical development (9–12). These noncompetitive ERα inhibitors and competitor antiestrogens are primarily cytostatic and act by preventing estrogen–ERα action; therefore, they are largely ineffective in therapy-resistant ERα containing cancer cells that no longer require estrogens and ERα for growth.To target the estrogen–ERα axis in therapy-resistant cancer cells, we developed (13) and implemented an unbiased pathway-directed screen of ∼150,000 small molecules. We identified ∼2,000 small molecule biomodulators of 17β-estradiol (E₂)–ERα-induced gene expression, evaluated these biomodulators for inhibition of E₂–ERα-induced cell proliferation, and performed simple follow-on assays to identify inhibitors with a novel mode of action. Here, we describe 3,3-bis(4-hydroxyphenyl)-7-methyl-1,3-dihydro-2H-indol-2-one (BHPI), our most promising small molecule ERα biomodulator.In response to stress, cancer cells often activate the endoplasmic reticulum (EnR) stress sensor, the unfolded protein response (UPR). We recently showed that as an essential component of the E₂–ERα proliferation program, estrogen induces a different mode of UPR activation, a weak anticipatory activation of the UPR before increased protein folding loads that accompany cell proliferation. This weak and transient E₂–ERα-mediated UPR activation is protective (14). BHPI distorts this normal action of E₂–ERα and induces a massive and sustained ERα-dependent activation of the UPR, converting UPR activation from cytoprotective to cytotoxic. Moreover, independent of its effect on the UPR and protein synthesis, BHPI rapidly suppresses E₂–ERα-regulated gene expression.     

Activated protein C cofactor function of protein S: a novel role for a γ-carboxyglutamic acid residue

Protein S has an important anticoagulant function by acting as a cofactor for activated protein C (APC). We recently reported that the EGF1 domain residue Asp95 is critical for APC cofactor function. In the present study, we examined whether additional interaction sites within the Gla domain of protein S might contribute to its APC cofactor function. We examined 4 residues, composing the previously reported "Face1" (N33S/P35T/E36A/Y39V) variant, as single point substitutions. Of these protein S variants, protein S E36A was found to be almost completely inactive using calibrated automated thrombography. In factor Va inactivation assays, protein S E36A had 89% reduced cofactor activity compared with wild-type protein S and was almost completely inactive in factor VIIIa inactivation; phospholipid binding was, however, normal. Glu36 lies outside the ω-loop that mediates Ca(2+)-dependent phospholipid binding. Using mass spectrometry, it was nevertheless confirmed that Glu36 is γ-carboxylated. Our finding that Gla36 is important for APC cofactor function, but not for phospholipid binding, defines a novel function (other than Ca(2+) coordination/phospholipid binding) for a Gla residue in vitamin K-dependent proteins. It also suggests that residues within the Gla and EGF1 domains of protein S act cooperatively for its APC cofactor function.