Systematic elucidation of neuron-astrocyte interaction in models of amyotrophic lateral sclerosis using multi-modal integrated bioinformatics workflow.

Cell-to-cell communications are critical determinants of pathophysiological phenotypes, but methodologies for their systematic elucidation are lacking. Herein, we propose an approach for the Systematic Elucidation and Assessment of Regulatory Cell-to-cell Interaction Networks (SEARCHIN) to identify ligand-mediated interactions between distinct cellular compartments. To test this approach, we selected a model of amyotrophic lateral sclerosis (ALS), in which astrocytes expressing mutant superoxide dismutase-1 (mutSOD1) kill wild-type motor neurons (MNs) by an unknown mechanism. Our integrative analysis that combines proteomics and regulatory network analysis infers the interaction between astrocyte-released amyloid precursor protein (APP) and death receptor-6 (DR6) on MNs as the top predicted ligand-receptor pair. The inferred deleterious role of APP and DR6 is confirmed in vitro in models of ALS. Moreover, the DR6 knockdown in MNs of transgenic mutSOD1 mice attenuates the ALS-like phenotype. Our results support the usefulness of integrative, systems biology approach to gain insights into complex neurobiological disease processes as in ALS and posit that the proposed methodology is not restricted to this biological context and could be used in a variety of other non-cell-autonomous communication mechanisms.

X-ray Structures of the Proprotein Convertase Furin Bound with Substrate Analogue Inhibitors Reveal Substrate Specificity Determinants beyond the S4 Pocket.

The proprotein convertase furin is a highly specific serine protease modifying and thereby activating proteins in the secretory pathway by proteolytic cleavage. Its substrates are involved in many diseases, including cancer and infections caused by bacteria and viruses. Understanding furin's substrate specificity is crucially important for the development of pharmacologically applicable inhibitors. Using protein X-ray crystallography, we investigated the extended substrate binding site of furin in complex with three peptide-derived inhibitors at up to 1.9 Å resolution. The structure of the protease bound with a hexapeptide inhibitor revealed molecular details of its S6 pocket, which remained completely unknown so far. The arginine residue at P6 induced an unexpected turnlike conformation of the inhibitor backbone, which is stabilized by intra- and intermolecular H-bonds. In addition, we confirmed the binding of arginine to the previously proposed S5 pocket (S51). An alternative S5 site (S52) could be utilized by shorter side chains as demonstrated for a 4-aminomethyl-phenylacetyl residue, which shows steric properties similar to those of a lysine side chain. Interestingly, we also observed binding of a peptide with citrulline at P4 substituting for the highly conserved arginine. The structural data might indicate an unusual protonation state of Asp264 maintaining the interaction with uncharged citrulline. The herein identified molecular interaction sites at P5 and P6 can be utilized to improve next-generation furin inhibitors. Our data will also help to predict furin substrates more precisely on the basis of the additional specificity determinants observed for P5 and P6.

Structural Studies Revealed Active Site Distortions of Human Furin by a Small Molecule Inhibitor.

Proprotein convertases (PCs) represent highly selective serine proteases that activate their substrates upon proteolytic cleavage. Their inhibition is a promising strategy for the treatment of several pathologies including cancer, atherosclerosis, hypercholesterolaemia, and infectious diseases. Here, we present the first experimental complex of furin with a non-substrate-like small molecule inhibitor, and the X-ray structure of the enzyme complexed to the small molecule inhibitor 1 at 1.9 Å resolution. Two molecules of inhibitor 1 were found to interact with furin. One is anchored at the S4 pocket of the enzyme and interferes directly with the conformation and function of the catalytic triade; the other molecule shows weaker binding and interacts with a distant, less conserved region of furin. The observed binding modes represent a new inhibition strategy of furin and imply the possibility to attain specificity among the PCs providing an innovative starting point of structure guided inhibitor development for furin.

The structure of a furin-antibody complex explains non-competitive inhibition by steric exclusion of substrate conformers.

Proprotein Convertases (PCs) represent highly selective serine proteases that activate their substrates upon proteolytic cleavage. Their inhibition is a promising strategy for the treatment of cancer and infectious diseases. Inhibitory camelid antibodies were developed, targeting the prototypical PC furin. Kinetic analyses of them revealed an enigmatic non-competitive mechanism, affecting the inhibition of large proprotein-like but not small peptidic substrates. Here we present the crystal structures of furin in complex with the antibody Nb14 and of free Nb14 at resolutions of 2.0 Å and 2.3 Å, respectively. Nb14 binds at a site distant to the substrate binding pocket to the P-domain of furin. Interestingly, no major conformational changes were observed upon complex formation, neither for the protease nor for the antibody. Inhibition of furin by Nb14 is instead explained by steric exclusion of specific substrate conformers, explaining why Nb14 inhibits the processing of bulky protein substrates but not of small peptide substrates. This mode of action was further supported by modelling studies with the ternary factor X-furin-antibody complex and a mutation that disrupted the interaction interface between furin and the antibody. The observed binding mode of Nb14 suggests a novel approach for the development of highly specific antibody-based proprotein convertase inhibitors.

Structure of the unliganded form of the proprotein convertase furin suggests activation by a substrate-induced mechanism.

Proprotein convertases (PCs) are highly specific proteases required for the proteolytic modification of many secreted proteins. An unbalanced activity of these enzymes is connected to pathologies like cancer, atherosclerosis, hypercholesterolaemia, and infectious diseases. Novel protein crystallographic structures of the prototypical PC family member furin in different functional states were determined to 1.8-2.0 Å. These, together with biochemical data and modeling by molecular dynamics calculations, suggest essential elements underlying its unusually high substrate specificity. Furin shows a complex activation mechanism and exists in at least four defined states: (i) the "off state," incompatible with substrate binding as seen in the unliganded enzyme; (ii) the active "on state" seen in inhibitor-bound furin; and the respective (iii) calcium-free and (iv) calcium-bound forms. The transition from the off to the on state is triggered by ligand binding at subsites S1 to S4 and appears to underlie the preferential recognition of the four-residue sequence motif of furin. The molecular dynamics simulations of the four structural states reflect the experimental observations in general and provide approximations of the respective stabilities. Ligation by calcium at the PC-specific binding site II influences the active-site geometry and determines the rotamer state of the oxyanion hole-forming Asn295, and thus adds a second level of the activity modulation of furin. The described crystal forms and the observations of different defined functional states may foster the development of new tools and strategies for pharmacological intervention targeting furin.

Engineering a Constrained Peptidic Scaffold towards Potent and Selective Furin Inhibitors.

We report the engineering of the monocyclic sunflower trypsin inhibitor (SFTI-1[1,14]) into a potent furin inhibitor. In a rational approach, we converted the native scaffold of this trypsin-like serine protease inhibitor into a subtilisin-like one by substitutions in the canonical and, particularly, in the substrate-binding loop. Although the substrate sequence for furin is Arg-X-Arg/Lys-Arg↓, the most potent inhibitor had a lysine at position P1. C-terminally truncated versions demonstrated the strongest activity, thus suggesting a lack of interaction between this motif and the surface of furin. This observation was further supported by molecular modeling. With an inhibition constant of 0.49 nm, the engineered peptide H-KRCKKSIPPICF-NH2 is a promising compound for further development of furin inhibitors aimed at controlling the activity of this protease in vitro and in vivo.

Novel insights into structure and function of factor XIIIa-inhibitor tridegin.

The inhibition of the final step in blood coagulation, the factor XIIIa (FXIIIa) catalyzed cross-linking of fibrin monomers, is currently still a challenge in medicinal chemistry. We report synthesis, recombinant expression, disulfide connectivity, and biological activity of tridegin, the sole existing peptide representative displaying inhibitory activity on FXIIIa. Inhibition of the enzyme by this 66-mer cysteine-rich peptide is mediated by its C-terminal sequence, while the N-terminal part comprises structural information and contributes to inhibitor binding. Either of the production strategies examined leads to the formation of different disulfide-bridged isomers indicating the requirement of the correct fold for inhibitory activity. Molecular modeling and docking studies confirm disulfide bond isomer preference with respect to binding to FXIIIa, in turn, the knowledge of the enzyme-inhibitor interactions might bring about comprehensive ideas for the design of a suitable lead structure for addressing FXIIIa.

X-ray structures of human furin in complex with competitive inhibitors.

Furin inhibitors are promising therapeutics for the treatment of cancer and numerous infections caused by bacteria and viruses, including the highly lethal Bacillus anthracis or the pandemic influenza virus. Development and improvement of inhibitors for pharmacological use require a detailed knowledge of the protease's substrate and inhibitor binding properties. Here we present a novel preparation of human furin and the first crystal structures of this enzyme in complex with noncovalent inhibitors. We show the inhibitor exchange by soaking, allowing the investigation of additional inhibitors and substrate analogues. Thus, our work provides a basis for the rational design of furin inhibitors.

Regulation of Son of sevenless by the membrane-actin linker protein ezrin.

Receptor tyrosine kinases participate in several signaling pathways through small G proteins such as Ras (rat sarcoma). An important component in the activation of these G proteins is Son of sevenless (SOS), which catalyzes the nucleotide exchange on Ras. For optimal activity, a second Ras molecule acts as an allosteric activator by binding to a second Ras-binding site within SOS. This allosteric Ras-binding site is blocked by autoinhibitory domains of SOS. We have reported recently that Ras activation also requires the actin-binding proteins ezrin, radixin, and moesin. Here we report the mechanism by which ezrin modulates SOS activity and thereby Ras activation. Active ezrin enhances Ras/MAPK signaling and interacts with both SOS and Ras in vivo and in vitro. Moreover, in vitro kinetic assays with recombinant proteins show that ezrin also is important for the activity of SOS itself. Ezrin interacts with GDP-Ras and with the Dbl homology (DH)/pleckstrin homology (PH) domains of SOS, bringing GDP-Ras to the proximity of the allosteric site of SOS. These actions of ezrin are antagonized by the neurofibromatosis type 2 tumor-suppressor protein merlin. We propose an additional essential step in SOS/Ras control that is relevant for human cancer as well as all physiological processes involving Ras.

Localization and orientation of heavy-atom cluster compounds in protein crystals using molecular replacement.

Heavy-atom clusters (HA clusters) containing a large number of specifically arranged electron-dense scatterers are especially useful for experimental phase determination of large complex structures, weakly diffracting crystals or structures with large unit cells. Often, the determination of the exact orientation of the HA cluster and hence of the individual heavy-atom positions proves to be the critical step in successful phasing and subsequent structure solution. Here, it is demonstrated that molecular replacement (MR) with either anomalous or isomorphous differences is a useful strategy for the correct placement of HA cluster compounds. The polyoxometallate cluster hexasodium α-metatungstate (HMT) was applied in phasing the structure of death receptor 6. Even though the HA cluster is bound in alternate partially occupied orientations and is located at a special position, its correct localization and orientation could be determined at resolutions as low as 4.9 Å. The broad applicability of this approach was demonstrated for five different derivative crystals that included the compounds tantalum tetradecabromide and trisodium phosphotungstate in addition to HMT. The correct placement of the HA cluster depends on the length of the intramolecular vectors chosen for MR, such that both a larger cluster size and the optimal choice of the wavelength used for anomalous data collection strongly affect the outcome.

Purification of the proprotein convertase furin by affinity chromatography based on PC-specific inhibitors.

In eucaryotes, many secreted proteins and peptides are proteolytically excised from larger precursor proteins by a specific class of serine proteases, the proprotein/prohormone convertases (PCs). This cleavage is essential for substrate activation, making the PCs very interesting pharmacological targets in cancer and infectious disease research. Correspondingly, their structure, function and inhibition are intensely studied - studies that require the respective target proteins in large amounts and at high purity. Here we describe the development of a novel purification protocol of furin, the best-studied member of the PC family. We combined the heterologous expression of furin from CHO cells with a novel purification scheme employing an affinity step that efficiently extracts only active furin from the conditioned medium by using furin-specific inhibitor moieties as bait. Several potential affinity tags were synthesized and their binding to furin characterized. The best compound, Biotin-(Adoa)(2)-Arg-Pro-Arg-4-Amba coupled to streptavidin-Sepharose beads, was used in a three-step chromatographic protocol and routinely resulted in a high yield of a homogeneous furin preparation with a specific activity of ~60 units/mg protein. This purification and the general strategy can easily be adapted to the efficient purification of other PC family members.

The crystal structure of death receptor 6 (DR6): a potential receptor of the amyloid precursor protein (APP).

Death receptors belong to the tumor necrosis factor receptor (TNFR) super family and are intimately involved in the signal transduction during apoptosis, stress response and cellular survival. Here we present the crystal structure of recombinantly expressed death receptor six (DR6), one family member that was recently shown to bind to the amyloid precursor protein (APP) and hence to be probably involved in the development of Alzheimer's disease. The extracellular cysteine rich region of DR6, the typical ligand binding region of all TNFRs, was refined to 2.2 Å resolution and shows that its four constituting cysteine rich domains (CRDs) are arranged in a rod-like overall structure, which presents DR6-specific surface patches responsible for the exclusive recognition of its ligand(s). Based on the structural data, the general ligand binding modes of TNFRs and molecular modeling experiments we were able to elucidate structural features of the potential DR6-APP signaling complex.

ALS-associated fused in sarcoma (FUS) mutations disrupt Transportin-mediated nuclear import.

Mutations in fused in sarcoma (FUS) are a cause of familial amyotrophic lateral sclerosis (fALS). Patients carrying point mutations in the C-terminus of FUS show neuronal cytoplasmic FUS-positive inclusions, whereas in healthy controls, FUS is predominantly nuclear. Cytoplasmic FUS inclusions have also been identified in a subset of frontotemporal lobar degeneration (FTLD-FUS). We show that a non-classical PY nuclear localization signal (NLS) in the C-terminus of FUS is necessary for nuclear import. The majority of fALS-associated mutations occur within the NLS and impair nuclear import to a degree that correlates with the age of disease onset. This presents the first case of disease-causing mutations within a PY-NLS. Nuclear import of FUS is dependent on Transportin, and interference with this transport pathway leads to cytoplasmic redistribution and recruitment of FUS into stress granules. Moreover, proteins known to be stress granule markers co-deposit with inclusions in fALS and FTLD-FUS patients, implicating stress granule formation in the pathogenesis of these diseases. We propose that two pathological hits, namely nuclear import defects and cellular stress, are involved in the pathogenesis of FUS-opathies.

Potent inhibitors of furin and furin-like proprotein convertases containing decarboxylated P1 arginine mimetics.

Furin belongs to the family of proprotein convertases (PCs) and is involved in numerous normal physiological and pathogenic processes, such as viral propagation, bacterial toxin activation, cancer, and metastasis. Furin and related furin-like PCs cleave their substrates at characteristic multibasic consensus sequences, preferentially after an arginine residue. By incorporating decarboxylated arginine mimetics in the P1 position of substrate analogue peptidic inhibitors, we could identify highly potent furin inhibitors. The most potent compound, phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide (15), inhibits furin with a K(i) value of 0.81 nM and has also comparable affinity to other PCs like PC1/3, PACE4, and PC5/6, whereas PC2 and PC7 or trypsin-like serine proteases were poorly affected. In fowl plague virus (influenza A, H7N1)-infected MDCK cells, inhibitor 15 inhibited proteolytic hemagglutinin cleavage and was able to reduce virus propagation in a long-term infection test. Molecular modeling revealed several key interactions of the 4-amidinobenzylamide residue in the S1 pocket of furin contributing to the excellent affinity of these inhibitors.

Secondary amides of sulfonylated 3-amidinophenylalanine. New potent and selective inhibitors of matriptase.

Matriptase is an epithelium-derived type II transmembrane serine protease and has been implicated in the activation of substrates such as pro-HGF/SF and pro-uPA, which are likely involved in tumor progression and metastasis. Through screening, we have identified bis-basic secondary amides of sulfonylated 3-amidinophenylalanine as matriptase inhibitors. X-ray analyses of analogues 8 and 31 in complex with matriptase revealed that these inhibitors occupy, in addition to part of the previously described S4-binding site, the cleft formed by the molecular surface and the unique 60 loop of matriptase. Therefore, optimization of the inhibitors included the incorporation of appropriate sulfonyl substituents that could improve binding of these inhibitors into both characteristic matriptase subsites. The most potent derivatives inhibit matriptase highly selective with K(i) values below 5 nM. Molecular modeling revealed that their improved affinity results from interaction with the S4 site of matriptase. Analogues 8 and 59 were studied in an orthotopic xenograft mouse model of prostate cancer. Compared to control, both inhibitors reduced tumor growth, as well as tumor dissemination.

X-ray structures of free and leupeptin-complexed human alphaI-tryptase mutants: indication for an alpha-->beta-tryptase transition.

Tryptases alpha and beta are trypsin-like serine proteinases expressed in large amounts by mast cells. Beta-tryptase is a tetramer that has enzymatic activity, but requires heparin binding to maintain functional and structural stability, whereas alpha-tryptase has little, if any, enzymatic activity but is a stable tetramer in the absence of heparin. As shown previously, these differences can be mainly attributed to the different conformations of the 214-220 segment. Interestingly, the replacement of Asp216 by Gly, which is present in beta-tryptase, results in enzymatically active but less stable alpha-tryptase mutants. We have solved the crystal structures of both the single (D216G) and the double (K192Q/D216G) mutant forms of recombinant human alphaI-tryptase in complex with the peptide inhibitor leupeptin, as well as the structure of the non-inhibited single mutant. The inhibited mutants exhibited an open functional substrate binding site, while in the absence of an inhibitor, the open (beta-tryptase-like) and the closed (alpha-tryptase-like) conformations were present simultaneously. This shows that both forms are in a two-state equilibrium, which is influenced by the residues in the vicinity of the active site and by inhibitor/substrate binding. Novel insights regarding the observed stability differences as well as a potential proteolytic activity of wild-type alpha-tryptase, which may possess a cryptic active site, are discussed.

Proprotein convertase models based on the crystal structures of furin and kexin: explanation of their specificity.

In eukaryotes, many secreted proteins and peptide hormones are excised from larger precursors by calcium-dependent serine proteinases, the proprotein/prohormone convertases (PCs). These PCs cleave their protein substrates very specifically following multiple basic residues. The seven mammalian PCs and their yeast orthologue kexin are multi-domain proteinases consisting of a subtilisin-related catalytic domain, a conserved P-domain and a variable, often cysteine-rich domain, which in some PCs is followed by an additional C-terminal trans-membrane domain and a short cytoplasmic domain. The recently published crystal structures of the soluble mouse furin and yeast kexin ectodomains have revealed the relative arrangement of catalytic and P domains, the exact domain fold and the detailed architecture of the substrate binding clefts. Based on these experimental structures, we now have modelled the structures of the other human/mouse PCs. According to topology and to structure-based sequence comparisons, these other PCs closely resemble furin, with PC4, PACE4 and PC5/6 being more similar, and PC1/3, PC2 and PC7 being less similar to furin. Except for PC1 and PC2, this order of similarity is valid for the catalytic as well as for the P domains, and is almost reversed using kexin as a reference molecule. A similar order results from the number and clustering of negative charges lining the non-prime subsites, explaining the gradually decreasing requirement for basic residues N-terminal to substrate cleavage sites. The preference of the different PCs for distinct substrates seems to be governed by overall charge compensation and matching of the detailed charge distribution pattern.

Inhibition of furin by polyarginine-containing peptides: nanomolar inhibition by nona-D-arginine.

Polyarginine-containing peptides represent potent inhibitors of furin, a mammalian endoprotease that plays an important role in metabolism, activation of pathogenic toxins, and viral proliferation. The therapeutic use of D-polyarginines is especially interesting because they are not cleaved by furin and possess inhibitory potency almost equal to L-polyarginines. In this study we attempted to determine the important elements within polyarginines that contribute to effective inhibition. Structure-function analyses of polyarginine peptides showed that inhibition by polyarginine-containing peptides appeared to depend on the total number of basic charges of the positively charged inhibitors bound to the negatively charged substrate binding pocket; peptide positioning did not appear to be rigorously determined. Screening of L- and D-decapeptide positional scanning combinatorial peptide libraries indicated a preference for basic residues in nearly all positions, similar to previous results with hexapeptide libraries. Length and terminal modification studies showed that the most potent D-polyarginine tested was nona-D-arginine (D9R) amide with a K(i) of 1.3 nm. D9R amide was shown to protect RAW264.7 cells against anthrax toxemia with an IC(50) of 3.7 microm. Because of its high stability, specificity, low toxicity, small molecular weight, and extremely low K(i) against furin, D9R amide or its derivatives may represent promising compounds for therapeutic use.

The 2.0 A crystal structure and substrate specificity of the KDEL-tailed cysteine endopeptidase functioning in programmed cell death of Ricinus communis endosperm.

In the senescing endosperm of germinating castor bean (Ricinus communis) a special organelle (the ricinosome) releases a papain-type cysteine endopeptidase (CysEP) during the final stages of cellular disintegration. Protein cleavage sites for the Ricinus CysEP were determined with fluorogenic peptides (Abz-Xaa-Arg-/-Gln-Gln-Tyr(NO2)-Asp). The highest kcat/Km values were obtained with neutral amino acid residues with large aliphatic and non-polar (Leu, Val, Ile, Met) or aromatic (Phe, Tyr, Trp) side-chains. A second series (Abz-Leu-Xaa-/Gln-Pro-Tyr(NO2)-Asp) was evaluated. Based on these results, the covalent binding inhibitor H-D-Val-Leu-Lys-chloromethylketone (CMK) was chosen as substrate analogue for replacement in the catalytic site. Unusually, CysEP cleaved beta-casein N and C-terminal to the amino acid proline. CysEP was crystallized, its structure was solved by molecular replacement at 2.0 A resolution and refined to a R-factor of 18.1% (Rfree=22.6%). The polypeptide chain folds as in papain into two domains divided by the active site cleft, an elongated surface depression harboring the active site. The non-primed specificity subsites of the proteinase are clearly defined by the H-D-Val-Leu-Lys-CMK-inhibitor covalently bound to the active site. The absence of the occluding loop, which blocks the active site of exopeptidases at the C-terminal side of the scissile bond, identifies CysEP as an endopeptidase. The more open pocket of the Ricinus CysEP correlates with the extended variety of substrate amino acid residues accommodated by this enzyme, including even proline at the P1 and P1' positions. This may allow the enzyme to attack a greater variety of proteins during programmed cell death.

The crystal structure of the proprotein processing proteinase furin explains its stringent specificity.

In eukaryotes, many essential secreted proteins and peptide hormones are excised from larger precursors by members of a class of calcium-dependent endoproteinases, the prohormone-proprotein convertases (PCs). Furin, the best-characterized member of the mammalian PC family, has essential functions in embryogenesis and homeostasis but is also implicated in various pathologies such as tumor metastasis, neurodegeneration and various bacterial and viral diseases caused by such pathogens as anthrax and pathogenic Ebola virus strains. Furin cleaves protein precursors with narrow specificity following basic Arg-Xaa-Lys/Arg-Arg-like motifs. The 2.6 A crystal structure of the decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk)-inhibited mouse furin ectodomain, the first PC structure, reveals an eight-stranded jelly-roll P domain associated with the catalytic domain. Contoured surface loops shape the active site by cleft, thus explaining furin's stringent requirement for arginine at P1 and P4, and lysine at P2 sites by highly charge-complementary pockets. The structure also explains furin's preference for basic residues at P3, P5 and P6 sites. This structure will aid in the rational design of antiviral and antibacterial drugs.