Identification of serpin determinants of specificity and selectivity for furin inhibition through studies of α1PDX (α1-protease inhibitor Portland)-serpin B8 and furin active-site loop chimeras.

α1-Protease inhibitor Portland (α1PDX) is an engineered serpin family inhibitor of the proprotein convertase (PC), furin, that exhibits high specificity but limited selectivity for inhibiting furin over other PC family proteases. Here, we characterize serpin B8, a natural inhibitor of furin, together with α1PDX-serpin B8 and furin-PC chimeras to identify determinants of serpin specificity and selectivity for furin inhibition. Replacing reactive center loop (RCL) sequences of α1PDX with those of serpin B8 demonstrated that both the P4-P1 RXXR recognition sequence as well as the P1'-P5' sequence are critical determinants of serpin specificity for furin. Alignments of PC catalytic domains revealed four variable active-site loops whose role in furin reactivity with serpin B8 was tested by engineering furin-PC loop chimeras. The furin(298-300) loop but not the other loops differentially affected furin reactivity with serpin B8 and α1PDX in a manner that depended on the serpin RCL-primed sequence. Modeling of the serpin B8-furin Michaelis complex identified serpin exosites in strand 3C close to the 298-300 loop whose substitution in α1PDX differentially affected furin reactivity depending on the furin loop and serpin RCL-primed sequences. These studies demonstrate that RCL-primed residues, strand 3C exosites, and the furin(298-300) loop are critical determinants of serpin reactivity with furin, which may be exploited in the design of specific and selective α1PDX inhibitors of PCs.

FGF21 can be mimicked in vitro and in vivo by a novel anti-FGFR1c/ß-Klotho bispecific protein.

The endocrine hormone FGF21 has attracted considerable interest as a potential therapeutic for treating diabetes and obesity. As an alternative to the native cytokine, we generated bispecific Avimer polypeptides that bind with high affinity and specificity to one of the receptor and coreceptor pairs used by FGF21, FGFR1c and ß-Klotho. These Avimers exhibit FGF21-like activity in in vitro assays with potency greater than FGF21. In a study conducted in obese male cynomolgus monkeys, animals treated with an FGFR1c/ß-Klotho bispecific Avimer showed improved metabolic parameters and reduced body weight comparable to the effects seen with FGF21. These results not only demonstrate the essential roles of FGFR1c and ß-Klotho in mediating the metabolic effects of FGF21, they also describe a first bispecific activator of this unique receptor complex and provide validation for a novel therapeutic approach to target this potentially important pathway for treating diabetes and obesity.

Analysis of microtubule plus-end-tracking proteins in cilia.

The microtubule (MT) plus-end-tracking proteins (+TIPs) belonging to the end binding (EB) protein family have been studied extensively in the context of cytoplasmic MTs and were shown to play pivotal roles in regulating MT dynamics and in recruiting other +TIPs to MT ends. Early studies in the green alga Chlamydomonas reinhardtii showed that EB1 localizes to the distal flagellum tip and the basal body, and subsequent studies using green fluorescent protein-tagged fusion proteins have demonstrated similar localization of EBs in other ciliated organisms, including mammalian cells as demonstrated here. Functional analysis of EBs in cultured mammalian cells revealed that EB1 and EB3 are required for biogenesis of primary cilia. Although mammalian EB3 localizes to the tip of some cilia and induces cilium elongation, EBs primarily seem to promote ciliogenesis via MT minus-end anchoring at the basal body, in turn facilitating vesicular trafficking to the cilium base. Moreover, mammalian EBs were shown to interact with several proteins implicated in MT minus-end anchoring and/or vesicular trafficking to cilia. Recent work suggests that apart from EBs, additional +TIPs may be present at the distal tip of cilia where they could regulate axoneme assembly, stability, or disassembly.

Structural basis of RIP1 inhibition by necrostatins.

Necroptosis is a cellular mechanism that mediates necrotic cell death. The receptor-interacting serine/threonine protein kinase 1 (RIP1) is an essential upstream signaling molecule in tumor-necrosis-factor-α-induced necroptosis. Necrostatins, a series of small-molecule inhibitors, suppress necroptosis by specifically inhibiting RIP1 kinase activity. Both RIP1 structure and the mechanisms by which necrostatins inhibit RIP1 remain unknown. Here, we report the crystal structures of the RIP1 kinase domain individually bound to necrostatin-1 analog, necrostatin-3 analog, and necrostatin-4. Necrostatin, caged in a hydrophobic pocket between the N- and C-lobes of the kinase domain, stabilizes RIP1 in an inactive conformation through interactions with highly conserved amino acids in the activation loop and the surrounding structural elements. Structural comparison of RIP1 with the inhibitor-bound oncogenic kinase B-RAF reveals partially overlapping binding sites for necrostatin and for the anticancer compound PLX4032. Our study provides a structural basis for RIP1 inhibition by necrostatins and offers insights into potential structure-based drug design.

Design of a superior cytokine antagonist for topical ophthalmic use.

IL-1 is a key inflammatory and immune mediator in many diseases, including dry-eye disease, and its inhibition is clinically efficacious in rheumatoid arthritis and cryopyrin-associated periodic syndromes. To treat ocular surface disease with a topical biotherapeutic, the uniqueness of the site necessitates consideration of the agent's size, target location, binding kinetics, and thermal stability. Here we chimerized two IL-1 receptor ligands, IL-1ß and IL-1Ra, to create an optimized receptor antagonist, EBI-005, for topical ocular administration. EBI-005 binds its target, IL-1R1, 85-fold more tightly than IL-1Ra, and this increase translates to an ∼100-fold increase in potency in vivo. EBI-005 preserves the affinity bias of IL-1Ra for IL-1R1 over the decoy receptor (IL-1R2), and, surprisingly, is also more thermally stable than either parental molecule. This rationally designed antagonist represents a unique approach to therapeutic design that can potentially be exploited for other ß-trefoil family proteins in the IL-1 and FGF families.

Zinc transporter 5 and zinc transporter 7 induced by high glucose protects peritoneal mesothelial cells from undergoing apoptosis.

Zinc is an essential micronutrient and cytoprotectant involved in many types of apoptosis. The zinc transporter family SLC30A (ZnTs) is an important factor in the regulation of zinc homeostasis; however, its function in apoptosis in peritoneal mesothelial cells (PMCs) remains unknown. This study explores the regulation of zinc transporters and how they play a role in cell survival, particularly in rat peritoneal mesothelial cells (RPMCs), surrounding glucose concentrations, and the molecular mechanism involved. The messenger RNA (mRNA) transcripts were quantitatively measured by real-time polymerase chain reaction for all known nine zinc transport exporters (SLC30A1-8,10), as well as in primary RPMCs and the cells cultured under nonstimulated and HG-stimulated conditions. While many zinc transporters were constitutively expressed, ZnT5 mRNA and ZnT7 mRNA were strongly induced by HG. Overexpression of ZnT5 and ZnT7 respectively resulted in a decrease in the expression of caspace 3, caspace 8, BAX, and AIF and coincided with cell survival in the presence of HG. Inhibition of ZnT5 and ZnT7 expression using considerable siRNA-mediated knockdown of RPMCs was examined and, afterwards, the impact on cell apoptosis was investigated. Increased levels of apoptosis were observed after knockdown of ZnT5 and ZnT7. Furthermore, overexpression of ZnT5 and ZnT7 is accompanied by activation of PI3K/Akt pathway and inhibiting HG-induced apoptosis. This study suggests that the zinc transporting system in RPMCs is influenced by exposure to HG, particularly ZnT5 and ZnT7. This may account for the inhibition of HG-induced RPMC apoptosis and peritoneum injury, likely through targeting PI3K/Akt pathway-mediated cell survival.

Ubiquitin-specific protease 4 promotes TNF-α-induced apoptosis by deubiquitination of RIP1 in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a common type of cancer. Better understanding of molecular aberrations associated with HNSCC might identify new diagnostic and therapeutic strategies for this disease. In this study, we found ubiquitin-specific protease 4 (USP4) was significantly upregulated in HNSCC. USP4 negatively regulates RIP1-mediated NF-κB activation and promotes TNF-α-induced apoptosis in FaDu cells. USP4 directly interacts with receptor-interacting protein 1 (RIP1) and deubiquitinates K63-linked ubiquitination from RIP1. Therefore, our results indicate that USP4 has tumor suppressor roles in HNSCC and suggest USP4 as a potential therapeutic target for HNSCC.

Matching biochemical and functional efficacies confirm ZIP as a potent competitive inhibitor of PKMζ in neurons.

PKMζ is an autonomously active, atypical protein kinase C (aPKC) isoform that is both necessary and sufficient for maintaining long-term potentiation (LTP) and long-term memory. The myristoylated ζ-pseudosubstrate peptide, ZIP, potently inhibits PKMζ biochemically in vitro, within cultured cells, and within neurons in hippocampal slices, and reverses LTP maintenance and erases long-term memory storage. A recent study (Wu-Zhang et al., 2012), however, suggested ZIP was not effective on a PKMζ fusion protein overexpressed in cultured cells. Chelerythrine, a redox-sensitive PKC inhibitor that inhibits PKMζ and disrupts LTP maintenance and memory storage, was also reported by Wu-Zhang et al. (2012) not to inhibit the expressed PKMζ fusion protein. However, the efficacy of inhibitors on endogenous enzymes in cells may not be adequately assessed in expression systems in which levels of expression of exogenous enzymes greatly exceed those of endogenous enzymes. Thus, we show, biochemically, that when PKMζ reaches a level beyond that necessary for substrate phosphorylation such that much of the enzyme is excess or 'spare' kinase, ZIP and chelerythrine do not effectively block substrate phosphorylation. We also show that the cellular overexpression techniques used by Wu-Zhang et al. (2012) increase kinase levels ~30-40 fold above normal levels in transfected cells. Using a mathematical model we show that at such level of overexpression, standard concentrations of inhibitor should have no noticeable effect. Furthermore, we demonstrate the standard concentrations of ZIP, but not scrambled ZIP, inhibit the ability of PKMζ to potentiate AMPAR responses at postsynaptic sites, the physiological function of the kinase. Wu-Zhang et al. (2012) had also claimed that staurosporine, a general kinase inhibitor that does not effectively inhibit PKMζ biochemically in vitro, nonetheless indirectly blocked the PKMζ fusion protein overexpressed in cultured cells by inhibiting phosphoinositide-dependent protein kinase-1 (PDK1). However, here we show that staurosporine does not affect PDK1 phosphorylation of the endogenous PKMζ in hippocampal slices. Thus, the biochemical in vitro effects of PKMζ inhibitors correspond with their intracellular effects, and ZIP and chelerythrine, together with scrambled ZIP and staurosporine as controls, are effective tools to examine the function of PKMζ in neurons. This article is part of a Special Issue entitled 'Cognitive Enhancers'.

Characterization, bioinformatic analysis and dithiocarbamate inhibition studies of two new α-carbonic anhydrases, CAH1 and CAH2, from the fruit fly Drosophila melanogaster.

Carbonic anhydrases (CAs) are essential and ubiquitous enzymes. Thus far, there are no articles on characterization of Drosophila melanogaster α-CAs. Data from invertebrate CA studies may provide opportunities for anti-parasitic drug development because α-CAs are found in many parasite or parasite vector invertebrates. We have expressed and purified D. melanogaster CAH1 and CAH2 as proteins of molecular weights 30kDa and 28kDa. CAH1 is cytoplasmic whereas CAH2 is a membrane-attached protein. Both are highly active enzymes for the CO2 hydration reaction, being efficiently inhibited by acetazolamide. CAH2 in the eye of D. melanogaster may provide a new animal model for CA-related eye diseases. A series of dithiocarbamates were also screened as inhibitors of these enzymes, with some representatives showing inhibition in the low nanomolar range.

The influence of flavonoid compounds on the in vitro inhibition study of a human fibroblast collagenase catalytic domain expressed in E. coli.

The human fibroblast collagenase catalytic domain (MMP1ca) that is considered a prototype for all interstitial collagenase and plays an important role in the turnover of collagen fibrils in the matrix was expressed as an inclusion body in the Escherichia coli. The purified enzyme displayed activity with substrate Dnp-Pro-Leu-Ala-Leu-Trp-Ala-Arg-OH with a K(m) value of 26.61±1.42 µM. The inhibition activity of the nine flavonoid compounds and gallic acid against MMP1ca was examined. Among the compounds tested, the IC(50) of seven flavonoid compounds were determined and ranged from 14.13 to 339.21 µM. Epigallocatechin gallate (EGCG) showed the highest inhibition toward MMP1ca with IC(50) values of 14.13±0.49 µM. EGCG showed a competitive inhibition pattern with a K(i) value of 10.47±0.51 µM. The free binding energy of EGCG against MMP1ca was -13.07 kcal mol(-1), which was calculated by using Autodock 3.0.5 software and showed numerous hydrophobic and hydrogen bond interactions. The galloyl group of EGCG, gallocatechin gallate and epicatechin gallate was determined to be important for inhibitory activity against MMP1ca.

DJ-1 expression modulates astrocyte-mediated protection against neuronal oxidative stress.

DJ-1 deficiency is a cause of genetic Parkinson's disease (PARK7 PD). In sporadic Parkinson's disease (PD), however, DJ-1 is abundantly expressed in reactive astrocytes. This may represent a compensatory protective response. In initial support of this hypothesis, we have shown in vitro that DJ-1-overexpressing astrocytes protect neurons against rotenone-induced death. Rotenone, a pesticide linked to increased PD risk, can stimulate oxidative stress. This process is implicated in PD pathogenesis. Since DJ-1 can enhance antioxidant systems, we hypothesized that augmenting its expression in astrocytes would protect cocultured neurons against oxidative stress. We report here that DJ-1-overexpressing astrocytes were significantly more protective against rotenone-induced neuronal thiol oxidation than wild-type astrocytes in neuron-astrocyte cocultures. DJ-1-knockdown astrocytes, on the other hand, were significantly impaired in their capacity to protect neurons against thiol oxidation. Each of these findings was replicated using astrocyte-conditioned media on neuron-enriched cultures. Thus, DJ-1-modulated, astrocyte-released soluble factors must be involved in the mechanism. This is the first demonstration that the manipulation of a PD-causing gene in astrocytes affects their ability to protect neurons against oxidative stress.

A phage display selected 7-mer peptide inhibitor of the Tannerella forsythia metalloprotease-like enzyme Karilysin can be truncated to Ser-Trp-Phe-Pro.

Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a likely drug target. In this study peptides were selected towards the catalytic domain from Karilysin (Kly18) by phage display. The peptides were linear with low micromolar binding affinities. The two best binders (peptide14 and peptide15), shared the consensus sequence XWFPXXXGGG. A peptide15 fusion with Maltose Binding protein (MBP) was produced with peptide15 fused to the N-terminus of MBP. The peptide15-MBP was expressed in E. coli and the purified fusion-protein was used to verify Kly18 specific binding. Chemically synthesised peptide15 (SWFPLRSGGG) could inhibit the enzymatic activity of both Kly18 and intact Karilysin (Kly48). Furthermore, peptide15 could slow down the autoprocessing of intact Kly48 to Kly18. The WFP motif was important for inhibition and a truncation study further demonstrated that the N-terminal serine was also essential for Kly18 inhibition. The SWFP peptide had a Ki value in the low micromolar range, which was similar to the intact peptide15. In conclusion SWFP is the first reported inhibitor of Karilysin and can be used as a valuable tool in structure-function studies of Karilysin.

The Substrate-Activity-Screening methodology applied to receptor tyrosine kinases: a proof-of-concept study.

Protein kinases are widely recognized as important therapeutic targets due to their involvement in signal transduction pathways. These pathways are tightly controlled and regulated, notably by the ability of kinases to selectively phosphorylate a defined set of substrates. A wide variety of disorders can arise as a consequence of abnormal kinase-mediated phosphorylation and numerous kinase inhibitors have earned their place as key components of the modern pharmacopeia. Although "traditional" kinase inhibitors typically act by preventing the interaction between the kinase and ATP, thus stopping substrate phosphorylation, an alternative approach consists in disrupting the protein-protein interaction between the kinase and its downstream partners. In order to facilitate the identification of potential chemical starting points for substrate-site inhibition approaches, we desired to investigate the application of Substrate Activity Screening to kinases. We herein report a proof-of-concept study demonstrating, on a model tyrosine kinase, that the key requirements of this methodology can be met. Namely, using peptides as model substrates, we show that a simple ADP-accumulation assay can be used to monitor substrate efficiency and that efficiency can be optimized in a modular manner. More importantly, we demonstrate that structure-efficiency relationships translate into structure-activity relationships upon conversion of the substrates into inhibitors.

Cell-free expression of human glucosamine 6-phosphate N-acetyltransferase (HsGNA1) for inhibitor screening.

Glucosamine 6-phosphate N-acetyltransferase (GNA1; EC 2.3.1.4) is required for the de novo synthesis of N-acetyl-d-glucosamine-6-phosphate (GlcNAc-6P), which is an essential precursor in Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) biosynthesis pathway. Therefore, GNA1 is indispensable for the viability of organisms. Here, a novel cell-free expression strategy was developed to efficiently produce large amounts of human GNA1(HsGNA1) and HsGNA1-sGFP for throughput inhibitor screening. The binding site of inhibitor glucose-6-phosphate (G6P) to hGNA was identified by simulated annealing. Subtle differences to the binding site of Aspergillius GNA1(AfGNA1) can be harnessed for inhibitor design. HsGNA1 may be also useful as an antimicrobial and chemotherapeutic target against cancer. Additionally HsGNA1 inhibitors/modulators can possibly be administered with other drugs in the next generation of personalized medicine.

ShRNA-targeted COMMD7 suppresses hepatocellular carcinoma growth.

BACKGROUND: COMMD7 is a newly identified gene overexpressed in hepatocellular carcinoma (HCC) and associated with tumor invasion and poor prognosis. We aim to examine the biological function of COMMD7 in HCC by shRNA silencing. METHODS: COMMD7 expressions were examined in human HCC cell lines HepG2, Huh7, Hep3B, HLE, HLF, SK-Hep-1 and PLC/PRF/5 cells. Recombinant pGenesil-COMMD7-shRNA was transfected into COMMD7-abundant HepG2 cells to silence COMMD7 expression. The effects of COMMD7 silencing on HepG2 cell proliferation in vitro and xenograft tumor growth in vivo were evaluated. Flow cytometry profiling was used to detect the presence of apoptosis in COMMD7-silenced HepG2 cells and to differentiate cell cycle distribution. Electrophoretic mobility shift assay and luciferase reporter assays to examine the activities of nuclear factor-kappaB (NF-κB) signaling pathways in response to tumor necrosis factor (TNF)-α in COMMD7-silenced HepG2 cells. RESULTS: COMMD7 expression level was abundance in HepG2 and SK-Hep-1 cells. COMMD7 was aberrantly overexpressed in HepG2 cells, whilst pGenesil-COMMD7-shRNA exhibited a maximal inhibition rate of 75%. COMMD7 silencing significantly reduced HepG2 cell proliferation and colony formation. The knockdown of COMMD7 resulted in an increased apoptosis and cell cycle arrest at S-phase. COMMD7 knockdown also exhibited an antineoplastic effect in vivo, which manifested as tumor xenograft growth retardation. COMMD7 silencing also suppressed the responsiveness of NF-κB signaling pathway to the stimulation with TNF-α in vitro. Moreover, the similar suppressive effects of COMMD7 silence on SK-Hep-1 cells were also observed. CONCLUSIONS: COMMD7 contributes to HCC progression by reducing cell apoptosis and overcoming cell cycle arrest. The proliferative and antiapoptotic effects of COMMD7 may be mediated by NF-κB signaling pathway.

Gating effects on picrotin block of glycine receptors.

Picrotoxin is a pore blocker that can differentiate ligand-gated inhibitory chloride channels. Even within one receptor type, such as the glycine receptor, picrotoxin block differs between subunits. The effect of subunit gating properties on block of the inhibitory glycine receptor (GlyR) was explored using heteromeric α subunit expression in voltage-clamped HEK293 cells. The α2 GlyR is more sensitive to picrotin block than the α1 GlyR, and this difference was used to explore whether mutations that interfered with gating of the α2 subunit would also interfere with picrotin block. Two mutations were used: one that decreased the glycine sensitivity of α2 by almost two log units and the other that was unresponsive to glycine. In both cases, the sensitivity to picrotin was essentially unaltered. The results indicated that α2 subunits can determine the picrotin sensitivity of α1α2-heteromeric receptors and that direct gating of the α2 subunit is not required for this picrotin inhibition.

Alterations in lipid signaling underlie lipodystrophy secondary to AGPAT2 mutations.

Congenital generalized lipodystrophy (CGL), secondary to AGPAT2 mutation is characterized by the absence of adipocytes and development of severe insulin resistance. In the current study, we investigated the adipogenic defect associated with AGPAT2 mutations. Adipogenesis was studied in muscle-derived multipotent cells (MDMCs) isolated from vastus lateralis biopsies obtained from controls and subjects harboring AGPAT2 mutations and in 3T3-L1 preadipocytes after knockdown or overexpression of AGPAT2. We demonstrate an adipogenic defect using MDMCs from control and CGL human subjects with mutated AGPAT2. This defect was rescued in CGL MDMCs with a retrovirus expressing AGPAT2. Both CGL-derived MDMCs and 3T3-L1 cells with knockdown of AGPAT2 demonstrated an increase in cell death after induction of adipogenesis. Lack of AGPAT2 activity reduces Akt activation, and overexpression of constitutively active Akt can partially restore lipogenesis. AGPAT2 modulated the levels of phosphatidic acid, lysophosphatidic acid, phosphatidylinositol species, as well as the peroxisome proliferator-activated receptor γ (PPARγ) inhibitor cyclic phosphatidic acid. The PPARγ agonist pioglitazone partially rescued the adipogenic defect in CGL cells. We conclude that AGPAT2 regulates adipogenesis through the modulation of the lipome, altering normal activation of phosphatidylinositol 3-kinase (PI3K)/Akt and PPARγ pathways in the early stages of adipogenesis.

Kinetic properties and small-molecule inhibition of human myosin-6.

Myosin-6 is an actin-based motor protein that moves its cargo towards the minus-end of actin filaments. Mutations in the gene encoding the myosin-6 heavy chain and changes in the cellular abundance of the protein have been linked to hypertrophic cardiomyopathy, neurodegenerative diseases, and cancer. Here, we present a detailed kinetic characterization of the human myosin-6 motor domain, describe the effect of 2,4,6-triiodophenol on the interaction of myosin-6 with F-actin and nucleotides, and show how addition of the drug reduces the number of myosin-6-dependent vesicle fusion events at the plasma membrane during constitutive secretion.

Methods applied to the study of protein arginine methylation.

Arginine methylation was discovered in the mid-1960s. About 15 years ago, the first protein arginine N-methyltransferase (PRMT) enzyme was described. The PRMT family now stands at nine members, and these enzymes play a key role in regulating a multitude of cellular events. The majority of the PRMTs have been deleted in mice, thus providing genetically tractable systems for in vivo and cell-based studies. These studies have implicated this posttranslational modification in chromatin remodeling, transcriptional regulation, RNA processing, protein/RNA trafficking, signal transduction, and DNA repair. In this chapter, we introduce different approaches that have been developed to assess protein arginine methylation levels and characterize PRMT substrates.

The combination of RAD001 and NVP-BKM120 synergistically inhibits the growth of lung cancer in vitro and in vivo.

This study focuses on determining whether the combination of NYP-BKM120 (BKM120) and RAD001 exerts enhanced therapeutic effect against lung cancer. The combination of BKM120 and RAD001 exerted synergistic inhibitory effects on the growth of lung cancer cells both in culture and in mouse xenograft model. This combination abrogated RAD001-induced Akt phosphorylation and exerted enhanced suppressive effect on 4EBP1 phosphorylation. Collectively, we suggest that the combination of RAD001 and BKM120 may be an effective regimen for treatment of lung cancer, hence warranting further evaluation of the combination in the clinic.