Benzothiazole Substitution Analogs of Rhodacyanine Hsp70 Inhibitors Modulate Tau Accumulation.

The accumulation and aggregation of the microtubule-associated protein tau (tau) into intracellular neuronal tangles are a hallmark of a range of progressive neurodegenerative tauopathies, including Alzheimer's disease (AD), frontotemporal dementia, Pick's disease, and progressive supranuclear palsy. The aberrant phosphorylation of tau is associated with tau aggregates in AD. Members of the heat shock protein 70 kDa (Hsp70) family of chaperones bind directly to tau and modulate tau clearance and aggregation. Small molecules that inhibit the Hsp70 family of chaperones have been shown to reduce the accumulation of tau, including phosphorylated tau. Here, eight analogs of the rhodacyanine inhibitor, JG-98, were synthesized and evaluated. Like JG-98, many of the compounds inhibited ATPase activity of the cytosolic heat shock cognate 70 protein (Hsc70) and reduced total, aggregated, and phosphorylated tau accumulation in cultured cells. Three compounds, representing divergent clogP values, were evaluated for in vivo blood-brain barrier penetration and tau reduction in an ex vivo brain slice model. AL69, the compound with the lowest clogP and the lowest membrane retention in a parallel artificial membrane permeability assay (PAMPA), reduced phosphorylated tau accumulation. Our results suggest that benzothiazole substitutions of JG-98 that increase hydrophilicity may increase the efficacy of these Hsp70 inhibitors to reduce phosphorylated tau.

Hsp22 with an N-Terminal Domain Truncation Mediates a Reduction in Tau Protein Levels.

Misfolding, aggregation and accumulation of proteins are toxic elements in the progression of a broad range of neurodegenerative diseases. Molecular chaperones enable a cellular defense by reducing or compartmentalizing these insults. Small heat shock proteins (sHsps) engage proteins early in the process of misfolding and can facilitate their proper folding or refolding, sequestration, or clearance. Here, we evaluate the effects of the sHsp Hsp22, as well as a pseudophosphorylated mutant and an N-terminal domain deletion (NTDΔ) variant on tau aggregation in vitro and tau accumulation and aggregation in cultured cells. Hsp22 wild-type (WT) protein had a significant inhibitory effect on heparin-induced aggregation in vitro and the pseudophosphorylated mutant Hsp22 demonstrated a similar effect. When co-expressed in a cell culture model with tau, these Hsp22 constructs significantly reduced soluble tau protein levels when transfected at a high ratio relative to tau. However, the Hsp22 NTDΔ protein drastically reduced the soluble protein expression levels of both tau WT and tau P301L/S320F even at lower transfection ratios, which resulted in a correlative reduction of the triton-insoluble tau P301L/S320F aggregates.

Small Heat Shock Proteins, Big Impact on Protein Aggregation in Neurodegenerative Disease.

Misfolding, aggregation, and aberrant accumulation of proteins are central components in the progression of neurodegenerative disease. Cellular molecular chaperone systems modulate proteostasis, and, therefore, are primed to influence aberrant protein-induced neurotoxicity and disease progression. Molecular chaperones have a wide range of functions from facilitating proper nascent folding and refolding to degradation or sequestration of misfolded substrates. In disease states, molecular chaperones can display protective or aberrant effects, including the promotion and stabilization of toxic protein aggregates. This seems to be dependent on the aggregating protein and discrete chaperone interaction. Small heat shock proteins (sHsps) are a class of molecular chaperones that typically associate early with misfolded proteins. These interactions hold proteins in a reversible state that helps facilitate refolding or degradation by other chaperones and co-factors. These sHsp interactions require dynamic oligomerization state changes in response to diverse cellular triggers and, unlike later steps in the chaperone cascade of events, are ATP-independent. Here, we review evidence for modulation of neurodegenerative disease-relevant protein aggregation by sHsps. This includes data supporting direct physical interactions and potential roles of sHsps in the stewardship of pathological protein aggregates in brain. A greater understanding of the mechanisms of sHsp chaperone activity may help in the development of novel therapeutic strategies to modulate the aggregation of pathological, amyloidogenic proteins. sHsps-targeting strategies including modulators of expression or post-translational modification of endogenous sHsps, small molecules targeted to sHsp domains, and delivery of engineered molecular chaperones, are also discussed.

Isoform-selective Hsp90 inhibition rescues model of hereditary open-angle glaucoma.

The heat shock protein 90 (Hsp90) family of molecular chaperones regulates protein homeostasis, folding, and degradation. The ER-resident Hsp90 isoform, glucose-regulated protein 94 (Grp94), promotes the aggregation of mutant forms of myocilin, a protein associated with primary open-angle glaucoma. While inhibition of Grp94 promotes the degradation of mutant myocilin in vitro, to date no Grp94-selective inhibitors have been investigated in vivo. Here, a Grp94-selective inhibitor facilitated mutant myocilin degradation and rescued phenotypes in a transgenic mouse model of hereditary primary open-angle glaucoma. Ocular toxicities previously associated with pan-Hsp90 inhibitors were not evident with our Grp94-selective inhibitor, 4-Br-BnIm. Our study suggests that selective inhibition of a distinct Hsp90 family member holds translational promise for ocular and other diseases associated with cell stress and protein misfolding.

Hsp90 activator Aha1 drives production of pathological tau aggregates.

The microtubule-associated protein tau (MAPT, tau) forms neurotoxic aggregates that promote cognitive deficits in tauopathies, the most common of which is Alzheimer's disease (AD). The 90-kDa heat shock protein (Hsp90) chaperone system affects the accumulation of these toxic tau species, which can be modulated with Hsp90 inhibitors. However, many Hsp90 inhibitors are not blood-brain barrier-permeable, and several present associated toxicities. Here, we find that the cochaperone, activator of Hsp90 ATPase homolog 1 (Aha1), dramatically increased the production of aggregated tau. Treatment with an Aha1 inhibitor, KU-177, dramatically reduced the accumulation of insoluble tau. Aha1 colocalized with tau pathology in human brain tissue, and this association positively correlated with AD progression. Aha1 overexpression in the rTg4510 tau transgenic mouse model promoted insoluble and oligomeric tau accumulation leading to a physiological deficit in cognitive function. Overall, these data demonstrate that Aha1 contributes to tau fibril formation and neurotoxicity through Hsp90. This suggests that therapeutics targeting Aha1 may reduce toxic tau oligomers and slow or prevent neurodegenerative disease progression.

18F-5-Fluoroaminosuberic Acid as a Potential Tracer to Gauge Oxidative Stress in Breast Cancer Models.

The cystine transporter (system xC-) is an antiporter of cystine and glutamate. It has relatively low basal expression in most tissues and becomes upregulated in cells under oxidative stress (OS) as one of the genes expressed in response to the antioxidant response element promoter. We have developed 18F-5-fluoroaminosuberic acid (FASu), a PET tracer that targets system xC- The goal of this study was to evaluate 18F-FASu as a specific gauge for system xC- activity in vivo and its potential for breast cancer imaging. Methods:18F-FASu specificity toward system xC- was studied by cell inhibition assay, cellular uptake after OS induction with diethyl maleate, with and without anti-xCT small interfering RNA knockdown, in vitro uptake studies, and in vivo uptake in a system xC--transduced xenograft model. In addition, radiotracer uptake was evaluated in 3 breast cancer models: MDA-MB-231, MCF-7, and ZR-75-1. Results: Reactive oxygen species-inducing diethyl maleate increased glutathione levels and 18F-FASu uptake, whereas gene knockdown with anti-xCT small interfering RNA led to decreased tracer uptake. 18F-FASu uptake was robustly inhibited by system xC- inhibitors or substrates, whereas uptake was significantly higher in transduced cells and tumors expressing xCT than in wild-type HEK293T cells and tumors (P < 0.0001 for cells, P = 0.0086 for tumors). 18F-FASu demonstrated tumor uptake in all 3 breast cancer cell lines studied. Among them, triple-negative breast cancer MDA-MB-231, which has the highest xCT messenger RNA level, had the highest tracer uptake (P = 0.0058 when compared with MCF-7; P < 0.0001 when compared with ZR-75-1). Conclusion:18F-FASu as a system xC- substrate is a specific PET tracer for functional monitoring of system xC- and OS imaging. By enabling noninvasive analysis of xC- responses in vivo, this biomarker may serve as a valuable target for the diagnosis and treatment monitoring of certain breast cancers.

A simple route to [11C]N-Me labeling of aminosuberic acid for proof of feasibility imaging of the x(C)⁻ transporter.

Oxidative stress has been implicated in a variety of conditions, including cancer, heart failure, diabetes, neurodegeneration and other diseases. A potential biomarker for oxidative stress is the cystine/glutamate transporter, system x(C)(-). L-Aminosuberic acid (L-ASu) has been identified as a system x(C)(-) substrate. Here we report a facile method for [(11)C]N-Me labeling of L-ASu, automation of the radiochemical process, and preliminary PET imaging with EL4 tumor bearing mice. The results demonstrate uptake in the tumor above background, warranting further studies on the use of radiolabeled analogs of L-ASu as a PET imaging agent for system x(C)(-).

Functional imaging of oxidative stress with a novel PET imaging agent, 18F-5-fluoro-L-aminosuberic acid.

UNLABELLED: Glutathione is the predominant endogenous cellular antioxidant, playing a critical role in the cellular defensive response to oxidative stress by neutralizing free radicals and reactive oxygen species. With cysteine as the rate-limiting substrate in glutathione biosynthesis, the cystine/glutamate transporter (system xc(-)) represents a potentially attractive PET biomarker to enable in vivo quantification of xc(-) activity in response to oxidative stress associated with disease. We have developed a system xc(-) substrate that incorporates characteristics of both natural substrates, L-cystine and L-glutamate (L-Glu). L-aminosuberic acid (L-ASu) has been identified as a more efficient system xc(-) substrate than L-Glu, leading to an assessment of a series of anionic amino acids as prospective PET tracers. Herein, we report the synthesis and in vitro and in vivo validation of a lead candidate, (18)F-5-fluoro-aminosuberic acid ((18)F-FASu), as a PET tracer for functional imaging of a cellular response to oxidative stress with remarkable tumor uptake and retention. METHODS: (18)F-FASu was identified as a potential PET tracer based on an in vitro screening of compounds similar to L-cystine and L-Glu. Affinity toward system xc(-) was determined via in vitro uptake and inhibition studies using oxidative stress-induced EL4 and SKOV-3 cells. In vivo biodistribution and PET imaging studies were performed in mice bearing xenograft tumors (EL4 and SKOV-3). RESULTS: In vitro assay results determined that L-ASu inhibited system xc(-) as well as or better than L-Glu. The direct comparison of uptake of tritiated compounds demonstrated more efficient system xc(-) uptake of L-ASu than L-Glu. Radiosynthesis of (18)F-FASu allowed the validation of uptake for the fluorine-bearing derivative in vitro. Evaluation in vivo demonstrated primarily renal clearance and uptake of approximately 8 percentage injected dose per gram in SKOV-3 tumors, with tumor-to-blood and tumor-to-muscle ratios of approximately 12 and approximately 28, respectively. (18)F-FASu uptake was approximately 5 times greater than (18)F-FDG uptake in SKOV-3 tumors. Dynamic PET imaging demonstrated uptake in EL4 tumor xenografts of approximately 6 percentage injected dose per gram and good tumor retention for at least 2 h after injection. CONCLUSION: (18)F-FASu is a potentially useful metabolic tracer for PET imaging of a functional cellular response to oxidative stress. (18)F-FASu may provide more sensitive detection than (18)F-FDG in certain tumors.

In vivo targeting of HER2-positive tumor using 2-helix affibody molecules.

Molecular imaging of human epidermal growth factor receptor type 2 (HER2) expression has drawn significant attention because of the unique role of the HER2 gene in diagnosis, therapy and prognosis of human breast cancer. In our previous research, a novel cyclic 2-helix small protein, MUT-DS, was discovered as an anti-HER2 Affibody analog with high affinity through rational protein design and engineering. MUT-DS was then evaluated for positron emission tomography (PET) of HER2-positive tumor by labeling with two radionuclides, 68Ga and 18F, with relatively short half-life (t1/2<2 h). In order to fully study the in vivo behavior of 2-helix small protein and demonstrate that it could be a robust platform for labeling with a variety of radionuclides for different applications, in this study, MUT-DS was further radiolabeled with 64Cu or 111In and evaluated for in vivo targeting of HER2-positive tumor in mice. Design 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated MUT-DS (DOTA-MUT-DS) was chemically synthesized using solid phase peptide synthesizer and I2 oxidation. DOTA-MUT-DS was then radiolabeled with 64Cu or 111In to prepare the HER2 imaging probe (64Cu/111In-DOTA-MUT-DS). Both biodistribution and microPET imaging of the probe were evaluated in nude mice bearing subcutaneous HER2-positive SKOV3 tumors. DOTA-MUT-DS could be successfully synthesized and radiolabeled with 64Cu or 111In. Biodistribution study showed that tumor uptake value of 64Cu or 111In-labeled DOTA-MUT-DS was 4.66±0.38 or 2.17±0.15%ID/g, respectively, in nude mice bearing SKOV3 xenografts (n=3) at 1 h post-injection (p.i.). Tumor-to-blood and tumor-to-muscle ratios for 64Cu-DOTA-MUT-DS were attained to be 3.05 and 3.48 at 1 h p.i., respectively, while for 111In-DOTA-MUT-DS, they were 2.04 and 3.19, respectively. Co-injection of the cold Affibody molecule ZHER2:342 with 64Cu-DOTA-MUT-DS specifically reduced the SKOV3 tumor uptake of the probe by 48%. 111In-DOTA-MUT-DS displayed lower liver uptake at all the time points investigated and higher tumor to blood ratios at 4 and 20 h p.i., when compared with 64Cu-DOTA-MUT-DS. This study demonstrates that the 2-helix protein based probes, 64Cu/111In DOTA-MUT-DS, are promising molecular probes for imaging HER2-positive tumor. Two-helix small protein scaffold holds great promise as a novel and robust platform for imaging and therapy applications.

A novel 18F-labeled two-helix scaffold protein for PET imaging of HER2-positive tumor.

PURPOSE: Two-helix scaffold proteins (~ 5 kDa) against human epidermal growth factor receptor type 2 (HER2) have been discovered in our previous work. In this research we aimed to develop an (18)F-labeled two-helix scaffold protein for positron emission tomography (PET) imaging of HER2-positive tumors. METHODS: An aminooxy-functionalized two-helix peptide (AO-MUT-DS) with high HER2 binding affinity was synthesized through conventional solid phase peptide synthesis. The purified linear peptide was cyclized by I(2) oxidation to form a disulfide bridge. The cyclic peptide was then conjugated with a radiofluorination synthon, 4-(18)F-fluorobenzyl aldehyde ((18)F-FBA), through the aminooxy functional group at the peptide N terminus (30% yield, non-decay corrected). The binding affinities of the peptides were analyzed by Biacore analysis. Cell uptake assay of the resulting PET probe, (18)F-FBO-MUT-DS, was performed at 37°C. (18)F-FBO-MUT-DS with high specific activity (20-32 MBq/nmol, 88-140 µCi/µg, end of synthesis) was injected into mice xenograft model bearing SKOV3 tumor. MicroPET and biodistribution and metabolic stability studies were then conducted. RESULTS: Cell uptake assays showed high and specific cell uptake (~12% applied activity at 1 h) by incubation of (18)F-FBO-MUT-DS with HER2 high-expressing SKOV3 ovarian cancer cells. The affinities (K(D)) of AO-MUT-DS and FBO-MUT-DS as tested by Biacore analysis were 2 and 1 nM, respectively. In vivo small animal PET demonstrated fast tumor targeting, high tumor accumulation, and good tumor to normal tissue contrast of (18)F-FBO-MUT-DS. Biodistribution studies further revealed that the probe had excellent tumor uptake (6.9%ID/g at 1 h post-injection) and was cleared through both liver and kidneys. Co-injection of the probe with 500 µg of HER2 Affibody protein reduced the tumor uptake (6.9 vs 1.8%ID/g, p < 0.05). CONCLUSION: F-FBO-MUT-DS displays excellent HER2 targeting ability and tumor PET imaging quality. The two-helix scaffold proteins are suitable for development of (18)F-based PET probes.

64Cu-labeled affibody molecules for imaging of HER2 expressing tumors.

INTRODUCTION: The development of molecular probes based on novel engineered protein constructs is under active investigation due to the great potential of this generalizable strategy for imaging a variety of tumor targets. DISCUSSION: In this report, human epidermal growth factor receptor type 2 (HER2)-binding Affibody molecules were radiolabeled with (64)Cu and their imaging ability was further evaluated in tumor mice models to understand the promise and limitations of such probes. The anti-HER2 Affibody molecules in monomeric (Z(HER2:477)) and dimeric [(Z(HER2:477))(2)] forms were site specifically modified with the maleimide-functionalized chelator, 1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid)-10-acetate mono (N-ethylmaleimide amide) (Mal-DOTA). The resulting DOTA-Affibody conjugates were radiolabeled with (64)Cu and evaluated in nude mice bearing subcutaneous SKOV3 tumors. Biodistribution experiments showed that tumor uptake values of (64)Cu-DOTA-Z(HER2:477) and (64)Cu-DOTA-(Z(HER2:477))(2) were 6.12 +/- 1.44% and 1.46 +/- 0.50% ID/g, respectively, in nude mice (n = 3 each) at 4 h postinjection. Moreover, (64)Cu-labeled monomer exhibited significantly higher tumor/blood ratio than that of radiolabeled dimeric counterpart at all time points examined in this study. MicroPET imaging of (64)Cu-DOTA-Z(HER2:477) in SKOV3 tumor mice clearly showed good and specific tumor localization. This study demonstrates that (64)Cu-labeled Z(HER2:477) is a promising targeted molecular probe for imaging HER2 receptor expression in living mice. Further work is needed to improve the excretion properties, hence dosimetry and imaging efficacy, of the radiometal-based probe.

A 2-helix small protein labeled with 68Ga for PET imaging of HER2 expression.

UNLABELLED: Affibody molecules are a class of scaffold proteins being developed into a generalizable approach to targeting tumors. Many 3-helix-based Affibody proteins have shown excellent in vivo properties for tumor imaging and therapy. By truncating one alpha-helix that is not responsible for receptor recognition in the Affibody and maturating the protein affinity through synthetic strategies, we have successfully identified in our previous research several small 2-helix proteins with excellent binding affinities to human epidermal growth factor receptor type 2 (HER2). With preferential properties such as faster blood clearance and tumor accumulation, lower immunogenic potential, and facile and economically viable synthetic schemes, we hypothesized that these 2-helix protein binders could become excellent molecular imaging probes for monitoring HER2 expression and modulation. METHODS: In this study, a 2-helix small protein, MUT-DS, was chemically modified with a metal chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). DOTA-MUT-DS was then site-specifically radiolabeled with an important PET radionuclide, (68)Ga. The resulting radiolabeled anti-HER2 2-helix molecule was further evaluated as a potential molecular probe for small-animal PET HER2 imaging in a SKOV3 tumor mouse model. RESULTS: The 2-helix DOTA-MUT-DS showed high HER2-binding affinity (dissociation constant, 4.76 nM). The radiolabeled probe displayed high stability in mouse serum and specificity toward HER2 in cell cultures. Biodistribution and small-animal PET studies further showed that (68)Ga-DOTA-MUT-DS had rapid and high SKOV3 tumor accumulation and quick clearance from normal organs. The specificity of (68)Ga-DOTA-MUT-DS for SKOV3 tumors was confirmed by monitoring modulation of HER2 protein on treatment of tumor mice with heat shock protein 90 inhibitor 17-N,N-dimethyl ethylene diamine-geldanamycin in vivo. CONCLUSION: This proof-of-concept research clearly demonstrated that synthetic 2-helix (68)Ga-DOTA-MUT-DS is a promising PET probe for imaging HER2 expression in vivo. The Affibody-derived small 2-helix protein scaffold has great potential for developing targeting agents for a variety of tumor-associated biomarkers.

Engineered two-helix small proteins for molecular recognition.

Less is more: By starting with a high-affinity HER2-binding 3-helix affibody molecule, we successfully developed 2-helix small protein binders with 5 nM affinities by using a combination of several different strategies. Our efforts clearly suggest that 2-helix small proteins against important tumor targets can be obtained by rational protein design and engineering.

Suppression of Huntington's disease pathology in Drosophila by human single-chain Fv antibodies.

Misfolded neuronal proteins have been identified in a number of neurodegenerative disorders and have been implicated in the pathogenesis of diseases that include Alzheimer's disease, Parkinson's disease, prion-based dementia, Huntington's disease (HD), and other polyglutamine diseases. Although underlying mechanisms remain the subject of ongoing research, it is clear that aberrant processing, protein degradation, and aggregate formation or spurious protein association of the abnormal neuronal proteins may be critical factors in disease progression. Recent work in these diseases has demonstrated in vitro that specific engineered antibody species, peptides, or other general agents may suppress the formation of aggregates. We have modified an approach with intracellularly expressed single-chain Fv (sFv) antibodies (intrabodies) that bind with unique HD protein epitopes. In cell and tissue culture models of HD, anti-N-terminal huntingtin intrabodies (C4 sFv) reduce aggregation and cellular toxicity. Here, we present the crucial experiment of intrabody-mediated in vivo suppression of neuropathology, using a Drosophila model of HD. In the presence of the C4 sFv intrabody, the proportion of HD flies surviving to adulthood increases from 23% to 100%, and the mean and maximum lifespan of adult HD flies is significantly prolonged. Neurodegeneration and formation of visible huntingtin aggregates are slowed. We conclude from this investigation that engineered intrabodies are a potential new class of therapeutic agents for the treatment of neurodegenerative diseases. They may also serve as tools for drug discovery and validation of sites on mutant neuronal proteins that could be exploited for rational drug design.

Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion in the number of polyglutamine-encoding CAG repeats in the gene that encodes the huntingtin (htt) protein. A property of the mutant protein that is intimately involved in the development of the disease is the propensity of the glutamine-expanded protein to misfold and generate an N-terminal proteolytic htt fragment that is toxic and prone to aggregation. Intracellular antibodies (intrabodies) against htt have been shown to reduce htt aggregation by binding to the toxic fragment and inactivating it or preventing its misfolding. Intrabodies may therefore be a useful gene-therapy approach to treatment of the disease. However, high levels of intrabody expression have been required to obtain even limited reductions in aggregation. We have engineered a single-domain intracellular antibody against htt for robust aggregation inhibition at low expression levels by increasing its affinity in the absence of a disulfide bond. Furthermore, the engineered intrabody variable light-chain (V(L))12.3, rescued toxicity in a neuronal model of HD. We also found that V(L)12.3 inhibited aggregation and toxicity in a Saccharomyces cerevisiae model of HD. V(L)12.3 is significantly more potent than earlier anti-htt intrabodies and is a potential candidate for gene therapy treatment for HD. To our knowledge, this is the first attempt to improve affinity in the absence of a disulfide bond to improve intrabody function. The demonstrated importance of disulfide bond-independent binding for intrabody potency suggests a generally applicable approach to the development of effective intrabodies against other intracellular targets.

Bortezomib rapidly suppresses ubiquitin thiolesterification to ubiquitin-conjugating enzymes and inhibits ubiquitination of histones and type I inositol 1,4,5-trisphosphate receptor.

The proteasome inhibitor bortezomib is an emerging anticancer agent. Although the proteasome is clearly its locus of action, the early biochemical consequences of bortezomib treatment are poorly defined. Here, we show in cultured cells that bortezomib and other proteasome inhibitors rapidly inhibit free ubiquitin levels and ubiquitin thiolesterification to ubiquitin-conjugating enzymes. Inhibition of thiolesterification correlated with a reduction in the ubiquitination of certain substrates, exemplified by a dramatic decline in histone monoubiquitination and a decrease in the rate of inositol 1,4,5-trisphosphate receptor polyubiquitination. Thus, in addition to the expected effect of blocking the degradation of polyubiquitinated substrates, bortezomib can also inhibit ubiquitination. The effect of bortezomib on histone monoubiquitination may contribute to its therapeutic actions.

Development of a human light chain variable domain (V(L)) intracellular antibody specific for the amino terminus of huntingtin via yeast surface display.

Intracellular antibodies (intrabodies) provide an attractive means for manipulating intracellular protein function, both for research and potentially for therapy. A challenge in the isolation of effective intrabodies is the ability to find molecules that exhibit sufficient binding affinity and stability when expressed in the reducing environment of the cytoplasm. Here, we have used yeast surface display of proteins to isolate novel scFv clones against huntingtin from a non-immune human antibody library. We then applied yeast surface display to affinity mature this scFv pool and analyze the location of the binding site of the mutant with the highest affinity. Interestingly, the paratope was mapped exclusively to the variable light chain domain of the scFv. A single domain antibody was constructed consisting solely of this variable light chain domain, and was found to retain full binding activity to huntingtin. Cytoplasmic expression levels in yeast of the single domain were at least fivefold higher than the scFv. The ability of the single-domain intrabody to inhibit huntingtin aggregation, which has been implicated in the pathogenesis of Huntington's disease (HD), was confirmed in a cell-free in vitro assay as well as in a mammalian cell culture model of HD. Significantly, a single-domain intrabody that is functionally expressable in the cytoplasm was derived from a non-functional scFv by performing affinity maturation and binding site analysis on the yeast cell surface, despite the differences between the cytoplasmic and extracellular environment. This approach may find application in the development of intrabodies to a wide variety of intracellular targets.

N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine inhibits ligand binding to certain G protein-coupled receptors.

N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) is used widely in biological systems to chelate certain heavy metals, particularly Zn2+. Here we show that TPEN inhibits ligand binding to certain G protein-coupled receptors and is an antagonist at muscarinic receptors. In intact human neuroblastoma SH-SY5Y cells, the binding of the muscarinic receptor ligand [N-methyl-3H]scopolamine methyl chloride was inhibited by TPEN (Ki approximately 26 microM), as was muscarinic receptor agonist-induced inositol 1,4,5-trisphosphate formation (Ki approximately 26 microM). This antagonism was not due to metal ion chelation, indicating that it resulted from a direct interaction of TPEN with muscarinic receptors. Examination of the effects of TPEN on other receptors in SH-SY5Y cell membrane preparations showed that the binding of the nonpeptide opioid receptor ligand [15,16-3H]diprenorphine was strongly inhibited, whereas binding of [125I]vasoactive intestinal polypeptide was not. This pattern of selectivity was also seen in AR4-2J rat pancreatoma cell membranes, in which TPEN inhibited ligand binding to muscarinic receptors, but not that to cholecystokinin receptors. In conclusion, these data show that TPEN inhibits ligand binding to certain G protein-coupled receptors and exhibits selectivity towards those receptors whose transmembrane helices form the predominant site for ligand interaction. TPEN may have widespread antagonistic activity towards G protein-coupled receptors of this kind.

Inositol 1,4,5-trisphosphate receptor ubiquitination is mediated by mammalian Ubc7, a component of the endoplasmic reticulum-associated degradation pathway, and is inhibited by chelation of intracellular Zn2+.

In response to activation of certain cell surface receptors, inositol 1,4,5-trisphosphate receptors (InsP3Rs), which are located in the endoplasmic reticulum, can be rapidly ubiquitinated and then degraded by the proteasome. Ubiquitination is mediated by the concerted action of ubiquitin-conjugating enzymes (Ubcs or E2s) and ubiquitin-protein ligases (E3s). In the present study we have examined the enzymology of ubiquitination of endogenous InsP3Rs in muscarinic agonist-stimulated SH-SY5Y human neuroblastoma cells, focusing our attention on two mammalian E2s, MmUbc6 and MmUbc7, that have been implicated in endoplasmic reticulum-associated degradation (ERAD) and are homologous to the yeast ERAD E2s, Ubc6p and Ubc7p. Analysis of SH-SY5Y cells stably expressing these enzymes and their dominant-negative mutants revealed that MmUbc7 mediates InsP3R ubiquitination and down-regulation, but that MmUbc6 does not. These data indicate that InsP3Rs are processed by a component of the ERAD pathway and suggest that MmUbc7 may be employed selectively to ubiquitinate proteins, like InsP3Rs, that are subject to regulated ERAD. Additional studies showed that the Zn2+ chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine blocked InsP3R ubiquitination, suggesting that a RING finger domain-containing E3 is also involved in this process. Finally, muscarinic agonist-induced InsP3R ubiquitination was seen in rat brain slices, indicating that the results obtained from SH-SY5Y cells reflect a physiological process.

Ubiquitination and proteasomal degradation of endogenous and exogenous inositol 1,4,5-trisphosphate receptors in alpha T3-1 anterior pituitary cells.

In alphaT3-1 mouse anterior pituitary gonadotropes, chronic activation of gonadotropin-releasing hormone (GnRH) receptors causes inositol 1,4,5-trisphosphate (InsP(3)) receptor down-regulation (Willars, G. B., Royall, J. E., Nahorski, S. R., El-Gehani, F., Everest, H. and McArdle, C. A. (2001) J. Biol. Chem. 276, 3123-3129). In the current study, we sought to define the mechanism behind this adaptive response. We show that GnRH induces a rapid and dramatic increase in InsP(3) receptor polyubiquitination and that proteasome inhibitors block InsP(3) receptor down-regulation and cause the accumulation of polyubiquitinated receptors. Thus, the ubiquitin/proteasome pathway is active in alphaT3-1 cells, and GnRH regulates the levels of InsP(3) receptors via this mechanism. Given these findings and further characterization of this system, we also examined the possibility that alphaT3-1 cells could be used to examine the ubiquitination of exogenous InsP(3) receptors introduced by cDNA transfection. This was found to be the case, since exogenous wild-type InsP(3) receptors, but not binding-defective mutant receptors, were polyubiquitinated in a GnRH-dependent manner, and agents that inhibited the polyubiquitination of endogenous receptors also inhibited the polyubiquitination of exogenous receptors. Further, we used this system to determine whether phosphorylation was involved in triggering InsP(3) receptor polyubiquitination. This was not the case, since mutation of serine residues 1588 and 1755 (the predominant phosphorylation sites in the type I receptor) did not inhibit polyubiquitination. In total, these data show that the ubiquitin/proteasome pathway is active in anterior pituitary cells, that this pathway targets both endogenous and exogenous InsP(3) receptors in GnRH-stimulated alphaT3-1 cells, and that, in contrast to the situation for many other substrates, phosphorylation does not trigger InsP(3) receptor polyubiquitination.