.

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.

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.

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.

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.

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.

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

The cystine transporter (system x) 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 F-5-fluoroaminosuberic acid (FASu), a PET tracer that targets system x The goal of this study was to evaluate F-FASu as a specific gauge for system x activity in vivo and its potential for breast cancer imaging.

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.

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).

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).

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.

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).

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).

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.

Small-animal PET imaging of human epidermal growth factor receptor type 2 expression with site-specific 18F-labeled protein scaffold molecules.

Unlabelled: Human epidermal growth factor receptor type 2 (HER2) is a well-established tumor biomarker that is overexpressed in a wide variety of cancers and that serves as a molecular target for therapeutic intervention. HER2 also serves as a prognostic indicator of patient survival and as a predictive marker of the response to antineoplastic therapy. The development of (18)F-labeled biomolecules for PET imaging of HER2 (HER2 PET) is very important because it may provide a powerful tool for the early detection of HER2-positive tumor recurrence and for the monitoring of HER2-based tumor treatment.

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.

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.

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.

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.

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).

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.

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.