Normal Soluble Protein Research Articles

Prions in Microbes: The Least in the Most.

Prions are infectious proteins that mostly replicate in self-propagating amyloid conformations (filamentous protein polymers) and consist of structurally altered normal soluble proteins. Prions can arise spontaneously in the cell without any clear reason and are generally considered fatal disease-causing agents that are only present in mammals. However, after the seminal discovery of two prions, [PSI+] and [URE3], in the eukaryotic model microorganism Saccharomyces cerevisiae, at least ten more prions have been discovered, and their biological and pathological effects on the host, molecular structure, and the relationship between prions and cellular components have been studied. In a filamentous fungus model, Podospora anserina, a vegetative incomparability-related [Het-s] prion that directly triggers cell death during anastomosis (hyphal fusion) was discovered. These prions in eukaryotic microbes have extended our understanding to overcome most fatal human prion/amyloid diseases. A prokaryotic microorganism (Clostridium botulinum) was reported to have a prion analog. The transcriptional regulators of C. botulinum-Rho can be converted into the self-replicating prion form ([RHO-X-C+]), which may affect global transcription. Here, we outline the major issues with prions in microbes and the lessons learned from the relatively uncovered microbial prion world.

A rare presentation of amyloid goiter with renal amyloidosis in a young female

Amyloidosis is a rare systemic disorder caused by abnormal folding of normal soluble proteins leading to fibril formation in one or more body organs, systems or soft tissues. Amyloid goiter is characterized by deposits of amyloid protein in the thyroid tissue. Amyloid infiltration of thyroid gland with development of secondary goiter is rare. Here we report a case of 36-year-old female presented with progressive painless swelling over neck. Thyroid profile was normal. Ultrasound neck showed enlarged bilateral thyroid gland and isthmus. Fine needle aspiration cytology suggestive of subacute thyroiditis (granulomatous thyroiditis). Total thyroidectomy was done and biopsy sample revealed amyloid goiter.

Protein-Based Biosensor for Screening of Tau Aggregation Inhibitors, a Pharmaceutical Application

Aggregation of tau proteins underlies several neurodegenerative diseases, notably Alzheimer’s disease (AD). Protein aggregation is a complex multi-step process where normal soluble proteins self-assemble in an ordered fashion into higher order conglomerates of low solubility. Experimental evidence suggests that not only the large tau aggregates, known as neurofibrillary tangles (NFTs), are involved in neuronal death, but also small tau oligomers are more likely contributed to the neuronal toxicity.1-3 While in the recent years substantial progress has been made in the design of drugs that interfere with tau aggregation, there is a lack of methods available that allow rapid screening of potential drug candidates which is critical for the future successful clinical trial.4-6 The heterogeneous nature of protein aggregation is the key challenge for testing the aggregation inhibition.Our previous studies showed the capability of surface-based electrochemistry to monitor the conformational changes of tau film on the gold surface by interrogating the electrochemical properties of tau-modified surface using the ferro/ferricyanide redox couple.7 We also clearly demonstrated that electrochemistry is a highly useful tool to monitor tau-metal and tau-tau interactions.7 Thus, we proposed that this approach can be expanded to monitor changes in the current/impedance as a result of the interaction of surface-linked tau proteins with tau protein in solution or with drug candidates. The former will provide information about tau dimerization and aggregation, while the latter will give information about tau-drug interactions and can potentially be further developed into a drug screening tool by following two different strategies. The first approach examines the affinity of the drug to bind to tau, followed by determining the effectiveness of the drug to prevent the tau dimerization. The second strategy monitors tau oligomerization and aggregation in an attempt to examine the inhibitory activity of potential drug candidates.We utilized Screen printed gold electrodes to reduce the sample size and the preparation time. A commercially available tau aggregation inhibitor, Cpd16 (amino thienopyridazine) was chosen to evaluate the developed biosensor. Since the orientation of the protein molecules on the surface has a significant impact on the efficiency and robustness of the biosensor, two different approaches were employed to anchor tau proteins on the electrode surface. First, full-length tau protein was chemically linked to the gold surface using lipoic acid N-hydroxysuccinimide ester (Lip-NHS), in which tau molecules randomly immobilize on the gold electrode. Although, results obtained from this approach was promising, non-specific adsorption is one of the biggest challenges that might have contributed to the false positive results. Therefore, biotin-tagged tau proteins were immobilized on the gold surface through the anchor NeutrAvidin to provide an oriented self-assembled monolayer as a biorecognition element. The non-specific adsorption can be minimized by this approach due to highly specific interaction of biotin with NeutrAvidin. This is the first report of using biotin-tagged tau for modification of a protein-based electrochemical biosensor. The oriented film has the advantages of higher sensitivity and reproducibility compared to the random orientation film on the gold surface. Also, the time for surface modification was significantly reduce by this approach.For both surface modification, a range of different electrochemical techniques has been exploited to study tau-tau interaction as well as tau-Cpd16 interaction by monitoring the redox activities of ferro/ferricyanide redox couple. Also changes in the film resistance as a result of such biomolecular interactions were conveniently monitored by electrochemical impedance spectroscopy. Circular dichroism spectroscopy and transmission electron microscopy were utilized as supporting techniques to monitor tau aggregation kinetics. The IC50 of Cpd16 obtained from the developed biosensors is comparable with the reported IC50, which shows the ability of this biosensor to predict the IC50 of the drug candidate for the in vitro model of AD.This project represents a significant achievement that contributes to our understanding of tau protein aggregation and provides a highly sensitive analytical tool that makes it possible to rapidly screen drug candidates that target tau aggregation. It can be expected that such a tool will be critical to identify the efficient drug candidates for AD therapy.

A Temperature-Controlled Stopped-Flow Droplet-Based Microfluidic Reactor for Fast Biomolecular Kinetics

Several important biomolecular reactions, including folding/unfolding, aggregation, and binding, occur on a time scale of sub-seconds or seconds. One relevant example is represented by microscopic reactions underlying, the formation of amyloid fibrils from normal soluble proteins and peptides, which is associated with the onset and progression of Alzheimer's and Parkinson's diseases. The protein aggregation process consists of a network of several microscopic steps of nucleation and growth. Each individual step has different contributions to the formation of the final fibrils and of the soluble intermediates generated during the reactions, which are currently thought to represent the most toxic species. The characterization of individual reactions in a sub-second time scale is challenging with the current available biophysical techniques. In this work, we leverage the power of droplet-based microfluidics to quantitatively explore aggregation kinetics within a time window of milliseconds to seconds. Herein we present a universal droplet-based microfluidic platform for rapid and high-precision kinetics using stroboscopic illumination and fluorescence detection within a single experiment. The microfluidic platform is also integrated with a heating element at the reactor zone, which is isolated from the droplet generation and mixing part. The droplets can be heated up rapidly to a set temperature (within a range of 24 to 90°C) once they enter the heating zone enabling fast biomolecular kinetics to be extracted at different temperatures. We prove the potential of this platform with two systems: the denaturation kinetics of hen egg white lysozyme under various temperatures, and the elongation rate of fibrils composed of the peptide Abeta42, the peptide associated with Alzheimer's disease.

Beta-sandwich – beta-barrel transformation may serve as a model of a conversion of 'normal' soluble proteins into amyloid aggregates

Many data suggest that specific sequence characteristics affect the ability to form amyloid fibrils. To pinpoint that structural variation in amino acids are responsible for transforming normal soluble proteins to insoluble amyloid aggregates, we propose that this process is similar to the interconversion of two types of beta proteins – sandwich proteins (SP) and barrel proteins (BP). We determine and compare conserved residues, which are common to SP and BP, which we call structural determinants of SP and BP. Common methods of sequence alignment for identification of conserved residues cannot be applied here because analysed sequences are very diverse. To uncover structural determinants, a new method of structural-based sequence alignment was applied, which allows comparing sequences with very low sequence similarity, but similar 3D structures. For analysis and comparison of structural determinants of BP and SP, a recently suggested scale of hydrophobicity was used. It allowed us to reveal delicate sequence features, which distinguish SP and BP and are likely responsible for amyloid aggregation.

Inhibition of Protein Misfolding and Aggregation by Small Rationally-Designed Peptides

Several human diseases are associated with the presence of toxic fibrillar protein deposits. These diseases called protein misfolding disorders, are characterized by the accumulation of misfolded protein aggregates in diverse tissues. Strong evidence indicates that the conversion of a normal soluble protein into a beta-sheet-rich oligomeric structure and further fibrillar aggregation are the key events in the disease pathogenesis. Therefore, a promising therapeutic target consists of the prevention and dissolution of misfolded protein aggregates. Peptides designed to specifically bind to the pathogenic protein and block and/or reverse its abnormal conformational change constitute a new class of drugs. This article reviews this approach, describing diverse compounds reported to have this activity.

A Neatly Pleated Sheet

BIOCHEMISTRY Amyloid diseases such as Alzheimer's disease are characterized by a buildup of insoluble protein aggregates in tissues. These aggregates are formed by the conversion of normal soluble proteins into insoluble self-assembling fibrils via a soluble oligomeric intermediate that may be toxic to cells. An antibody that binds specifically to the oligomeric intermediates of several different amyloid proteins blocks toxicity, suggesting that the intermediates may share a common structure. To identify what this structure might be, Armen et al. have modeled the conformational changes of four amyloid proteins under the low pH conditions that favor amyloid fibril formation. From their molecular dynamics simulations, they conclude that a key step in oligomeric intermediate formation is the acquisition of an α-pleated sheet that could be the target of the toxicity-blocking antibody. The α-pleated sheet, a secondary structural motif proposed more than 50 years ago by Pauling and Corey, has garnered little attention because it is rarely found in proteins. The α-pleated sheet has a residue length of 3.0 A compared to 3.3 A for the more common β-sheet conformation found in many proteins. The hunt is on to find this α-pleated sheet structure in the test tube; if it exists, such an unusual structure would be a valuable target for designing therapeutics. — OMS Proc. Natl. Acad. Sci. U.S.A. 101 , 11622 (2004).

Possible involvement of copper(II) in Alzheimer disease.

The beta-amyloid (Abeta) peptide is a principal component of insoluble amyloid plaques that are characteristic neuropathological features of Alzheimer disease (AD). The amyloid peptide also exists as a normal soluble protein that undergoes a pathogenic transition to an aggregated, fibrous form. This transition can be affected by extraneous proteinaceous elements and nonproteinaceous elements such as copper ions, which may promote aggregation and/or stabilization of the fibrils. Copper has been found in abnormally high concentrations in amyloid plaques and AD-affected neuropil, and copper-selective chelators have been shown to dissolve Abeta peptide from postmortem brain specimens. Although Cu(2+) is an essential element for life and the function of numerous enzymes is basic to neurobiology, free or incorrectly bound Cu(2+) can also catalyze generation of the most damaging radicals, such as hydroxyl radical, giving a chemical modification of the protein, alternations in protein structure and solubility, and oxidative damage to surrounding tissue.

Examining the zinc binding site of the amyloid-beta peptide.

The amyloid beta-peptide (Abeta) is a principal component of insoluble amyloid plaques which are characteristic neuropathological features of Alzheimer's disease. Abeta also exists as a normal soluble protein that undergoes a pathogenic transition to an aggregated, fibrous form. This transition can be affected by extraneous proteinaceous and nonproteinaceous elements, such as zinc ions, which may promote aggregation and/or stabilization of the fibrils. Protein chelation of zinc is typically mediated by histidines, cysteines and carboxylates. Previous studies have demonstrated that the Abeta-Zn2+ binding site is localized within residues 6-28 and that histidines may serve as the principal sites of interaction. To localize key residues within this region, a series of Abeta peptides (residues 1-28) were synthesized that contained systematic His/Ala substitutions. Circular dichroism and electron microscopy were used to monitor the effects of Zn2+ on the peptide beta-sheet conformation and fibril aggregation. Our results indicate that substitution of either His13 or His14 but not His6 eliminates the zinc-mediated effects. These observations indicate a specific zinc binding site within Abeta that involves these central histidine residues.

Amyloid β binding proteins in vitro and in normal human cerebrospinal fluid

A major neuropathological feature of Alzheimer's disease (AD) is the deposition of amyloid β (Aβ) in the form of senile plaques. The Aβ peptide exists both in a β-pleated sheet fibrillar form in amyloid deposits and as a normal soluble protein in biological fluids. Numerous proteins have been identified immunohistochemically to be associated with senile plaques, where Aβ is the major constituent. Some of the latter have also been suggested to be carrier of the normal soluble Aβ (sAβ) including apolipoprotein J (apoJ), apolipoprotein E (apoE) and transthyretin (TTR). We have found, using several different methods, that numerous proteins can bind synthetic Aβ peptides when high concentrations are used; however, using an affinity anti-sAβ column we confirm that apoJ is the major binding protein in pooled human cerebrospinal fluid. On the other hand it is known that apoE co-purifies with Aβ biochemically extracted from senile plaques. In AD tissue there may be a change in the major apolipoprotein binding Aβ from apoJ to apoE.

Unifying features of systemic and cerebral amyloidosis

Amyloidosis is a generic term for a group of clinically and biochemically diverse diseases that are characterized by the deposition of an insoluble fibrillar protein in the extracellular space. Over 16 biochemically distinct amyloids are known. Despite this diversity, all amyloids have a particular ultrastructural and tinctorial appearance, a beta-pleated sheet structure, and are codeposited with a group of amyloid-associated proteins. The most common amyloidosis is Alzheimer's disease (AD), where A beta is the main component of the amyloid. Recently it has been found that A beta exists as a normal soluble protein (sA beta) in biological fluids. This links AD more closely to some of the systemic amyloidoses, where the amyloid precursor is found in the circulation normally. Numerous mutations have been found in the A beta precursor (beta PP) gene, associated with familial AD. Many mutations are also found in some of the hereditary systemic amyloidoses. For example, over 40 mutations in the transthyretin (TTR) gene are associated with amyloid. However, both A beta and TTR related amyloid deposition can occur with no mutation. The pathogenesis of amyloid is complex, and appears to be associated with genetic and environmental risk factors that can be similar in the systemic and cerebral amyloidoses.

Association of heparan sulfate proteoglycan with the neurofibrillary tangles of Alzheimer's disease

The major intracytoplasmic lesion of Alzheimer's disease is the neurofibrillary tangle (NFT), which is primarily composed of paired helical filaments (PHFs). The mechanism responsible for the formation of PHFs, as well as their insolubility and apparent heterogeneity, is unknown. We found that basic fibroblast growth factor (bFGF) binds to heparinase-sensitive sites in NFTs. bFGF binding is due to a heparan sulfate proteoglycan (HSPG) immunocytochemically identified in NFTs. In the presence of polycations (e.g., Ca2+), HSPG will bind to free carboxyl groups in NFT proteins. HSPG binding may play a role in transforming normal soluble proteins into insoluble PHFs.

Agar electrophoresis of normal soluble proteins in guinea pig liver.

HITHERTO, neither free electrophoresis1 nor electrophoresis on filter paper of the soluble proteins in liver (ref. 2, and Smetana, R., and Kořinek, J., personal communication) has afforded satisfactory delimitation of a sufficient number of fractions. After obtaining a number of unsatisfactory results on filter paper, we turned our attention to electrophoresis on agar gel.