Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer''s, Parkinson''s, and Huntington''s diseases, hereditary amyotrophic lateral sclerosis, and type 2 diabetes. Owing to the metastable nature of these protein assemblies, it is difficult to assess their oligomer size distribution quantitatively using classical methods, such as electrophoresis, chromatography, fluorescence, or dynamic light scattering. Oligomers of amyloidogenic proteins exist as metastable mixtures, in which the oligomers dissociate into monomers and associate into larger assemblies simultaneously. PICUP stabilizes oligomer populations by covalent cross-linking and when combined with fractionation methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size-exclusion chromatography (SEC), PICUP provides snapshots of the oligomer size distributions that existed before cross-linking. Hence, PICUP enables visualization and quantitative analysis of metastable protein populations and can be used to monitor assembly and decipher relationships between sequence modifications and oligomerization¹. Mechanistically, PICUP involves photo-oxidation of Ru²⁺ in a tris(bipyridyl)Ru(II) complex (RuBpy) to Ru³⁺ by irradiation with visible light in the presence of an electron acceptor. Ru³⁺ is a strong one-electron oxidizer capable of abstracting an electron from a neighboring protein molecule, generating a protein radical^1,2. Radicals are unstable, highly-reactive species and therefore disappear rapidly through a variety of intra- and intermolecular reactions. A radical may utilize the high energy of an unpaired electron to react with another protein monomer forming a dimeric radical, which subsequently loses a hydrogen atom and forms a stable, covalently-linked dimer. The dimer may then react further through a similar mechanism with monomers or other dimers to form higher-order oligomers. Advantages of PICUP relative to other photo- or chemical cross-linking methods^3,4 include short (≤1 s) exposure to non-destructive visible light, no need for pre facto modification of the native sequence, and zero-length covalent cross-linking. In addition, PICUP enables cross-linking of proteins within wide pH and temperature ranges, including physiologic parameters. Here, we demonstrate application of PICUP to cross-linking of three amyloidogenic proteins the 40- and 42-residue amyloid β-protein variants (Aβ40 and Aβ42), and calcitonin, and a control protein, growth-hormone releasing factor (GRF).     

Hacking the Code of Amyloid Formation: The Amyloid Stretch Hypothesis

Many research efforts in the last years have been directed towards understanding the factors determining protein misfolding and amyloid formation. Protein stability and amino acid composition have been identified as the two major factors in vitro. The research of our group has been focused on understanding the relationship between amino acid sequence and amyloid formation. Our approach has been the design of simple model systems that reproduce the biophysical properties of natural amyloids. An amyloid sequence pattern was extracted that can be used to detect amyloidogenic hexapeptide stretches in proteins. We have added evidence supporting that these amyloidogenic stretches can trigger amyloid formation by nonamyloidogenic proteins. Some experimental results in other amyloid proteins will be analyzed under the conclusions obtained in these studies. Our conclusions together with evidences from other groups suggest that amyloid formation is the result of the interplay between a decrease of protein stability, and the presence of highly amyloidogenic regions in proteins. As many of these results have been obtained in vitro, the challenge for the next years will be to demonstrate their validity in in vivo systems.     

When amyloids become prions

The conformational diseases, linked to protein aggregation into amyloid conformations, range from non-infectious neurodegenerative disorders, such as Alzheimer''s disease (AD), to highly infectious ones, such as human transmissible spongiform encephalopathies (TSEs). They are commonly known as prion diseases. However, since all amyloids could be considered prions (from those involved in cell-to-cell transmission to those responsible for real neuronal invasion), it is necessary to find an underlying cause of the different capacity to infect that each of the proteins prone to form amyloids has. As proposed here, both the intrinsic cytotoxicity and the number of nuclei of aggregation per cell could be key factors in this transmission capacity of each amyloid.     

Using radial basis function on the general form of Chou's pseudo amino acid composition and PSSM to predict subcellular locations of proteins with both single and multiple sites

Prediction of protein subcellular location is a meaningful task which attracted much attention in recent years. A lot of protein subcellular location predictors which can only deal with the single-location proteins were developed. However, some proteins may belong to two or even more subcellular locations. It is important to develop predictors which will be able to deal with multiplex proteins, because these proteins have extremely useful implication in both basic biological research and drug discovery. Considering the circumstance that the number of methods dealing with multiplex proteins is limited, it is meaningful to explore some new methods which can predict subcellular location of proteins with both single and multiple sites. Different methods of feature extraction and different models of predict algorithms using on different benchmark datasets may receive some general results. In this paper, two different feature extraction methods and two different models of neural networks were performed on three benchmark datasets of different kinds of proteins, i.e. datasets constructed specially for Gram-positive bacterial proteins, plant proteins and virus proteins. These benchmark datasets have different number of location sites. The application result shows that RBF neural network has apparently superiorities against BP neural network on these datasets no matter which type of feature extraction is chosen.     

A Comparison of Protein Extraction Methods Suitable for Gel-Based Proteomic Studies of Aphid Proteins

Protein extraction methods can vary widely in reproducibility and in representation of the total proteome, yet there are limited data comparing protein isolation methods. The methodical comparison of protein isolation methods is the first critical step for proteomic studies. To address this, we compared three methods for isolation, purification, and solubilization of insect proteins. The aphid Schizaphis graminum, an agricultural pest, was the source of insect tissue. Proteins were extracted using TCA in acetone (TCA-acetone), phenol, or multi-detergents in a chaotrope solution. Extracted proteins were solubilized in a multiple chaotrope solution and examined using 1-D and 2-D electrophoresis and compared directly using 2-D Difference Gel Electrophoresis (2-D DIGE). Mass spectrometry was used to identify proteins from each extraction type. We were unable to ascribe the differences in the proteins extracted to particular physical characteristics, cell location, or biological function. The TCA-acetone extraction yielded the greatest amount of protein from aphid tissues. Each extraction method isolated a unique subset of the aphid proteome. The TCA-acetone method was explored further for its quantitative reliability using 2-D DIGE. Principal component analysis showed that little of the variation in the data was a result of technical issues, thus demonstrating that the TCA-acetone extraction is a reliable method for preparing aphid proteins for a quantitative proteomics experiment. These data suggest that although the TCA-acetone method is a suitable method for quantitative aphid proteomics, a combination of extraction approaches is recommended for increasing proteome coverage when using gel-based separation techniques.     

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.     

Annotation of hypothetical proteins orthologous in Pongo abelii and Sus scrofa

A hypothetical protein is predicted to be expressed from an open reading frame without known experimental evidence of translation. They constitute a substantial fraction of proteomes. Domain extraction from these hypothetical sequences helps to search for protein coding genes for protein structural and functional annotation. We describe the analysis of prediction data in a sequence dataset of hypothetical protein orthologs of Pongo abelii (orangutan) and Sus scrofa (pig). It should be noted that these orangutan-pig orthologs are also non-homologous to human proteins. These predicted data find application in the genome wide annotation of proteins in poorly understood genomes.

Abbreviations

PDB - Protein Data Bank, DEG - Database of Essential Genes, CDD - Conserved Domain Database, IUCN - International Union for Conservation of Nature.     

Should I stay or should I go: VCP/p97-mediated chromatin extraction in the DNA damage response

The ordered assembly of DNA repair factors on chromatin has been studied in great detail, whereas we are only beginning to realize that selective extraction of proteins from chromatin plays a central role in the DNA damage response. Interestingly, the protein modifier ubiquitin not only regulates the well-documented recruitment of repair proteins, but also governs the temporally and spatially controlled extraction of proteins from DNA lesions. The facilitator of protein extraction is the ubiquitin-dependent ATPase valosin-containing protein (VCP)/p97 complex, which, through its segregase activity, directly extracts ubiquitylated proteins from chromatin. In this review, we summarize recent studies that uncovered this important role of VCP/p97 in the cellular response to genomic insults and discuss how ubiquitin regulates two intuitively counteracting activities at sites of DNA damage.     

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.     

Probing the mechanism of amyloidogenesis through a tandem repeat of the PI3-SH3 domain suggests a generic model for protein aggregation and fibril formation

Aggregation of the SH3 domain of the PI3 kinase, both as a single domain and as a tandem repeat in which the C terminus of one domain is linked to the N terminus of another by a flexible linker of ten glycine/serine residues, has been studied under a range of conditions in order to investigate the mechanism of protein aggregation and amyloid formation. The tandem repeat was found to form amyloid fibrils much more readily than the single domain under the acidic conditions used here, and the fibrils themselves have higher morphological homogeneity. The folding-unfolding transition of the PI3-SH3 domain shows two-state behaviour and is pH dependent; at pH 3.6, which is near the pH mid-point for folding and only slightly below the isoelectric point of the protein, both the single domain and the tandem repeat spontaneously form broad distributions of soluble oligomers without requirement for nucleation. Under prolonged incubation under these conditions, the oligomers convert into thin, curly fibrils that interact with thioflavin-T, suggesting that they contain an organised beta-sheet structure. Under more acidic conditions (pH 2.0) where the proteins are fully denatured and carry a positive net charge, long, straight fibrils are formed in a process having a pronounced lag phase. The latter was found to be reduced dramatically by the addition of oligomers exceeding a critical size of approximately 20 molecules. The results suggest that the process of aggregation of these SH3 domains can take place by a variety of mechanisms, ranging from downhill formation of relatively amorphous species to nucleated formation of highly organised structures, the relative importance of which varies greatly with solution conditions. Comparison with the behaviour of other amyloidogenic systems suggests that the general mechanistic features outlined here are likely to be common to at least a wide variety of peptides and proteins.     

The mouse nicotinamide mononucleotide adenylyltransferase chaperones diverse pathological amyloid client proteins

Molecular chaperones safeguard cellular protein homeostasis and obviate proteotoxicity. In the process of aging, as chaperone networks decline, aberrant protein amyloid aggregation accumulates in a mechanism that underpins neurodegeneration, leading to pathologies such as Alzheimer’s disease and Parkinson’s disease. Thus, it is important to identify and characterize chaperones for preventing such protein aggregation. In this work, we identified that the NAD⁺ synthase–nicotinamide mononucleotide adenylyltransferase (NMNAT) 3 from mouse (mN3) exhibits potent chaperone activity to antagonize aggregation of a wide spectrum of pathological amyloid client proteins including α-synuclein, Tau (K19), amyloid β, and islet amyloid polypeptide. By combining NMR spectroscopy, cross-linking mass spectrometry, and computational modeling, we further reveal that mN3 uses different region of its amphiphilic surface near the active site to directly bind different amyloid client proteins. Our work demonstrates a client recognition mechanism of NMNAT via which it chaperones different amyloid client proteins against pathological aggregation and implies a potential protective role for NMNAT in different amyloid-associated diseases.     

Life cycle of cytosolic prions

Prions are self-templating protein aggregates that were originally identified as the causative agent of prion diseases in mammals, but have since been discovered in other kingdoms. Mammalian prions represent a unique class of infectious agents that are composed of misfolded prion protein. Prion proteins usually exist as soluble proteins but can refold and assemble into highly ordered, self-propagating prion polymers. The prion concept is also applicable to a growing number of non-Mendelian elements of inheritance in lower eukaryotes. While prions identified in mammals are clearly pathogens, prions in lower eukaryotes can be either detrimental or beneficial to the host. Prion phenotypes in fungi are transmitted vertically from mother to daughter cells during cell division and horizontally during mating or abortive mating, but extracellular phases have not been reported. Recent findings now demonstrate that in a mammalian cell environment, protein aggregates derived from yeast prion domains exhibit a prion life cycle similar to mammalian prions propagated ex vivo. This life cycle includes a soluble state of the protein, an induction phase by exogenous prion fibrils, stable replication of prion entities, vertical transmission to progeny and natural horizontal transmission to neighboring cells. Our data reveal that mammalian cells contain all co-factors required for cytosolic prion propagation and dissemination. This has important implications for understanding prion-like properties of disease-related protein aggregates. In light of the growing number of identified functional amyloids, cell-to-cell propagation of cytosolic protein conformers might not only be relevant for the spreading of disease-associated proteins, but might also be of more general relevance under non-disease conditions.     

Phosphorylation of muscle plasma membrane protein by a membrane-bound protein kinase

Protein kinase activity has been demonstrated in purified plasma membranes from rat diaphragm by measuring the incorporation of ³²P from [³²P]-ATP into endogenous membrane proteins and into histone, in vitro. Histone appears to be a better substrate than the endogenous membrane proteins; however, the properties of the enzyme are similar when phosphorylating endogenous or exogenous proteins. The activity of this membrane-associated protein kinase is not significantly affected by cyclic adenosine 3′,5′-monophosphate or by cyclic guanosine 3′,5′-monophosphate, but is inhibited by theophylline. The ³²P incorporated into membrane proteins is alkali-labile and is released from the membrane by protease digestion, but it is not removed by phospholipase C, by hydroxylamine, or by chloroform—methanoll extraction. Solubilization of ³²P-labeled membranes by sodium dodecylsulfate and fractionation by sodium dodecylsulfate polyacrylamide gel electrophoresis reveals that the radioactivity is predominantly associated with a single protein band with an apparent molecular weight of about 51 000. The phosphoprotein is a minor membrane component as judged by Coomassie blue staining.     

Prediction of aggregation rate and aggregation-prone segments in polypeptide sequences

The reliable identification of beta-aggregating stretches in protein sequences is essential for the development of therapeutic agents for Alzheimer's and Parkinson's diseases, as well as other pathological conditions associated with protein deposition. Here, a model based on physicochemical properties and computational design of beta-aggregating peptide sequences is shown to be able to predict the aggregation rate over a large set of natural polypeptide sequences. Furthermore, the model identifies aggregation-prone fragments within proteins and predicts the parallel or anti-parallel beta-sheet organization in fibrils. The model recognizes different beta-aggregating segments in mammalian and nonmammalian prion proteins, providing insights into the species barrier for the transmission of the prion disease.     

The role of protein stability, solubility, and net charge in amyloid fibril formation

Ribonuclease Sa and two charge-reversal variants can be converted into amyloid in vitro by the addition of 2,2,2-triflouroethanol (TFE). We report here amyloid fibril formation for these proteins as a function of pH. The pH at maximal fibril formation correlates with the pH dependence of protein solubility, but not with stability, for these variants. Additionally, we show that the pH at maximal fibril formation for a number of well-characterized proteins is near the pI, where the protein is expected to be the least soluble. This suggests that protein solubility is an important determinant of fibril formation.     

The effect on Raman spectra of extraction of peripheral proteins from human erythrocyte membranes

We compare the Raman spectra of intact erythrocyte membranes with spectra of membranes from which essentially all peripheral proteins have been extracted. The results indicate that the extraction procedure causes considerable alteration in the environment of peptide bonds and a marked change in the environment of the phenylalanine and tryptophan ring moieties of the integral membrane proteins. We infer from our data that the lipid portion of the bilayer is little affected by extraction. Finally, we have observed that the total decrease in the heights of the protein-associated peaks of the extracted vesicles is generally less than that expected from the mass of protein removed by extraction.     

Detecting hidden sequence propensity for amyloid fibril formation

The preponderance of evidence implicates protein misfolding in many unrelated human diseases. In all cases, normal correctly folded proteins transform from their proper native structure into an abnormal beta-rich structure known as amyloid fibril. Here we introduce a computational algorithm to detect nonnative (hidden) sequence propensity for amyloid fibril formation. Analyzing sequence-structure relationships in terms of tertiary contact (TC), we find that the hidden beta-strand propensity of a query local sequence can be quantitatively estimated from the secondary structure preferences of template sequences of known secondary structure found in regions of high TC. The present method correctly pinpoints the minimal peptide fragment shown experimentally as the likely local mediator of amyloid fibril formation in beta-amyloid peptide, islet amyloid polypeptide (hIAPP), alpha-synuclein, and human acetylcholinesterase (AChE). It also found previously unrecognized beta-strand propensities in the prototypical helical protein myoglobin that has been reported as amyloidogenic. Analysis of 2358 nonhomologous protein domains provides compelling evidence that most proteins contain sequences with significant hidden beta-strand propensity. The present method may find utility in many medically relevant applications, such as the engineering of protein sequences and the discovery of therapeutic agents that specifically target these sequences for the prevention and treatment of amyloid diseases.     

DNA aptamers detecting generic amyloid epitopes

Amyloids are fibrillar protein aggregates resulting from non-covalent autocatalytic polymerization of various structurally and functionally unrelated proteins. Previously we have selected DNA aptamers, which bind specifically to the in vitro assembled amyloid fibrils of the yeast prionogenic protein Sup35. Here we show that such DNA aptamers can be used to detect SDS-insoluble amyloid aggregates of the Sup35 protein, and of some other amyloidogenic proteins, including mouse PrP, formed in yeast cells. The obtained data suggest that these aggregates and the Sup35 amyloid fibrils assembled in vitro possess common conformational epitopes recognizable by aptamers. The described DNA aptamers may be used for detection of various amyloid aggregates in yeast and, presumably, other organisms.     

Towards a structural understanding of the fibrillization pathway in Machado-Joseph's disease: trapping early oligomers of non-expanded ataxin-3

Machado-Joseph's disease is caused by a CAG trinucleotide repeat expansion that is translated into an abnormally long polyglutamine tract in the protein ataxin-3. Except for the polyglutamine region, proteins associated with polyglutamine diseases are unrelated, and for all of these diseases aggregates containing these proteins are the major components of the nuclear proteinaceous deposits found in the brain. Aggregates of the expanded proteins display amyloid-like morphological and biophysical properties. Human ataxin-3 containing a non-pathological number of glutamine residues (14Q), as well as its Caenorhabditis elegans (1Q) orthologue, showed a high tendency towards self-interaction and aggregation, under near-physiological conditions. In order to understand the discrete steps in the assembly process leading to ataxin-3 oligomerization, we have separated chromatographically high molecular mass oligomers as well as medium mass multimers of non-expanded ataxin-3. We show that: (a) oligomerization occurs independently of the poly(Q)-repeat and it is accompanied by an increase in beta-structure; and (b) the first intermediate in the oligomerization pathway is a Josephin domain-mediated dimer of ataxin-3. Furthermore, non-expanded ataxin-3 oligomers are recognized by a specific antibody that targets a conformational epitope present in soluble cytotoxic species found in the fibrillization pathway of expanded polyglutamine proteins and other amyloid-forming proteins. Imaging of the oligomeric forms of the non-pathological protein using electron microscopy reveals globular particles, as well as short chains of such particles that likely mimic the initial stages in the fibrillogenesis pathway occurring in the polyglutamine-expanded protein. Thus, they constitute potential targets for therapeutic approaches in Machado-Joseph's disease, as well as valuable diagnostic markers in disease settings.     

Adenosine 3′,5′-monophosphate-dependent phosphorylation of a 6000 and A 22000 dalton protein from cardiac sarcoplasmic reticulum

In canine cardiac sarcoplasmic reticulum, adenosine 3′,5′-monophosphate (cyclic AMP)-dependent protein kinase specifically phosphorylates two proteins, as seen by sodium dodecyl sulfate-slab gel electrophoresis and autoradiography. One protein has a molecular weight ranging between 22 000 and 24 000 daltons and has previously been identified and named phospholamban (Tada, M., Kirchberger, M.A. and Katz, A.M. (1975) J. Biol. Chem. 250, 2640–2647). The other protein that the ³²P label incorporates into has a molecular weight of approximately 6000. Like the 22 000 dalton protein, the 6000 dalton protein has characteristic of phosphoester bonding. The time-dependent course of phosphorylation shows that initially the ³²P label is incorporated more rapidly into the 22 000 dalton protein than the 6000 dalton protein, with both proteins reaching a steady-state level of phosphorylation after 10 min of incubation. When both protein kinase and cyclic AMP are eliminated from the incubation medium, both the 22 000 and the 6000 dalton protein are still phosphorylated but only to about a quarter of the activity found when cyclic AMP and protein kinase are included in the incubation mixture. The addition of phosphodiesterase completely eliminates the phosphorylation of both proteins. Treating the microsomes with trypsin prevents subsequent phosphorylation of either protein. Phosphorylating the microsomes first, then treating with trypsin, renders both the 22 000 and the 6000 dalton proteins resistant to even prolonged trypsin attack. Unphosphorylated, both proteins are solubilized by a very low concentration of deoxycholate. After phosphorylation the proteins cannot be solubilized by deoxycholate. Phosphorylation appears to alter greatly the physical properties of these proteins.Control experiments exclude the possibility that a lipid is being phosphorylated. After phosphorylation, the phosphorylated 22 000 dalton protein is separated from the 6000 dalton protein by proteolipid extraction. After first treating the microsomes with methanol, the 22 000 dalton protein is then soluble in acidified chloroform/methanol, while the 6000 dalton protein remains insoluble. The finding that both proteins have much different biochemical properties when phosphorylated than when not, may be relevant in how they regulate calcium transport in the sarcoplasmic reticulum.