An optimized Western blot assay provides a comprehensive assessment of the physiological endoproteolytic processing of the prion protein.

The prion protein (PrPC) is subjected to several conserved endoproteolytic events producing bioactive fragments that are of increasing interest for their physiological functions and their implication in the pathogenesis of prion diseases and other neurodegenerative diseases. However, systematic and comprehensive investigations on the full spectrum of PrPC proteoforms have been hampered by the lack of methods able to identify all PrPC-derived proteoforms. Building on previous knowledge of PrPC endoproteolytic processing, we thus developed an optimized Western blot assay able to obtain the maximum information about PrPC constitutive processing and the relative abundance of PrPC proteoforms in a complex biological sample. This approach led to the concurrent identification of the whole spectrum of known endoproteolytic-derived PrPC proteoforms in brain homogenates, including C-terminal, N-terminal and, most importantly, shed PrPC-derived fragments. Endoproteolytic processing of PrPC was remarkably similar in the brain of widely used wild type and transgenic rodent models, with α-cleavage-derived C1 representing the most abundant proteoform and ADAM10-mediated shedding being an unexpectedly prominent proteolytic event. Interestingly, the relative amount of shed PrPC was higher in WT mice than in most other models. Our results indicate that constitutive endoproteolytic processing of PrPC is not affected by PrPC overexpression or host factors other than PrPC but can be impacted by PrPC primary structure. Finally, this method represents a crucial step in gaining insight into pathophysiological roles, biomarker suitability, and therapeutic potential of shed PrPC and for a comprehensive appraisal of PrPC proteoforms in therapies, drug screening, or in the progression of neurodegenerative diseases.

A conserved domain of Drosophila RNA-binding protein Pumilio interacts with multiple CCR4-NOT deadenylase complex subunits to repress target mRNAs.

Pumilio is a sequence-specific RNA-binding protein that controls development, stem cell fate, and neurological functions in Drosophila. Pumilio represses protein expression by destabilizing target mRNAs in a manner dependent on the CCR4-NOT deadenylase complex. Three unique repression domains in the N-terminal region of Pumilio were previously shown to recruit CCR4-NOT, but how they do so was not well understood. In this study, we identified the motifs that are necessary and sufficient for the activity of the third repression domain of Pumilio, designated RD3, which is present in all isoforms and has conserved regulatory function. We identified multiple conserved regions of RD3 that are important for repression activity in cell-based reporter gene assays. Using yeast two-hybrid assays, we show that RD3 contacts specific regions of the Not1, Not2, and Not3 subunits of the CCR4-NOT complex. Our results indicate that RD3 makes multivalent interactions with CCR4-NOT mediated by conserved short linear interaction motifs. Specifically, two phenylalanine residues in RD3 make crucial contacts with Not1 that are essential for its repression activity. Using reporter gene assays, we also identify three new target mRNAs that are repressed by Pumilio and show that RD3 contributes to their regulation. Together, these results provide important insights into the mechanism by which Pumilio recruits CCR4-NOT to regulate the expression of target mRNAs.

Human Variability in Carboxylesterases and carboxylesterase-related Uncertainty Factors for Chemical Risk Assessment.

Carboxylesterases (CES) are an important class of enzymes involved in the hydrolysis of a range of chemicals and show large inter-individual variability in vitro. An extensive literature search was performed to identify in vivo probe substrates for CES1 and CES2 together with their protein content and enzymatic activity. Human pharmacokinetic (PK) data on Cmax, clearance, and AUC were extracted from 89 publications and Bayesian meta-analysis was performed using a hierarchical model to derive CES-related variability distributions and related uncertainty factors (UF). The CES-related variability indicated that 97.5% of healthy adults are covered by the kinetic default UF (3.16), except for clopidogrel and dabigatran etexilate. Clopidogrel is metabolised for a small amount by the polymorphic CYP2C19, which can have an impact on the overall pharmacokinetics, while the variability seen for dabigatran etexilate might be due to differences in the absorption, since this can be influenced by food intake. The overall CES-related variability was moderate to high in vivo (

Characterization of Chenopodin Isoforms from Quinoa Seeds and Assessment of Their Potential Anti-Inflammatory Activity in Caco-2 Cells.

Several food-derived molecules, including proteins and peptides, can show bioactivities toward the promotion of well-being and disease prevention in humans. There is still a lack of information about the potential effects on immune and inflammatory responses in mammalian cells following the ingestion of seed storage proteins. This study, for the first time, describes the potential immunomodulation capacity of chenopodin, the major protein component of quinoa seeds. After characterizing the molecular features of the purified protein, we were able to separate two different forms of chenopodin, indicated as LcC (Low charge Chenopodin, 30% of total chenopodin) and HcC (High charge Chenopodin, 70% of total chenopodin). The biological effects of LcC and HcC were investigated by measuring NF-κB activation and IL-8 expression studies in undifferentiated Caco-2 cells. Inflammation was elicited using IL-1ß. The results indicate that LcC and HcC show potential anti-inflammatory activities in an intestinal cell model, and that the proteins can act differently, depending on their structural features. Furthermore, the molecular mechanisms of action and the structural/functional relationships of the protein at the basis of the observed bioactivity were investigated using in silico analyses and structural predictions.

Risk assessment of the antifungal and insecticidal peptide Jaburetox and its parental protein the Jack bean (Canavalia ensiformis) urease.

Jaburetox (JBTX) is an insecticidal and antifungal peptide derived from jack bean (Canavalia ensiformis) urease that has been considered a candidate for developing genetically modified crops. This study aimed to perform the risk assessment of the peptide JBTX following the general recommendations of the two-tiered, weight-of-evidence approach proposed by International Life Sciences Institute. The urease of C. ensiformis (JBU) and its isoform JBURE IIb (the JBTX parental protein) were assessed. The history of safe use revealed no hazard reports for the studied proteins. The available information shows that JBTX possesses selective activity against insects and fungi. JBTX and JBU primary amino acids sequences showed no relevant similarity to toxic, antinutritional or allergenic proteins. Additionally, JBTX and JBU were susceptible to in vitro digestibility, and JBU was also susceptible to heat treatment. The results did not identify potential risks of adverse effects and reactions associated to JBTX. However, further allergen (e.g. serum IgE binding test) and toxicity (e.g. rodent toxicity tests) experimentation can be done to gather additional safety information on JBTX, and to meet regulatory inquiries for commercial approval of transgenic cultivars expressing this peptide.

Protein knots provide mechano-resilience to an AAA+ protease-mediated proteolysis with profound ATP energy expenses.

Knotted proteins are some of the most fascinating examples of how linear polypeptide chains can achieve intricate topological arrangements efficiently and spontaneously. The entanglements of polypeptide chains could potentially enhance their folding stabilities. We recently reported the unprecedented mechanostability of the Gordian (52) knotted family of human ubiquitin C-terminal hydrolases (UCHs) in the context of withstanding the mechanical unfolding of the bacterial AAA+ proteasome, ClpXP; a green fluorescence protein (GFP) was fused to the N-terminus of various UCHs as a reporter of the unfolding and degradation of these topologically knotted substrates, but it also limited the ability to examine the effect of untying the knotted topology via N-terminal truncation. In this study, we directly monitored the ClpXP-mediated degradation of UCH variants by electrophoresis and quantitative imaging analyses. We demonstrated that untying of the 52 knot in UCHL1 via N-terminal truncation (UCHL1Δ11) significantly reduces its mechanostability. We further quantified the ATP expenditures of degrading different UCH variants by ClpXP. The unknotted UCHL1Δ11 underwent accelerated ClpXP-dependent proteolysis, with a 30-fold reduction in ATP consumption compared to the knotted wild type. Unlike all other known ClpXP substrates, UCHL5, which is the most resilient substrate known to date, significantly slowed down the ATP turnover rate by ClpXP. Furthermore, UCHL5 required 1000-fold more ATP to be fully degraded by ClpXP compared to GFP. Our results underscored how the complex, knotted folding topology in UCHs may interfere with the mechano-unfolding processes of the AAA+ unfoldase, ClpX.

Structural Lesions of Proteins Connected to Lipid Membrane Damages Caused by Radical Stress: Assessment by Biomimetic Systems and Raman Spectroscopy.

Model systems constituted by proteins and unsaturated lipid vesicles were used to gain more insight into the effects of the propagation of an initial radical damage on protein to the lipid compartment. The latter is based on liposome technology and allows measuring the trans unsaturated fatty acid content as a result of free radical stress on proteins. Two kinds of sulfur-containing proteins were chosen to connect their chemical reactivity with membrane lipid transformation, serum albumins and metallothioneins. Biomimetic systems based on radiation chemistry were used to mimic the protein exposure to different kinds of free radical stress and Raman spectroscopy to shed light on protein structural changes caused by the free radical attack. Among the amino acid residues, Cys is one of the most sensitive residues towards the attack of free radicals, thus suggesting that metal-Cys clusters are good interceptors of reactive species in metallothioneins, together with disulfides moieties in serum albumins. Met is another important site of the attack, in particular under reductive conditions. Tyr and Phe are sensitive to radical stress too, leading to electron transfer reactions or radical-induced modifications of their structures. Finally, modifications in protein folding take place depending on reactive species attacking the protein.

Identification of cytochrome P450 isoforms involved in the metabolism of artocarpin and assessment of its drug-drug interaction.

Artocarpin isolated from an agricultural plant Artocarpus communis has shows anti-inflammation and anticancer activities. In this study, we utilized recombinant human UDP-glucuronosyltransferasesupersomes (UGTs) and human liver microsomes to explore its inhibitory effect on UGTs and cytochrome p450 enzymes (CYPs). Chemical inhibition studies and screening assays with recombinant human CYPs were used to identify if CYP isoform is involved in artocarpin metabolism. Artocarpin showed strong inhibition against UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, CYP2C8 and CYP3A4. In particular, artocarpin exhibited competitive inhibition against CYP3A4 and noncompetitive inhibition against UGT1A3 and UGT1A7. The half inhibition concentration values for CYP3A4, UGT1A3 and UGT1A7 were 4.67, 3.82 and 4.82 µm, and the inhibition kinetic parameters for them were 0.78, 2.67 and 3.14 µm, respectively. After artocarpin was incubated in human liver microsomes and determined by HPLC, we observed its main metabolites (M1 and M2). In addition, we proved that CYP2D6 played the key role in the biotransformation of artocarpin in human liver microsomes. The result of molecular docking further confirmed that artocarpin interacted with CYP2D6, CYP2C8 and CYP3A4 through hydrogen bonds. This study provided preliminary results for further research on artocarpin or artocarpin-containing herbs.

Atomistic Insights into Structural Differences between E3 and E4 Isoforms of Apolipoprotein E.

Among various isoforms of Apolipoprotein E (ApoE), the E4 isoform (ApoE4) is considered to be the strongest risk factor for Alzheimer's disease, whereas the E3 isoform (ApoE3) is neutral to the disease. Interestingly, the sequence of ApoE4 differs from its wild-type ApoE3 by a single amino acid C112R in the 299-amino-acid-long sequence. Hence, the puzzle remains: how a single-amino-acid difference between the ApoE3 and ApoE4 sequences can give rise to structural dissimilarities between the two isoforms, which can potentially lead to functional differences with significant pathological consequences. The major obstacle in addressing this question has been the lack of a 3D atomistic structure of ApoE4 to date. In this work, we resolve the issue by computationally modeling a plausible atomistic 3D structure of ApoE4. Our microsecond-long atomistic simulations elucidate key structural differences between monomeric ApoE3 and ApoE4, which renders ApoE4 thermodynamically less stable, less structured, and topologically less rigid compared to ApoE3. Consistent with an experimental report of the molten globule state of ApoE4, simulations identify multiple partially folded intermediates for ApoE4, which are implicated in the stronger aggregation propensity of ApoE4.

A single Markov-type kinetic model accounting for the macroscopic currents of all human voltage-gated sodium channel isoforms.

Modelling ionic channels represents a fundamental step towards developing biologically detailed neuron models. Until recently, the voltage-gated ion channels have been mainly modelled according to the formalism introduced by the seminal works of Hodgkin and Huxley (HH). However, following the continuing achievements in the biophysical and molecular comprehension of these pore-forming transmembrane proteins, the HH formalism turned out to carry limitations and inconsistencies in reproducing the ion-channels electrophysiological behaviour. At the same time, Markov-type kinetic models have been increasingly proven to successfully replicate both the electrophysiological and biophysical features of different ion channels. However, in order to model even the finest non-conducting molecular conformational change, they are often equipped with a considerable number of states and related transitions, which make them computationally heavy and less suitable for implementation in conductance-based neurons and large networks of those. In this purely modelling study we develop a Markov-type kinetic model for all human voltage-gated sodium channels (VGSCs). The model framework is detailed, unifying (i.e., it accounts for all ion-channel isoforms) and computationally efficient (i.e. with a minimal set of states and transitions). The electrophysiological data to be modelled are gathered from previously published studies on whole-cell patch-clamp experiments in mammalian cell lines heterologously expressing the human VGSC subtypes (from NaV1.1 to NaV1.9). By adopting a minimum sequence of states, and using the same state diagram for all the distinct isoforms, the model ensures the lightest computational load when used in neuron models and neural networks of increasing complexity. The transitions between the states are described by original ordinary differential equations, which represent the rate of the state transitions as a function of voltage (i.e., membrane potential). The kinetic model, developed in the NEURON simulation environment, appears to be the simplest and most parsimonious way for a detailed phenomenological description of the human VGSCs electrophysiological behaviour.

Subunit NDUFV3 is present in two distinct isoforms in mammalian complex I.

Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the electron transport chain in mammalian mitochondria. Extensive proteomic and structural analyses of complex I from Bos taurus heart mitochondria have shown it comprises 45 subunits encoded on both the nuclear and mitochondrial genomes; 44 of them are different and one is present in two copies. The bovine heart enzyme has provided a model for studying the composition of complex I in other mammalian species, including humans, but the possibility of additional subunits or isoforms in other species or tissues has not been explored. Here, we describe characterization of the complexes I purified from five rat tissues and from a rat hepatoma cell line. We identify a~50kDa isoform of subunit NDUFV3, for which the canonical isoform is only ~10kDa in size. We combine LC-MS and MALDI-TOF mass spectrometry data from two different purification methods (chromatography and immuno-purification) with information from blue native PAGE analyses to show the long isoform is present in the mature complex, but at substoichiometric levels. It is also present in complex I in cultured human cells. We describe evidence that the long isoform is more abundant in both the mitochondria and purified complexes from brain (relative to in heart, liver, kidney and skeletal muscle) and more abundant still in complex I in cultured cells. We propose that the long 50kDa isoform competes with its canonical 10kDa counterpart for a common binding site on the flavoprotein domain of complex I.

Food safety assessment of an antifungal protein from Moringa oleifera seeds in an agricultural biotechnology perspective.

Mo-CBP3 is an antifungal protein produced by Moringa oleifera which has been investigated as potential candidate for developing transgenic crops. Before the use of novel proteins, food safety tests must be conducted. This work represents an early food safety assessment of Mo-CBP3, using the two-tiered approach proposed by ILSI. The history of safe use, mode of action and results for amino acid sequence homology using the full-length and short contiguous amino acids sequences indicate low risk associated to this protein. Mo-CBP3 isoforms presented a reasonable number of alignments (>35% identity) with allergens in a window of 80 amino acids. This protein was resistant to pepsin degradation up to 2 h, but it was susceptible to digestion using pancreatin. Many positive attributes were presented for Mo-CBP3. However, this protein showed high sequence homology with allergens and resistance to pepsin digestion that indicates that further hypothesis-based testing on its potential allergenicity must be done. Additionally, animal toxicity evaluations (e.g. acute and repeated dose oral exposure assays) must be performed to meet the mandatory requirements of several regulatory agencies. Finally, the approach adopted here exemplified the importance of performing an early risk assessment of candidate proteins for use in plant transformation programs.

Total and isoform-specific quantitative assessment of circulating fibulin-1 using selected reaction monitoring MS and time-resolved immunofluorometry.

PURPOSE: Targeted proteomics using SRM-MS combined with stable-isotope dilution has emerged as a promising quantitative technique for the study of circulating protein biomarkers. The purpose of this study was to develop and characterize robust quantitative assays for the emerging cardiovascular biomarker fibulin-1 and its circulating isoforms in human plasma. EXPERIMENTAL DESIGN: We used bioinformatics analysis to predict total and isoform-specific tryptic peptides for absolute quantitation using SRM-MS. Fibulin-1 was quantitated in plasma by nanoflow-LC-SRM-MS in undepleted plasma and time-resolved immunofluorometric assay (TRIFMA). Both methods were validated and compared to a commercial ELISA (CircuLex). Molecular size determination was performed under native conditions by SEC analysis coupled to SRM-MS and TRIFMA. RESULTS: Absolute quantitation of total fibulin-1, isoforms -1C, and -1D was performed by SRM-MS. Fibulin-1C was the most abundant isoform in plasma. Circulating fibulin-1 isoforms were homo -or hetero multimeric complexes (range 318-364 kDa). Good correlation was obtained between SRM-MS and TRIFMA but not CircuLex. CONCLUSIONS AND CLINICAL RELEVANCE: For biomarker studies using smaller cohorts, SRM-MS provides an alternative measure of total and specific fibulin-1 isoforms in undepleted plasma. For larger cohorts TRIFMA provides a faster platform for fibulin-1 quantitation in plasma. While the correlation between these methods was acceptable, low correlation was obtained between the commercial CircuLex assay and SRM-MS or TRIFMA.

Purification and characterization of parvalbumin isotypes from grass carp (Ctenopharyngodon idella).

The prevalence of fish allergy is rapidly increasing because of a growing fish consumption driven mainly by a positive image of the fish and health relationship. The purpose of this study was to characterize parvalbumin isotypes from grass carp (Ctenopharyngodon idella), one of the most frequently consumed freshwater fish in China. Three parvalbumin isotypes were purified using consecutive gel filtration and reverse-phase chromatography and denoted as PVI, PVII, and PVIII. The molecular weights of the isotypes were determined to be 11.968, 11.430, and 11.512 kDa, respectively. PVI showed 74% matched amino acids sequence with PV isotype 4a from Danio rerio, while PVII and PVIII showed 46% matched amino acids sequence with PV isotypes from Hypophthalmichthys molitrix. PVII is the dominant allergen, but it was liable to gastrointestinal enzymes as PVIII; however, PVI was resistant to pepsin digestion. A further study is to characterize the epitopes of PVII, the dominant allergen.

Assessment of peptide chemical modifications on the development of an accurate and precise multiplex selected reaction monitoring assay for apolipoprotein e isoforms.

Apolipoprotein E (ApoE) is a polymorphic protein that plays a major role in lipid metabolism in the central nervous system and periphery. It has three common allelic isoforms, ApoE2, ApoE3, and ApoE4, that differ in only one or two amino acids. ApoE isoforms have been associated with the occurrence and progression of several pathological conditions, such as coronary atherosclerosis and Alzheimer's disease. The aim of this study was to develop a mass spectrometry (MS)-based assay for absolute quantification of ApoE isoforms in cerebrospinal fluid and plasma samples using isotope-labeled peptides. The assay included five tryptic peptides: CLAVYQAGAR (ApoE2), LGADMEDVCGR (ApoE2 and 3), LAVYQAGAR (ApoE3 and 4), LGADMEDVR (ApoE4), and LGPLVEQGR (total ApoE). Both cerebrospinal fluid and plasma samples were assayed to validate the method. The digestion yield and the extension of chemical modifications in selected amino acid residues (methionine oxidation, glutamine deamidation, and cyclization of N-terminus carbamidomethylcysteine) were also studied. The ApoE phenotype was successfully assigned to all samples analyzed in a blinded manner. The method showed good linearity (R(2) > 0.99) and reproducibility (within laboratory imprecision <13%). The comparison of the MS-based assay with an ELISA for total ApoE concentration showed a moderate correlation (R(2) = 0.59). This MS-based assay can serve as an important tool in clinical studies aiming to elucidate the association between ApoE genotype, total ApoE, and ApoE isoform concentrations in various disorders related to ApoE polymorphisms.

Assessment of naturally occurring covalent and total dimer levels in human IgG1 and IgG2.

Antibody dimers, two self-associated monomers, have been detected on both recombinantly expressed and endogenous human IgG proteins. Nearly 10 years ago, Yoo et al. (2003) described low levels of IgG2 covalent dimer, in human serum, but did not quantify the levels. Here we quantify the total and covalent dimer levels of IgG2 and IgG1 in human blood, and study the origin of covalent dimer formation. Low levels (<1%) of total IgG1 and IgG2 dimers were measured in freshly prepared human plasma. Both IgG1 and IgG2 covalent dimers were also found in plasma. Whereas IgG1 covalent dimer levels were significantly reduced by steps intended to eliminate artifacts during sample preparation, IgG2 covalent dimer levels remain stable in such conditions. About 0.4% of IgG2 in plasma was in a covalent dimer form, yet very little (<0.03%) of IgG1 covalent dimer could be considered naturally occurring. IgG2 dimer also formed in vitro under conditions designed to mimic those in blood, suggesting that formation occurs in vivo during circulation. Thus, small amounts of covalent IgG2 dimer do appear to form naturally.

Assessment of adiponectin and its isoforms in Polish centenarians.

BACKGROUND: The physiological mechanisms that promote longevity remain unclear. It has been suggested that insulin sensitivity is preserved in centenarians, whereas typical aging is accompanied by increasing insulin resistance. The oldest-old individuals display raised total adiponectin levels, despite the potential correlation between enhanced adiponectin and all-cause and cardiovascular mortality. AIM: To evaluate the level of adiponectin and its isoforms in sera of centenarians and to assess associations between adiponectin and metabolic parameters. PARTICIPANTS: A group of 58 Polish centenarians (50 women and 8 men, mean age 101±1.34 years) and 68 elderly persons (55 women and 13 men, mean age 70±5.69 years) as controls. MEASUREMENTS: Serum samples were analyzed to evaluate the following parameters: adiponectin array (total adiponectin, HWM-, MMW- and LMW-adiponectin; all by ELISA methods), insulin (by IRMA methods), glucose and lipid profiles. HOMA-IR was calculated. Clinical data were collected. Statistical analyses were performed. RESULTS: The concentrations of all adiponectin isoforms were significantly higher in the oldest-old participants. In the centenarian group, total adiponectin positively correlated with age and HDL-cholesterol, and HMW-adiponectin was negatively associated with insulin and triglycerides. The long-lived participants had a lower incidence of hypertension, type 2 diabetes, overweight and obesity, with lower concentrations of serum glucose and insulin, and reduced HOMA-IR. CONCLUSION: Our findings support the thesis that centenarians possess a different adiponectin isoform pattern and have a favorable metabolic phenotype in comparison with elderly individuals. However, additional work is necessary to understand the relevance of these findings to longevity.

Bioinformatic approaches for the identification of serpin genes with multiple reactive site loop coding exons.

In several branches of the tree of life, alternative splicing of a single primary transcript may give rise to multiple serpin isoforms exhibiting different target enzyme specificities. Though the continuously increasing number of genome sequencing projects has been paralleled by a rapidly rising number of serpin genes, the full spectrum of isoforms that some of these genes can encode has often not been recognized in routine database searches. In this chapter, we introduce procedures that enable the systematic extraction of multi-isoform generating serpin genes from genomic sequences. Spot checking of a model organism demonstrates that the phyletic distribution of such genes appears to be largely underestimated. The bioinformatic approach presented here may help to dissect the complete antiproteolytic spectrum of a genome's serpin complement and to register the occurrence of multitasking serpin genes in eukaryotes for functional and evolutionary studies.

Baking a mass-spectrometry data PIE with McMC and simulated annealing: predicting protein post-translational modifications from integrated top-down and bottom-up data.

MOTIVATION: Post-translational modifications are vital to the function of proteins, but are hard to study, especially since several modified isoforms of a protein may be present simultaneously. Mass spectrometers are a great tool for investigating modified proteins, but the data they provide is often incomplete, ambiguous and difficult to interpret. Combining data from multiple experimental techniques-especially bottom-up and top-down mass spectrometry-provides complementary information. When integrated with background knowledge this allows a human expert to interpret what modifications are present and where on a protein they are located. However, the process is arduous and for high-throughput applications needs to be automated. RESULTS: This article explores a data integration methodology based on Markov chain Monte Carlo and simulated annealing. Our software, the Protein Inference Engine (the PIE) applies these algorithms using a modular approach, allowing multiple types of data to be considered simultaneously and for new data types to be added as needed. Even for complicated data representing multiple modifications and several isoforms, the PIE generates accurate modification predictions, including location. When applied to experimental data collected on the L7/L12 ribosomal protein the PIE was able to make predictions consistent with manual interpretation for several different L7/L12 isoforms using a combination of bottom-up data with experimentally identified intact masses. AVAILABILITY: Software, demo projects and source can be downloaded from http://pie.giddingslab.org/

Monte carlo simulations of tau proteins: effect of phosphorylation.

We perform Monte Carlo simulations of tau proteins bound to a cylinder that mimics a microtubule (MT), and then study them in solution. Tau protein binds to a highly anionic MT surface to stabilize the cylindrical structure of MT. The negatively charged tail domain floats away from the anionic MT surface while positively charged tau segments localize near the MT surface. Monte Carlo simulations demonstrate that, in 3RS tau isoform (which has three imperfect repeats (R) short (S) isoform), amino acids are more condensed near a highly charged interface compared to 4RL isoform (which has four imperfect repeats (R) long (L) isoform). In 4RL isoform, amino acids in tail domain stay mostly apart from the MT surface. In the bulk solution, dephosphorylated taus are separated due to Coulomb repulsion between similarly charged isoforms. Moderate phosphorylation of 3RS isoform decreases average intermolecular distance between dephosphorylated and phosphorylated taus and lead to their overlap. Further phosphorylation does not change noticeably the intermolecular distances.