Unfolded Monomers Research Articles

Fibrin fiber deformation mechanisms: insights from phenomenological modeling to molecular details.

The deformation mechanism of fibrin fibers has been a long-standing challenge to uncover due to the fiber's complex structure and mechanical behaviors. In this paper, a phenomenological, bilinear, force-strain model is derived to accurately reproduce the fibrin fiber force-strain curve, and then, the phenomenological model is converted to a mechanistic model using empirical relationships developed from particle simulation data. The mechanistic model assumes that the initial linear fibrin fiber force-strain response is due to entropic extension of polypeptide chains, and the final linear response is due to enthalpic extension of protofibrils. This model is the first fibrin fiber tensile force-strain equation to simultaneously (1) reproduce the bilinear force-strain curve of fibrin fibers in tension; (2) explicitly include the number of protofibrils through the fibrin fiber cross section, persistence length of [Formula: see text]-regions, and stiffness of fibrin protofibrils; and (3) make demonstrably reasonable/accurate predictions of fibrin fiber mechanics when tempered against experimental results. The model predicted that the count of protofibrils through the cross section for the analyzed fibrin fibers is between 207 and 421, the persistence length of [Formula: see text]-regions is [Formula: see text], and the stiffness of protofibrils in a deforming fiber is [Formula: see text]. The predicted [Formula: see text]-region persistence length is within the range typical of amino acid residue lengths [Formula: see text] and the predicted protofibril stiffness is shown to correspond to half-staggered protofibrils of unfolded fibrin monomers. Our analysis supports the proposition that entropic extension of [Formula: see text]-regions could be responsible for fibrin fiber's initial force-strain stiffness and suggests a structural change in fibrin protofibrils during fibrin fiber deformation. The results from the model are compared to those from five candidate deformation mechanisms reported in the literature. Our work provides (1) strong quantitative support to a deformation mechanism that was previously supported by anecdote and qualitative argument, and (2) a model for rigorously analyzing fibrin fiber force-strain data and simulating fibrin fibers in tension.

Mechanistic insights into monomer level prevention of amyloid aggregation of lysozyme by glycyrrhizic acid

As the primary bioactive compound of glycyrrhiza rhizome, the triterpene glycoside conjugate Glycyrrhizic acid (GA) has demonstrated neuroprotective effects in vivo. This study evaluates the effectiveness of GA as an inhibitor of GuHCl-induced amyloid aggregation of hen egg white lysozyme (HEWL). Fibril formation as measured by Thioflavin-T fluorescence, 900 light scattering, and 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence illustrated ∼90 % prevention of fibrils at [GA]/[HEWL] ≥2:1. Images of Transmission electron microscopy evidence for the absence of any fibril or amorphous aggregation products. The spectral characteristics of soluble HEWL were in close resemblance to unfolded monomer. Computational and fluorescence investigations performed to analyse GA-HEWL interactions demonstrated slightly higher affinity of GA to unfolded HEWL and aggregation-prone regions. The likely mechanism of monomer level aggregation prevention by GA as dermined by computational, stability, and ANS experiments illustrated that GA modulated the compactness, solvent-accessible surface, and solvent-exposed hydrophobic surfaces of aggregation-prone state of HEWL. Our findings corroborate GA as an effective inhibitor of HEWL amyloid formation. To our knowledge, GA interaction-induced inhibition of aggregation-prone states has not been previously discussed. GA's modulation of aggregation-prone states of disease-related proteins will successfully develop GA as an amyloid inhibitor for clinical trials of amyloidosis and neurodegenerative illnesses.

Parkinson-causing mutations in LRRK2 impair the physiological tetramerization of endogenous α-synuclein in human neurons

α-Synuclein (αSyn) aggregation in Lewy bodies and neurites defines both familial and ‘sporadic’ Parkinson’s disease. We previously identified α-helically folded αSyn tetramers, in addition to the long-known unfolded monomers, in normal cells. PD-causing αSyn mutations decrease the tetramer:monomer (T:M) ratio, associated with αSyn hyperphosphorylation and cytotoxicity in neurons and a motor syndrome of tremor and gait deficits in transgenic mice that responds in part to L-DOPA. Here, we asked whether LRRK2 mutations, the most common genetic cause of cases previously considered sporadic PD, also alter tetramer homeostasis. Patient neurons carrying G2019S, the most prevalent LRRK2 mutation, or R1441C each had decreased T:M ratios and pSer129 hyperphosphorylation of their endogenous αSyn along with increased phosphorylation of Rab10, a widely reported substrate of LRRK2 kinase activity. Two LRRK2 kinase inhibitors normalized the T:M ratio and the hyperphosphorylation in the G2019S and R1441C patient neurons. An inhibitor of stearoyl-CoA desaturase, the rate-limiting enzyme for monounsaturated fatty acid synthesis, also restored the αSyn T:M ratio and reversed pSer129 hyperphosphorylation in both mutants. Coupled with the recent discovery that PD-causing mutations of glucocerebrosidase in Gaucher’s neurons also decrease T:M ratios, our findings indicate that three dominant genetic forms of PD involve life-long destabilization of αSyn physiological tetramers as a common pathogenic mechanism that can occur upstream of progressive neuronal synucleinopathy. Based on αSyn’s finely-tuned interaction with certain vesicles, we hypothesize that the fatty acid composition and fluidity of membranes regulate αSyn’s correct binding to highly curved membranes and subsequent assembly into metastable tetramers.

Brain region-specific susceptibility of Lewy body pathology in synucleinopathies is governed by \u03b1-synuclein conformations

The protein α-synuclein, a key player in Parkinson’s disease (PD) and other synucleinopathies, exists in different physiological conformations: cytosolic unfolded aggregation-prone monomers and helical aggregation-resistant multimers. It has been shown that familial PD-associated missense mutations within the α-synuclein gene destabilize the conformer equilibrium of physiologic α-synuclein in favor of unfolded monomers. Here, we characterized the relative levels of unfolded and helical forms of cytosolic α-synuclein in post-mortem human brain tissue and showed that the equilibrium of α-synuclein conformations is destabilized in sporadic PD and DLB patients. This disturbed equilibrium is decreased in a brain region-specific manner in patient samples pointing toward a possible “prion-like” propagation of the underlying pathology and forms distinct disease-specific patterns in the two different synucleinopathies. We are also able to show that a destabilization of multimers mechanistically leads to increased levels of insoluble, pathological α-synuclein, while pharmacological stabilization of multimers leads to a “prion-like” aggregation resistance. Together, our findings suggest that these disease-specific patterns of α-synuclein multimer destabilization in sporadic PD and DLB are caused by both regional neuronal vulnerability and “prion-like” aggregation transmission enabled by the destabilization of local endogenous α-synuclein protein.

Inhibiting mTTR Aggregation/Fibrillation by a Chaperone-like Hydrophobic Amino Acid-Conjugated SPION.

Transthyretin (TTR) aggregation via misfolding of a mutant or wild-type protein leads to systemic or partial amyloidosis (ATTR). Here, we utilized variable biophysical assays to characterize two distinct aggregation pathways for mTTR (a synthesized monomer TTR incapable of association into a tetramer) at pH 4.3 and also pH 7.4 with agitation, referred to as mTTR aggregation and fibrillation, respectively. The findings suggest that early-stage conformational changes termed monomer activation here determine the aggregation pathway, resulting in developing either amorphous aggregates or well-organized fibrils. Less packed partially unfolded monomers consisting of more non-regular secondary structures that were rapidly produced via a mildly acidic condition form amorphous aggregates. Meanwhile, more hydrophobic and packed monomers consisting of rearranged β sheets and increased helical content developed well-organized fibrils. Conjugating superparamagnetic iron oxide nanoparticles (SPIONs) with leucine and glutamine (L-SPIONs and G-SPIONs in order) via a trimethoxysilane linker provided the chance to study the effect of hydrophobic/hydrophilic surfaces on mTTR aggregation. The results indicated a powerful inhibitory effect of hydrophobic L-SPIONs on both mTTR aggregation and fibrillation. Monomer depletion was introduced as the governing mechanism for inhibiting mTTR aggregation, while a chaperone-like property of L-SPIONs by maintaining an mTTR native structure and adsorbing oligomers suppressed the progression of further fibril formation.

β-Barrels and Amyloids: Structural Transitions, Biological Functions, and Pathogenesis.

Insoluble protein aggregates with fibrillar morphology called amyloids and β-barrel proteins both share a β-sheet-rich structure. Correctly folded β-barrel proteins can not only function in monomeric (dimeric) form, but also tend to interact with one another—followed, in several cases, by formation of higher order oligomers or even aggregates. In recent years, findings proving that β-barrel proteins can adopt cross-β amyloid folds have emerged. Different β-barrel proteins were shown to form amyloid fibrils in vitro. The formation of functional amyloids in vivo by β-barrel proteins for which the amyloid state is native was also discovered. In particular, several prokaryotic and eukaryotic proteins with β-barrel domains were demonstrated to form amyloids in vivo, where they participate in interspecies interactions and nutrient storage, respectively. According to recent observations, despite the variety of primary structures of amyloid-forming proteins, most of them can adopt a conformational state with the β-barrel topology. This state can be intermediate on the pathway of fibrillogenesis (“on-pathway state”), or can be formed as a result of an alternative assembly of partially unfolded monomers (“off-pathway state”). The β-barrel oligomers formed by amyloid proteins possess toxicity, and are likely to be involved in the development of amyloidoses, thus representing promising targets for potential therapy of these incurable diseases. Considering rapidly growing discoveries of the amyloid-forming β-barrels, we may suggest that their real number and diversity of functions are significantly higher than identified to date, and represent only “the tip of the iceberg”. Here, we summarize the data on the amyloid-forming β-barrel proteins, their physicochemical properties, and their biological functions, and discuss probable means and consequences of the amyloidogenesis of these proteins, along with structural relationships between these two widespread types of β-folds.

Physiological Metals Can Induce Conformational Changes in Transthyretin Structure: Neuroprotection or Misfolding Induction?

Transthyretin (TTR) is a plasma homotetrameric protein that transports thyroxine and retinol. TTR itself, under pathological conditions, dissociates into partially unfolded monomers that aggregate and form fibrils. Metal ions such as Zn2+, Cu2+, Fe2+, Mn2+ and Ca2+ play a controversial role in the TTR amyloidogenic pathway. TTR is also present in cerebrospinal fluid (CSF), where it behaves as one of the major Aβ-binding-proteins. The interaction between TTR and Aβ is stronger in the presence of high concentrations of Cu2+. Crystals of TTR, soaked in solutions of physiological metals such as Cu2+ and Fe2+, but not Mn2+, Zn2+, Fe3+, Al3+, Ni2+, revealed an unusual conformational change. Here, we investigate the effects that physiological metals have on TTR, in order to understand if metals can induce a specific and active conformation of TTR that guides its Aβ-scavenging role. The capability of certain metals to induce and accelerate its amyloidogenic process is also discussed.

Brazilin Removes Toxic Alpha-Synuclein and Seeding Competent Assemblies from Parkinson Brain by Altering Conformational Equilibrium

Alpha-synuclein (α-syn) fibrils, a major constituent of the neurotoxic Lewy Bodies in Parkinson’s disease, form via nucleation dependent polymerization and can replicate by a seeding mechanism. Brazilin, a small molecule derived from red cedarwood trees in Brazil, has been shown to inhibit the fibrillogenesis of amyloid-beta (Aβ) and α-syn as well as remodel mature fibrils and reduce cytotoxicity. Here we test the effects of Brazilin on both seeded and unseeded α-syn fibril formation and show that the natural polyphenol inhibits fibrillogenesis of α-syn by a unique mechanism that alters conformational equilibria in two separate points of the assembly mechanism: Brazilin preserves the natively unfolded state of α-syn by specifically binding to the compact conformation of the α-syn monomer. Brazilin also eliminates seeding competence of α-syn assemblies from Parkinson’s disease patient brain tissue, and reduces toxicity of pre-formed assemblies in primary neurons by inducing the formation of large fibril clusters. Molecular docking of Brazilin shows the molecule to interact both with unfolded α-syn monomers and with the cross-β sheet structure of α-syn fibrils. Our findings suggest that Brazilin has substantial potential as a neuroprotective and therapeutic agent for Parkinson’s disease.

Maturation of neuronal AD-tau pathology involves site-specific phosphorylation of cytoplasmic and synaptic tau preceding conformational change and fibril formation

In Alzheimer's disease (AD), tau-protein undergoes a multi-step process involving the transition from a natively unfolded monomer to large, aggregated structures such as neurofibrillary tangles (NFTs). However, it is not yet clear which events initiate the early preclinical phase of AD tauopathy and whether they have impact on the propagation of tau pathology in later disease stages. To address this question, we analyzed the distribution of tau species phosphorylated at T231, S396/S404 and S202/T205, conformationally modified at the MC1 epitope and fibrillary tau detected by the Gallyas method (Gallyas-tau), in the brains of 15 symptomatic and 20 asymptomatic cases with AD pathology as well as of 19 nonAD cases. As initial tau lesions, we identified phosphorylated-T231-tau diffusely distributed within the somatodendritic compartment (IC-tau) and phosphorylated-S396/pS404-tau in axonal lesions of the white matter and in the neuropil (IN-tau). The subcellular localization of pT231-tau in the cell body and pS396/pS404-tau in the presynapse was confirmed in hP301L mutant Drosophila larvae. Phosphorylated-S202/T205-tau, MC1-tau and Gallyas-tau were negative for these lesions. IC- and IN-tau were observed in all analyzed regions of the human brain, including early affected regions in nonAD cases (entorhinal cortex) and late affected regions in symptomatic AD cases (cerebellum), indicating that tau pathology initiation follows similar processes when propagating into previously unaffected regions. Furthermore, a sequence of AD-related maturation of tau-aggregates was observed, initiated by the appearance of IC- and IN-tau, followed by the formation of pretangles exhibiting pT231-tau, pS396/pS404-tau and pS202/pT205-tau, then by MC1-conformational tau, and, finally, by the formation of Gallyas-positive NFTs. Since cases classified as nonAD [Braak NFT stages < I (including a-1b)] already showed IC- and IN-tau, our findings suggest that these lesions are a prerequisite for the development of AD.

How epigallocatechin gallate binds and assembles oligomeric forms of human alpha-synuclein

The intrinsically disordered human protein α-synuclein (αSN) can self-associate into oligomers and amyloid fibrils. Several lines of evidence suggest that oligomeric αSN is cytotoxic, making it important to devise strategies to either prevent oligomer formation and/or inhibit the ensuing toxicity. (−)-epigallocatechin gallate (EGCG) has emerged as a molecular modulator of αSN self-assembly, as it reduces the flexibility of the C-terminal region of αSN in the oligomer and inhibits the oligomer's ability to perturb phospholipid membranes and induce cell death. However, a detailed structural and kinetic characterization of this interaction is still lacking. Here, we use liquid-state NMR spectroscopy to investigate how EGCG interacts with monomeric and oligomeric forms of αSN. We find that EGCG can bind to all parts of monomeric αSN but exhibits highest affinity for the N-terminal region. Monomeric αSN binds ∼54 molecules of EGCG in total during oligomerization. Furthermore, kinetic data suggest that EGCG dimerization is coupled with the αSN association reaction. In contrast, preformed oligomers only bind ∼7 EGCG molecules per protomer, in agreement with the more compact nature of the oligomer compared with the natively unfolded monomer. In previously conducted cell assays, as little as 0.36 EGCG per αSN reduce oligomer toxicity by 50%. Our study thus demonstrates that αSN cytotoxicity can be inhibited by small molecules at concentrations at least an order of magnitude below full binding capacity. We speculate this is due to cooperative binding of protein-stabilized EGCG dimers, which in turn implies synergy between protein association and EGCG dimerization.

Concentration-Dependent Structural Transition of the HIV-1 gp41 MPER Peptide into α-Helical Trimers.

The membrane proximal external region (MPER) of HIV-1 gp41 contains epitopes for at least four broadly neutralizing antibodies. Depending on solution conditions and construct design, different structures have been reported for this segment. We show that in aqueous solution the MPER fragment (gp160660-674 ) exists in a monomer-trimer equilibrium with an association constant in the micromolar range. Thermodynamic analysis reveals that the association is exothermic, more favorable in D2 O than H2 O, and increases with ionic strength, indicating hydrophobically driven intermolecular interactions. Circular dichroism, 13 Cα chemical shifts, NOE, and hydrogen exchange rates reveal that MPER undergoes a structural transition from predominately unfolded monomer at low concentrations to an α-helical trimer at high concentrations. This result has implications for antibody recognition of MPER prior to and during the process where gp41 switches from a pre-hairpin intermediate to its post-fusion 6-helical bundle state.

Concentration‐Dependent Structural Transition of the HIV‐1 gp41 MPER Peptide into α‐Helical Trimers

AbstractThe membrane proximal external region (MPER) of HIV‐1 gp41 contains epitopes for at least four broadly neutralizing antibodies. Depending on solution conditions and construct design, different structures have been reported for this segment. We show that in aqueous solution the MPER fragment (gp160660–674) exists in a monomer‐trimer equilibrium with an association constant in the micromolar range. Thermodynamic analysis reveals that the association is exothermic, more favorable in D2O than H2O, and increases with ionic strength, indicating hydrophobically driven intermolecular interactions. Circular dichroism, 13Cα chemical shifts, NOE, and hydrogen exchange rates reveal that MPER undergoes a structural transition from predominately unfolded monomer at low concentrations to an α‐helical trimer at high concentrations. This result has implications for antibody recognition of MPER prior to and during the process where gp41 switches from a pre‐hairpin intermediate to its post‐fusion 6‐helical bundle state.

Altered Conformation of α-Synuclein Drives Malfunction of Synaptic Vesicles in a Model of Parkinson's Disease

Misfolded, aggregated forms of alpha-synuclein (αSyn) are the principal protein component of Lewy bodies, the defining lesions of Parkinson’s disease (PD) and dementia with Lewy bodies. Increased copy number, missense mutations, and specific 5’ and 3’ regulatory elements of αSyn can all cause familial PD. Although αSyn participates in the regulation of synaptic vesicles, fundamental questions about the relationship between its structure and function in the presynaptic terminal are unanswered. Here, we systematically examined brain synaptosomes from non-transgenic (NTG) mice and transgenic mice expressing wild-type (WT) human αSyn or an amplified form of the E46K PD mutation (called 3K) that impairs the normal α-helical tetramerization of WT αSyn and increases its membrane affinity. Synaptosomes from transgenic mice expressing the 3K mutant had reduced Ca2+-dependent vesicle exocytosis, altered synaptic vesicle ultrastructure, impaired SNARE-complex assembly, and abnormal levels of select proteins involved in synaptic activity compared with NTG and WT human αSyn mice. We also developed a method to perform intra-synaptosomal NMR that revealed that WT αSyn participates in heterogeneous interactions with synaptic components. We show that these interactions depend on the presence of endogenous αSyn and that the 3K mutation diminishes the interplay between αSyn and the synapse. We conclude that the microenvironment inside presynaptic terminals is necessary for the proper conformation and interaction network of αSyn, where unfolded monomers of αSyn no longer represent the dominant conformation. We speculate that the inability to populate dynamically different conformational ensembles likely represents the early steps in αSyn misfunction that result in the neurotoxicity observed in PD and related synucleinopathies. With the advance of techniques such as cryo-electron tomography, in-cell single-molecule FRET, and in-cell NMR, in situ approaches like that presented here have the potential to address questions about the physiology of proteins such as αSyn that show diverse and changing conformations in their native, highly complex environments.

A structure-function study of C-terminal residues predicted to line the export channel in Salmonella Flagellin

BackgroundStructural studies of a Salmonella Typhimurium flagellin protein indicated that four polar or charged C-terminal amino acid residues line the inner channel of the flagellum. The hydrophilic character of these putative channel-lining residues was predicted to be essential to facilitate the transport of unfolded flagellin monomers during flagellar assembly. The structure-function relationship of these putative channel-lining residues was investigated by site-directed mutagenesis to examine effects of side chain polarity and size on flagella assembly and function. MethodsChannel-lining residue variants were generated using site-directed mutagenesis to substitute alanine and other residues to examine the effects of altered side-chain polarity on export and assembly. The export, in vivo motility function, and flagellar structure of variants was characterized by agar motility, video microscopy, immunofluorescence, and SDS-PAGE. ResultsAlanine substitution yielded decreased motility and flagellar assembly for three of the four residues. However, alanine substitution of residue Arg 494 did not alter export, although substitution with negatively charged glutamate decreased motility and flagellar filament length. Furthermore, many of the C-terminal mutations affected flagellar filament morphology and stability, often resulting in more tightly coiled and/or more brittle flagella than the wild type. ConclusionsThe four channel-lining C-terminal residues may facilitate monomer protein transport but also have structural roles in determining the stability and morphology of the flagellum. General significanceThese results provide further insight into the complex process of bacterial flagellin export and flagellar assembly and provide evidence of previously unknown structural functions for the four putative channel-lining residues.

Trifluoroethanol Partially Unfolds G93A SOD1 Leading to Protein Aggregation: A Study by Native Mass Spectrometry and FPOP Protein Footprinting.

Misfolding of Cu, Zn superoxide dismutase (SOD1) variants may lead to protein aggregation and ultimately amyotrophic lateral sclerosis (ALS). The mechanism and protein conformational changes during this process are complex and remain unclear. To study SOD1 variant aggregation at the molecular level and in solution, we chemically induced aggregation of a mutant variant (G93A SOD1) with trifluoroethanol (TFE) and used both native mass spectrometry (MS) to analyze the intact protein and fast photochemical oxidation of proteins (FPOP) to characterize the structural changes induced by TFE. We found partially unfolded G93A SOD1 monomers prior to oligomerization and identified regions of the N-terminus, C-terminus, and strands β5, β6 accountable for the partial unfolding. We propose that exposure of hydrophobic interfaces of these unstructured regions serves as a precursor to aggregation. Our results provide a possible mechanism and molecular basis for ALS-linked SOD1 misfolding and aggregation.

Comprehensive Structural and Thermodynamic Analysis of Prefibrillar WT α-Synuclein and Its G51D, E46K, and A53T Mutants by a Combination of Small-Angle X-ray Scattering and Variational Bayesian Weighting.

The in solution synchrotron small-angle X-ray scattering SAXS technique has been used to investigate an intrinsically disordered protein (IDP) related to Parkinson’s disease, the α-synuclein (α-syn), in prefibrillar diluted conditions. SAXS experiments have been performed as a function of temperature and concentration on the wild type (WT) and on the three pathogenic mutants G51D, E46K, and A53T. To identify the conformers that populate WT α-syn and the pathogenic mutants in prefibrillar conditions, scattering data have been analyzed by a new variational bayesian weighting method (VBWSAS) based on an ensemble of conformers, which includes unfolded monomers, trimers, and tetramers, both in helical-rich and strand-rich forms. The developed VBWSAS method uses a thermodynamic scheme to account for temperature and concentration effects and considers long-range protein–protein interactions in the framework of the random phase approximation. The global analysis of the whole set of data indicates that WT α-syn is mostly present as unfolded monomers and trimers (helical-rich trimers at low T and strand-rich trimers at high T), but not tetramers, as previously derived by several studies. On the contrary, different conformer combinations characterize mutants. In the α-syn G51D mutant, the most abundant aggregates at all the temperatures are strand-rich tetramers. Strand-rich tetramers are also the predominant forms in the A53T mutant, but their weight decreases with temperature. Only monomeric conformers, with a preference for the ones with the smallest sizes, are present in the E46K mutant. The derived conformational behavior then suggests a different availability of species prone to aggregate, depending on mutation, temperature, and concentration and accounting for the different neurotoxicity of α-syn variants. Indeed, this approach may be of pivotal importance to describe conformational and aggregational properties of other IDPs.

A CFD model for predicting protein aggregation in low-pH virial inactivation for mAb production.

Significant amounts of soluble product aggregates were observed in the low-pH viral inactivation (VI) operation during an initial scale-up run for an immunoglobulin-G 4 (IgG4) monoclonal antibody (mAb IgG4-N1). Being earlier in development, a scale-down model did not exist, nor was it practical to use costly Protein A eluate (PAE) for testing the VI process at scale, thus, a computational fluid dynamics (CFD)-based high-molecular weight (HMW) prediction model was developed for troubleshooting and risk mitigation. It was previously reported that the IgG4-N1 molecules upon exposure to low pH tend to change into transient and partially unfolded monomers during VI acidification (i.e., VIA) and form aggregates after neutralization (i.e., VIN). Therefore, the CFD model reported here focuses on the VIA step. The model mimics the continuous addition of acid to PAE and tracks acid distribution during VIA. Based on the simulated low-pH zone (≤pH 3.3) profiles and PAE properties, the integrated low-pH zone (ILPZ) value was obtained to predict HMW level at the VI step. The simulations were performed to examine the operating parameters, such as agitation speed, acid addition rate, and protein concentration of PAE, of the pilot scale (50-200 L) runs. The conditions with predictions of no product aggregation risk were recommended to the real scale-up runs, resulted in 100% success rate of the consecutive 12 pilot-scale runs. This study demonstrated that the CFD-based HMW prediction model could be used as a tool to facilitate the scale up of the low-pH VI process directly from bench to pilot/production scale.

Muscle myosins form folded monomers, dimers, and tetramers during filament polymerization in vitro

Muscle contraction depends on the cyclical interaction of myosin and actin filaments. Therefore, it is important to understand the mechanisms of polymerization and depolymerization of muscle myosins. Muscle myosin 2 monomers exist in two states: one with a folded tail that interacts with the heads (10S) and one with an unfolded tail (6S). It has been thought that only unfolded monomers assemble into bipolar and side-polar (smooth muscle myosin) filaments. We now show by electron microscopy that, after 4 s of polymerization in vitro in both the presence (smooth muscle myosin) and absence of ATP, skeletal, cardiac, and smooth muscle myosins form tail-folded monomers without tail-head interaction, tail-folded antiparallel dimers, tail-folded antiparallel tetramers, unfolded bipolar tetramers, and small filaments. After 4 h, the myosins form thick bipolar and, for smooth muscle myosin, side-polar filaments. Nonphosphorylated smooth muscle myosin polymerizes in the presence of ATP but with a higher critical concentration than in the absence of ATP and forms only bipolar filaments with bare zones. Partial depolymerization in vitro of nonphosphorylated smooth muscle myosin filaments by the addition of MgATP is the reverse of polymerization.

A Characterization of Misfolded Alpha‐Synuclein in Parkinson’s Disease

The Moeller SMART Team in conjunction with Dr. JiaJie Diao at the University of Cincinnati Medical College and MSOE Center for Biomolecular Modeling used 3‐D modeling and printing technology to study the role of misfolded alpha‐synuclein proteins in Parkinson’s Disease (PD). Neurodegenerative diseases are all characterized by the aggregation of misfolded proteins that form an insoluble substrate. Misfolded alpha‐synuclein is thought to be the protein involved in PD. This protein is natively found as an unfolded monomer that can take on many different conformations. It consists of 140 amino acids and is divided into three regions: the N‐terminal region (residues 1–60), non‐amyloid beta component (NAC) (residues 61–95), and the C‐terminus (residues 96–140). Alpha‐synuclein shows the ability to form amyloid fibrils which are associated with toxicity. Subunits within the fibrils adopt a beta‐strand conformation with hydrogen bonding between adjacent beta strands to form long linear polymers. The N‐terminal and C‐terminal residues display a random coil arrangement that are not involved in hydrogen bonding. The central beta sheet core is comprised of an inner hydrophobic region that interlocks into compact right‐angled spirals. Each fibril can form a dimer with rotational symmetry about the bonding interface. Within the beta sheet region, the NAC and pre‐NAC regions are responsible for fibril formation. The NAC region is the central hydrophobic region that when removed from alpha‐synuclein leads to the inability of aggregation.

Tracking Oligomerization of Alpha-Synuclein Demonstrates Pivotal Role of Mitochondria in Seeding

Aggregation of α-synuclein, which normally exists as an unfolded monomer, in human brain contributes to the major pathogenesis of the synucleinopathies, Parkinson's disease and Lewybody dementia. However, the initial triggers to form early aggregates are unknown. Of note, visualizing the dynamic process of aggregation in neurons has been limited due to the inability to detect the early soluble toxic intermediates, oligomers, inside cells. Here we report a Foster resonance energy transfer (FRET) based method that enables the kinetics of intracellular oligomerization to be tracked in live cells. We first visualized that neurons uptake externally applied monomers and intracellularly form early and late oligomers, dependent on the initial monomer concentration. Then we utilized mutant α-synuclein carrying A53T point mutation that causes early onset progressive Parkinson's disease to investigate triggers of aggregation. We demonstrated that the A53T mutant monomer progressively oligomerizes due to the fast intracellular uptake than WT and other α-synuclein mutants. In addition, correlative light and electron microscopy (CLEM) allowed us to identify cellular regions in human neurons in which oligomerization is detected, such as mitochondria and lysosome. Importantly, A53T monomer produces excess reactive oxygen species (ROS) and opens mitochondria permeabilization pore (PTP) prior to cell death unlike the WT monomer. Importantly prevention of mitochondrial ROS inhibits the oligomerization of the A53T monomer and maintains the protein in its monomeric state, and this subsequently prevents cell death. In summary, our study provides evidence that mitochondria seed the aggregation of α-synuclein in neurons and mitochondrial stress in the form of enhanced ROS production is a major trigger for oligomerization.