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Spontaneous locomotion is a common feature of most metazoan cells, generally attributed to the properties of actomyosin networks. This force-producing machinery has been studied down to the most minute molecular details, especially in lamellipodium-driven migration. Nevertheless, how actomyosin networks work inside contraction-driven amoeboid cells still lacks unifying principles. Here, using stable motile blebs from HeLa cells as a model amoeboid motile system, we imaged the dynamics of the actin cortex at the single filament level and revealed the co-existence of three distinct rheological phases. We introduce "advected percolation," a process where rigidity percolation and active advection synergize, spatially organizing the actin network's mechanical properties into a minimal and generic locomotion mechanism. Expanding from our observations on simplified systems, we speculate that this model could explain, down to the single actin filament level, how amoeboid cells, such as cancer or immune cells, can propel efficiently through complex 3D environments., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The natural history of cancers can be understood through the lens of evolution given that the driving forces of cancer development are mutation and selection of fitter clones. Cancer growth and progression are spatial processes that involve the breakdown of normal tissue organization, invasion and metastasis. For these reasons, spatial patterns are an integral part of histological tumour grading and staging as they measure the progression from normal to malignant disease. Furthermore, tumour cells are part of an ecosystem of tumour cells and their surrounding tumour microenvironment. A range of new spatial genomic, transcriptomic and proteomic technologies offers new avenues for the study of cancer evolution with great molecular and spatial detail. These methods enable precise characterizations of the tumour microenvironment, cellular interactions therein and micro-anatomical structures. In conjunction with spatial genomics, it emerges that tumours and microenvironments co-evolve, which helps explain observable patterns of heterogeneity and offers new routes for therapeutic interventions., (© 2022. Springer Nature Limited.)

Genome sequencing of cancers often reveals mosaics of different subclones present in the same tumour 1-3 . Although these are believed to arise according to the principles of somatic evolution, the exact spatial growth patterns and underlying mechanisms remain elusive 4,5 . Here, to address this need, we developed a workflow that generates detailed quantitative maps of genetic subclone composition across whole-tumour sections. These provide the basis for studying clonal growth patterns, and the histological characteristics, microanatomy and microenvironmental composition of each clone. The approach rests on whole-genome sequencing, followed by highly multiplexed base-specific in situ sequencing, single-cell resolved transcriptomics and dedicated algorithms to link these layers. Applying the base-specific in situ sequencing workflow to eight tissue sections from two multifocal primary breast cancers revealed intricate subclonal growth patterns that were validated by microdissection. In a case of ductal carcinoma in situ, polyclonal neoplastic expansions occurred at the macroscopic scale but segregated within microanatomical structures. Across the stages of ductal carcinoma in situ, invasive cancer and lymph node metastasis, subclone territories are shown to exhibit distinct transcriptional and histological features and cellular microenvironments. These results provide examples of the benefits afforded by spatial genomics for deciphering the mechanisms underlying cancer evolution and microenvironmental ecology., (© 2022. The Author(s).)

Spatial transcriptomic technologies promise to resolve cellular wiring diagrams of tissues in health and disease, but comprehensive mapping of cell types in situ remains a challenge. Here we present сell2location, a Bayesian model that can resolve fine-grained cell types in spatial transcriptomic data and create comprehensive cellular maps of diverse tissues. Cell2location accounts for technical sources of variation and borrows statistical strength across locations, thereby enabling the integration of single-cell and spatial transcriptomics with higher sensitivity and resolution than existing tools. We assessed cell2location in three different tissues and show improved mapping of fine-grained cell types. In the mouse brain, we discovered fine regional astrocyte subtypes across the thalamus and hypothalamus. In the human lymph node, we spatially mapped a rare pre-germinal center B cell population. In the human gut, we resolved fine immune cell populations in lymphoid follicles. Collectively, our results present сell2location as a versatile analysis tool for mapping tissue architectures in a comprehensive manner., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Although textbook pictures depict the cell nucleus as a simple ovoid object, it is now clear that it adopts a large variety of shapes in tissues. When cells deform, because of cell crowding or migration through dense matrices, the nucleus is subjected to large constraints that alter its shape. In this review, we discuss recent studies related to nuclear fragility, focusing on the surprising finding that the nuclear envelope can form blebs. Contrary to the better-known plasma membrane blebs, nuclear blebs are unstable and almost systematically lead to nuclear envelope opening and uncontrolled nucleocytoplasmic mixing. They expand, burst, and repair repeatedly when the nucleus is strongly deformed. Although blebs are a major source of nuclear instability, they are poorly understood so far, which calls for more in-depth studies of these structures., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The WAVE regulatory complex (WRC) is crucial for assembly of the peripheral branched actin network constituting one of the main drivers of eukaryotic cell migration. Here, we uncover an essential role of the hematopoietic-specific WRC component HEM1 for immune cell development. Germline-encoded HEM1 deficiency underlies an inborn error of immunity with systemic autoimmunity, at cellular level marked by WRC destabilization, reduced filamentous actin, and failure to assemble lamellipodia. Hem1 -/- mice display systemic autoimmunity, phenocopying the human disease. In the absence of Hem1, B cells become deprived of extracellular stimuli necessary to maintain the strength of B cell receptor signaling at a level permissive for survival of non-autoreactive B cells. This shifts the balance of B cell fate choices toward autoreactive B cells and thus autoimmunity., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Alzheimer's disease (AD) is the most common form of dementia around the world (~ 65%). Here, we portray the neuropathology of AD, biomarkers, and classification of amyloid plaques (diffuse, non-cored, dense core, compact). Tau pathology and its involvement with Aβ plaques and cell death are discussed. Amyloid cascade hypotheses, aggregation mechanisms, and molecular species formed in vitro and in vivo (on- and off-pathways) are described. Aβ42/Aβ40 monomers, dimers, trimers, Aβ-derived diffusible ligands, globulomers, dodecamers, amylospheroids, amorphous aggregates, protofibrils, fibrils, and plaques are characterized (structure, size, morphology, solubility, toxicity, mechanistic steps). An update on AD-approved drugs by regulatory agencies, along with new Aβ-based therapies, is presented. Beyond prescribing Aβ plaque disruptors, cholinergic agonists, or NMDA receptor antagonists, other therapeutic strategies (RNAi, glutaminyl cyclase inhibitors, monoclonal antibodies, secretase modulators, Aβ aggregation inhibitors, and anti-amyloid vaccines) are already under clinical trials. New drug discovery approaches based on "designed multiple ligands", "hybrid molecules", or "multitarget-directed ligands" are also being put forward and may contribute to tackling this highly debilitating and fatal form of human dementia., (© 2024. The Author(s).)

Nuclear pore complexes tightly regulate nucleo-cytoplasmic transport, controlling the nuclear concentration of several transcription factors. In a recent issue of Cell, Elosegui-Artola et al. (2017) show that nuclear deformation modulates the nuclear entry rates of YAP/TAZ via nuclear pore stretching, clarifying how forces affect gene transcription., (Copyright © 2017. Published by Elsevier Inc.)

Oligomeric peptides exist widely in living organisms and play a role in a broad range of biological functions. We report the first observation of liquid-liquid phase separation (LLPS) in peptide solutions, in particular, solutions of peptides consisting of noncovalent oligomers. We determined the binary phase boundary of the oligomeric peptide solution and compared the result to the well-established phase diagram of globular proteins. We also provide simple theoretical interpretations of the similarities and differences between the phase diagrams of peptides and proteins. Finally, by tuning inter-oligomer interactions using a crowding agent, we demonstrated that LLPS is a universal phenomenon that can be observed under different solution conditions for a variety of peptides.

Phase transformation in antibody solutions is of growing interest in both academia and the pharmaceutical industry. Recent experimental studies have shown that, as in near-spherical proteins, antibodies can undergo a liquid-liquid phase separation under conditions metastable with respect to crystallization. However, the phase diagram of the Y-shaped antibodies exhibits unique features that differ substantially from those of spherical proteins. Specifically, antibody solutions have an exceptionally low critical volume fraction (CVF) and a broader and more asymmetric liquid-liquid coexistence curve than those of spherical proteins. Using molecular dynamics simulation on a series of trimetric Y-shaped coarse-grained models, we investigate the phase behavior of antibody solutions and compare the results with the experimental phase diagram of human immunoglobulin G (IgG), one of the most common Y-shape typical of antibody molecules. With the fitted size of spheres, our simulation reproduces both the low CVF and the asymmetric shape of the experimental coexistence curve of IgG antibodies. The broadness of the coexistence curve can be attributed to the anisotropic nature of the inter-protein interaction. In addition, the repulsion between the inner parts of the spherical domains of IgG dramatically expands the coexistence region in the scaled phase diagram, while the hinge length has only a minor effect on the CVF and the overall shape of the coexistence curve. We thus propose a seven-site model with empirical parameters characterizing the exclusion volume and the hinge length of the IgG molecules, which provides a base for simulation studies of the phase behavior of IgG antibodies.

Colloidal stability of IgG antibody solutions is important for pharmaceutical and medicinal applications. Solution pH and ionic strength are two key factors that affect the colloidal stability of protein solutions. In this work, we use a method based on the PEG-induced liquid-liquid phase separation to examine the effects of pH and ionic strength on the colloidal stability of IgG solutions. We found that at high ionic strength (≥0.25M), the colloidal stability of most of our IgGs is insensitive to pH, and at low ionic strength (≤0.15M), all IgG solutions are much more stable at pH 5 than at pH 7. In addition, the PEG-induced depletion force is less efficient in causing phase separation at pH 5 than at pH 7. In contrast to the native inter-protein interaction of IgGs, the effect of depletion force on phase separation of the antibody solutions is insensitive to ionic strength. Our results suggest that the long-range electrostatic inter-protein repulsion at low ionic strength stabilizes the IgG solutions at low pH. At high ionic strength, the short-range electrostatic interactions do not make a significant contribution to the colloidal stability for most IgGs with a few exceptions. The weaker effect of depletion force at lower pH indicates a reduction of protein concentration in the condensed phase. This work advances our basic understanding of the colloidal stability of IgG solutions and also introduces a practical approach to measuring protein colloidal stability under various solution conditions.

Hybrid incretin peptides are a new generation of drugs for the treatment of diabetes and obesity. Despite their biological potency, the effectiveness of these peptides as drugs is limited by their short circulation time in blood (typically within minutes). In this work, we show that lipid conjugated forms of a GLP-1/GIP/glucagon hybrid peptides stay in circulation for hours. We studied the oligomerization and albumin-binding of the unconjugated hybrid peptide as well as its lipidated variants. These lipidated peptides differ in the N-terminal mutation, the position of lipidation and the linkage to lipid. We found that these lipidated peptides form stable oligomers at concentrations above 1mg/mL. This concentration range is relevant to formulation and storage of the peptides. We observed no binding between the peptide oligomers and human serum albumin. However, at the expected therapeutic concentration range (~10-100ng/mL), the oligomers dissociate into monomers. The monomers of lipidated peptides bind to albumin. We have determined the dissociation constants of binding between the lipidated peptides and serum albumin. The dissociation constants of albumin-binding of our lipidated peptides are all very close and similar to that of the fatty acid binding of albumin. Our findings suggest that the monomeric lipidated peptides bind to HSA mainly by the fatty acid chain. Therefore, albumin binding is likely to be a universal mechanism of the prolonged circulating duration of lipidated pharmaceutical peptides., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A variety of species express the amyloid β-protein (Aβ (the term "Aβ" refers both to Aβ40 and Aβ42, whereas "Aβ40" and "Aβ42" refer to each isoform specifically). Those species expressing Aβ with primary structure identical to that expressed in humans have been found to develop amyloid deposits and Alzheimer's disease-like neuropathology. In contrast, the Aβ sequence in mice and rats contains three amino acid substitutions, Arg5Gly, His13Arg, and Tyr10Phe, which apparently prevent the development of AD-like neuropathology. Interestingly, the brush-tailed rat, Octodon degus, expresses Aβ containing only one of these substitutions, His13Arg, and does develop AD-like pathology. We investigate here the biophysical and biological properties of Aβ peptides from humans, mice (Mus musculus), and rats (Octodon degus). We find that each peptide displays statistical coil → β-sheet secondary structure transitions, transitory formation of hydrophobic surfaces, oligomerization, formation of annuli, protofibrils, and fibrils, and an inverse correlation between rate of aggregation and aggregate size (faster aggregation produced smaller aggregates). The rank order of assembly rate was mouse > rat > Aβ42. The rank order of neurotoxicity of assemblies formed by each peptide immediately after preparation was Aβ42 > mouse ≈ rat. These data do not support long-standing hypotheses that the primary factor controlling development of AD-like neuropathology in rodents is Aβ sequence. Instead, the data support a hypothesis that assembly quaternary structure and organismal responses to toxic peptide assemblies mediate neuropathogenetic effects. The implication of this hypothesis is that a valid understanding of disease causation within a given system (organism, tissue, etc.) requires the coevaluation of both biophysical and cell biological properties of that system.

Oligomerization of lipidated peptides is of general scientific interest and is important in biomedical and pharmaceutical applications. We investigated the solution properties of a lipidated peptide, Liraglutide, which is one of the glucagon-like peptide-1 (GLP-1) agonists used for the treatment of type II diabetes. Liraglutide can serve as a model system for studying biophysical and biochemical properties of micelle-like self-assemblies of the lipidated peptides. Here, we report a transformation induced in Liraglutide oligomers by changing pH in the vicinity of pH 7. This fully reversible transformation is characterized by changes in the size and aggregation number of the oligomer and an associated change in the secondary structure of the constituent peptides. This transformation has quite slow kinetics: the equilibrium is reached in a course of several days. Interestingly, while the transformation is induced by changing pH, its kinetics is essentially independent of the final pH. We interpreted these findings using a model in which desorption of the monomer from the oligomer is the rate-limiting step in the transformation, and we determined the rate constant of the monomer desorption.

The early detection of lung cancer has the potential to greatly impact disease burden through the timely identification and treatment of affected individuals at a manageable stage of development. The insufficient specificity demonstrated by currently used screening and diagnostic techniques has led to intense investigation into biomarkers as diagnostic tools. Urine may represent a noninvasive alternative matrix for diagnostic biomarker development. We performed an analysis of 242 biomarkers in urines obtained from 83 patients with non-small cell lung carcinomas (NSCLC), 74 patients diagnosed with benign pulmonary conditions, and 77 healthy donors. A large number of significant alterations were observed between the NSCLC and control groups. A multivariate analysis identified a three-biomarker panel consisting of IGFBP-1, sIL-1Ra, CEACAM-1, which discriminated NSCLC from healthy controls with a sensitivity/specificity of 84/95 in an initial training set and 72/100 in an independent validation set. This panel performed well among multiple subtypes of NSCLC and early-stage disease but demonstrated only limited efficacy for the discrimination of NSCLC from benign controls and limited specificity for patients with several other cancers and tuberculosis. These findings demonstrate that urine biomarkers may provide screening and diagnostic properties which exceed those reported for serum biomarkers and approach a level necessary for further clinical development., (©2014 American Association for Cancer Research.)

Dimers of Aβ (amyloid β-protein) are believed to play an important role in Alzheimer's disease. In the absence of sufficient brain-derived dimers, we studied one of the only possible dimers that could be produced in vivo, [Aβ](DiY) (dityrosine cross-linked Aβ). For comparison, we used the Aβ monomer and a design dimer cross-linked by replacement of Ser²⁶ with cystine [AβS26C]₂. We showed that similar to monomers, unaggregated dimers lack appreciable structure and fail to alter long-term potentiation. Importantly, dimers exhibit subtly different structural propensities from monomers and each other, and can self-associate to form larger assemblies. Although [Aβ](DiY) and [AβS26C]₂ have distinct aggregation pathways, they both populate bioactive soluble assemblies for longer durations than Aβ monomers. Our results indicate that the link between Aβ dimers and Alzheimer's disease results from the ability of dimers to further assemble and form synaptotoxic assemblies that persist for long periods of time.

One of the earliest events in amyloid β-protein (Aβ) self-association is nucleation of Aβ monomer folding through formation of a turn at Gly25-Lys28. We report here the effects of structural changes at the center of the turn, Gly25-Ser26, on Aβ42 conformational dynamics and assembly. We used "click peptide" chemistry to quasi-synchronously create Aβ42 from 26-O-acyliso-Aβ42 (iAβ42) through a pH jump from 3 to 7.4. We also synthesized Nα-acetyl-Ser26-iAβ42 (Ac-iAβ42), which cannot undergo O→N acyl chemistry, to study the behavior of this ester form of Aβ42 itself at neutral pH. Data from experiments monitoring increases in β-sheet formation (thioflavin T, CD), hydrodynamic radius (RH), scattering intensity (quasielastic light scattering spectroscopy), and extent of oligomerization (ion mobility spectroscopy-mass spectrometry) were quite consistent. A rank order of Ac-iAβ42>iAβ42>Aβ42 was observed. Photochemically cross-linked iAβ42 displayed an oligomer distribution with a prominent dimer band that was not present with Aβ42. These dimers also were observed selectively in iAβ42 in ion mobility spectrometry experiments. The distinct biophysical behaviors of iAβ42 and Aβ42 appear to be due to the conversion of iAβ42 into "pure" Aβ42 monomer, a nascent form of Aβ42 that does not comprise the variety of oligomeric and aggregated states present in pre-existent Aβ42. These results emphasize the importance of the Gly25-Ser26 dipeptide in organizing Aβ42 monomer structure and thus suggest that drugs altering the interactions of this dipeptide with neighboring side-chain atoms or with the peptide backbone could be useful in therapeutic strategies targeting formation of Aβ oligomers and higher-order assemblies., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Colloidal stability of antibody solutions, i.e., the propensity of the folded protein to precipitate, is an important consideration in formulation development of therapeutic monoclonal antibodies. In a protein solution, different pathways including crystallization, colloidal aggregation, and liquid-liquid phase separation (LLPS) can lead to the formation of precipitates. The kinetics of crystallization and aggregation are often slow and vary from protein to protein. Due to the diverse mechanisms of these protein condensation processes, it is a challenge to develop a standardized test for an early evaluation of the colloidal stability of antibody solutions. LLPS would normally occur in antibody solutions at sufficiently low temperature, provided that it is not preempted by freezing of the solution. Poly(ethylene glycol) (PEG) can be used to induce LLPS at temperatures above the freezing point. Here, we propose a colloidal stability test based on inducing LLPS in antibody solutions and measuring the antibody concentration of the dilute phase. We demonstrate experimentally that such a PEG-induced LLPS test can be used to compare colloidal stability of different antibodies in different solution conditions and can be readily applied to high-throughput screening. We have derived an equation for the effects of PEG concentration and molecular weight on the results of the LLPS test. Finally, this equation defines a binding energy in the condensed phase, which can be determined in the PEG-induced LLPS test. This binding energy is a measure of attractive interactions between antibody molecules and can be used for quantitative characterization of the colloidal stability of antibody solutions.

Background: The clinical management of pancreatic cancer is severely hampered by the absence of effective screening tools., Methods: Sixty-seven biomarkers were evaluated in prediagnostic sera obtained from cases of pancreatic cancer enrolled in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO)., Results: The panel of CA 19-9, OPN, and OPG, identified in a prior retrospective study, was not effective. CA 19-9, CEA, NSE, bHCG, CEACAM1 and PRL were significantly altered in sera obtained from cases greater than 1 year prior to diagnosis. Levels of CA 19-9, CA 125, CEA, PRL, and IL-8 were negatively associated with time to diagnosis. A training/validation study using alternate halves of the PLCO set failed to identify a biomarker panel with significantly improved performance over CA 19-9 alone. When the entire PLCO set was used for training at a specificity (SP) of 95%, a panel of CA 19-9, CEA, and Cyfra 21-1 provided significantly elevated sensitivity (SN) levels of 32.4% and 29.7% in samples collected <1 and >1 year prior to diagnosis, respectively, compared to SN levels of 25.7% and 17.2% for CA 19-9 alone., Conclusions: Most biomarkers identified in previously conducted case/control studies are ineffective in prediagnostic samples, however several biomarkers were identified as significantly altered up to 35 months prior to diagnosis. Two newly derived biomarker combinations offered advantage over CA 19-9 alone in terms of SN, particularly in samples collected >1 year prior to diagnosis. However, the efficacy of biomarker-based tools remains limited at present. Several biomarkers demonstrated significant velocity related to time to diagnosis, an observation which may offer considerable potential for enhancements in early detection.

Protein condensations, such as crystallization, liquid-liquid phase separation, aggregation, and gelation, have been observed in concentrated antibody solutions under various solution conditions. While most IgG antibodies are quite soluble, a few outliers can undergo condensation under physiological conditions. Condensation of IgGs can cause serious consequences in some human diseases and in biopharmaceutical formulations. The phase transitions underlying protein condensations in concentrated IgG solutions is also of fundamental interest for the understanding of the phase behavior of non-spherical protein molecules. Due to the high solubility of generic IgGs, the phase behavior of IgG solutions has not yet been well studied. In this work, we present an experimental approach to study IgG solutions in which the phase transitions are hidden below the freezing point of the solution. Using this method, we have investigated liquid-liquid phase separation of six human myeloma IgGs and two recombinant pharmaceutical human IgGs. We have also studied the relation between crystallization and liquid-liquid phase separation of two human cryoglobulin IgGs. Our experimental results reveal several important features of the generic phase behavior of IgG solutions: (1) the shape of the coexistence curve is similar for all IgGs but quite different from that of quasi-spherical proteins; (2) all IgGs have critical points located at roughly the same protein concentration at ~100 mg/ml while their critical temperatures vary significantly; and (3) the liquid-liquid phase separation in IgG solutions is metastable with respect to crystallization. These features of phase behavior of IgG solutions reflect the fact that all IgGs have nearly identical molecular geometry but quite diverse net inter-protein interaction energies. This work provides a foundation for further experimental and theoretical studies of the phase behavior of generic IgGs as well as outliers with large propensity to condense. The investigation of the phase diagram of IgG solutions is of great importance for the understanding of immunoglobulin deposition diseases as well as for the understanding of the colloidal stability of IgG pharmaceutical formulations.

Oligomerization of the amyloid β-protein (Aβ) is a seminal event in Alzheimer's disease. Aβ42, which is only two amino acids longer than Aβ40, is particularly pathogenic. Why this is so has not been elucidated fully. We report here results of computational and experimental studies revealing a C-terminal turn at Val36-Gly37 in Aβ42 that is not present in Aβ40. The dihedral angles of residues 36 and 37 in an Ile31-Ala42 peptide were consistent with β-turns, and a β-hairpin-like structure was indeed observed that was stabilized by hydrogen bonds and by hydrophobic interactions between residues 31-35 and residues 38-42. In contrast, Aβ(31-40) mainly existed as a statistical coil. To study the system experimentally, we chemically synthesized Aβ peptides containing amino acid substitutions designed to stabilize or destabilize the hairpin. The triple substitution Gly33Val-Val36Pro-Gly38Val ("VPV") facilitated Aβ42 hexamer and nonamer formation, while inhibiting formation of classical amyloid-type fibrils. These assemblies were as toxic as were assemblies from wild-type Aβ42. When substituted into Aβ40, the VPV substitution caused the peptide to oligomerize similarly to Aβ42. The modified Aβ40 was significantly more toxic than Aβ40. The double substitution d-Pro36-l-Pro37 abolished hexamer and dodecamer formation by Aβ42 and produced an oligomer size distribution similar to that of Aβ40. Our data suggest that the Val36-Gly37 turn could be the sine qua non of Aβ42. If true, this structure would be an exceptionally important therapeutic target., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Elevated cerebrospinal fluid concentrations of tau discriminate Alzheimer's disease from other neurodegenerative conditions. The reasons for this are unclear. While commercial assay kits are widely used to determine total-tau concentrations, little is known about their ability to detect different aggregation states of tau. We demonstrate that the leading commercial enzyme-linked immunosorbent assay reliably detects aggregated and monomeric tau and evinces good recovery of both species when added into cerebrospinal fluid. Hence, the disparity between total-tau levels encountered in Alzheimer's disease and other neurodegenerative conditions is not due to differential recognition of tau assembly forms or the extent of degeneration.

Background: More than a million diagnostic cardiac catheterizations are performed annually in the US for evaluation of coronary artery anatomy and the presence of atherosclerosis. Nearly half of these patients have no significant coronary lesions or do not require mechanical or surgical revascularization. Consequently, the ability to rule out clinically significant coronary artery disease (CAD) using low cost, low risk tests of serum biomarkers in even a small percentage of patients with normal coronary arteries could be highly beneficial., Methods: Serum from 359 symptomatic subjects referred for catheterization was interrogated for proteins involved in atherogenesis, atherosclerosis, and plaque vulnerability. Coronary angiography classified 150 patients without flow-limiting CAD who did not require percutaneous intervention (PCI) while 209 required coronary revascularization (stents, angioplasty, or coronary artery bypass graft surgery). Continuous variables were compared across the two patient groups for each analyte including calculation of false discovery rate (FDR ≤ 1%) and Q value (P value for statistical significance adjusted to ≤ 0.01)., Results: Significant differences were detected in circulating proteins from patients requiring revascularization including increased apolipoprotein B100 (APO-B100), C-reactive protein (CRP), fibrinogen, vascular cell adhesion molecule 1 (VCAM-1), myeloperoxidase (MPO), resistin, osteopontin, interleukin (IL)-1β, IL-6, IL-10 and N-terminal fragment protein precursor brain natriuretic peptide (NT-pBNP) and decreased apolipoprotein A1 (APO-A1). Biomarker classification signatures comprising up to 5 analytes were identified using a tunable scoring function trained against 239 samples and validated with 120 additional samples. A total of 14 overlapping signatures classified patients without significant coronary disease (38% to 59% specificity) while maintaining 95% sensitivity for patients requiring revascularization. Osteopontin (14 times) and resistin (10 times) were most frequently represented among these diagnostic signatures. The most efficacious protein signature in validation studies comprised osteopontin (OPN), resistin, matrix metalloproteinase 7 (MMP7) and interferon γ (IFNγ) as a four-marker panel while the addition of either CRP or adiponectin (ACRP-30) yielded comparable results in five protein signatures., Conclusions: Proteins in the serum of CAD patients predominantly reflected (1) a positive acute phase, inflammatory response and (2) alterations in lipid metabolism, transport, peroxidation and accumulation. There were surprisingly few indicators of growth factor activation or extracellular matrix remodeling in the serum of CAD patients except for elevated OPN. These data suggest that many symptomatic patients without significant CAD could be identified by a targeted multiplex serum protein test without cardiac catheterization thereby eliminating exposure to ionizing radiation and decreasing the economic burden of angiographic testing for these patients.

Condensation of Igs has been observed in pharmaceutical formulations and in vivo in cases of cryoglobulinemia. We report a study of monoclonal IgG cryoglobulins overexpressed by two patients with multiple myeloma. These cryoglobulins form crystals, and we measured their solubility lines. Depending on the supersaturation, we observed a variety of condensate morphologies consistent with those reported in clinical investigations. Remarkably, the crystallization can occur at quite low concentrations. This suggests that, even within the regular immune response to infections, cryoprecipitation of Ig can be possible.

Quasielastic light scattering (QLS) spectroscopy is a noninvasive optical method for studying the dynamic properties of macromolecular solutions. Its most important application is the determination of diffusion coefficients, from which the sizes of particles in solution may be estimated. The technique thus is particularly useful for monitoring assembly (polymerization and aggregation) reactions without the need for removing aliquots from the assembly system or disrupting the assembly process in any other way. We discuss here two of the most important aspects of QLS: (1) measurement of the correlation function of the scattered light intensity and (2) the use of this correlation function to reconstruct the distribution of sizes of the scattering particles. The ability to monitor the temporal evolution of particle size provides a powerful tool for studying protein assembly. We illustrate here how QLS has been applied to elucidate features of the oligomerization and fibrillogenesis of the amyloid β-protein, Aβ, thought to be the causative agent of Alzheimer's disease.

Amyloidoses are diseases characterized by abnormal protein folding and self-assembly, for which no cure is available. Inhibition or modulation of abnormal protein self-assembly, therefore, is an attractive strategy for prevention and treatment of amyloidoses. We examined Lys-specific molecular tweezers and discovered a lead compound termed CLR01, which is capable of inhibiting the aggregation and toxicity of multiple amyloidogenic proteins by binding to Lys residues and disrupting hydrophobic and electrostatic interactions important for nucleation, oligomerization, and fibril elongation. Importantly, CLR01 shows no toxicity at concentrations substantially higher than those needed for inhibition. We used amyloid β-protein (Aβ) to further explore the binding site(s) of CLR01 and the impact of its binding on the assembly process. Mass spectrometry and solution-state NMR demonstrated binding of CLR01 to the Lys residues in Aβ at the earliest stages of assembly. The resulting complexes were indistinguishable in size and morphology from Aβ oligomers but were nontoxic and were not recognized by the oligomer-specific antibody A11. Thus, CLR01 binds already at the monomer stage and modulates the assembly reaction into formation of nontoxic structures. The data suggest that molecular tweezers are unique, process-specific inhibitors of aberrant protein aggregation and toxicity, which hold promise for developing disease-modifying therapy for amyloidoses.

We report the observation of liquid-liquid phase separation in a solution of human monoclonal antibody, IgG2, and the effects of human serum albumin, a major blood protein, on this phase separation. We find a significant reduction of phase separation temperature in the presence of albumin, and a preferential partitioning of the albumin into the antibody-rich phase. We provide a general thermodynamic analysis of the antibody-albumin mixture phase diagram and relate its features to the magnitude of the effective interprotein interactions. Our analysis suggests that additives (HSA in this report), which have moderate attraction with antibody molecules, may be used to forestall undesirable protein condensation in antibody solutions. Our findings are relevant to understanding the stability of pharmaceutical solutions of antibodies and the mechanisms of cryoglobulinemia.

A panel of biomarkers may improve predictive performance over individual markers. Although many biomarker panels have been described for ovarian cancer, few studies used prediagnostic samples to assess the potential of the panels for early detection. We conducted a multisite systematic evaluation of biomarker panels using prediagnostic serum samples from the Prostate, Lung, Colorectal, and Ovarian Cancer (PLCO) screening trial. Using a nested case-control design, levels of 28 biomarkers were measured laboratory-blinded in 118 serum samples obtained before cancer diagnosis and 951 serum samples from matched controls. Five predictive models, each containing 6 to 8 biomarkers, were evaluated according to a predetermined analysis plan. Three sequential analyses were conducted: blinded validation of previously established models (step 1); simultaneous split-sample discovery and validation of models (step 2); and exploratory discovery of new models (step 3). Sensitivity, specificity, sensitivity at 98% specificity, and AUC were computed for the models and CA125 alone among 67 cases diagnosed within one year of blood draw and 476 matched controls. In step 1, one model showed comparable performance to CA125, with sensitivity, specificity, and AUC at 69.2%, 96.6%, and 0.892, respectively. Remaining models had poorer performance than CA125 alone. In step 2, we observed a similar pattern. In step 3, a model derived from all 28 markers failed to show improvement over CA125. Thus, biomarker panels discovered in diagnostic samples may not validate in prediagnostic samples; utilizing prediagnostic samples for discovery may be helpful in developing validated early detection panels.

Background: Computed tomography (CT) scanning has emerged as an effective means of early detection for lung cancer. Despite marked improvement over earlier methodologies, the low level of specificity demonstrated by CT scanning has limited its clinical implementation as a screening tool. A minimally-invasive biomarker-based test that could further characterize CT-positive patients based on risk of malignancy would greatly enhance its clinical efficacy., Methods: We performed an analysis of 81 serum proteins in 92 patients diagnosed with lung cancer and 172 CT-screened control individuals. We utilize a series of bioinformatics algorithms including Metropolis-Monte Carlo, artificial neural networks, Naïve Bayes, and additive logistic regression to identify multimarker panels capable of discriminating cases from controls with high levels of sensitivity and specificity in distinct training and independent validation sets., Results: A three-biomarker panel comprised of MIF, prolactin, and thrombospondin identified using the Metropolis-Monte Carlo algorithm provided the best classification with a %Sensitivity/Specificity/Accuracy of 74/90/86 in the training set and 70/93/82 in the validation set. This panel was effective in the classification of control individuals demonstrating suspicious pulmonary nodules and stage I lung cancer patients., Conclusions: The selected serum biomarker panel demonstrated a high diagnostic utility in the current study and performance characteristics which compare favorably with previous reports. Further advancements may lead to the development of a diagnostic tool useful as an adjunct to CT-scanning.

Oligomeric forms of amyloid beta-protein (Abeta) are key neurotoxins in Alzheimer's disease (AD). Previously, we found that C-terminal fragments (CTFs) of Abeta42 interfered with assembly of full-length Abeta42 and inhibited Abeta42-induced toxicity. To decipher the mechanism(s) by which CTFs affect Abeta42 assembly and neurotoxicity, here, we investigated the interaction between Abeta42 and CTFs using photoinduced cross-linking and dynamic light scattering. The results demonstrate that distinct parameters control CTF inhibition of Abeta42 assembly and Abeta42-induced toxicity. Inhibition of Abeta42-induced toxicity was found to correlate with stabilization of oligomers with a hydrodynamic radius (R(H)) of 8-12 nm and attenuation of formation of oligomers with an R(H) of 20-60 nm. In contrast, inhibition of Abeta42 paranucleus formation correlated with CTF solubility and the degree to which CTFs formed amyloid fibrils themselves but did not correlate with inhibition of Abeta42-induced toxicity. Our findings provide important insight into the mechanisms by which different CTFs inhibit the toxic effect of Abeta42 and suggest that stabilization of nontoxic Abeta42 oligomers is a promising strategy for designing inhibitors of Abeta42 neurotoxicity.

We have experimentally determined the coexistence surface characterizing the phase behavior of gammaD-betaB1-water ternary solutions. The coexistence surface fully describes the solution conditions, i.e., temperature, protein concentration, and protein composition, at which liquid-liquid phase separation occurs in a ternary solution. We have observed a significant demixing of gammaD and betaB1 i.e., large difference of composition in the two coexisting phases. This demixing suggests that the energy of the gammaD-betaB1 attractive interaction is significantly smaller than the energy of the gammaD-gammaD attractive interaction. We also observed the lowering of the phase separation temperature upon increasing of the fraction of betaB1 in solution. We provide a theoretical analysis of our experimental data, which enables a quantitative description of our principal experimental findings. In this way, we have evaluated the magnitude and temperature dependence of the relevant interprotein interaction energies. Our findings provide insight into the factors essential for maintaining lens proteins in a single homogeneous phase, thereby enabling lens transparency.

Objectives: The diagnosis of an adnexal mass is a prevalent issue among women in the United States, although current methods of identifying those at high risk of malignancy remain insufficient. Ineffective triage of women with malignant masses is associated with delayed or inappropriate treatment and a negative effect on disease outcome., Methods: We performed an evaluation of 65 ovarian cancer-related biomarkers in the circulation of women diagnosed with an adnexal mass. Our subject group consisted of women diagnosed with benign masses and early- and late-stage ovarian cancer., Results: More than half of the biomarkers tested were found to differ significantly between benign and malignant cases. As individual markers, HE4 and CA-125 provided the greatest level of discrimination between benign and malignant cases, and the combination of these two biomarkers provided a higher level of discriminatory power than either marker considered alone. Multivariate statistical analysis identified several multimarker panels that could discriminate early-stage, late-stage, and combined ovarian cancers from benign cases with similar or slightly improved SN/SP levels to the CA-125/HE4 combination; however, these larger panels could not outperform the 2-biomarker panel in an independent validation set. We also identified a 3-biomarker panel with particular utility in premenopausal women., Conclusions: Our findings serve to advance the development of blood-based screening methods for the discrimination of benign and malignant ovarian masses by confirming and expanding upon the superior utility of the CA-125/HE4 combination., (Copyright 2010 Elsevier Inc. All rights reserved.)

An important component of the pathologic process underlying Alzheimer's disease is oxidative stress. Met(35) in amyloid beta-protein (A beta) is prone to participating in redox reactions promoting oxidative stress, and therefore is believed to contribute significantly A beta-induced toxicity. Thus, substitution of Met(35) by residues that do not participate in redox chemistry would be expected to decrease A beta toxicity. Indeed, substitution of Met(35) by norleucine (Nle) was reported to reduce A beta toxicity. Surprisingly, however, substitution of Met(35) by Val was reported to increase toxicity. A beta toxicity is known to be strongly related to its self-assembly. However, neither substitution is predicted to affect A beta assembly substantially. Thus, the effect of these substitutions on toxicity is difficult to explain. We revisited this issue and compared A beta 40 and A beta 42 with analogs containing Met(35)-->Nle or Met(35)-->Val substitutions using multiple biophysical and toxicity assays. We found that substitution of Met(35) by Nle or Val had moderate effects on A beta assembly. Surprisingly, despite these effects, neither substitution changed A beta neurotoxicity significantly in three different assays. These results suggest that the presence of Met(35) in A beta is not important for A beta toxicity, challenging to the prevailing paradigm, which suggests that redox reactions involving Met(35) contribute substantially to A beta-induced toxicity.

Purpose: Early detection of ovarian cancer has great promise to improve clinical outcome., Patients and Methods: Ninety-six serum biomarkers were analyzed in sera from healthy women and from patients with ovarian cancer, benign pelvic tumors, and breast, colorectal, and lung cancers, using multiplex xMAP bead-based immunoassays. A Metropolis algorithm with Monte Carlo simulation (MMC) was used for analysis of the data., Results: A training set, including sera from 139 patients with early-stage ovarian cancer, 149 patients with late-stage ovarian cancer, and 1,102 healthy women, was analyzed with MMC algorithm and cross validation to identify an optimal biomarker panel discriminating early-stage cancer from healthy controls. The four-biomarker panel providing the highest diagnostic power of 86% sensitivity (SN) for early-stage and 93% SN for late-stage ovarian cancer at 98% specificity (SP) was comprised of CA-125, HE4, CEA, and VCAM-1. This model was applied to an independent blinded validation set consisting of sera from 44 patients with early-stage ovarian cancer, 124 patients with late-stage ovarian cancer, and 929 healthy women, providing unbiased estimates of 86% SN for stage I and II and 95% SN for stage III and IV disease at 98% SP. This panel was selective for ovarian cancer showing SN of 33% for benign pelvic disease, SN of 6% for breast cancer, SN of 0% for colorectal cancer, and SN of 36% for lung cancer., Conclusion: A panel of CA-125, HE4, CEA, and VCAM-1, after additional validation, could serve as an initial stage in a screening strategy for epithelial ovarian cancer.

Using quantitative phase microscopy, we have discovered a quadratic relationship between the radius R and the thickness t of helical ribbons that form spontaneously in multicomponent cholesterol-surfactant mixtures. These helical ribbons may serve as mesoscopic springs to measure or to exert forces on nanoscale biological objects. The spring constants of these helices depend on their submicroscopic thickness. The quadratic relationship (R proportional to t(2)) between radius and thickness is a consequence of the crystal structure of the ribbons and enables a determination of the spring constant of any of our helices solely in terms of its observable geometrical dimensions.

Aggregation and accumulation of amyloid-β peptide (Aβ) are a critical trigger for the onset of Alzheimer's disease (AD). While the plaques are the most outstanding Aβ pathological feature, much of the recent research emphasis has been on soluble Aβ species because of their diffusible, proinflammatory, and toxic properties. The focus on soluble aggregated Aβ species has also increased the interest in antibodies that are selective for different Aβ conformations. In the current study, we developed and characterized a new class of monoclonal antibodies (referred to as mAbSL) that are selective for Aβ protofibrils. Cloning and sequencing of the heavy and light chain variable regions for multiple antibodies identified sequence characteristics that may impart the conformational selectivity by the antibodies. Transfection of FreeStyle 293F cells with the plasmids permitted in-house expression and purification of mAbSL antibodies along with non-conformation-selective Aβ monoclonal antibodies (Aβ mAbs). Several of the purified mAbSL antibodies demonstrated significant affinity and selectivity for Aβ42 protofibrils compared with Aβ42 monomers and Aβ42 fibrils. Competition ELISA assays assessing the best overall antibody, mAbSL 113, yielded affinity constants of 7 nM for the antibody-Aβ42 protofibril interaction, while the affinity for either Aβ42 monomers or Aβ42 fibrils was roughly 80 times higher. mAbSL 113 significantly inhibited Aβ42 monomer aggregation by a unique mechanism compared with the inhibition displayed by Aβ mAb 513. Aβ42 protofibril dynamics were also markedly altered in the presence of mAbSL 113, whereby insoluble complex formation and protofibril deposition were stimulated by the antibody at low substoichiometric molar ratios. As the field contemplates the therapeutic effectiveness of Aβ conformation-selective antibodies, the findings presented here demonstrate new information on a monoclonal antibody that selectively targets Aβ protofibrils and impacts Aβ dynamics., (© 2023 International Society for Neurochemistry.)

Alzheimer's disease (AD) is an age-related disorder that threatens to become an epidemic as the world population ages. Neurotoxic oligomers of Abeta42 are believed to be the main cause of AD; therefore, disruption of Abeta oligomerization is a promising approach for developing therapeutics for AD. Formation of Abeta42 oligomers is mediated by intermolecular interactions in which the C terminus plays a central role. We hypothesized that peptides derived from the C terminus of Abeta42 may get incorporated into oligomers of Abeta42, disrupt their structure, and thereby inhibit their toxicity. We tested this hypothesis using Abeta fragments with the general formula Abeta(x-42) (x = 28-39). A cell viability screen identified Abeta(31-42) as the most potent inhibitor. In addition, the shortest peptide, Abeta(39-42), also had high activity. Both Abeta(31-42) and Abeta(39-42) inhibited Abeta-induced cell death and rescued disruption of synaptic activity by Abeta42 oligomers at micromolar concentrations. Biophysical characterization indicated that the action of these peptides likely involved stabilization of Abeta42 in nontoxic oligomers. Computer simulations suggested a mechanism by which the fragments coassembled with Abeta42 to form heterooligomers. Thus, Abeta(31-42) and Abeta(39-42) are leads for obtaining mechanism-based drugs for treatment of AD using a systematic structure-activity approach.

The P23T mutant of human gammaD-crystallin (HGD) is associated with cataract. We have previously investigated the solution properties of this mutant, as well as those of the closely related P23V and P23S mutants, and shown that although mutations at site 23 of HGD do not produce a significant structural change in the protein, they nevertheless profoundly alter the solubility of the protein. Remarkably, the solubility of the mutants decreases with increasing temperature, in sharp contrast to the behavior of the native protein. This inverted solubility corresponds to a strong increase in the binding energy with temperature. Here we have investigated the liquid-liquid coexistence curve and the diffusivity of the P23V mutant and find that these solution properties are unaffected by the mutation. This means that the chemical potentials in the solution phase are essentially unaltered. The apparent discrepancy between the interaction energies in the solution phase, as compared with the solid phase, is explicable in terms of highly anisotropic interprotein interactions, which are averaged out in the solution phase but are fully engaged in the solid phase.

Amyloid beta-protein (Abeta) oligomers may be the proximate neurotoxins in Alzheimer's disease (AD). Recently, to elucidate the oligomerization pathway, we studied Abeta monomer folding and identified a decapeptide segment of Abeta, (21)Ala-(22)Glu-(23)Asp-(24)Val-(25)Gly-(26)Ser-(27)Asn-(28)Lys-(29)Gly-(30)Ala, within which turn formation appears to nucleate monomer folding. The turn is stabilized by hydrophobic interactions between Val-24 and Lys-28 and by long-range electrostatic interactions between Lys-28 and either Glu-22 or Asp-23. We hypothesized that turn destabilization might explain the effects of amino acid substitutions at Glu-22 and Asp-23 that cause familial forms of AD and cerebral amyloid angiopathy. To test this hypothesis, limited proteolysis, mass spectrometry, and solution-state NMR spectroscopy were used here to determine and compare the structure and stability of the Abeta(21-30) turn within wild-type Abeta and seven clinically relevant homologues. In addition, we determined the relative differences in folding free energies (DeltaDeltaG(f)) among the mutant peptides. We observed that all of the disease-associated amino acid substitutions at Glu-22 or Asp-23 destabilized the turn and that the magnitude of the destabilization correlated with oligomerization propensity. The Ala21Gly (Flemish) substitution, outside the turn proper (Glu-22-Lys-28), displayed a stability similar to that of the wild-type peptide. The implications of these findings for understanding Abeta monomer folding and disease causation are discussed.

Objective: Endometrial carcinoma is the most common gynecologic cancer. Although the prognosis for endometrial cancer is generally good, cancers identified at late stages are associated with high levels of morbidity and mortality. Therefore, prevention and early detection may further reduce the burden of this challenging disease., Methods: A panel of 64 serum biomarkers was analyzed in sera of patients with stages I-III endometrial cancer and age-matched healthy women, utilizing a multiplex xMAP bead-based immunoassay. For multivariate analysis, four different statistical classification methods were used: logistic regression (LR), separating hyperplane (SHP), k nearest neighbors (KNN), and classification tree (CART). For each of these classifiers, a diagnostic model was created based on the cross-validation set consisting of sera from 115 patients with endometrial cancer and 135 healthy women., Results: Our data have demonstrated that patients with endometrial cancer have significantly different expression patterns of several serum biomarkers as compared to healthy controls. Prolactin was the strongest discriminative biomarker for endometrial cancer providing 98.3% sensitivity and 98.0% specificity alone. Our results have revealed that serum concentration of cancer antigens, including CA 125, CA 15-3, and CEA are higher in patients with Stage III endometrial cancer as compared to those with Stage I. In addition, we have shown that the expression of CA 125, AFP, and ACTH is elevated in women with tumor grade 3 vs. grade 1. Furthermore, five-biomarker panel (prolactin, GH, Eotaxin, E-selectin, and TSH) identified in this study was able to discriminate endometrial cancer from ovarian and breast cancers with high sensitivity and specificity., Conclusions: The ability of prolactin to accurately discriminate between cancer and control groups indicates that this biomarker could potentially be used for development of blood-based test for the early detection of endometrial cancer in high-risk populations. Combining the information on multiple serum markers using flexible statistical methods allows for achieving high cancer selectivity.

We report the results of x-ray-scattering studies of individual helical ribbons formed in multicomponent solutions of cholesterol solubilized by various surfactants. The solutions were chemically defined lipid concentrate (CDLC) and model bile. In these and many analogous multicomponent surfactant-cholesterol solutions, helical ribbons of two well defined pitch angles, namely 11 degrees and 54 degrees, are formed. We have suggested previously that this remarkable stability results from an underlying crystalline structure of the sterol ribbon strips. Using a synchrotron x-ray source, we have indeed observed Bragg reflections from individual ribbons having 11 degrees pitch angle. We have been able to deduce the parameters of the unit cell. The crystal structure of these ribbons is similar to that of cholesterol monohydrate, with the important difference that the length of the unit cell perpendicular to the cholesterol layers is tripled. We discuss possible origins for this triplication as well as the connection between the crystalline structure and the geometrical form of the helical ribbons.

Quasielastic light scattering spectroscopy (QLS) is an optical method for the determination of diffusion coefficients of particles in solution. Here we discuss the principles of QLS and explain how the distribution of particle sizes can be reconstructed from the measured correlation function of scattered light. Non-invasive observation of the temporal evolution of particle sizes provides a powerful tool for studying protein assembly. We illustrate practical applications of QLS with examples from studies of fibril formation of the amyloid beta-protein.

Quasielastic light scattering (QLS) spectroscopy is an optical method for the determination of diffusion coefficients of particles in solution. In this chapter, we discuss the principles and practice of QLS with respect to protein assembly reactions. Particles undergoing Brownian motion produce fluctuations in scattered light intensity. We describe how the temporal correlation function of these fluctuations can be measured and how this correlation function provides information about the distribution of diffusion coefficients of the particles in solution. We discuss the intricacies of deconvolution of the correlation function and the assumptions incorporated into data analysis procedures. We explain how the Stokes-Einstein relationship can be used to convert distributions of diffusion coefficients into distributions of particle size. Noninvasive observation of the temporal evolution of particles sizes provides a powerful tool for studying protein aggregation and self-assembly. We use examples from studies of Abeta fibrillogenesis to illustrate QLS application for understanding the molecular mechanisms of the nucleation and growth of amyloid fibrils.

It is generally assumed that crystallization begins with a small, crystalline nucleus. For proteins this paradigm may not be valid. Our numerical simulations show that under conditions typically used to produce protein crystals, small clusters of model proteins (particles with short-range, attractive interactions) cannot maintain a crystalline structure. Protein crystal nucleation is therefore an indirect, two-step process. A nucleus first forms and grows as a disordered, liquid-like aggregate. Once the aggregate grows beyond a critical size (about a few hundred particles) crystal nucleation becomes possible.

Amyloid beta-protein (Abeta) is linked to neuronal injury and death in Alzheimer's disease (AD). Of particular relevance for elucidating the role of Abeta in AD is new evidence that oligomeric forms of Abeta are potent neurotoxins that play a major role in neurodegeneration and the strong association of the 42-residue form of Abeta, Abeta42, with the disease. Detailed knowledge of the structure and assembly dynamics of Abeta thus is important for the development of properly targeted AD therapeutics. Recently, we have shown that Abeta oligomers can be cross-linked efficiently, and their relative abundances quantified, by using the technique of photo-induced cross-linking of unmodified proteins (PICUP). Here, PICUP, size-exclusion chromatography, dynamic light scattering, circular dichroism spectroscopy, and electron microscopy have been combined to elucidate fundamental features of the early assembly of Abeta40 and Abeta42. Carefully prepared aggregate-free Abeta40 existed as monomers, dimers, trimers, and tetramers, in rapid equilibrium. In contrast, Abeta42 preferentially formed pentamerhexamer units (paranuclei) that assembled further to form beaded superstructures similar to early protofibrils. Addition of Ile-41 to Abeta40 was sufficient to induce formation of paranuclei, but the presence of Ala-42 was required for their further association. These data demonstrate that Abeta42 assembly involves formation of several distinct transient structures that gradually rearrange into protofibrils. The strong etiologic association of Abeta42 with AD may thus be a result of assemblies formed at the earliest stages of peptide oligomerization.

We have studied the effect of polyethylene glycol (PEG) on the liquid-liquid phase separation (LLPS) of aqueous solutions of bovine gammaD-crystallin (gammaD), a protein in the eye lens. We observe that the phase separation temperature increases with both PEG concentration and PEG molecular weight. PEG partitioning, which is the difference between the PEG concentration in the two coexisting phases, has been measured experimentally and observed to increase with PEG molecular weight. The measurements of both LLPS temperature and PEG partitioning in the ternary gammaD-PEG-water systems are used to successfully predict the location of the liquid-liquid phase boundary of the binary gammaD-water system. We show that our LLPS measurements can be also used to estimate the protein solubility as a function of the concentration of crystallizing agents. Moreover, the slope of the tie-lines and the dependence of LLPS temperature on polymer concentration provide a powerful and sensitive check of the validity of excluded volume models. Finally, we show that the increase of the LLPS temperature with PEG concentration is due to attractive protein-protein interactions.

Increasing evidence supports the hypothesis that amyloid beta-protein (Abeta) assembly is a key pathogenic feature of Alzheimer's disease. Thus, understanding the assembly process offers opportunities for the development of strategies for treating this devastating disease. In prior studies, Abeta was found to form micelle-like aggregates under acidic conditions. These structures exhibited an average observed hydrodynamic radius of 7 nm. They were found to be in rapid equilibrium with Abeta monomers or low molecular weight oligomers, and were centers of fibril nucleation. Here the technique of small angle neutron scattering has been used to determine the structure of these Abeta micelles. The data reveal that the micellar assemblies comprise 30-50 Abeta monomers and have elongated geometries. The best fit of the data to a uniform spherocylinder yields a radius approximately 2.4 nm and cylinder length approximately 11 nm. These structure parameters remain constant over more than a decade in concentration range. The concentration independence of the length of the cylindrical aggregate indicates the presence of an internal nonrepetitive structure that spans the entire length of the Abeta assembly.

The cysteine residues of the gamma crystallins, a family of ocular lens proteins, are involved in the aggregation and phase separation of these proteins. Both these phenomena are implicated in cataract formation. We have used bovine gammaB crystallin as a model system to study the role of the individual cysteine residues in the aggregation and phase separation of the gamma crystallins. Here, we compare the thermodynamic and kinetic behavior of the recombinant wild-type protein (WT) and the Cys18 to Ser (C18S) mutant. We find that the solubilities of the two proteins are similar. The kinetics of crystallization, however, are different. The WT crystallizes slowly enough for the metastable liquid-liquid coexistence to be easily observed. C18S, on the other hand, crystallizes rapidly; the metastable coexisting liquid phases of the pure mutant do not form. Nevertheless, the coexistence curve of C18S can be determined provided that crystallization is kinetically suppressed. In this way we found that the coexistence curve coincides with that of the WT. Despite the difference in the kinetics of crystallization, the two proteins were found to have the same crystal forms and almost identical X-ray structures. Our results demonstrate that even conservative point mutations can bring about dramatic changes in the kinetics of crystallization. The implications of our findings for cataract formation and protein crystallization are discussed., (Copyright 2001 Academic Press.)

Assembly of the amyloid beta-protein (Abeta) into neurotoxic oligomers and fibrils is a seminal event in Alzheimer's disease. Understanding the earliest phases of Abeta assembly, including prenucleation and nucleation, is essential for the development of rational therapeutic strategies. We have applied a powerful new method, photoinduced cross-linking of unmodified proteins (PICUP), to the study of Abeta oligomerization. Significant advantages of this method include an extremely short reaction time, enabling the identification and quantification of short lived metastable assemblies, and the fact that no pre facto structural modification of the native peptide is required. Using PICUP, the distribution of Abeta oligomers existing prior to assembly was defined. A rapid equilibrium was observed involving monomer, dimer, trimer, and tetramer. A similar distribution was seen in studies of an unrelated amyloidogenic peptide, whereas nonamyloidogenic peptides yielded distributions indicative of a lack of monomer preassociation. These results suggest that simple nucleation-dependent polymerization models are insufficient to describe the dynamic equilibria associated with prenucleation phases of Abeta assembly.