Arresting Amyloid with Coulomb’s Law: Acetylation of ALS-Linked SOD1 by Aspirin Impedes Aggregation

Abdolvahabi, Alireza; Shi, Yulei; Rhodes, Nicholas R.; Cook, Nathan P.; Martí, Ángel A.; Shaw, Bryan F.

doi:10.1016/j.bpj.2015.01.014

Cited by 47 publications

(100 citation statements)

References 61 publications

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“…[1,4] Deoxyribonuclease and ovalbumin, for example, have identical isoelectricp oints of pI = 5.1, but the formal and measured net charge of both proteins differ by approximately 7u nits at pH 8.4. [4] The systematic absence of experimentally determinedv alues of Z has likely impeded ar igorous understanding of most chemicalp rocesses in which proteinsa re involved including aggregation and self-assembly, [20][21][22][23][24][25][26] ligand binding, [27][28][29][30][31][32][33][34] catalysis, [35][36][37][38][39] electron transfer, [3,6,[40][41][42][43][44][45][46][47] protein crystallization, [14,48] analytical separation, [49,50] and protein engineering. [51][52][53][54][55][56] It is tempting to assume that the formal net chargeo faprotein predicted from generalized residue pK a values (Z seq )issosimilar to the actual net charge that any difference is irrelevant, and the isoelectric point tells us all we need to know about ap rotein's net charge.…”

Section: Introductionmentioning

confidence: 99%

What Are We Missing by Not Measuring the Net Charge of Proteins?

Zahler

Shaw

2019

Chemistry A European J

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The net electrostatic charge (Z) of a folded protein in solution represents a bird's eye view of its surface potentials—including contributions from tightly bound metal, solvent, buffer, and cosolvent ions—and remains one of its most enigmatic properties. Few tools are available to the average biochemist to rapidly and accurately measure Z at pH≠pI. Tools that have been developed more recently seem to go unnoticed. Most scientists are content with this void and estimate the net charge of a protein from its amino acid sequence, using textbook values of pKa. Thus, Z remains unmeasured for nearly all folded proteins at pH≠pI. When marveling at all that has been learned from accurately measuring the other fundamental property of a protein—its mass—one wonders: what are we missing by not measuring the net charge of folded, solvated proteins? A few big questions immediately emerge in bioinorganic chemistry. When a single electron is transferred to a metalloprotein, does the net charge of the protein change by approximately one elementary unit of charge or does charge regulation dominate, that is, do the pKa values of most ionizable residues (or just a few residues) adjust in response to (or in concert with) electron transfer? Would the free energy of charge regulation (ΔΔGz) account for most of the outer sphere reorganization energy associated with electron transfer? Or would ΔΔGz contribute more to the redox potential? And what about metal binding itself? When an apo‐metalloprotein, bearing minimal net negative charge (e.g., Z=−2.0) binds one or more metal cations, is the net charge abolished or inverted to positive? Or do metalloproteins regulate net charge when coordinating metal ions? The author's group has recently dusted off a relatively obscure tool—the “protein charge ladder”—and used it to begin to answer these basic questions.

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Section: Introductionmentioning

confidence: 99%

What Are We Missing by Not Measuring the Net Charge of Proteins?

Zahler

Shaw

2019

Chemistry A European J

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“…To accelerate amyloidogenesis and reduce stochasticity ( 9 ), solutions are gyrated in microtiter plates at >100 rpm with millimeter-sized stirring beads placed in each sample well ( 9 , 12 , 13 ). Beads are typically composed of polytetrafluoroethylene (Teflon), but borosilicate glass and polymethylmethylacrylate (PMMA) have also been used ( 8 , 9 , 12 , 14 , 15 , 16 ). Establishing a few basic rules of thumb for how different bead types do or do not affect formation rates (and morphologies) of amyloid fibrils will help optimize assays; help discern the growing (and often inconsistent) list of reported rates of amyloid nucleation and elongation ( 10 , 14 , 17 , 18 ), and might uncover clues about the mechanical, chemical, and surface forces that drive fibrillization.…”

Section: Introductionmentioning

confidence: 99%

How Do Gyrating Beads Accelerate Amyloid Fibrillization?

Abdolvahabi

Shi

Rasouli

et al. 2017

Biophysical Journal

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The chemical and physical mechanisms by which gyrating beads accelerate amyloid fibrillization in microtiter plate assays are unclear. Identifying these mechanisms will help optimize high-throughput screening assays for molecules and mutations that modulate aggregation and might explain why different research groups report different rates of aggregation for identical proteins. This article investigates how the rate of superoxide dismutase-1 (SOD1) fibrillization is affected by 12 different beads with a wide range of hydrophobicity, mass, stiffness, and topology but identical diameter. All assays were performed on D90A apo-SOD1, which is a stable and wild-type-like variant of SOD1. The most significant and uniform correlation between any material property of each bead and that bead's effect on SOD1 fibrillization rate was with regard to bead mass. A linear correlation existed between bead mass and rate of fibril elongation (R = 0.7): heavier beads produced faster rates and shorter fibrils. Nucleation rates (lag time) also correlated with bead mass, but only for non-polymeric beads (i.e., glass, ceramic, metallic). The effect of bead mass on fibrillization correlated (R = 0.96) with variations in buoyant forces and contact forces (between bead and microplate well), and was not an artifact of residual momentum during intermittent gyration. Hydrophobic effects were observed, but only for polymeric beads: lag times correlated negatively with contact angle of water and degree of protein adhesion (surface adhesion and hydrophobic effects were negligible for non-polymeric beads). These results demonstrate that contact forces (alone) explain kinetic variation among non-polymeric beads, whereas surface hydrophobicity and contact forces explain kinetic variation among polymeric beads. This study also establishes conditions for high-throughput amyloid assays of SOD1 that enable the control over fibril morphologies and produce eightfold faster lag times and fourfold less stochasticity than in previous studies.

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“…Abdolvahabi and co-workers demonstrated that acylation of Lys residues in superoxide dismutase 1 with aspirin increases the net negative charge of the polypeptide and slows the rate of aggregation. 50 In a similar fashion, Patil and Alexandrescu have recently applied a charge repulsion approach to inhibiting amylin self-assembly by preparing “charge-loaded” analogs of full-length amylin. 51 In this study, strings of Arg or Asp residues were strategically incorporated into the native amylin sequence to produce aggregation inhibitors.…”

Section: Resultsmentioning

confidence: 99%

Peptide Conjugates of Benzene Carboxylic Acids as Agonists and Antagonists of Amylin Aggregation

2017

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Human islet amyloid polypeptide (hIAPP), also known as amylin, is a 37 residue peptide hormone that is stored and co-secreted with insulin. hIAPP plays a pivotal role in type 2 diabetes and is the major component of amyloid deposits found in the pancreas of patients afflicted with the disease. The self-assembly of hIAPP and the formation of amyloid is linked to the death of insulin producing β-cells. Recent findings suggest that soluble hIAPP oligomers are the cytotoxic species responsible for β-cell loss whereas amyloid fibrils themselves may indeed be innocuous. Potential avenues of therapeutic intervention include the development of compounds that prevent hIAPP self-assembly as well as those that reduce or eliminate lag time and rapidly accelerate the formation of amyloid fibrils. Both of these approaches minimize temporal exposure to soluble cytotoxic hIAPP oligomers. Toward this end our laboratory has pursued an electrostatic repulsion approach to the development of potential inhibitors and modulators of hIAPP self-assembly. Peptide conjugates were constructed in which benzene carboxylic acids of varying charge were employed as electrostatic disrupting elements and appended to the N-terminal of the hIAPP22–29 (NFGAILSS) self-recognition sequence. The self-assembly kinetics of conjugates were characterized by turbidity measurements and the structure of aggregates probed by Raman and CD spectroscopy while the morphology was assessed using transmission electron microscopy. Several benzene carboxylic acid peptide conjugates failed to self-assemble and some were found to inhibit the aggregation of full-length amylin while others served to enhance the rate of amyloid formation and/or increase the yield of amyloid produced. Studies reveal that the geometric display of free carboxylates on the benzene ring of the conjugates plays an important role in the activity of conjugates. In addition, a number of free benzene carboxylic acids were found to modulate amylin self-assembly on their own. The results of these investigations confirm the viability of the electrostatic repulsion approach to the modulation of amyloid formation and may aid the design and development of potential therapeutic agents.

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Arresting Amyloid with Coulomb’s Law: Acetylation of ALS-Linked SOD1 by Aspirin Impedes Aggregation

Cited by 47 publications

References 61 publications

What Are We Missing by Not Measuring the Net Charge of Proteins?

What Are We Missing by Not Measuring the Net Charge of Proteins?

How Do Gyrating Beads Accelerate Amyloid Fibrillization?

Peptide Conjugates of Benzene Carboxylic Acids as Agonists and Antagonists of Amylin Aggregation

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