The effects of shear flow on protein structure and function

Bekard, Innocent B.; Asimakis, Peter; Bertolini, Joseph; Dunstan, Dave E.

doi:10.1002/bip.21646

Cited by 137 publications

(136 citation statements)

References 151 publications

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“…However, neither wild-type TTR nor the other TTR variants we tested (Val30Met, Leu55-Pro, and Val122Ile) were similarly cleaved in vitro, and thus they are notably more stable to proteolysis. Nevertheless, it is plausible that in in vivo conditions, in particular shear flow and exposure to hydrophobic surfaces in the dynamic environment of the interstitial space, may promote local structural destabilization (21,22) and enable proteolytic cleavage. Indeed focal mechanical destabilization of the polypeptide chain is a well-known mechanism for priming limited physiological proteolytic cleavage in the homeostasis of hemostatic proteins.…”

Section: Discussionmentioning

confidence: 99%

“…Although this hydrogen bond might be sustained by the new N terminus generated by cleavage, the separation of this N-terminal end from the new carboxylate of Lys48 must widen from 1.2 to at least 2.8 Å and promote displacement of the CD turn. Departure of the 1-48 peptide caused by shear forces in solution would remove important dimer-dimer interaction regions (19)(20)(21)(22)(23) and promote dissociation of the remaining 49-127 fragment.…”

Section: Limited Proteolysis Of Recombinant Wild-type and Variant Ttrmentioning

confidence: 99%

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Proteolytic cleavage of Ser52Pro variant transthyretin triggers its amyloid fibrillogenesis

Mangione

Porcari²,

Gillmore³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

136

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The Ser52Pro variant of transthyretin (TTR) produces aggressive, highly penetrant, autosomal-dominant systemic amyloidosis in persons heterozygous for the causative mutation. Together with a minor quantity of full-length wild-type and variant TTR, the main component of the ex vivo fibrils was the residue 49-127 fragment of the TTR variant, the portion of the TTR sequence that previously has been reported to be the principal constituent of type A, cardiac amyloid fibrils formed from wild-type TTR and other TTR variants [Bergstrom J, et al. (2005) J Pathol 206(2):224-232]. This specific truncation of Ser52Pro TTR was generated readily in vitro by limited proteolysis. In physiological conditions and under agitation the residue 49-127 proteolytic fragment rapidly and completely self-aggregates into typical amyloid fibrils. The remarkable susceptibility to such cleavage is likely caused by localized destabilization of the β-turn linking strands C and D caused by loss of the wildtype hydrogen-bonding network between the side chains of residues Ser52, Glu54, Ser50, and a water molecule, as revealed by the high-resolution crystallographic structure of Ser52Pro TTR. We thus provide a structural basis for the recently hypothesized, crucial pathogenic role of proteolytic cleavage in TTR amyloid fibrillogenesis. Binding of the natural ligands thyroxine or retinol-binding protein (RBP) by Ser52Pro variant TTR stabilizes the native tetrameric assembly, but neither protected the variant from proteolysis. However, binding of RBP, but not thyroxine, inhibited subsequent fibrillogenesis.misfolding | protein aggregation

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Section: Discussionmentioning

confidence: 99%

Section: Limited Proteolysis Of Recombinant Wild-type and Variant Ttrmentioning

confidence: 99%

Proteolytic cleavage of Ser52Pro variant transthyretin triggers its amyloid fibrillogenesis

Mangione

Porcari²,

Gillmore³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

136

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“…For the constituent milk proteins, shear has the potential to cause conformational changes, in particular unfolding. Indeed, it has been recognized from a series of studies on a diversity of proteins that shear can cause unfolding of polypeptide chains to varying degrees (reviewed in Bekard et al, 2011).…”

Section: Shear Stress and Casein Protein Aggregationmentioning

confidence: 99%

Invited review: Caseins and the casein micelle: Their biological functions, structures, and behavior in foods

Holt

Carver

Ecroyd

et al. 2013

Journal of Dairy Science

360

231

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A typical casein micelle contains thousands of casein molecules, most of which form thermodynamically stable complexes with nanoclusters of amorphous calcium phosphate. Like many other unfolded proteins, caseins have an actual or potential tendency to assemble into toxic amyloid fibrils, particularly at the high concentrations found in milk. Fibrils do not form in milk because an alternative aggregation pathway is followed that results in formation of the casein micelle. As a result of forming micelles, nutritious milk can be secreted and stored without causing either pathological calcification or amyloidosis of the mother's mammary tissue. The ability to sequester nanoclusters of amorphous calcium phosphate in a stable complex is not unique to caseins. It has been demonstrated using a number of noncasein secreted phosphoproteins and may be of general physiological importance in preventing calcification of other biofluids and soft tissues. Thus, competent noncasein phosphoproteins have similar patterns of phosphorylation and the same type of flexible, unfolded conformation as caseins. The ability to suppress amyloid fibril formation by forming an alternative amorphous aggregate is also not unique to caseins and underlies the action of molecular chaperones such as the small heat-shock proteins. The open structure of the protein matrix of casein micelles is fragile and easily perturbed by changes in its environment. Perturbations can cause the polypeptide chains to segregate into regions of greater and lesser density. As a result, the reliable determination of the native structure of casein micelles continues to be extremely challenging. The biological functions of caseins, such as their chaperone activity, are determined by their composition and flexible conformation and by how the casein polypeptide chains interact with each other. These same properties determine how caseins behave in the manufacture of many dairy products and how they can be used as functional ingredients in other foods. aBStraCtA typical casein micelle contains thousands of casein molecules, most of which form thermodynamically stable complexes with nanoclusters of amorphous calcium phosphate. Like many other unfolded proteins, caseins have an actual or potential tendency to assemble into toxic amyloid fibrils, particularly at the high concentrations found in milk. Fibrils do not form in milk because an alternative aggregation pathway is followed that results in formation of the casein micelle. As a result of forming micelles, nutritious milk can be secreted and stored without causing either pathological calcification or amyloidosis of the mother's mammary tissue. The ability to sequester nanoclusters of amorphous calcium phosphate in a stable complex is not unique to caseins. It has been demonstrated using a number of noncasein secreted phosphoproteins and may be of general physiological importance in preventing calcification of other biofluids and soft tissues. Thus, competent noncasein phosphoproteins have similar patterns of phosp...

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“…The force applied onto a protein as a consequence of hydrodynamic flow has also been observed to trigger protein aggregation and has fundamental (3), medical (4), and industrial relevance, especially in the manufacture of biopharmaceuticals (5-8). Although a wealth of studies have been performed (7,(9)(10)(11)(12)(13), no consensus has emerged on the ability of hydrodynamic flow to induce protein aggregation (7,14,15). This is due to the wide variety of proteins used (ranging from lysozyme, BSA, and alcohol dehydrogenase to IgGs), differences in the type of flow field generated (e.g., shear, extensional, or mixtures of these), and to the presence or absence of an interface (16).…”

mentioning

confidence: 99%

Inducing protein aggregation by extensional flow

Dobson

Kumar

Willis

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

118

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Relative to other extrinsic factors, the effects of hydrodynamic flow fields on protein stability and conformation remain poorly understood. Flow-induced protein remodeling and/or aggregation is observed both in Nature and during the large-scale industrial manufacture of proteins. Despite its ubiquity, the relationships between the type and magnitude of hydrodynamic flow, a protein's structure and stability, and the resultant aggregation propensity are unclear. Here, we assess the effects of a defined and quantified flow field dominated by extensional flow on the aggregation of BSA, β 2 -microglobulin (β 2 m), granulocyte colony stimulating factor (G-CSF), and three monoclonal antibodies (mAbs). We show that the device induces protein aggregation after exposure to an extensional flow field for 0.36-1.8 ms, at concentrations as low as 0.5 mg mL −1 . In addition, we reveal that the extent of aggregation depends on the applied strain rate and the concentration, structural scaffold, and sequence of the protein. Finally we demonstrate the in situ labeling of a buried cysteine residue in BSA during extensional stress. Together, these data indicate that an extensional flow readily unfolds thermodynamically and kinetically stable proteins, exposing previously sequestered sequences whose aggregation propensity determines the probability and extent of aggregation.extensional flow | aggregation | unfolding | bioprocessing | antibody P roteins are dynamic and metastable and consequently have conformations that are highly sensitive to the environment (1). Over the last 50 y the effect of changes in temperature, pH, and the concentration of kosmatropic/chaotropic agents on the conformational energy landscape of proteins has become well understood (1). This, in turn, has allowed a link to be established between the partial or full unfolding of proteins and their propensity to aggregate (2). The force applied onto a protein as a consequence of hydrodynamic flow has also been observed to trigger protein aggregation and has fundamental (3), medical (4), and industrial relevance, especially in the manufacture of biopharmaceuticals (5-8). Although a wealth of studies have been performed (7,(9)(10)(11)(12)(13), no consensus has emerged on the ability of hydrodynamic flow to induce protein aggregation (7,14,15). This is due to the wide variety of proteins used (ranging from lysozyme, BSA, and alcohol dehydrogenase to IgGs), differences in the type of flow field generated (e.g., shear, extensional, or mixtures of these), and to the presence or absence of an interface (16). A shearing flow field (Fig. 1A, Top) is caused by a gradient in velocity perpendicular to the direction of travel and is characterized by the shear rate (s −1 ). This results in a weak rotating motion of a protein alongside translation in the direction of the flow. An extensional flow field (Fig. 1A, Bottom) is generated by a gradient in velocity in the direction of travel and is characterized by the strain rate (s −1 ). A protein in this type of flow would experien...

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The effects of shear flow on protein structure and function

Cited by 137 publications

References 151 publications

Proteolytic cleavage of Ser52Pro variant transthyretin triggers its amyloid fibrillogenesis

Proteolytic cleavage of Ser52Pro variant transthyretin triggers its amyloid fibrillogenesis

Invited review: Caseins and the casein micelle: Their biological functions, structures, and behavior in foods

Inducing protein aggregation by extensional flow

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