Peptides or proteins convert under some conditions from their soluble forms into highly ordered fibrillar aggregates. Such transitions can give rise to pathological conditions ranging from neurodegenerative disorders to systemic amyloidoses. In this review, we identify the diseases known to be associated with formation of fibrillar aggregates and the specific peptides and proteins involved in each case. We describe, in addition, that living organisms can take advantage of the inherent ability of proteins to form such structures to generate novel and diverse biological functions. We review recent advances toward the elucidation of the structures of amyloid fibrils and the mechanisms of their formation at a molecular level. Finally, we discuss the relative importance of the common main-chain and side-chain interactions in determining the propensities of proteins to aggregate and describe some of the evidence that the oligomeric fibril precursors are the primary origins of pathological behavior.

/pdf/protein-misfolding-functional-amyloid-and-human-disease-2w386pwb7k.pdf

Protein Misfolding, Functional Amyloid, and Human Disease

The binding of thioflavin-T to amyloid fibrils: localisation and implications.

Because understanding amyloid fibrillation in molecular detail is essential for development of strategies to control amyloid formation and overcome neurodegenerative disorders, increased understanding of present molecular probes as well as development of new probes are of utmost importance. To date, the binding modes of these molecular probes to amyloid fibrils are by no means adequately described or understood, and the large number of studies on Thioflavin T (ThT) and Congo Red (CR) binding have resulted in models that are incomplete and conflicting. Different types of binding sites are likely to be present in amyloid fibrils with differences in binding modes. ThT may bind in channels running parallel to the long axis of the fibril. In the channels, ThT may bind in either a monomeric or dimeric form of which the molecular conformation is likely to be planar. CR may bind in grooves formed along the β-sheets as a planar molecule in either a monomeric or supramolecular form.

/pdf/binding-mode-of-thioflavin-t-and-other-molecular-probes-in-2jznhxuyet.pdf

Binding mode of Thioflavin T and other molecular probes in the context of amyloid fibrils—current status

Synchrotron-based infrared and X-ray imaging shows focalized accumulation of Cu and Zn co-localized with beta-amyloid deposits in Alzheimer's disease.

Small heat shock proteins: Role in cellular functions and pathology.

New advances in polarized light microscopy were used to image Congo red-stained cerebral amyloidosis in sharp relief. The rotating-polarizer method was used to separate the optical effects of transmission, linear birefringence, extinction, linear dichroism, and orientation of the electric dipole transition moments and to display them as false-color maps. These effects are typically convolved in an ordinary polarized light microscope. In this way, we show that the amyloid deposits in Alzheimer's disease plaques contain structurally disordered centers, providing clues to mechanisms of crystallization of amyloid in vivo. Comparisons are made with plaques from tissues of subjects having Down's syndrome and a prion disease. In plaques characteristic of each disease, the Congo red molecules are oriented radially. The optical orientation in amyloid deposited in blood vessels from subjects having cerebral amyloid angiopathy was 90° out of phase from that in the plaques, suggesting that the fibrils run tangentially with respect to the circumference of the blood vessels. Our result supports an early model in which Congo red molecules are aligned along the long fiber axis and is in contrast to the most recent binding models that are based on computation. This investigation illustrates that the latest methods for the optical analysis of heterogeneous substances are useful for in situ study of amyloid.

/pdf/imaging-linear-birefringence-and-dichroism-in-cerebral-892h8zcha6.pdf

Imaging linear birefringence and dichroism in cerebral amyloid pathologies

Protein pin arrays identified seven interactive sequences for chaperone activity in human alphaB crystallin using natural lens proteins, beta(H) crystallin and gammaD crystallin, and in vitro chaperone target proteins, alcohol dehydrogenase and citrate synthase. The N-terminal domain contained two interactive sequences, (9)WIRRPFFPFHSP(20) and (43)SLSPFYLRPPSFLRAP(58). The alpha crystallin core domain contained four interactive sequences, (75)FSVNLDVK(82) (beta3), (113)FISREFHR(120), (131)LTITSSLS(138) (beta8), and (141)GVLTVNGP(148) (beta9). The C-terminal domain contained one interactive sequence, (157)RTIPITRE(164), that included the highly conserved I-X-I/V motif. Two interactive sequences, (73)DRFSVNLDVKHFS(85) and (131)LTITSSLSDGV(141), belonging to the alpha crystallin core domain were synthesized as peptides and assayed for chaperone activity in vitro. Both synthesized peptides inhibited the thermal aggregation of beta(H) crystallin, alcohol dehydrogenase, and citrate synthase in vitro. Five of the seven chaperone sequences identified by the pin arrays overlapped with sequences identified previously as sequences for subunit-subunit interactions in human alphaB crystallin. The results suggested that interactive sequences in human alphaB crystallin have dual roles in subunit-subunit assembly and chaperone activity.

Interactive domains for chaperone activity in the small heat shock protein, human αB crystallin

Knowledge of the interactive domains on the surface of small heat shock proteins (sHSPs) is necessary for understanding the assembly of complexes and the activity as molecular chaperones. The primary sequences of 26 sHSP molecular chaperones were aligned and compared. In the interactive β3 sequence, 73DRFSVNLDVKHFS85 of human αB crystallin, Ser-76, Asn-78, Lys-82, and His-83 were identified as nonconserved residues on the exposed surface of the α crystallin core domain. Site-directed mutagenesis produced the mutant αB crystallins: S76E, N78G, K82Q, and H83F. Domain swapping with homologous β3 sequences, 32EKFEVGLDVQFFT44 from Caenorhabditis elegans sHSP12.2 or 69DKFVIFLDVKHFS81 from αA crystallin, resulted in the mutant αB crystallins, CE1 and αA1, respectively. Decreased chaperone activity was observed with the point mutants N78G, K82Q, and H83F and with the mutant, CE1, in aggregation assays using βL crystallin, alcohol dehydrogenase (ADH), or citrate synthase (CS). The S76E mutant had minimal effect on chaperone activity, and domain swapping with αA crystallin had no effect on chaperone activity. The mutations that resulted in altered chaperone activity, produced minimal modification to the secondary, tertiary, and quaternary structure of human αB crystallin as determined by ultraviolet circular dichroism spectroscopy, chymotrypsin proteolysis, and size exclusion chromatography. Chaperone activity was influenced by the amount of unfolding of the target proteins and independent of complex size. The results characterized the importance of the exposed side chains of Glu-78, Lys-82, and His-83 in the interactive β3 sequence of the α crystallin core domain in αB crystallin for chaperone function.

/pdf/the-function-of-the-b3-interactive-domain-in-the-small-heat-2mhkzxztw0.pdf

The function of the β3 interactive domain in the small heat shock protein and molecular chaperone, human αB crystallin

The functional importance of the β8 sequence (131LTITSSLS138), which is on the surface of the α crystallin core domain of human αB crystallin, was evaluated using site-directed mutagenesis. Ultraviolet circular dichroism determined that mutating the surface-exposed, nonconserved residues, Leu-131, Thr-132, Thr-134, Ser-135, Ser-136, and Ser-138 individually or in combination (αAβ8 and CEβ8), had no measurable effect on secondary and tertiary structure. Size exclusion chromatography determined the size of the complexes formed by the β8 mutants to be 6−8 subunits larger than wt αB crystallin. In chaperone assays, the protective effect of the L131S, T132A, and S135C mutants of the β8 sequence was similar to wt αB crystallin when βL crystallin and alcohol dehydrogenase were the chaperone substrates and decreased to 66% when citrate synthase was the chaperone substrate. In contrast, the chaperone activity for all three substrates was dramatically reduced for the T134K, S138A, S136H, and CEβ8 mutants. The promi...

Structure-based analysis of the β8 interactive sequence of human αB crystallin

https://doi.org/10.1016/j.vaccine.2022.07.017

Marcus R. Estrada

Papers

Imaging linear birefringence and dichroism in cerebral amyloid pathologies

Interactive domains for chaperone activity in the small heat shock protein, human αB crystallin

The function of the β3 interactive domain in the small heat shock protein and molecular chaperone, human αB crystallin

Structure-based analysis of the β8 interactive sequence of human αB crystallin

Concordance of in vitro and in vivo measures of non-replicating rotavirus vaccine potency