Structural Organization of Insulin Fibrils Based on Polarized Raman Spectroscopy: Evaluation of Existing Models.

Many different proteins undergo misfolding and self-assemble into amyloid fibrils, resulting in a range of neurodegenerative diseases. The limitations of conventional methods of structural biology for fibril characterization have led to the use of polarized Raman spectroscopy for obtaining quantitative structural information regarding the organization of amyloid fibrils. Herein, we report the orientation of selected chemical groups and secondary structure elements in aligned insulin fibrils, including ß-sheets, which possess a high level of orientation in the cross-ß core, and α-helices in the disordered portions of the fibrils. Strong orientation of disulfide bonds in amyloid fibrils was also revealed, indicating their association with the fibril core. The determined orientation of chemical groups provides strong constraints for modeling the overall structure of amyloid fibrils, including the core and disordered parts. The developed methodology allows for the validation of structural models proposed in the literature for amyloid fibrils. Specifically, the polarized Raman data obtained herein strongly agreed with two insulin fibril models (Jiménez et al., Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 9196-9201 and Ivanova et al., Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 18990-18995) yet revealed significant qualitative and quantitative differences. This work demonstrates the great potential of polarized Raman spectroscopy for structural characterization of anisotropic biological species.

Rapid degradation kinetics of amyloid fibrils under mild conditions by an archaeal chaperonin.

Amyloid depositions containing exceptionally stable ß-sheet rich protein aggregates, called fibrils are associated with prevalent and incurable neurodegenerative diseases. Chaperones are proteins that facilitate protein folding in both eukaryotes and prokaryotes. We found that a cold-adapted mutant ATP-dependant chaperonins (Hsp60) from a hyperthermophilic archaeon binds to and fragments insulin fibrils very rapidly with local targeted entry points. Individual fragments swell and the fibrillar ß-sheet is quickly transformed into a mix of α-helical and unordered protein structures. After further incubation, the fragments coalesced, forming large amorphous aggregates with poly-disperse topologies. This finding represents a new approach to the disassembly of refractory protein aggregates under physiological conditions.

Structure of putative tumor suppressor ALDH1L1.

Putative tumor suppressor ALDH1L1, the product of natural fusion of three unrelated genes, regulates folate metabolism by catalyzing NADP+-dependent conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. Cryo-EM structures of tetrameric rat ALDH1L1 revealed the architecture and functional domain interactions of this complex enzyme. Highly mobile N-terminal domains, which remove formyl from 10-formyltetrahydrofolate, undergo multiple transient inter-domain interactions. The C-terminal aldehyde dehydrogenase domains, which convert formyl to CO2, form unusually large interfaces with the intermediate domains, homologs of acyl/peptidyl carrier proteins (A/PCPs), which transfer the formyl group between the catalytic domains. The 4'-phosphopantetheine arm of the intermediate domain is fully extended and reaches deep into the catalytic pocket of the C-terminal domain. Remarkably, the tetrameric state of ALDH1L1 is indispensable for catalysis because the intermediate domain transfers formyl between the catalytic domains of different protomers. These findings emphasize the versatility of A/PCPs in complex, highly dynamic enzymatic systems.

CHIP E3 ligase mediates proteasomal degradation of the proliferation regulatory protein ALDH1L1 during the transition of NIH3T3 fibroblasts from G0/G1 to S-phase.

ALDH1L1 is a folate-metabolizing enzyme abundant in liver and several other tissues. In human cancers and cell lines derived from malignant tumors, the ALDH1L1 gene is commonly silenced through the promoter methylation. It was suggested that ALDH1L1 limits proliferation capacity of the cell and thus functions as putative tumor suppressor. In contrast to cancer cells, mouse cell lines NIH3T3 and AML12 do express the ALDH1L1 protein. In the present study, we show that the levels of ALDH1L1 in these cell lines fluctuate throughout the cell cycle. During S-phase, ALDH1L1 is markedly down regulated at the protein level. As the cell cultures become confluent and cells experience increased contact inhibition, ALDH1L1 accumulates in the cells. In agreement with this finding, NIH3T3 cells arrested in G1/S-phase by a thymidine block completely lose the ALDH1L1 protein. Treatment with the proteasome inhibitor MG-132 prevents such loss in proliferating NIH3T3 cells, suggesting the proteasomal degradation of the ALDH1L1 protein. The co-localization of ALDH1L1 with proteasomes, demonstrated by confocal microscopy, supports this mechanism. We further show that ALDH1L1 interacts with the chaperone-dependent E3 ligase CHIP, which plays a key role in the ALDH1L1 ubiquitination and degradation. In NIH3T3 cells, silencing of CHIP by siRNA halts, while transient expression of CHIP promotes, the ALDH1L1 loss. The downregulation of ALDH1L1 is associated with the accumulation of the ALDH1L1 substrate 10-formyltetrahydrofolate, which is required for de novo purine biosynthesis, a key pathway activated in S-phase. Overall, our data indicate that CHIP-mediated proteasomal degradation of ALDH1L1 facilitates cellular proliferation.

Two Mechanisms of Tip Enhancement of Raman Scattering by Protein Aggregates.

Tip-enhanced Raman spectroscopy (TERS) is a powerful tool for probing the surface of biological species with nanometer spatial resolution. Here, we report the TER spectra of an individual insulin fibril, the protein cast film and a short peptide (LVEALYL) microcrystal mimicking the fibril core. Two different types of TER spectra were acquired depending on the "roughness" of the probed surface at the molecular level. A fully reproducible, low-intensity, normal Raman-type spectrum was characteristic of the top flat surface of the microcrystal while highly variable, higher intensity TER spectra were obtained for the edges of the microcrystal, cast film, and fibril. As a result, two tip enhancement mechanisms of Raman scattering, long- and short-range, were proposed by analogy with the physical and chemical enhancement mechanisms, respectively, known for surface-enhanced Raman spectroscopy.

Polarized Raman Spectroscopy for Determining the Orientation of di-D-phenylalanine Molecules in a Nanotube.

Self-assembly of short peptides into nanostructures has become an important strategy for the bottom-up fabrication of nanomaterials. Significant interest to such peptide-based building blocks is due to the opportunity to control the structure and properties of well-structured nanotubes, nanofibrils, and hydrogels. X-ray crystallography and solution NMR, two major tools of structural biology, have significant limitations when applied to peptide nanotubes because of their non-crystalline structure and large weight. Polarized Raman spectroscopy was utilized for structural characterization of well-aligned D-Diphenylalanine nanotubes. The orientation of selected chemical groups relative to the main axis of the nanotube was determined. Specifically, the C-N bond of CNH3+groups is oriented parallel to the nanotube axis, the peptides' carbonyl groups are tilted at approximately 54° from the axis and the COO- groups run perpendicular to the axis. The determined orientation of chemical groups allowed the understanding of the orientation of D-diphenylalanine molecule that is consistent with its equilibrium conformation. The obtained data indicate that there is only one orientation of D-diphenylalanine molecules with respect to the nanotube main axis.

Hydrogen sulfide inhibits amyloid formation.

Amyloid fibrils are large aggregates of misfolded proteins, which are often associated with various neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, and vascular dementia. The amount of hydrogen sulfide (H2S) is known to be significantly reduced in the brain tissue of people diagnosed with Alzheimer's disease relative to that of healthy individuals. These findings prompted us to investigate the effects of H2S on the formation of amyloids in vitro using a model fibrillogenic protein hen egg white lysozyme (HEWL). HEWL forms typical ß-sheet rich fibrils during the course of 70 min at low pH and high temperatures. The addition of H2S completely inhibits the formation of ß-sheet and amyloid fibrils, as revealed by deep UV resonance Raman (DUVRR) spectroscopy and ThT fluorescence. Nonresonance Raman spectroscopy shows that disulfide bonds undergo significant rearrangements in the presence of H2S. Raman bands corresponding to disulfide (RSSR) vibrational modes in the 550-500 cm(-1) spectral range decrease in intensity and are accompanied by the appearance of a new 490 cm(-1) band assigned to the trisulfide group (RSSSR) based on the comparison with model compounds. The formation of RSSSR was proven further using a reaction with TCEP reduction agent and LC-MS analysis of the products. Intrinsic tryptophan fluorescence study shows a strong denaturation of HEWL containing trisulfide bonds. The presented evidence indicates that H2S causes the formation of trisulfide bridges, which destabilizes HEWL structure, preventing protein fibrillation. As a result, small spherical aggregates of unordered protein form, which exhibit no cytotoxicity by contrast with HEWL fibrils.

Polarized Raman Spectroscopy of Aligned Insulin Fibrils.

Amyloid fibrils are associated with many neurodegenerative diseases. The application of conventional techniques of structural biology, X-ray crystallography and solution NMR, for fibril characterization is limited because of the non-crystalline and insoluble nature of the fibrils. Here, polarized Raman spectroscopy was used to determine the orientation of selected chemical groups in aligned insulin fibrils, specifically of peptide carbonyls. The methodology is solely based on the measurement of the change in Raman scattered intensity as a function of the angle between the incident laser polarization and the aligned fibrils. The order parameters 〈 P2 〉 and 〈 P4 〉 of the orientation distribution function were obtained, and the most probable distribution of C=O group orientation was calculated. The results indicate that the peptides' carbonyl groups are oriented at an angle of 13±5° from the fibril axis, which is in consistent with previously reported qualitative descriptions of an almost parallel orientation of the C=O groups relative to the main fibril axis.

Deconstruction of stable cross-Beta fibrillar structures into toxic and nontoxic products using a mutated archaeal chaperonin.

Our group recently determined that a mutant archaeal chaperonin (Hsp 60) exhibited substantially enhanced protein folding activity at low temperatures and was able to deconstruct refractory protein aggregates. ATP dependent conversion of fibril structures into amorphous aggregates was observed in insulin amyloid preparations (Kurouski et al. Biochem. Biophys. Res. Commun. 2012). In the current study, mechanistic insights into insulin fibril deconstruction were obtained by examination of early stage complexes between Hsp60 and fibrils in the absence of ATP. Activity of the Hsp60 was significantly curtailed without ATP; however, some fibril deconstruction occurred, which is consistent with some models of the folding cycle that predict initial removal of unproductive protein folds. Chaperonin molecules adsorbed on the fibril surface and formed chaperonin clusters with no ATP present. We propose that there are specific locations on the fibril surface where chaperonin can unravel the fibril to release short fragments. Spontaneous coagulation of these fibril fragments resulted in the formation of amorphous aggregates without the release of insulin into solution. The addition of ATP significantly increased the toxicity of the insulin fibril-chaperonin reaction products toward mammalian cells.