In situ characterization of protein aggregates in human tissues affected by light chain amyloidosis: a FTIR microspectroscopy study

Abstract

Light chain (AL) amyloidosis, caused by deposition of amyloidogenic immunoglobulin light chains (LCs), is the most common systemic form in industrialized countries. Still open questions, and premises for developing targeted therapies, concern the mechanisms of amyloid formation in vivo and the bases of organ targeting and dysfunction. Investigating amyloid material in its natural environment is crucial to obtain new insights on the molecular features of fibrillar deposits at individual level. To this aim, we used Fourier transform infrared (FTIR) microspectroscopy for studying in situ unfixed tissues (heart and subcutaneous abdominal fat) from patients affected by AL amyloidosis. We compared the infrared response of affected tissues with that of ex vivo and in vitro fibrils obtained from the pathogenic LC derived from one patient, as well as with that of non amyloid-affected tissues. We demonstrated that the IR marker band of intermolecular β-sheets, typical of protein aggregates, can be detected in situ in LC amyloid-affected tissues, and that FTIR microspectroscopy allows exploring the inter- and intra-sample heterogeneity. We extended the infrared analysis to the characterization of other biomolecules embedded within the amyloid deposits, finding an IR pattern that discloses a possible role of lipids, collagen and glycosaminoglycans in amyloid deposition in vivo.

Amyloid β-Sheet Secondary Structure Identified in UV-Induced Cataracts of Porcine Lenses using 2D IR Spectroscopy

“…1636 cm −1 is a typical frequency for short β-sheets found in many soluble proteins. 29 In the case of γD-crystallin, the 1636 cm −1 frequency is formed by 2 Greek Key folds (4 strands of antiparallel β-sheets) found in each of the two domains (Fig. 2).…”

“…In a slice of lens tissue, no other biomolecules absorb appreciably in the same frequency range as the amide I modes, so we attribute all the signals in our 2D IR spectra to proteins. 28; 29 As we explain further in the Discussion, only 4 or 5 β-strands in the tight molecular geometry of amyloid fibers are necessary to form the characteristic 2D IR features. Thus, 2D IR spectroscopy provides a diagnostic for amyloid β-sheet secondary structure even if fibers formed by those amyloid β-sheets are in a size regime that may be difficult to detect with other approaches such as TEM.…”

Conformational stability and dynamics in crystals recapitulate protein behaviour in solution

“…Figure 3A shows the second derivatives in the Amide I region of the FTIR absorption spectra collected at room temperature for the wt β2m protein in solution and in the crystalline state. The Amide I band is due to the C=O stretching mode of the peptide bond and it is particularly sensitive to the protein secondary structures, including the intermolecular β-sheet structures in protein aggregates (26,27,29,(35)(36)(37). The second derivative spectrum of crystalline wt β2m displays sharper Amide I peaks suggesting more rigid protein molecules compared to what observed in the solution spectra ( Figure 3A).…”

“…Moreover, protein stability was assessed for the set of the three mutations (D76N, wt and the highly stable W60G), using Fourier transform infrared (FTIR) spectroscopy, a biophysical technique which is amenable for monitoring both solid and liquid samples by following the changes in secondary structure. FTIR spectroscopy has been widely adopted for the analysis of protein samples in different conditions, resulting a well suitable technique to compare the secondary structures of proteins both in solution and in crystals as well as in intact cells, tissues and entire organisms (22)(23)(24)(25)(26)(27)(28). In particular, a recent work published by our group showed the feasibility of collecting FTIR spectra on single protein crystals and highlighted the high quality of the FTIR spectra collected on β2m crystallin samples (29).…”

Dynamics of amyloid-like aggregation and gel formation of hen egg-white lysozyme in highly concentrated ethanol solution

“…Recently, amyloid fiber formation has been investigated using various techniques including far-UV CD [11][12][13][14][15], IR absorption [13][14][15][16][17][18], nuclear magnetic resonance (NMR) *E-mail: rkita@keyaki.cc.u-tokai.ac.jp spectroscopy [16,19,20], and small-angle X-ray and neutron scattering [10,21,22]. Although these spectroscopic techniques yield detailed information about the structures of protein molecules, they cannot provide information about their state of aggregation.…”