Light chain (AL) amyloidosis is a devastating, complex, and incurable protein misfolding disease. It is characterized by an abnormal proliferation of plasma cells (fully differentiated B cells) producing an excess of monoclonal immunoglobulin light chains that are secreted into circulation, where the light chains misfold, aggregate as amyloid fibrils in target organs, and cause organ dysfunction, organ failure, and death. In this article, we will review the factors that contribute to AL amyloidosis complexity, the findings by our laboratory from the last 16 years and the work from other laboratories on understanding the structural, kinetics, and thermodynamic contributions that drive immunoglobulin light chain-associated amyloidosis. We will discuss the role of cofactors and the mechanism of cellular damage. Last, we will review our recent findings on the high resolution structure of AL amyloid fibrils. AL amyloidosis is the best example of protein sequence diversity in misfolding diseases, as each patient has a unique combination of germline donor sequences and multiple amino acid mutations in the protein that forms the amyloid fibril.

Background: Collagenous tissues are composed of precisely oriented, tightly packed collagen fibril bundles to confer the maximal strength within the smallest volume. While this compact form benefits mobility, it consequentially restricts vascularity and cell density to a minimally viable level in some regions. These tissues reside in a homeostatic state with an unstable equilibrium, where perturbations to structure or molecular milieu cause descension into a long-term compromised state. Several studies have shown that glycosaminoglycans are key molecules required for healthy tissue maintenance. Our long-term goal is to determine if glycosaminoglycans serve a critical function of stabilizing soluble monomeric collagen in the interstitial fluid that bathes tissue for immediate availability in tissue development and repair in vivo. Materials and Methods: To test glycosaminoglycan and collagen interactions at the most fundamental level, we have explored the effect of the monosaccharides that populate the glycosaminoglycans of the extracellular matrix on collagen assembly kinetics, pre-established matrix stability, and collagen incorporation into a preassembled matrix. Results: Results showed that monosaccharides increased the threshold concentration required for spontaneous polymerization by at least three orders of magnitude. When the monosaccharides were introduced to a pre-existing collagen network, fibrillar dissociation was undetectable. Fluorescent-labeling studies illustrated that in the presence of the saccharide solution, soluble collagen maintains the functional capacity to integrate into a pre-existing network. Conclusion: This work demonstrates a feasible role for glycosaminoglycans in supporting tissue remodeling and highlights the potential importance of age-related deterioration of glycosaminoglycan biosynthesis in reference to the homeostasis of collagen-based tissues.

Crystallins comprise the protein-rich tissue of the eye lens. Of the three most common vertebrate subtypes, β-crystallins exhibit the widest degree of polydispersity due to their complex multimerization properties in situ. While polydispersity enables precise packing densities across the concentration gradient of the lens for vision, it is unclear why there is such a high degree of structural complexity within the β-crystallin subtype and what the role of this feature is in the lens. To investigate this, we first characterized β-crystallin polydispersity and then established a method to dynamically disrupt it in a process that is dependent on isoform composition and the presence of divalent cationic salts (CaCl 2 or MgCl 2 ). We used size-exclusion chromatography together with dynamic light scattering and mass spectrometry to show how high concentrations of divalent cations dissociate β-crystallin oligomers, reduce polydispersity, and shift the overall protein surface charge—properties that can be reversed when salts are removed. While the direct, physiological relevance of these divalent cations in the lens is still under investigation, our results support that specific isoforms of β-crystallin modulate polydispersity through multiple chemical equilibria and that this native state is disrupted by cation binding. This dynamic process may be essential to facilitating the molecular packing and optical function of the lens.

The use and incorporation of type I collagen (COL) in biomaterials and regenerative medicine have remained challenging due to COL’s ability to spontaneously polymerize at physiological pH in vitro. Previous work has shown that the addition of monosaccharides can delay COL polymerization and increase its solubility under neutral conditions by three orders of magnitudetwo features that enable structure retention and integration into pre-existing fibrous networks. We expand on these findings and describe the thermodynamic effects of these monosaccharides on the growth phase of COL polymerization. We derive van’t Hoff plots for each experimental condition and use these data to support an indirect mechanism for delayed COL assembly profiles based on solvent ordering. We observe that the presence of monosaccharides in a COL solution neither altered nor permanently inhibited the formation of COL fibrils. Finally, we demonstrate the utility of our findings through a proof-of-concept study which showed how the presence of these monosaccharides aided in the delivery of a high (2 mg/mL) concentration of neutralized, monomeric COL into simple patterns without disrupting COL structure formation. Our findings support the application of entropy-regulating systems such as monosaccharides in manipulating the dynamics of some self-assembling proteins to aid in biomaterials design.

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