Macromolecular crowding favors the fibrillization of ß2-microglobulin by accelerating the nucleation step and inhibiting fibril disassembly.

Hemodialysis-associated amyloidosis (HAA) involves the fibrillization of ß2-microglobulin (ß2M) and occurs in crowded physiological environments. However, how macromolecular crowding affects amyloid formation of ß2M remains elusive. Here we study the effects of macromolecular crowding on amyloid formation and fibril disassembly of wild-type human ß2M and its pathogenic mutant ΔN6. At strongly acidic pH2.5, the presence of a strong crowding agent (Ficoll 70 or dextran 70) not only dramatically accelerates the fibrillization of both wild-type ß2M and its ΔN6 variant by reducing the lag time to a large extent, indicating the acceleration of the nucleation phase, but also remarkably increases the amount of ß2M fibrils. At weakly acidic pH6.2, such an enhancing effect of macromolecular crowding on fibril formation is only observed for pathogenic mutant ΔN6, but not for wild-type ß2M which does not form amyloid fibrils in the absence and presence of a crowding agent. Thus, we propose that the monomers of ß2M form the nuclei, which is enhanced by macromolecular crowding, followed by the step of fibril elongation. Furthermore, at physiological pH, macromolecular crowding remarkably inhibits ß2M fibril disassembly by decreasing rate constants corresponding to fast and slow stages of fibril disaggregation. Our data demonstrate that macromolecular crowding favors the fibrillization of ß2M by accelerating the nucleation step and inhibiting fibril disassembly. Our findings provide clear evidence for the pathology of HAA that macromolecular crowding should be taken into account.

d-Ribose glycates ß(2)-microglobulin to form aggregates with high cytotoxicity through a ROS-mediated pathway.

ß(2)-Microglobulin (ß(2)M) modified with advanced glycation end products (AGEs) is a major component of the amyloid deposits in hemodialysis-associated amyloidosis (HAA). However, the effect of glycation on the misfolding and aggregation of ß(2)M has not been studied so far. Here we examine the molecular mechanism of aggregate formation of HAA-related ribosylated ß(2)M in vitro. We find that the glycating agent d-ribose interacts with human ß(2)M to generate AGEs that form aggregates in a time-dependent manner. Ribosylated ß(2)M molecules are highly oligomerized compared with unglycated ß(2)M, and have granular morphology. Furthermore, such ribosylated ß(2)M aggregates show significant cytotoxicity to both human SH-SY5Y neuroblastoma and human foreskin fibroblast FS2 cells and induce intracellular reactive oxygen species (ROS). Presence of the antioxidant N-acetylcysteine (1.0mM) attenuated intracellular ROS and prevented cell death induction in both SH-SY5Y and FS2 cells, indicating that the cytotoxicity of ribosylated ß(2)M aggregates depends on a ROS-mediated pathway in both cell lines. In other words, d-ribose reacts with ß(2)M and induces the ribosylated protein to form granular aggregates with high cytotoxicity through a ROS-mediated pathway. These findings suggest that ribosylated ß(2)M aggregates could contribute to the dysfunction and death of cells and could play an important role in the pathogenesis of ß(2)M-associated diseases such as HAA.

dbCRID: a database of chromosomal rearrangements in human diseases.

Chromosomal rearrangement (CR) events result from abnormal breaking and rejoining of the DNA molecules, or from crossing-over between repetitive DNA sequences, and they are involved in many tumor and non-tumor diseases. Investigations of disease-associated CR events can not only lead to important discoveries about DNA breakage and repair mechanisms, but also offer important clues about the pathologic causes and the diagnostic/therapeutic targets of these diseases. We have developed a database of Chromosomal Rearrangements In Diseases (dbCRID, http://dbCRID.biolead.org), a comprehensive database of human CR events and their associated diseases. For each reported CR event, dbCRID documents the type of the event, the disease or symptoms associated, and--when possible--detailed information about the CR event including precise breakpoint positions, junction sequences, genes and gene regions disrupted and experimental techniques applied to discover/analyze the CR event. With 2643 records of disease-associated CR events curated from 1172 original studies, dbCRID is a comprehensive and dynamic resource useful for studying DNA breakage and repair mechanisms, and for analyzing the genetic basis of human tumor and non-tumor diseases.