THE LYSOSOMAL AMYLOID AGGREGATION OF B2-MICROGLOBULIN PROMOTES MULTIPLE MYELOMA PROGRESSION: A MOLECULAR FOCUS ON THIS PRO-CANCER MECHANISM

Maintenance of native folding is crucial for ensuring protein biological activity. Despite cells have evolved several control mechanisms to preserve folding, when they fail, proteins undergo destabilization or partial unfolding, events that favor the onset of protein misfolding diseases. These conditions are characterized by formation of protein aggregates, which impair tissues and organ functionalities. A widely studied example is β2-microglobulin (β2m), whose aggregation into amyloid fibrils underlies dialysis-related amyloidosis. Beyond this role, β2m also serves as prognostic factor for several hematological malignancies, including multiple myeloma (MM). Recent studies demonstrated that β2m actively contributes to MM pathogenesis by aggregating inside the lysosomes of tumor-associated macrophages. The resulting lysosomal damage induces NLRP3 inflammasome activation which sustain inflammation and malignant plasma cell proliferation. This thesis investigates the link between β2m fold stability and aggregation propensity under lysosomal-like conditions, and the resulting inflammatory response in macrophage-like cells. Wild-type and four β2m variants, namely D76N, ΔN6, D59P, and W60G were expressed, purified, and biophysically characterized in terms of pH and temperature stability, flexibility and aggregation propensity under physiological and lysosomal-like pH. In parallel, cellular studies were performed on THP-1-derived macrophages to monitor the effect of β2m uptake on lysosomal integrity, inflammasome activation, cytokine release, and cell viability. The biophysical analysis revealed three stability groups: ΔN6 and D59P, strongly destabilized by lysosomal pH and highly aggregation-prone; wt and D76N, with intermediate stability; and W60G, remarkably resistant to pH destabilization and aggregation. In cells, aggregation-prone variants formed large intracellular foci that increased in number and size over time. Furthermore, a prolonged NLRP3 and caspase-1 activation, as well as elevated IL-1β release was detected. In contrast, W60G, by remaining stable under lysosomal conditions, did not lead to aggregate formation, and neither to inflammasome activation and inflammatory cytokine release. Taken together, these results establish a direct link between β2m structural stability and its capacity to modulate macrophage-driven inflammation, supporting the evidence that β2m is not only a biomarker but also an active pathogenic factor in MM. Additionally, two distinct side projects were developed. The first aiming at the characterization of affinity molecules designed to stabilize β2m fold under lysosomal-like conditions; in the second a site-directed mutagenesis was applied on reactive intermediate deaminase to characterize the remarkable stability and enzymatic potential of this enzyme.

Grantor dc:publisher

Università degli Studi di Milano

Year dc:date

2025

Author dc:creator

• RIZZI, GIULIA

Contributors dc:contributor

• supervisor: S. Ricagno ; phd coordinator: S. Ricagno
• G. Rizzi
• RICAGNO, STEFANO

• Beta2-microglobulin
• multiple myeloma
• protein aggregation
• lysosome
• Settore BIOS-07/A - Biochimica

Statement dc:rights

• info:eu-repo/semantics/openAccess
• license:Creative commons
• license uri:http://creativecommons.org/licenses/by-sa/4.0/

Language dc:language

eng

Handle dc:identifier

https://hdl.handle.net/2434/1205155

OAI identifier oai:identifier

oai:air.unimi.it:2434/1205155