β2-Microglobulin on Edge: Stress-induced conformational switch

Research Summary: We characterize stages of β2-microglobulin amyloid assembly under low pH and varying salt concentrations. Our results illustrate that oligomers and protofibrils follow a nucleation-independent pathway, whereas amyloids follow nucleation process.

Author interview: Khushboo Rani, a 5th-year PhD candidate (thesis submitted) in Dr. Neha Jain’s lab at IIT Jodhpur, is interested in understanding the crosstalk between amyloidogenic proteins in Parkinson’s disease progression.

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Lab: Dr. Neha Jain, Indian Institute of Technology Jodhpur

What was the core problem you aimed to solve with this research?

β2-Microglobulin (β2m) is a long polypeptide, non-covalently linked to the heavy chain of Major Histocompatibility Complex (MHC) class I, located on all nucleated cells. During the normal catabolic cycle, β2m dissociates from the heavy chain of the MHC Class I molecule, is shed into the plasma, and is transported to the kidney, where it is primarily degraded and excreted. In patients undergoing dialysis due to kidney failure, β2m from plasma does not get removed; therefore, its concentration increases to ~60-fold. The increased accumulation of β2m initiates its aggregation and amyloid formation, which contributes to various kidney-related diseases such as chronic kidney disease (CKD), end-stage renal disease (ESRD), and dialysis-related amyloidosis (DRA). However, how β2m assembles into amyloids in patients undergoing long-term renal dialysis remains elusive. Moreover, it is difficult to capture the conformational transition as the intermediate species are transient and heterogeneous. Our research aimed to fill this gap and identify the different stages during the amyloid assembly of β2m under in vitro conditions. We believe that unraveling the process may provide insights into the amyloid formation and help us understand the initiation of the disease at an early stage.

How did you go about solving this problem?

Establishing the different stages of amyloid formation by β2m was the first and most crucial part of this study. After a series of experiments, we identified conditions where we were able to form three different conformers of β2m, namely oligomers, protofibrils, and amyloids, at low pH with varying salt concentrations. We used various biophysical and biochemical techniques to establish the formation of three distinct conformations of β2m. Through electron microscopy images, we were able to distinguish between oligomers, protofibrils, and amyloids. The on-pathway intermediates during amyloid formation are usually unstable and heterogeneous and therefore pose a difficulty for characterization. However, in our experimental conditions, we were able to prove that the conformers were stable up to four days, enabling us to interrogate the detailed analysis.

How would you explain your research outcomes (Key findings) to the non-scientific community?

Biomolecules such as proteins are fundamental molecules essential for the functioning of all living organisms. Unfavorable conditions can disrupt the protein structure, causing them to lose shape and lead to the accumulation of misfolded protein aggregates, known as amyloids, in various parts of the body. These aggregates are a hallmark of several amyloidogenic diseases, including dialysis-related amyloidosis (DRA), Parkinson’s disease, and Type 2 diabetes mellitus. In most disease conditions, amyloid formation involves different on-pathway intermediates such as oligomers and protofibrils. However, the precise mechanism by which monomers convert into these species and drive disease progression is still actively being investigated.

Our lab is deeply interested in understanding the process of amyloid formation in disease initiation and progression. The present work was rooted in curiosity and led to unexpected yet meaningful and insightful research outcomes. In an effort to understand the underlying process of amyloid formation during DRA, we identified and characterized three stages of β2-microglobulin (β2m) aggregation, namely, oligomers, protofibrils, and fibrils, while varying salt concentrations and agitation under low pH conditions. We found that when subjected to specific conditions, the protein underwent a profound change in its structure that allowed it to transform into various conformers. Once we had the three conformers at our disposal, we were poised to understand their specific features that differentiated them from each other. We observed them under a microscope and found that all three appeared to be very different. Oligomers were just blobs, protofibrils were tiny ribbons, whereas amyloids had rope-like structures. All of them have varied potential to bind to membranes and generate free reactive oxygen species, which are harmful to the cells. Although the conformers displayed contrasting features, they all showed similar stability and potential to transform into amyloid fibrils, similar to those seen in diseased conditions. Altogether, we successfully deciphered the different intermediates during the β2m amyloid assembly that may resemble the disease scenario.

Every step towards understanding the mechanism brings us closer to winning the battle against deadly diseases.

What are the potential implications of your findings for the field and society?

It is imperative to understand the mechanism of disease progression for early diagnosis and better treatment. We directed our efforts to mimic the disease condition and capture the process in laboratory conditions. We succeeded in achieving our goal to understand the amyloid formation by β2m via different intermediate conformers. We believe that the results will have several significant implications in the scientific field as well as in society. Since we have characterized three stages, now we can target them specifically to prevent the disease at different stages. The study will also aid in designing interventions for the early diagnosis of the disease. Since we have broken down our complete findings into simpler explanations, our results would be more accessible to laymen in understanding the amyloid disease in general. The insights into the mechanism of β2m conformers gained from this study may be beneficial to guide future possible avenues for designing inhibitors and attractive therapeutic strategies for the treatment of DRA and other amyloidogenic diseases.

What was the exciting moment during your research?

Throughout my research, every outcome, whether positive or negative, served as a stepping stone. These experiences enriched my understanding, sparked excitement, and deepened my learning. I have learned from my supervisor, Dr. Neha Jain, in designing and executing the experiments, which inspired and illuminated our discussions. During this study, the most exciting moment for us was to identify the three different stages of β2m, oligomers, protofibrils, and amyloids under controlled conditions, which not only distinguishes the mechanistic difference but also provides crucial insights into early, potentially more toxic intermediates in amyloid formation. It was an important step toward understanding disease initiation at an early stage. Even though the journey was filled with challenges due to the heterogeneous nature of the conformers, her unwavering encouragement and constant support played a crucial role in surpassing the obstacles. Beyond the excitement of research, overcoming obstacles, collaborative efforts, and ongoing learning made the journey truly rewarding.

Paper reference: Rani K, Gurnani B, Jain N., (2025), Probing a salt-induced conformational switch in β2-microglobulin under low pH conditions. FEBS J, doi: 10.1111/febs.70142, PMID: 40418633. https://febs.onlinelibrary.wiley.com/doi/10.1111/febs.70142

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