022 – Using subcellular fractionation and immunoprecipitation to investigate the cellular location and protein-binding partners of the paraneoplastic Yo antigens (CDR2 and CDR2L)
Ida Herdlevær (1) – Torbjørn Kråkenes (2) – Mette Haugen (1) – Manja Schubert (1) – Christian A. Vedeler (1)
Haukeland University Hospital, Department of Neurology, Bergen, Norway (1) – University of Bergen, Department of Clinical Medicine, Bergen, Norway (2)
Background: Paraneoplastic cerebellar degeneration (PCD) is a rare autoimmune-mediated neurodegenerative disease that can occur in patients with gynecological or breast cancers. PCD is characterized by cerebellar ataxia and paraneoplastic autoantibodies, such as anti-Yo. Paraneoplastic autoantibodies are initiated as an immune response against the underlying tumor, but can also bind to neuronal proteins, if they cross the blood-brain barrier. In the Purkinje neurons of the cerebellum, anti-Yo can bind to the cerebellar degeneration-related (CDR) proteins, CDR2 and CDR2Like. By today, the knowledge in terms of function and structure of CDR2 and CDR2L is quite limited. However, recent studies performed by our group imply that calcium signaling from the plasma membrane to the mitochondria is negatively impacted when anti-Yo is internalized by Purkinje neurons. Not knowing neither the exact function of CDR2 and CDR2L, nor the signaling pathways that are altered by anti-Yo binding, makes it difficult to develop new treatment strategies for PCD. By identifying CDR2/CDR2L location, function, and interaction partners, we aim to draw a clearer picture of the anti-Yo induced neurodegeneration.
Aim: To address the cellular- and functional properties of CDR2 and CDR2L in cancer cell lines.
Methods: Use subcellular fractionation to first, pinpoint the cellular location of CDR2/CDR2L to the nucleus, mitochondria, Golgi, endoplasmic reticulum (ER), cytosol or plasma membrane, and second concentrate the candidate proteins for further identification of protein-protein interactions. The CDR2/2L location is confirmed by Western blot analysis, whereas protein-protein interactions are identified by immunoprecipitation followed by Western blot analysis.
Preliminary results: We have found that the cellular location of CDR2 is to the cytosol and nucleus, whereas CDR2L is associated with membranous organelles, such as mitochondria, mitochondria-associated membranes (MAM) and ER.
Conclusion: We have demonstrated a cytoplasmic and nuclear cellular location of CDR2, suggesting a functional role in signal transduction and/or gene transcription. CDR2L localizes to cell organelles that are associated with cellular calcium homeostasis, such as the mitochondria, (MAM) and ER, implying a functional role in calcium signaling regulation.