DIORA1 in molecular signaling and cellular function

<p dir="ltr">Autoimmune diseases are a complex and diverse group of disorders arising from an interplay between genetic predisposition and environmental triggers. Genome-wide association studies have identified the link between polymorphisms in the FAM167A-BLK locus and several rheumatic autoimmune diseases, including Sjögren's disease, systemic lupus erythematosus, rheumatoid arthritis, and systemic sclerosis. This locus encodes two genes: BLK, which encodes B-lymphocyte kinase, and FAM167A. Expression quantitative trait loci analyses show that disease-associated variants are strongly associated with an increased FAM167A expression, while effects on BLK are modest. Elevated levels of the FAM167A-encoded protein DIORA1 have also been observed in inflamed salivary glands of Sjögren's disease patients. Despite these findings, the molecular, cellular, and systemic functions of DIORA1 remain unknown. To address this, this thesis combines molecular, cellular, and in vivo approaches to elucidate the role of DIORA1 in immune regulation and its potential contribution to autoimmunity.</p><p dir="ltr">Using proximity biotinylation and co-immunoprecipitation assays, we identified a direct interaction between DIORA1 and three conserved regions of the cytoskeleton-regulating MRCK kinase family - the KIM motif, C1-PH, and CNH domains. AlphaFold-based modeling guided further mapping experiments, confirming that the C-terminal half of DIORA1 mediates MRCK binding. Functionally, DIORA1 knockdown in human cells reduced phosphorylation of MRCK substrates, upregulated epithelial-mesenchymal transition signature, and increased cell invasion in our functional assays. This phenotype was reversed by pharmacological inhibition of MRCK activity, confirming the role of the DIORA1-MRCK interaction in regulating cytoskeletal reorganization and cell motility.</p><p dir="ltr">Further investigation into MRCK regulation revealed a previously unrecognized family of DIORA1-related proteins containing a conserved MRCK-KIM-binding motif, which we named KIMURA. This motif was both necessary and sufficient for MRCK interaction, with three key amino acid residues critical for binding. While most KIMURA proteins depended on these residues, some retained MRCK binding despite their mutation, suggesting functional specialization within the family. Transcriptomic profiling after systematic knockdown of KIMURA family members revealed that KIMURA-containing proteins may regulate both shared and distinct gene programs and that this can depend on cellular context. Their co-expression with specific MRCK isoforms across tissues, along with genome-wide associations to anthropometric, autoimmune, and cancer traits, implies their physiological relevance in modulating MRCK signaling in tissue-specific and disease-relevant contexts.</p><p dir="ltr">To explore the role of DIORA1 in immune regulation in vivo, we generated Fam167a knockout (Dioral^-/-) mice and performed detailed characterization. Knockout animals exhibited cell-intrinsic alterations in B and T cell subsets, including expanded effector/memory and T follicular helper (Tfh) cells. Transcriptomic analysis of Dioral^-/- Tfh cells revealed elevated activation signatures but also signs of reduced cellular fitness. These T cell alterations were accompanied by impaired antigen-specific IgA production, driven by B cell-intrinsic defects following T-dependent immunization. Furthermore, phosphoproteomic analysis of Diora1-deficient B cells revealed disrupted TGF-β signaling, a pathway essential for IgA class switching and regulation of T helper cell responses, suggesting a mechanistic link between Diora1 loss and altered immune homeostasis.</p><p dir="ltr">To support future work dissecting DIORA1 and KIMURA functions with spatiotemporal precision, we developed an optogenetic CRISPR platform, BLU-VIPR, that uses blue light to induce expression of ribozyme-flanked guide RNAs. This genetically encoded system enabled multiple gene editing approaches, including CRISPR knockout, CRISPR activation, and base editing. Coupled with fiber-optic illumination, BLU-VIPR enabled precise spatiotemporal genome editing of T cells within a single lymph node in living mice, demonstrating targeted in vivo perturbation in otherwise inaccessible immune settings.</p><p dir="ltr">In summary, this thesis identifies DIORA1 as a regulator of MRCK kinases, suggesting a role in cytoskeleton regulation. By integrating molecular interaction mapping, structural modeling, transcriptomic and proteomic profiling, and in vivo immunological studies, we uncover a mechanistic role for DIORA1 in shaping immune responses, possibly through MRCK modulation. The discovery of the KIMURA protein family expands the repertoire of MRCK regulators and offers new insight into how cytoskeletal signaling networks are tuned in health and disease. Together, these findings may help explain the genetic risk associated with the FAM167A-BLK locus and suggest a role for DIORA1 in maintaining cellular homeostasis and immune regulation.</p><h3>List of scientific papers</h3><p dir="ltr">I. Autoimmunity-associated DIORA1 binds the MRCK family of serine/threonine kinases and controls cell motility. <b>Tršelič, T.</b>, Pelo, N., Martin de Fremont, G., lyer, V. S., Richardsdotter Andersson, E., Ottosson, V., Frei, D. A., Baas, E., Nyberg, W. A., Thorlacius, G. E., Mentlein, L., Boddul, S. V., Sandu, I., Velasquez Pulgarin, D., Végvári, Á., Gerlach, C., Wermeling, F., Sunnerhagen, M., Wallner, B., Espinosa, A., Wahren-Herlenius, M. Proc Natl Acad Sci U S A. 2025 Oct 7;122(40):e2426917122. <a href="https://doi.org/10.1073/pnas.2426917122" rel="noreferrer" target="_blank">https://doi.org/10.1073/pnas.2426917122</a></p><p dir="ltr">II. The KIMURA motif defines a family of MRCK-binding proteins. <b>Tršelič, T.</b>, lyer, V. S., Richardsdotter Möller, E., Ottosson, V., Ottosson, L., Thorlacius, G. E., Espinosa, A., Wahren-Herlenius, M. [Manuscript]</p><p dir="ltr">III. DIORA1 regulates T follicular helper cell differentiation and immunoglobulin class switch. Richardsdotter Möller, E., <b>Tršelič, T.</b>, Gerstner, C., Meneghel, L., Schurz, H., Martin de Fremont, G., Margenat Arqué, C., Iyer, V. S., Ottosson, V., Thorlacius, G. E., Boddul, S. V., Bharaj, T. K., Mentlein, L., Pelo, N., Velasquez Pulgarin, D., Liu, J., Adner, M., Skarstein, K., Gerlach, C., Noble, J., Reinius, B., Wermeling, F., Espinosa, A., Wahren-Herlenius, M. [Manuscript]</p><p dir="ltr">IV. Light-induced expression of gRNA allows for optogenetic gene editing of T lymphocytes in vivo. Velasquez Pulgarin, D., Pelo, N., Ferrandiz, L., <b>Tršelič, T.</b>, Nyberg, W. A., Bowlin, G., Espinosa, A. Nucleic Acids Res. 2025 Mar 20;53(6):gkaf213. <a href="https://doi.org/10.1093/nar/gkaf213" rel="noreferrer" target="_blank">https://doi.org/10.1093/nar/gkaf213</a></p>