PKCdelta as a key molecule in human (auto)immunity

Human autoimmune disorders present in various forms, often with a life long burden of high morbidity and even mortality. Many different ways lead to the loss of immune tolerance and often the origin is multifactorial impeding our good comprehension of it. Recently, patients with a mutation in PRKCD encoding Protein kinase c delta (PKCd) have been identified as a monogenic prototype for one of the most prominent forms of humoral systemic autoimmune diseases, systemic lupus erythematosus (SLE). PKCd is a signaling protein with multiple downstream target proteins and functions in various signaling pathways. Interestingly, mouse models have indicated a special role of the ubiquitously expressed protein in the control of B cell tolerance revealed by the severe autoimmunity in Prkcd knock out mice as the major phenotype. Therefore the newly identified PKC- deficient patients and their lymphocytes grant a unique opportunity to investigate the role of PKCd in the control of immune tolerance in humans. Based on their complementary long-term expertise in the field of human immune mediated diseases the laboratories of K. Warnatz (CCI Freiburg, Germany) and K. Boztug (CeMM Vienna, Austria) will screen large European SLE cohorts for mutations in PRKCD. We will investigate lymphocyte and especially B cell development and homeostasis in the affected patients. The analysis of signaling pathways in the absence of PKCd will shed light on the role of PKCd in the signaling networks downstream of different receptors in human B-, T- and NK cells. New interaction partners shall be identified by proteomics. This altered signaling cumulates in a disturbed cell cycle control, survival and proliferation of B cells possibly underlying the autoimmune process in PKCd deficiency. Functional studies of primary B cells will be used to dissect the contributing factors. Finally, the combined genetic and proteomic analysis of this study allows for the discovery of new PKC-related proteins involved in human autoimmunity. Integrating the signaling, proteomic and functional data this human model of systemic autoimmune disease will not only provide a better insight into the underlying pathomechanism in the enhanced B cell growth and loss of tolerance in PKC deficiency, but also into the more general pathomechanisms relevant for the development of SLE and implicate thereby new targeted therapeutic options for human autoimmune disease in order to re-establish tolerance.

In our project FWF I2250-B28 in collaboration with Professor Dr. Warnatz (Freiburg, Germany) we have set out to unravel the complex machinery behind the function of PKCd, thereby gaining fundamental understanding of PKCd signaling and systemic lupus erythematosus (SLE) pathogenesis through an example of monogenic gene defect underlying SLE. Our further goals were to identify and characterize novel defects causing SLE, thereby further expanding our knowledge of SLE pathogenesis. In the course of the project, we have successfully established a cohort of more than 40 patients from multiple (inter)national collaboration partners form Austria, Hungary, France, and Iran. We have done extensive exclusion of known SLE-causing gene defects, resulting in a genetic diagnosis for 15 patients. Furthermore, we have used exome sequencing to analyze around 20 patient cases, using state- of-the-art bioinformatics approaches, including network-based variant prioritization currently developed in-house. The analysis of potentially causal gene defects from this dataset lead to the identification of four novel SLE candidate genes. We have performed functional assays to establish causality and to describe the pathomechanisms behind the gene defects. As well as focusing on novel gene defects, we have simultaneously analyzed proteomics pull- down data on PKCd with RNA-seq and phosphoproteomics data from the group of Prof. Warnatz using complementary methods. This analysis has revealed interesting synergies and discrepancies, identified functional subgroups within all realms of data and gave useful insights in the pleiotropic effects of PKCd function on multiple biological levels. To further elucidate the complex machinery behind PKCd function, we have combined our in-house proteomics pull-down data that identified interaction partners of PKCd with RNA-seq and global and phosphoproteomics data from the group of Professor Dr. Warnatz. We have integrated this multi-dimensional data in a bipartite PKCdome network, with the use of co- expression and protein-protein interaction data. Analysis of the PRCdome has revealed insights by identifying key drivers and hubs that contribute to PKCd-related pathogenesis. The combination of identification of single gene defects causing an SLE-like phenotype, as well as a systematic approach to integrate and analyze multi-dimensional data to create the PKCdome has revealed valuable insights and allows for a better understanding of SLE pathogenesis through key players of the disease.