Human IRAK kinases differentially alter metabolic regulation and mitochondrial function in Saccharomyces cerevisiae

Interleukin-1 receptor-associated kinases (IRAKs) are Ser/Thr protein kinases characterized by an N-terminal Death domain (DD). Upon stimulation of Toll-like receptors (TLRs) or the interleukin-1 receptor (IL-1R), IRAKs are recruited to supramolecular signalling complexes, known as myddosomes, through interactions between their DDs and the adaptor protein MyD88. Myddosomes are essential for the activation of nuclear factor kappa B (NF-{kappa}B) in response to diverse pathogen- and damage-associated molecular patterns (PAMPs and DAMPs), and they contribute to inflammation, cell survival, and proliferation. In the hierarchical assembly of the myddosome, MyD88 first recruits IRAK4, which serves as a scaffold for the subsequent binding of IRAK1 and/or IRAK2. To explore alternative models for studying IRAK function, we expressed human IRAK1, IRAK2 and IRAK4 individually in Saccharomyces cerevisiae and performed a comparative analysis. Heterologous expression of these kinases, especially IRAK4, impaired yeast growth; an effect dependent on its kinase activity. Transcriptomic and biochemical assays revealed that IRAK1 and IRAK4, but not IRAK2, differentially impacted metabolic regulation. Notably, IRAK4 induced mitochondrial fragmentation and mitochondrial membrane potential depolarization, whereas IRAK1 had the opposite effect. Additionally, IRAK4 led to actin depolarization and vacuole fragmentation. Based on these findings, we develop two yeast-based bioassays to screen for IRAK4 kinase inhibitors: one based on growth recovery and another using a fluorescent reporter. We provide proof-of-concept that both assays are suitable for evaluating IRAK4 function and its pharmacological inhibition. ImportanceIRAK kinases are essential components of the myddosome signalling complex, a key mediator of the innate immune response, with IRAK4 playing a pivotal role in the recruitment of IRAK1 and/or IRAK2. Although the precise cellular functions of IRAK-dependent phosphorylation remain incompletely understood, IRAK inhibitors are emerging as promising therapeutic candidates for the treatment of autoimmune disorders, such as rheumatoid arthritis, and various cancers, including acute myeloid leukemia. To date, most insights into IRAK function have been derived from studies in animal models, particularly mice. Our work establishes Saccharomyces cerevisiae as a convenient and genetically tractable platform for in vivo analyses of human IRAKs. We demonstrate that IRAK kinases induce metabolic deregulation and growth inhibition in yeast. Engineered S. cerevisiae strains could therefore be exploited for the preclinical screening of anti-inflammatory and antitumor compounds targeting IRAK signalling, potentially contributing to the development of therapies for severe human diseases.