UNDERSTANDING THE NON-CANONICAL REGULATION OF SREBP IN ASPERGILLUS FUMIGATUS

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive pulmonary aspergillosis (IPA), with high mortality rates in immunocompromised individuals. Adaptation to the hypoxic microenvironment of IPA is crucial for fungal virulence. The transcription factor SrbA, a Sterol Regulatory Element-Binding Protein (SREBP) homolog, plays a key role in this hypoxic response and regulates genes involved in hypoxic growth, sterol biosynthesis, and azole resistance, making it an attractive therapeutic target. However, SrbA activation in A. fumigatus lacks critical components of the canonical SREBP pathway, such as SCAP, Site-1 protease (S1P), and Site-2 protease (S2P). Instead, the rhomboid protease RbdB, signal peptide peptidase SppA, and Dsc E3 ubiquitin ligase complex have been identified as essential for SrbA activation through regulated intramembrane proteolysis (RIP). This study aimed to elucidate the molecular mechanisms governing SrbA regulation and function. SrbA's post-translational modification profile and membrane topology were characterized, revealing its type II transmembrane orientation within the endoplasmic reticulum (ER). Site-directed mutagenesis identified the core mutations in the Ub3 mutant: alone among the five mutants within SrbAUb3, the F443L mutation within the transmembrane domain, exhibited a complete growth defect under hypoxia and the absence of the cleaved, transcriptionally active SrbA fragment. This suggested F443L may disrupt SrbA cleavage by RbdB and SppA, a crucial activation step. Overcoming challenges like a lack of characterized glycoproteins and specific antibodies, a rigorous approach involving cross-validation and protocol optimization was adopted to ensure reliable results. This study advanced the understanding of SrbA regulation, laying the groundwork for exploring transcriptional regulation and virulence mechanisms in pathogenic fungi, paving the way for novel therapeutic strategies targeting the non-canonical SREBP activation pathway in invasive aspergillosis.