Deciphering the role of HAX1 in neutrophil differentiation

Fan, Yanxin (2022): Deciphering the role of HAX1 in neutrophil differentiation. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Loss-of-function mutations in HAX1 (HS1-associated protein X1) in humans result in autosomal recessive severe congenital neutropenia (SCN), yet the exact molecular function of HAX1 remains unclear. Here, we aimed to determine its specific cellular function and elucidate potential pathophysiology related to HAX1 deficiency. Using mitochondrial swelling and carbonate extraction methods, we demonstrate that HAX1 is located in the mitochondrial intermembrane space. We performed mass-spectrometry (MS) studies and identified Caseinolytic peptidase B protein homolog (CLPB) as a novel interacting protein of HAX1 in HEK293T cells. CLPB is a member of the ATPase superfamily associated with diverse cellular activities (AAA+), which was previously shown to serve as a mitochondrial disaggregase in mammalian cells. Biallelic mutations in CLPB cause a rare neurological disorder associated with impaired cognitive development, 3-methylglutaconic aciduria, and congenital neutropenia. We show that human mutations leading to SCN in either HAX1 or CLPB disrupt the mutual interactions between both proteins of the corresponding proteins. Using an approach combining SILAC and MS, we reveal that both HAX1 and CLPB deficiencies in myeloid leukemia cell lines cause perturbed mitochondrial protein turnover, mainly in the respiratory complex I, respiratory complex III and tricarboxylic acid cycle (TCA cycle). As a result, mtROS production is enhanced in the absence of HAX1. In comparative studies of the proteome (WT vs HAX1 deficient PLB-985 cells), HSP27 is found to be mostly dysregulated in HAX1-/- PLB-985 cells. Intriguingly, through a series of biochemical methods, such as Western blotting of cellular extracts upon fractionation, as well as by confocal microscopy experiments, we found a reduced phosphorylation state and reduced solubility of HSP27 in HAX1-/- cells. Further interactome assay of HSP27 demonstrate that HSP27 is associated with respiratory complexes and ribosomal subunits in mitochondria. Thus, these data indicate a critical involvement of the CLPB/HAX1 axis in maintaining mitochondrial complex stability and mitochondrial protein synthesis. Importantly, HSP27’s reconstitution reverses the elevated ROS production in HAX1-/- PLB-985 cells and the perturbed neutrophil differentiation in HAX1-/- iPSCs model. Our study discovers a new and essential role of HAX1 in maintenance of mitochondrial proteostasis.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 21. März 2022
• 1. Bericht­erstatter:in: Förstemann, Klaus