Cellular and Molecular Features of Gene Mutations in Primary Ciliary Dyskinesia

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Summary The genetic syndrome primary ciliary dyskinesia (PCD) is characterized by defects in cilia motility resulting in bronchiectasis, chronic sinusitis, infertility and cardiac malformations. Intense efforts over the past decade have uncovered pathways required for cilia assembly and discovered genes that are mutant in PCD. The next goal is to understand the specific cellular consequences of different classes of mutations to identify therapeutic avenues, similar to the approach used for cystic fibrosis. Cilia motility is dependent on dynein motors, which are fixed in large complexes on the skeletal axoneme of cilia. Genes that code for these dynein motors commonly harbor PCD mutations. However, studies in model organisms and human cells have revealed that dyneins must be prepared in the cytoplasm by dynein axoneme assembly proteins (DyAPs). We found that DyAPs colocalize with dyneins in cytoplasmic foci of multiciliated cells. Mutation in DyAPs results in an absence of dynein motors within the cilia and consequentially, impaired cilia function, also resulting in PCD. We have recently identified a subset of DyAPs composed of HEATR2/SPAG1/DNAAF2, which form an early scaffold to function in an initiation phase of dynein assembly. We propose that this scaffold engages with a second group of DyAPs, that we term the folding phase complex, which carries out dynein assembly for transport to the cilia. We observed that mutations in the initiation phase DyAPs result in the formation of cytoplasmic aggregates tagged with the proteostasis adapter SQSTM1/p62, suggesting that abnormal protein processing leads to pathway interruption. Consistent with this concept, these aggregates contain all three proteins of the initiation phase complex. We hypothesize that mutations in DyAPs interrupt the complex function, lead to the formation of intracellular aggregates of non- functioning machinery and a failure to move dynein motors to the cilia. We address this question with these aims: (1) A functional analysis of human mutations of the initiation phase DyAPs to determine their effect on the pathway related to the formation of aggregates, intracellular trafficking, and protein interactions with other DyAPs and (2) a biochemical analysis to identify the composition of the aggregates and the associated activity of the cellular proteostasis pathways to mitigate formation. Our goal is to identify factors related to the formation of DyAP aggregates, determine how the DyAP pathway is interrupted and ask if aggregates formation can be manipulated to rescue sufficient amounts of protein for function, as a first step toward conceptualizing a specific treatment for one class of PCD mutations.