Regulation and Function of the Nuclear Transport Factor KPNA7

Genetic material in eukaryotic cells is isolated inside the double membrane of the nucleus. To facilitate the myriad processes which occur within, cells need a mechanism to transport important nuclear proteins through the semipermeable pore complexes of the nuclear membrane. While multiple transport pathways exist, the best characterized relies on a short amino acid sequence termed a nuclear localization signal (NLS). These signals are diverse but typically consist of one or two clusters of basic amino acids. Transport of NLS containing proteins is mediated by a pair of transport receptors, Importin-α and Importin-β. Importin-α is an adapter protein which contains separate domains to recognize NLS and bind to Importin β. Facilitation of transport through the nuclear pore is mediated by interactions of Importin-β with proteins in the nuclear pore complex.
Nuclear transport is highly controlled through coordination of expression of the transport machinery and dysregulation is implicated in many disease states. One level of this control is through the Importin-α family of transport receptors. The human genome encodes seven isoforms of Import-α which have a conserved structure consisting of an N-terminal, auto-inhibitory, Importin-β-binding domain, and a C-terminal core made up of alpha-helical armadillo repeats used for binding nuclear localization signals. Despite high conservation of primary, secondary and tertiary protein structures, small variations between the isoforms of the Importin-α family result in differing affinities of each isoform for different NLS. Through differential expression of the Imp-α isoforms, the differences in NLS affinity between them functions to regulate the overall nuclear import capacity for a given cell. This mechanism of regulation is utilized to help promote various developmental programs and alteration of the expression of even a single isoform can negatively impact cellular processes and promote disease.
In this study, we have characterized the biochemical function and regulation of the most recently discovered Importin-α isoform, KPNA7. Our analysis in Chapter II has identified differential regulation of KPNA7 by the Importin-β binding (IBB) domain it contains. The canonical function of the IBB domain, which features clusters of basic amino acids, is to bind in the NLS binding groove in the body of the receptor and prevent the association of an NLS in the absence of Importin-β. Only in the presence of both Importin-β and an NLS containing protein is there coordinated binding and formation of a heterotrimeric transport complex. In contrast to the other members of the Importin-α family, we have determined the IBB domain of KPNA7 displays a weaker auto-inhibitory function. KPNA7 binds strongly to Importin-β in the absence of an NLS and may adopt an open conformation in the nucleus. Furthermore, KPNA7 binds weakly to the Importin-α nuclear export factor CAS. We identified an Importin-β-dependent enhancement of NLS binding by KPNA7 but believe this is to be a mechanism which functions outside of relief from auto-inhibition. Together, these data suggest that, in addition to acting as a transport factor, KPNA7 may modulate the nuclear activity of NLS containing proteins.
In Chapter III of this work, we evaluated how mutations in KPNA7 identified in neurodevelopmental disease affect the NLS binding and transport capabilities of the receptor. These mutations result in amino acid substitutions proximal to the NLS binding groove of the receptor, and one, Glu344Gln, was determined to significantly reduce NLS binding and transport by KPNA7. This reduction was found to apply to both monopartite and bipartite NLS. We identified neuronal KPNA7 interacting proteins and characterized an interaction of KPNA7 with two heterogeneous nuclear ribonuclear proteins, hnRNP R and hnRNP U. The Glu344Gln substitution disrupted KPNA7 binding to each of the proteins. A functional bipartite NLS was identified in hnRNP R which is required for its nuclear localization. Binding and transport of this NLS, as well as a monopartite NLS in hnRNP U, were similarly reduced by the disease-associated mutation. We identified induction of KPNA7 expression during neurogenesis and evidence that regulation of KPNA7 expression in important for multiple developmental programs. Finally, investigation of the KPNA7 gene revealed a potential secondary effect of the disease-associated mutations via disruption of a binding site for the transcriptional insulator CTCF. Our data suggest a neuronal function for KPNA7 which is disrupted by disease-associated mutation.
In addition to our studies of KPNA7, in Chapter IV of this work, we investigated the use of the E. coli biotin-protein ligase BirA for identifying protein-protein interactions in cells. The commonly used BioID method utilizes a BirA mutant with an Arg118Gly amino acid substitution. We generated new BirA mutants with amino acid substitutions at position 118 of the enzyme. We utilized a set of biochemical and cell biological methods to investigate the biotinylation characteristics of these BirA mutants, and identified BirA(Arg118Lys) as a mutant with qualities useful for proximity labeling experiments.
Overall, this work furthers the understanding of the biochemical regulation and function of KPNA7 protein and determines how mutations associated with neurodevelopmental disease affect the characteristics we have described.