Kapβ2 recognition of cargos involved in neurodegenerative/neurodevelopmental diseases

Ut Southwestern Medical Center, Dallas TX

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Abstract

Karyopherin ?2 (Kap?2) is a nuclear import factor that transports many heterogeneous ribonucleoproteins (hnRNPs) into the nucleus by recognizing the nuclear localization signal (NLS) known as PY-NLS. Many mutations in Kap?2 cargoes are linked to neurological diseases by causing protein mislocalization to the cytoplasm, followed by aggregate formation and fibrillization. In this proposal, I seek to understand the Kap?2- mediated nuclear import mechanism of cargoes Fused in Sarcoma (FUS) and hnRNP H2, which are involved in neurodegenerative and neurodevelopmental disease, respectively. Aim 1. Many mutations in the FUS proteins are associated with familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. One mutation results in the mutant FUS495X protein, which is truncated at the C-terminus and missing its PY-NLS and causes a severe type of familial ALS. When FUS495X is expressed in cells, it is localized to both the cytoplasm and the nucleus, suggesting a fraction is still imported into the nucleus despite missing its PY-NLS. Preliminary studies suggest that Kap?2 uses its PY-NLS binding site to bind FUS495X. Our goaI here is to 1) map the region(s) of FUS495X that binds Kap?2, 2) use isothermal titration calorimetry (ITC) to measure binding affinities of different FUS495X regions for Kap?2, 3) test if these FUS region(s) can be transported into the nucleus in cells, and 3) solve the crystal structure of Kap?2 bound to FUS495X. Using qualitative pull-down binding assays and quantitative ITC experiments, I have mapped Kap?2-binding to the C-terminal 130 residues of FUS495X, which spans its RGG2, ZnFinger and RGG3 domains/regions and binds Kap?2 with a KD of 200 nM. I will test cellular localization of this region of FUS495X to assess if it can be imported into the nucleus by Kap?2. I will express FUS 371-495 in a few human cell lines to determine if it can localize to the nucleus. I will also attempt to solve the crystal structure of Kap?2 complexed this FUS fragment to understand how it binds the importin. Aim 2. Three mutations in hnRNP H2 have been found in patients with MRXSB (mental retardation, X-linked, syndromic, Bain type) diseases. The goal here is to understand how these mutations affect Kap?2-binding. I have generated various hnRNP H2 constructs and used both qualitative pull- down binding assays and ITC to map Kap?2-binding to a fragment containing RRM2 and a PY-NLS. ITC using this hnRNP H2 fragment showed that notable losses of affinity for Kap?2-binding of the MRXSB mutants and these results correlate well with hnRNP H2 mutants localization to stress granules and with disease severity. The interactions between hnRNP H2 and Kap?2 is highly unusual in that it involves a folded RRM domain in addition to a PY-NLS. I will determine the structure of Kap?2 complexed with hnRNP H2 RRM-PY-NLS to understand this novel mode of interaction and also to understand how MRXSB disease mutations result in loss of Kap?2-binding.