# Kapβ2 recognition of cargos involved in neurodegenerative/neurodevelopmental diseases

> **NIH NIH F31** · UT SOUTHWESTERN MEDICAL CENTER · 2020 · $39,120

## Abstract

Karyopherin β2 (Kapβ2) 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 Kapβ2 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 Kapβ2-
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 Kapβ2 uses its PY-NLS binding site to bind FUS495X. Our goaI here is to 1)
map the region(s) of FUS495X that binds Kapβ2, 2) use isothermal titration calorimetry (ITC) to measure
binding affinities of different FUS495X regions for Kapβ2, 3) test if these FUS region(s) can be transported into
the nucleus in cells, and 3) solve the crystal structure of Kapβ2 bound to FUS495X. Using qualitative pull-down
binding assays and quantitative ITC experiments, I have mapped Kapβ2-binding to the C-terminal 130
residues of FUS495X, which spans its RGG2, ZnFinger and RGG3 domains/regions and binds Kapβ2 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 Kapβ2. 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 Kapβ2 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 Kapβ2-binding. I have generated various hnRNP H2 constructs and used both qualitative pull-
down binding assays and ITC to map Kapβ2-binding to a fragment containing RRM2 and a PY-NLS. ITC using
this hnRNP H2 fragment showed that notable losses of affinity for Kapβ2-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 Kapβ2 is highly unusual in that it involves a folded RRM domain in
addition to a PY-NLS. I will determine the structure of Kapβ2 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 Kapβ2-binding.

## Key facts

- **NIH application ID:** 10141737
- **Project number:** 1F31NS120465-01
- **Recipient organization:** UT SOUTHWESTERN MEDICAL CENTER
- **Principal Investigator:** Abner Gonzalez-Castro
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $39,120
- **Award type:** 1
- **Project period:** 2020-09-29 → 2022-09-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10141737

## Citation

> US National Institutes of Health, RePORTER application 10141737, Kapβ2 recognition of cargos involved in neurodegenerative/neurodevelopmental diseases (1F31NS120465-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10141737. Licensed CC0.

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