# Engineering KapB2 to Disaggregate Disease-Linked Proteins

> **NIH NIH F31** · UNIVERSITY OF PENNSYLVANIA · 2021 · $46,036

## Abstract

Project Summary
 Protein aggregation underlies many neurodegenerative diseases, and previous studies indicate that
mutations to proteins involved in stress-granule formation can increase the likelihood that neurodegenerative
diseases will develop. For example, mutations to FUS have been shown to lead to the development of
amyotrophic lateral sclerosis (ALS), a disease that results in the specific degeneration of motor neurons. FUS
is an RNA-binding protein (RBP) that carries proline-tyrosine nuclear-localization signal (PY-NLS), and when
the NLS is compromised by mutation, FUS accumulates in the cytoplasm. Cytoplasmic accumulation of FUS
leads to high local concentrations of aggregation-prone protein, promoting protein misfolding. Our lab has
shown that the nuclear-import receptor Karyopherin-b2 (Kapb2) can recognize wild-type FUS, prevent its
aggregation, and promote its disaggregation both in vitro and in vivo. Similarly, Kapb2 can dissolve aggregates
comprised of other PY-NLS-bearing proteins such as hnRNPA1. However, when the NLS of either protein is
mutated (e.g. FUSP525L and hnRNPA1A288S), Kapb2 is significantly less effective. I have found that by making
targeted mutations to the cargo-binding interface, I can partially recover the ability of Kapb2 to prevent the
aggregation of FUSP525L. Additionally, it has been shown that Kapb2 is capable of trafficking proteins that lack a
PY-NLS, suggesting that there are multiple factors that contribute to defining Kapb2 cargo. Based on these
preliminary data, I hypothesize that (1) making targeted compensatory mutations to Kapb2 will enable Kapb2
to bind and disaggregate disease-linked protein, and that (2) Kapb2 can be evolved to disaggregate disease-
linked cargo which lack or harbor a mutated PY-NLS. To test these hypotheses, my aims are to engineer
Kapb2 to (1) disaggregate ALS-linked FUS variants and (2) disaggregate ALS-linked hnRNPA1 variants. I will
do this by taking both a structure-based rational approach and an unbiased screen to generate a library of
mutant Kapb2 proteins to be tested in vitro and in vivo. These studies will demonstrate that a nuclear-import
receptor can be engineered to recognize and disaggregate a diverse repertoire of cargos, including cargos that
are misfolded in disease. Furthermore, engineering Kapb2 to recognize disease-linked protein introduces a
new strategy for disaggregating pathologically misfolded oligomers using a human protein disaggregation
system.

## Key facts

- **NIH application ID:** 10227152
- **Project number:** 5F31NS111870-03
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** CHARLOTTE MIRIAM FARE
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $46,036
- **Award type:** 5
- **Project period:** 2019-09-01 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10227152

## Citation

> US National Institutes of Health, RePORTER application 10227152, Engineering KapB2 to Disaggregate Disease-Linked Proteins (5F31NS111870-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10227152. Licensed CC0.

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