# Synthetically-evolved and engineered Nanobodies selective for Cb isoforms of PKA

> **NIH NIH R21** · UNIVERSITY OF CALIFORNIA, SAN DIEGO · 2022 · $434,500

## Abstract

ABSTRACT. Our collaboration builds on the protein kinase expertise of the Taylor lab and the expertise of the
Chang lab to isolate Synthetically-evolved and engineered Nanobodies (SENs). Our goal is to isolate SENs
that will discriminate between the isoforms of the catalytic (C) subunits of cAMP-dependent protein kinase
(PKA). This builds on the realization that >50% of PKA signaling in the brain is mediated by C subunits.
Although the C subunit of PKA was the second to be discovered, the first to be sequenced, and the first to be
crystallized, virtually nothing is known about the C proteins, which represent a family of proteins that differ
only in their N-terminal sequence encoded for by Exon 1. The C3/4 isoforms are expressed exclusively in
brain while C2 is expressed in lymphoid tissues and T cells. Using the retina as a “Window into the Brain”
Taylor showed that C is highly expressed in these terminally differentiated neurons and that it localizes
differently from C, which supports the functional non-redundancy of these isoforms. The surprising discovery
that C, but not C, RII, or RII, localizes to mitochondria adds further credence to the importance of C and
its link to neurodegenerative diseases. To validate the hypothesis that imaging of the retina could serve as a
window into the more complex signaling in brain and to determine if PKA signaling changes as a function of the
onset and progression of AD, Taylor, in collaboration with the Alzheimer Disease Research Center at UCSD
(UCSD ADRC) Neuropathology Core and Robert Rissman who directs this core is now comparing AD brains
from individuals with mild and advanced AD. The preliminary results, coupled with imaging of pTau, a hallmark
of advanced AD, confirm the hypothesis and highlight the importance of having tools that will discriminate
between the various C isoforms. To achieve this essential next step we turned to the newly developed SENS
technology. Using C-subunit proteins, purified in the Taylor lab, the Chang lab will fluorescently tag the proteins
and then select for SENs that will discriminate between the different C-subunit isoforms. Initially they will focus
on C4/C4ab as these, based on RNA scope, appeared to be the best candidates for association with
mitochondria, and C2 which is expressed in T cells and has the potential to serve as a Biomarker for early
onset PD. In Aim I Taylor will express C-subunit isoforms as well as peptide fragments that correspond to the
N-terminal tails of each subunit. In Aim II Chang will fluorescently tag these proteins and through multiple
rounds of screening will isolate selective high affinity SENs. These SENs will be analyzed for their ability to
detect specific isoforms by Western blots and by imaging in both human and mouse tissues. Inhibition of
kinase activity will also be assessed. A long-term goal will be to crystallize SENs: C-subunit complexes.
Because this toolbox of reagents can be widely used by both clinicians and ...

## Key facts

- **NIH application ID:** 10525796
- **Project number:** 1R21AG079330-01
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN DIEGO
- **Principal Investigator:** GEOFFREY A CHANG
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $434,500
- **Award type:** 1
- **Project period:** 2022-09-15 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10525796

## Citation

> US National Institutes of Health, RePORTER application 10525796, Synthetically-evolved and engineered Nanobodies selective for Cb isoforms of PKA (1R21AG079330-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10525796. Licensed CC0.

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