# Discovery of cryptic open reading frames in Alzheimer's brain

> **NIH NIH R21** · JOHNS HOPKINS UNIVERSITY · 2020 · $204,688

## Abstract

PROJECT SUMMARY
Since the beginning of -omics era, reference protein databases have defined the list of proteins are believed to
be expressed in the cells. However, most proteins in the databases have been constructed by a conceptual
translation of mRNAs based on a limited set of predictions rules such as that a protein translation starts at
AUG that can make the longest open reading frame (ORF). Recent mass spectrometry-based proteomics and
ribosome profiling studies discovered that protein translation can start at non-AUG codons, noncanonical
translation initiation sites (TISs), generating small ORFs or N-terminal extension, collectively called cryptic
ORFs. Strikingly, a growing number of studies have started reporting that cryptic ORFs are involved in
pathogeneses of many diseases including Alzheimer’s disease (AD) that is the most prevalent
neurodegenerative disease. In AD, one of the translation initiation factors, eIF2α, is inactivated by a
phosphorylation on it. Consequently, eIF2A replaces the role of eIF2α switching general translation to gene-
specific translation. Because eIF2A is less strict in using AUG at a TIS, when eIF2A is involved in the
translation initiation, the usage of non-AUG codon is increased expressing more cryptic ORFs. Therefore, the
discovery of cryptic ORFs differentially expressed in AD would have great importance in deeper understanding
the AD pathogenesis mechanism. Moreover, this established method can be applicable to other neurological
diseases as well. Nevertheless, the detection of those proteins has been missed due to the incomplete
reference databases and technical limitations. Since ~90% of human proteins have N-terminal acetylation, this
can serve as a signature modification for TISs and an in-depth identification of N-terminal acetylated peptides
enable us to achieve in-depth identification of cryptic ORFs. Our group already identified over 120 cryptic
ORFs from human samples using an N-terminal peptide enrichment technology, but even deeper proteome
analysis for cryptic ORFs is required to cover most of the differentially expressed cryptic ORFs in AD brains.
To achieve these goals, we propose three aims in this proposal. In Aim 1, we will develop a new method for
the identification of N-terminal acetylated peptides by combining two different methods that were originally
developed for the identification of proteolytic cleavage sites, the TAILS method and the subtiligase-based
method. In Aim 2, we will determine whether acetylated peptides mapping to non-cognate codons are bona
fide TISs or post-cleavage acetylated peptides using a nascent protein enrichment method. In Aim 3, we will
discovery cryptic ORFs differentially expressed in AD brain using the method developed in Aim 1 as well as an
in-depth total proteome analysis strategy. This project with expand and optimize our prior work on cryptic TISs,
and lead to the discovery of cryptic ORFs expressed in AD brains. We hypothesize those novel cryptic O...

## Key facts

- **NIH application ID:** 9985692
- **Project number:** 5R21AG064536-02
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Chan-Hyun Na
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $204,688
- **Award type:** 5
- **Project period:** 2019-08-01 → 2022-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9985692

## Citation

> US National Institutes of Health, RePORTER application 9985692, Discovery of cryptic open reading frames in Alzheimer's brain (5R21AG064536-02). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/9985692. Licensed CC0.

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