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<meta name="description" content="In ribosome-associated quality control (RQC), nascent polypeptides produced by interrupted translation are modified with C-terminal polyalanine tails (&quot;Ala-tails&quot;) that function outside ribosomes to induce ubiquitylation by E3 ligases Pirh2 (p53-induced RING-H2 domain-containing) or CRL2 (Cullin-2 R …"><meta name="keywords" content="pmid:37676773, PMC10591846, doi:10.1016/j.celrep.2023.113100, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov&#x27;t, Pratik Rajendra Patil, A Maxwell Burroughs, Claudio A P Joazeiro, Alanine / metabolism, Binding Sites, Carrier Proteins* / metabolism, Humans, Proteolysis, Ubiquitin-Protein Ligases* / metabolism, Ubiquitination, PubMed Abstract, NIH, NLM, NCBI, National Institutes of Health, National Center for Biotechnology Information, National Library of Medicine, MEDLINE"><meta name="robots" content="index,nofollow,noarchive"><meta property="og:title" content="Mechanism and evolutionary origins of alanine-tail C-degron recognition by E3 ligases Pirh2 and CRL2-KLHDC10 - PubMed"><meta property="og:url" content="https://pubmed.ncbi.nlm.nih.gov/37676773/"><meta property="og:description" content="In ribosome-associated quality control (RQC), nascent polypeptides produced by interrupted translation are modified with C-terminal polyalanine tails (&quot;Ala-tails&quot;) that function outside ribosomes to induce ubiquitylation by E3 ligases Pirh2 (p53-induced RING-H2 domain-containing) or CRL2 (Cullin-2 R …"><meta property="og:image" content="https://cdn.ncbi.nlm.nih.gov/pubmed/persistent/pubmed-meta-image-v2.jpg"><meta property="og:image:secure_url" content="https://cdn.ncbi.nlm.nih.gov/pubmed/persistent/pubmed-meta-image-v2.jpg"><meta property="og:type" content="website"><meta property="og:site_name" content="PubMed"><meta name="twitter:domain" content="pubmed.ncbi.nlm.nih.gov"><meta name="twitter:card" content="summary_large_image"><meta name="twitter:title" content="Mechanism and evolutionary origins of alanine-tail C-degron recognition by E3 ligases Pirh2 and CRL2-KLHDC10 - PubMed"><meta name="twitter:url" content="https://pubmed.ncbi.nlm.nih.gov/37676773/"><meta name="twitter:description" content="In ribosome-associated quality control (RQC), nascent polypeptides produced by interrupted translation are modified with C-terminal polyalanine tails (&quot;Ala-tails&quot;) that function outside ribosomes to induce ubiquitylation by E3 ligases Pirh2 (p53-induced RING-H2 domain-containing) or CRL2 (Cullin-2 R …"><meta name="twitter:image" content="https://cdn.ncbi.nlm.nih.gov/pubmed/persistent/pubmed-meta-image-v2.jpg">
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<div class="action-panel-control-wrap">
<label for="email-from" class="action-panel-label">
From:
</label>
<input type="email" aria-label="Sender Email Address" placeholder="email@example.com" id="email-from" class="email-from" pattern="^[a-zA-Z0-9_.+-]+@[a-zA-Z0-9-]+\.[a-zA-Z0-9-.]+$" maxlength="256">
</div>
<div class="action-panel-control-wrap">
<label for="email-citation-format" class="action-panel-label">
Format:
</label>
<select id="email-citation-format" name="citation-format" class="action-panel-selector email-citation-format">
<option selected="selected" value="summary">Summary</option>
<option value="summary-text">Summary (text)</option>
<option value="abstract">Abstract</option>
<option value="abstract-text">Abstract (text)</option>
</select>
</div>
<div class="include-supplemental-container">
<input type="checkbox" aria-label="Include MeSH and other data" name="include-supplemental" id="email-include-supplemental" class="email-include-supplemental">
<label for="email-include-supplemental" class="email-include-supplemental-label">MeSH and other data</label>
</div>
<div class="form-field recaptcha ">
<div class="g-recaptcha" id="id-recaptcha" data-sitekey="6LfsWHMdAAAAAClKbtOpjQ2pMjgsGxvv7NdZW9uI"></div>
</div>
<div id="captcha-error-message" class="usa-input-error-message captcha-validation-message" role="alert"></div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Sending..."
data-ga-category="save_share"
data-ga-action="email"
data-ga-label="send">
Send email
</button>
<button class="action-panel-cancel"
aria-label="Close 'Email citations' panel"
ref="linksrc=close_email_panel"
aria-controls="email-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="email"
data-ga-label="cancel">
Cancel
</button>
</div>
<input type="hidden" name="email-search-details" value="" />
<input type="hidden" name="email-search-details-hash" value="0e42663a6c3bd85498fcb88798998fed7bfdc45d457db35281e41afe13cc0524" />
</form>
</div>
</div>
<div id="collections-action-panel"
class="collections-action-panel action-panel in-progress-dots-panel"
aria-hidden="true"
data-collections-open-panel-enabled="false"
data-collections-open-panel-url-hash="#open-collections-panel">
<div class="inner-wrap">
<h3 class="action-panel-heading">
Add to Collections
</h3>
<form id="collections-action-panel-form"
class="collections-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-existing-collections-url="/list-existing-collections/"
data-add-to-existing-collection-url="/add-to-existing-collection/"
data-create-and-add-to-new-collection-url="/create-and-add-to-new-collection/"
data-get-article-ids-by-search-url="/get-article-ids-by-search/"
data-myncbi-max-collection-name-length="100"
data-add-to-collection-max-amount="1000"
data-collections-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/">
<input type="hidden" name="csrfmiddlewaretoken" value="OVuUDtUDOqH4CJ7muVElEDSOwFossggF5IeZgjnZo1uywadc7ehn8Los4J6ElROZ">
<div class="choice-group" role="radiogroup">
<ul class="radio-group-items">
<li>
<input type="radio"
id="collections-action-panel-new"
class="collections-new"
name="collections"
value="new"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="collections_radio_new">
<label for="collections-action-panel-new">Create a new collection</label>
</li>
<li>
<input type="radio"
id="collections-action-panel-existing"
class="collections-existing"
name="collections"
value="existing"
checked="true"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="collections_radio_existing">
<label for="collections-action-panel-existing">Add to an existing collection</label>
</li>
</ul>
</div>
<div class="controls-wrapper">
<div class="action-panel-control-wrap new-collections-controls">
<label for="collections-action-panel-add-to-new" class="action-panel-label required-field-asterisk">
Name your collection:
</label>
<input
type="text"
name="add-to-new-collection"
id="collections-action-panel-add-to-new"
class="collections-action-add-to-new"
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/"
maxlength="100"
data-ga-category="save_share"
data-ga-action="create_collection"
data-ga-label="non_favorties_collection">
<div class="collections-new-name-too-long usa-input-error-message selection-validation-message">
Name must be less than 100 characters
</div>
</div>
<div class="action-panel-control-wrap existing-collections-controls">
<label for="collections-action-panel-add-to-existing" class="action-panel-label">
Choose a collection:
</label>
<select id="collections-action-panel-add-to-existing"
class="action-panel-selector collections-action-add-to-existing"
data-ga-category="save_share"
data-ga-action="select_collection"
data-ga-label="($('#collections-action-add-to-existing').val() === 'Favorites') ? 'Favorites' : 'non_favorites_collection'">
</select>
<div class="collections-retry-load-on-error usa-input-error-message selection-validation-message">
Unable to load your collection due to an error<br>
<a href="#">Please try again</a>
</div>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Adding..."
data-pinger-ignore
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="add">
Add
</button>
<button class="action-panel-cancel"
aria-label="Close 'Add to Collections' panel"
ref="linksrc=close_collections_panel"
aria-controls="collections-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="collections"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="bibliography-action-panel"
class="bibliography-action-panel action-panel in-progress-dots-panel"
aria-hidden="true"
data-bibliography-open-panel-enabled="false"
data-bibliography-open-panel-url-hash="#open-bibliography-panel">
<div class="inner-wrap">
<h3 class="action-panel-heading">
Add to My Bibliography
</h3>
<form id="bibliography-action-panel-form"
class="bibliography-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-add-to-bibliography-max-amount="100"
data-add-to-bibliography-batch-size="10"
data-bibliography-delegates-url="/list-bibliography-delegates/"
data-add-to-bibliography-url="/add-to-bibliography/"
data-get-article-ids-by-search-url="/get-article-ids-by-search/"
data-mybib-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/mybibliography/">
<input type="hidden" name="csrfmiddlewaretoken" value="OVuUDtUDOqH4CJ7muVElEDSOwFossggF5IeZgjnZo1uywadc7ehn8Los4J6ElROZ">
<div class="action-panel-control-wrap bibliographies-controls">
<div class="choice-group">
<ul class="bibliographies-action-add radio-group-items">
<li>
<input name="bibliography" id="my-bibliography" class="my-bibliography" type="radio" checked/>
<label for="my-bibliography">My Bibliography</label>
</li>
</ul>
</div>
</div>
<div class="bibliographies-retry-load-on-error usa-input-error-message selection-validation-message">
Unable to load your delegates due to an error<br>
<a href="#">Please try again</a>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Adding..."
data-pinger-ignore>
Add
</button>
<button class="action-panel-cancel"
aria-label="Close 'Add to bibliography' panel"
ref="linksrc=close_bibliography_panel"
aria-controls="bibliography-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="mybib"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="saved-search-action-panel" class="saved-search-action-panel action-panel " aria-hidden="true"
data-saved-search-open-panel-enabled="false"
data-saved-search-open-panel-url-hash="#open-saved-search-panel">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Your saved search
</h2>
<form id="saved-search-action-panel-form"
class="saved-search-action-panel-form action-panel-content action-form"
data-create-saved-search-url="/create-saved-search/"
data-try-search-terms-url="/try-search-term/"
data-saved-search-root-url="https://www.ncbi.nlm.nih.gov/myncbi/searches/">
<input type="hidden" name="csrfmiddlewaretoken" value="OVuUDtUDOqH4CJ7muVElEDSOwFossggF5IeZgjnZo1uywadc7ehn8Los4J6ElROZ">
<div class="action-panel-control-wrap">
<label for="saved-search-name" class="action-panel-label saved-search-name-label required-field-asterisk">
Name of saved search:
</label>
<input maxlength="200"
type="text"
name="saved-search-name"
id="saved-search-name"
class="saved-search-name"
value=""
required
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/">
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-term" class="action-panel-label required-field-asterisk">
Search terms:
</label>
<textarea name="saved-search-term" id="saved-search-term" class="saved-search-term" required></textarea>
</div>
<div class="test-search-term-wrap">
<a href="#" class="try-search-term">Test search terms</a>
</div>
<div class="choice-group action-panel-extra-margin-top">
<span class="action-panel-label" id="fieldset-label">
Would you like email updates of new search results?
</span>
<fieldset id="saved-search-alert" aria-describedby="fieldset-label">
<legend class="usa-sr-only">Saved Search Alert Radio Buttons</legend>
<ul class="radio-group-items">
<li>
<input type="radio" id="saved-search-alert-yes" class="saved-search-alert-yes" name="saved-search-alert" value="yes" checked>
<label for="saved-search-alert-yes" class="action-panel-label">Yes</label>
</li>
<li>
<input aria-label="No radio input" type="radio" id="saved-search-alert-no" class="saved-search-alert-no" name="saved-search-alert" value="no">
<label for="saved-search-alert-no" class="action-panel-label">No</label>
</li>
</ul>
</fieldset>
</div>
<div class="alert-schedule-wrap">
<div class="action-panel-control-wrap">
<label class="action-panel-label">
Email:
</label>
<span aria-label="Email address" id="saved-search-email" class="action-panel-label"><span class="action-panel-label-bold"></span> (<a class="myncbi-account-settings" href="https://www.ncbi.nlm.nih.gov/account/settings/">change</a>)</span>
</div>
<div class="action-panel-control-wrap action-panel-extra-margin-top">
<label for="saved-search-frequency" class="action-panel-label">
Frequency:
</label>
<select id="saved-search-frequency" class="no-border-panel-selector saved-search-frequency">
<option value="monthly">Monthly</option>
<option value="weekly">Weekly</option>
<option value="daily">Daily</option>
</select>
</div>
<div class="action-panel-control-wrap saved-search-monthly-additional">
<label for="saved-search-monthly-on-day" class="action-panel-label">
Which day?
</label>
<select id="saved-search-monthly-on-day" class="no-border-panel-selector">
<option value="Sunday">The first Sunday</option>
<option value="Monday">The first Monday</option>
<option value="Tuesday">The first Tuesday</option>
<option value="Wednesday">The first Wednesday</option>
<option value="Thursday">The first Thursday</option>
<option value="Friday">The first Friday</option>
<option value="Saturday">The first Saturday</option>
<option value="day">The first day</option>
<option value="weekday">The first weekday</option>
</select>
</div>
<div class="action-panel-control-wrap saved-search-weekly-additional">
<label for="saved-search-weekly-on-day" class="action-panel-label">
Which day?
</label>
<select id="saved-search-weekly-on-day" class="no-border-panel-selector saved-search-weekly-on-day">
<option value="Sunday">Sunday</option>
<option value="Monday">Monday</option>
<option value="Tuesday">Tuesday</option>
<option value="Wednesday">Wednesday</option>
<option value="Thursday">Thursday</option>
<option value="Friday">Friday</option>
<option value="Saturday">Saturday</option>
</select>
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-report" class="action-panel-label">
Report format:
</label>
<select id="saved-search-report" class="no-border-panel-selector saved-search-report">
<option value="DocSum">Summary</option>
<option value="DocSumText">Summary (text)</option>
<option value="Abstract">Abstract</option>
<option value="AbstractText">Abstract (text)</option>
<option value="MEDLINE">PubMed</option>
</select>
</div>
<div class="action-panel-control-wrap">
<label for="saved-search-amount" class="action-panel-label">
Send at most:
</label>
<select id="saved-search-amount" class="no-border-panel-selector saved-search-amount">
<option value="1">1 item</option>
<option value="5" selected>5 items</option>
<option value="10">10 items</option>
<option value="20">20 items</option>
<option value="50">50 items</option>
<option value="100">100 items</option>
<option value="200">200 items</option>
</select>
</div>
<div>
<input type="checkbox" id="saved-search-send-if-no-result" class="saved-search-send-if-no-result" name="saved-search-send-if-no-result">
<label for="saved-search-send-if-no-result" class="action-panel-label smaller-checkbox">
Send even when there aren't any new results
</label>
</div>
<div class="action-panel-control-wrap option-text-in-email-wrap">
<label for="saved-search-email-text" class="action-panel-label">
Optional text in email:
</label>
<textarea name="saved-search-email-text"
id="saved-search-email-text"
class="saved-search-email-text"></textarea>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Saving..."
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="save">
Save
</button>
<button class="action-panel-cancel"
aria-label="Close 'Your saved search' panel"
ref="linksrc=close_saved_search_panel"
aria-controls="saved-search-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="citation-manager-action-panel" class="citation-manager-action-panel action-panel" aria-hidden="true">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Create a file for external citation management software
</h2>
<form id="citation-manager-action-panel-form"
class="action-panel-content action-form"
action="/results-export-ids/"
data-by-search-action="/results-export-search-data/"
data-by-ids-action="/results-export-ids/"
method="post"
data-by-search-method="post"
data-by-ids-method="post">
<input type="hidden" name="csrfmiddlewaretoken" value="OVuUDtUDOqH4CJ7muVElEDSOwFossggF5IeZgjnZo1uywadc7ehn8Los4J6ElROZ">
<input name="results-format" type="hidden" value="pubmed"/>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Sending..."
data-ga-category="save_share"
data-ga-action="citation_manager"
data-ga-label="save">
Create file
</button>
<button class="action-panel-cancel"
aria-label="Close 'Send citations to citation manager' panel"
ref="linksrc=close_citation_manager_panel"
aria-controls="citation-manager-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="citation_manager"
data-ga-label="cancel">
Cancel
</button>
</div>
</form>
</div>
</div>
<div id="rss-action-panel" class="rss-action-panel action-panel " aria-hidden="true">
<div class="inner-wrap">
<h2 class="action-panel-heading">
Your RSS Feed
</h2>
<form id="rss-action-panel-form"
class="rss-action-panel-form action-panel-content action-form"
data-create-rss-feed-url="/create-rss-feed-url/"
data-search-form-term-value="">
<input type="hidden" name="csrfmiddlewaretoken" value="OVuUDtUDOqH4CJ7muVElEDSOwFossggF5IeZgjnZo1uywadc7ehn8Los4J6ElROZ">
<div class="action-panel-control-wrap">
<label for="rss-name" class="action-panel-label required-field-asterisk">
Name of RSS Feed:
</label>
<input maxlength="200"
placeholder="Name"
type="text"
name="rss-name"
id="rss-name"
class="rss-name"
value=''
required
pattern="[^&quot;&amp;=&lt;&gt;\/]*" title="The following characters are not allowed in the Name field: &quot;&amp;=&lt;&gt;/">
</div>
<div class="rss-limit-wrap">
<div class="action-panel-control-wrap action-panel-extra-margin-top">
<label for="rss-limit" class="action-panel-label">
Number of items displayed:
</label>
<select id="rss-limit" class="no-border-panel-selector rss-limit">
<option value="5">5</option>
<option value="10">10</option>
<option value="15" selected="selected">15</option>
<option value="20">20</option>
<option value="50">50</option>
<option value="100">100</option>
</select>
</div>
</div>
<div class="action-panel-actions">
<button class="action-panel-submit"
type="submit"
data-loading-label="Creating..."
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="save">
Create RSS
</button>
<button class="action-panel-cancel"
aria-label="Close 'Your RSS' panel"
ref="linksrc=close_rss_panel"
aria-controls="rss-action-panel"
aria-expanded="false"
data-ga-category="save_share"
data-ga-action="alert"
data-ga-label="cancel">
Cancel
</button>
</div>
<div class="action-panel-control-wrap rss-link-copy-wrap">
<label for="rss-link" class="usa-sr-only">RSS Link</label>
<input placeholder="Your RSS Feed Link" type="text" name="rss-link" id="rss-link" class="rss-link" title="RSS Link">
<button
type="button"
disabled
class="rss-link-copy-button disabled"
data-ga-category="save_share"
data-ga-action="rss"
data-ga-label="copy">
Copy
</button>
</div>
</form>
</div>
</div>
</div>
</div>
<div class="article-page" id="article-page" data-article-pmid="37676773">
<aside class="page-sidebar">
<div class="inner-wrap">
<div class="full-text-links">
<div class="full-view">
<h3 class="title">
Full text links
</h3>
<div class="full-text-links-list">
<a class="link-item
dialog-focus"
href="https://linkinghub.elsevier.com/retrieve/pii/S2211-1247(23)01111-7"
target="_blank"
rel="noopener"
ref="linksrc=fulltextorjournal_fulltext&amp;is_pmc=False&amp;PrId=3048&amp;itool=Abstract-def&amp;log$=linkouticon&amp;uid=37676773&amp;db=pubmed&amp;nlmid=101573691"
title="See full text options at Elsevier Science"
data-ga-category="full_text"
data-ga-action="Elsevier Science"
data-ga-label="37676773"
><img src="https://cdn.ncbi.nlm.nih.gov/corehtml/query/egifs/https:--linkinghub.elsevier.com-ihub-images-celloa.png" alt="Elsevier Science full text link"><span class="text">
Elsevier Science
</span></a><a class="link-item
pmc
"
href="https://pmc.ncbi.nlm.nih.gov/articles/pmid/37676773/"
target="_blank"
rel="noopener"
ref="linksrc=fulltextorjournal_fulltext&amp;is_pmc=True&amp;PrId=3494&amp;itool=Abstract-def&amp;log$=linkouticon&amp;uid=37676773&amp;db=pubmed&amp;nlmid=101573691"
title="Free full text at PubMed Central"
data-ga-category="full_text"
data-ga-action="PMC"
data-ga-label="37676773"
><span class="text">
Free PMC article
</span></a>
</div>
</div>
<div class="short-view">
<a href="#" class="full-text-links-button full-text-links-dialog-trigger">
Full text links
</a>
</div>
</div>
<div class="actions-buttons sidebar"><h3 class="title">Actions</h3><div class="inner-wrap"><button class="citation-button citation-dialog-trigger"
aria-label="Open dialog with citation text in different styles"
data-ga-category="save_share"
data-ga-action="cite"
data-ga-label="open"
data-all-citations-url="/37676773/citations/"
data-citation-style="nlm"
data-pubmed-format-link="/37676773/export/"><span class="button-label">Cite</span></button><link type="text/css" href="ncbi-overlay-block/src/overlay-block.css"><div class="collections-button-container" data-article-id="37676773" data-article-db="pubmed"><button class="collections-button collections-dialog-trigger"
aria-label="Save article in MyNCBI collections."
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_button"
data-collections-open-dialog-enabled="false"
data-collections-open-dialog-url="https://account.ncbi.nlm.nih.gov/?back_url=https%3A%2F%2Fpubmed.ncbi.nlm.nih.gov%2F37676773%2F%23open-collections-dialog"
data-in-collections="false"><span class="button-label">Collections</span></button><div class="overlay" role="dialog"><div id="collections-action-dialog"
class="dialog collections-dialog"
aria-hidden="true"><div class="title">Add to Collections</div><div class="collections-action-panel action-panel"><form id="collections-action-dialog-form"
class="collections-action-panel-form action-panel-content action-form action-panel-smaller-selectors"
data-existing-collections-url="/list-existing-collections/"
data-add-to-existing-collection-url="/add-to-existing-collection/"
data-create-and-add-to-new-collection-url="/create-and-add-to-new-collection/"
data-myncbi-max-collection-name-length="100"
data-collections-root-url="https://www.ncbi.nlm.nih.gov/myncbi/collections/"><input type="hidden" name="csrfmiddlewaretoken" value="OVuUDtUDOqH4CJ7muVElEDSOwFossggF5IeZgjnZo1uywadc7ehn8Los4J6ElROZ"><div class="choice-group" role="radiogroup"><ul class="radio-group-items"><li><input type="radio"
id="collections-action-dialog-new"
class="collections-new"
name="collections"
value="new"
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_radio_new"><label for="collections-action-dialog-new">Create a new collection</label></li><li><input type="radio"
id="collections-action-dialog-existing"
class="collections-existing"
name="collections"
value="existing"
checked="true"
data-ga-category="collections_button"
data-ga-action="click"
data-ga-label="collections_radio_existing"><label for="collections-action-dialog-existing">Add to an existing collection</label></li></ul></div><div class="controls-wrapper"><div class="action-panel-control-wrap new-collections-controls"><label for="collections-action-dialog-add-to-new" class="action-panel-label required-field-asterisk">
Name your collection:
</label><input
type="text"
name="add-to-new-collection"
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><sup class="key">1</sup> Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH-Alliance, 69120 Heidelberg, Germany.</li>
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><sup class="key">2</sup> National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.</li>
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><sup class="key">3</sup> Institute of Biochemistry II, Goethe University Faculty of Medicine, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany; Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Max-von-Laue-Strasse 15, 60438 Frankfurt am Main, Germany.</li>
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><sup class="key">4</sup> Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH-Alliance, 69120 Heidelberg, Germany; Department of Molecular Medicine, UF Scripps Biomedical Research, Jupiter, FL 33458, USA. Electronic address: c.joazeiro@zmbh.uni-heidelberg.de.</li>
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<p>
In ribosome-associated quality control (RQC), nascent polypeptides produced by interrupted translation are modified with C-terminal polyalanine tails ("Ala-tails") that function outside ribosomes to induce ubiquitylation by E3 ligases Pirh2 (p53-induced RING-H2 domain-containing) or CRL2 (Cullin-2 RING ligase2)-KLHDC10. Here, we investigate the molecular basis of Ala-tail function using biochemical and in silico approaches. We show that Pirh2 and KLHDC10 directly bind to Ala-tails and that structural predictions identify candidate Ala-tail-binding sites, which we experimentally validate. The degron-binding pockets and specific pocket residues implicated in Ala-tail recognition are conserved among Pirh2 and KLHDC10 homologs, suggesting that an important function of these ligases across eukaryotes is in targeting Ala-tailed substrates. Moreover, we establish that the two Ala-tail-binding pockets have convergently evolved, either from an ancient module of bacterial provenance (Pirh2) or via tinkering of a widespread C-degron-recognition element (KLHDC10). These results shed light on the recognition of a simple degron sequence and the evolution of Ala-tail proteolytic signaling.
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Keywords:
</strong>
Ala-tail; C-end degron; CP: Molecular biology; E3 ubiquitin ligase; KLHDC10; Pirh2; polyalanine; protein degradation; protein quality control; protein-protein interaction; structure prediction.
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<p xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:p1="http://pubmed.gov/pub-one">Declaration of interests The authors declare no competing interests.</p>
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<a class="figure-link"
href="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a82e/10591846/3860d8c0915e/nihms-1933911-f0001.jpg"
itemprop="contentUrl"
aria-describedby="figure-caption-0"
role="button"
data-image-width="1050"
data-image-height="991"
data-image-alt="Figure 1."
data-pmc-id="PMC10591846"
data-figure-id="F1">
<img class="figure-thumb" itemprop="thumbnail"
id="article-image-0"
src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a82e/10591846/70a798debd91/nihms-1933911-f0001.gif"
alt="Figure 1." />
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<strong class="figure-label">
<p> Figure 1.. Pirh2 and KLHDC10 directly bind… </p>
</strong>
</div>
</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Figure 1.. Pirh2 and KLHDC10 directly bind to Ala-tails </p>
</strong>
<div class="figure-caption-contents"><p> (A) Domain arrangement of human Pirh2:… </p></div>
</div>
<figcaption id="figure-caption-0" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Figure 1.. Pirh2 and KLHDC10 directly bind to Ala-tails
</strong>
<div class="figure-caption-contents">(A) Domain arrangement of human Pirh2: NTM, N-terminal module; RING, really interesting new gene domain; CTD, C-terminal domain. (B) <i>In vitro</i> GST pull-down assay using the specified GST-Pirh2 truncation constructs and recombinantly purified GFP or GFP-Ala<sub>6</sub>. Anti-GST and anti-GFP immunoblots are indicated. (C) AlphaScreen assay to assess Pirh2-Ala-tail binding. Reactants were GST fusions with Pirh2 fragments containing both the NTM and RING domains (Pirh2<sub>1195</sub>) or the NTM alone (Pirh2<sub>1137</sub>) and a biotinylated Ala-tail peptide (MDELYKAAAAAA). Proximity-induced fluorescence signal (see STAR Methods) was monitored in the presence of increasing amounts of a competing, non-biotinylated Ala-tail peptide to determine IC<sub>50</sub> values from the dose-response curve. Each data point is a technical triplicate, and error bars represent ± SD. The y axis presents the normalized AlphaScreen signal as arbitrary fluorescence units (AFUs). (D) Domain arrangement of human KLHDC10. (E) <i>In vitro</i> GST pull-down assay using the GST-KLHDC10 and GST-KLHDC2 constructs and recombinantly purified GFP or GFP-Ala<sub>6</sub>. Anti-GST and anti-GFP immunoblots are indicated. (F) AlphaScreen assay to assess KLHDC10-Ala-tail binding. As in (C) but using GST-KLHDC10 and GST-KLHDC10<sub>β1-88442</sub> instead. See also Figures S1, S2, S10, and S11.</div>
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aria-describedby="figure-caption-1"
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data-image-width="793"
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data-image-alt="Figure 2."
data-pmc-id="PMC10591846"
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<img class="figure-thumb" itemprop="thumbnail"
id="article-image-1"
src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a82e/10591846/e1823c786df0/nihms-1933911-f0002.gif"
alt="Figure 2." />
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<strong class="figure-label">
<p> Figure 2.. Four Ala residues at the… </p>
</strong>
</div>
</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Figure 2.. Four Ala residues at the C terminus and the terminal carboxyl group are… </p>
</strong>
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<figcaption id="figure-caption-1" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Figure 2.. Four Ala residues at the C terminus and the terminal carboxyl group are essential for Ala-tail degron binding by Pirh2 and KLHDC10
</strong>
<div class="figure-caption-contents">(AJ) As in Figure 1C, but using GST-Pirh2<sub>15-195</sub> (A, C, E, G, and I), GST-KLHDC10<sub>β1-88442</sub> (B, D, F, H, and J), and competing peptides, as indicated, to measure the effects of C-terminal amide substitution (A and B); 15 Ala residues at the C terminus (C and D); shortened N-terminal sequences in Ala<sub>5</sub>-containing peptides (E and F); 5 Ala followed by a C-terminal Gly, Asp, or Leu (G and H); and Leu substitution at each of the six terminal positions (I and J). See also Figure S3. Each data point is a technical triplicate, and error bars represent ± SD.</div>
</figcaption>
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data-image-width="1050"
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data-image-alt="Figure 3."
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<img class="figure-thumb" itemprop="thumbnail"
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src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a82e/10591846/a35a6263f813/nihms-1933911-f0003.gif"
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<strong class="figure-label">
<p> Figure 3.. Identification of a candidate Ala-tail… </p>
</strong>
</div>
</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Figure 3.. Identification of a candidate Ala-tail C-degron-binding site in Pirh2 </p>
</strong>
<div class="figure-caption-contents"><p> (A) AlphaFold2 prediction of… </p></div>
</div>
<figcaption id="figure-caption-2" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Figure 3.. Identification of a candidate Ala-tail C-degron-binding site in Pirh2
</strong>
<div class="figure-caption-contents">(A) AlphaFold2 prediction of Pirh2<sub>1195</sub> in complex with an Ala-tail peptide. The highest-ranking among predicted models is shown. Ribbon representation of Pirh2 NTM and RING domains colored according to Figure 1. The Ala-tail peptide is shown in green. The inset shows the Ala-tail degron-binding site in close-up view. (B and C) Electrostatic surface potential (B) and surface hydrophobicity (C) represented for the focused region shown in (A); Ala-tail peptide in green. (D) A close-up view of the Ala-tail peptide in complex with Pirh2<sub>1195</sub>, with the C-terminal Ala residues displayed in stick representation and colored in green. Pirh2<sub>1195</sub> is represented as a transparent surface, with residues in the vicinity of the peptide labeled and sides chains in ball-stick representation. See also Figures S4, S5, and S12.</div>
</figcaption>
</figure>
<figure class="figure-item "
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itemprop="contentUrl"
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data-image-width="1050"
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data-pmc-id="PMC10591846"
data-figure-id="F4">
<img class="figure-thumb" itemprop="thumbnail"
id="article-image-3"
src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a82e/10591846/22d187e54fac/nihms-1933911-f0004.gif"
alt="Figure 4." />
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<strong class="figure-label">
<p> Figure 4.. Identification of a candidate Ala-tail… </p>
</strong>
</div>
</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Figure 4.. Identification of a candidate Ala-tail C-degron-binding site in KLHDC10 </p>
</strong>
<div class="figure-caption-contents"><p> (A) As in Figure… </p></div>
</div>
<figcaption id="figure-caption-3" class="figure-caption-full figure-caption-text" itemtype="http://schema.org/ImageObject" itemprop="description">
<strong class="figure-label">
Figure 4.. Identification of a candidate Ala-tail C-degron-binding site in KLHDC10
</strong>
<div class="figure-caption-contents">(A) As in Figure 3, but for KLHDC10<sub>β1-88442</sub>. KLHDC10 is colored as in Figure 1D. Top predictions of KLHDC10<sub>β1-88442</sub> in complex with an LYKAAAAAA peptide (left) or fused to the LYKAAAAAA peptide through a Gly50 linker (right). (B and C) Electrostatic surface potential (B) and surface hydrophobicity (C) represented for the focused region shown in (A), left; Ala-tail peptide in green. (D) Close-up view of the left panel shown in (A). Two orientations of Ala-tail peptide (green) are presented, in stick and wire representations. (E) Close-up of the right panel shown in (A). See also Figures S6 and S12.</div>
</figcaption>
</figure>
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<img class="figure-thumb" itemprop="thumbnail"
id="article-image-4"
src="https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a82e/10591846/37ea60aeeee2/nihms-1933911-f0005.gif"
alt="Figure 5." />
</a>
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<p> Figure 5.. Validation of residues implicated in… </p>
</strong>
</div>
</div>
<div class="figure-caption-medium figure-caption-text" aria-hidden="true">
<strong class="figure-label">
<p> Figure 5.. Validation of residues implicated in Ala-tail degron recognition by Pirh2 and KLHDC10 </p>
</strong>
<div class="figure-caption-contents"><p> (A)… </p></div>
</div>
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Figure 5.. Validation of residues implicated in Ala-tail degron recognition by Pirh2 and KLHDC10
</strong>
<div class="figure-caption-contents">(A) <i>In vitro</i> GST pull-down assay using GST-Pirh2<sub>1195</sub> WT and mutant constructs as indicated and recombinantly purified GFP or GFP-Ala<sub>6</sub>. Anti-GST and anti-GFP immunoblots are shown. (B) GFP or GFP-Ala<sub>6</sub> and WT or mutant full-length FLAG-Pirh2 were expressed in HeLa cells. Interaction between GFP-Ala<sub>6</sub> with Pirh2 and GFP-Ala<sub>6</sub> ubiquitylation was analyzed by GFP IP followed by anti-FLAG and anti-GFP immunoblots. The dashed line indicates that the blot image has been spliced. (C) As in (A), but for GST-KLHDC10<sub>β1-88442</sub> WT and indicated mutant constructs. (D) As in (B), but for WT and mutant full-length KLHDC10 constructs in HeLa Pirh2-knockout cells and including anti-ubiquitin blot.</div>
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<p> Figure 6.. Conservation of residues implicated in… </p>
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<p> Figure 6.. Conservation of residues implicated in Ala-tail binding among Pirh2 homologs </p>
</strong>
<div class="figure-caption-contents"><p> (A) Multiple sequence… </p></div>
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Figure 6.. Conservation of residues implicated in Ala-tail binding among Pirh2 homologs
</strong>
<div class="figure-caption-contents">(A) Multiple sequence alignment for Pirh2 homologs. The blue boxes highlight residues predicted to stabilize the Ala-tail peptide at the binding pocket in Pirh2 that are shown in Figure 3D and corresponding residues in the homologs. (B) Residues conserved in Pirh2 homologs mapped onto the AlphaFold structure of human Pirh2 (Uni-Prot: Q96PM5) using ConSurf webserver. The color key for variable to conserved residues is shown below. The Ala<sub>6</sub> peptide is shown in stick representation at the Ala-tail-binding site. (C) AlphaFold models for Pirh2 homologs in (A) are superposed. Backbone of each protein is in ribbon representation in different colors, and human Pirh2 is represented with transparent surface. (D) <i>In vitro</i> GST pull-down assay using GST-tagged full-length Pirh2 homolog from <i>S. pombe</i> and recombinantly purified GFP, GFP-Ala<sub>6</sub>, GFP-Thr<sub>6</sub>, or GFP-(Ala-Thr)<sub>3</sub>. Anti-GST and anti-GFP immunoblots are indicated. See also Figures S10 and S11.</div>
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<p> Figure 7.. Conservation of residues implicated in… </p>
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<p> Figure 7.. Conservation of residues implicated in Ala-tail binding among KLHDC10 homologs </p>
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Figure 7.. Conservation of residues implicated in Ala-tail binding among KLHDC10 homologs
</strong>
<div class="figure-caption-contents">(AC) As in Figure 6, but for the KLHDC10 Kelch domain. In (A), blue boxes highlight residues implicated in Ala-tail binding as shown in Figure 4D. See also Figures S7S9.</div>
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