Cargando…

Self-Cleavage of Human Chloride Channel Accessory 2 Causes a Conformational Shift That Depends on Membrane Anchorage and Is Required for Its Regulation of Store-Operated Calcium Entry

Human CLCA2 regulates store-operated calcium entry (SOCE) by interacting with Orai1 and STIM1. It is expressed as a 943aa type I transmembrane protein that is cleaved at amino acid 708 to produce a diffusible 100 kDa product. The N-terminal ectodomain contains a hydrolase-like subdomain with a conse...

Descripción completa

Detalles Bibliográficos
Autores principales: Ramena, Grace T., Sharma, Aarushi, Chang, Yan, Pan, Zui, Elble, Randolph C.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10669480/
https://www.ncbi.nlm.nih.gov/pubmed/38001916
http://dx.doi.org/10.3390/biomedicines11112915
_version_ 1785139706517782528
author Ramena, Grace T.
Sharma, Aarushi
Chang, Yan
Pan, Zui
Elble, Randolph C.
author_facet Ramena, Grace T.
Sharma, Aarushi
Chang, Yan
Pan, Zui
Elble, Randolph C.
author_sort Ramena, Grace T.
collection PubMed
description Human CLCA2 regulates store-operated calcium entry (SOCE) by interacting with Orai1 and STIM1. It is expressed as a 943aa type I transmembrane protein that is cleaved at amino acid 708 to produce a diffusible 100 kDa product. The N-terminal ectodomain contains a hydrolase-like subdomain with a conserved HEXXH zinc-binding motif that is proposed to cleave the precursor autoproteolytically. Here, we tested this hypothesis and its link to SOCE. We first studied the conditions for autocleavage in isolated membranes and then in a purified protein system. Cleavage was zinc-dependent and abolished by mutation of the E in the HEXXH motif to Q, E165Q. Cleavage efficiency increased with CLCA2 concentration, implying that it occurs in trans. Accordingly, the E165Q mutant was cleaved by co-transfected wildtype CLCA2. Moreover, CLCA2 precursors with different epitope tags co-immunoprecipitated. In a membrane-free system utilizing immunopurified protease and target, no cleavage occurred unless the target was first denatured, implying that membranes provide essential structural or conformational cues. Unexpectedly, cleavage caused a conformational shift: an N-terminal antibody that immunoprecipitated the precursor failed to precipitate the N-terminal product unless the product was first denatured with an ionic detergent. The E165Q mutation abolished the stimulation of SOCE caused by wildtype CLCA2, establishing that the metalloprotease activity is required for this regulatory function.
format Online
Article
Text
id pubmed-10669480
institution National Center for Biotechnology Information
language English
publishDate 2023
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-106694802023-10-28 Self-Cleavage of Human Chloride Channel Accessory 2 Causes a Conformational Shift That Depends on Membrane Anchorage and Is Required for Its Regulation of Store-Operated Calcium Entry Ramena, Grace T. Sharma, Aarushi Chang, Yan Pan, Zui Elble, Randolph C. Biomedicines Article Human CLCA2 regulates store-operated calcium entry (SOCE) by interacting with Orai1 and STIM1. It is expressed as a 943aa type I transmembrane protein that is cleaved at amino acid 708 to produce a diffusible 100 kDa product. The N-terminal ectodomain contains a hydrolase-like subdomain with a conserved HEXXH zinc-binding motif that is proposed to cleave the precursor autoproteolytically. Here, we tested this hypothesis and its link to SOCE. We first studied the conditions for autocleavage in isolated membranes and then in a purified protein system. Cleavage was zinc-dependent and abolished by mutation of the E in the HEXXH motif to Q, E165Q. Cleavage efficiency increased with CLCA2 concentration, implying that it occurs in trans. Accordingly, the E165Q mutant was cleaved by co-transfected wildtype CLCA2. Moreover, CLCA2 precursors with different epitope tags co-immunoprecipitated. In a membrane-free system utilizing immunopurified protease and target, no cleavage occurred unless the target was first denatured, implying that membranes provide essential structural or conformational cues. Unexpectedly, cleavage caused a conformational shift: an N-terminal antibody that immunoprecipitated the precursor failed to precipitate the N-terminal product unless the product was first denatured with an ionic detergent. The E165Q mutation abolished the stimulation of SOCE caused by wildtype CLCA2, establishing that the metalloprotease activity is required for this regulatory function. MDPI 2023-10-28 /pmc/articles/PMC10669480/ /pubmed/38001916 http://dx.doi.org/10.3390/biomedicines11112915 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Ramena, Grace T.
Sharma, Aarushi
Chang, Yan
Pan, Zui
Elble, Randolph C.
Self-Cleavage of Human Chloride Channel Accessory 2 Causes a Conformational Shift That Depends on Membrane Anchorage and Is Required for Its Regulation of Store-Operated Calcium Entry
title Self-Cleavage of Human Chloride Channel Accessory 2 Causes a Conformational Shift That Depends on Membrane Anchorage and Is Required for Its Regulation of Store-Operated Calcium Entry
title_full Self-Cleavage of Human Chloride Channel Accessory 2 Causes a Conformational Shift That Depends on Membrane Anchorage and Is Required for Its Regulation of Store-Operated Calcium Entry
title_fullStr Self-Cleavage of Human Chloride Channel Accessory 2 Causes a Conformational Shift That Depends on Membrane Anchorage and Is Required for Its Regulation of Store-Operated Calcium Entry
title_full_unstemmed Self-Cleavage of Human Chloride Channel Accessory 2 Causes a Conformational Shift That Depends on Membrane Anchorage and Is Required for Its Regulation of Store-Operated Calcium Entry
title_short Self-Cleavage of Human Chloride Channel Accessory 2 Causes a Conformational Shift That Depends on Membrane Anchorage and Is Required for Its Regulation of Store-Operated Calcium Entry
title_sort self-cleavage of human chloride channel accessory 2 causes a conformational shift that depends on membrane anchorage and is required for its regulation of store-operated calcium entry
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10669480/
https://www.ncbi.nlm.nih.gov/pubmed/38001916
http://dx.doi.org/10.3390/biomedicines11112915
work_keys_str_mv AT ramenagracet selfcleavageofhumanchloridechannelaccessory2causesaconformationalshiftthatdependsonmembraneanchorageandisrequiredforitsregulationofstoreoperatedcalciumentry
AT sharmaaarushi selfcleavageofhumanchloridechannelaccessory2causesaconformationalshiftthatdependsonmembraneanchorageandisrequiredforitsregulationofstoreoperatedcalciumentry
AT changyan selfcleavageofhumanchloridechannelaccessory2causesaconformationalshiftthatdependsonmembraneanchorageandisrequiredforitsregulationofstoreoperatedcalciumentry
AT panzui selfcleavageofhumanchloridechannelaccessory2causesaconformationalshiftthatdependsonmembraneanchorageandisrequiredforitsregulationofstoreoperatedcalciumentry
AT elblerandolphc selfcleavageofhumanchloridechannelaccessory2causesaconformationalshiftthatdependsonmembraneanchorageandisrequiredforitsregulationofstoreoperatedcalciumentry