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Papers by Yubin ZHOU

Research paper thumbnail of Identification of Molecular Determinants That Modulate Trafficking of ΔF508 CFTR, the Mutant ABC Transporter Associated With Cystic Fibrosis

Cell Biochemistry and Biophysics, 2005

ABSTRACT

Research paper thumbnail of Molecular Determinants for STIM1 Activation During Store- Operated Ca2+ Entry

Current Molecular Medicine, 2017

Background-STIM/ORAI-mediated store-operated Ca 2+ entry (SOCE) mediates a myriad of Ca 2+-depend... more Background-STIM/ORAI-mediated store-operated Ca 2+ entry (SOCE) mediates a myriad of Ca 2+-dependent cellular activities in mammals. Genetic defects in STIM1/ORAI1 lead to devastating severe combined immunodeficiency; whereas gain-of-function mutations in STIM1/ ORAI1 are intimately associated with tubular aggregate myopathy. At molecular level, a decrease in the Ca 2+ concentrations within the lumen of endoplasmic reticulum (ER) initiates multimerization of the STIM1 luminal domain to switch on the STIM1 cytoplasmic domain to engage and gate ORAI channels, thereby leading to the ultimate Ca 2+ influx from the extracellular space into the cytosol. Despite tremendous progress made in dissecting functional STIM1-ORAI1 coupling, the activation mechanism of SOCE remains to be fully characterized. Objective and Methods-Building upon a robust fluorescence resonance energy transfer assay designed to monitor STIM1 intramolecular autoinhibition, we aimed to systematically dissect the molecular determinants required for the activation and oligomerization of STIM1. Results-Here we showed that truncation of the STIM1 luminal domain predisposes STIM1 to adopt a more active conformation. Replacement of the single transmembrane (TM) domain of

Research paper thumbnail of Inside-out Ca2+ signalling prompted by STIM1 conformational switch

Nature Communications, 2015

Store-operated Ca 2 þ entry mediated by STIM1 and ORAI1 constitutes one of the major Ca 2 þ entry... more Store-operated Ca 2 þ entry mediated by STIM1 and ORAI1 constitutes one of the major Ca 2 þ entry routes in mammalian cells. The molecular choreography of STIM1-ORAI1 coupling is initiated by endoplasmic reticulum (ER) Ca 2 þ store depletion with subsequent oligomerization of the STIM1 ER-luminal domain, followed by its redistribution towards the plasma membrane to gate ORAI1 channels. The mechanistic underpinnings of this inside-out Ca 2 þ signalling were largely undefined. By taking advantage of a unique gain-of-function mutation within the STIM1 transmembrane domain (STIM1-TM), here we show that local rearrangement, rather than alteration in the oligomeric state of STIM1-TM, prompts conformational changes in the cytosolic juxtamembrane coiled-coil region. Importantly, we further identify critical residues within the cytoplasmic domain of STIM1 (STIM1-CT) that entail autoinhibition. On the basis of these findings, we propose a model in which STIM1-TM reorganization switches STIM1-CT into an extended conformation, thereby projecting the ORAI-activating domain to gate ORAI1 channels.

Book Reviews by Yubin ZHOU

Research paper thumbnail of Patch-Clamp Recording of the CRAC Channel Current in STIM-Orai Overexpressing Cells

Induced by the depletion of ER calcium store, the calcium influx through calcium release-activate... more Induced by the depletion of ER calcium store, the calcium influx through calcium release-activated calcium (CRAC) channels is an ubiquitous way of Ca 2+ influx for most cell types. This process is mediated by STIM protein, ER calcium sensor, and PM localized Orai calcium channels. Biophysical characterization of this STIM-Orai-mediated current, or I CRAC , with whole-cell patch-clamp technique is essential for revealing the molecular mechanisms underlying the process of STIM-Orai activation or modulation. Here we describe one commonly used procedure of monitoring CRAC activity with whole-cell patch-clamp technique.

Research paper thumbnail of Chapter 2 Fluorescence-Based Ratiometric Measurement of CRAC Channel Activity in STIM-Orai-Overexpressing HEK-293 Cells

Calcium influx through store-operated Ca 2+ entry (SOCE), mediated by STIM-operated Orai channels... more Calcium influx through store-operated Ca 2+ entry (SOCE), mediated by STIM-operated Orai channels, is crucial for many cellular functions. To dissect the molecular mechanisms underlying the process of STIM-Orai activation and identify regulators that modify this process, ratiometric imaging of SOCE responses in HEK cells overexpressing STIM and Orai is a routinely used method. Here we describe one commonly used procedure of monitoring SOCE activity with a ratiometric membrane-permeable dye fura-2-AM. Other ratiometric indicators suitable for SOCE measurements are also discussed.

Research paper thumbnail of Engineered Cross-Linking to Study the Pore Architecture of the CRAC Channel

ORAI1 constitutes the pore-forming subunit of the calcium release-activated calcium (CRAC) channe... more ORAI1 constitutes the pore-forming subunit of the calcium release-activated calcium (CRAC) channel, a prototypical store-operated channel that is essential for the activation of cells of the immune system. Here we describe a convenient yet powerful cross-linking approach to examine the pore architecture of CRAC channels using ORAI1 proteins engineered to contain one or two cysteine residues. The generalizable cross-linking in situ approach can also be readily extended to study other integral membrane proteins expressed in various types of cells.

Research paper thumbnail of Identification of Molecular Determinants That Modulate Trafficking of ΔF508 CFTR, the Mutant ABC Transporter Associated With Cystic Fibrosis

Cell Biochemistry and Biophysics, 2005

ABSTRACT

Research paper thumbnail of Molecular Determinants for STIM1 Activation During Store- Operated Ca2+ Entry

Current Molecular Medicine, 2017

Background-STIM/ORAI-mediated store-operated Ca 2+ entry (SOCE) mediates a myriad of Ca 2+-depend... more Background-STIM/ORAI-mediated store-operated Ca 2+ entry (SOCE) mediates a myriad of Ca 2+-dependent cellular activities in mammals. Genetic defects in STIM1/ORAI1 lead to devastating severe combined immunodeficiency; whereas gain-of-function mutations in STIM1/ ORAI1 are intimately associated with tubular aggregate myopathy. At molecular level, a decrease in the Ca 2+ concentrations within the lumen of endoplasmic reticulum (ER) initiates multimerization of the STIM1 luminal domain to switch on the STIM1 cytoplasmic domain to engage and gate ORAI channels, thereby leading to the ultimate Ca 2+ influx from the extracellular space into the cytosol. Despite tremendous progress made in dissecting functional STIM1-ORAI1 coupling, the activation mechanism of SOCE remains to be fully characterized. Objective and Methods-Building upon a robust fluorescence resonance energy transfer assay designed to monitor STIM1 intramolecular autoinhibition, we aimed to systematically dissect the molecular determinants required for the activation and oligomerization of STIM1. Results-Here we showed that truncation of the STIM1 luminal domain predisposes STIM1 to adopt a more active conformation. Replacement of the single transmembrane (TM) domain of

Research paper thumbnail of Inside-out Ca2+ signalling prompted by STIM1 conformational switch

Nature Communications, 2015

Store-operated Ca 2 þ entry mediated by STIM1 and ORAI1 constitutes one of the major Ca 2 þ entry... more Store-operated Ca 2 þ entry mediated by STIM1 and ORAI1 constitutes one of the major Ca 2 þ entry routes in mammalian cells. The molecular choreography of STIM1-ORAI1 coupling is initiated by endoplasmic reticulum (ER) Ca 2 þ store depletion with subsequent oligomerization of the STIM1 ER-luminal domain, followed by its redistribution towards the plasma membrane to gate ORAI1 channels. The mechanistic underpinnings of this inside-out Ca 2 þ signalling were largely undefined. By taking advantage of a unique gain-of-function mutation within the STIM1 transmembrane domain (STIM1-TM), here we show that local rearrangement, rather than alteration in the oligomeric state of STIM1-TM, prompts conformational changes in the cytosolic juxtamembrane coiled-coil region. Importantly, we further identify critical residues within the cytoplasmic domain of STIM1 (STIM1-CT) that entail autoinhibition. On the basis of these findings, we propose a model in which STIM1-TM reorganization switches STIM1-CT into an extended conformation, thereby projecting the ORAI-activating domain to gate ORAI1 channels.

Research paper thumbnail of Patch-Clamp Recording of the CRAC Channel Current in STIM-Orai Overexpressing Cells

Induced by the depletion of ER calcium store, the calcium influx through calcium release-activate... more Induced by the depletion of ER calcium store, the calcium influx through calcium release-activated calcium (CRAC) channels is an ubiquitous way of Ca 2+ influx for most cell types. This process is mediated by STIM protein, ER calcium sensor, and PM localized Orai calcium channels. Biophysical characterization of this STIM-Orai-mediated current, or I CRAC , with whole-cell patch-clamp technique is essential for revealing the molecular mechanisms underlying the process of STIM-Orai activation or modulation. Here we describe one commonly used procedure of monitoring CRAC activity with whole-cell patch-clamp technique.

Research paper thumbnail of Chapter 2 Fluorescence-Based Ratiometric Measurement of CRAC Channel Activity in STIM-Orai-Overexpressing HEK-293 Cells

Calcium influx through store-operated Ca 2+ entry (SOCE), mediated by STIM-operated Orai channels... more Calcium influx through store-operated Ca 2+ entry (SOCE), mediated by STIM-operated Orai channels, is crucial for many cellular functions. To dissect the molecular mechanisms underlying the process of STIM-Orai activation and identify regulators that modify this process, ratiometric imaging of SOCE responses in HEK cells overexpressing STIM and Orai is a routinely used method. Here we describe one commonly used procedure of monitoring SOCE activity with a ratiometric membrane-permeable dye fura-2-AM. Other ratiometric indicators suitable for SOCE measurements are also discussed.

Research paper thumbnail of Engineered Cross-Linking to Study the Pore Architecture of the CRAC Channel

ORAI1 constitutes the pore-forming subunit of the calcium release-activated calcium (CRAC) channe... more ORAI1 constitutes the pore-forming subunit of the calcium release-activated calcium (CRAC) channel, a prototypical store-operated channel that is essential for the activation of cells of the immune system. Here we describe a convenient yet powerful cross-linking approach to examine the pore architecture of CRAC channels using ORAI1 proteins engineered to contain one or two cysteine residues. The generalizable cross-linking in situ approach can also be readily extended to study other integral membrane proteins expressed in various types of cells.

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