Efficient identification of phosphatidylserine-binding proteins by ORF phage display

Caberoy, Nora B.; Zhou, Yixiong; Alvarado, Gabriela; Fan, Xianqun; Li, Wei

doi:10.1016/j.bbrc.2009.06.010

Cited by 34 publications

(41 citation statements)

References 20 publications

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“…2G). TMEM24 bound acidic liposomes containing either phosphatidylserine (PS) [see also (26)] or phosphoinositides, without preference for a specific phosphoinositide, including PI(4,5)P 2 , a key determinant of PM identity. Supporting this hypothesis, depletion of this phosphoinositide in the PM by light-induced recruitment of the 5-phosphatase domain of Lowe oculocerebrorenal syndrome protein (OCRL) fused to cryptochrome 2 (CRY2) (27, 28) did not affect the localization of TMEM24 to contact sites in COS-7 cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

Lipid transport by TMEM24 at ER–plasma membrane contacts regulates pulsatile insulin secretion

Lees

Messa

Sun

et al. 2017

Science

178

241

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INTRODUCTION Insulin is secreted by pancreatic β cells in response to glucose stimulation. Its release is controlled by the interplay of calcium and phosphoinositide signaling pathways. A rapid release phase, in which insulin containing granules that are already docked and primed at the plasma membrane (PM) undergo exocytosis, is followed by slow release. In this second phase, granules are docked and primed and then released in a series of bursts, each triggered by a spike in cytosolic Ca2+. RATIONALE To better understand the molecular basis underlying insulin secretion, we characterized TMEM24, a protein enriched in neuroendocrine cells previously suggested to be required for a normal secretory response. RESULTS We found that TMEM24 is an endoplasmic reticulum (ER) protein that concentrates at ER-PM contact sites, where it tethers the two bilayers. TMEM24 binding “in trans” to the PM is negatively regulated by phosphorylation in response to elevation of cytosolic Ca2+, so that TMEM24 transiently dissociates from the PM as Ca2+ concentration spikes and then reassociates with this membrane upon dephosphorylation. Additionally, TMEM24 contains a lipid transport module of the synaptotagmin-like, mitochondrial and lipid-binding protein (SMP) family, which we structurally characterized and showed to bind glycerolipids with a preference for phosphatidylinositol (PI). Thus, TMEM24 helps deliver PI, which is synthesized in the ER, to the PM, where it is converted to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] to replenish pools of this lipid hydrolyzed during glucose-stimulated signaling. Supporting a key role of TMEM24 in the coordination of Ca2+ and phosphoinositide signaling, the lipid transport function of TMEM24 is essential for sustaining the intracellular Ca2+ oscillations that trigger bursts of insulin granule release and hence insulin secretion. PI(4,5)P2 is required for Ca2+-dependent exocytosis. It also controls the activity of PM ion channels that regulate cytosolic Ca2+ levels and is the precursor of IP3, which also helps to modulate cytosolic Ca2+ by triggering Ca2+ release from the ER. Thus, in insulin-secreting cells, TMEM24 participates in coordinating Ca2+ and phosphoinositide signaling pathways to cause pulsatile insulin secretion (see the figure). CONCLUSION Our findings implicate ER-PM contact sites and an ER resident lipid-transfer protein in the direct regulation of PM phosphoinositide pools, offering fresh insights into the mechanisms of cellular phosphoinositide dynamics. More specifically, they elaborate the mechanisms underlying insulin secretion, which is impaired in patients with type II diabetes, and may ultimately have therapeutic ramifications. TMEM24 activity cycle at ER-PM contacts Glucose stimulation of insulin-secreting cells triggers Ca2+ influx, phospholipase C–dependent PI(4,5)P2 cleavage, and granule exocytosis. Ca2+-stimulated phosphorylation causes TMEM24 dissociation from the PM and interruption of SMP-mediated PI transfer that allows PI(4,5)P2 resynthesis. Lower...

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Section: Resultsmentioning

confidence: 99%

Lipid transport by TMEM24 at ER–plasma membrane contacts regulates pulsatile insulin secretion

Lees

Messa

Sun

et al. 2017

Science

178

241

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“…A method of constructing ORF phage display cDNA libraries was described in detail in our recent publications [20,21]. The general procedures and special technical considerations are summarized here (Fig.…”

Section: Description Of Methodsmentioning

confidence: 99%

“…NotI and XhoI restriction sites with rare frequencies in mammalian genomes are imbedded in the upstream and downstream primers, respectively. The PCR products are resolved on agarose gel or by other fractionation methods to purify cDNA fragments of 300–1,500 bp, digested with NotI and XhoI, and ligated into an engineered T7Bio3C T7 phage vector at the same cutting sites [20,21]. The ligated DNAs are packaged into phages using T7 package extract (EMD Bioscience).…”

Section: Description Of Methodsmentioning

confidence: 99%

ORF phage display to identify cellular proteins with different functions

2012

Methods

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Open reading frame (ORF) phage display is a new branch of phage display aimed at improving its efficiency to identify cellular proteins with specific binding or functional activities. Despite the success of phage display with antibody libraries and random peptide libraries, phage display with cDNA libraries of cellular proteins identifies a high percentage of non-ORF clones encoding unnatural short peptides with minimal biological implications. This is mainly because of the uncontrollable reading frames of cellular proteins in conventional cDNA libraries. ORF phage display solves this problem by eliminating non-ORF clones to generate ORF cDNA libraries. Here I summarize the procedures of ORF phage display, discuss the factors influencing its efficiency, present examples of its versatile applications, and highlight evidence of its capability of identifying biologically relevant cellular proteins. ORF phage display coupled with different selection strategies is capable of delineating diverse functions of cellular proteins with unique advantages.

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“…The solution to this problem was to use ORF phage display, which is a strategy to identify proteins that has proved efficiency, sensitivity and versatility to help elucidate protein-protein interaction pathways, defense mechanisms and therapeutic targets [53,134]. The ORF phage display cDNA library technique is based on the fact that ORF cDNA does not include a stop codon, since high frequency of stop codons are found in non-ORF cDNA [135,136]. The engineered ORF phage display system was developed from the T7 phage vector with a biotinylation tag in the C-terminus of cDNA library proteins Fig.…”

Section: Orf Phage Display Patentsmentioning

confidence: 99%

The Phage Display Technique: Advantages and Recent Patents

Almeida

Magalhaes²,

Soares³

et al. 2011

DNAG

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Phage display technology has advanced considerably since its creation, and the number of research projects using this technique is constantly increasing, generating numerous antibody and antigen libraries. These libraries, besides expediting library screening, improving selection methods and allowing evaluation of novel applications, have great potential for the development of new vaccines, drugs and diagnosis tests. Consequently, patent registries for the protection of these sequences are essential.

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Efficient identification of phosphatidylserine-binding proteins by ORF phage display

Cited by 34 publications

References 20 publications

Lipid transport by TMEM24 at ER–plasma membrane contacts regulates pulsatile insulin secretion

Lipid transport by TMEM24 at ER–plasma membrane contacts regulates pulsatile insulin secretion

ORF phage display to identify cellular proteins with different functions

The Phage Display Technique: Advantages and Recent Patents

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