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Exploring and Exploiting Ion Channel Function

Using MiniGenes™ to Make Peptides


The Critical Role of Channels in Cellular Signaling and Homeostasis

Intracellular Ca2+ release channels, gap-junctional channels, and hemichannels regulate the flux of important signaling molecules, such as Ca2+ and ATP [1,2]. Hence, they play a critical role in how cells behave, differentiate, and signal one another. Misregulation of these ion channels can affect cellular homeostasis and function, resulting in uncontrolled cell survival and death that can lead to disease [3].

Using Peptides to Study Ion Channel Function

After his post-doc with Dr Cyert (Stanford, CA) [4], Dr Geert Bultynck joined the Laboratory of Molecular and Cellular Signaling (Drs Himpens, Parys, Missiaen, De Smedt; Katholieke Universiteit Leuven, Belgium). The central paradigm of Dr Geert Bultynck’s research is based on the finding that the function of ion channels is critically regulated by proteinprotein interactions and formation of multiprotein complexes [5,6,7]. Together with an extensive network of national (e.g., Dr Leybaert, Ghent University, Belgium) and international collaborators (e.g., Dr Distelhorst, Case Western Reserve University, OH), his team works to identify and characterize the key molecules controlling channel function, to elucidate how changes in these complexes are involved in human pathologies, and to develop novel therapeutic strategies by exploiting or restoring these molecular determinants [8].

The lab focuses on the role channel complexes play in polycystic kidney disease, B-cell lymphoma, and ischemic reperfusion in the brain and heart. They do this by developing channel peptide fragments to identify their critical determinants. While the laboratory uses chemically synthesized peptides from commercial sources (Thermo Fisher and LifeTein), these peptides are expensive. Since each sequence requires a new synthesis reaction, costs accumulate rapidly, especially when several variants or modifications of the same peptide are needed. Prices typically go down for larger batches, but stability can become an issue for some peptide sequences due to deterioration during prolonged storage.

 Development of BH4-domain Peptide Fused to TAT and HA via Custom MiniGene Synthesis
Figure 1. Development of BH4-domain Peptide Fused to TATand HA via Custom MiniGene™ Synthesis. IDT provided a custom MiniGene (IDT) in which the nucleotides encoding the TAT sequence (YGRKKRRQRRR), HA tag (YPYDVPDYA), and BH4 domain of Bcl-2 (RTGYDNREIVMKYIHYKLSQRGYEW) were tandem fused and cloned into the BamHI/EcoRI-restriction sites of the pGEX6p2 vector (GE Healthcare). A short linker (GG) was inserted between all functional domains. This vector contains a PreScission-protease recognition site (LEVLFQ¦GP), separating the GST from the protein/peptide of interest, and allowing removal of the GST tag (¦ symbol = protease-cleavage site). It is important to remove these proteases (or at least inactivate them) from the purified peptide preparation. Some amino acids will remain attached to the expressed peptide (e.g., as when using a TEV-cleavage site). Additional tags provide for further purifications (e.g., a 6x His tag). Here the GST tag allowed purification of the GSTfusion peptide. The other tags were used to promote cell entry of the purified peptides, to perform immunolocalization experiments using anti-HA antibodies, or to perform interaction studies with purified fragments of the IP3R, an intracellular Ca2+-release channel. The researchers developed these tools for the BH4 domain of the antiapoptotic Bcl-2 and Bcl-Xl.

Custom Genes to Study Protein:Protein Interactions

Custom MiniGenes—plasmids synthesized by IDT—now provide an alternate approach that is more cost effective and allows greater experimental flexibility. Researchers can remake peptides at any time, the renewable stock alleviating stability issues. Mutant variants are easily made and, if designed properly, the peptides can be used for affinity purifications, immunofluorescence approaches, binding studies, and western-blotting approaches.

Custom Gene Synthesis

IDT offers a confidential custom gene synthesis service that includes miniGenes (up to 400 bp) and Genes (401–1500 bp). By ordering MiniGenes and Genes from IDT, researchers not only save money spent on reagents necessary for construction, cloning, and sequencing but can also save time by outsourcing the manufacturing of hard-to-clone gene sequences which often result in repeated failures. At IDT, all genes are constructed using Ultramer™ Oligos, the highest fidelity next generation synthesis technology available. MiniGenes and Genes arrive in a plasmid cloning vector and are ready for use in a variety of applications.

Generating Peptides

Dr Bultynck’s group cloned the MiniGene (117 bp), encoding a wild type or mutated peptide sequence of interest into a bacterial expression vector (Figure 1). The backbone vector (pGEX6p2; GE Healthcare) contained a GST-tag for purification on Glutathione-Sepharose 4B beads. The MiniGene sequence included three functional units: a TAT sequence (to promote cellular uptake of the peptides), an HA tag (to facilitate detection in western blotting assays or in immunofluorescence assays) and the BH4 domain of Bcl-2. The group also included a short flexible sequence between all functional units. This vector allows removal of the GST-tag from the peptide using the PreScission Protease (GE Healthcare), yielding the peptide of interest: TAT-HA-BH4-Bcl-2. As indicated in Figure 1, additional protease-cleavage sites may be introduced as can other tags (e.g., FLAG, 6xHis, etc.). The rapid turnaround for delivery by IDT, plus the endless possibilities and flexibility of the Custom Genes are all great advantages.

This method does require additional laboratory time, including affinity purification from BL21(DE3) E. coli strain extracts and subsequent protease digestion. Furthermore, the lab performs additional controls to address, for example, whether the resulting peptides are expressed in inclusion bodies. For other research groups considering a similar approach, Dr Bultynck highly recommends using a control construct that contains the reverse or scrambled sequence that has undergone all the same processing in the bacteria.

Despite this extra hands-on time, Dr Bultynck notes, “The MiniGenes-to-Peptide" approach is an excellent alternative to chemical peptide synthesis, providing the researcher a lot of freedom and flexibility. One can easily create new mutations, or change or insert a removable tag. And by subcloning the construct into a mammalian expression vector or adenoviral vector, the peptide can be delivered to and expressed in mammalian cells”.

Using Peptides to Intervene with Cellular Signaling

The Katholieke Universiteit Leuven team has treated cultured cell lines and primary cells from living animals or human patients with these peptides, and studied Ca2+ signaling dynamics and cell survival/cell-death responses [8]. Currently, they assess Ca2+ responses in peptide-treated cells using the FlexStation3 (Molecular Devices), an automated 96-well plate reader [9], and are implementing this method for ATP-release experiments. This is quite unique, as this research group is among the few that have implemented this technique in their routine laboratory studies. In the future, these scientists also plan to apply this method to analysis of apoptosis and autophagy.

Results Obtained Using GST-TAT-HA-BH4
Figure 2. Results Obtained Using GST-TAT-HA-BH4. (A) GST-fusion peptides were expressed in BL21(DE3) after IPTG induction and were purified from the bacterial lysates using Glutathione-Sepharose 4B beads (GE Healthcare). GST-TAT-HA-BH4 was eluted using reduced Glutathione and dialyzed against PBS. Purified proteins were analyzed via SDS-PAGE and stained using SyproOrange™ (BioRad). Molecular weight markers (Mark12; Invitrogen) are indicated on the left of the gels. (B) Sample treated with PreScission Protease™ to remove the GST tag from the TAT-HA-BH4 peptide. (C) First, purification using Centricon® Centrifugal Filter Units (Millipore) with a 30 kDa cut-off. Second, concentration of the remaining sample using a cut-off of 3 kDa. The presence of the HA-tag was confirmed using western blotting analysis and anti-HA antibodies.

A Specific Example: The BH4 Domain

Dr Bultynck is currently using the MiniGene-to-peptide approach to study the BH4 domains of Bcl-2, an accessory protein that inhibits ER Ca2+ release and apoptosis by binding the regulatory and coupling domains of the IP3 receptor channel [4]. He has found the expressed BH4 domains to be insoluble in aqueous buffers (like their commercially purchased counterparts), making them difficult to work with, but Dr Bultynck is working on ways to overcome this problem. He notes, “We knew from the chemically synthesized peptides that this system would be a challenge, but certainly the MiniGene-to-peptide approach is valid for peptide stretches typically soluble in aqueous solutions.” Nevertheless, Giovanni Monaco, a PhD student of the lab, was able to express and purify the GST-TAT-HA-BH4 domain and to remove the GST-tag using the PreScission Protease™ system (Figure 2). Using this method, they obtained the TAT-HA-BH4 peptide at ~ 0.2 μg/μL and a purity of ~70%.

Other Approaches: qPCR Screening and RNA Interference

In addition to the peptide targeting work, the group is systematically screening cancer cell lines for different channel proteins using IDT PrimeTime qPCR Assays. “The IDT qPCR system generates very reliable results, is cheaper than the other supplier we were using, and we are totally happy with the data we are getting”, explains Santeri Kiviluoto, a PhD student in Dr Bultynck’s lab. “We also use IDT oligos as sequencing primers and for cloning—we get them quickly and the quality is very good.”

Dr Bultynck uses RNA interference to knock down expression of channel accessory proteins in mouse embryonic fibroblasts from knockout mice, as well as HeLa and 293HeK cells, which contain high levels of anti-apoptotic proteins, where siRNA interference is easily and very accurately detected.

Recently, with the help of Dr Vanoevelen, the group introduced zebrafish as a model system for studying the development and differentiation of evolutionarily conserved proteins that regulate Ca2+ signaling.

Looking Ahead…

As it becomes clearer which proteins are regulating intracellular Ca2+-release channels, polycystins, and connexin channels, and how they are involved in the control of cellular homeostasis, proliferation, differentiation, and death, novel therapeutic strategies will evolve. Here, the access to samples from human patients via the University Hospital (UK) Leuven (Dr Levtchenko, Dr Janssens, Dr Vandenberghe) and U.Z. Ghent (Dr Offner) is crucial and provides the lab with a translational approach. Cells from blood samples from chronic lymphocytic leukemia patients, and urine from patients diagnosed with polycystic kidney disease allow the
lab to address how channel accessory proteins contribute to fundamental signaling processes and how they influence the diseased state and the efficacy of drug regimes.

Researcher Profile

Dr. Geert Bultynck and team Prof. Geert Bultynck received his PhD in 2001 for work on binding of immunophilines to intracellular Ca2+ release channel proteins (Humbert De Smedt laboratory). After several postdoctoral fellowships, including one at Stanford University (USA), to address calcineurin and apoptosis targets and Ca2+ channels in neurons, he returned to Belgium to start his own research group. In October 2008, he was appointed Professor at Katholieke Universiteit Leuven, in Belgium. His recent work involves the characterization of i) anti-apoptotic Bcl-2-family members in pro-survival Ca2+ signaling, ii) Bax Inhibitor-1 in ER Ca2+, iii) IP3R/Bcl-2 complexes in cancer cells, iv) IP3Rs in autophagy, and v) connexin hemi-channel activity. Along with 3 other principle investigators, Dr Bultynck leads a very motivated team of 2 post docs, 5 PhD students, and 2 technicians. His work is supported by numerous competitive grants from The Research Foundation Flanders and The Research Council of Katholieke Universiteit Leuven. Dr Bultynck is on the left end of the first row.

References
1.  Sammels E, Parys JB, et al. (2010) Intracellular Ca2+ storage in health and disease: a dynamic equilibrium. Cell Calcium, 47(4):297−314.

2.  D’hondt C, Ponsaerts R, et al. (2011) Pannexin channels in ATP release and beyond: an unexpected rendezvous at the endoplasmic reticulum. Cell Signal, 23(2):305−316.

3.  Decuypere JP, Monaco G, et al. (2010) The IP(3) receptor-mitochondria connection in apoptosis and autophagy. Biochim Biophys Acta. Epub.

4.  Bultynck G, Heath VL, et al. (2006) Slm1 and slm2 are novel substrates of the calcineurin phosphatase required for heat stress-induced endocytosis of the yeast uracil permease. Mol Cell Biol, 26(12):4729−4745.

5.  Ponsaerts R, De Vuyst E, et al. (2010) Intramolecular loop/tail interactions are essential for connexin 43-hemichannel activity. FASEB J, 24(11):4378−4395.

6.  Rong YP, Aromolaran AS, et al. (2008) Targeting Bcl-2-IP3 receptor interaction to reverse Bcl-2’s inhibition of apoptotic calcium signals. Mol Cell, 31(2):255−265.

7.  Sammels E, Devogelaere B, et al. (2010) Polycystin-2 activation by inositol 1,4,5-trisphosphate-induced Ca2+ release requires its direct association with the inositol 1,4,5-trisphosphate receptor in a signaling microdomain. J Biol Chem. 285(24):18794−18805.

8.  Zhong F, Harr MW, et al. (2010) Induction of Ca2+-driven apoptosis in chronic lymphocytic leukemia cells by peptide-mediated disruption of Bcl-2-IP3 receptor interaction. Blood, 117(10):2924−2934.

9.  Tovey SC, Sun Y, Taylor CW. (2006) Rapid functional assays of intracellular Ca2+ channels. Nat Protoc, 1(1):259−266.

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