Biology subjects

Pike, A.

Publications and source records attributed to Pike, A..

4 recordsLinked to original sources

bioRxiv2026

Deep mutational scan of the pore of the cold-sensing TRPM8 channel

Members of the Transient Receptor Potential (TRP) family of ion channels have a nearly ubiquitous role in human physiology, tuning cell signaling to remarkably diverse physical and chemical stimuli. Although there is extensive structural data on TRP channels, a systematic and unbiased interrogation of structure-function relations in these proteins is required to fully elucidate their mechanisms of function. By focusing on a critical pore region of the TRPM8 channel, which is the main detector of cold and cooling agents in sensory neurons, we show how deep mutational scanning can be used in combination with the available structural data to understand how TRP channels respond to stimuli. We define a novel mechanism whereby the extracellular pore loop, which has only been resolved in structures representing desensitized states of the channel, plays an essential role in the response of TRPM8 to menthol or cold by coordinating the movement of the S6 helices that line and gate the pore, and the ion-selectivity filter that binds permeant cations. Moreover, our screen reveals sequence determinants along the S6 helices that explain how their architecture sustains gating and, together, provide strong support for a structural mechanism of TRPM8 pore opening in response to menthol and cold.

biophysics↗

bioRxiv2024

A simple, robust, broadly applicable insertion mutagenesis method to create random fluorescent protein - target protein fusions.

A simple broadly applicable method was developed using an in vitro transposition reaction followed by transformation into Escherichia coli and screening plates for fluorescent colonies. The transposition reaction catalyzes the random insertion of a fluorescent protein open reading frame into a target gene on a plasmid. The transposition reaction is employed directly in an E. coli transformation with no further procedures. Plating at high colony density yields fluorescent colonies. Plasmids purified from fluorescent colonies contain random, in-frame fusion proteins into the target gene. The plate screen also results in expressed, stable proteins. A large library of chimeric proteins was produced that was useful for downstream research. The effect of using different fluorescent proteins was investigated as well as the dependence of linker sequence between the target and fluorescent protein open reading frames. The utility and simplicity of the method was demonstrated by the fact that it has been employed in an undergraduate biology laboratory class without failure over dozens of class sections. This suggests that the method will be useful in high impact research at small liberal arts colleges with limited resources. However, in frame fusion proteins were obtained from eight different targets suggesting that the method is broadly applicable in any research setting. SummaryThis report describes a simple screen to obtain random insertions of fluorescent proteins into a target protein of interest. The screen results in a useful library of mutated and functional tagged proteins that can be employed to investigate protein biochemical activity, protein structure, protein function and protein cellular localization. The screen is robust, generally applicable, and has been employed in an undergraduate laboratory class. It is also useful for studies in advanced research-intensive projects carried out in institutions of all types. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/578020v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@22dc94org.highwire.dtl.DTLVardef@bf789dorg.highwire.dtl.DTLVardef@87c53forg.highwire.dtl.DTLVardef@1a6c0e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2019

Lipid Interactions of a Ciliary Membrane TRP Channel: Simulation and Structural Studies of Polycystin-2 (PC2)

Polycystin-2 (PC2) is a member of the TRPP subfamily of TRP channels and is present in ciliary membranes of the kidney. PC2 can be either homo-tetrameric, or heterotetrameric with PC1. PC2 shares a common transmembrane fold with other TRP channels, in addition to having a novel extracellular domain. Several TRP channels have been suggested to be regulated by lipids, including phosphatidylinositol phosphates (PIPs). We have combined molecular dynamics simulations with cryoelectron microscopy to explore possible lipid interactions sites on PC2. We propose that PC2 has a PIP-binding site close to the equivalent vanilloid/lipid-binding site in the TRPV1 channel. A 3.0 [A] cryoelectron microscopy map reveals a binding site for cholesterol on PC2. Cholesterol interactions with the channel at this site are further characterized by MD simulations. These results help to position PC2 within an emerging model of the complex roles of lipids in the regulation and organization of ciliary membranes.

biophysics↗

bioRxiv2017

BRD4 inhibitors block telomere elongation

Cancer cells maintain telomere length equilibrium to avoid senescence and apoptosis induced by short telomeres, which are triggered by the DNA damage response. Limiting the potential for telomere maintenance in cancer cells has been long been proposed as a therapeutic target. Using an unbiased shRNA screen targeting known kinases, we identified bromodomain 4 (BRD4) as a telomere length regulator. Four independent BRD4 inhibitors blocked telomere elongation, in a dose dependent manner, in mouse cells overexpressing telomerase. Long-term treatment with BRD4 inhibitors caused telomere shortening in both mouse and human cells, suggesting BRD4 plays a role in telomere maintenance in vivo. Telomerase enzymatic activity was not directly affected by BRD4 inhibition. BRD4 is in clinical trials for a number of cancers, but its effects on telomere maintenance have not been previously reported investigated.

molecular biology↗