Analysis of novel candidate 'BH3-only' proteins

Apoptosis is a form of programmed cell death common to metazoan species. BH3-only proteins can be thought of as ‘linchpins’ in the apoptotic cell death pathway. This is because of their ability to respond to a variety of cell stress signals and to promote apoptosis by interacting with both pro- and anti-apoptotic members of the BCL-2 protein family, making them master regulators of apoptosis. Apart from the short BH3 motif found in BCL-2 homologs, there are eight canonical human BH3-only proteins that share no amino acid sequence similarity with other members of the BCL-2 family or with each other. The development of chemical or peptide mimetics of the BH3 motif to promote apoptotic cell death of infected or transformed cells as drugs against viruses, parasites like schistosomes as well as against cancer has become an important goal for both pharmaceutical industries and in academic settings (Shamas-Din, Brahmbhatt, Leber, & Andrews, 2011). However, because of the absence of a ‘signature sequence’ that rigorously defines the BH3 motif, it has not been possible to systematically screen for BH3-only proteins in the proteome. BH3-only proteins function by either neutralizing anti-apoptotic BCL-2 proteins or directly activating the pro-apoptotic BCL-2 proteins. Th BH3 motif can be considered as bearing Short Linear Motifs (SLiMs) that is 8-12 amino acids long with two ‘hot-spot’ residues: Asp (D) and Leu (L), which are essential for binding to the hydrophobic cleft of BCL-2 family member proteins (Aouacheria, Combet, Tompa, & Hardwick, 2015). This thesis project focuses on 10 candidate novel BH3-only proteins that were identified by Dr. Abdel Aouacheria and his colleagues using proprietary computational methods to search the entire available proteome. Plasmids expressing these candidate BH3-only proteins, both wild type (WT) as well as mutants (M) that have point mutations in the two hotspot residues, intended to abolish their binding activity to the hydrophobic groove of members of the BCL-2 family, were provided from Dr. Abdel Aouacheria. These candidate novel BH3-only proteins stem from diverse non-mammalian organisms including parasites and bacteria. Candidate BH3-only proteins were tested to determine if they have any pro-death function when expressed in mammalian cells. Three of the ten proteins were cytotoxic and localized to mitochondria, similar to many mammalian apoptosis regulators. Importantly, this cytotoxicity was reduced by the hot-spot mutations, despite wild type and mutant proteins being expressed at similar levels. These candidates were also tested to determine if their cytotoxicity could be inhibited by their putative mammalian binding partners, namely the anti-apoptotic BCL-2 family proteins BCL-xL and BCL-W. I found that both BCL-xL and BCL-W could rescue mammalian cells from the cytotoxic activity of the three killer BH3-only proteins, implying a functional interaction. The remaining seven non-toxic candidate BH3-containing proteins did not localize to mitochondria and could not be activated to induce cell death by the presence of the mammalian BH3-only protein tBID, which itself is highly toxic. These seven candidates also did not prevent anti-death proteins BCL-xL and BCL-W from rescuing cells from the tBID death signal. Overall, these results provide evidence for the existence of functional BH3 motifs in at least three of ten candidate proteins of diverse origin. These findings raise the possibility of evolutionary conservation of the BH3 motif as an ancient protein-protein interaction motif. More importantly, the algorithm opens the door to the possibility of accurately predicting short sequence motifs of degenerate nature but that have functional significance not previously possible

Improvement of human keratinocyte migration by a redox active bioelectric dressing.

Exogenous application of an electric field can direct cell migration and improve wound healing; however clinical application of the therapy remains elusive due to lack of a suitable device and hence, limitations in understanding the molecular mechanisms. Here we report on a novel FDA approved redox-active Ag/Zn bioelectric dressing (BED) which generates electric fields. To develop a mechanistic understanding of how the BED may potentially influence wound re-epithelialization, we direct emphasis on understanding the influence of BED on human keratinocyte cell migration. Mapping of the electrical field generated by BED led to the observation that BED increases keratinocyte migration by three mechanisms: (i) generating hydrogen peroxide, known to be a potent driver of redox signaling, (ii) phosphorylation of redox-sensitive IGF1R directly implicated in cell migration, and (iii) reduction of protein thiols and increase in integrinαv expression, both of which are known to be drivers of cell migration. BED also increased keratinocyte mitochondrial membrane potential consistent with its ability to fuel an energy demanding migration process. Electric fields generated by a Ag/Zn BED can cross-talk with keratinocytes via redox-dependent processes improving keratinocyte migration, a critical event in wound re-epithelialization

Improvement of Human Keratinocyte Migration by a Redox Active Bioelectric Dressing

<div><p>Exogenous application of an electric field can direct cell migration and improve wound healing; however clinical application of the therapy remains elusive due to lack of a suitable device and hence, limitations in understanding the molecular mechanisms. Here we report on a novel FDA approved redox-active Ag/Zn bioelectric dressing (BED) which generates electric fields. To develop a mechanistic understanding of how the BED may potentially influence wound re-epithelialization, we direct emphasis on understanding the influence of BED on human keratinocyte cell migration. Mapping of the electrical field generated by BED led to the observation that BED increases keratinocyte migration by three mechanisms: (i) generating hydrogen peroxide, known to be a potent driver of redox signaling, (ii) phosphorylation of redox-sensitive IGF1R directly implicated in cell migration, and (iii) reduction of protein thiols and increase in integrin_αv expression, both of which are known to be drivers of cell migration. BED also increased keratinocyte mitochondrial membrane potential consistent with its ability to fuel an energy demanding migration process. Electric fields generated by a Ag/Zn BED can cross-talk with keratinocytes via redox-dependent processes improving keratinocyte migration, a critical event in wound re-epithelialization.</p></div

Increased rate of migration of human keratinocytes under an Ag/Zn bioelectric dressing (BED).

<p>(<b>A</b>) HaCaT cells were treated with Ag/Zn BED or polyester printer printed with only silver or only zinc for 24 h followed by scratch assay and migration of cells was observed at 6 h and 9 h following scratch. (<b>B</b>) % closure at 6 h, (<b>C</b>) % closure at 9 h. BED significantly increased rate of migration while placebo or silver alone or zinc alone did not change cell migration. (n = 4).</p

Experimental determination of electric fields generated by the bioelectric dressing.

<p>(<b>A</b>) Voltage measurements from an experiment with a Zn (99.994% pure) and silver (99.998% pure) foil immersed in 100 mL of de-ionized water. (<b>B</b>) Schematic showing the computational grid and boundary conditions used to calculate the magnitude and extent of the electric fields under the bioelectric dressing presumed to be in contact with H₂O. (<b>C, D</b>) Contour plots of calculated values of the electric potential for 0.2 V and 1 V potential difference, respectively. (<b>E, F</b>) Electric fields from the solution of the 2-D Laplace equation for the particular case of 0.2 V and 1 V potential difference, respectively between the silver and the zinc dots. (<b>G,H</b>) Expanded view of the electric field vectors between adjacent pairs of electrodes for potential difference of 0.2 V and 1 V respectively.</p

Summary figure.

<p>Summary figure.</p

BED induces IGF1 receptor phosphorylation which helps in migration.

<p>(<b>A</b>) Validation of increased phosphorylation of IGF1R using ELISA. (<b>B–C</b>) IGF1R inhibitor attenuates Ag/Zn BED induced faster keratinocyte migration (n = 3).</p

Characterization of the Ag/Zn BED.

<p>(<b>A, B</b>) Schematic diagram of the design, application and electric fields generated by the bioelectric dressing, (<b>C</b>) EDS spectrum of a silver dot on a BED (<b>D</b>) EDS spectrum of a zinc dot on a BED. (<b>E</b>) Photographs of silver and (<b>F</b>) zinc dots of the BED mounted using carbon tape in an aluminum holder for energy dispersive spectroscopic (EDS) X-ray analysis. (<b>G</b>) Scanning electron microscope image of a silver dot on a BED (<b>H</b>) Scanning electron microscope image of a zinc dot on a BED.</p