Directly light-regulated binding of RGS-LOV photoreceptors to anionic membrane phospholipids

We report natural light–oxygen–voltage (LOV) photoreceptors with a blue light-switched, high-affinity (KD ∼ 10−7 M), and direct electrostatic interaction with anionic phospholipids. Membrane localization of one such photoreceptor, BcLOV4 from Botrytis cinerea, is directly coupled to its flavin photocycle, and is mediated by a polybasic amphipathic helixinthelinker regionbetween the LOV sensor and its C-terminal domain of unknown function (DUF), as revealed through a combination of bioinformatics, computational protein modeling, structure–function studies, and optogenetic assays in yeast and mammalian cell line expression systems. In model systems, BcLOV4 rapidly translocates from the cytosol to plasma membrane (∼1 second). The reversible electrostatic interaction is nonselective among anionic phospholipids, exhibiting binding strengths dependent on the total anionic content of the membrane without preference for a specific headgroup. The in vitro and cellular responses were also observed with a BcLOV4 homolog and thus are likely to be general across the dikarya LOV class, whose members are associated with regulator of G-protein signaling (RGS) domains. Natural photoreceptors are not previously known to directly associate with membrane phospholipids in a light-dependent manner, and thus this work establishes both a photosensory signal transmission mode and a single-component optogenetic tool with rapid membrane localization kinetics that approaches the diffusion limit

The Structural and Functional Role of Photosensing in RGS-LOV Proteins

Light provides organisms with energy and spatiotemporal information. To survive and adapt, organisms have developed the ability to sense light to drive biochemical effects that underlie vision, entrainment of circadian rhythm, stress response, virulence, and many other important molecularly driven responses. Blue-light sensing Light-Oxygen-Voltage (LOV) domains are ubiquitous across multiple kingdoms of life and modulate various physiological events via diverse effector domains. Using a small molecule flavin chromophore, the LOV domain undergoes light-dependent structural changes leading to activation or repression of these catalytic and non-catalytic effectors. In silico analyses of high-throughput genomic sequencing data has led to the marked expansion in the collection of known LOV proteins. Many of these proteins contain new LOV-effector pairings, each of which can be predicted to have novel light-controlled function. Of these combinations, RGS (regulator of G protein signaling)-LOV proteins are an exciting new system for light regulation of cellular process and provide an interesting test case to study. Successfully establishing the structural role of LOV photoreceptors with RGS effectors will provide insights into LOV photoreceptor biochemistry that will both enhance understanding of the generality of LOV signaling and yield new optogenetic tools for manipulating biological circuitry. In this dissertation, I build on a previous report in collaboration with Brian Chow’s group (detailed in Chapter 2) of RGS-LOV proteins utilizing a light-regulated and reversible electrostatic interaction between a polybasic amphipathic helix in the linker between the LOV and DUF (Domain of Unknown Function) domains to mediate binding to anionic plasma membrane phospholipids. With a combination of biophysical and biochemical methods, including cryo-EM, limited proteolysis, and HDX-MS, I report the first structural characterization of bcLOV4, a Botrytis cinerea RGS-LOV photoreceptor (Chapter 3). This study establishes the three-dimensional domain arrangement between the RGS, LOV, and DUF domains and gives insights into the domain-level light-triggered signal propagation, membrane recruitment, and function activation as a GTPase accelerating protein (GAP). To reveal more information about the well-conserved DUF domain that flanks the LOV at the C-terminus, I have tried both reductionist approaches as well as comparative analysis (outlined in Chapter 4). I characterized five different RGS-LOVs using bioinformatic techniques followed by heterologous overexpression in bacteria, purification, and initial solution evaluation. For the first time, I describe apRGS and ffRGS, which to date have not yet been experimentally characterized. This comparative study will aid in understanding general trends of the RGS-LOV class of proteins and give insights into their role in fungal photobiology. In this work, I showed a potent combination of results here to help enhance our understanding on the RGS-LOV class and the role that the DUF plays in the overall structure-function. This work has substantial implications on understanding the ways that such proteins might be regulated in natural settings for control of GPCR signaling and further, for the engineering of RGS-LOVs as valuable components for creating optogenetic tools to perturb cellular signaling and physiology

The Effect of Different Ester Chain Modifications of Two Guaianolides for Inhibition of Colorectal Cancer Cell Growth

Several sesquiterpene lactones (STLs) have been tested as lead drugs in cancer clinical trials. Salograviolide-A (Sal-A) and salograviolide-B (Sal-B) are two STLs that have been isolated from Centaurea ainetensis, an indigenous medicinal plant of the Middle Eastern region. The parent compounds Sal-A and Sal-B were modified and successfully prepared into eight novel guaianolide-type STLs (compounds 1–8) bearing ester groups of different geometries. Sal-A, Sal-B, and compounds 1–8 were tested against a human colorectal cancer cell line model with differing p53 status; HCT116 with wild-type p53 and HCT116 p53−/− null for p53, and the normal-like human colon mucosa cells with wild-type p53, NCM460. IC50 values indicated that derivatization of Sal-A and Sal-B resulted in potentiation of HCT116 cell growth inhibition by 97% and 66%, respectively. The effects of the different molecules on cancer cell growth were independent of p53 status. Interestingly, the derivatization of Sal-A and Sal-B molecules enhanced their anti-growth properties versus 5-Fluorouracil (5-FU), which is the drug of choice in colorectal cancer. Structure-activity analysis revealed that the enhanced molecule potencies were mainly attributed to the position and number of the hydroxy groups, the lipophilicity, and the superiority of ester groups over hydroxy substituents in terms of their branching and chain lengths. The favorable cytotoxicity and selectivity of the potent molecules, to cancer cells versus their normal counterparts, pointed them out as promising leads for anti-cancer drug design

Global economic burden of unmet surgical need for appendicitis

Background There is a substantial gap in provision of adequate surgical care in many low- and middle-income countries. This study aimed to identify the economic burden of unmet surgical need for the common condition of appendicitis. Methods Data on the incidence of appendicitis from 170 countries and two different approaches were used to estimate numbers of patients who do not receive surgery: as a fixed proportion of the total unmet surgical need per country (approach 1); and based on country income status (approach 2). Indirect costs with current levels of access and local quality, and those if quality were at the standards of high-income countries, were estimated. A human capital approach was applied, focusing on the economic burden resulting from premature death and absenteeism. Results Excess mortality was 4185 per 100 000 cases of appendicitis using approach 1 and 3448 per 100 000 using approach 2. The economic burden of continuing current levels of access and local quality was US $92 492 million using approach 1 and$73 141 million using approach 2. The economic burden of not providing surgical care to the standards of high-income countries was $95 004 million using approach 1 and$75 666 million using approach 2. The largest share of these costs resulted from premature death (97.7 per cent) and lack of access (97.0 per cent) in contrast to lack of quality. Conclusion For a comparatively non-complex emergency condition such as appendicitis, increasing access to care should be prioritized. Although improving quality of care should not be neglected, increasing provision of care at current standards could reduce societal costs substantially

Global economic burden of unmet surgical need for appendicitis

Background There is a substantial gap in provision of adequate surgical care in many low- and middle-income countries. This study aimed to identify the economic burden of unmet surgical need for the common condition of appendicitis. Methods Data on the incidence of appendicitis from 170 countries and two different approaches were used to estimate numbers of patients who do not receive surgery: as a fixed proportion of the total unmet surgical need per country (approach 1); and based on country income status (approach 2). Indirect costs with current levels of access and local quality, and those if quality were at the standards of high-income countries, were estimated. A human capital approach was applied, focusing on the economic burden resulting from premature death and absenteeism. Results Excess mortality was 4185 per 100 000 cases of appendicitis using approach 1 and 3448 per 100 000 using approach 2. The economic burden of continuing current levels of access and local quality was US $92 492 million using approach 1 and$73 141 million using approach 2. The economic burden of not providing surgical care to the standards of high-income countries was $95 004 million using approach 1 and$75 666 million using approach 2. The largest share of these costs resulted from premature death (97.7 per cent) and lack of access (97.0 per cent) in contrast to lack of quality. Conclusion For a comparatively non-complex emergency condition such as appendicitis, increasing access to care should be prioritized. Although improving quality of care should not be neglected, increasing provision of care at current standards could reduce societal costs substantially