Protein evolution

On the origin of proteins A series of mistakes over the past 3.7 billion years or so has left us with a spectacular array of protein structures and functions, which are responsible for nothing less than life itself, writes Bea Perk

Theophany and Chaoskampf : the interpretation of Theophanic imagery in the Baal Epic, Isaiah, and the Twelve

This dissertation concerns the interpretation of theophanic imagery in the Prophets. In essence, my argument is that the imagery which attends and describes theophany in the poetic books of the Hebrew Bible is mythic and not metaphorical-that it should be interpreted in relation to the Chaoskampf myth, as Yahweh defeats chaos and restores order, rather than being understood as a metaphorical comparison with natural phenomena. In arguing this way, however, I am taking a new approach to theophanic imagery, for such imagery has almost uniformly been understood in Old Testament scholarship as metaphor. Before examining in detail this traditional and more widespread approach to theophanic imagery and the alternative to it which I wish to suggest, however, it will be helpful to state at the outset two foundations upon which my argument rests and the specific texts which will be enlisted to support it

Vestigialization of an Allosteric Switch: Genetic and Structural Mechanisms for the Evolution of Constitutive Activity in a Steroid Hormone Receptor

An important goal in molecular evolution is to understand the genetic and physical mechanisms by which protein functions evolve and, in turn, to characterize how a protein's physical architecture influences its evolution. Here we dissect the mechanisms for an evolutionary shift in function in the mollusk ortholog of the steroid hormone receptors (SRs), a family of biologically essential transcription factors. In vertebrates, the activity of SRs allosterically depends on binding a hormonal ligand; in mollusks, however, the SR ortholog (called ER, because of high sequence similarity to vertebrate estrogen receptors) activates transcription in the absence of ligand and does not respond to steroid hormones. To understand how this shift in regulation evolved, we combined evolutionary, structural, and functional analyses. We first determined the X-ray crystal structure of the ER of the Pacific oyster Crassostrea gigas (CgER), and found that its ligand pocket is filled with bulky residues that prevent ligand occupancy. To understand the genetic basis for the evolution of mollusk ERs' unique functions, we resurrected an ancient SR progenitor and characterized the effect of historical amino acid replacements on its functions. We found that reintroducing just two ancient replacements from the lineage leading to mollusk ERs recapitulates the evolution of full constitutive activity and the loss of ligand activation. These substitutions stabilize interactions among key helices, causing the allosteric switch to become “stuck” in the active conformation and making activation independent of ligand binding. Subsequent changes filled the ligand pocket without further affecting activity; by degrading the allosteric switch, these substitutions vestigialized elements of the protein's architecture required for ligand regulation and made reversal to the ancestral function more complex. These findings show how the physical architecture of allostery enabled a few large-effect mutations to trigger a profound evolutionary change in the protein's function and shaped the genetics of evolutionary reversibility.</p

Crystallization and preliminary X-ray diffraction analysis of Sfh3, a member of the Sec14 protein superfamily

Sec14 is the major phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer protein in the yeast Saccharomyces cerevisiae and is the founding member of the Sec14 protein superfamily. Recent functional data suggest that Sec14 functions as a nanoreactor for PtdCho-regulated presentation of PtdIns to PtdIns kinase to affect membrane trafficking. Extrapolation of this concept to other members of the Sec14 superfamily suggests a mechanism by which a comprehensive cohort of Sec14-like nanoreactors sense correspondingly diverse pools of lipid metabolites. In turn, metabolic information is translated to signaling circuits driven by phosphoinositide metabolism. Sfh3, one of five Sec14 homologs in yeast, exhibits several interesting functional features, including its unique localization to lipid particles and microsomes. This localization forecasts novel regulatory interfaces between neutral lipid metabolism and phosphoinositide signaling. To launch a detailed structural and functional characterization of Sfh3, the recombinant protein was purified to homogeneity, diffraction-quality crystals were produced and a native X-ray data set was collected to 2.2 Å resolution. To aid in phasing, SAD X-ray diffraction data were collected to 1.93 Å resolution from an SeMet-labeled crystal at the Southeast Regional Collaborative Access Team at the Advanced Photon Source. Here, the cloning and purification of Sfh3 and the preliminary diffraction of Sfh3 crystals are reported, enabling structural analyses that are expected to reveal novel principles governing ligand binding and functional specificity for Sec14-superfamily proteins

A phospholipid mimetic targeting LRH-1 ameliorates colitis.

Phospholipids are ligands for nuclear hormone receptors (NRs) that regulate transcriptional programs relevant to normal physiology and disease. Here, we demonstrate that mimicking phospholipid-NR interactions is a robust strategy to improve agonists of liver receptor homolog-1 (LRH-1), a therapeutic target for colitis. Conventional LRH-1 modulators only partially occupy the binding pocket, leaving vacant a region important for phospholipid binding and allostery. Therefore, we constructed a set of molecules with elements of natural phospholipids appended to a synthetic LRH-1 agonist. We show that the phospholipid-mimicking groups interact with the targeted residues in crystal structures and improve binding affinity, LRH-1 transcriptional activity, and conformational changes at a key allosteric site. The best phospholipid mimetic markedly improves colonic histopathology and disease-related weight loss in a murine T&nbsp;cell transfer model of colitis. This evidence of in&nbsp;vivo efficacy for an LRH-1 modulator in colitis represents a leap forward in agonist development

Rapid, High Throughput, Automated Detection of SARS-CoV-2 Neutralizing Antibodies Against Wuhan-Wt, Delta and Omicron BA1, BA2 Spike Trimers

Traditional cellular and live-virus methods for detection of SARS-CoV-2 neutralizing antibodies (nAbs) are labor- and time-intensive, and thus not suited for routine use in the clinical lab to predict vaccine efficacy and natural immune protection. Here, we report the development and validation of a rapid, high throughput method for measuring SARS-CoV-2 nAbs against native-like trimeric spike proteins. This assay uses a blockade of human angiotensin converting enzyme 2 (hACE-2) binding (BoAb) approach in an automated digital immunoassay on the Quanterix HD-X platform. BoAb assays using Wuhan-WT (vaccine strain), delta (B.1.167.2), omicron BA1 and BA2 variant viral strains showed strong correlation with cell-based pseudovirus neutralization activity (PNA) and live-virus neutralization activity. Importantly, we were able to detect similar patterns of delta and omicron variant resistance to neutralization in samples with paired vaccine strain and delta variant BoAb measurements. Finally, we screened clinical samples from patients with or without evidence of SARS-CoV-2 exposure by a single-dilution screening version of our assays, finding significant nAb activity only in exposed individuals. Importantly, this completely automated assay can be performed in 4 h to measure neutralizing antibody titers for 16 samples over 8 serial dilutions or, 128 samples at a single dilution with replicates. In principle, these assays offer a rapid, robust, and scalable alternative to time-, skill-, and cost-intensive standard methods for measuring SARS-CoV-2 nAb levels

Protein Evolution by Molecular Tinkering: Diversification of the Nuclear Receptor Superfamily from a Ligand-Dependent Ancestor

Phylogenetic reconstruction of the structure and function of the ancestor of the nuclear receptor protein family reveals how functional diversity evolves by subtle tinkering with an ancestral template

Mechanisms for the Evolution of a Derived Function in the Ancestral Glucocorticoid Receptor

Understanding the genetic, structural, and biophysical mechanisms that caused protein functions to evolve is a central goal of molecular evolutionary studies. Ancestral sequence reconstruction (ASR) offers an experimental approach to these questions. Here we use ASR to shed light on the earliest functions and evolution of the glucocorticoid receptor (GR), a steroid-activated transcription factor that plays a key role in the regulation of vertebrate physiology. Prior work showed that GR and its paralog, the mineralocorticoid receptor (MR), duplicated from a common ancestor roughly 450 million years ago; the ancestral functions were largely conserved in the MR lineage, but the functions of GRs—reduced sensitivity to all hormones and increased selectivity for glucocorticoids—are derived. Although the mechanisms for the evolution of glucocorticoid specificity have been identified, how reduced sensitivity evolved has not yet been studied. Here we report on the reconstruction of the deepest ancestor in the GR lineage (AncGR1) and demonstrate that GR's reduced sensitivity evolved before the acquisition of restricted hormone specificity, shortly after the GR–MR split. Using site-directed mutagenesis, X-ray crystallography, and computational analyses of protein stability to recapitulate and determine the effects of historical mutations, we show that AncGR1's reduced ligand sensitivity evolved primarily due to three key substitutions. Two large-effect mutations weakened hydrogen bonds and van der Waals interactions within the ancestral protein, reducing its stability. The degenerative effect of these two mutations is extremely strong, but a third permissive substitution, which has no apparent effect on function in the ancestral background and is likely to have occurred first, buffered the effects of the destabilizing mutations. Taken together, our results highlight the potentially creative role of substitutions that partially degrade protein structure and function and reinforce the importance of permissive mutations in protein evolution

A phosphatidylinositol transfer protein integrates phosphoinositide signaling with lipid droplet metabolism to regulate a developmental program of nutrient stress-induced membrane biogenesis

The Sec14-like phosphatidylinositol transfer protein Sfh3 associates with bulk LDs in vegetative cells but targets to a neutral lipid hydrolase-rich LD pool during sporulation. Sfh3 inhibits LD utilization by a PtdIns-4-phosphate–dependent mechanism, and this inhibition prevents prospore membrane biogenesis in sporulating cells.Lipid droplet (LD) utilization is an important cellular activity that regulates energy balance and release of lipid second messengers. Because fatty acids exhibit both beneficial and toxic properties, their release from LDs must be controlled. Here we demonstrate that yeast Sfh3, an unusual Sec14-like phosphatidylinositol transfer protein, is an LD-associated protein that inhibits lipid mobilization from these particles. We further document a complex biochemical diversification of LDs during sporulation in which Sfh3 and select other LD proteins redistribute into discrete LD subpopulations. The data show that Sfh3 modulates the efficiency with which a neutral lipid hydrolase-rich LD subclass is consumed during biogenesis of specialized membrane envelopes that package replicated haploid meiotic genomes. These results present novel insights into the interface between phosphoinositide signaling and developmental regulation of LD metabolism and unveil meiosis-specific aspects of Sfh3 (and phosphoinositide) biology that are invisible to contemporary haploid-centric cell biological, proteomic, and functional genomics approaches

Physiological Adaptations to Progressive Endurance Exercise Training in Adult and Aged Rats: Insights from the Molecular Transducers of Physical Activity Consortium (MoTrPAC)

While regular physical activity is a cornerstone of health, wellness, and vitality, the impact of endurance exercise training on molecular signaling within and across tissues remains to be delineated. The Molecular Transducers of Physical Activity Consortium (MoTrPAC) was established to characterize molecular networks underlying the adaptive response to exercise. Here, we describe the endurance exercise training studies undertaken by the Preclinical Animal Sites Studies component of MoTrPAC, in which we sought to develop and implement a standardized endurance exercise protocol in a large cohort of rats. To this end, Adult (6-mo) and Aged (18-mo) female (n&nbsp;=&nbsp;151) and male (n&nbsp;=&nbsp;143) Fischer 344 rats were subjected to progressive treadmill training (5 d/wk, ∼70%-75% VO2max) for 1, 2, 4, or 8 wk; sedentary rats were studied as the control group. A total of 18 solid tissues, as well as blood, plasma, and feces, were collected to establish a publicly accessible biorepository and for extensive omics-based analyses by MoTrPAC. Treadmill training was highly effective, with robust improvements in skeletal muscle citrate synthase activity in as little as 1-2 wk and improvements in maximum run speed and maximal oxygen uptake by 4-8 wk. For body mass and composition, notable age- and sex-dependent responses were observed. This work in mature, treadmill-trained rats represents the most comprehensive and publicly accessible tissue biorepository, to date, and provides an unprecedented resource for studying temporal-, sex-, and age-specific responses to endurance exercise training in a preclinical rat model