The lncRNA Growth Arrest Specific 5 regulates cell survival via distinct structural modules with independent functions

There is increasing evidence that the architecture of long non-coding RNAs (lncRNAs) just like that of proteins-is hierarchically organized into independently folding sub-modules with distinct functions. Studies characterizing the cellular activities of such modules, however, are rare. The lncRNA growth arrest specific 5 (GAS5) is a key regulator of cell survival in response to stress and nutrient availability. We use SHAPE-MaP to probe the structure of GAS5 and identify three separate structural modules that act independently in leukemic T cells. The 5' terminal module with low secondary structure content affects basal survival and slows the cell cycle, whereas the highly structured core module mediates the effects of mammalian target of rapamycin (mTOR) inhibition on cell growth. These results highlight the central role of GAS5 in regulating cell survival and reveal how a single lncRNA transcript utilizes a modular structure-function relationship to respond to a variety of cellular stresses under various cellular conditions

Crystal Structure of an Ancient Protein: Evolution by Conformational Epistasis

The structural mechanisms by which proteins have evolved new functions are known only indirectly. We report x-ray crystal structures of a resurrected ancestral protein—the ∼450 million year-old precursor of vertebrate glucocorticoid (GR) and mineralocorticoid (MR) receptors. Using structural, phylogenetic, and functional analysis, we identify the specific set of historical mutations that recapitulate the evolution of GR’s hormone specificity from an MR-like ancestor. These substitutions repositioned crucial residues to create new receptor-ligand and intra-protein contacts. Strong epistatic interactions occur because one substitution changes the conformational position of another site. “Permissive” mutations—substitutions of no immediate consequence, which stabilize specific elements of the protein and allow it to tolerate subsequent function-switching changes—played a major role in determining GR’s evolutionary trajectory

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

The Orphan Nuclear Receptor LRH-1 and ERα Activate GREB1 Expression to Induce Breast Cancer Cell Proliferation

BACKGROUND: Liver Receptor Homolog 1 (LRH-1, NR5A2) is an orphan nuclear receptor that is over-expressed in cancers in tissues such as the breast, colon and pancreas. LRH-1 plays important roles in embryonic development, steroidogenesis and cholesterol homeostasis. In tumor cells, LRH-1 induces proliferation and cell cycle progression. High LRH-1 expression is demonstrated in breast cancers, positively correlating with ERα status and aromatase activity. LRH-1 dependent cellular mechanisms in breast cancer epithelial cells are poorly defined. Hence in the present study we investigated the actions of LRH-1 in estrogen receptor α (ERα) positive breast cancer cells. RESULTS: The study aimed to investigate LRH-1 dependent mechanisms that promote breast cancer proliferation. We identified that LRH-1 regulated the expression of Growth Regulation by Estrogen in Breast Cancer 1 (GREB1) in MCF-7 and MDA-MB-231 cells. Over-expression of LRH-1 increased GREB1 mRNA levels while knockdown of LRH-1 reduced its expression. GREB1 is a well characterised ERα target gene, with three estrogen response elements (ERE) located on its promoter. Chromatin immunoprecipitation studies provided evidence of the co-localisation of LRH-1 and ERα at all three EREs. With electrophoretic mobility shift assays, we demonstrated direct binding of LRH-1 to EREs located on GREB1 and Trefoil Factor 1 (TFF1, pS2) promoters. LRH-1 and ERα co-operatively activated transcription of ERE luciferase reporter constructs suggesting an overlap in regulation of target genes in breast cancer cells. Over-expression of LRH-1 resulted in an increase in cell proliferation. This effect was more pronounced with estradiol treatment. In the presence of ICI 182,780, an ERα antagonist, LRH-1 still induced proliferation. CONCLUSIONS: We conclude that in ER-positive breast cancer cells, LRH-1 promotes cell proliferation by enhancing ERα mediated transcription of target genes such as GREB-1. Collectively these findings indicate the importance of LRH-1 in the progression of hormone-dependent breast cancer and implicate LRH-1 as a potential avenue for drug development

Evolution of a Signaling Nexus Constrained by Protein Interfaces and Conformational States

Heterotrimeric G proteins act as the physical nexus between numerous receptors that respond to extracellular signals and proteins that drive the cytoplasmic response. The Gα subunit of the G protein, in particular, is highly constrained due to its many interactions with proteins that control or react to its conformational state. Various organisms contain differing sets of Gα-interacting proteins, clearly indicating that shifts in sequence and associated Gα functionality were acquired over time. These numerous interactions constrained much of Gα evolution; yet Gα has diversified, through poorly understood processes, into several functionally specialized classes, each with a unique set of interacting proteins. Applying a synthetic sequence-based approach to mammalian Gα subunits, we established a set of seventy-five evolutionarily important class-distinctive residues, sites where a single Gα class is differentiated from the three other classes. We tested the hypothesis that shifts at these sites are important for class-specific functionality. Importantly, we mapped known and well-studied class-specific functionalities from all four mammalian classes to sixteen of our class-distinctive sites, validating the hypothesis. Our results show how unique functionality can evolve through the recruitment of residues that were ancestrally functional. We also studied acquisition of functionalities by following these evolutionarily important sites in non-mammalian organisms. Our results suggest that many class-distinctive sites were established early on in eukaryotic diversification and were critical for the establishment of new Gα classes, whereas others arose in punctuated bursts throughout metazoan evolution. These Gα class-distinctive residues are rational targets for future structural and functional studies

Correlated Evolution of Nearby Residues in Drosophilid Proteins

Here we investigate the correlations between coding sequence substitutions as a function of their separation along the protein sequence. We consider both substitutions between the reference genomes of several Drosophilids as well as polymorphisms in a population sample of Zimbabwean Drosophila melanogaster. We find that amino acid substitutions are “clustered” along the protein sequence, that is, the frequency of additional substitutions is strongly enhanced within ≈10 residues of a first such substitution. No such clustering is observed for synonymous substitutions, supporting a “correlation length” associated with selection on proteins as the causative mechanism. Clustering is stronger between substitutions that arose in the same lineage than it is between substitutions that arose in different lineages. We consider several possible origins of clustering, concluding that epistasis (interactions between amino acids within a protein that affect function) and positional heterogeneity in the strength of purifying selection are primarily responsible. The role of epistasis is directly supported by the tendency of nearby substitutions that arose on the same lineage to preserve the total charge of the residues within the correlation length and by the preferential cosegregation of neighboring derived alleles in our population sample. We interpret the observed length scale of clustering as a statistical reflection of the functional locality (or modularity) of proteins: amino acids that are near each other on the protein backbone are more likely to contribute to, and collaborate toward, a common subfunction

LRH-1 agonism favours an immune-islet dialogue which protects against diabetes mellitus

Type 1 diabetes mellitus (T1DM) is due to the selective destruction of islet beta cells by immune cells. Current therapies focused on repressing the immune attack or stimulating beta cell regeneration still have limited clinical efficacy. Therefore, it is timely to identify innovative targets to dampen the immune process, while promoting beta cell survival and function. Liver receptor homologue-1 (LRH-1) is a nuclear receptor that represses inflammation in digestive organs, and protects pancreatic islets against apoptosis. Here, we show that BL001, a small LRH-1 agonist, impedes hyperglycemia progression and the immune-dependent inflammation of pancreas in murine models of T1DM, and beta cell apoptosis in islets of type 2 diabetic patients, while increasing beta cell mass and insulin secretion. Thus, we suggest that LRH-1 agonism favors a dialogue between immune and islet cells, which could be druggable to protect against diabetes mellitus.the Juvenile Diabetes Research Foundation (17-2013-372 to B.R.G.), the Consejeria de Salud, Fundacion Publica Andaluza Progreso y Salud, Junta de Andalucia (PI-0727-2010 to B.R.G. and P10CTS6505 to B.S.), Consejeria de Economia, Innovacion y Ciencia (P10.CTS.6359 to B.R.G.), the Ministerio de Economia y Competidividad cofunded by Fondos FEDER (PI10/00871, PI13/00593, and BFU2017-83588-P to B.R.G.; PI14/01015, RD12/0019/0028, and RD16/0011/0034 to B.S.; PI16/00259 to A. H.) and Deutsche Forschungsgemeinschaft (GRK-1789 ´CEMMA´ and DFG SCHI-505/ 6-1 to R.S.). Special thanks to the families of the DiabetesCero Foundation that graciously supported this work (to B.R.G.). A.M.M. is a recipient of a Miguel Servet grant (CP14/ 00105) from the Instituto de Salud Carlos III co-funded by Fondos FEDER whereas E.F. M. is a recipient of a Juan de la Cierva Fellowship. I.G.H.G. is supported by a fellowship from Amarna Therapeutics. In some instances, human islets were procured through the European Consortium for Islet Transplantation funded by Juvenile Diabetes Research Foundation (3-RSC-2016-162-I-X)

The Rodin-Ohno hypothesis that two enzyme superfamilies descended from one ancestral gene: an unlikely scenario for the origins of translation that will not be dismissed

Background Because amino acid activation is rate-limiting for uncatalyzed protein synthesis, it is a key puzzle in understanding the origin of the genetic code. Two unrelated classes (I and II) of contemporary aminoacyl-tRNA synthetases (aaRS) now translate the code. Observing that codons for the most highly conserved, Class I catalytic peptides, when read in the reverse direction, are very nearly anticodons for Class II defining catalytic peptides, Rodin and Ohno proposed that the two superfamilies descended from opposite strands of the same ancestral gene. This unusual hypothesis languished for a decade, perhaps because it appeared to be unfalsifiable. Results The proposed sense/antisense alignment makes important predictions. Fragments that align in antiparallel orientations, and contain the respective active sites, should catalyze the same two reactions catalyzed by contemporary synthetases. Recent experiments confirmed that prediction. Invariant cores from both classes, called Urzymes after Ur = primitive, authentic, plus enzyme and representing ~20% of the contemporary structures, can be expressed and exhibit high, proportionate rate accelerations for both amino-acid activation and tRNA acylation. A major fraction (60%) of the catalytic rate acceleration by contemporary synthetases resides in segments that align sense/antisense. Bioinformatic evidence for sense/antisense ancestry extends to codons specifying the invariant secondary and tertiary structures outside the active sites of the two synthetase classes. Peptides from a designed, 46-residue gene constrained by Rosetta to encode Class I and II ATP binding sites with fully complementary sequences both accelerate amino acid activation by ATP ~400 fold. Conclusions Biochemical and bioinformatic results substantially enhance the posterior probability that ancestors of the two synthetase classes arose from opposite strands of the same ancestral gene. The remarkable acceleration by short peptides of the rate-limiting step in uncatalyzed protein synthesis, together with the synergy of synthetase Urzymes and their cognate tRNAs, introduce a new paradigm for the origin of protein catalysts, emphasize the potential relevance of an operational RNA code embedded in the tRNA acceptor stems, and challenge the RNA-World hypothesis. Reviewers This article was reviewed by Dr. Paul Schimmel (nominated by Laura Landweber), Dr. Eugene Koonin and Professor David Ardell

Lipidome Changes Associated with a Diet-Induced Reduction in Hepatic Fat among Adolescent Boys with Metabolic Dysfunction-Associated Steatotic Liver Disease

Little is known about lipid changes that occur in the setting of metabolic-dysfunction-associated steatotic liver disease (MASLD) regression. We previously reported improvements in hepatic steatosis, de novo lipogenesis (DNL), and metabolomic profiles associated with oxidative stress, inflammation, and selected lipid metabolism in 40 adolescent boys (11–16 y) with hepatic steatosis ≥5% (98% meeting the definition of MASLD). Participants were randomized to a low-free-sugar diet (LFSD) (n = 20) or usual diet (n = 20) for 8 weeks. Here, we employed untargeted/targeted lipidomics to examine lipid adaptations associated with the LFSD and improvement of hepatic steatosis. Our LC-MS/MS analysis revealed decreased triglycerides (TGs), diacylglycerols (DGs), cholesteryl esters (ChE), lysophosphatidylcholine (LPC), and phosphatidylcholine (PC) species with the diet intervention (p p < 0.05). Overall, we observed reductions in TGs, DGs, ChE, PC, and LPC species among participants in the LFSD group. These same lipids have been associated with MASLD progression; therefore, our findings may indicate normalization of key biological processes, including lipid metabolism, insulin resistance, and lipotoxicity. Additionally, our targeted oxylipins assay revealed novel changes in eicosanoids, suggesting improvements in oxidative stress. Future studies are needed to elucidate the mechanisms of these findings and prospects of these lipids as biomarkers of MASLD regression