The designability of protein switches by chemical rescue of structure: mechanisms of inactivation and reactivation

This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Journal of the American Chemical Society, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/ja407644b.The ability to selectively activate function of particular proteins via pharmacological agents is a longstanding goal in chemical biology. Recently, we reported an approach for designing a de novo allosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. However, rationally identifying analogous de novo binding sites in other enzymes represents a key challenge for extending this approach to introduce allosteric control into other enzymes. Here we show that mutation sites leading to protein inactivation via tryptophan-to-glycine substitution and allowing (partial) reactivation by the subsequent addition of indole are remarkably frequent. Through a suite of methods including a cell-based reporter assay, computational structure prediction and energetic analysis, fluorescence studies, enzymology, pulse proteolysis, x-ray crystallography and hydrogen-deuterium mass spectrometry we find that these switchable proteins are most commonly modulated indirectly, through control of protein stability. Addition of indole in these cases rescues activity not by reverting a discrete conformational change, as we had observed in the sole previously reported example, but rather rescues activity by restoring protein stability. This important finding will dramatically impact the design of future switches and sensors built by this approach, since evaluating stability differences associated with cavity-forming mutations is a far more tractable task than predicting allosteric conformational changes. By analogy to natural signaling systems, the insights from this study further raise the exciting prospect of modulating stability to design optimal recognition properties into future de novo switches and sensors built through chemical rescue of structure

Brief Report: Long‐Term Functional Engraftment of Mesenchymal Progenitor Cells in a Mouse Model of Accelerated Aging

Age‐related osteoporosis is characterized by a decrease in bone‐forming capacity mediated by defects in the number and function of osteoblasts. An important cellular mechanism that may in part explain osteoblast dysfunction that occurs with aging is senescence of mesenchymal progenitor cells (MPCs). In the telomere‐based Wrn −/− Terc −/− model of accelerated aging, the osteoporotic phenotype of these mice is also associated with a major decline in MPC differentiation into osteoblasts. To investigate the role of MPC aging as a cell‐autonomous mechanism in senile bone loss, transplantation of young wild‐type whole bone marrow into Wrn −/− Terc −/− mutants was performed and the ability of engrafted cells to differentiate into cells of the osteoblast lineage was assessed. We found that whole bone marrow transplantation in Wrn −/− Terc −/− mice resulted in functional engraftment of MPCs up to 42 weeks, which was accompanied by a survival advantage as well as delays in microarchitectural features of skeletal aging. S TEM C ELLS 2013;31:607–611Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/96667/1/sc-12-0760_sm_SupplFigure1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96667/2/1294_ftp.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96667/3/sc-12-0760_sm_SupplFigure2.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96667/4/sc-12-0760_sm_SupplFigure3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96667/5/sc-12-0760_sm_SupplInform.pd

Conserved and Differential Effects of Dietary Energy Intake on the Hippocampal Transcriptomes of Females and Males

The level of dietary energy intake influences metabolism, reproductive function, the development of age-related diseases, and even cognitive behavior. Because males and females typically play different roles in the acquisition and allocation of energy resources, we reasoned that dietary energy intake might differentially affect the brains of males and females at the molecular level. To test this hypothesis, we performed a gene array analysis of the hippocampus in male and female rats that had been maintained for 6 months on either ad libitum (control), 20% caloric restriction (CR), 40% CR, intermittent fasting (IF) or high fat/high glucose (HFG) diets. These diets resulted in expected changes in body weight, and circulating levels of glucose, insulin and leptin. However, the CR diets significantly increased the size of the hippocampus of females, but not males. Multiple genes were regulated coherently in response to energy restriction diets in females, but not in males. Functional physiological pathway analyses showed that the 20% CR diet down-regulated genes involved in glycolysis and mitochondrial ATP production in males, whereas these metabolic pathways were up-regulated in females. The 40% CR diet up-regulated genes involved in glycolysis, protein deacetylation, PGC-1α and mTor pathways in both sexes. IF down-regulated many genes in males including those involved in protein degradation and apoptosis, but up-regulated many genes in females including those involved in cellular energy metabolism, cell cycle regulation and protein deacetylation. Genes involved in energy metabolism, oxidative stress responses and cell death were affected by the HFG diet in both males and females. The gender-specific molecular genetic responses of hippocampal cells to variations in dietary energy intake identified in this study may mediate differential behavioral responses of males and females to differences in energy availability

Deconvolution of Images from BLAST 2005: Insight into the K3-50 and IC 5146 Star-Forming Regions

We present an implementation of the iterative flux-conserving Lucy-Richardson (L-R) deconvolution method of image restoration for maps produced by the Balloon-borne Large Aperture Submillimeter Telescope (BLAST). We have analyzed its performance and convergence extensively through simulations and cross-correlations of the deconvolved images with available highresolution maps. We present new science results from two BLAST surveys, in the Galactic regions K3-50 and IC 5146, further demonstrating the benefits of performing this deconvolution. We have resolved three clumps within a radius of 4.'5 inside the star-forming molecular cloud containing K3-50. Combining the well-resolved dust emission map with available multi-wavelength data, we have constrained the Spectral Energy Distributions (SEDs) of five clumps to obtain masses (M), bolometric luminosities (L), and dust temperatures (T). The L-M diagram has been used as a diagnostic tool to estimate the evolutionary stages of the clumps. There are close relationships between dust continuum emission and both 21-cm radio continuum and 12CO molecular line emission. The restored extended large scale structures in the Northern Streamer of IC 5146 have a strong spatial correlation with both SCUBA and high resolution extinction images. A dust temperature of 12 K has been obtained for the central filament. We report physical properties of ten compact sources, including six associated protostars, by fitting SEDs to multi-wavelength data. All of these compact sources are still quite cold (typical temperature below ~ 16 K) and are above the critical Bonner-Ebert mass. They have associated low-power Young Stellar Objects (YSOs). Further evidence for starless clumps has also been found in the IC 5146 region.Comment: 13 pages, 12 Figures, 3 Table

The Balloon-Borne Large Aperture Submillimeter Telescope (BLAST) 2005: A 10 deg^2 Survey of Star Formation in Cygnus X

We present Cygnus X in a new multi-wavelength perspective based on an unbiased BLAST survey at 250, 350, and 500 micron, combined with rich datasets for this well-studied region. Our primary goal is to investigate the early stages of high mass star formation. We have detected 184 compact sources in various stages of evolution across all three BLAST bands. From their well-constrained spectral energy distributions, we obtain the physical properties mass, surface density, bolometric luminosity, and dust temperature. Some of the bright sources reaching 40 K contain well-known compact H II regions. We relate these to other sources at earlier stages of evolution via the energetics as deduced from their position in the luminosity-mass (L-M) diagram. The BLAST spectral coverage, near the peak of the spectral energy distribution of the dust, reveals fainter sources too cool (~ 10 K) to be seen by earlier shorter-wavelength surveys like IRAS. We detect thermal emission from infrared dark clouds and investigate the phenomenon of cold ``starless cores" more generally. Spitzer images of these cold sources often show stellar nurseries, but these potential sites for massive star formation are ``starless" in the sense that to date there is no massive protostar in a vigorous accretion phase. We discuss evolution in the context of the L-M diagram. Theory raises some interesting possibilities: some cold massive compact sources might never form a cluster containing massive stars; and clusters with massive stars might not have an identifiable compact cold massive precursor.Comment: 42 pages, 31 Figures, 6 table

SRT1720 improves survival and healthspan of obese mice

Sirt1 is an NAD+-dependent deacetylase that extends lifespan in lower organisms and improves metabolism and delays the onset of age-related diseases in mammals. Here we show that SRT1720, a synthetic compound that was identified for its ability to activate Sirt1 in vitro, extends both mean and maximum lifespan of adult mice fed a high-fat diet. This lifespan extension is accompanied by health benefits including reduced liver steatosis, increased insulin sensitivity, enhanced locomotor activity and normalization of gene expression profiles and markers of inflammation and apoptosis, all in the absence of any observable toxicity. Using a conditional SIRT1 knockout mouse and specific gene knockdowns we show SRT1720 affects mitochondrial respiration in a Sirt1- and PGC-1α-dependent manner. These findings indicate that SRT1720 has long-term benefits and demonstrate for the first time the feasibility of designing novel molecules that are safe and effective in promoting longevity and preventing multiple age-related diseases in mammals

The High-Energy Radiation Environment Around a 10 Gyr M Dwarf: Habitable at Last?

High levels of X-ray and UV activity on young M dwarfs may drive rapid atmospheric escape on temperate, terrestrial planets orbiting within the liquid water habitable zone. However, secondary atmospheres on planets orbiting older, less active M dwarfs may be stable and present more promising candidates for biomarker searches. We present new HST and Chandra observations of Barnard's Star (GJ 699), a 10 Gyr old M3.5 dwarf, acquired as part of the Mega-MUSCLES program. Despite the old age and long rotation period of Barnard's star, we observe two FUV ($\delta_{130}$ $\approx$ 5000s; $E_{130}$ $\approx$ 10$^{29.5}$ erg each) and one X-ray ($E_{X}$ $\approx$ 10$^{29.2}$ erg) flares, and estimate a high-energy flare duty cycle (defined here as the fraction of the time the star is in a flare state) of $\sim$ 25\%. A 5 A - 10 $\mu$m SED of GJ 699 is created and used to evaluate the atmospheric stability of a hypothetical, unmagnetized terrestrial planet in the habitable zone ($r_{HZ}$ $\sim$ 0.1 AU). Both thermal and non-thermal escape modeling indicate (1) the $quiescent$ stellar XUV flux does not lead to strong atmospheric escape: atmospheric heating rates are comparable to periods of high solar activity on modern Earth, and (2) the $flare$ environment could drive the atmosphere into a hydrodynamic loss regime at the observed flare duty cycle: sustained exposure to the flare environment of GJ 699 results in the loss of $\approx$ 87 Earth atmospheres Gyr$^{-1}$ through thermal processes and $\approx$ 3 Earth atmospheres Gyr$^{-1}$ through ion loss processes, respectively. These results suggest that if rocky planet atmospheres can survive the initial $\sim$ 5 Gyr of high stellar activity, or if a second generation atmosphere can be formed or acquired, the flare duty cycle may be the controlling stellar parameter for the stability of Earth-like atmospheres around old M stars.Comment: Accepted to A

Domestication-induced reduction in eye size revealed in multiple common garden experiments: The case of Atlantic salmon (Salmo salar L.)

Domestication leads to changes in traits that are under directional selection in breeding programmes, though unintentional changes in nonproduction traits can also arise. In offspring of escaping fish and any hybrid progeny, such unintentionally altered traits may reduce fitness in the wild. Atlantic salmon breeding programmes were established in the early 1970s, resulting in genetic changes in multiple traits. However, the impact of domestication on eye size has not been studied. We measured body size corrected eye size in 4000 salmon from six common garden experiments conducted under artificial and natural conditions, in freshwater and saltwater environments, in two countries. Within these common gardens, offspring of domesticated and wild parents were crossed to produce 11 strains, with varying genetic backgrounds (wild, domesticated, F1 hybrids, F2 hybrids and backcrosses). Size-adjusted eye size was influenced by both genetic and environmental factors. Domesticated fish reared under artificial conditions had smaller adjusted eye size when compared to wild fish reared under identical conditions, in both the freshwater and marine environments, and in both Irish and Norwegian experiments. However, in parr that had been introduced into a river environment shortly after hatching and sampled at the end of their first summer, differences in adjusted eye size observed among genetic groups were of a reduced magnitude and were nonsignificant in 2-year-old sea migrating smolts sampled in the river immediately prior to sea entry. Collectively, our findings could suggest that where natural selection is present, individuals with reduced eye size are maladapted and consequently have reduced fitness, building on our understanding of the mechanisms that underlie a well-documented reduction in the fitness of the progeny of domesticated salmon, including hybrid progeny, in the wild

Immunogenicity of Self-Associated Aggregates and Chemically Cross-Linked Conjugates of the 42 kDa Plasmodium falciparum Merozoite Surface Protein-1

Self-associated protein aggregates or cross-linked protein conjugates are, in general, more immunogenic than oligomeric or monomeric forms. In particular, the immunogenicity in mice of a recombinant malaria transmission blocking vaccine candidate, the ookinete specific Plasmodium falciparum 25 kDa protein (Pfs25), was increased more than 1000-fold when evaluated as a chemical cross-linked protein-protein conjugate as compared to a formulated monomer. Whether alternative approaches using protein complexes improve the immunogenicity of other recombinant malaria vaccine candidates is worth assessing. In this work, the immunogenicity of the recombinant 42 kDa processed form of the P. falciparum merozoite surface protein 1 (MSP142) was evaluated as a self-associated, non-covalent aggregate and as a chemical cross-linked protein-protein conjugate to ExoProtein A, which is a recombinant detoxified form of Pseudomonas aeruginosa exotoxin A. MSP142 conjugates were prepared and characterized biochemically and biophysically to determine their molar mass in solution and stoichiometry, when relevant. The immunogenicity of the MSP142 self-associated aggregates, cross-linked chemical conjugates and monomers were compared in BALB/c mice after adsorption to aluminum hydroxide adjuvant, and in one instance in association with the TLR9 agonist CPG7909 with an aluminum hydroxide formulation. Antibody titers were assessed by ELISA. Unlike observations made for Pfs25, no significant enhancement in MSP142 specific antibody titers was observed for any conjugate as compared to the formulated monomer or dimer, except for the addition of the TLR9 agonist CPG7909. Clearly, enhancing the immunogenicity of a recombinant protein vaccine candidate by the formation of protein complexes must be established on an empirical basis

SRT1720 improves survival and healthspan of obese mice

Sirt1 is an NAD1-dependent deacetylase that extends lifespan in lower organisms and improves metabolism and delays the onset of age-related diseases in mammals. Here we show that SRT1720, a synthetic compound that was identified for its ability to activate Sirt1 in vitro, extends both mean and maximum lifespan of adult mice fed a high-fat diet. This lifespan extension is accompanied by health benefits including reduced liver steatosis, increased insulin sensitivity, enhanced locomotor activity and normalization of gene expression profiles and markers of inflammation and apoptosis, all in the absence of any observable toxicity. Using a conditional SIRT1 knockout mouse and specific gene knockdowns we show SRT1720 affects mitochondrial respiration in a Sirt1- and PGC-1a-dependent manner. These findings indicate that SRT1720 has long-term benefits and demonstrate for the first time the feasibility of designing novel molecules that are safe and effective in promoting longevity and preventing multiple age-related diseases in mammals