Triosephosphate isomerase I170V alters catalytic site, enhances stability and induces pathology in a Drosophila model of TPI deficiency ☆

Triosephosphate isomerase (TPI) is a glycolytic enzyme which homodimerizes for full catalytic activity. Mutations of the TPI gene elicit a disease known as TPI Deficiency, a glycolytic enzymopathy noted for its unique severity of neurological symptoms. Evidence suggests that TPI Deficiency pathogenesis may be due to conformational changes of the protein, likely affecting dimerization and protein stability. In this report, we genetically and physically characterize a human disease-associated TPI mutation caused by an I170V substitution. Human TPI I170V elicits behavioral abnormalities in Drosophila. An examination of hTPI I170V enzyme kinetics revealed this substitution reduced catalytic turnover, while assessments of thermal stability demonstrated an increase in enzyme stability. The crystal structure of the homodimeric I170V mutant reveals changes in the geometry of critical residues within the catalytic pocket. Collectively these data reveal new observations of the structural and kinetic determinants of TPI Deficiency pathology, providing new insights into disease pathogenesis

CMB-S4: Forecasting Constraints on Primordial Gravitational Waves

CMB-S4---the next-generation ground-based cosmic microwave background (CMB) experiment---is set to significantly advance the sensitivity of CMB measurements and enhance our understanding of the origin and evolution of the Universe, from the highest energies at the dawn of time through the growth of structure to the present day. Among the science cases pursued with CMB-S4, the quest for detecting primordial gravitational waves is a central driver of the experimental design. This work details the development of a forecasting framework that includes a power-spectrum-based semi-analytic projection tool, targeted explicitly towards optimizing constraints on the tensor-to-scalar ratio, $r$, in the presence of Galactic foregrounds and gravitational lensing of the CMB. This framework is unique in its direct use of information from the achieved performance of current Stage 2--3 CMB experiments to robustly forecast the science reach of upcoming CMB-polarization endeavors. The methodology allows for rapid iteration over experimental configurations and offers a flexible way to optimize the design of future experiments given a desired scientific goal. To form a closed-loop process, we couple this semi-analytic tool with map-based validation studies, which allow for the injection of additional complexity and verification of our forecasts with several independent analysis methods. We document multiple rounds of forecasts for CMB-S4 using this process and the resulting establishment of the current reference design of the primordial gravitational-wave component of the Stage-4 experiment, optimized to achieve our science goals of detecting primordial gravitational waves for $r > 0.003$ at greater than $5\sigma$, or, in the absence of a detection, of reaching an upper limit of $r < 0.001$ at $95\%$ CL.Comment: 24 pages, 8 figures, 9 tables, submitted to ApJ. arXiv admin note: text overlap with arXiv:1907.0447

CMB-S4: Forecasting Constraints on Primordial Gravitational Waves

Abstract: CMB-S4—the next-generation ground-based cosmic microwave background (CMB) experiment—is set to significantly advance the sensitivity of CMB measurements and enhance our understanding of the origin and evolution of the universe. Among the science cases pursued with CMB-S4, the quest for detecting primordial gravitational waves is a central driver of the experimental design. This work details the development of a forecasting framework that includes a power-spectrum-based semianalytic projection tool, targeted explicitly toward optimizing constraints on the tensor-to-scalar ratio, r, in the presence of Galactic foregrounds and gravitational lensing of the CMB. This framework is unique in its direct use of information from the achieved performance of current Stage 2–3 CMB experiments to robustly forecast the science reach of upcoming CMB-polarization endeavors. The methodology allows for rapid iteration over experimental configurations and offers a flexible way to optimize the design of future experiments, given a desired scientific goal. To form a closed-loop process, we couple this semianalytic tool with map-based validation studies, which allow for the injection of additional complexity and verification of our forecasts with several independent analysis methods. We document multiple rounds of forecasts for CMB-S4 using this process and the resulting establishment of the current reference design of the primordial gravitational-wave component of the Stage-4 experiment, optimized to achieve our science goals of detecting primordial gravitational waves for r > 0.003 at greater than 5σ, or in the absence of a detection, of reaching an upper limit of r < 0.001 at 95% CL

Direct Detection of Dark Matter -- APPEC Committee Report

This Report provides an extensive review of the experimental programme of direct detection searches of particle dark matter. It focuses mostly on European efforts, both current and planned, but does it within a broader context of a worldwide activity in the field. It aims at identifying the virtues, opportunities and challenges associated with the different experimental approaches and search techniques. It presents scientific and technological synergies, both existing and emerging, with some other areas of particle physics, notably collider and neutrino programmes, and beyond. It addresses the issue of infrastructure in light of the growing needs and challenges of the different experimental searches. Finally, the Report makes a number of recommendations from the perspective of a long-term future of the field. They are introduced, along with some justification, in the opening Overview and Recommendations section and are next summarised at the end of the Report. Overall, we recommend that the direct search for dark matter particle interactions with a detector target should be given top priority in astroparticle physics, and in all particle physics, and beyond, as a positive measurement will provide the most unambiguous confirmation of the particle nature of dark matter in the Universe

Structural and Genetic Studies Demonstrate Neurologic Dysfunction in Triosephosphate Isomerase Deficiency Is Associated with Impaired Synaptic Vesicle Dynamics

<div><p>Triosephosphate isomerase (TPI) deficiency is a poorly understood disease characterized by hemolytic anemia, cardiomyopathy, neurologic dysfunction, and early death. TPI deficiency is one of a group of diseases known as glycolytic enzymopathies, but is unique for its severe patient neuropathology and early mortality. The disease is caused by missense mutations and dysfunction in the glycolytic enzyme, TPI. Previous studies have detailed structural and catalytic changes elicited by disease-associated TPI substitutions, and samples of patient erythrocytes have yielded insight into patient hemolytic anemia; however, the neuropathophysiology of this disease remains a mystery. This study combines structural, biochemical, and genetic approaches to demonstrate that perturbations of the TPI dimer interface are sufficient to elicit TPI deficiency neuropathogenesis. The present study demonstrates that neurologic dysfunction resulting from TPI deficiency is characterized by synaptic vesicle dysfunction, and can be attenuated with catalytically inactive TPI. Collectively, our findings are the first to identify, to our knowledge, a functional synaptic defect in TPI deficiency derived from molecular changes in the TPI dimer interface.</p></div

hTPI^M82T elicits a conformational change in TPI resulting in reduced dimerization.

<p>(A) Sequence alignment of <i>D</i>.<i>melanogaster</i> and <i>H</i>.<i>sapiens</i> TPI protein sequence with asterisks highlighting residues of interest. (B) The hTPI^M82T mutation confers a reduction in mean protein hydrodynamic radius as measured by dynamic light scattering. (C) Intensity correlation plots reveal a largely monodisperse hTPI^WT population and polydisperse hTPI^M82T population. (D) Gel filtration indicates a change in monomer:dimer ratios elicited by hTPI^M82T with relative quantification (inset). n≥3, comparisons were made using Student’s T test, *** indicates p<0.001.</p

hTPI^Δcat crystal structure reveals a catalytically incompetent enzyme with an unaltered dimer interface.

<p>(A) The hTPI^Δcat adopts an open lid conformation. Superposition of hTPI^WT (grey) and hTPI^Δcat (blue) structures. Loop3 (tan), the position of the lid covering the TPI active site (magenta and pink), and the locations of bromide and phosphate ions from the active site pocket of hTPI^WT are indicated. New hydrophobic interactions that help to reposition M13 are shown (green). (B) The dimer interface of hTPI^Δcat is unchanged relative to hTPI^WT. Superposition of hTPI^WT (grey) and hTPI^Δcat shown as in (A), with monomer subunits of hTPI^Δcat dimer in blue and tan. Key dimer interface residues and loops are indicated.</p

<i>dTPI</i>^<i>T73R</i> impairs NMJ synaptic vesicle dynamics.

<p>(A) An FM1-43 timecourse at the NMJ with loading times of 15, 30, and 60 sec., (B) with quantification of 60 sec. at 38°C, and (C) 60 sec. at room temperature (RT). (D) Representative images of <i>dTPI</i>^<i>WT</i>, <i>dTPI</i>^<i>T73R</i>, <i>dTPI</i>^{<i>T73R/Δcat</i>} and <i>Shi</i>^<i>ts1</i>, n = 6. (E) FM1-43 unloading is unchanged between <i>dTPI</i>^<i>WT</i> and <i>dTPI</i>^<i>T73R</i> at 38°C with animal replicates indicated, and (F) representative images. Comparisons were made with a One-way ANOVA using Tukey’s post hoc test, ***p<0.001. Scale bars = 5μm.</p