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

Recombination and microdiversity in coastal marine cyanophages

Genetic exchange is an important process in bacteriophage evolution. Here, we examine the role of homologous recombination in the divergence of closely related cyanophage isolates from natural marine populations. Four core-viral genes (coliphage T4 homologues g20, g23, g43 and a putative tail fibre gene) and four viral-encoded bacterial-derived genes (psbA, psbD, cobS and phoH) were analysed for 60 cyanophage isolates belonging to five Rhode Island Myovirus (RIM) strains. Phylogenetic analysis of the 60 concatenated sequences revealed well-resolved sequence clusters corresponding to the RIM strain designations. Viral isolates within a strain shared an average nucleotide identity of 99.3-99.8%. Nevertheless, extensive microdiversity was observed within each cyanophage strain; only three of the 60 isolates shared the same nucleotide haplotype. Microdiversity was generated by point mutations, homologous recombination within a strain, and intragenic recombination between RIM strains. Intragenic recombination events between distinct RIM strains were detected most often in host-derived photosystem II psbA and psbD genes, but were also identified in some major capsid protein g23 genes. Within a strain, more variability was observed at the psbA locus than at any of the other seven loci. Although most of the microdiversity within a strain was neutral, some amino acid substitutions were identified, and thus microdiversity within strains has the potential to influence the population dynamics of viral-host interactions. © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd

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

TPI dimer interface substitutions do not alter NMJ development and morphology.

<p>(A) NMJ morphology of segment A2 muscle 6/7 was characterized for (B) bouton number and (C) branching. Boutons were defined as varicosities greater than 2 μm in diameter. Neither parameter showed significant differences elicited by the mutations, relative to either <i>dTPI</i>^<i>WT</i> or <i>dTPI</i>^{<i>WT-CFP</i>}<i>/TPI</i>^<i>WT</i>. CFP tags did not alter the behavioral deficits of the animals (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005941#pgen.1005941.s006" target="_blank">S6 Fig</a>). n = 10. Comparisons were made with a One-way ANOVA using Tukey’s post hoc test, <i>ns</i> indicated no significance. Scale bar = 10 μm.</p

Heterodimerization of inactive TPI and dimer interface mutations.

<p>(A) dTPI^Δcat-CFP interacts modestly with dTPI^M80T, dTPI^T73R, and dTPI^T73R,G74E, yet robustly with dTPI^G74E. Representative immunoprecipitation and input blots are shown with (B) IP:anti-GFP quantification n = 3. Quantification represents 25kD TPI IP signal, with negative control subtracted, normalized to the lysate β-tubulin loading control, and compared to WT. Comparisons were made with a One-way ANOVA using Tukey’s post hoc test, <i>ns</i> indicates no significance, ** p<0.01, and *** p<0.001.</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

Mutations at the TPI dimer interface reduce TPI levels <i>in vivo</i> without aggregation.

<p>(A) <i>dTPI</i>^<i>T73R</i> and <i>dTPI</i>^<i>G74E</i> homozygote animal lysates display reduced protein levels with (B<b>)</b> quantification normalized to WT and an ATPalpha loading control, n = 3. (C) The reduction in SDS-soluble TPI is not caused by protein aggregation; increasing amounts of lysate were loaded and show no differences in filter-trapped TPI across all genotypes, (D) 10μg of huntingtin exon1-GFP lysate displayed robust retention on the filter, n = 2. Comparisons were made with a One-way ANOVA using Tukey’s post hoc test, *** p<0.001 relative to WT.</p

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

Mutations affecting the TPI dimer interface recapitulate <i>dTPI</i>^<i>M80T</i> phenotypes.

<p>(A) <i>dTPI</i>^<i>T73R</i> and <i>dTPI</i>^<i>G74E</i> homozygotes display severely reduced lifespans, n>150. (B) Dimer interface mutations exhibit severe mechanical stress at Day 1 and (C) thermal stress sensitivity at Day 2, n>30. Thermal stress paralysis times at 360 sec. represent wild type behavior, the assay was stopped at 6 min. (D) Both <i>dTPI</i>^<i>T73R</i> and <i>dTPI</i>^<i>G74E</i> homozygotes display reduced lysate isomerase activity, n≥3. Comparisons were made with a One-way ANOVA using Tukey’s post hoc test, and lifespans by a Log-rank (Mantel-Cox) survival test, ** indicated p<0.01, *** p<0.001.</p

Crystallographic Data collection and Refinement statistics.

<p>Crystallographic Data collection and Refinement statistics.</p