Papers by Lalita Oonthonpan
Molecular and Cellular Biochemistry, Nov 2, 2011
Pediatric cataract of the congenital type is the most common form of childhood blindness and it i... more Pediatric cataract of the congenital type is the most common form of childhood blindness and it is clinically and genetically heterogeneous. Mutations in 22 different genes have been identified to be associated with congenital cataracts, and among them, eight mutants belong to A-crystallin. To explain how mutations in A-crystallin lead to the development of cataract, quaternary structural parameters, and chaperone function have been investigated in A-wt and in the following mutants: R12C, R21L, R21W, R49C, R54C, R116C, and R116H. Average molar mass, mass at the RI peak, mass across the peak, hydrodynamic radius (Rh), and polydispersity index (PDI) were determined by dynamic light-scattering measurements. The average molar mass and mass across the peak showed major increase in R116C and R116H, moderate increase in R12C, R21W, and R54C, and no increase in R21L and R49C as compared to A-wt. PDI and Rh values were significantly increased only in R116C and R116H. Significant secondary structural changes, as determined by CD measurements, were seen in R21W, R21L, R116C, and R116H, and tertiary structural changes were evident in R21W, R54C, R116C, and R116H. Non-reducing SDSPAGE has shown the presence of dimers presumably formed by inter-polypeptide disulfide bonds. Chaperone activity, as measured with ADH as the target protein, appeared normal in R49C and R54C, while R12C, R21L, and R21W showed moderate loss and R116C and R116H showed significant loss. Although a specific change in the A-crystallin behavior that is common to all the mutants was not evident, each mutant showed one or more perturbation as the end effect that leads to cataract.
<p>A representative emission spectra of αB-crystallin excited at 336 nm were recorded at 0 ... more <p>A representative emission spectra of αB-crystallin excited at 336 nm were recorded at 0 (a), 2 (b), 4 (c), 6 (d), 8 (e), 10 (f), 20 (g), 45 (h), 75 (i) and 120 (j) minutes after mixing of SITS-labeled αB-wt and LYI-labeled αA-wt or mutants. The decrease in fluorescence intensity at 426 nm of the SITS-labeled αB-wt protein and the concomitant increase in fluorescence intensity at 515 nm of the LYI-labeled αA-wt or mutants proteins are the indicative of energy transfer between two labeled populations. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031421#pone-0031421-g001" target="_blank"><i>Fig. 1A</i></a>: Time dependent spectral changes in the FRET due to subunit exchange of SITS labeled αB-wt and LYI-labeled αA-wt. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031421#pone-0031421-g001" target="_blank"><i>Fig. 1B</i></a>: Time dependent spectral changes in the FRET due to subunit exchange of SITS labeled αB-wt and LYI-labeled αA-R12C. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031421#pone-0031421-g001" target="_blank"><i>Fig. 1C</i></a>: The emission spectra of αB-crystallin excited at 336 nm were recorded at 0 (a), 2 (b), 4 (c), 6 (d), 8 (e), 10 (f), 20 (g), 45 (h), 75 (i) and 120 (j) minutes after mixing of SITS-labeled αB-wt and LYI-labeled R21L. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031421#pone-0031421-g001" target="_blank"><i>Fig. 1D</i></a>: The emission spectra of αB-crystallin excited at 336 nm were recorded at 0 (a), 2 (b), 4 (c), 6 (d), 8 (e), 10 (f), 20 (g), 45 (h), 75 (i) and 120 (j) minutes after mixing of SITS-labeled αB-wt and LYI-labeled R21W mutants. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031421#pone-0031421-g001" target="_blank"><i>Fig. 1E</i></a>: The emission spectra of αB-crystallin excited at 336 nm were recorded at 0 (a), 2 (b), 4 (c), 6 (d), 8 (e), 10 (f), 20 (g), 45 (h), 75 (i) and 120 (j) minutes after mixing of SITS-labeled αB-wt and LYI-labeled R49C. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031421#pone-0031421-g001" target="_blank"><i>Fig. 1F</i></a>: The emission spectra of αB-crystallin excited at 336 nm were recorded at 0 (a), 2 (b), 4 (c), 6 (d), 8 (e), 10 (f), 20 (g), 45 (h), 75 (i) and 120 (j) minutes after mixing of SITS-labeled αB-wt and LYI-labeled R54C. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031421#pone-0031421-g001" target="_blank"><i>Fig. 1G</i></a>: The emission spectra of αB-crystallin excited at 336 nm were recorded at 0 (a), 2 (b), 4 (c), 6 (d), 8 (e), 10 (f), 20 (g), 45 (h), 75 (i) and 120 (j) minutes after mixing of SITS-labeled αB-wt and LYI-labeled R116C. Only 10 spectral curves were shown because of RF-5301PC software is not allowing more than 10.</p
<p>Examples of the acceptor photobleaching method for determining the FRET efficiency. In t... more <p>Examples of the acceptor photobleaching method for determining the FRET efficiency. In this example, CFPαBwt (donor) was co-expressed with either YFPαA-wt or YFPαA-R2116C (acceptor). The acceptor fluorescence was bleached by high intensity argon laser light. This resulted in an increase in donor fluorescence intensity and a decrease in acceptor fluorescence. The actual FRET efficiency was measured from at least 50 regions of interest (ROI) in the cell images obtained in three independent experiments for each pair.</p
<p>Rate constants (k) of heteroaggregates of αB-wt and αA-wt and its mutants at the accepto... more <p>Rate constants (k) of heteroaggregates of αB-wt and αA-wt and its mutants at the acceptor, 515 nm energy.</p
<p>FRET efficiency demonstrates the interaction between the αA and αB subunits of α-crystal... more <p>FRET efficiency demonstrates the interaction between the αA and αB subunits of α-crystallin. The interaction was strong between the wild-types of αA and αB subunits. The interaction between the mutated constructs, αA-R21W and αA-R116C with αB-wt was lower to αAwt+αBwt and also lower to other mutants group. The results were expressed as mean ± SD. Two-tailed unpaired Student's t-test was used and the p value for αB-wt+αA-R21W is <0.001 and for αB-wt+αA-R116C is <0.0008 compared to αB-wt+αA-wt group.</p
<p>Graph depicts time dependent increases in emissions intensity due to subunit exchange of... more <p>Graph depicts time dependent increases in emissions intensity due to subunit exchange of αB-wt and αA-wt or mutants subunits. Increase in the relative fluorescence intensity at 515 nm due to fluorescence energy transfer from the SITS-labeled to the LYI-labeled proteins. Each curve represents the best statistical fit of the data to the exponential function of Ft/F0 = A<sub>1</sub>+A<sub>2</sub>e<sup>−kt</sup>. The two-tailed unpaired Student's t-test was used to determine the significance. The p value is <0.0001 for αB-wt + αA-wt Vs αB-wt + αA-R21W and αB-wt + αA-wt Vs αB-wt + αA-R116C.</p
JCI insight, Jul 11, 2019
The Mitochondrial Pyruvate Carrier (MPC) occupies a central metabolic node by transporting cytoso... more The Mitochondrial Pyruvate Carrier (MPC) occupies a central metabolic node by transporting cytosolic pyruvate into the mitochondrial matrix and linking glycolysis with mitochondrial metabolism. Two reported human MPC1 mutations cause developmental abnormalities, neurological problems, metabolic deficits, and for one patient, early death. We aimed to understand biochemical mechanisms by which the human patient C289T and T236A MPC1 alleles disrupt MPC function. MPC1 C289T encodes two protein variants, a mis-spliced, truncation mutant (A58G) and a full length point mutant (R97W). MPC1 T236A encodes a full length point mutant (L79H). Using human patient fibroblasts and complementation of CRISPR-deleted, MPC1 null mouse C2C12 cells, we investigated how MPC1 mutations cause MPC deficiency. Truncated MPC1 A58G protein was intrinsically unstable and failed to form MPC complexes. The MPC1 R97W protein was less stable but when overexpressed formed complexes with MPC2 that retained pyruvate transport activity. Conversely, MPC1 L79H protein formed stable complexes with MPC2, but these complexes failed to transport pyruvate. These findings inform MPC structure-function relationships and delineate three distinct biochemical pathologies resulting from two human patient MPC1 mutations. They also illustrate an efficient gene pass-through system for mechanistically investigating human inborn errors in pyruvate metabolism.
Cell Reports, Sep 1, 2019
Highlights d The MPC is retained in HCC, supporting TCA cycle pyruvate metabolism d MPC disruptio... more Highlights d The MPC is retained in HCC, supporting TCA cycle pyruvate metabolism d MPC disruption directs glutamine to the TCA cycle away from glutathione synthesis d Glutathione synthesis is diminished, impairing hepatocellular tumorigenesis
Diabetes, Jun 1, 2019
The mitochondrial pyruvate carrier (MPC) occupies a carbohydrate metabolism nexus and is a potent... more The mitochondrial pyruvate carrier (MPC) occupies a carbohydrate metabolism nexus and is a potential target of thiazolidinediones (TZDs) prescribed to treat the metabolic syndrome. MSDC-0602, a TZD-like, PPARγ sparing molecule, is currently in phase 2B clinical for NASH treatment following positive results in animal studies. Previous research demonstrates that MSDC-0602 inhibits mitochondrial pyruvate oxidation and physically interacts with the MPC. However, the extent to which MSDC-0602 directly inhibits mitochondrial pyruvate uptake has not been fully resolved. Here, we aimed to comprehensively test the action of MSDC-0206 as a specific MPC inhibitor. We examined MSDC-0602 treatment effects on pyruvate oxidation and pyruvate transport by isolated liver mitochondria from normal chow (NCD)- and high fat diet (HFD)-fed mice. MSDC-0602 inhibited pyruvate-driven State III and State IV+Uncoupled respiration in a dose dependent manner. MSDC-0602 reduced pyruvate-driven respiration to levels observed for liver mitochondria isolated from MPC liver knockout (MPC LivKO) mice. NCD and HFD mice showed similar response to MSDC-0602. These data confirm that MSDC-0602 inhibits pyruvate-driven respiration. However, pyruvate oxidation is an indirect measure of MPC activity because it depends upon pyruvate-oxidizing enzymes and electron transport. To directly test the action of MSDC-0602 on mitochondrial pyruvate transport, we performed isolated mitochondria 14C-pyruvate uptake assays. Pyruvate uptake was significantly decreased by MSDC-0602 to rates observed for MPC LivKO mouse liver mitochondria. Finally, we examined effects of MSDC-0602 metabolites and found them to also inhibit isolated liver mitochondria pyruvate oxidation. Together, these data demonstrate that MSDC-0602 directly and specifically inhibits MPC activity as a mechanism for impairing downstream pyruvate utilization. The therapeutic effects of MSDC-0602 may be conferred by MPC inhibition and resulting metabolic adaptations. Disclosure A.J. Rauckhorst: None. L. Oonthonpan: None. E.B. Taylor: Research Support; Self; Cirius Therapeutics. Funding American Diabetes Association (1-18-PDF-060 to A.J.R.); National Institutes of Health (DK104998); Cirius Therapeutics
for your helpful suggestions and valuable insights. Your guidance shaped and helped me with my pe... more for your helpful suggestions and valuable insights. Your guidance shaped and helped me with my personal and career development. Thank you for always pushing me to think critically and taking the time to be on my committee. Thanks to all members of the Taylor lab, past and present, for all of your help, your kindness, and meaningful discussions. You are a fun and productive work family. I will cherish our friendships and hope you all know that you have a special place in my heart. Thanks to Dr.
Background: Mutation in aA-crystallin contributes to the development of congenital cataract in hu... more Background: Mutation in aA-crystallin contributes to the development of congenital cataract in humans. Heterooligomerization of aA-crystallin and aB-crystallin is essential for maintaining transparency in the eye lens. The effect of congenital cataract causing mutants of aA-crystallin on subunit exchange and interaction with aB-crystallin is unknown. In the present study, interaction of the mutants of aA-crystallin with aB-crystallin was studied both in vitro and in situ by the fluorescence resonance energy transfer (FRET) technique. Methodology/Principal Findings: In vitro FRET technique was used to demonstrate the rates of subunit exchange of aB-wt with the following aA-crystallin mutants: R12C, R21L, R21W, R49C, R54C, and R116C. The subunit exchange rates (k values) of R21W and R116C with aB-wt decreased drastically as compared to aA-wt interacting with aB-wt. Moderately decreased k values were seen with R12C, R49C and R54C while R21L showed nearly normal k value. The interaction...
Journal of Psychiatric Research
for your helpful suggestions and valuable insights. Your guidance shaped and helped me with my pe... more for your helpful suggestions and valuable insights. Your guidance shaped and helped me with my personal and career development. Thank you for always pushing me to think critically and taking the time to be on my committee. Thanks to all members of the Taylor lab, past and present, for all of your help, your kindness, and meaningful discussions. You are a fun and productive work family. I will cherish our friendships and hope you all know that you have a special place in my heart. Thanks to Dr.
Cell Reports
Highlights d The MPC is retained in HCC, supporting TCA cycle pyruvate metabolism d MPC disruptio... more Highlights d The MPC is retained in HCC, supporting TCA cycle pyruvate metabolism d MPC disruption directs glutamine to the TCA cycle away from glutathione synthesis d Glutathione synthesis is diminished, impairing hepatocellular tumorigenesis
eLife
Metabolic cycles are a fundamental element of cellular and organismal function. Among the most cr... more Metabolic cycles are a fundamental element of cellular and organismal function. Among the most critical in higher organisms is the Cori Cycle, the systemic cycling between lactate and glucose. Here, skeletal muscle-specific Mitochondrial Pyruvate Carrier (MPC) deletion in mice diverted pyruvate into circulating lactate. This switch disinhibited muscle fatty acid oxidation and drove Cori Cycling that contributed to increased energy expenditure. Loss of muscle MPC activity led to strikingly decreased adiposity with complete muscle mass and strength retention. Notably, despite decreasing muscle glucose oxidation, muscle MPC disruption increased muscle glucose uptake and whole-body insulin sensitivity. Furthermore, chronic and acute muscle MPC deletion accelerated fat mass loss on a normal diet after high fat diet-induced obesity. Our results illuminate the role of the skeletal muscle MPC as a whole-body carbon flux control point. They highlight the potential utility of decreasing muscl...
JCI Insight
The Mitochondrial Pyruvate Carrier (MPC) occupies a central metabolic node by transporting cytoso... more The Mitochondrial Pyruvate Carrier (MPC) occupies a central metabolic node by transporting cytosolic pyruvate into the mitochondrial matrix and linking glycolysis with mitochondrial metabolism. Two reported human MPC1 mutations cause developmental abnormalities, neurological problems, metabolic deficits, and for one patient, early death. We aimed to understand biochemical mechanisms by which the human patient C289T and T236A MPC1 alleles disrupt MPC function. MPC1 C289T encodes two protein variants, a mis-spliced, truncation mutant (A58G) and a full length point mutant (R97W). MPC1 T236A encodes a full length point mutant (L79H). Using human patient fibroblasts and complementation of CRISPR-deleted, MPC1 null mouse C2C12 cells, we investigated how MPC1 mutations cause MPC deficiency. Truncated MPC1 A58G protein was intrinsically unstable and failed to form MPC complexes. The MPC1 R97W protein was less stable but when overexpressed formed complexes with MPC2 that retained pyruvate transport activity. Conversely, MPC1 L79H protein formed stable complexes with MPC2, but these complexes failed to transport pyruvate. These findings inform MPC structure-function relationships and delineate three distinct biochemical pathologies resulting from two human patient MPC1 mutations. They also illustrate an efficient gene pass-through system for mechanistically investigating human inborn errors in pyruvate metabolism.
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Papers by Lalita Oonthonpan