Mis-targeting of the mitochondrial protein LIPT2 leads to apoptotic cell death

<div><p>Lipoyl(Octanoyl) Transferase 2 (LIPT2) is a protein involved in the post-translational modification of key energy metabolism enzymes in humans. Defects of lipoic acid synthesis and transfer start to emerge as causes of fatal or severe early-onset disease. We show that the first 31 amino acids of the N-terminus of LIPT2 represent a mitochondrial targeting sequence and inhibition of the transit of LIPT2 to the mitochondrion results in apoptotic cell death associated with activation of the apoptotic volume decrease (AVD) current in normotonic conditions, as well as over-activation of the swelling-activated chloride current (IClswell), mitochondrial membrane potential collapse, caspase-3 cleavage and nuclear DNA fragmentation. The findings presented here may help elucidate the molecular mechanisms underlying derangements of lipoic acid biosynthesis.</p></div

Removal of amino acids 1–31 prevents the targeting of LIPT2 to the mitochondrion.

<p>(A) From left to right: fluorescent signal of EYFP (green) and the mitochondrial marker (magenta) in HeLa cells expressing LIPT2-EYFP void of the amino acids 1–31 (ΔmitotagLIPT2-EYFP) for 72 hours, corresponding merge image and scatter plot. Scale bar: 20 μm. (B) Pearson’s correlation coefficient, overlap coefficient and co-localization rate (%) referred to the co-localization of ΔmitotagLIPT2-EYFP and the mitochondrion determined in HeLa cells 72 hours after transfection. (n) indicates the number of cells. ***: p<0.001, one-way ANOVA with Bonferroni’s post-test.</p

IClswell current is not affected following alteration of LIPT2 expression.

<p>(A) Current density (pA/pF) to voltage (mV) relationship measured in hypertonic solution and after a 10 minutes exposure to a hypotonic solution (left) and current density (pA/pF) to time (sec) relationship measured in hypotonic solution (right) in HEK 293 Phoenix cells transfected for 48 hours with LIPT2 or a transfection marker (control). (B) Levels of the transcript of <i>LIPT2</i> detected by RT-PCR in HEK 293 Phoenix cells transfected for 48 hours with siRNA #1, 2 and 3 or control siRNA (control) and normalized for the levels of the transcript of the housekeeping gene <i>β-ACTIN</i>. Gene silencing of 7, 48 and 25% was obtained, respectively. (C) Current density (pA/pF) to voltage (mV) relationship measured in hypertonic solution and after a 10 minutes exposure to a hypotonic solution (left) and current density (pA/pF) to time (sec) relationship measured in hypotonic solution (right) in cells transfected with siRNA #2 or a control siRNA (control). n.s., not significant, **, ***: p<0.01, p<0.001, unpaired Student’s t test. (n) in A and C indicates the number of cells. In B, n = 3 for each condition.</p

Apoptosis is induced in EYFP-LIPT2 and ΔmitotagLIPT2 expressing cells.

<p>(A) Representative immunodetection of calreticulin in whole cell lysates from HEK 293 Phoenix cells 48 hours after transfection with the indicated constructs or from cells treated with the vehicle or 20 μM staurosporine for 4 hours. The lower panels correspond to the signal of the housekeeping protein GAPDH. (B) The expression levels of calreticulin were quantified by densitometry and normalized to GAPDH. n = 3, *: p<0.05, **: p<0.01, ***: p<0.001, two-tailed, unpaired Student’s t test. (C) Immunodetection of cleaved caspase-3 and the housekeeping protein β-actin in whole cell lysates from HEK 293 Phoenix cells 48 hours after transfection with the indicated constructs or from cells treated with the vehicle or 20 μM staurosporine for 4 hours. The image is representative of three independent experiments. (D) From left to right: fluorescent signal of DAPI (cyan), TUNEL (magenta) and corresponding merge image of HEK 293 Phoenix cells expressing EYFP-LIPT2 (top), LIPT2-EYFP (middle), and ΔmitotagLIPT2 (bottom) for 48 hours. Scale bar: 50 μm. (E) Normalized TUNEL signal intensity of cells transfected with the indicated constructs or treated overnight with the vehicle or 2 μM staurosporine. 12≤n≤25 indicates the number of imaging fields from 3 independent experiments. *: p<0.05, one-way ANOVA with Bonferroni’s post-test.</p

IClswell current is over-stimulated in EYFP-LIPT2 and ΔmitotagLIPT2 expressing cells.

<p>(A) Original current recordings obtained in whole-cell configuration from HEK 293 Phoenix cells in hypertonic (hyper) and hypotonic (hypo) bath solution following stimulation with the pulse protocol represented in the inset. Cells were transfected for 48 hours with EYFP-LIPT2 or LIPT2-EYFP, as indicated. (B) Current density (pA/pF) to voltage (mV) relationship measured in hypertonic solution and after a 10 minutes exposure to a hypotonic solution (left) and current density (pA/pF) to time (sec) relationship measured in hypotonic solution (right) in cells expressing EYFP-LIPT2 or LIPT2-EYFP. C, current density (pA/pF) to voltage (mV) relationship measured in hypertonic solution and after a 10 minutes exposure to a hypotonic solution (left) and current density (pA/pF) to time (sec) relationship measured in hypotonic solution (right) in cells expressing LIPT2 or ΔmitotagLIPT2. In B, left, ***: p<0.001 at all applied voltages except for 0 mV compared to LIPT2-EYFP, hypo, unpaired Student’s t test, and right, p<0.001 when time >160 sec, unpaired Student’s t test. In C, left, *: p<0.05 at all applied voltages except for 0 mV compared to LIPT2, hypo, unpaired Student’s t test, and right, p<0.05 when time >360 sec and <660 sec, unpaired Student’s t test. (n) indicates the number of cells.</p

Experimental Study on a Coiled Tube Solar Receiver under Variable Solar Radiation Condition

K. Wang, H. Wu, D. Wang, Y. Wang, Z. Tong, F. Lin, and A. G. Olabi, 'Experimental study on a coiled tube solar receiver under variable solar radiation condition', Solar Energy, Vol. 122, pp.. 1080-1090, December 2015, doi: https://doi.org/10.1016/j.solener.2015.10.004.This article presents an experimental investigation of the heat transfer characteristics as well as energy and exergy performance for a coil type solar dish receiver under variable solar radiation conditions. During a 4.5 h period of continuous operation in the morning, the solar irradiance fluctuates in the range between 160 W/m2 and 650 W/m2, and the concentrated solar flux is approximately 1000 kW/m2 at aperture. Experimental results show that: (1) the efficiency of the solar receiver is found to be over 58% and the highest efficiency can be reached to 64%. Generally, the efficiency is maintained at around 60% under steady state condition. (2) A very low value of the heat loss factor (0.035 kW/K) could be achieved during steady state operation. (3) The highest value of the exergy rate is approximately 7.3 kW, whereas the maximum energy rate can reach 16.2 kW. (4) The highest exergy efficiency is approximately 23%, whereas the highest energy efficiency is around 64%.Peer reviewe

Lipoic acid biosynthesis.

<p>mtFAS generates octanoyl-ACP, that enters the lipoic acid biosynthesis pathway. The octanoyl moiety is then transferred from ACP to H or E2 proteins. Subsequently, insertion of two sulfur atoms occurs on the octanoyl moiety to generate lipoylated H or E2 proteins. 2-KGDH, α-ketoglutarate dehydrogenase; 2-OADH, 2-oxoadipate dehydrogenase; ACP, acyl carrier protein; BCKDH, branched-chain ketoacid dehydrogenase; GCS, glycine cleavage system; LA, lipoic acid; LIAS, lipoic acid synthetase; LIPT1, lipoyl(octanoyl) transferase 1; LIPT2, lipoyl(octanoyl) transferase 2; mtFAS, mitochondrial fatty acidynthesis; PDH, pyruvate dehydrogenase.</p

LIPT2 and LIPT2-EYFP target the mitochondrion.

<p>(A) Immunodetection of endogenous LIPT2 (~25 kDa) in mitochondrial, cytosolic and microsomal fractions obtained from HEK 293 Phoenix cellular proteins. Alpha-tubulin (55 kDa) and ox-Phos-complex IV (35–40 kDa) were used as markers of the non-mitochondrial (cytosolic and microsomal) and mitochondrial fractions, respectively. The image is representative of three independent experiments. (B) From left to right: fluorescent signal of EYFP (green) and the mitochondrial marker (magenta) in HeLa cells expressing EYFP, EYFP-LIPT2 or LIPT2-EYFP for 72 hours, corresponding merge image and scatter plot. Scale bar: 20 μm. Pearson’s correlation coefficient, overlap coefficient and co-localization rate (%) referred to the co-localization of EYFP, EYFP-LIPT2 or LIPT2-EYFP and the mitochondrion determined in HeLa cells (C), 24, (D), 48 and (E), 72 hours after transfection. (n) indicates the number of cells. **: p<0.01, ***: p<0.001, one-way ANOVA with Bonferroni’s post-test.</p

Amino acids 1–31 of LIPT2 target EYFP to the mitochondrion.

<p>(A) Fluorescent signal of EYFP, mitotag-EYFP, mitotag-ΔATG-EYFP after 24 (left), 48 (middle) and 72 hours (right) transfection in HeLa cells. Scale bar: 20 μm. (B) Pearson’s correlation coefficient, overlap coefficient and co-localization rate (%) referred to the co-localization of EYFP, mitotag-EYFP, mitotag-ΔATG-EYFP and the mitochondrion determined in HeLa cells 72 hours after transfection. (n) indicates the number of cells. **: p<0.01, ***: p<0.001, one-way ANOVA with Bonferroni’s post-test.</p

I_Cl,swell characterisation in cells over-expressing 4.1R^80/135.

<p>(A) Representative whole-cell traces recorded in control cells (over-expressing GFP) or cells over-expressing the 4.1R⁸⁰ or the 4.1R¹³⁵ protein exposed to hypertonic (<i>Hyper</i>) and hypotonic (<i>Hypo</i>) extracellular solutions. (B) Relationship between mean current density, <i>d (pA/pF)</i>, and membrane voltage, <i>V (mV)</i>, in cells over-expressing the indicated proteins and exposed to the hypotonic extracellular solution for 10 min (GFP: n = 22, 4.1R⁸⁰: n = 15, 4.1R¹³⁵: n = 14). (C) Chloride current activation during hypotonic exposure (GFP: n = 24, 4.1R⁸⁰: n = 17, 4.1R¹³⁵: n = 14. (D,E) Relationship between mean current density and membrane voltage in control cells or cells over-expressing the 4.1R⁸⁰ (C) or the 4.1R¹³⁵ protein (D) in the hypertonic extracellular solution (GFP: n = 43, 4.1R⁸⁰: n = 38, 4.1R¹³⁵: n = 27. *p<0.05; ***p<0.001. Two-way ANOVA.</p