Structures and functions of mitochondrial ABC transporters

A small number of physiologically important ATP-binding cassette (ABC) transporters are found in mitochondria. Most are half transporters of the B group forming homodimers and their topology suggests they function as exporters. The results of mutant studies point towards involvement in iron cofactor biosynthesis. In particular, ABC subfamily B member 7 (ABCB7) and its homologues in yeast and plants are required for iron-sulfur (Fe-S) cluster biosynthesis outside of the mitochondria, whereas ABCB10 is involved in haem biosynthesis. They also play a role in preventing oxidative stress. Mutations in ABCB6 and ABCB7 have been linked to human disease. Recent crystal structures of yeast Atm1 and human ABCB10 have been key to identifying substrate-binding sites and transport mechanisms. Combined with in vitro and in vivo studies, progress is being made to find the physiological substrates of the different mitochondrial ABC transporters

RT-qPCR Demonstrates Light-Dependent AtRBCS1A and AtRBCS3B mRNA Expressions in Arabidopsis Thaliana Leaves

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is widely used in diagnosis and research to determine specific mRNA expressions in cells. As RT-qPCR applications increase, it’s necessary to provide undergraduates hands-on experience of this modern technique. Here, we report a 3-week laboratory exercise using RT-qPCR to demonstrate the light-dependent expressions of AtRBCS1A and AtRBCS3B genes encoding two Arabidopsis thaliana small subunits of the ribulose 1,5-bisphosphate carboxylase/ oxygenase (Rubisco). In the first week, students purified and quantified total RNA from leaves of A. thaliana pretreated in the dark for 96 hr and untreated controls. In the second week, RNA samples were separated by formaldehyde gel electrophoresis and used for RT-qPCR. Students calculated expressions of the two genes in dark treated leaves as percentages of those of the controls by using the 22DDC T method and the collected CTs. In the third week, class CTs, melting curves, students’ calculations, and factors affecting the reliability of RT-qPCR results were summarized and discussed. Students’ results show that (i) relatively pure and intact RNA samples are obtained; (ii) ACTIN2 is a better reference gene than the 18S rRNA; (iii) the dark treatment reduces both gene expressions to \u3c 1%; (iv) the reduction in the expression of AtRBCS3B is significantly more than that of the AtRBCS1A. Results from preand post-lab tests indicate that besides the theory, this exercise helps students learn the applications and associated techniques of RT-qPCR. Future modifications and new experiments that can be developed based on students’ learning outcomes and assessment are also discussed

Essential histidine pairs indicate conserved haem binding in epsilonproteobacterial cytochrome c haem lyases

Bacterial cytochrome c maturation occurs at the outside of the cytoplasmic membrane, requires transport of haem b across the membrane, and depends on membrane-bound cytochrome c haem lyase (CCHL), an enzyme that catalyses covalent attachment of haem b to apocytochrome c. Epsilonproteobacteria such as Wolinella succinogenes use the cytochrome c biogenesis system II and contain unusually large CCHL proteins of about 900 amino acid residues that appear to be fusions of the CcsB and CcsA proteins found in other bacteria. CcsBA-type CCHLs have been proposed to act as haem transporters that contain two haem b coordination sites located at different sides of the membrane and formed by histidine pairs. W. succinogenes cells contain three CcsBA-type CCHL isoenzymes (NrfI, CcsA1 and CcsA2) that are known to differ in their specificity for apocytochromes and apparently recognize different haem c binding motifs such as CX2CH (by CcsA2), CX2CK (by NrfI) and CX15CH (by CcsA1). In this study, conserved histidine residues were individually replaced by alanine in each of the W. succinogenes CCHLs. Characterization of NrfI and CcsA1 variants in W. succinogenes demonstrated that a set of four histidines is essential for maturing the dedicated multihaem cytochromes c NrfA and MccA, respectively. The function of W. succinogenes CcsA2 variants produced in Escherichia coli was also found to depend on each of these four conserved histidine residues. The presence of imidazole in the growth medium of both W. succinogenes and E. coli rescued the cytochrome c biogenesis activity of most histidine variants, albeit to different extents, thereby implying the presence of two functionally distinct histidine pairs in each CCHL. The data support a model in which two conserved haem b binding sites are involved in haem transport catalysed by CcsBA-type CCHLs

Persistent ER Stress Induces the Spliced Leader RNA Silencing Pathway (SLS), Leading to Programmed Cell Death in Trypanosoma brucei

Trypanosomes are parasites that cycle between the insect host (procyclic form) and mammalian host (bloodstream form). These parasites lack conventional transcription regulation, including factors that induce the unfolded protein response (UPR). However, they possess a stress response mechanism, the spliced leader RNA silencing (SLS) pathway. SLS elicits shut-off of spliced leader RNA (SL RNA) transcription by perturbing the binding of the transcription factor tSNAP42 to its cognate promoter, thus eliminating trans-splicing of all mRNAs. Induction of endoplasmic reticulum (ER) stress in procyclic trypanosomes elicits changes in the transcriptome similar to those induced by conventional UPR found in other eukaryotes. The mechanism of up-regulation under ER stress is dependent on differential stabilization of mRNAs. The transcriptome changes are accompanied by ER dilation and elevation in the ER chaperone, BiP. Prolonged ER stress induces SLS pathway. RNAi silencing of SEC63, a factor that participates in protein translocation across the ER membrane, or SEC61, the translocation channel, also induces SLS. Silencing of these genes or prolonged ER stress led to programmed cell death (PCD), evident by exposure of phosphatidyl serine, DNA laddering, increase in reactive oxygen species (ROS) production, increase in cytoplasmic Ca2+, and decrease in mitochondrial membrane potential, as well as typical morphological changes observed by transmission electron microscopy (TEM). ER stress response is also induced in the bloodstream form and if the stress persists it leads to SLS. We propose that prolonged ER stress induces SLS, which serves as a unique death pathway, replacing the conventional caspase-mediated PCD observed in higher eukaryotes

Intracellular Ca2+ Imbalance Critically Contributes to Paraptosis

Paraptosis is a type of programmed cell death that is characterized by dilation of the endoplasmic reticulum (ER) and/or mitochondria. Since paraptosis is morphologically and biochemically different from apoptosis, understanding its regulatory mechanisms may provide a novel therapeutic strategy in malignant cancer cells that have proven resistant to conventional pro-apoptotic treatments. Relatively little is known about the molecular basis of paraptosis, but perturbations of cellular proteostasis and ion homeostasis appear to critically contribute to the process. Ca2+ transport has been shown to be important in the paraptosis induced by several natural products, metal complexes, and co-treatment with proteasome inhibitors and certain Ca2+-modulating agents. In particular, the Ca2+-mediated communication between the ER and mitochondria plays a crucial role in paraptosis. Mitochondrial Ca2+ overload from the intracellular Ca2+-flux system located at the ER-mitochondrial axis can induce mitochondrial dilation during paraptosis, while the accumulation of misfolded proteins within the ER lumen is believed to exert an osmotic force and draw water from the cytoplasm to distend the ER lumen. In this process, Ca2+ release from the ER also critically contributes to aggravating ER stress and ER dilation. This review focuses on the role of Ca2+ transport in paraptosis by summarizing the recent findings related to the actions of Ca2+-modulating paraptosis-inducing agents and discussing the potential cancer therapeutic strategies that may effectively induce paraptosis via Ca2+ signaling

Hypercontractive semigroups and Sobolev’s inequality

If H ⩾ 0 H \geqslant 0 is the generator of a hypercontractive semigroup (HCSG), it is known that ( H + 1 ) − 1 / 2 {(H + 1)^{ - 1/2}} is a bounded operator from L p {L^p} to L p , 1 ⩽ p ⩽ ∞ {L^p},1 \leqslant p \leqslant \infty . We prove that ( H + 1 ) − 1 / 2 {(H + 1)^{ - 1/2}} is bounded from L 2 {L^2} to the Orlicz space L 2  I n + L {L^2}{\text { I}}{{\text {n}}^ + }L , basing the proof on the uniform semiboundedness of the operator H + V H + V , for suitable V V . We also prove by an interpolation argument, that ( H + 1 ) − 1 / 2 {(H + 1)^{ - 1/2}} is bounded from L p {L^p} to L p  I n + L , 2 ⩽ p &gt; ∞ {L^p}{\text { I}}{{\text {n}}^ + }L,2 \leqslant p &gt; \infty . Another interpolation argument shows that ( H + 1 ) − 1 / 2 {(H + 1)^{ - 1/2}} is bounded from L p ( I n + L ) m {L^p}{({\text {I}}{{\text {n}}^ + }L)^m} to L p ( I n + L ) m + 1 {L^p}{({\text {I}}{{\text {n}}^ + }L)^{m + 1}} and m m a positive integer. Finally, we identify the topological duals of the spaces mentioned above.</p

Mitochondrial Ca2+ and ROS take center stage to orchestrate TNF-α–mediated inflammatory responses

Proinflammatory stimuli induce inflammation that may progress to sepsis or chronic inflammatory disease. The cytokine TNF-α is an important endotoxin-induced inflammatory glycoprotein produced predominantly by macrophages and lymphocytes. TNF-α plays a major role in initiating signaling pathways and pathophysiological responses after engaging TNF receptors. In this issue of JCI, Rowlands et al. demonstrate that in lung microvessels, soluble TNF-α (sTNF-α) promotes the shedding of the TNF-α receptor 1 ectodomain via increased mitochondrial Ca2+ that leads to release of mitochondrial ROS. Shedding mediated by TNF-α–converting enzyme (TACE) results in an unattached TNF receptor, which participates in the scavenging of sTNF-α, thus limiting the propagation of the inflammatory response. These findings suggest that mitochondrial Ca2+, ROS, and TACE might be therapeutically targeted for treating pulmonary endothelial inflammation