Prostate cancer proliferation is affected by the subcellular localization of MCT2 and accompanied by significant peroxisomal alterations

Reprogramming of lipid metabolism directly contributes to malignant transformation and progression. The increased uptake of circulating lipids, the transfer of fatty acids from stromal adipocytes to cancer cells, the de novo fatty acid synthesis, and the fatty acid oxidation support the central role of lipids in many cancers, including prostate cancer (PCa). Fatty acid ß-oxidation is the dominant bioenergetic pathway in PCa and recent evidence suggests that PCa takes advantage of the peroxisome transport machinery to target monocarboxylate transporter 2 (MCT2) to peroxisomes in order to increase ß-oxidation rates and maintain the redox balance. Here we show evidence suggesting that PCa streamlines peroxisome metabolism by upregulating distinct pathways involved in lipid metabolism. Moreover, we show that MCT2 is required for PCa cell proliferation and, importantly, that its specific localization at the peroxisomal membranes is essential for this role. Our results highlight the importance of peroxisomes in PCa development and uncover different cellular mechanisms that may be further explored as possible targets for PCa therapy.This work was supported by the Portuguese Foundation for Science and Technology (FCT): PTDC/IMI-MIC/0828/2012, PTDC/BIA-CEL/31378/2017, CEECIND/03747/2017, SFRH/BPD/77619/2011, SFRH/BD/101942/2014, UIDB/04501/2020, under the scope of the Operational Program “Competitiveness and internationalization”, in its FEDER/FNR component. It was also funded by the Comissão da Região Centro CCDRC and FEDER through the integrated project pAGE-CENTRO-01-0145-FEDER-000003. This work was also supported by national funds (OE), through FCT, I.P., in the scope of the framework contract foreseen in the numbers 4, 5, and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19

Mutagenesis separates ATPase and thioesterase activities of the peroxisomal ABC transporter, Comatose

The peroxisomal ABC transporter, Comatose (CTS), a full length transporter from Arabidopsis has intrinsic acyl-CoA thioesterase (ACOT) activity, important for physiological function. We used molecular modelling, mutagenesis and biochemical analysis to identify amino acid residues important for ACOT activity. D863, Q864 and T867 lie within transmembrane helix 9. These residues are orientated such that they might plausibly contribute to a catalytic triad similar to type II Hotdog fold thioesterases. When expressed in Saccharomyces cerevisiae, mutation of these residues to alanine resulted in defective of β-oxidation. All CTS mutants were expressed and targeted to peroxisomes and retained substrate-stimulated ATPase activity. When expressed in insect cell membranes, Q864A and S810N had similar ATPase activity to wild type but greatly reduced ACOT activity, whereas the Walker A mutant K487A had greatly reduced ATPase and no ATP-dependent ACOT activity. In wild type CTS, ATPase but not ACOT was stimulated by non-cleavable C14 ether-CoA. ACOT activity was stimulated by ATP but not by non-hydrolysable AMPPNP. Thus, ACOT activity depends on functional ATPase activity but not vice versa, and these two activities can be separated by mutagenesis. Whether D863, Q864 and T867 have a catalytic role or play a more indirect role in NBD-TMD communication is discussed

A functional SMAD2/3 binding site in the PEX11β promoter identifies a role for TGFβ in peroxisome proliferation in humans

This is the final version. Available on open access from Frontiers Media via the DOI in this recordData availability: The research data supporting this publication are provided within this paper and as supplementary information.In mammals, peroxisomes perform crucial functions in cellular metabolism, signalling and viral defense which are essential to the viability of the organism. Molecular cues triggered by changes in the cellular environment induce a dynamic response in peroxisomes, which manifests itself as a change in peroxisome number, altered enzyme levels and adaptations to the peroxisomal morphology. How the regulation of this process is integrated into the cell’s response to different stimuli, including the signalling pathways and factors involved, remains unclear. Here, a cell-based peroxisome proliferation assay has been applied to investigate the ability of different stimuli to induce peroxisome proliferation. We determined that serum stimulation, long-chain fatty acid supplementation and TGFβ application all increase peroxisome elongation, a prerequisite for proliferation. Time-resolved mRNA expression during the peroxisome proliferation cycle revealed a number of peroxins whose expression correlated with peroxisome elongation, including the β isoform of PEX11, but not the α or γ isoforms. An initial map of putative regulatory motif sites in the respective promoters showed a difference between binding sites in PEX11α and PEX11β, suggesting that these genes may be regulated by distinct pathways. A functional SMAD2/3 binding site in PEX11β points to the involvement of the TGFβ signalling pathway in expression of this gene and thus peroxisome proliferation/dynamics in humans.Biotechnology & Biological Sciences Research Council (BBSRC)Swiss National Science Foundation (SNSF)European Union FP7Medical Research Council (MRC

HIBCH mutations can cause Leigh-like disease with combined deficiency of multiple mitochondrial respiratory chain enzymes and pyruvate dehydrogenase

Background: Deficiency of 3-hydroxy-isobutyryl-CoA hydrolase (HIBCH) caused by HIBCH mutations is a rare cerebral organic aciduria caused by disturbance of valine catabolism. Multiple mitochondrial respiratory chain (RC) enzyme deficiencies can arise from a number of mechanisms, including defective maintenance or expression of mitochondrial DNA. Impaired biosynthesis of iron-sulphur clusters and lipoic acid can lead to pyruvate dehydrogenase complex (PDHc) deficiency in addition to multiple RC deficiencies, known as the multiple mitochondrial dysfunctions syndrome. Methods: Two brothers born to distantly related Pakistani parents presenting in early infancy with a progressive neurodegenerative disorder, associated with basal ganglia changes on brain magnetic resonance imaging, were investigated for suspected Leigh-like mitochondrial disease. The index case had deficiencies of multiple RC enzymes and PDHc in skeletal muscle and fibroblasts respectively, but these were normal in his younger brother. The observation of persistently elevated hydroxy-C4-carnitine levels in the younger brother led to suspicion of HIBCH deficiency, which was investigated by biochemical assay in cultured skin fibroblasts and molecular genetic analysis. Results: Specific spectrophotometric enzyme assay revealed HIBCH activity to be below detectable limits in cultured skin fibroblasts from both brothers. Direct Sanger sequence analysis demonstrated a novel homozygous pathogenic missense mutation c.950G <A; p.Gly317Glu in the HIBCH gene, which segregated with infantile-onset neurodegeneration within the family. Conclusions: HIBCH deficiency, a disorder of valine catabolism, is a novel cause of the multiple mitochondrial dysfunctions syndrome, and should be considered in the differential diagnosis of patients presenting with multiple RC deficiencies and/or pyruvate dehydrogenase deficiency

Differential roles for ACBD4 and ACBD5 in peroxisome-ER interactions and lipid metabolism

This is the author accepted manuscript. The final version is available on open access from Elsevier via the DOI in this record Data availability: The research data supporting this publication are provided within this paper, or as supplementary information.Peroxisomes and the endoplasmic reticulum (ER) are intimately linked subcellular organelles, physically connected at membrane contact sites. As well as collaborating in lipid metabolism, e.g. of very long chain fatty acids (VLCFAs) and plasmalogens, the ER also plays a role in peroxisome biogenesis. Recent work has identified tethering complexes on the ER and peroxisome membranes which connect the organelles. These include membrane contacts formed via interactions between the ER protein VAPB (vesicle-associated membrane proteinassociated protein B) and the peroxisomal proteins ACBD4 and ACBD5 (acyl-coenzyme Abinding domain protein). Loss of ACBD5 has been shown to cause a significant reduction in peroxisome-ER contacts and accumulation of VLCFAs. However, the role of ACBD4, and the relative contribution these two proteins make to contact site formation and recruitment of VLCFAs to peroxisomes remains unclear. Here, we address these questions, using a combination of molecular cell biology, biochemical and lipidomics analyses following loss of ACBD4 or ACBD5 in HEK293 cells. We show that the tethering function of ACBD5 is not absolutely required for efficient peroxisomal β-oxidation of VLCFAs. We demonstrate that loss of ACBD4 does not reduce peroxisome-ER connections or result in accumulation of VLCFAs. Instead, the loss of ACBD4 resulted in an increase in the rate of β-oxidation of VLCFAs. Finally, we observe interaction between ACBD5 and ACBD4, independent of VAPB binding. Overall, our findings suggest that ACBD5 may act as a primary tether and VLCFA recruitment factor, whereas ACBD4 may have regulatory functions in peroxisomal lipid metabolism at the peroxisome-ER interface.Biotechnology & Biological Sciences Research Council (BBSRC)UK Research and InnovationRoyal SocietyEuropean Union Horizon 2020Medical Research Council (MRC

Extension of Yeast Chronological Lifespan by Methylamine

Background: Chronological aging of yeast cells is commonly used as a model for aging of human post-mitotic cells. The yeast Saccharomyces cerevisiae grown on glucose in the presence of ammonium sulphate is mainly used in yeast aging research. We have analyzed chronological aging of the yeast Hansenula polymorpha grown at conditions that require primary peroxisome metabolism for growth. Methodology/Principal Findings: The chronological lifespan of H. polymorpha is strongly enhanced when cells are grown on methanol or ethanol, metabolized by peroxisome enzymes, relative to growth on glucose that does not require peroxisomes. The short lifespan of H. polymorpha on glucose is mainly due to medium acidification, whereas most likely ROS do not play an important role. Growth of cells on methanol/methylamine instead of methanol/ammonium sulphate resulted in further lifespan enhancement. This was unrelated to medium acidification. We show that oxidation of methylamine by peroxisomal amine oxidase at carbon starvation conditions is responsible for lifespan extension. The methylamine oxidation product formaldehyde is further oxidized resulting in NADH generation, which contributes to increased ATP generation and reduction of ROS levels in the stationary phase. Conclusion/Significance: We conclude that primary peroxisome metabolism enhanced chronological lifespan of H. polymorpha. Moreover, the possibility to generate NADH at carbon starvation conditions by an organic nitrogen source supports further extension of the lifespan of the cell. Consequently, the interpretation of CLS analyses in yeast should include possible effects on the energy status of the cell.

Compromized geranylgeranylation of RhoA and Rac1 in mevalonate kinase deficiency

Mevalonate kinase deficiency (MKD) is an autoinflammatory disorder caused by mutations in the MVK gene resulting in decreased activity of the enzyme mevalonate kinase (MK). Although MK is required for biosynthesis of all isoprenoids, in MKD, in particular, the timely synthesis of geranylgeranyl pyrophosphate appears to be compromised. Because small guanosine triphosphatases (GTPases) depend on geranylgeranylation for their proper signaling function, we studied the effect of MK deficiency on geranylgeranylation and activation of the two small GTPases, RhoA and Rac1. We demonstrate that both geranylgeranylation and activation of the two GTPases are more easily disturbed in MKD cells than in control cells when the flux though the isoprenoid biosynthesis pathway is suppressed by low concentrations of simvastatin. The limited capacity of geranylgeranylation in MKD cells readily leads to markedly increased levels of nonisoprenylated and activated GTPases, which will affect proper signaling by these GTPases

Characterisation of a Desmosterol Reductase Involved in Phytosterol Dealkylation in the Silkworm, Bombyx mori

Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C29 and C28) yielding cholesterol (C27). The final step of this dealkylation pathway involves desmosterol reductase (DHCR24)-catalysed reduction of desmosterol to cholesterol. We now report the molecular characterisation in the silkworm, Bombyx mori, of such a desmosterol reductase involved in production of cholesterol from phytosterol, rather than in de novo synthesis of cholesterol. Phylogenomic analysis of putative desmosterol reductases revealed the occurrence of various clades that allowed for the identification of a strong reductase candidate gene in Bombyx mori (BGIBMGA 005735). Following PCR-based cloning of the cDNA (1.6 kb) and its heterologous expression in Saccharomyces cerevisae, the recombinant protein catalysed reduction of desmosterol to cholesterol in an NADH- and FAD- dependent reaction

Targeting and Function of the Mitochondrial Fission Factor GDAP1 Are Dependent on Its Tail-Anchor

Proteins controlling mitochondrial dynamics are often targeted to and anchored into the mitochondrial outer membrane (MOM) by their carboxyl-terminal tail-anchor domain (TA). However, it is not known whether the TA modulates protein function. GDAP1 is a mitochondrial fission factor with two neighboring hydrophobic domains each flanked by basic amino acids (aa). Here we define GDAP1 as TA MOM protein. GDAP1 carries a single transmembrane domain (TMD) that is, together with the adjacent basic aa, critical for MOM targeting. The flanking N-terminal region containing the other hydrophobic domain is located in the cytoplasm. TMD sequence, length, and high hydrophobicity do not influence GDAP1 fission function if MOM targeting is maintained. The basic aa bordering the TMD in the cytoplasm, however, are required for both targeting of GDAP1 as part of the TA and GDAP1-mediated fission. Thus, this GDAP1 region contains critical overlapping motifs defining intracellular targeting by the TA concomitant with functional aspects