Genome-scale requirements for dynein-based transport revealed by a high-content arrayed CRISPR screen.

The microtubule motor dynein plays a key role in cellular organization. However, little is known about how dynein's biosynthesis, assembly, and functional diversity are orchestrated. To address this issue, we have conducted an arrayed CRISPR loss-of-function screen in human cells using the distribution of dynein-tethered peroxisomes and early endosomes as readouts. From a genome-wide gRNA library, 195 validated hits were recovered and parsed into those impacting multiple dynein cargoes and those whose effects are restricted to a subset of cargoes. Clustering of high-dimensional phenotypic fingerprints revealed co-functional proteins involved in many cellular processes, including several candidate novel regulators of core dynein functions. Further analysis of one of these factors, the RNA-binding protein SUGP1, indicates that it promotes cargo trafficking by sustaining functional expression of the dynein activator LIS1. Our data represent a rich source of new hypotheses for investigating microtubule-based transport, as well as several other aspects of cellular organization captured by our high-content imaging.

A novel peroxisome-related gene signature predicts clinical prognosis and is associated with immune microenvironment in low-grade glioma.

Low-grade glioma (LGG), a common primary tumor, mainly originates from astrocytes and oligodendrocytes. Increasing evidence has shown that peroxisomes function in the regulation of tumorigenesis and development of cancer. However, the prognostic value of peroxisome-related genes (PRGs) in LGG has not been reported. Therefore, it is necessary to construct a prognostic risk model for LGG patients based on the expression profiles of peroxisome-related genes. Our study mainly concentrated on developing a peroxisome-related gene signature for overall survival (OS) prediction in LGG patients. First, according to these peroxisome-related genes, all LGG patients from The Cancer Genome Atlas (TCGA) database could be divided into two subtypes. Univariate Cox regression analysis was used to find prognostic peroxisome-related genes in TCGA_LGG dataset, and least absolute shrinkage and selection operator Cox regression analysis was employed to establish a 14-gene signature. The risk score based on the signature was positively associated with unfavorable prognosis. Then, multivariate Cox regression incorporating additional clinical characteristics showed that the 14-gene signature was an independent predictor of LGG. Time-dependent ROC curves revealed good performance of this prognostic signature in LGG patients. The performance about predicting OS of LGG was validated using the GSE107850 dataset derived from the Gene Expression Omnibus (GEO) database. Furethermore, we constructed a nomogram model based on the gene signature and age, which showed a better prognostic power. Gene ontology (GO) and Kyoto Encylopedia of Genes and Genomes (KEGG) analyses showed that neuroactive ligand-receptor interaction and phagosome were enriched and that the immune status was decreased in the high-risk group. Finally, cell counting kit-8 (CCK8) were used to detect cell proliferation of U251 and A172 cells. Inhibition of ATAD1 (ATPase family AAA domain-containing 1) and ACBD5 (Acyl-CoA binding-domain-containing-5) expression led to significant inhibition of U251 and A172 cell proliferation. Flow cytometry detection showed that ATAD1 and ACBD5 could induce apoptosis of U251 and A172 cells. Therefore, through bioinformatics methods and cell experiments, our study developed a new peroxisome-related gene signature that migh t help improve personalized OS prediction in LGG patients.

Genetic variants in DDO and PEX5L in peroxisome-related pathways predict non-small cell lung cancer survival.

Peroxisomes play a role in lipid metabolism and regulation of reactive oxygen species, but its role in development and progression of non-small cell lung cancer (NSCLC) is not well understood. Here, we investigated the associations between 9708 single-nucleotide polymorphisms (SNPs) in 113 genes in the peroxisome-related pathways and survival of NSCLC patients from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) and the Harvard Lung Cancer Susceptibility (HLCS) study. In 1185 NSCLC patients from the PLCO trial, we found that 213 SNPs were significantly associated with NSCLC overall survival (OS) (p ≤ 0.05, Bayesian false discovery probability [BFDP] ≤ 0.80), of which eight SNPs were validated in the HLCS data set. In a multivariate Cox proportional hazards regression model, two independent SNPs (rs9384742 DDO and rs9825224 PEX5L) were significantly associated with NSCLC survival (hazards ratios [HR] of 1.17 with 95% CI [confidence interval] of 1.06-1.28 and 0.86 with 95% CI of 0.77-0.96, respectively). Patients with one or two protective genotypes had a significantly higher OS (HR: 0.787 [95% CI: 0.620-0.998] and 0.691 [95% CI: 0.543-0.879], respectively). Further expression quantitative trait loci analysis using whole blood and lung tissue showed that the minor allele of rs9384742 DDO was significantly associated with decreased messenger RNA (mRNA) expression levels and that DDO expression was also decreased in NSCLC tumor tissue. Additionally, high PEX5L expression levels were significantly associated with lower survival of NSCLC. Our data suggest that variants in these peroxisome-related genes may influence gene regulation and are potential predictors of NSCLC OS, once validated by additional studies.

Overexpression of Peroxisome-Localized GmABCA7 Promotes Seed Germination in Arabidopsis thaliana.

Peroxisome is one of the important organelles for intracellular lipid metabolism in plant cells and ß-oxidation of fatty acids in peroxisomes provides the energy for oil-containing seed germination. In this study, we identified an ATP-binding cassette (ABC) transporter gene, GmABCA7 from soybean, which is highly expressed in the different developmental stages of seeds. Transient expression of GmABCA7 in tobacco epidermal cells showed that GmABCA7 was specifically localized at the peroxisomes. Overexpression of GmABCA7 in Arabidopsis does not change seed phenotypes, or the overall levels of lipid, protein and sugar stored in the seeds; however, the transgenic seeds produced more gluconeogenic pathway precursors such as succinate and malate and germinated earlier compared to the wild type seeds. These results suggest that GmABCA7 may affect the ß-oxidation of fatty acids and play an important role in seed germination.

Identification of an at-risk subpopulation with high immune infiltration based on the peroxisome pathway and TIM3 in colorectal cancer.

BACKGROUND: Peroxisomes are pivotal metabolic organelles that exist in almost all eukaryote cells. A reduction in numbers and enzymatic activities of peroxisomes was found in colon adenocarcinomas. However, the role of peroxisomes or the peroxisome pathway in colorectal cancer (CRC) is not defined. METHODS: In the current study, a peroxisome score was calculated to indicate the activity of the peroxisome pathway using gene set variant analysis based on transcriptomic datasets. CIBERSORTx was chosen to infer enriched immune cells for tumors among subgroups. The SubMap algorithm was applied to predict its sensitivity to immunotherapy. RESULTS: The patients with a relatively low peroxisome score and high level of T-cell immunoglobulin and mucin domain 3 (TIM-3) presented the worse overall survival than others. Moreover, low peroxisome scores were associated with high infiltration of lymphocytes and poor prognosis in those CRC patients. Thus, a PERLowTIM3High CRC risk subpopulation was identified and characterized by high immune infiltration. The results also showed that CD8 T cells and macrophages highly infiltrated tumors of the PERLowTIM3High group, regardless of consortium molecular subtype and microsatellite instability status. This subgroup had the highest tumor mutational burden and overexpression of immune checkpoint genes. Further, the PERLowTIM3High group showed a higher probability of responding to programmed cell death protein-1-based immunotherapy. In addition, genes involved in peroxisomal metabolic processes in CRC were also investigated since peroxisome is a rather pleiotropic and highly metabolic organelle in cell. The results indicated that only those genes involved in fatty acid alpha oxidation could be used to stratify CRC patients as similar as peroxisome pathway genes. CONCLUSIONS: We revealed the favorable prognostic value of the peroxisome pathway in CRC and provided a new CRC stratification based on peroxisomes and TIM3, which might be helpful for CRC diagnostics and personalized treatment.

Peroxisome proliferator-activated receptor δ rescues xCT-deficient cells from ferroptosis by targeting peroxisomes.

Ferroptosis is a recently recognized process of cell death characterized by accumulation of iron-dependent lipid peroxides. Herein, we demonstrate that peroxisome proliferator-activated receptor δ (PPARδ) inhibits ferroptosis of mouse embryonic fibroblasts (MEFs) derived from cysteine/glutamate transporter (xCT)-knockout mice. Activation of PPARδ by the specific ligand GW501516 led to a dose-dependent decrease in ferroptotic cell death triggered by xCT deficiency, along with decreased levels of intracellular iron accumulation and lipid peroxidation. These effects of GW501516 were abolished by PPARδ-targeting small interfering RNA (siRNA) and the PPARδ inhibitor GSK0660, indicating that PPARδ inhibits xCT deficiency-induced ferroptosis. In addition, GW501516-activated PPARδ time- and dose-dependently upregulated catalase expression at both the mRNA and protein levels. This PPARδ-mediated upregulation of catalase was markedly attenuated in cells treated with PPARδ-targeting siRNA and GSK0660, indicating that expression of catalase is dependent on PPARδ. Consistently, the effects of GW501516 on ferroptosis of xCT-deficient MEFs were counteracted in the presence of 3-amino-1,2,4-triazole, a specific inhibitor of catalase, suggesting that catalase is essential for the effect of PPARδ on ferroptosis triggered by xCT deficiency. GW501516-activated PPARδ stabilized peroxisomes through catalase upregulation by targeting peroxisomal hydrogen peroxide-mediated lysosomal rupture, which led to ferroptosis of xCT-deficient MEFs. Collectively, these results demonstrate that PPARδ modulates ferroptotic signals in xCT-deficient MEFs by regulating catalase expression.

Nuclear receptors NHR-49 and NHR-79 promote peroxisome proliferation to compensate for aldehyde dehydrogenase deficiency in C. elegans.

The intracellular level of fatty aldehydes is tightly regulated by aldehyde dehydrogenases to minimize the formation of toxic lipid and protein adducts. Importantly, the dysregulation of aldehyde dehydrogenases has been implicated in neurologic disorder and cancer in humans. However, cellular responses to unresolved, elevated fatty aldehyde levels are poorly understood. Here, we report that ALH-4 is a C. elegans aldehyde dehydrogenase that specifically associates with the endoplasmic reticulum, mitochondria and peroxisomes. Based on lipidomic and imaging analysis, we show that the loss of ALH-4 increases fatty aldehyde levels and reduces fat storage. ALH-4 deficiency in the intestine, cell-nonautonomously induces NHR-49/NHR-79-dependent hypodermal peroxisome proliferation. This is accompanied by the upregulation of catalases and fatty acid catabolic enzymes, as indicated by RNA sequencing. Such a response is required to counteract ALH-4 deficiency since alh-4; nhr-49 double mutant animals are sterile. Our work reveals unexpected inter-tissue communication of fatty aldehyde levels and suggests pharmacological modulation of peroxisome proliferation as a therapeutic strategy to tackle pathology related to excess fatty aldehydes.

Peroxisomal ABC Transporters: An Update.

ATP-binding cassette (ABC) transporters constitute one of the largest superfamilies of conserved proteins from bacteria to mammals. In humans, three members of this family are expressed in the peroxisomal membrane and belong to the subfamily D: ABCD1 (ALDP), ABCD2 (ALDRP), and ABCD3 (PMP70). These half-transporters must dimerize to form a functional transporter, but they are thought to exist primarily as tetramers. They possess overlapping but specific substrate specificity, allowing the transport of various lipids into the peroxisomal matrix. The defects of ABCD1 and ABCD3 are responsible for two genetic disorders called X-linked adrenoleukodystrophy and congenital bile acid synthesis defect 5, respectively. In addition to their role in peroxisome metabolism, it has recently been proposed that peroxisomal ABC transporters participate in cell signaling and cell control, particularly in cancer. This review presents an overview of the knowledge on the structure, function, and mechanisms involving these proteins and their link to pathologies. We summarize the different in vitro and in vivo models existing across the species to study peroxisomal ABC transporters and the consequences of their defects. Finally, an overview of the known and possible interactome involving these proteins, which reveal putative and unexpected new functions, is shown and discussed.

A new functional role of mitochondria-anchored protein ligase in peroxisome morphology in mammalian cells.

Mitochondria and peroxisomes are metabolically interconnected and functionally active subcellular organelles. These two dynamic organelles, share a number of common biochemical functions such as ß-oxidation of fatty acids and detoxification of peroxides. The biogenesis and morphology of both these organelles in the mammalian cells is controlled by common transcription factors like PGC1α, and by a common fission machinery comprising of fission proteins like DRP1, Mff, and hFis1, respectively. In addition, the outer membrane mitochondria-anchored protein ligase (MAPL), the first mitochondrial SUMO E3 ligase with a RING-finger domain, also regulates mitochondrial morphology inducing mitochondrial fragmentation upon its overexpression. This fragmentation is dependent on both the RING domain of MAPL and the presence of the mitochondrial fission GTPase dynamin-related protein-1 (DRP1). Earlier studies have demonstrated that mitochondrial-derived vesicles are formed independently of the known mitochondrial fission GTPase, DRP1 are enriched for MAPL and are targeted to peroxisomes. The current study shows that MAPL regulates morphology of peroxisomes in a cell-type specific manner. Fascinatingly, the peroxisome elongation caused either due to silencing of DRP1 or by addition of polyunsaturated fatty acid, docosahexaenoic acid was blocked by overexpressing MAPL in mammalian cell lines. Furthermore, the transfection and colocalisation studies of MAPL with peroxisome membrane marker, PMP70, in different cell lines clearly revealed a cell-type specificity of transport of MAPL to peroxisomes. Previous work has placed the Vps35 (retromer component) as vital for delivery of MAPL to peroxisomes, placing the retromer as critical for the formation of MAPL-positive mitochondrial-derived vesicles. The results of polyethylene glycol-based cell-cell fusion assay signified that the enrichment of MAPL in peroxisomes is through vesicles and a retromer dependent phenomenon. Thus, a novel function for MAPL in peroxisomes is established to regulate peroxisome elongation and morphology under growth conditions and thus possibly modulate peroxisome fission.

PI5P4Ks drive metabolic homeostasis through peroxisome-mitochondria interplay.

PI5P4Ks are a class of phosphoinositide kinases that phosphorylate PI-5-P to PI-4,5-P2. Distinct localization of phosphoinositides is fundamental for a multitude of cellular functions. Here, we identify a role for peroxisomal PI-4,5-P2 generated by the PI5P4Ks in maintaining energy balance. We demonstrate that PI-4,5-P2 regulates peroxisomal fatty acid oxidation by mediating trafficking of lipid droplets to peroxisomes, which is essential for sustaining mitochondrial metabolism. Using fluorescent-tagged lipids and metabolite tracing, we show that loss of the PI5P4Ks significantly impairs lipid uptake and ß-oxidation in the mitochondria. Further, loss of PI5P4Ks results in dramatic alterations in mitochondrial structural and functional integrity, which under nutrient deprivation is further exacerbated, causing cell death. Notably, inhibition of the PI5P4Ks in cancer cells and mouse tumor models leads to decreased cell viability and tumor growth, respectively. Together, these studies reveal an unexplored role for PI5P4Ks in preserving metabolic homeostasis, which is necessary for tumorigenesis.

Acquired deficiency of peroxisomal dicarboxylic acid catabolism is a metabolic vulnerability in hepatoblastoma.

Metabolic reprogramming provides transformed cells with proliferative and/or survival advantages. Capitalizing on this therapeutically, however, has been only moderately successful because of the relatively small magnitude of these differences and because cancers may further adapt their metabolism to evade metabolic pathway inhibition. Mice lacking the peroxisomal bifunctional enzyme enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase (Ehhadh) and supplemented with the 12-carbon fatty acid lauric acid (C12) accumulate the toxic metabolite dodecanedioic acid (DDDA), which causes acute hepatocyte necrosis and liver failure. We noted that, in a murine model of pediatric hepatoblastoma (HB) and in primary human HBs, downregulation of Ehhadh occurs in association with the suppression of mitochondrial ß- and endosomal/peroxisomal ω-fatty acid oxidation pathways. This suggested that HBs might be more susceptible than normal liver tissue to C12 dietary intervention. Indeed, HB-bearing mice provided with C12- and/or DDDA-supplemented diets survived significantly longer than those on standard diets. In addition, larger tumors developed massive necrosis following short-term DDDA administration. In some HBs, the eventual development of DDDA resistance was associated with 129 transcript differences, ∼90% of which were downregulated, and approximately two-thirds of which correlated with survival in numerous human cancers. These transcripts often encoded extracellular matrix components, suggesting that DDDA resistance arises from reduced Ehhadh uptake. Lower Ehhadh expression was also noted in murine hepatocellular carcinomas and in subsets of certain human cancers, supporting the likely generality of these results. Our results demonstrate the feasibility of C12 or DDDA dietary supplementation that is nontoxic, inexpensive, and likely compatible with more standard chemotherapies.

Plasticity of ether lipids promotes ferroptosis susceptibility and evasion.

Ferroptosis-an iron-dependent, non-apoptotic cell death process-is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers1. The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions2-5. However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR-Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome-ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis.

Ultrastructural, Cytochemical, and Comparative Genomic Evidence of Peroxisomes in Three Genera of Pathogenic Free-Living Amoebae, Including the First Morphological Data for the Presence of This Organelle in Heteroloboseans.

Peroxisomes perform various metabolic processes that are primarily related to the elimination of reactive oxygen species and oxidative lipid metabolism. These organelles are present in all major eukaryotic lineages, nevertheless, information regarding the presence of peroxisomes in opportunistic parasitic protozoa is scarce and in many cases it is still unknown whether these organisms have peroxisomes at all. Here, we performed ultrastructural, cytochemical, and bioinformatic studies to investigate the presence of peroxisomes in three genera of free-living amoebae from two different taxonomic groups that are known to cause fatal infections in humans. By transmission electron microscopy, round structures with a granular content limited by a single membrane were observed in Acanthamoeba castellanii, Acanthamoeba griffini, Acanthamoeba polyphaga, Acanthamoeba royreba, Balamuthia mandrillaris (Amoebozoa), and Naegleria fowleri (Heterolobosea). Further confirmation for the presence of peroxisomes was obtained by treating trophozoites in situ with diaminobenzidine and hydrogen peroxide, which showed positive reaction products for the presence of catalase. We then performed comparative genomic analyses to identify predicted peroxin homologues in these organisms. Our results demonstrate that a complete set of peroxins-which are essential for peroxisome biogenesis, proliferation, and protein import-are present in all of these amoebae. Likewise, our in silico analyses allowed us to identify a complete set of peroxins in Naegleria lovaniensis and three novel peroxin homologues in Naegleria gruberi. Thus, our results indicate that peroxisomes are present in these three genera of free-living amoebae and that they have a similar peroxin complement despite belonging to different evolutionary lineages.

Neural-specific distribution of transmembrane protein TMEM240 and formation of TMEM240-Body.

Mutation in TMEM240 is suggested to cause SCA21, but the specific mechanism has not been clarified. The subcellular localization, specific biological function, and corresponding mechanism of action of TMEM240 have also not been delineated. In this study, the mRNA and protein expression of TMEM240 were assessed using qPCR and western blotting, respectively. Live cell imaging was used to establish the sub-cellular location of TMEM240, and electron microscopy was used to determine the morphology and distribution of TMEM240 in the cell. TMEM240 was specifically expressed in the neurons. Exogenous TMEM240 formed a multilayered cell structure, which we refer to as TMEM240-Body (T240-Body). T240-Body was separated and purified by centrifugation and filtration. An anchor protein His-tagged-GFP-BP on Ni-NTA agarose was used to pull down T240-GFP binding proteins. Both the N-terminal and the C-terminal of TMEM240 were confirmed to be inside the T240-Body. Co-localization experiments suggested that peroxisomes might contribute to T240-Body formation, and the two transmembrane regions of TMEM240 appear to be essential for formation of the T240-Body. Emerin protein contributed to formation of T240-Body when combined with TMEM240. Overall, this study provides new insights into TMEM240, which inform future research to further our understanding of its biological function.

The AAA+ ATPase Msp1 is a processive protein translocase with robust unfoldase activity.

Msp1 is a conserved eukaryotic AAA+ ATPase localized to the outer mitochondrial membrane, where it is thought to extract mislocalized tail-anchored proteins. Despite recent in vivo and in vitro studies supporting this function, a mechanistic understanding of how Msp1 extracts its substrates is still lacking. Msp1's ATPase activity depends on its hexameric state, and previous characterizations of the cytosolic AAA+ domain in vitro had proved challenging due to its monomeric nature in the absence of the transmembrane domain. Here, we used a hexamerization scaffold to study the substrate-processing mechanism of the soluble Msp1 motor, the functional homo-hexameric state of which was confirmed by negative-stain electron microscopy. We demonstrate that Msp1 is a robust bidirectional protein translocase that is able to unfold diverse substrates by processive threading through its central pore. This unfoldase activity is inhibited by Pex3, a membrane protein proposed to regulate Msp1 at the peroxisome.

miR-142 induces accumulation of reactive oxygen species (ROS) by inhibiting pexophagy in aged bone marrow mesenchymal stem cells.

Elevation of the levels of reactive oxygen species (ROS) is a major tissue-degenerative phenomenon involved in aging and aging-related diseases. The detailed mechanisms underlying aging-related ROS generation remain unclear. Presently, the expression of microRNA (miR)-142-5p was significantly upregulated in bone marrow mesenchymal stem cells (BMMSCs) of aged mice. Overexpression of miR-142 and subsequent observation revealed that miR-142 involved ROS accumulation through the disruption of selective autophagy for peroxisomes (pexophagy). Mechanistically, attenuation of acetyltransferase Ep300 triggered the upregulation of miR-142 in aged BMMSCs, and miR-142 targeted endothelial PAS domain protein 1 (Epas1) was identified as a regulatory protein of pexophagy. These findings support a novel molecular mechanism relating aging-associated ROS generation and organelle degradation in BMMSCs, and suggest a potential therapeutic target for aging-associated disorders that are accompanied by stem cell degeneration.

Impaired Peroxisomal Fitness in Obese Mice, a Vicious Cycle Exacerbating Adipocyte Dysfunction via Oxidative Stress.

Aims: Peroxisome is a critical organelle for fatty acid oxidation (FAO) and metabolism of reactive oxygen species (ROS). Increased oxidative stress in adipose tissue contributes to the development of insulin resistance and metabolic syndrome in obesity. This study aimed to investigate the role of peroxisomal fitness in maintaining adipocyte function, which has been under-rated in the obesity research area. Results: Reduced peroxisomal gene expressions in white adipose tissue (WAT) of obese mice suggested a close correlation between peroxisomes and obesity. Peroxisomal biogenesis factor 5 siRNA increased cellular ROS and inflammatory mediators in 3T3-L1 adipocytes. On the contrary, hydrogen peroxide or tumor necrosis factor-α treatment significantly decreased biogenesis- and function-related peroxisomal proteins, suggesting a positive feedback loop of ROS/inflammation and peroxisomal dysfunction. Correspondingly, catalase (a major peroxisomal antioxidant)-knockout mice fed with high-fat diet (HFD) exhibited suppressed peroxisomal proteins along with increased oxidative stress and accelerated obesity. In response to fenofibrate (a peroxisomal proliferator) treatment, WAT of HFD-fed wild-type mice showed not only increases in peroxisomal biogenesis and FAO but also attenuated features of adipocyte dysfunction and obesity. However, these results were not observed in peroxisome proliferator-activated receptor-alpha null obese mice. Innovation: Impaired peroxisomal fitness enhanced oxidative stress and inflammation in adipocytes, which exacerbates obesity. Conclusion: Adipose tissue peroxisomal homeostasis plays an important role in attenuating the features of obesity, and it can be a potential therapeutic target of obesity.

Peroxisomal gene and protein expression increase in response to a high-lipid challenge in human skeletal muscle.

Peroxisomes are essential for lipid metabolism and disruption of liver peroxisomal function results in neonatal death. Little is known about how peroxisomal content and activity respond to changes in the lipid environment in human skeletal muscle (HSkM). AIMS: We hypothesized and tested that increased peroxisomal gene/protein expression and functionality occur in HSkM as an adaptive response to lipid oversupply. MATERIALS AND METHODS: HSkM biopsies, derived from a total of sixty-two subjects, were collected for 1) examining correlations between peroxisomal proteins and intramyocellular lipid content (IMLC) as well as between peroxisomal functionality and IMLC, 2) assessing peroxisomal gene expression in response to acute- or 7-day high fat meal (HFM), and in human tissue derived primary myotubes for 3) treating with high fatty acids to induce peroxisomal adaptions. IMLC were measured by both biochemical analyses and fluorescent staining. Peroxisomal membrane protein PMP70 and biogenesis gene (PEX) expression were assessed using western blotting and realtime qRT-PCR respectively. 1-14C radiolabeled lignocerate and palmitate oxidation assays were performed for peroxisomal and mitochondrial functionality respectively. RESULTS: 1) Under fasting conditions, HSkM tissue demonstrated a significant correlation (P ≪ 0.05) between IMCL and the peroxisomal biogenesis factor 19 (PEX19) protein as well as between lipid content and palmitate and lignocerate complete oxidation. 2) Similarly, post-HFM, additional PEX genes (Pex19, PEX11A, and PEX5) were significantly (P ≪ 0.05) upregulated. 3) Increments in PMP70, carnitine octanoyl transferase (CrOT), PGC-1α, and ERRα mRNA were observed post-fatty acid incubation in HSkM cells. PMP70 protein was significantly (P ≪ 0.05) elevated 48-h post lipid treatment. CONCLUSIONS: These results are the first to associate IMLC with peroxisomal gene/protein expression and function in HSkM suggesting an adaptive role for peroxisomes in lipid metabolism in this tissue.

Hepatic symptoms and histology in 13 patients with a Zellweger spectrum disorder.

Patients with a Zellweger spectrum disorder (ZSD) have a defect in the assembly or maintenance of peroxisomes, leading to a multisystem disease with variable outcome. Liver disease is an important feature in patients with severe and milder phenotypes and a frequent cause of death. However, the course and histology of liver disease in ZSD patients are ill-defined. We reviewed the hepatic symptoms and histological findings of 13 patients with a ZSD in which one or several liver biopsies have been performed (patient age 0.2-39 years). All patients had at least some histological liver abnormalities, ranging from minor fibrosis to cirrhosis. Five patients demonstrated significant disease progression with liver failure and early death. In others, liver-related symptoms were absent, although some still silently developed cirrhosis. Patients with peroxisomal mosaicism had a better prognosis. In addition, we show that patients are at risk to develop a hepatocellular carcinoma (HCC), as one patient developed a HCC at the age of 36 years and one patient a precancerous lesion at the age of 18 years. Thus, regular examination to detect fibrosis or cirrhosis should be included in the standard care of ZSD patients. In case of advanced fibrosis/cirrhosis expert consultation and HCC screening should be initiated. This study further delineates the spectrum and significance of liver involvement in ZSDs.

A facile forward-genetic screen for Arabidopsis autophagy mutants reveals twenty-one loss-of-function mutations disrupting six ATG genes.

Macroautophagy is a process through which eukaryotic cells degrade large substrates including organelles, protein aggregates, and invading pathogens. Over 40 autophagy-related (ATG) genes have been identified through forward-genetic screens in yeast. Although homology-based analyses have identified conserved ATG genes in plants, only a few atg mutants have emerged from forward-genetic screens in Arabidopsis thaliana. We developed a screen that consistently recovers Arabidopsis atg mutations by exploiting mutants with defective LON2/At5g47040, a protease implicated in peroxisomal quality control. Arabidopsis lon2 mutants exhibit reduced responsiveness to the peroxisomally-metabolized auxin precursor indole-3-butyric acid (IBA), heightened degradation of several peroxisomal matrix proteins, and impaired processing of proteins harboring N-terminal peroxisomal targeting signals; these defects are ameliorated by preventing autophagy. We optimized a lon2 suppressor screen to expedite recovery of additional atg mutants. After screening mutagenized lon2-2 seedlings for restored IBA responsiveness, we evaluated stabilization and processing of peroxisomal proteins, levels of several ATG proteins, and levels of the selective autophagy receptor NBR1/At4g24690, which accumulates when autophagy is impaired. We recovered 21 alleles disrupting 6 ATG genes: ATG2/At3g19190, ATG3/At5g61500, ATG5/At5g17290, ATG7/At5g45900, ATG16/At5g50230, and ATG18a/At3g62770. Twenty alleles were novel, and 3 of the mutated genes lack T-DNA insertional alleles in publicly available repositories. We also demonstrate that an insertional atg11/At4g30790 allele incompletely suppresses lon2 defects. Finally, we show that NBR1 is not necessary for autophagy of lon2 peroxisomes and that NBR1 overexpression is not sufficient to trigger autophagy of seedling peroxisomes, indicating that Arabidopsis can use an NBR1-independent mechanism to target peroxisomes for autophagic degradation. Abbreviations: ATG: autophagy-related; ATI: ATG8-interacting protein; Col-0: Columbia-0; DSK2: dominant suppressor of KAR2; EMS: ethyl methanesulfonate; GFP: green fluorescent protein; IAA: indole-3-acetic acid; IBA: indole-3-butyric acid; ICL: isocitrate lyase; MLS: malate synthase; NBR1: Next to BRCA1 gene 1; PEX: peroxin; PMDH: peroxisomal malate dehydrogenase; PTS: peroxisomal targeting signal; thiolase: 3-ketoacyl-CoA thiolase; UBA: ubiquitin-associated; WT: wild type.