Peroxisome population control by phosphoinositide signaling at the endoplasmic reticulum-plasma membrane interface.

Phosphoinositides are lipid signaling molecules acting at the interface of membranes and the cytosol to regulate membrane trafficking, lipid transport and responses to extracellular stimuli. Peroxisomes are multicopy organelles that are highly responsive to changes in metabolic and environmental conditions. In yeast, peroxisomes are tethered to the cell cortex at defined focal structures containing the peroxisome inheritance protein, Inp1p. We investigated the potential impact of changes in cortical phosphoinositide levels on the peroxisome compartment of the yeast cell. Here we show that the phosphoinositide, phosphatidylinositol-4-phosphate (PI4P), found at the junction of the cortical endoplasmic reticulum and plasma membrane (cER-PM) acts to regulate the cell's peroxisome population. In cells lacking a cER-PM tether or the enzymatic activity of the lipid phosphatase Sac1p, cortical PI4P is elevated, peroxisome numbers and motility are increased, and peroxisomes are no longer firmly tethered to Inp1p-containing foci. Reattachment of the cER to the PM through an artificial ER-PM "staple" in cells lacking the cER-PM tether does not restore peroxisome populations to the wild-type condition, demonstrating that integrity of PI4P signaling at the cell cortex is required for peroxisome homeostasis.

The peroxisome: an up-and-coming organelle in immunometabolism.

Peroxisomes are essential metabolic organelles, well known for their roles in the metabolism of complex lipids and reactive ionic species. In the past 10 years, peroxisomes have also been cast as central regulators of immunity. Lipid metabolites of peroxisomes, such as polyunsaturated fatty acids (PUFAs), are precursors for important immune mediators, including leukotrienes (LTs) and resolvins. Peroxisomal redox metabolism modulates cellular immune signaling such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. Additionally, peroxisomal ß-oxidation and ether lipid synthesis control the development and aspects of the activation of both innate and adaptive immune cells. Finally, peroxisome number and metabolic activity have been linked to inflammatory diseases. These discoveries have opened avenues of investigation aimed at targeting peroxisomes for therapeutic intervention in immune disorders, inflammation, and cancer.

Modulation of the cell membrane lipid milieu by peroxisomal ß-oxidation induces Rho1 signaling to trigger inflammatory responses.

Phagocytosis, signal transduction, and inflammatory responses require changes in lipid metabolism. Peroxisomes have key roles in fatty acid homeostasis and in regulating immune function. We find that Drosophila macrophages lacking peroxisomes have perturbed lipid profiles, which reduce host survival after infection. Using lipidomic, transcriptomic, and genetic screens, we determine that peroxisomes contribute to the cell membrane glycerophospholipid composition necessary to induce Rho1-dependent signals, which drive cytoskeletal remodeling during macrophage activation. Loss of peroxisome function increases membrane phosphatidic acid (PA) and recruits RhoGAPp190 during infection, inhibiting Rho1-mediated responses. Peroxisome-glycerophospholipid-Rho1 signaling also controls cytoskeleton remodeling in mouse immune cells. While high levels of PA in cells without peroxisomes inhibit inflammatory phenotypes, large numbers of peroxisomes and low amounts of cell membrane PA are features of immune cells from patients with inflammatory Kawasaki disease and juvenile idiopathic arthritis. Our findings reveal potential metabolic markers and therapeutic targets for immune diseases and metabolic disorders.

A Small Molecule Inhibitor of Pex3-Pex19 Interaction Disrupts Glycosome Biogenesis and Causes Lethality in Trypanosoma brucei.

Trypanosomatid parasites, including Trypanosoma and Leishmania, are infectious zoonotic agents for a number of severe diseases such as African sleeping sickness and American trypanosomiasis (Chagas disease) that affect millions of people, mostly in the emergent world. The glycosome is a specialized member of the peroxisome family of organelles found in trypanosomatids. These organelles compartmentalize essential enzymes of the glycolytic pathway, making them a prime target for drugs that can kill these organisms by interfering with either their biochemical functions or their formation. Glycosome biogenesis, like peroxisome biogenesis, is controlled by a group of proteins called peroxins (Pex). Pex3 is an early acting peroxin that docks Pex19, the receptor for peroxisomal membrane proteins, to initiate biogenesis of peroxisomes from the endoplasmic reticulum. Identification of Pex3 as the essential master regulator of glycosome biogenesis has implications in developing small molecule inhibitors that can impede Pex3-Pex19 interaction. Low amino acid sequence conservation between trypanosomatid Pex3 and human Pex3 (HsPex3) would aid in the identification of small molecule inhibitors that selectively interfere with the trypanosomatid Pex3-Pex19 interaction. We tested a library of pharmacologically active compounds in a modified yeast two-hybrid assay and identified a compound that preferentially inhibited the interaction of Trypanosoma brucei Pex3 and Pex19 versus HsPex3 and Pex19. Addition of this compound to either the insect or bloodstream form of T. brucei disrupted glycosome biogenesis, leading to mislocalization of glycosomal enzymes to the cytosol and lethality for the parasite. Our results show that preferential disruption of trypanosomal Pex3 function by small molecule inhibitors could help in the accelerated development of drugs for the treatment of trypanosomiases.

The Nitric Oxide Donor, S-Nitrosoglutathione, Rescues Peroxisome Number and Activity Defects in PEX1G843D Mild Zellweger Syndrome Fibroblasts.

Peroxisome biogenesis disorders (PBDs) are a group of metabolic developmental diseases caused by mutations in one or more genes encoding peroxisomal proteins. Zellweger syndrome spectrum (PBD-ZSS) results from metabolic dysfunction caused by damaged or non-functional peroxisomes and manifests as a multi-organ syndrome with significant morbidity and mortality for which there is no current drug therapy. Mild PBD-ZSS patients can exhibit a more progressive disease course and could benefit from the identification of drugs to improve the quality of life and extend the lifespan of affected individuals. Our study used a high-throughput screen of FDA-approved compounds to identify compounds that improve peroxisome function and biogenesis in human fibroblast cells carrying the mild PBD-ZSS variant, PEX1G843D. Our screen identified the nitrogen oxide donor, S-nitrosoglutathione (GSNO), as a potential therapeutic for this mild form of PBD-ZSS. Further biochemical characterization showed that GSNO enhances both peroxisome number and function in PEX1G843D mutant fibroblasts and leads to increased survival and longer lifespan in an in vivo humanized Drosophila model carrying the PEX1G843D mutation. GSNO is therefore a strong candidate to be translated to clinical trials as a potential therapeutic for mild PBD-ZSS.

Peroxisomes exhibit compromised structure and matrix protein content in SARS-CoV-2-infected cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that has triggered global health and economic crises. Here we report the effects of SARS-CoV-2 infection on peroxisomes of human cell lines Huh-7 and SK-N-SH. Peroxisomes undergo dramatic changes in morphology in SARS-CoV-2-infected cells. Rearrangement of peroxisomal membranes is followed by redistribution of peroxisomal matrix proteins to the cytosol, resulting in a dramatic decrease in the number of mature peroxisomes. The SARS-CoV-2 ORF14 protein was shown to interact physically with human PEX14, a peroxisomal membrane protein required for matrix protein import and peroxisome biogenesis. Given the important roles of peroxisomes in innate immunity, SARS-CoV-2 may directly target peroxisomes, resulting in loss of peroxisome structural integrity, matrix protein content and ability to function in antiviral signaling.

Peroxisome prognostications: Exploring the birth, life, and death of an organelle.

Peroxisomes play a central role in human health and have biochemical properties that promote their use in many biotechnology settings. With a primary role in lipid metabolism, peroxisomes share a niche with lipid droplets within the endomembrane-secretory system. Notably, factors in the ER required for the biogenesis of peroxisomes also impact the formation of lipid droplets. The dynamic interface between peroxisomes and lipid droplets, and also between these organelles and the ER and mitochondria, controls their metabolic flux and their dynamics. Here, we review our understanding of peroxisome biogenesis to propose and reframe models for understanding how peroxisomes are formed in cells. To more fully understand the roles of peroxisomes and to take advantage of their many properties that may prove useful in novel therapeutics or biotechnology applications, we recast mechanisms controlling peroxisome biogenesis in a framework that integrates inference from these models with experimental data.

The Canadian Rare Diseases Models and Mechanisms (RDMM) Network: Connecting Understudied Genes to Model Organisms.

Advances in genomics have transformed our ability to identify the genetic causes of rare diseases (RDs), yet we have a limited understanding of the mechanistic roles of most genes in health and disease. When a novel RD gene is first discovered, there is minimal insight into its biological function, the pathogenic mechanisms of disease-causing variants, and how therapy might be approached. To address this gap, the Canadian Rare Diseases Models and Mechanisms (RDMM) Network was established to connect clinicians discovering new disease genes with Canadian scientists able to study equivalent genes and pathways in model organisms (MOs). The Network is built around a registry of more than 500 Canadian MO scientists, representing expertise for over 7,500 human genes. RDMM uses a committee process to identify and evaluate clinician-MO scientist collaborations and approve 25,000 Canadian dollars in catalyst funding. To date, we have made 85 clinician-MO scientist connections and funded 105 projects. These collaborations help confirm variant pathogenicity and unravel the molecular mechanisms of RD, and also test novel therapies and lead to long-term collaborations. To expand the impact and reach of this model, we made the RDMM Registry open-source, portable, and customizable, and we freely share our committee structures and processes. We are currently working with emerging networks in Europe, Australia, and Japan to link international RDMM networks and registries and enable matches across borders. We will continue to create meaningful collaborations, generate knowledge, and advance RD research locally and globally for the benefit of patients and families living with RD.

The early-acting glycosome biogenic protein Pex3 is essential for trypanosome viability.

Trypanosomatid parasites are infectious agents for diseases such as African sleeping sickness, Chagas disease, and leishmaniasis that threaten millions of people, mostly in the emerging world. Trypanosomes compartmentalize glycolytic enzymes to an organelle called the glycosome, a specialized peroxisome. Functionally intact glycosomes are essential for trypanosomatid viability, making glycosomal proteins as potential drug targets against trypanosomatid diseases. Peroxins (Pex), of which Pex3 is the master regulator, control glycosome biogenesis. Although Pex3 has been found throughout the eukaryota, its identity has remained stubbornly elusive in trypanosomes. We used bioinformatics predictive of protein secondary structure to identify trypanosomal Pex3. Microscopic and biochemical analyses showed trypanosomal Pex3 to be glycosomal. Interaction of Pex3 with the peroxisomal membrane protein receptor Pex19 observed for other eukaryotes is replicated by trypanosomal Pex3 and Pex19. Depletion of Pex3 leads to mislocalization of glycosomal proteins to the cytosol, reduced glycosome numbers, and trypanosomatid death. Our findings are consistent with Pex3 being an essential gene in trypanosomes.

Pex20p functions as the receptor for non-PTS1/non-PTS2 acyl-CoA oxidase import into peroxisomes of the yeast Yarrowia lipolytica.

Most soluble proteins targeted to the peroxisomal matrix contain a C-terminal peroxisome targeting signal type 1 (PTS1) or an N-terminal PTS2 that is recognized by the receptors Pex5p and Pex7p, respectively. These receptors cycle between the cytosol and peroxisome and back again for multiple rounds of cargo delivery to the peroxisome. A small number of peroxisomal matrix proteins, including all six isozymes of peroxisomal fatty acyl-CoA oxidase (Aox) of the yeast Yarrowia lipolytica, contain neither a PTS1 nor a PTS2. Pex20p has been shown to function as a co-receptor for Pex7p in the import of PTS2 cargo into peroxisomes. Here we show that cells of Y. lipolytica deleted for the PEX20 gene fail to import not only the PTS2-containing protein 3-ketoacyl-CoA thiolase (Pot1p) but also the non-PTS1/non-PTS2 Aox isozymes. Pex20p binds directly to Aox isozymes Aox3p and Aox5p, which requires the C-terminal Wxxx(F/Y) motif of Pex20p. A W411G mutation in the C-terminal Wxxx(F/Y) motif causes Aox isozymes to be mislocalized to the cytosol. Pex20p interacts physically with members of the peroxisomal import docking complex, Pex13p and Pex14p. Our results are consistent with a role for Pex20p as the receptor for import of the non-PTS1/non-PTS2 Aox isozymes into peroxisomes.

Author Correction: Genome-wide SWAp-Tag yeast libraries for proteome exploration.

The version of Supplementary Table 1 originally published online with this article contained incorrect localization annotations for one plate. This error has been corrected in the online Supplementary Information.

Determinants of the assembly, integrity and maintenance of the endoplasmic reticulum-peroxisome tether.

Organelle tethering and intercommunication are crucial for proper cell function. We previously described a tether between peroxisomes and the endoplasmic reticulum (ER) that acts in peroxisome population control in the yeast, Saccharomyces cerevisiae. Components of this tether are Pex3p, an integral membrane protein of both peroxisomes and the ER and Inp1p, a connector that links peroxisomes to the ER. Here, we report the analysis of random Inp1p mutants that enabled identification of regions in Inp1p required for the assembly and maintenance of the ER-peroxisome tether. Interaction analysis between Inp1p mutants and known Inp1p-binding proteins demonstrated that Pex3p and Inp1p do not constitute the sole components of the ER-peroxisome tether. Deletion of these Inp1p interactors whose steady-state localization is outside of ER-peroxisome tethers affected peroxisome dynamics. Our findings are consistent with the presence of regulatory cues that act on ER-peroxisome tethers and point to the existence of membrane contact sites between peroxisomes and organelles other than the ER.

Distinct Roles for Peroxisomal Targeting Signal Receptors Pex5 and Pex7 in Drosophila.

Peroxisomes are ubiquitous membrane-enclosed organelles involved in lipid processing and reactive oxygen detoxification. Mutations in human peroxisome biogenesis genes (Peroxin, PEX, or Pex) cause developmental disabilities and often early death. Pex5 and Pex7 are receptors that recognize different peroxisomal targeting signals called PTS1 and PTS2, respectively, and traffic proteins to the peroxisomal matrix. We characterized mutants of Drosophila melanogaster Pex5 and Pex7 and found that adult animals are affected in lipid processing. Pex5 mutants exhibited severe developmental defects in the embryonic nervous system and muscle, similar to what is observed in humans with PEX5 mutations, while Pex7 fly mutants were weakly affected in brain development, suggesting different roles for fly Pex7 and human PEX7. Of note, although no PTS2-containing protein has been identified in Drosophila, Pex7 from Drosophila can function as a bona fide PTS2 receptor because it can rescue targeting of the PTS2-containing protein thiolase to peroxisomes in PEX7 mutant human fibroblasts.

Dysfunctional peroxisomes compromise gut structure and host defense by increased cell death and Tor-dependent autophagy.

The gut has a central role in digestion and nutrient absorption, but it also serves in defending against pathogens, engages in mutually beneficial interactions with commensals, and is a major source of endocrine signals. Gut homeostasis is necessary for organismal health and changes to the gut are associated with conditions like obesity and diabetes and inflammatory illnesses like Crohn's disease. We report that peroxisomes, organelles involved in lipid metabolism and redox balance, are required to maintain gut epithelium homeostasis and renewal in Drosophila and for survival and development of the organism. Dysfunctional peroxisomes in gut epithelial cells activate Tor kinase-dependent autophagy that increases cell death and epithelial instability, which ultimately alter the composition of the intestinal microbiota, compromise immune pathways in the gut in response to infection, and affect organismal survival. Peroxisomes in the gut effectively function as hubs that coordinate responses from stress, metabolic, and immune signaling pathways to maintain enteric health and the functionality of the gut-microbe interface.

Reconstitution of human peroxisomal ß-oxidation in yeast.

We report the permanent introduction of the human peroxisomal ß-oxidation enzymatic machinery required for straight chain degradation of fatty acids into the yeast, Saccharomyces cerevisiae. Peroxisomal ß-oxidation encompasses four sequential reactions that are confined to three enzymes. The genes encoding human acyl-CoA oxidase 1, peroxisomal multifunctional enzyme type 2 and 3-ketoacyl-CoA thiolase were introduced into the genomic loci of their yeast gene equivalents. The human ß-oxidation genes were individually tagged with sequence coding for GFP and expression of the protein chimeras as well as their targeting to peroxisomes was confirmed. Functional complementation of the ß-oxidation pathway was assessed by growth on media containing fatty acids of different chain lengths. Yeast cells exhibited distinctive substrate specificities depending on whether they expressed the human or their endogenous ß-oxidation machinery. The genetic engineering of yeast to contain a 'humanized' organelle is a first step for the in vivo study of human peroxisome disorders in a model organism.

Genome-wide SWAp-Tag yeast libraries for proteome exploration.

Yeast libraries revolutionized the systematic study of cell biology. To extensively increase the number of such libraries, we used our previously devised SWAp-Tag (SWAT) approach to construct a genome-wide library of ~5,500 strains carrying the SWAT NOP1promoter-GFP module at the N terminus of proteins. In addition, we created six diverse libraries that restored the native regulation, created an overexpression library with a Cherry tag, or enabled protein complementation assays from two fragments of an enzyme or fluorophore. We developed methods utilizing these SWAT collections to systematically characterize the yeast proteome for protein abundance, localization, topology, and interactions.

ESCRT-III is required for scissioning new peroxisomes from the endoplasmic reticulum.

Dynamic control of peroxisome proliferation is integral to the peroxisome's many functions. The endoplasmic reticulum (ER) serves as a source of preperoxisomal vesicles (PPVs) that mature into peroxisomes during de novo peroxisome biogenesis and support growth and division of existing peroxisomes. However, the mechanism of PPV formation and release from the ER remains poorly understood. In this study, we show that endosomal sorting complexes required for transport (ESCRT)-III are required to release PPVs budding from the ER into the cytosol. Absence of ESCRT-III proteins impedes de novo peroxisome formation and results in an aberrant peroxisome population in vivo. Using a cell-free PPV budding assay, we show that ESCRT-III proteins Vps20 and Snf7 are necessary to release PPVs from the ER. ESCRT-III is therefore a positive effector of membrane scission for vesicles budding both away from and toward the cytosol. These findings have important implications for the evolutionary timing of emergence of peroxisomes and the rest of the internal membrane architecture of the eukaryotic cell.

Involvement of SNARE protein Ykt6 in glycosome biogenesis in Trypanosoma brucei.

The kinetoplastid parasites Trypanosoma and Leishmania are etiologic agents of diseases like African sleeping sickness, Chagas and leishmaniasis that inflict many tropical and subtropical parts of the world. These parasites are distinctive in that they compartmentalize most of the usually cytosolic enzymes of the glycolytic pathway within a peroxisome-like organelle called the glycosome. Functional glycosomes are essential in both the procyclic and bloodstream forms of trypanosomatid parasites, and mislocalization of glycosomal enzymes to the cytosol is fatal for the parasite. The life cycle of these parasites is intimately linked to their efficient protein and vesicular trafficking machinery that helps them in immune evasion, host-pathogen interaction and organelle biogenesis and integrity. Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins play important roles in vesicular trafficking and mediate a wide range of protein-protein interactions in eukaryotes. We show here that the SNARE protein Ykt6 is necessary for glycosome biogenesis and function in Trypanosoma brucei. RNAi-mediated depletion of Ykt6 in both the procyclic and bloodstream forms of T. brucei leads to mislocalization of glycosomal matrix proteins to the cytosol, pronounced reduction in glycosome number, and cell death. GFP-tagged Ykt6 appears as punctate structures in the T. brucei cell and colocalizes in part to glycosomes. Our results constitute the first demonstration of a role for SNARE proteins in the biogenesis of peroxisomal organelles.

Extreme genome diversity in the hyper-prevalent parasitic eukaryote Blastocystis.

Blastocystis is the most prevalent eukaryotic microbe colonizing the human gut, infecting approximately 1 billion individuals worldwide. Although Blastocystis has been linked to intestinal disorders, its pathogenicity remains controversial because most carriers are asymptomatic. Here, the genome sequence of Blastocystis subtype (ST) 1 is presented and compared to previously published sequences for ST4 and ST7. Despite a conserved core of genes, there is unexpected diversity between these STs in terms of their genome sizes, guanine-cytosine (GC) content, intron numbers, and gene content. ST1 has 6,544 protein-coding genes, which is several hundred more than reported for ST4 and ST7. The percentage of proteins unique to each ST ranges from 6.2% to 20.5%, greatly exceeding the differences observed within parasite genera. Orthologous proteins also display extreme divergence in amino acid sequence identity between STs (i.e., 59%-61% median identity), on par with observations of the most distantly related species pairs of parasite genera. The STs also display substantial variation in gene family distributions and sizes, especially for protein kinase and protease gene families, which could reflect differences in virulence. It remains to be seen to what extent these inter-ST differences persist at the intra-ST level. A full 26% of genes in ST1 have stop codons that are created on the mRNA level by a novel polyadenylation mechanism found only in Blastocystis. Reconstructions of pathways and organellar systems revealed that ST1 has a relatively complete membrane-trafficking system and a near-complete meiotic toolkit, possibly indicating a sexual cycle. Unlike some intestinal protistan parasites, Blastocystis ST1 has near-complete de novo pyrimidine, purine, and thiamine biosynthesis pathways and is unique amongst studied stramenopiles in being able to metabolize α-glucans rather than ß-glucans. It lacks all genes encoding heme-containing cytochrome P450 proteins. Predictions of the mitochondrion-related organelle (MRO) proteome reveal an expanded repertoire of functions, including lipid, cofactor, and vitamin biosynthesis, as well as proteins that may be involved in regulating mitochondrial morphology and MRO/endoplasmic reticulum (ER) interactions. In sharp contrast, genes for peroxisome-associated functions are absent, suggesting Blastocystis STs lack this organelle. Overall, this study provides an important window into the biology of Blastocystis, showcasing significant differences between STs that can guide future experimental investigations into differences in their virulence and clarifying the roles of these organisms in gut health and disease.