Mammalian circadian clock proteins form dynamic interacting microbodies distinct from phase separation.

Liquid-liquid phase separation (LLPS) underlies diverse biological processes. Because most LLPS studies were performed in vitro using recombinant proteins or in cells that overexpress protein, the physiological relevance of LLPS for endogenous protein is often unclear. PERIOD, the intrinsically disordered domain-rich proteins, are central mammalian circadian clock components and interact with other clock proteins in the core circadian negative feedback loop. Different core clock proteins were previously shown to form large complexes. Circadian clock studies often rely on experiments that overexpress clock proteins. Here, we show that when Per2 transgene was stably expressed in cells, PER2 protein formed nuclear phosphorylation-dependent slow-moving LLPS condensates that recruited other clock proteins. Super-resolution microscopy of endogenous PER2, however, revealed formation of circadian-controlled, rapidly diffusing nuclear microbodies that were resistant to protein concentration changes, hexanediol treatment, and loss of phosphorylation, indicating that they are distinct from the LLPS condensates caused by protein overexpression. Surprisingly, only a small fraction of endogenous PER2 microbodies transiently interact with endogenous BMAL1 and CRY1, a conclusion that was confirmed in cells and in mice tissues, suggesting an enzyme-like mechanism in the circadian negative feedback process. Together, these results demonstrate that the dynamic interactions of core clock proteins are a key feature of mammalian circadian clock mechanism and the importance of examining endogenous proteins in LLPS and circadian clock studies.

The biochemistry and physiology of long-chain dicarboxylic acid metabolism.

Mitochondrial ß-oxidation is the most prominent pathway for fatty acid oxidation but alternative oxidative metabolism exists. Fatty acid ω-oxidation is one of these pathways and forms dicarboxylic acids as products. These dicarboxylic acids are metabolized through peroxisomal ß-oxidation representing an alternative pathway, which could potentially limit the toxic effects of fatty acid accumulation. Although dicarboxylic acid metabolism is highly active in liver and kidney, its role in physiology has not been explored in depth. In this review, we summarize the biochemical mechanism of the formation and degradation of dicarboxylic acids through ω- and ß-oxidation, respectively. We will discuss the role of dicarboxylic acids in different (patho)physiological states with a particular focus on the role of the intermediates and products generated through peroxisomal ß-oxidation. This review is expected to increase the understanding of dicarboxylic acid metabolism and spark future research.

Peroxisomal ß-oxidation stimulates cholesterol biosynthesis in the liver in diabetic mice.

Although diabetes normally causes an elevation of cholesterol biosynthesis and induces hypercholesterolemia in animals and human, the mechanism linking diabetes to the dysregulation of cholesterol biosynthesis in the liver is not fully understood. As liver peroxisomal ß-oxidation is induced in the diabetic state and peroxisomal oxidation of fatty acids generates free acetate, we hypothesized that peroxisomal ß-oxidation might play a role in liver cholesterol biosynthesis in diabetes. Here, we used erucic acid, a specific substrate for peroxisomal ß-oxidation, and 10,12-tricosadiynoic acid, a specific inhibitor for peroxisomal ß-oxidation, to specifically induce and suppress peroxisomal ß-oxidation. Our results suggested that induction of peroxisomal ß-oxidation increased liver cholesterol biosynthesis in streptozotocin-induced diabetic mice. We found that excessive oxidation of fatty acids by peroxisomes generated considerable free acetate in the liver, which was used as a precursor for cholesterol biosynthesis. In addition, we show that specific inhibition of peroxisomal ß-oxidation decreased cholesterol biosynthesis by reducing acetate formation in the liver in diabetic mice, demonstrating a crosstalk between peroxisomal ß-oxidation and cholesterol biosynthesis. Based on these results, we propose that induction of peroxisomal ß-oxidation serves as a mechanism for a fatty acid-induced upregulation in cholesterol biosynthesis and also plays a role in diabetes-induced hypercholesterolemia.

Asymmetric horseshoe-like assembly of peroxisomal yeast oxalyl-CoA synthetase.

Oxalyl-CoA synthetase from Saccharomyces cerevisiae is one of the most abundant peroxisomal proteins in yeast and hence has become a model to study peroxisomal translocation. It contains a C-terminal Peroxisome Targeting Signal 1, which however is partly dispensable, suggesting additional receptor bindings sites. To unravel any additional features that may contribute to its capacity to be recognized as peroxisomal target, we determined its assembly and overall architecture by an integrated structural biology approach, including X-ray crystallography, single particle cryo-electron microscopy and small angle X-ray scattering. Surprisingly, it assembles into mixture of concentration-dependent dimers, tetramers and hexamers by dimer self-association. Hexameric particles form an unprecedented asymmetric horseshoe-like arrangement, which considerably differs from symmetric hexameric assembly found in many other protein structures. A single mutation within the self-association interface is sufficient to abolish any higher-level oligomerization, resulting in a homogenous dimeric assembly. The small C-terminal domain of yeast Oxalyl-CoA synthetase is connected by a partly flexible hinge with the large N-terminal domain, which provides the sole basis for oligomeric assembly. Our data provide a basis to mechanistically study peroxisomal translocation of this target.

Biogenesis and metabolic homeostasis of trypanosomatid glycosomes: New insights and new questions.

Kinetoplastea and Diplonemea possess peroxisome-related organelles that, uniquely, contain most of the enzymes of the glycolytic pathway and are hence called glycosomes. Enzymes of several other core metabolic pathways have also been located in glycosomes, in addition to some characteristic peroxisomal systems such as pathways of lipid metabolism. A considerable amount of research has been performed on glycosomes of trypanosomes since their discovery four decades ago. Not only the role of the glycosomal enzyme systems in the overall cell metabolism appeared to be unique, but also the organelles display remarkable features regarding their biogenesis and structural properties. These features are similar to those of the well-studied peroxisomes of mammalian and plant cells and yeasts yet exhibit also differences reflecting the large evolutionary distance between these protists and the representatives of other major eukaryotic lineages. Despite all research performed, many questions remain about various properties and the biological roles of glycosomes and peroxisomes. Here, we review the current knowledge about glycosomes, often comparing it with information about peroxisomes. Furthermore, we highlight particularly many questions that remain about the biogenesis, and the heterogeneity in structure and content of these enigmatic organelles, and the properties of their boundary membrane.

Suramin exposure alters cellular metabolism and mitochondrial energy production in African trypanosomes.

Introduced about a century ago, suramin remains a frontline drug for the management of early-stage East African trypanosomiasis (sleeping sickness). Cellular entry into the causative agent, the protozoan parasite Trypanosoma brucei, occurs through receptor-mediated endocytosis involving the parasite's invariant surface glycoprotein 75 (ISG75), followed by transport into the cytosol via a lysosomal transporter. The molecular basis of the trypanocidal activity of suramin remains unclear, but some evidence suggests broad, but specific, impacts on trypanosome metabolism (i.e. polypharmacology). Here we observed that suramin is rapidly accumulated in trypanosome cells proportionally to ISG75 abundance. Although we found little evidence that suramin disrupts glycolytic or glycosomal pathways, we noted increased mitochondrial ATP production, but a net decrease in cellular ATP levels. Metabolomics highlighted additional impacts on mitochondrial metabolism, including partial Krebs' cycle activation and significant accumulation of pyruvate, corroborated by increased expression of mitochondrial enzymes and transporters. Significantly, the vast majority of suramin-induced proteins were normally more abundant in the insect forms compared with the blood stage of the parasite, including several proteins associated with differentiation. We conclude that suramin has multiple and complex effects on trypanosomes, but unexpectedly partially activates mitochondrial ATP-generating activity. We propose that despite apparent compensatory mechanisms in drug-challenged cells, the suramin-induced collapse of cellular ATP ultimately leads to trypanosome cell death.

Glycosome heterogeneity in kinetoplastids.

Kinetoplastid parasites have essential organelles called glycosomes that are analogous to peroxisomes present in other eukaryotes. While many of the processes that regulate glycosomes are conserved, there are several unique aspects of their biology that are divergent from other systems and may be leveraged as therapeutic targets for the treatment of kinetoplastid diseases. Glycosomes are heterogeneous organelles that likely exist as sub-populations with different protein composition and function in a given cell, between individual cells, and between species. However, the limitations posed by the small size of these organelles makes the study of this heterogeneity difficult. Recent advances in the analysis of small vesicles by flow-cytometry provide an opportunity to overcome these limitations. In this review, we describe studies that document the diverse nature of glycosomes and propose an approach to using flow cytometry and organelle sorting to study the diverse composition and function of these organelles. Because the cellular machinery that regulates glycosome protein import and biogenesis is likely to contribute, at least in part, to glycosome heterogeneity we highlight some ways in which the glycosome protein import machinery differs from that of peroxisomes in other eukaryotes.

Effect of lysine acetylation on the regulation of Trypanosoma brucei glycosomal aldolase activity.

Post-translational modifications provide suitable mechanisms for cellular adaptation to environmental changes. Lysine acetylation is one of these modifications and occurs with the addition of an acetyl group to Nε-amino chain of this residue, eliminating its positive charge. Recently, we found distinct acetylation profiles of procyclic and bloodstream forms of Trypanosoma brucei, the agent of African Trypanosomiasis. Interestingly, glycolytic enzymes were more acetylated in the procyclic, which develops in insects and uses oxidative phosphorylation to obtain energy, compared with the bloodstream form, whose main source of energy is glycolysis. Here, we investigated whether acetylation regulates the T. brucei fructose 1,6-bisphosphate aldolase. We found that aldolase activity was reduced in procyclic parasites cultivated in the absence of glucose and partial recovered by in vitro deacetylation. Similarly, acetylation of protein extracts from procyclics cultivated in glucose-rich medium, caused a reduction in the aldolase activity. In addition, aldolase acetylation levels were higher in procyclics cultivated in the absence of glucose compared with those cultivated in the presence of glucose. To further confirm the role of acetylation, lysine residues near the catalytic site were substituted by glutamine in recombinant T. brucei aldolase. These replacements, especially K157, inhibited enzymatic activity, changed the electrostatic surface potential, decrease substrate binding and modify the catalytic pocket structure of the enzyme, as predicted by in silico analysis. Taken together, these data confirm the role of acetylation in regulating the activity of an enzyme from the glycolytic pathway of T. brucei, expanding the factors responsible for regulating important pathways in this parasite.

A novel signal sequence negative multimeric glycosomal protein required for cell cycle progression of Leishmania donovani parasites.

Expression of the intracellular form amastigote specific genes in the Leishmania donovani parasite plays a major role in parasite replication in the macrophage. In the current work, we have characterized a novel hypothetical gene, Ld30b that is specifically transcribed in the intracellular stage of the parasite. The recombinant Ld30b protein exists as a pentamer in solution as identified by native-PAGE and size exclusion gel chromatography. Structural analysis using circular dichroism and molecular modeling indicate that Ld30b belongs to family of cAMP-dependent protein kinase type I-alpha regulatory subunit. Co-localization immunofluorescence microscopy and western blot analyses (using anti-Ld30b antibody and anti-hypoxanthine-guanine phosphoribosyl transferase, a glycosome marker) on the isolated parasite glycosome organelle fractions show that Ld30b is localized in glycosome, though lacked a glycosome targeting PTS1/2 signal in the protein sequence. Episomal expression of Ld30b in the parasite caused the arrest of promastigotes and amastigotes growth in vitro. Cell cycle analysis using flow cytometry indicates that these parasites are arrested in 'sub G0/G1' phase of the cell cycle. Single allele knockout of Ld30b in the parasite similarly attenuated its growth by accumulation of cells in the S phase of cell cycle, thus confirming the probable importance of appropriate level of protein in the cells. Studying such intracellular stage expressing genes might unravel novel regulatory pathways for the development of drugs or vaccine candidates against leishmaniasis.

The dynamic life of the glycogen granule.

Glycogen, the primary storage form of glucose, is a rapid and accessible form of energy that can be supplied to tissues on demand. Each glycogen granule, or "glycosome," is considered an independent metabolic unit composed of a highly branched polysaccharide and various proteins involved in its metabolism. In this Minireview, we review the literature to follow the dynamic life of a glycogen granule in a multicompartmentalized system, i.e. the cell, and how and where glycogen granules appear and the factors governing its degradation. A better understanding of the importance of cellular compartmentalization as a regulator of glycogen metabolism is needed to unravel its role in brain energetics.

Inorganic polyphosphate interacts with nucleolar and glycosomal proteins in trypanosomatids.

Inorganic polyphosphate (polyP) is a polymer of three to hundreds of phosphate units bound by high-energy phosphoanhydride bonds and present from bacteria to humans. Most polyP in trypanosomatids is concentrated in acidocalcisomes, acidic calcium stores that possess a number of pumps, exchangers, and channels, and are important for their survival. In this work, using polyP as bait we identified > 25 putative protein targets in cell lysates of both Trypanosoma cruzi and Trypanosoma brucei. Gene ontology analysis of the binding partners found a significant over-representation of nucleolar and glycosomal proteins. Using the polyphosphate-binding domain (PPBD) of Escherichia coli exopolyphosphatase (PPX), we localized long-chain polyP to the nucleoli and glycosomes of trypanosomes. A competitive assay based on the pre-incubation of PPBD with exogenous polyP and subsequent immunofluorescence assay of procyclic forms (PCF) of T. brucei showed polyP concentration-dependent and chain length-dependent decrease in the fluorescence signal. Subcellular fractionation experiments confirmed the presence of polyP in glycosomes of T. brucei PCF. Targeting of yeast PPX to the glycosomes of PCF resulted in polyP hydrolysis, alteration in their glycolytic flux and increase in their susceptibility to oxidative stress.

Cytochemical Localization of Polyphenol Oxidase Activity in K2-Bodies of Saprolegnia ferax Secondary Zoospores.

Zoospores of the oomycete Saprolegnia ferax release adhesive material from K-bodies at the onset of attachment to substrates. To understand more fully how K-bodies function in adhesion, enzyme activity was investigated cytochemically in secondary zoospores. Presence of catalase, a marker enzyme for microbodies, was explored in the diaminobenzidine (DAB) reaction. Although pH 9.2 DAB-staining characteristic of catalase activity was detected in the granular matrix regions of K-bodies, reaction controls indicated that the reaction was due to oxidative enzyme activity other than catalase. Because polyphenol oxidase (PPO) is another metal-containing enzyme capable of oxidizing DAB, activity of this enzyme was tested with a more specific substrate, dihydroxyphenylalanine (DOPA). In the DOPA procedure, reaction product was exclusively localized within K-bodies, indicating the presence of PPO. Results with three methods of reaction controls (elimination of substrate, addition of a PPO enzyme inhibitor, and heat-inactivation of enzymes) all supported the presence of PPO in K-bodies. This study highlights potential roles for K-body PPO in stabilization of adhesion bodies by: cross-linking matrix phenolic proteins or glycoproteins as K-bodies discharge adhesives onto substrates, or polymerizing phenolics protective against microbial attacks of the adhesion pad.

The hydrophobic region of the Leishmania peroxin 14: requirements for association with a glycosome mimetic membrane.

Protein import into the Leishmania glycosome requires docking of the cargo-loaded peroxin 5 (PEX5) receptor to the peroxin 14 (PEX14) bound to the glycosome surface. To examine the LdPEX14-membrane interaction, we purified L. donovani promastigote glycosomes and determined the phospholipid and fatty acid composition. These membranes contained predominately phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol (PG) modified primarily with C18 and C22 unsaturated fatty acid. Using large unilamellar vesicles (LUVs) with a lipid composition mimicking the glycosomal membrane in combination with sucrose density centrifugation and fluorescence-activated cell sorting technique, we established that the LdPEX14 membrane-binding activity was dependent on a predicted transmembrane helix found within residues 149-179. Monolayer experiments showed that the incorporation of PG and phospholipids with unsaturated fatty acids, which increase membrane fluidity and favor a liquid expanded phase, facilitated the penetration of LdPEX14 into biological membranes. Moreover, we demonstrated that the binding of LdPEX5 receptor or LdPEX5-PTS1 receptor-cargo complex was contingent on the presence of LdPEX14 at the surface of LUVs.

Proteomic Profiling of Leishmania donovani Promastigote Subcellular Organelles.

To facilitate a greater understanding of the biological processes in the medically important Leishmania donovani parasite, a combination of differential and density-gradient ultracentrifugation techniques were used to achieve a comprehensive subcellular fractionation of the promastigote stage. An in-depth label-free proteomic LC-MS/MS analysis of the density gradients resulted in the identification of ∼50% of the Leishmania proteome (3883 proteins detected), which included ∼645 integral membrane proteins and 1737 uncharacterized proteins. Clustering and subcellular localization of proteins was based on a subset of training Leishmania proteins with known subcellular localizations that had been determined using biochemical, confocal microscopy, or immunoelectron microscopy approaches. This subcellular map will be a valuable resource that will help dissect the cell biology and metabolic processes associated with specific organelles of Leishmania and related kinetoplastids.

A novel phosphatase cascade regulates differentiation in Trypanosoma brucei via a glycosomal signaling pathway.

In the mammalian bloodstream, the sleeping sickness parasite Trypanosoma brucei is held poised for transmission by the activity of a tyrosine phosphatase, TbPTP1. This prevents differentiation of the transmissible "stumpy forms" until entry into the tsetse fly, whereupon TbPTP1 is inactivated and major changes in parasite physiology are initiated to allow colonization of the arthropod vector. Using a substrate-trapping approach, we identified the downstream step in this developmental signaling pathway as a DxDxT phosphatase, TbPIP39, which is activated upon tyrosine phosphorylation, and hence is negatively regulated by TbPTP1. In vitro, TbPIP39 promotes the activity of TbPTP1, thereby reinforcing its own repression, this being alleviated by the trypanosome differentiation triggers citrate and cis-aconitate, generating a potentially bistable regulatory switch. Supporting a role in signal transduction, TbPIP39 becomes rapidly tyrosine-phosphorylated during differentiation, and RNAi-mediated transcript ablation in stumpy forms inhibits parasite development. Interestingly, TbPIP39 localizes in glycosomes, peroxisome-like organelles that compartmentalize the trypanosome glycolytic reactions among other enzymatic activities. Our results invoke a phosphatase signaling cascade in which the developmental signal is trafficked to a unique metabolic organelle in the parasite: the glycosome. This is the first characterized environmental signaling pathway targeted directly to a peroxisome-like organelle in any eukaryotic cell.

Arginase Is Essential for Survival of Leishmania donovani Promastigotes but Not Intracellular Amastigotes.

Studies of Leishmania donovani have shown that both ornithine decarboxylase and spermidine synthase, two enzymes of the polyamine biosynthetic pathway, are critical for promastigote proliferation and required for maximum infection in mice. However, the importance of arginase (ARG), the first enzyme of the polyamine pathway in Leishmania, has not been analyzed in L. donovani To test ARG function in intact parasites, we generated Δarg null mutants in L. donovani and evaluated their ability to proliferate in vitro and trigger infections in mice. The Δarg knockout was incapable of growth in the absence of polyamine supplementation, but the auxotrophic phenotype could be bypassed by addition of either millimolar concentrations of ornithine or micromolar concentrations of putrescine or by complementation with either glycosomal or cytosolic versions of ARG. Spermidine supplementation of the medium did not circumvent the polyamine auxotrophy of the Δarg line. Although ARG was found to be essential for ornithine and polyamine synthesis, ornithine decarboxylase appeared to be the rate-limiting enzyme for polyamine production. Mouse infectivity studies revealed that the Δarg lesion reduced parasite burdens in livers by an order of magnitude but had little impact on the numbers of parasites recovered from spleens. Thus, ARG is essential for proliferation of promastigotes but not intracellular amastigotes. Coupled with previous studies, these data support a model in which L. donovani amastigotes readily salvage ornithine and have some access to host spermidine pools, while host putrescine appears to be unavailable for salvage by the parasite.

Biogenesis, maintenance and dynamics of glycosomes in trypanosomatid parasites.

Peroxisomes of organisms belonging to the protist group Kinetoplastea, which include trypanosomatid parasites of the genera Trypanosoma and Leishmania, are unique in playing a crucial role in glycolysis and other parts of intermediary metabolism. They sequester the majority of the glycolytic enzymes and hence are called glycosomes. Their glycosomal enzyme content can vary strongly, particularly quantitatively, between different trypanosomatid species, and within each species during its life cycle. Turnover of glycosomes by autophagy of redundant ones and biogenesis of a new population of organelles play a pivotal role in the efficient adaptation of the glycosomal metabolic repertoire to the sudden, major nutritional changes encountered during the transitions in their life cycle. The overall mechanism of glycosome biogenesis is similar to that of peroxisomes in other organisms, but the homologous peroxins involved display low sequence conservation as well as variations in motifs mediating crucial protein-protein interactions in the process. The correct compartmentalisation of enzymes is essential for the regulation of the trypanosomatids' metabolism and consequently for their viability. For Trypanosoma brucei it was shown that glycosomes also play a crucial role in its life-cycle regulation: a crucial developmental control switch involves the translocation of a protein phosphatase from the cytosol into the organelles. Many glycosomal proteins are differentially phosphorylated in different life-cycle stages, possibly indicative of regulation of enzyme activities as an additional means to adapt the metabolic network to the different environmental conditions encountered.

Tdrkh is essential for spermatogenesis and participates in primary piRNA biogenesis in the germline.

Piwi proteins and Piwi-interacting RNAs (piRNAs) repress transposition, regulate translation, and guide epigenetic programming in the germline. Here, we show that an evolutionarily conserved Tudor and KH domain-containing protein, Tdrkh (a.k.a. Tdrd2), is required for spermatogenesis and involved in piRNA biogenesis. Tdrkh partners with Miwi and Miwi2 via symmetrically dimethylated arginine residues in Miwi and Miwi2. Tdrkh is a mitochondrial protein often juxtaposed to pi-bodies and piP-bodies and is required for Tdrd1 cytoplasmic localization and Miwi2 nuclear localization. Tdrkh mutants display meiotic arrest at the zygotene stage, attenuate methylation of Line1 DNA, and upregulate Line1 RNA and protein, without inducing apoptosis. Furthermore, Tdrkh mutants have severely reduced levels of mature piRNAs but accumulate a distinct population of 1'U-containing, 2'O-methylated 31-37 nt RNAs that largely complement the missing mature piRNAs. Our results demonstrate that the primary piRNA biogenesis pathway involves 3'→5' processing of 31-37 nt intermediates and that Tdrkh promotes this final step of piRNA biogenesis but not the ping-pong cycle. These results shed light on mechanisms underlying primary piRNA biogenesis, an area in which information is conspicuously absent.

Localization of a Trypanosome Peroxin to the Endoplasmic Reticulum.

Trypanosoma brucei is the causative agent of diseases that affect 30,000-50,000 people annually. Trypanosoma brucei harbors unique organelles named glycosomes that are essential to parasite survival, which requires growth under fluctuating environmental conditions. The mechanisms that govern the biogenesis of these organelles are poorly understood. Glycosomes are evolutionarily related to peroxisomes, which can proliferate de novo from the endoplasmic reticulum or through the growth and division of existing organelles depending on the organism and environmental conditions. The effect of environment on glycosome biogenesis is unknown. Here, we demonstrate that the glycosome membrane protein, TbPex13.1, is localized to glycosomes when cells are cultured under high glucose conditions and to the endoplasmic reticulum in low glucose conditions. This localization in low glucose was dependent on the presence of a C-terminal tripeptide sequence. Our findings suggest that glycosome biogenesis is influenced by extracellular glucose levels and adds to the growing body of evidence that de novo glycosome biogenesis occurs in trypanosomes. Because the movement of peroxisomal membrane proteins is a hallmark of ER-dependent peroxisome biogenesis, TbPex13.1 may be a useful marker for the study such processes in trypanosomes.

Glycosomal bromodomain factor 1 from Trypanosoma cruzi enhances trypomastigote cell infection and intracellular amastigote growth.

Acetylation is a ubiquitous protein modification present in prokaryotic and eukaryotic cells that participates in the regulation of many cellular processes. The bromodomain is the only domain known to bind acetylated lysine residues. In the last few years, many bromodomain inhibitors have been developed in order to treat diseases caused by aberrant acetylation of lysine residues and have been tested as anti-parasitic drugs. In the present paper, we report the first characterization of Trypanosoma cruzi bromodomain factor 1 (TcBDF1). TcBDF1 is expressed in all life cycle stages, but it is developmentally regulated. It localizes in the glycosomes directed by a PTS2 (peroxisome-targeting signal 2) sequence. The overexpression of wild-type TcBDF1 is detrimental for epimastigotes, but it enhances the infectivity rate of trypomastigotes and the replication of amastigotes. On the other hand, the overexpression of a mutated version of TcBDF1 has no effect on epimastigotes, but it does negatively affect trypomastigotes' infection and amastigotes' replication.