Host cell lipids control cholesteryl ester synthesis and storage in intracellular Toxoplasma

The intracellular protozoan Toxoplasma gondii lacks a de novo mechanism for cholesterol synthesis and therefore must scavenge this essential lipid from the host environment. In this study, we demonstrated that T. gondii diverts cholesterol from low-density lipoproteins for cholesteryl ester synthesis and storage in lipid bodies. We identified and characterized two isoforms of acyl-CoA:cholesterol acyltransferase (ACAT)-related enzymes, designated TgACAT1alpha and TgACAT1beta in T. gondii. Both proteins are coexpressed in the parasite, localized to the endoplasmic reticulum and participate in cholesteryl ester synthesis. In contrast to mammalian ACAT, TgACAT1alpha and TgACAT1beta preferentially incorporate palmitate into cholesteryl esters and present a broad sterol substrate affinity. Mammalian ACAT-deficient cells transfected with either TgACAT1alpha or TgACAT1beta are restored in their capability of cholesterol esterification. TgACAT1alpha produces steryl esters and forms lipid bodies after transformation in a Saccharomyces cerevisiae mutant strain lacking neutral lipids. In addition to their role as ACAT substrates, host fatty acids and low-density lipoproteins directly serve as Toxoplasma ACAT activators by stimulating cholesteryl ester synthesis and lipid droplet biogenesis. Free fatty acids significantly increase TgACAT1alpha mRNA levels. Selected cholesterol esterification inhibitors impair parasite growth by rapid disruption of plasma membrane. Altogether, these studies indicate that host lipids govern neutral lipid synthesis in Toxoplasma and that interference with mechanisms of host lipid storage is detrimental to parasite survival in mammalian cells.     

Insights into unique physiological features of neutral lipids in Apicomplexa: from storage to potential mediation in parasite metabolic activities

The fast intracellular multiplication of apicomplexan parasites including Toxoplasma and Plasmodium, requires large amounts of lipids necessary for the membrane biogenesis of new progenies. Hence, the study of lipids is fundamental in order to understand the biology and pathogenesis of these deadly organisms. Much has been reported on the importance of polar lipids, e.g. phospholipids in Plasmodium. Comparatively, little attention has been paid to the metabolism of neutral lipids, including sterols, steryl esters and acylglycerols. In eukaryotic cells, free sterols are membrane components whereas steryl esters and acylglycerols are stored in cytosolic lipid inclusions. The first part of this review describes the recent advances in neutral lipid synthesis and storage in Toxoplasma and Plasmodium. New potential pharmacological targets in the pathways producing neutral lipids are outlined. In addition to lipid bodies, Apicomplexa contain unique secretory organelles involved in parasite invasion named rhoptries. These compartments appear to sequester most of the cholesterol found in the exocytic pathway. The second part of the review focuses on rhoptry cholesterol and its potential roles in the biogenesis, structural organisation and function of these unique organelles among eukaryotes.     

Characterization of a second sterol‐esterifying enzyme in Toxoplasma highlights the importance of cholesterol storage pathways for the parasite

Lipid bodies are eukaryotic structures for temporary storage of neutral lipids such as acylglycerols and steryl esters. Fatty acyl‐CoA and cholesterol are two substrates for cholesteryl ester (CE) synthesis via the ACAT reaction. The intracellular parasite Toxoplasma gondii is incapable of sterol synthesis and unremittingly scavenges cholesterol from mammalian host cells. We previously demonstrated that the parasite expresses a cholesteryl ester‐synthesizing enzyme, TgACAT1. In this article, we identified and characterized a second ACAT‐like enzyme, TgACAT2, which shares 56% identity with TgACAT1. Both enzymes are endoplasmic reticulum‐associated and contribute to CE formation for storage in lipid bodies. While TgACAT1 preferentially utilizes palmitoyl‐CoA, TgACAT2 has broader fatty acid specificity and produces more CE. Genetic ablation of each individual ACAT results in parasite growth impairment whereas dual ablation of ACAT1 and ACAT2 is not tolerated by Toxoplasma. ΔACAT1 and ΔACAT2 parasites have reduced CE levels, fewer lipid bodies, and accumulate free cholesterol, which causes injurious membrane effects. Mutant parasites are particularly vulnerable to ACAT inhibitors. This study underlines the important physiological role of ACAT enzymes to store cholesterol in a sterol‐auxotrophic organism such as Toxoplasma, and furthermore opens up possibilities of exploiting TgACAT as targets for the development of antitoxoplasmosis drugs.     

Lipidomic analysis of Toxoplasma gondii tachyzoites rhoptries: further insights into the role of cholesterol

Rhoptries are secretory organelles involved in the virulence of the human pathogen Toxoplasma gondii. In the present study we have used HPLC and capillary GLC to isolate and quantify lipids from whole Toxoplasma cells and their purified rhoptries. This comparative lipidomic analysis revealed an enrichment of cholesterol, sphingomyelin and, most of all, saturated fatty acids in the rhoptries. These lipids are known, when present in membranes, to contribute to their rigidity and, interestingly, fluorescence anisotropy measurements confirmed that rhoptry-derived membranes have a lower fluidity than membranes from whole T. gondii cells. Moreover, although rhoptries were initially thought to be highly enriched in cholesterol, we demonstrated that cholesterol is present in lower proportions, and we have provided additional evidence towards a lack of involvement of rhoptry cholesterol in the process of host-cell invasion by the parasite. Indeed, depleting the cholesterol content of the parasites did not prevent the secretion of protein-containing rhoptry-derived vesicles and the parasites could still establish a structure called the moving junction, which is necessary for invasion. Instead, the crucial role of host cholesterol for invasion, which has already been demonstrated [Coppens and Joiner (2003) Mol. Biol. Cell 14, 3804-3820], might be explained by the need of a cholesterol-rich region of the host cell we could visualize at the point of contact with the attached parasite, in conditions where parasite motility was blocked.     

Toxoplasma gondii sequesters lysosomes from mammalian hosts in the vacuolar space

The intracellular compartment harboring Toxoplasma gondii satisfies the parasite's nutritional needs for rapid growth in mammalian cells. We demonstrate that the parasitophorous vacuole (PV) of T. gondii accumulates material coming from the host mammalian cell via the exploitation of the host endo-lysosomal system. The parasite actively recruits host microtubules, resulting in selective attraction of endo-lysosomes to the PV. Microtubule-based invaginations of the PV membrane serve as conduits for the delivery of host endo-lysosomes within the PV. These tubular conduits are decorated by a parasite coat, including the tubulogenic protein GRA7, which acts like a garrote that sequesters host endocytic organelles in the vacuolar space. These data define an unanticipated process allowing the parasite intimate and concentrated access to a diverse range of low molecular weight components produced by the endo-lysosomal system. More generally, they identify a unique mechanism for unidirectional transport and sequestration of host organelles.     

Regulation of ATG8 membrane association by ATG4 in the parasitic protist Toxoplasma gondii

In the process of autophagy, the Atg8 protein is conjugated, through a ubiquitin-like system, to the lipid phosphatidylethanolamine (PE) to associate with the membrane of forming autophagosomes. There, it plays a crucial role in the genesis of these organelles and in autophagy in general. In most eukaryotes, the cysteine peptidase Atg4 processes the C terminus of cytosolic Atg8 to regulate its association with autophagosomal membranes and also delipidates Atg8 to release this protein from membranes. The parasitic protist Toxoplasma gondii contains a functional, yet apparently reduced, autophagic machinery. T. gondii Atg8 homolog, in addition to a cytosolic and occasionally autophagosomal localization, also localizes to the apicoplast, a nonphotosynthetic plastid bounded by four membranes. Our attempts to interfere with TgATG8 function showed that it appears to be essential for parasite multiplication inside its host cell. This protein also displays a peculiar C terminus that does not seem to necessitate processing prior to membrane association and yet an unusually large Toxoplasma homolog of ATG4 is predicted in the parasite genome. A TgATG4 conditional expression mutant that we have generated is severely affected in growth, and displays significant alterations at the organellar level, noticeably with a fragmentation of the mitochondrial network and a loss of the apicoplast. TgATG4-depleted parasites appear to be defective in the recycling of membrane-bound TgATG8. Overall, our data highlight a role for the TgATG8 conjugation pathway in maintaining the homeostasis of the parasite’s organelles and suggest that Toxoplasma has evolved a specialized autophagic machinery with original regulation.     

Role of an Ancestral D-Bifunctional Protein Containing Two Sterol-Carrier Protein-2 Domains in Lipid Uptake and Trafficking in Toxoplasma

The inability to synthesize cholesterol is universal among protozoa. The intracellular pathogen Toxoplasma depends on host lipoprotein-derived cholesterol to replicate in mammalian cells. Mechanisms of cholesterol trafficking in this parasite must be important for delivery to proper organelles. We characterized a unique d-bifunctional protein variant expressed by Toxoplasma consisting of one N-terminal d-3-hydroxyacyl-CoA dehydrogenase domain fused to two tandem sterol carrier protein-2 (SCP-2) domains. This multidomain protein undergoes multiple cleavage steps to release free SCP-2. The most C-terminal SCP-2 carries a PTS1 that directs the protein to vesicles before processing. Abrogation of this signal results in SCP-2 accumulation in the cytoplasm. Cholesterol specifically binds to parasite SCP-2 but with 10-fold lower affinity than phosphatidylcholine. In mammalian cells and Toxoplasma, the two parasite SCP-2 domains promote the circulation of various lipids between organelles and to the surface. Compared with wild-type parasites, TgHAD-2SCP-2–transfected parasites replicate faster and show enhanced uptake of cholesterol and oleate, which are incorporated into neutral lipids that accumulate at the basal end of Toxoplasma. This work provides the first evidence that the lipid transfer capability of an ancestral eukaryotic SCP-2 domain can influence the lipid metabolism of an intracellular pathogen to promote its multiplication in mammalian cells.     

A yeast model system for functional analysis of the Niemann-Pick type C protein 1 homolog, Ncr1p

Niemann-Pick disease type C (NP-C) is a progressive, ultimately fatal, autosomal recessive neurodegenerative disorder. The major biochemical hallmark of the disease is the endocytic accumulation of low-density lipoprotein-derived cholesterol. The majority of NP-C patients have mutations in the Niemann-Pick type C1 gene, NPC1. This study focuses on the Saccharomyces cerevisiae homolog of the human NPC1 protein encoded by the NCR1 gene. Ncr1p localizes to the vacuole, the yeast equivalent to the mammalian endosome-lysosome system. Here, we identify the first phenotype caused by deletion of NCR1 from the yeast genome, resistance to the ether lipid drug, edelfosine. Our results indicate that edelfosine has a cytotoxic, rather than cytostatic, effect on wildtype yeast cells. We exploit the edelfosine resistance phenotype to assess the function of yeast Ncr1 proteins carrying amino acid changes corresponding to human NPC1 patient mutations. We find that one of these amino acid changes severely compromises Ncr1p function as assessed using the edelfosine resistance assay. These findings establish S. cerevisiae as a model system that can be exploited to analyze the molecular consequences of patient mutations in NPC1 and provide the basis for future genetic studies using yeast.     

Conditional expression of Toxoplasma gondii apical membrane antigen-1 (TgAMA1) demonstrates that TgAMA1 plays a critical role in host cell invasion

Toxoplasma gondii is an obligate intracellular parasite and an important human pathogen. Relatively little is known about the proteins that orchestrate host cell invasion by T. gondii or related apicomplexan parasites (including Plasmodium spp., which cause malaria), due to the difficulty of studying essential genes in these organisms. We have used a recently developed regulatable promoter to create a conditional knockout of T. gondii apical membrane antigen-1 (TgAMA1). TgAMA1 is a transmembrane protein that localizes to the parasite's micronemes, secretory organelles that discharge during invasion. AMA1 proteins are conserved among apicomplexan parasites and are of intense interest as malaria vaccine candidates. We show here that T. gondii tachyzoites depleted of TgAMA1 are severely compromised in their ability to invade host cells, providing direct genetic evidence that AMA1 functions during invasion. The TgAMA1 deficiency has no effect on microneme secretion or initial attachment of the parasite to the host cell, but it does inhibit secretion of the rhoptries, organelles whose discharge is coupled to active host cell penetration. The data suggest a model in which attachment of the parasite to the host cell occurs in two distinct stages, the second of which requires TgAMA1 and is involved in regulating rhoptry secretion.     

Rapid invasion of host cells by Toxoplasma requires secretion of the MIC2-M2AP adhesive protein complex

Vertebrate cells are highly susceptible to infection by obligate intracellular parasites such as Toxoplasma gondii, yet the mechanism by which these microbes breach the confines of their target cell is poorly understood. While it is thought that Toxoplasma actively invades by secreting adhesive proteins from internal organelles called micronemes, no genetic evidence is available to support this contention. Here, we report successful disruption of M2AP, a microneme protein tightly associated with an adhesive protein called MIC2. M2AP knockout parasites were >80% impaired in host cell entry. This invasion defect was likely due to defective expression of MIC2, which partially accumulated in the parasite endoplasmic reticulum and Golgi. M2AP knockout parasites were also unable to rapidly secrete MIC2, an event that normally accompanies parasite attachment to a target cell. These findings indicate a critical role for the MIC2-M2AP protein complex in parasite invasion.     

Mutagenesis of the putative sterol-sensing domain of yeast Niemann Pick C-related protein reveals a primordial role in subcellular sphingolipid distribution

Lipid movement between organelles is a critical component of eukaryotic membrane homeostasis. Niemann Pick type C (NP-C) disease is a fatal neurodegenerative disorder typified by lysosomal accumulation of cholesterol and sphingolipids. Expression of yeast NP-C-related gene 1 (NCR1), the orthologue of the human NP-C gene 1 (NPC1) defective in the disease, in Chinese hamster ovary NPC1 mutant cells suppressed lipid accumulation. Deletion of NCR1, encoding a transmembrane glycoprotein predominantly residing in the vacuole of normal yeast, gave no phenotype. However, a dominant mutation in the putative sterol-sensing domain of Ncr1p conferred temperature and polyene antibiotic sensitivity without changes in sterol metabolism. Instead, the mutant cells were resistant to inhibitors of sphingolipid biosynthesis and super sensitive to sphingosine and C2-ceramide. Moreover, plasma membrane sphingolipids accumulated and redistributed to the vacuole and other subcellular membranes of the mutant cells. We propose that the primordial function of these proteins is to recycle sphingolipids and that defects in this process in higher eukaryotes secondarily result in cholesterol accumulation.     

Toxoplasma gondii Rab5 enhances cholesterol acquisition from host cells

The role of endocytosis in nutrient uptake by Toxoplasma gondii is unknown. To explore this issue, we characterized an endosomal compartment by identifying a T. gondii Rab5 homologue, a molecular marker for early endosomes in eukaryotic cells. The deduced amino acid sequence of the T. gondii Rab5 gene encodes a protein of 240 amino acids, which we termed TgRab51. TgRab51 was epitope-tagged at the N-terminus, expressed in the parasite, and localized by immunofluorescence and immunoelectron microscopy to tubulovesicular structures anterior to the parasite nucleus and adjacent to, but distinct from the Golgi. By immunofluorescence analysis, TgRab51wt-HA staining partially overlapped with Golgi/TGN markers, but not with the T. gondii secretory organelles. A dominant positive mutant, TgRab51Q103L-HA, enhanced uptake of exogenous cholesterol analogues in intracellular parasites, augmented formation of lipid droplets and accelerated parasite growth. Brefeldin A disrupted the TgRab51 compartment, and altered the distribution of fluorescent exogenous cholesterol in cells expressing TgRab51Q103L-HA. These results suggest that TgRab51 facilitates sterol uptake, possibly through a Golgi-dependent pathway.     

Dynamic movements of organelles containing Niemann-Pick C1 protein: NPC1 involvement in late endocytic events

People homozygous for mutations in the Niemann-Pick type C1 (NPC1) gene have physiological defects, including excess accumulation of intracellular cholesterol and other lipids, that lead to drastic neural and liver degeneration. The NPC1 multipass transmembrane protein is resident in late endosomes and lysosomes, but its functions are unknown. We find that organelles containing functional NPC1-fluorescent protein fusions undergo dramatic movements, some in association with extending strands of endoplasmic reticulum. In NPC1 mutant cells the NPC1-bearing organelles that normally move at high speed between perinuclear regions and the periphery of the cell are largely absent. Pulse-chase experiments with dialkylindocarbocyanine low-density lipoprotein showed that NPC1 organelles function late in the endocytic pathway; NPC1 protein may aid the partitioning of endocytic and lysosomal compartments. The close connection between NPC1 and the drug U18666A, which causes NPC1-like organelle defects, was established by rescuing drug-treated cells with overproduced NPC1. U18666A inhibits outward movements of NPC1 organelles, trapping membranes and cholesterol in perinuclear organelles similar to those in NPC1 mutant cells, even when cells are grown in lipoprotein-depleted serum. We conclude that NPC1 protein promotes the creation and/or movement of particular late endosomes, which rapidly transport materials to and from the cell periphery.     

The roles of Centrin 2 and Dynein Light Chain 8a in apical secretory organelles discharge of Toxoplasma gondii

To efficiently enter host cells, apicomplexan parasites such as Toxoplasma gondii rely on an apical complex composed of tubulin‐based structures as well as two sets of secretory organelles named micronemes and rhoptries. The trafficking and docking of these organelles to the apical pole of the parasite is crucial for the discharge of their contents. Here, we describe two proteins typically associated with microtubules, Centrin 2 (CEN2) and Dynein Light Chain 8a (DLC8a), that are required for efficient host cell invasion. CEN2 localizes to four different compartments, and remarkably, conditional depletion of the protein occurs in stepwise manner, sequentially depleting the protein pools from each location. This phenomenon allowed us to discern the essential function of the apical pool of CEN2 for microneme secretion, motility, invasion and egress. DLC8a localizes to the conoid, and its depletion also perturbs microneme exocytosis in addition to the apical docking of the rhoptry organelles, causing a severe defect in host cell invasion. Phenotypic characterization of CEN2 and DLC8a indicates that while both proteins participate in microneme secretion, they likely act at different steps along the cascade of events leading to organelle exocytosis.     

The Role of Clathrin in Post-Golgi Trafficking in Toxoplasma gondii

Apicomplexan parasites are single eukaryotic cells with a highly polarised secretory system that contains unique secretory organelles (micronemes and rhoptries) that are required for host cell invasion. In contrast, the role of the endosomal system is poorly understood in these parasites. With many typical endocytic factors missing, we speculated that endocytosis depends exclusively on a clathrin-mediated mechanism. Intriguingly, in Toxoplasma gondii we were only able to observe the endogenous clathrin heavy chain 1 (CHC1) at the Golgi, but not at the parasite surface. For the functional characterisation of Toxoplasma gondii CHC1 we generated parasite mutants conditionally expressing the dominant negative clathrin Hub fragment and demonstrate that CHC1 is essential for vesicle formation at the trans-Golgi network. Consequently, the functional ablation of CHC1 results in Golgi aberrations, a block in the biogenesis of the unique secretory microneme and rhoptry organelles, and of the pellicle. However, we found no morphological evidence for clathrin mediating endocytosis in these parasites and speculate that they remodelled their vesicular trafficking system to adapt to an intracellular lifestyle.     

Trypanosoma cruzi Epimastigotes Are Able to Manage Internal Cholesterol Levels under Nutritional Lipid Stress Conditions

Trypanosoma cruzi epimastigotes store high amounts of cholesterol and cholesteryl esters in reservosomes. These unique organelles are responsible for cellular digestion by providing substrates for homeostasis and parasite differentiation. Here we demonstrate that under nutritional lipid stress, epimastigotes preferentially mobilized reservosome lipid stocks, instead of lipid bodies, leading to the consumption of parasite cholesterol reservoirs and production of ergosterol. Starved epimastigotes acquired more LDL-NBD-cholesterol by endocytosis and distributed the exogenous cholesterol to their membranes faster than control parasites. Moreover, the parasites were able to manage internal cholesterol levels, alternating between consumption and accumulation. With normal lipid availability, parasites esterified cholesterol exhibiting an ACAT-like activity that was sensitive to Avasimibe in a dose-dependent manner. This result also implies that exogenous cholesterol has a role in lipid reservoirs in epimastigotes.     

Identification of the moving junction complex of Toxoplasma gondii: a collaboration between distinct secretory organelles

Apicomplexan parasites, including Toxoplasma gondii and Plasmodium sp., are obligate intracellular protozoa. They enter into a host cell by attaching to and then creating an invagination in the host cell plasma membrane. Contact between parasite and host plasma membranes occurs in the form of a ring-shaped moving junction that begins at the anterior end of the parasite and then migrates posteriorly. The resulting invagination of host plasma membrane creates a parasitophorous vacuole that completely envelops the now intracellular parasite. At the start of this process, apical membrane antigen 1 (AMA1) is released onto the parasite surface from specialized secretory organelles called micronemes. The T. gondii version of this protein, TgAMA1, has been shown to be essential for invasion but its exact role has not previously been determined. We identify here a trio of proteins that associate with TgAMA1, at least one of which associates with TgAMA1 at the moving junction. Surprisingly, these new proteins derive not from micronemes, but from the anterior secretory organelles known as rhoptries and specifically, for at least two, from the neck portion of these club-shaped structures. Homologues for these AMA1-associated proteins are found throughout the Apicomplexa strongly suggesting that this moving junction apparatus is a conserved feature of this important class of parasites. Differences between the contributing proteins in different species may, in part, be the result of selective pressure from the different niches occupied by these parasites.     

Ncr1p, the yeast ortholog of mammalian Niemann Pick C1 protein, is dispensable for endocytic transport

The Niemann Pick C1 protein localizes to late endosomes and plays a key role in the intracellular transport of cholesterol in mammalian cells. Cholesterol and other lipids accumulate in a lysosomal or late endosomal compartment in cells lacking normal NPC1 function. Other than accumulation of lipids, defects in lysosomal retroendocytosis, sorting of a multifunctional receptor and endosomal movement have also been detected in NPC1 mutant cells. Ncr1p is an ortholog of NPC1 in the budding yeast Saccharomyces cerevisiae. In this study, we show that Ncr1p is a vacuolar membrane protein that transits through the biosynthetic vacuolar protein sorting pathway, and that it can be solubilized by Triton X-100 at 4 degrees C. Using well-established assays, we demonstrate that the absence of Ncr1p had no effect on fluid phase and receptor- mediated endocytosis, biosynthetic delivery to the vacuole, retrograde transport from endosome to Golgi and ubiquitin- and nonubiquitin-dependent multivesicular body sorting. We conclude that Ncr1p does not have an essential role in known endocytic transport pathways in yeast.     

Characterization of the Niemann-Pick C pathway in alveolar type II cells and lamellar bodies of the lung

The Niemann-Pick C (NPC) pathway plays an essential role in the intracellular trafficking of cholesterol by facilitating the release of lipoprotein-derived sterol from the lumen of lysosomes. Regulation of cellular cholesterol homeostasis is of particular importance to lung alveolar type II cells because of the need for production of surfactant with an appropriate lipid composition. We performed microscopic and biochemical analysis of NPC proteins in isolated rat type II pneumocytes. NPC1 and NPC2 proteins were present in the lung, isolated type II cells in culture, and alveolar macrophages. The glycosylated and nonglycosylated forms of NPC1 were prominent in the lung and the lamellar body organelles. Immunocytochemical analysis of isolated type II pneumocytes showed localization of NPC1 to the limiting membrane of lamellar bodies. NPC2 and lysosomal acid lipase were found within these organelles, as confirmed by z-stack analysis of confocal images. All three proteins also were identified in small, lysosome-like vesicles. In the presence of serum, pharmacological inhibition of the NPC pathway with compound U18666A resulted in doubling of the cholesterol content of the type II cells. Filipin staining revealed a striking accumulation of cholesterol within lamellar bodies. Thus the NPC pathway functions to control cholesterol accumulation in lamellar bodies of type II pneumocytes and, thereby, may play a role in the regulation of surfactant cholesterol content.     

CRISPR screens for lipid regulators reveal a role for ER-bound SNX13 in lysosomal cholesterol export

We report here two genome-wide CRISPR screens performed to identify genes that, when knocked out, alter levels of lysosomal cholesterol or bis(monoacylglycero)phosphate. In addition, these screens were also performed under conditions of NPC1 inhibition to identify modifiers of NPC1 function in lysosomal cholesterol export. The screens confirm tight coregulation of cholesterol and bis(monoacylglycero)phosphate in cells and reveal an unexpected role for the ER-localized SNX13 protein as a negative regulator of lysosomal cholesterol export and contributor to ER–lysosome membrane contact sites. In the absence of NPC1 function, SNX13 knockdown redistributes lysosomal cholesterol and is accompanied by triacylglycerol-rich lipid droplet accumulation and increased lysosomal bis(monoacylglycero)phosphate. These experiments provide unexpected insight into the regulation of lysosomal lipids and modification of these processes by novel gene products.