FusionPro, a Versatile Proteogenomic Tool for Identification of Novel Fusion Transcripts and Their Potential Translation Products in Cancer Cells.

Fusion proteoforms are translation products derived from gene fusion. Although very rare, the fusion proteoforms play important roles in biomedical science. For example, fusion proteoforms influence the development of tumors by serving as cancer markers or cell cycle regulators. Although numerous studies have reported bioinformatics tools that can predict fusion transcripts, few proteogenomic tools are available that can predict and identify proteoforms. In this study, we develop a versatile proteogenomic tool "FusionPro," which facilitates the identification of fusion transcripts and their potential translatable peptides. FusionPro provides an independent gene fusion prediction module and can build sequence databases for annotated fusion proteoforms. FusionPro shows greater sensitivity than the available fusion finders when analyzing simulated or real RNA sequencing data sets. We use FusionPro to identify 18 fusion junction peptides and three potential fusion-derived peptides by MS/MS-based analysis of leukemia cell lines (Jurkat and K562) and ovarian cancer tissues from the Clinical Proteomic Tumor Analysis Consortium. Among the identified fusion proteins, we molecularly validate two fusion junction isoforms and a translation product of FAM133B:CDK6. Moreover, sequence analysis suggests that the fusion protein participates in the cell cycle progression. In addition, our prediction results indicate that fusion transcripts often have multiple fusion junctions and that these fusion junctions tend to be distributed in a nonrandom pattern at both the chromosome and gene levels. Thus, FusionPro allows users to detect various types of fusion translation products using a transcriptome-informed approach and to gain a comprehensive understanding of the formation and biological roles of fusion proteoforms.

A Molecular Basis for Reciprocal Regulation between Pheromones and Hormones in Response to Dietary Cues in C. elegans.

Under stressful conditions, the early larvae of C. elegans enter dauer diapause, a non-aging period, driven by the seemingly opposite influence of ascaroside pheromones (ASCRs) and steroid hormone dafachronic acids (DAs). However, the molecular basis of how these small molecules engage in competitive crosstalk in coordination with insulin/IGF-1 signaling (IIS) remains elusive. Here we report a novel transcriptional regulatory pathway that seems to operate between the ASCR and DA biosynthesis under ad libitum (AL) feeding conditions or bacterial deprivation (BD). Although expression of the ASCR and DA biosynthetic genes reciprocally inhibit each other, ironically and interestingly, such dietary cue-mediated modulation requires the presence of the competitors. Under BD, induction of ASCR biosynthetic gene expression required DA, while ASCR suppresses the expression of the DA biosynthetic gene daf-36. The negative regulation of DA by ASCR was IIS-dependent, whereas daf-36 regulation appeared to be independent of IIS. These observations suggest that the presence of ASCR determines the IIS-dependency of DA gene expression regardless of dietary conditions. Thus, our work defines a molecular basis for a novel reciprocal gene regulation of pheromones and hormones to cope with stressful conditions during development and aging.

Ascaroside Pheromones: Chemical Biology and Pleiotropic Neuronal Functions.

Pheromones are neuronal signals that stimulate conspecific individuals to react to environmental stressors or stimuli. Research on the ascaroside (ascr) pheromones in Caenorhabditis elegans and other nematodes has made great progress since ascr#1 was first isolated and biochemically defined in 2005. In this review, we highlight the current research on the structural diversity, biosynthesis, and pleiotropic neuronal functions of ascr pheromones and their implications in animal physiology. Experimental evidence suggests that ascr biosynthesis starts with conjugation of ascarylose to very long-chain fatty acids that are then processed via peroxisomal ß-oxidation to yield diverse ascr pheromones. We also discuss the concentration and stage-dependent pleiotropic neuronal functions of ascr pheromones. These functions include dauer induction, lifespan extension, repulsion, aggregation, mating, foraging and detoxification, among others. These roles are carried out in coordination with three G protein-coupled receptors that function as putative pheromone receptors: SRBC-64/66, SRG-36/37, and DAF-37/38. Pheromone sensing is transmitted in sensory neurons via DAF-16-regulated glutamatergic neurotransmitters. Neuronal peroxisomal fatty acid ß-oxidation has important cell-autonomous functions in the regulation of neuroendocrine signaling, including neuroprotection. In the future, translation of our knowledge of nematode ascr pheromones to higher animals might be beneficial, as ascr#1 has some anti-inflammatory effects in mice. To this end, we propose the establishment of pheromics (pheromone omics) as a new subset of integrated disciplinary research area within chemical ecology for system-wide investigation of animal pheromones.

HSF-1 is involved in regulation of ascaroside pheromone biosynthesis by heat stress in Caenorhabditis elegans.

The nematode worm Caenorhabditis elegans survives by adapting to environmental stresses such as temperature extremes by increasing the concentrations of ascaroside pheromones, termed ascarosides or daumones, which signal early C. elegans larvae to enter a non-aging dauer state for long-term survival. It is well known that production of ascarosides is stimulated by heat stress, resulting in enhanced dauer formation by which worms can adapt to environmental insults. However, the molecular mechanism by which ascaroside pheromone biosynthesis is stimulated by heat stress remains largely unknown. In the present study, we show that the heat-shock transcription factor HSF-1 can mediate enhanced ascaroside pheromone biosynthesis in response to heat stress by activating the peroxisomal fatty acid ß-oxidation genes in C. elegans. To explore the potential molecular mechanisms, we examined the four major genes involved in the ascaroside biosynthesis pathway and then quantified the changes in both the expression of these genes and ascaroside production under heat-stress conditions. The transcriptional activation of ascaroside pheromone biosynthesis genes by HSF-1 was quite notable, which is not only supported by chromatin immunoprecipitation assays, but also accompanied by the enhanced production of chemically detectable major ascarosides (e.g. daumones 1 and 3). Consequently, the dauer formation rate was significantly increased by the ascaroside pheromone extracts from N2 wild-type but not from hsf-1(sy441) mutant animals grown under heat-stress conditions. Hence heat-stress-enhanced ascaroside production appears to be mediated at least in part by HSF-1, which seems to be important in adaptation strategies for coping with heat stress in this nematode.

Genetic deficiency in neuronal peroxisomal fatty acid ß-oxidation causes the interruption of dauer development in Caenorhabditis elegans.

Although peroxisomal fatty acid (FA) ß-oxidation is known to be critical for animal development, the cellular mechanisms that control the manner in which its neuronal deficiency causes developmental defects remain unclear. To elucidate the potential cellular consequences of neuronal FA metabolic disorder for dauer development, an alternative developmental process in Caenorhabditis elegans that occurs during stress, we investigated the sequential effects of its corresponding genetic deficiency. Here, we show that the daf-22 gene in peroxisomal FA ß-oxidation plays a distinct role in ASK neurons, and its deficiency interrupts dauer development even in the presence of the exogenous ascaroside pheromones that induce such development. Un-metabolized FAs accumulated in ASK neurons of daf-22 mutants stimulate the endoplasmic reticulum (ER) stress response, which may enhance the XBP-1 activity that promotes the transcription of neuronal insulin-like peptides. These sequential cell-autonomous reactions in ASK neurons then activate insulin/IGF-1 signaling, which culminates in the suppression of DAF-16/FOXO activity. This suppression results in the interruption of dauer development, independently of pheromone presence. These findings suggest that neuronal peroxisomal FA ß-oxidation is indispensable for animal development by regulating the ER stress response and neuroendocrine signaling.

A conserved neuronal DAF-16/FoxO plays an important role in conveying pheromone signals to elicit repulsion behavior in Caenorhabditis elegans.

Animals use pheromones as a conspecific chemical language to respond appropriately to environmental changes. The soil nematode Caenorhabditis elegans secretes ascaroside pheromones throughout the lifecycle, which influences entry into dauer phase in early larvae, in addition to sexual attraction and aggregation. In adult hermaphrodites, pheromone sensory signals perceived by worms usually elicit repulsion as an initial behavioral signature. However, the molecular mechanisms underlying neuronal pheromone sensory process from perception to repulsion in adult hermaphrodites remain poorly understood. Here, we show that pheromone signals perceived by GPA-3 is conveyed through glutamatergic neurotransmission in which neuronal DAF-16/FoxO plays an important modulatory role by controlling glutaminase gene expression. We further provide evidence that this modulatory role for DAF-16/FoxO seems to be conserved evolutionarily by electro-physiological study in mouse primary hippocampal neurons that are responsible for glutamatergic neurotransmission. These findings provide the basis for understanding the nematode pheromone signaling, which seems crucial for adaptation of adult hermaphrodites to changes in environmental condition for survival.