Host-Microbe-Drug-Nutrient Screen Identifies Bacterial Effectors of Metformin Therapy.

Metformin is the first-line therapy for treating type 2 diabetes and a promising anti-aging drug. We set out to address the fundamental question of how gut microbes and nutrition, key regulators of host physiology, affect the effects of metformin. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we developed a high-throughput four-way screen to define the underlying host-microbe-drug-nutrient interactions. We show that microbes integrate cues from metformin and the diet through the phosphotransferase signaling pathway that converges on the transcriptional regulator Crp. A detailed experimental characterization of metformin effects downstream of Crp in combination with metabolic modeling of the microbiota in metformin-treated type 2 diabetic patients predicts the production of microbial agmatine, a regulator of metformin effects on host lipid metabolism and lifespan. Our high-throughput screening platform paves the way for identifying exploitable drug-nutrient-microbiome interactions to improve host health and longevity through targeted microbiome therapies. VIDEO ABSTRACT.

Mass spectrometric evidence for neuropeptide-amidating enzymes in Caenorhabditis elegans.

Neuropeptides constitute a vast and functionally diverse family of neurochemical signaling molecules and are widely involved in the regulation of various physiological processes. The nematode Caenorhabditis elegans is well-suited for the study of neuropeptide biochemistry and function, as neuropeptide biosynthesis enzymes are not essential for C. elegans viability. This permits the study of neuropeptide biosynthesis in mutants lacking certain neuropeptide-processing enzymes. Mass spectrometry has been used to study the effects of proprotein convertase and carboxypeptidase mutations on proteolytic processing of neuropeptide precursors and on the peptidome in C. elegans However, the enzymes required for the last step in the production of many bioactive peptides, the carboxyl-terminal amidation reaction, have not been characterized in this manner. Here, we describe three genes that encode homologs of neuropeptide amidation enzymes in C. elegans and used tandem LC-MS to compare neuropeptides in WT animals with those in newly generated mutants for these putative amidation enzymes. We report that mutants lacking both a functional peptidylglycine α-hydroxylating monooxygenase and a peptidylglycine α-amidating monooxygenase had a severely altered neuropeptide profile and also a decreased number of offspring. Interestingly, single mutants of the amidation enzymes still expressed some fully processed amidated neuropeptides, indicating the existence of a redundant amidation mechanism in C. elegans All MS data are available via ProteomeXchange with the identifier PXD008942. In summary, the key steps in neuropeptide processing in C. elegans seem to be executed by redundant enzymes, and loss of these enzymes severely affects brood size, supporting the need of amidated peptides for C. elegans reproduction.

Exploring neuropeptide signalling through proteomics and peptidomics.

INTRODUCTION: Neuropeptides are neuro-endocrine signaling molecules capable of signaling as neurotransmitters, neuromodulators or neurohormones. Studying how neuropeptide signaling is integrated in endocrine pathways and how neuropeptides regulate endogenous processes is crucial to understanding how multicellular organisms respond to environmental and internal cues. Areas covered: This review will cover proteomics and peptidomics approaches used in researching peptide signaling systems and breakthroughs that were achieved in this field. Both differential mass spectrometry and reverse genetic approaches are commonly used to study neuropeptidergic signaling. The field of proteomics quickly developed in the past decades and expanded from gel-based approaches to include advanced liquid chromatography and mass spectrometry. We explore how proteomics is used to reveal neuropeptide maturation and identify downstream targets of neuropeptide signaling pathways. We show how peptidomics differs from standard proteomics approaches and how it is used to study both neuropeptide processing and signal pathway identification. Expert commentary: Thanks to recent advancements in isolation techniques and increased sensitivity of equipment, proteomics and peptidomics studies of neuropeptide signaling are contributing increasingly to elucidating functional implications of endocrine signaling. Further technical progress should allow for full peptidomic profiling of single neurons, eventually providing us with a complete comprehension of endocrine signaling.

Metformin promotes lifespan through mitohormesis via the peroxiredoxin PRDX-2.

The antiglycemic drug metformin, widely prescribed as first-line treatment of type II diabetes mellitus, has lifespan-extending properties. Precisely how this is achieved remains unclear. Via a quantitative proteomics approach using the model organism Caenorhabditis elegans, we gained molecular understanding of the physiological changes elicited by metformin exposure, including changes in branched-chain amino acid catabolism and cuticle maintenance. We show that metformin extends lifespan through the process of mitohormesis and propose a signaling cascade in which metformin-induced production of reactive oxygen species increases overall life expectancy. We further address an important issue in aging research, wherein so far, the key molecular link that translates the reactive oxygen species signal into a prolongevity cue remained elusive. We show that this beneficial signal of the mitohormetic pathway is propagated by the peroxiredoxin PRDX-2. Because of its evolutionary conservation, peroxiredoxin signaling might underlie a general principle of prolongevity signaling.

Fast and Reliable Quantitative Peptidomics with labelpepmatch.

The use of stable isotope tags in quantitative peptidomics offers many advantages, but the laborious identification of matching sets of labeled peptide peaks is still a major bottleneck. Here we present labelpepmatch, an R-package for fast and straightforward analysis of LC-MS spectra of labeled peptides. This open-source tool offers fast and accurate identification of peak pairs alongside an appropriate framework for statistical inference on quantitative peptidomics data, based on techniques from other -omics disciplines. A relevant case study on the desert locust Schistocerca gregaria proves our pipeline to be a reliable tool for quick but thorough explorative analyses.

Functional neuropeptidomics in invertebrates.

Neuropeptides are key messengers in almost all physiological processes. They originate from larger precursors and are extensively processed to become bioactive. Neuropeptidomics aims to comprehensively identify the collection of neuropeptides in an organism, organ, tissue or cell. The neuropeptidome of several invertebrates is thoroughly explored since they are important model organisms (and models for human diseases), disease vectors and pest species. The charting of the neuropeptidome is the first step towards understanding peptidergic signaling. This review will first discuss the latest developments in exploring the neuropeptidome. The physiological roles and modes of action of neuropeptides can be explored in two ways, which are largely orthogonal and therefore complementary. The first way consists of inferring the functions of neuropeptides by a forward approach where neuropeptide profiles are compared under different physiological conditions. Second is the reverse approach were neuropeptide collections are used to screen for receptor-binding. This is followed by localization studies and functional tests. This review will focus on how these different functional screening methods contributed to the field of invertebrate neuropeptidomics and expanded our knowledge of peptidergic signaling. This article is part of a Special Issue entitled: Neuroproteomics: Applications in Neuroscience and Neurology.

The essence of insect metamorphosis and aging: electrical rewiring of cells driven by the principles of juvenile hormone-dependent Ca(2+)-homeostasis.

In holometabolous insects the fall to zero of the titer of Juvenile Hormone ends its still poorly understood "status quo" mode of action in larvae. Concurrently it initiates metamorphosis of which the programmed cell death of all internal tissues that actively secrete proteins, such as the fat body, midgut, salivary glands, prothoracic glands, etc. is the most drastic aspect. These tissues have a very well developed rough endoplasmic reticulum, a known storage site of intracellular Ca(2+). A persistent high [Ca(2+)]i is toxic, lethal and causal to apoptosis. Metamorphosis becomes a logical phenomenon if analyzed from: (1) the causal link between calcium toxicity and apoptosis; (2) the largely overlooked fact that at least some isoforms of Ca(2+)-ATPases have a binding site for farnesol-like endogenous sesquiterpenoids (FRS). The Ca(2+)-ATPase blocker thapsigargin, like JH a sesquiterpenoid derivative, illustrates how absence of JH might work. The Ca(2+)-homeostasis system is concurrently extremely well conserved in evolution and highly variable, enabling tissue-, developmental-, and species specificity. As long as JH succeeds in keeping [Ca(2+)]i low by keeping the Ca(2+)-ATPases pumping, it acts as "the status quo" hormone. When it disappears, its various inhibitory effects are lifted. The electrical wiring system of cells, in particular in the regenerating tissues, is subject to change during metamorphosis. The possibility is discussed that in vertebrates an endogenous farnesol-like sesquiterpenoid, probably farnesol itself, acts as a functional, but hitherto completely overlooked Juvenile anti-aging "Inbrome", a novel concept in signaling.

Structure-activity studies of Drosophila adipokinetic hormone (AKH) by a cellular expression system of dipteran AKH receptors.

Structure-activity studies for the adipokinetic hormone receptor of insects were for the first time performed in a cellular expression system. A series of single amino acid replacement analogues for the endogenous adipokinetic hormone of Drosophila melanogaster (pGlu-Leu-Thr-Phe-Ser-Pro-Asp-Trp-NH(2)) were screened for activity with a bioluminescence cellular assay, expressing the G-protein coupled receptor. For this series of peptide analogues, one amino acid of the N-terminal tetrapeptide was successively replaced by alanine, while those of the C-terminal tetrapeptide were successively substituted by glycine; other modifications included the blocked N- and C-termini that were replaced by an acetylated alanine and a hydroxyl group, respectively. The analogue series was tested on the AKH receptors of two dipteran species, D. melanogaster and Anopheles gambiae. The blocked termini of the AKH peptide probably play a minor role in receptor interaction and activation, but are considered functionally important elements to protect the peptide against exopeptidases. In contrast, the amino acids at positions 2, 3, 4 and 5 from the N-terminus all seem to be crucial for receptor activation. This can be explained by the potential presence of a ß-strand in this part of the peptide that interacts with the receptor. The inferred ß-strand is probably followed by a ß-turn in which the amino acids at positions 5-8 are involved. In this ß-turn, the residues at positions 6 and 8 seem to be essential, as their substitutions induce only a very low degree of receptor activation. Replacement of Asp(7), by contrast, does not influence receptor activation at all. This implies that its side chain is folded inside the ß-turn so that no interaction with the receptor occurs.

RPamide neuropeptides NLP-22 and NLP-2 act through GnRH-like receptors to promote sleep and wakefulness in C. elegans.

Sleep and wakefulness are fundamental behavioral states of which the underlying molecular principles are becoming slowly elucidated. Transitions between these states require the coordination of multiple neurochemical and modulatory systems. In Caenorhabditis elegans sleep occurs during a larval transition stage called lethargus and is induced by somnogenic neuropeptides. Here, we identify two opposing neuropeptide/receptor signaling pathways: NLP-22 promotes behavioral quiescence, whereas NLP-2 promotes movement during lethargus, by signaling through gonadotropin-releasing hormone (GnRH) related receptors. Both NLP-2 and NLP-22 belong to the RPamide neuropeptide family and share sequence similarities with neuropeptides of the bilaterian GnRH, adipokinetic hormone (AKH) and corazonin family. RPamide neuropeptides dose-dependently activate the GnRH/AKH-like receptors GNRR-3 and GNRR-6 in a cellular receptor activation assay. In addition, nlp-22-induced locomotion quiescence requires the receptor gnrr-6. By contrast, wakefulness induced by nlp-2 overexpression is diminished by deletion of either gnrr-3 or gnrr-6. nlp-2 is expressed in a pair of olfactory AWA neurons and cycles with larval periodicity, as reported for nlp-22, which is expressed in RIA. Our data suggest that the somnogenic NLP-22 neuropeptide signals through GNRR-6, and that both GNRR-3 and GNRR-6 are required for the wake-promoting action of NLP-2 neuropeptides.

Quantitative Peptidomics with Isotopic and Isobaric Tags.

In differential peptidomics, peptide profiles are compared between biological samples and the resulting expression levels are correlated to a phenotype of interest. This, in turn, allows us insight into how peptides may affect the phenotype of interest. In quantitative differential peptidomics, both label-based and label-free techniques are often employed. Label-based techniques have several advantages over label-free methods, primarily that labels allow for various samples to be pooled prior to liquid chromatography-mass spectrometry (LC-MS) analysis, reducing between-run variation. Here, we detail a method for performing quantitative peptidomics using stable amine-binding isotopic and isobaric tags.

Beyond ROS clearance: Peroxiredoxins in stress signaling and aging.

Antioxidants were long predicted to have lifespan-promoting effects, but in general this prediction has not been well supported. While some antioxidants do seem to have a clear effect on longevity, this may not be primarily as a result of their role in the removal of reactive oxygen species, but rather mediated by other mechanisms such as the modulation of intracellular signaling. In this review we discuss peroxiredoxins, a class of proteinaceous antioxidants with redox signaling and chaperone functions, and their involvement in regulating longevity and stress resistance. Peroxiredoxins have a clear role in the regulation of lifespan and survival of many model organisms, including the mouse, Caenorhabditis elegans and Drosophila melanogaster. Recent research on peroxiredoxins - in these models and beyond - has revealed surprising new insights regarding the interplay between peroxiredoxins and longevity signaling, which will be discussed here in detail. As redox signaling is emerging as a potentially important player in the regulation of longevity and aging, increased knowledge of these fascinating antioxidants and their mode(s) of action is paramount.

Signaling properties of the human chemokine receptors CXCR4 and CXCR7 by cellular electric impedance measurements.

The chemokine receptor 4 (CXCR4) and 7 (CXCR7) are G-protein-coupled receptors involved in various diseases including human cancer. As such, they have become important targets for therapeutic intervention. Cell-based receptor assays, able to detect agents that modulate receptor activity, are of key importance for drug discovery. We evaluated the potential of cellular electric impedance for this purpose. Dose-dependent and specific stimulation of CXCR4 was detected upon addition of its unique chemokine ligand CXCL12. The response magnitude correlated with the CXCR4 expression level. Gαi coupling and signaling contributed extensively to the impedance response, whereas Gαq- and Gßγ-related events had only minor effects on the impedance profile. CXCR7 signaling could not be detected using impedance measurements. However, increasing levels of CXCR7 expression significantly reduced the CXCR4-mediated impedance readout, suggesting a regulatory role for CXCR7 on CXCR4-mediated signaling. Taken together, cellular electric impedance spectroscopy can represent a valuable alternative pharmacological cell-based assay for the identification of molecules targeting CXCR4, but not for CXCR7 in the absence of CXCR4.

SKN-1-independent transcriptional activation of glutathione S-transferase 4 (GST-4) by EGF signaling.

In C. elegans research, transcriptional activation of glutathione S-transferase 4 (gst-4) is often used as a read-out for SKN-1 activity. While many heed an assumed non-exclusivity of the GFP reporter signal driven by the gst-4 promoter to SKN-1, this is also often ignored. We here show that gst-4 can also be transcriptionally activated by EOR-1, a transcription factor mediating effects of the epidermal growth factor (EGF) pathway. Along with enhancing exogenous oxidative stress tolerance, EOR-1 inde-pendently of SKN-1 increases gst-4 transcription in response to augmented EGF signaling. Our findings caution researchers within the C. elegans community to always rely on sufficient experimental controls when assaying SKN-1 transcriptional activity with a gst-4p::gfp reporter, such as SKN-1 loss-of-function mutants and/or additional target genes next to gst-4.

Integrating -Omics: Systems Biology as Explored Through C. elegans Research.

-Omics data have become indispensable to systems biology, which aims to describe the full complexity of functional cells, tissues, organs and organisms. Generating vast amounts of data via such methods, researchers have invested in ways of handling and interpreting these. From the large volumes of -omics data that have been gathered over the years, it is clear that the information derived from one -ome is usually far from complete. Now, individual techniques and methods for integration are maturing to the point that researchers can focus on network-based integration rather than simply interpreting single -ome studies. This review evaluates the application of integrated -omics approaches with a focus on Caenorhabditis elegans studies, intending to direct researchers in this field to useful databases and inspiring examples.

Life-prolonging measures for a dead theory?

In a recent review article, Selman and colleagues (Trends Ecol Evol 27:570-577, 2012) discuss the status quo of the oxidative stress theory of aging (OSTA) and how it links to life history evolution. They suggest that the OSTA should be tested in wild populations which might show effects masked in laboratory settings. We disagree with their propositions for several reasons. We argue that there is increasing evidence that reactive oxygen species (ROS) are not causally linked with aging and that ROS do not play a straightforward role in shaping life history evolution. We propose that laboratory animals and semi-wild populations rather than wild animals are suited best to test any hypothesized effect of reactive oxygen species. This is because data from controlled manipulative experiments rather than observational correlations are preferred to solve this issue. In addition, nonconventional model organisms will be useful in answering the question how relevant the OSTA could be for life history evolution.

Royalactin extends lifespan of Caenorhabditis elegans through epidermal growth factor signaling.

Royalactin is a glycoprotein essential for the development of long-lived queen honeybees. Only larvae fed with royal jelly, containing royalactin, develop into queens. Royalactin plays a central role in this process by switching on the epidermal growth factor (EGF) receptor signaling pathway which ultimately leads to epigenetic changes and a long-lived queen phenotype. Recently it was shown that royalactin by itself also extends lifespan in Drosophila melanogaster. Yet, the mechanism by which royalactin promotes longevity remains largely unknown. We set out to characterize the effects of royalactin on Caenorhabditis elegans lifespan, and clarify the possible involvement of EGF signaling in this process. We demonstrate that royalactin extends lifespan of this nematode and that both EGF (LIN-3) and its receptor (LET-23) are essential to this process. To our knowledge, this is the first report of royalactin-mediated lifespan extension in a non-insect species. Additionally, we show that royalactin enhances locomotion in adult nematodes, implying that royalactin also influences healthspan. Our results suggest that royalactin is an important lifespan-extending factor in royal jelly and acts by promoting EGF signaling in C. elegans. Further work will now be needed to clarify which (secondary) signaling pathways are activated by royalactin, and how this ultimately translates into an extended health- and lifespan.

The Fading Electricity Theory of Ageing: the missing biophysical principle?

Since a few years convincing data are accumulating showing that some of the premises of the master integrative theory of ageing, namely Harman's Reactive Oxygen Species or free radical theory, are less well founded than originally assumed. In addition, none of the about another dozen documented ageing mechanisms seems to hold the final answer as to the ultimate cause and evolutionary significance of ageing. This review raises the question whether, perhaps, something important has been overlooked, namely a biophysical principle, electrical in nature. The first cell on earth started to be alive when its system for generating its own electricity, carried by inorganic ions, became operational. Any cell dies at the very moment that this system irreversibly collapses. In between birth and death, the system is subject to wear and tear because any cell's overall repair system is not 100 percent waterproof; otherwise adaptation would not be an option. The Fading Electricity Theory of Ageing has all necessary properties for acting as a universal major integrative concept. The advent of novel methods will facilitate the study of bioelectrical phenomena with molecular biological methods in combination with optogenetics, thereby offering challenging possibilities for innovative research in evo-gero.

A pharmacological study of NLP-12 neuropeptide signaling in free-living and parasitic nematodes.

NLP-12a and b have been identified as cholecystokinin/sulfakinin-like neuropeptides in the free-living nematode Caenorhabditis elegans. They are suggested to play an important role in the regulation of digestive enzyme secretion and fat storage. This study reports on the identification and characterization of an NLP-12-like peptide precursor gene in the rat parasitic nematode Strongyloides ratti. The S. ratti NLP-12 peptides are able to activate both C. elegans CKR-2 receptor isoforms in a dose-dependent way with affinities in the same nanomolar range as the native C. elegans NLP-12 peptides. The C-terminal RPLQFamide sequence motif of the NLP-12 peptides is perfectly conserved between free-living and parasitic nematodes. Based on systemic amino acid replacements the Arg-, Leu- and Phe- residues appear to be critical for high-affinity receptor binding. Finally, a SAR analysis revealed the essential pharmacophore in C. elegans NLP-12b to be the pentapeptide RPLQFamide.