Origins, Technological Advancement, and Applications of Peptidomics.

Peptidomics is the comprehensive characterization of peptides from biological sources instead of heading for a few single peptides in former peptide research. Mass spectrometry allows to detect a multitude of peptides in complex mixtures and thus enables new strategies leading to peptidomics. The term was established in the year 2001, and up to now, this new field has grown to over 3000 publications. Analytical techniques originally developed for fast and comprehensive analysis of peptides in proteomics were specifically adjusted for peptidomics. Although it is thus closely linked to proteomics, there are fundamental differences with conventional bottom-up proteomics. Fundamental technological advancements of peptidomics since have occurred in mass spectrometry and data processing, including quantification, and more slightly in separation technology. Different strategies and diverse sources of peptidomes are mentioned by numerous applications, such as discovery of neuropeptides and other bioactive peptides, including the use of biochemical assays. Furthermore, food and plant peptidomics are introduced similarly. Additionally, applications with a clinical focus are included, comprising biomarker discovery as well as immunopeptidomics. This overview extensively reviews recent methods, strategies, and applications including links to all other chapters of this book.

Current Challenges and Future Directions in Peptidomics.

The field of peptidomics has been under development since its start more than 20 years ago. In this chapter we provide a personal outlook for future directions in this field. The applications of peptidomics technologies are spreading more and more from classical research of peptide hormones and neuropeptides towards commercial applications in plant and food-science. Many clinical applications have been developed to analyze the complexity of biofluids, which are being addressed with new instrumentation, automization, and data processing. Additionally, the newly developed field of immunopeptidomics is showing promise for cancer therapies. In conclusion, peptidomics will continue delivering important information in classical fields like neuropeptides and peptide hormones, benefiting from improvements in state-of-the-art technologies. Moreover, new directions of research such as immunopeptidomics will further complement classical omics technologies and may become routine clinical procedures. Taken together, discoveries of new substances, networks, and applications of peptides can be expected in different disciplines.

The peroxisome: an update on mysteries 3.0.

Peroxisomes are highly dynamic, oxidative organelles with key metabolic functions in cellular lipid metabolism, such as the ß-oxidation of fatty acids and the synthesis of myelin sheath lipids, as well as the regulation of cellular redox balance. Loss of peroxisomal functions causes severe metabolic disorders in humans. Furthermore, peroxisomes also fulfil protective roles in pathogen and viral defence and immunity, highlighting their wider significance in human health and disease. This has sparked increasing interest in peroxisome biology and their physiological functions. This review presents an update and a continuation of three previous review articles addressing the unsolved mysteries of this remarkable organelle. We continue to highlight recent discoveries, advancements, and trends in peroxisome research, and address novel findings on the metabolic functions of peroxisomes, their biogenesis, protein import, membrane dynamics and division, as well as on peroxisome-organelle membrane contact sites and organelle cooperation. Furthermore, recent insights into peroxisome organisation through super-resolution microscopy are discussed. Finally, we address new roles for peroxisomes in immune and defence mechanisms and in human disorders, and for peroxisomal functions in different cell/tissue types, in particular their contribution to organ-specific pathologies.

Differential roles for ACBD4 and ACBD5 in peroxisome-ER interactions and lipid metabolism.

Peroxisomes and the endoplasmic reticulum (ER) are intimately linked subcellular organelles, physically connected at membrane contact sites. While collaborating in lipid metabolism, for example, of very long-chain fatty acids (VLCFAs) and plasmalogens, the ER also plays a role in peroxisome biogenesis. Recent work identified tethering complexes on the ER and peroxisome membranes that connect the organelles. These include membrane contacts formed via interactions between the ER protein VAPB (vesicle-associated membrane protein-associated protein B) and the peroxisomal proteins ACBD4 and ACBD5 (acyl-coenzyme A-binding domain protein). Loss of ACBD5 has been shown to cause a significant reduction in peroxisome-ER contacts and accumulation of VLCFAs. However, the role of ACBD4 and the relative contribution these two proteins make to contact site formation and recruitment of VLCFAs to peroxisomes remain unclear. Here, we address these questions using a combination of molecular cell biology, biochemical, and lipidomics analyses following loss of ACBD4 or ACBD5 in HEK293 cells. We show that the tethering function of ACBD5 is not absolutely required for efficient peroxisomal ß-oxidation of VLCFAs. We demonstrate that loss of ACBD4 does not reduce peroxisome-ER connections or result in the accumulation of VLCFAs. Instead, the loss of ACBD4 resulted in an increase in the rate of ß-oxidation of VLCFAs. Finally, we observe an interaction between ACBD5 and ACBD4, independent of VAPB binding. Overall, our findings suggest that ACBD5 may act as a primary tether and VLCFA recruitment factor, whereas ACBD4 may have regulatory functions in peroxisomal lipid metabolism at the peroxisome-ER interface.

Organelle Membrane Extensions in Mammalian Cells.

Organelles within eukaryotic cells are not isolated static compartments, instead being morphologically diverse and highly dynamic in order to respond to cellular needs and carry out their diverse and cooperative functions. One phenomenon exemplifying this plasticity, and increasingly gaining attention, is the extension and retraction of thin tubules from organelle membranes. While these protrusions have been observed in morphological studies for decades, their formation, properties and functions are only beginning to be understood. In this review, we provide an overview of what is known and still to be discovered about organelle membrane protrusions in mammalian cells, focusing on the best-characterised examples of these membrane extensions arising from peroxisomes (ubiquitous organelles involved in lipid metabolism and reactive oxygen species homeostasis) and mitochondria. We summarise the current knowledge on the diversity of peroxisomal/mitochondrial membrane extensions, as well as the molecular mechanisms by which they extend and retract, necessitating dynamic membrane remodelling, pulling forces and lipid flow. We also propose broad cellular functions for these membrane extensions in inter-organelle communication, organelle biogenesis, metabolism and protection, and finally present a mathematical model that suggests that extending protrusions is the most efficient way for an organelle to explore its surroundings.

Immunolabeling for Detection of Endogenous and Overexpressed Peroxisomal Proteins in Mammalian Cells.

Peroxisomes are dynamic subcellular organelles in mammals, playing essential roles in cellular lipid metabolism and redox homeostasis. They perform a wide spectrum of functions in human health and disease, with new roles, mechanisms, and regulatory pathways still being discovered. Recently elucidated biological roles of peroxisomes include as antiviral defense hubs, intracellular signaling platforms, immunomodulators, and protective organelles in sensory cells. Furthermore, peroxisomes are part of a complex inter-organelle interaction network, which involves metabolic cooperation and cross talk via membrane contacts. The detection of endogenous and/or overexpressed proteins within a cell by immunolabelling informs us about the organellar and even sub-organellar localization of both known and putative peroxisomal proteins. In turn, this can be exploited to characterize the effects of experimental manipulations on the morphology, distribution, and/or number of peroxisomes in a cell, which are key properties controlling peroxisome function. Here, we present a protocol used successfully in our laboratory for the immunolabelling of peroxisomal proteins in cultured mammalian cells. We present immunofluorescence and transfection techniques as well as reagents to determine the localization of endogenous and overexpressed peroxisomal proteins.

Ultrastructural Analysis and Quantification of Peroxisome-Organelle Contacts.

Transmission electron microscopy (TEM) has long been a vital technology to visualize the interaction of cellular compartments at the highest possible resolution. While this paved the way to describing organelles within the cellular context in detail, TEM has long been underused to generate quantitative data, analyzing those interactions as well as underlying mechanisms leading to their formation and modification. Here we describe a simple stereological method to unbiasedly assess the extent of organelle-organelle membrane contact sites, able to efficiently generate accurate and reproducible quantitative data from cultured mammalian cells prepared for TEM.

Proximity-Ligation Assay to Detect Peroxisome-Organelle Interaction.

Peroxisomes are essential organelles in mammals, which contribute to cellular lipid metabolism and redox homeostasis. They do not function as isolated entities but cooperate and interact with other subcellular organelles, in particular the endoplasmic reticulum, mitochondria, and lipid droplets. Those interactions are often mediated by membrane contact sites. Tether proteins at those sites bring the organelles in close proximity to facilitate metabolite and lipid transfer as well as organelle communication. There is great interest in the investigation of the physiological functions of peroxisome-organelle contacts and how they are regulated. Here, we present an antibody- and fluorescence-based proximity ligation approach used successfully in our laboratory for the detection and quantification of peroxisome-organelle interactions in cultured mammalian cells.

Generation of Reporter Cell Lines for Endogenous Expression Analysis of Peroxisomal Proteins.

Peroxisomes are multifunctional, ubiquitous, and dynamic organelles. They are responsible for diverse metabolic and physiological functions and communicate with other organelles, including the ER, mitochondria, lipid droplets, and lysosomes, through membrane contact sites. However, despite their importance for healthy cell function, remarkably, little is known about how peroxisomes and peroxisomal proteins are regulated under physiological conditions in human cells. Here, we present a method to generate reporter cell lines to measure endogenous expression of peroxisomal proteins of interest. By CRISPR-mediated knock-in of an easily detectable protein-coding tag in-frame into the relevant genomic loci, endogenous levels of the protein of interest in a cell population can be quantified in a high-throughput manner under different conditions. This has important implications for the fundamental understanding of how peroxisomal proteins are regulated and may reveal the therapeutic potential of modulating peroxisomal protein expression to improve cell performance.

Assessing Peroxisomal Protein Interaction by Immunoprecipitation.

Organelles physically interact with each other via protein tethering complexes that bridge the opposing membranes. Organelle membrane contacts are highly dynamic, implying dynamism of the tethering complexes. Alterations in the binding of the tethering proteins can be assessed by immunoprecipitation. Antibody-conjugated beads allow for purification of the target protein with its binding partners, which can subsequently be examined by western blot analysis. We present immunoprecipitation methods and strategies to examine protein interaction domains, and for the identification of residues important for the regulation of the interaction, here focusing on phosphorylation. We use the peroxisomal membrane protein ACBD5 and its paralog ACBD4, which both bind ER membrane protein VAPB to mediate peroxisome-ER contacts, as example. However, this method can be applied to other peroxisomal and non-peroxisomal (membrane) proteins.

Phosphonylated tyrosine and lysine residues as biomarkers of local exposure of human hair to the organophosphorus nerve agents sarin and VX.

We herein present for the first time a micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HR MS) procedure to detect phosphonylated tyrosine (Tyr) and lysine (Lys) residues obtained from human hair exposed to organophosphorus nerve agents (OPNA). In general, toxic OPNA react with endogenous blood proteins causing the formation of adducts representing well-known targets for biomedical analysis to prove exposure. In contrast, no protein-derived biomarker has been introduced so far to document local exposure of hair. Accordingly, we developed and characterized a µLC-ESI MS/HR MS method for the analysis of scalp hair exposed to OPNA in vitro. Type I and Type II keratin from hair was dissolved during lysis, precipitated and subjected to pronase-catalyzed hydrolysis yielding single adducted Lys and in a much higher amount Tyr residues. Exposure to sarin caused the adduction of an isopropyl methylphosphonic acid moiety and exposure to VX yielded adducts of ethyl methylphosphonic acid, well suited as biomarkers of exposure. These were of appropriate stability in the autosampler for 24 h. The biomarker yield obtained from hair of six individuals as well as from hair of six different parts of the body of one individual (armpit, beard, leg, arm, scalp, and pubic) differed reasonably indicating the variable individual protein composition and structure of hair. Exposed hair stored at ambient temperature for 9 weeks with contact to air and daylight showed stability of all adducts and therefore their suitability for verification of exposure.

Controlling contacts-Molecular mechanisms to regulate organelle membrane tethering.

In recent years, membrane contact sites (MCS), which mediate interactions between virtually all subcellular organelles, have been extensively characterized and shown to be essential for intracellular communication. In this review essay, we focus on an emerging topic: the regulation of MCS. Focusing on the tether proteins themselves, we discuss some of the known mechanisms which can control organelle tethering events and identify apparent common regulatory hubs, such as the VAP interface at the endoplasmic reticulum (ER). We also highlight several currently hypothetical concepts, including the idea of tether oligomerization and redox regulation playing a role in MCS formation. We identify gaps in our current understanding, such as the identity of the majority of kinases/phosphatases involved in tether modification and conclude that a holistic approach-incorporating the formation of multiple MCS, regulated by interconnected regulatory modulators-may be required to fully appreciate the true complexity of these fascinating intracellular communication systems.

VAP Proteins - From Organelle Tethers to Pathogenic Host Interactors and Their Role in Neuronal Disease.

Vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) are ubiquitous ER-resident tail-anchored membrane proteins in eukaryotic cells. Their N-terminal major sperm protein (MSP) domain faces the cytosol and allows them to interact with a wide variety of cellular proteins. Therefore, VAP proteins are vital to many cellular processes, including organelle membrane tethering, lipid transfer, autophagy, ion homeostasis and viral defence. Here, we provide a timely overview of the increasing number of VAPA/B binding partners and discuss the role of VAPA/B in maintaining organelle-ER interactions and cooperation. Furthermore, we address how viruses and intracellular bacteria hijack VAPs and their binding partners to induce interactions between the host ER and pathogen-containing compartments and support pathogen replication. Finally, we focus on the role of VAP in human disease and discuss how mutated VAPB leads to the disruption of cellular homeostasis and causes amyotrophic lateral sclerosis.

Fission Impossible (?)-New Insights into Disorders of Peroxisome Dynamics.

Peroxisomes are highly dynamic and responsive organelles, which can adjust their morphology, number, intracellular position, and metabolic functions according to cellular needs. Peroxisome multiplication in mammalian cells involves the concerted action of the membrane-shaping protein PEX11ß and division proteins, such as the membrane adaptors FIS1 and MFF, which recruit the fission GTPase DRP1 to the peroxisomal membrane. The latter proteins are also involved in mitochondrial division. Patients with loss of DRP1, MFF or PEX11ß function have been identified, showing abnormalities in peroxisomal (and, for the shared proteins, mitochondrial) dynamics as well as developmental and neurological defects, whereas the metabolic functions of the organelles are often unaffected. Here, we provide a timely update on peroxisomal membrane dynamics with a particular focus on peroxisome formation by membrane growth and division. We address the function of PEX11ß in these processes, as well as the role of peroxisome-ER contacts in lipid transfer for peroxisomal membrane expansion. Furthermore, we summarize the clinical phenotypes and pathophysiology of patients with defects in the key division proteins DRP1, MFF, and PEX11ß as well as in the peroxisome-ER tether ACBD5. Potential therapeutic strategies for these rare disorders with limited treatment options are discussed.

PEX11ß and FIS1 cooperate in peroxisome division independently of mitochondrial fission factor.

Peroxisome membrane dynamics and division are essential to adapt the peroxisomal compartment to cellular needs. The peroxisomal membrane protein PEX11ß (also known as PEX11B) and the tail-anchored adaptor proteins FIS1 (mitochondrial fission protein 1) and MFF (mitochondrial fission factor), which recruit the fission GTPase DRP1 (dynamin-related protein 1, also known as DNML1) to both peroxisomes and mitochondria, are key factors of peroxisomal division. The current model suggests that MFF is essential for peroxisome division, whereas the role of FIS1 is unclear. Here, we reveal that PEX11ß can promote peroxisome division in the absence of MFF in a DRP1- and FIS1-dependent manner. We also demonstrate that MFF permits peroxisome division independently of PEX11ß and restores peroxisome morphology in PEX11ß-deficient patient cells. Moreover, targeting of PEX11ß to mitochondria induces mitochondrial division, indicating the potential for PEX11ß to modulate mitochondrial dynamics. Our findings suggest the existence of an alternative, MFF-independent pathway in peroxisome division and report a function for FIS1 in the division of peroxisomes. This article has an associated First Person interview with the first authors of the paper.

Multiple Ways to Keep FFAT Under Control!

Peroxisomes and the ER are closely inter-connected organelles, which collaborate in the metabolism of lipids. In a recent research paper in the Journal of Cell Biology, we describe a novel mechanism by which peroxisome-ER membrane contact sites are regulated, via phosphorylation of the peroxisomal protein ACBD5. We found that the interaction between ACBD5 and the ER protein VAPB, which we have previously shown to form a tether complex at peroxisome-ER contacts, is controlled by phosphorylation of ACBD5 at two different sites of its FFAT motif - the VAPB binding site. We also identify the kinase GSK3-ß as being responsible for direct phosphorylation of ACBD5 to negatively regulate interaction with VAPB, leading to reduced peroxisome-ER contacts. In this article we provide additional insights into how this work, in combination with other studies on phosphorylation of VAP interactors, suggests a complex system of both positive and negative regulation of the FFAT motif via phosphorylation.

Insights Into the Peroxisomal Protein Inventory of Zebrafish.

Peroxisomes are ubiquitous, oxidative subcellular organelles with important functions in cellular lipid metabolism and redox homeostasis. Loss of peroxisomal functions causes severe disorders with developmental and neurological abnormalities. Zebrafish are emerging as an attractive vertebrate model to study peroxisomal disorders as well as cellular lipid metabolism. Here, we combined bioinformatics analyses with molecular cell biology and reveal the first comprehensive inventory of Danio rerio peroxisomal proteins, which we systematically compared with those of human peroxisomes. Through bioinformatics analysis of all PTS1-carrying proteins, we demonstrate that D. rerio lacks two well-known mammalian peroxisomal proteins (BAAT and ZADH2/PTGR3), but possesses a putative peroxisomal malate synthase (Mlsl) and verified differences in the presence of purine degrading enzymes. Furthermore, we revealed novel candidate peroxisomal proteins in D. rerio, whose function and localisation is discussed. Our findings confirm the suitability of zebrafish as a vertebrate model for peroxisome research and open possibilities for the study of novel peroxisomal candidate proteins in zebrafish and humans.

Determinants of Peroxisome Membrane Dynamics.

Organelles within the cell are highly dynamic entities, requiring dramatic morphological changes to support their function and maintenance. As a result, organelle membranes are also highly dynamic, adapting to a range of topologies as the organelle changes shape. In particular, peroxisomes-small, ubiquitous organelles involved in lipid metabolism and reactive oxygen species homeostasis-display a striking plasticity, for example, during the growth and division process by which they proliferate. During this process, the membrane of an existing peroxisome elongates to form a tubule, which then constricts and ultimately undergoes scission to generate new peroxisomes. Dysfunction of this plasticity leads to diseases with developmental and neurological phenotypes, highlighting the importance of peroxisome dynamics for healthy cell function. What controls the dynamics of peroxisomal membranes, and how this influences the dynamics of the peroxisomes themselves, is just beginning to be understood. In this review, we consider how the composition, biophysical properties, and protein-lipid interactions of peroxisomal membranes impacts on their dynamics, and in turn on the biogenesis and function of peroxisomes. In particular, we focus on the effect of the peroxin PEX11 on the peroxisome membrane, and its function as a major regulator of growth and division. Understanding the roles and regulation of peroxisomal membrane dynamics necessitates a multidisciplinary approach, encompassing knowledge across a range of model species and a number of fields including lipid biochemistry, biophysics and computational biology. Here, we present an integrated overview of our current understanding of the determinants of peroxisome membrane dynamics, and reflect on the outstanding questions still remaining to be solved.