Prevalent bee venom genes evolved before the aculeate stinger and eusociality.

BACKGROUND: Venoms, which have evolved numerous times in animals, are ideal models of convergent trait evolution. However, detailed genomic studies of toxin-encoding genes exist for only a few animal groups. The hyper-diverse hymenopteran insects are the most speciose venomous clade, but investigation of the origin of their venom genes has been largely neglected. RESULTS: Utilizing a combination of genomic and proteo-transcriptomic data, we investigated the origin of 11 toxin genes in 29 published and 3 new hymenopteran genomes and compiled an up-to-date list of prevalent bee venom proteins. Observed patterns indicate that bee venom genes predominantly originate through single gene co-option with gene duplication contributing to subsequent diversification. CONCLUSIONS: Most Hymenoptera venom genes are shared by all members of the clade and only melittin and the new venom protein family anthophilin1 appear unique to the bee lineage. Most venom proteins thus predate the mega-radiation of hymenopterans and the evolution of the aculeate stinger.

Venomics of the milos viper (Macrovipera schweizeri) unveils patterns of venom composition and exochemistry across blunt-nosed viper venoms.

Introduction: Snakebite is a neglected tropical disease and a globally important driver of death and morbidity. Vipers of the genus Macrovipera (Viperidae: Viperinae) are among the snakes of higher medical importance in the Old World. Despite the medical relevance of Macrovipera venoms, the knowledge regarding them is heterogeneously distributed with virtually all works conducted so far focusing on subspecies of Macrovipera lebetinus, while other species within the genus are largely overlooked. Here we present the first proteomic evaluation of the venom from the Greek endemic Milos viper (Macrovipera schweizeri). In line with clinical symptoms typically elicited by Macrovipera envenomations, Milos viper venom primarily comprises coagulotoxic and cytotoxic protein families, such as metalloproteinases (svMP) and serine proteases (svSP). Methods: We conducted comparative bioactivity assays on venoms from M. schweizeri and the M. lebetinus subspecies M. lebetinus cernovi, M. lebetinus obtusa, and M. lebetinus turanica, and showed that they all exhibit similarities in levels of cytotoxicity proteolytic activity, and inhibition of prokaryotic growth. Lastly, we compared Macrovipera venom profiles by 1D-SDS-PAGE and RP-HPLC, as well as our proteomic data with previously published Macrovipera venom proteomes. Results and discussion: The analyzes performed to reveal that a general venom profile seems to be conserved across blunt-nosed vipers, and that, M. schweizeri envenomations, similarly to those caused by other blunt-nosed vipers, are able to cause significant tissue damage. The present work represents an important starting point for the development of comparative studies across the full taxonomic range of the genus Macrovipera and can potentially help optimize the treatment of envenomations caused by M. schweizeri.

Neutrophil Extracellular Traps Drive Dacryolithiasis.

Mucopeptide concretions, previously called dacryoliths, are macroscopic stones that commonly obstruct the lacrimal sac. The mechanism behind dacryolithiasis remains unclear; however, the involvement of various immune cells, including neutrophils, has been confirmed. These findings remain limited, and no information on neutrophil extracellular traps (NETs), essentially involved in the pathogenesis of other lithiases, is available yet. Here, we employ microcomputed tomography, magnetic resonance tomography, histochemistry, mass spectrometry, and enzyme activity analyses to investigate the role of neutrophils and NETs in dacryolithiasis. We classify mucopeptide concretions into three types, with respect to the quantity of cellular and acellular material, polysaccharides, and mucosubstances. We propose the role of neutrophils and NETs within the existing model of gradual formation and growth of mucopeptide concretions, with neutrophils contributing to the initial stages of dacryolithiasis, as they localized on the inner (older) parts of the tissue. As NETs localized on the outer (newer) parts of the tissue, we link their role to the late stages of dacryolithiasis, presumably maintaining the proinflammatory environment and preventing efficient clearance. An abundance of IgG on the surface indicates the involvement of the adaptive immune system later as well. These findings bring new perspectives on dacryolithiasis, in which the innate and adaptive immune system are essentially involved.

Functional Characterization of Lysophospholipids by Proteomic and Lipidomic Analysis of Fibroblast-like Synoviocytes.

Synovial fluid (SF) from human knee joints with osteoarthritis (OA) has elevated levels of lysophosphatidylcholine (LPC) species, but their functional role is not well understood. This in vitro study was designed to test the hypothesis that various LPCs found elevated in OA SF and their metabolites, lysophosphatidic acids (LPAs), modulate the abundance of proteins and phospholipids (PLs) in human fibroblast-like synoviocytes (FLSs), with even minute chemical variations in lysophospholipids determining the extent of regulation. Cultured FLSs (n = 5-7) were treated with one of the LPC species, LPA species, IL-1ß, or a vehicle. Tandem mass tag peptide labeling coupled with LC-MS/MS/MS was performed to quantify proteins. The expression of mRNA from regulated proteins was analyzed using RT-PCR. PL synthesis was determined via ESI-MS/MS, and the release of radiolabeled PLs was determined by means of liquid scintillation counting. In total, 3960 proteins were quantified using multiplexed MS, of which 119, 8, and 3 were significantly and reproducibly regulated by IL-1ß, LPC 16:0, and LPC 18:0, respectively. LPC 16:0 significantly inhibited the release of PLs and the synthesis of phosphatidylcholine, LPC, and sphingomyelin. Neither LPC metabolite-LPA 16:0 nor LPA 18:0-had any reproducible effect on the levels of each protein. In conclusion, small chemical variations in LPC species can result in the significantly altered expression and secretion of proteins and PLs from FLSs. IL-1ß influenced all proteins that were reproducibly regulated by LPC 16:0. LPC species are likely to modulate FLS protein expression only in more advanced OA stages with low IL-1ß levels. None of the eight proteins being significantly regulated by LPC 16:0 have been previously reported in OA. However, our in vitro findings show that the CD81 antigen, calumenin, and B4E2C1 are promising candidates for further study, focusing in particular on their potential ability to modulate inflammatory and catabolic mechanisms.

Identification and characterisation of the tegument-expressed aldehyde dehydrogenase SmALDH_312 of Schistosoma mansoni, a target of disulfiram.

Schistosomiasis is an infectious disease caused by blood flukes of the genus Schistosoma and affects approximately 200 million people worldwide. Since Praziquantel (PZQ) is the only drug for schistosomiasis, alternatives are needed. By a biochemical approach, we identified a tegumentally expressed aldehyde dehydrogenase (ALDH) of S. mansoni, SmALDH_312. Molecular analyses of adult parasites showed Smaldh_312 transcripts in both genders and different tissues. Physiological and cell-biological experiments exhibited detrimental effects of the drug disulfiram (DSF), a known ALDH inhibitor, on larval and adult schistosomes in vitro. DSF also reduced stem-cell proliferation and caused severe tegument damage in treated worms. In silico-modelling of SmALDH_312 and docking analyses predicted DSF binding, which we finally confirmed by enzyme assays with recombinant SmALDH_312. Furthermore, we identified compounds of the Medicine for Malaria Venture (MMV) pathogen box inhibiting SmALDH_312 activity. Our findings represent a promising starting point for further development towards new drugs for schistosomiasis.

Venomics of the Central European Myrmicine Ants Myrmica rubra and Myrmica ruginodis.

Animal venoms are a rich source of novel biomolecules with potential applications in medicine and agriculture. Ants are one of the most species-rich lineages of venomous animals. However, only a fraction of their biodiversity has been studied so far. Here, we investigated the venom components of two myrmicine (subfamily Myrmicinae) ants: Myrmica rubra and Myrmica ruginodis. We applied a venomics workflow based on proteotranscriptomics and found that the venoms of both species are composed of several protein classes, including venom serine proteases, cysteine-rich secretory protein, antigen 5 and pathogenesis-related 1 (CAP) superfamily proteins, Kunitz-type serine protease inhibitors and venom acid phosphatases. Several of these protein classes are known venom allergens, and for the first time we detected phospholipase A1 in the venom of M. ruginodis. We also identified two novel epidermal growth factor (EGF) family toxins in the M. ruginodis venom proteome and an array of additional EGF-like toxins in the venom gland transcriptomes of both species. These are similar to known toxins from the related myrmicine ant, Manica rubida, and the myrmecine (subfamily Myrmeciinae) Australian red bulldog ant Myrmecia gullosa, and are possibly deployed as weapons in defensive scenarios or to subdue prey. Our work suggests that M.rubra and M. ruginodis venoms contain many enzymes and other high-molecular-weight proteins that cause cell damage. Nevertheless, the presence of EGF-like toxins suggests that myrmicine ants have also recruited smaller peptide components into their venom arsenal. Although little is known about the bioactivity and function of EGF-like toxins, their presence in myrmicine and myrmecine ants suggests they play a key role in the venom systems of the superfamily Formicoidea. Our work adds to the emerging picture of ant venoms as a source of novel bioactive molecules and highlights the need to incorporate such taxa in future venom bioprospecting programs.

A Spider Toxin Exemplifies the Promises and Pitfalls of Cell-Free Protein Production for Venom Biodiscovery.

Arthropod venoms offer a promising resource for the discovery of novel bioactive peptides and proteins, but the limited size of most species translates into minuscule venom yields. Bioactivity studies based on traditional fractionation are therefore challenging, so alternative strategies are needed. Cell-free synthesis based on synthetic gene fragments is one of the most promising emerging technologies, theoretically allowing the rapid, laboratory-scale production of specific venom components, but this approach has yet to be applied in venom biodiscovery. Here, we tested the ability of three commercially available cell-free protein expression systems to produce venom components from small arthropods, using U2-sicaritoxin-Sdo1a from the six-eyed sand spider Hexophtalma dolichocephala as a case study. We found that only one of the systems was able to produce an active product in low amounts, as demonstrated by SDS-PAGE, mass spectrometry, and bioactivity screening on murine neuroblasts. We discuss our findings in relation to the promises and limitations of cell-free synthesis for venom biodiscovery programs in smaller invertebrates.

Tandem mass tag-based proteomics for studying the effects of a biotechnologically produced oyster mushroom against hepatic steatosis in obese Zucker rats.

Hepatic steatosis is a very common response to liver injury and often attributed to metabolic disorders. Prior studies have demonstrated the efficacy of a biotechnologically produced oyster mushroom (Pleurotus sajor-caju, PSC) in alleviating hepatic steatosis in obese Zucker rats. This study aims to elucidate molecular events underlying the anti-steatotic effects of PSC. Tandem mass tag (TMT) peptide labeling coupled with LC-MS/MS/MS was used to quantify and compare proteins in the livers of lean Zucker rats fed a control diet (LC), obese Zucker rats fed the same control diet (OC) and obese Zucker rats fed the control diet supplemented with 5% PSC (OPSC) for 4 weeks. Using this technique 3128 proteins could be quantified, out of which 108 were differentially abundant between the OPSC and OC group. Functional enrichment analysis of the up-regulated proteins showed that these proteins were mainly involved in metabolic processes, while the down-regulated proteins were involved in inflammatory processes. Results from proteomic analysis were successfully validated for two up-regulated (carbonic anhydrase 3, regucalcin) and two down-regulated (cadherin-17, ceruloplasmin) proteins by means of immunoblotting. SIGNIFICANCE: Valorization of low-grade agricultural waste by edible fungi, such as the mushroom Pleurotus sajor-caju (PSC), represents a promising strategy for the production of protein rich biomass since they boast of a unique enzyme system that has the ability to recover nutrients and energy from biodegradable waste. Herein, we describe the metabolic effects of PSC feeding using a combined quantitative proteomics and bioinformatics approach. In total, 108 proteins were identified to be regulated by PSC feeding in the liver of the obese rats. Complementary usage of a bioinformatics approach allowed us to decipher the mechanisms underlying the recently observed lipid-lowering and anti-inflammatory activity of PSC feeding in obese Zucker rats, namely a reduction of fatty acid synthesis, an improvement of hepatoprotective mechanisms and an enhancement of anti-inflammatory effects.

Proteo-Transcriptomic Analysis Identifies Potential Novel Toxins Secreted by the Predatory, Prey-Piercing Ribbon Worm Amphiporus lactifloreus.

Nemerteans (ribbon worms) employ toxins to subdue their prey, but research thus far has focused on the small-molecule components of mucus secretions and few protein toxins have been characterized. We carried out a preliminary proteotranscriptomic analysis of putative toxins produced by the hoplonemertean Amphiporus lactifloreus (Hoplonemertea, Amphiporidae). No variants were found of known nemertean-specific toxin proteins (neurotoxins, cytotoxins, parbolysins or nemertides) but several toxin-like transcripts were discovered, expressed strongly in the proboscis, including putative metalloproteinases and sequences resembling sea anemone actitoxins, crown-of-thorn sea star plancitoxins, and multiple classes of inhibitor cystine knot/knottin family proteins. Some of these products were also directly identified in the mucus proteome, supporting their preliminary identification as secreted toxin components. Two new nemertean-typical toxin candidates could be described and were named U-nemertotoxin-1 and U-nemertotoxin-2. Our findings provide insight into the largely overlooked venom system of nemerteans and support a hypothesis in which the nemertean proboscis evolved in several steps from a flesh-melting organ in scavenging nemerteans to a flesh-melting and toxin-secreting venom apparatus in hunting hoplonemerteans.

An Economic Dilemma Between Molecular Weapon Systems May Explain an Arachno-atypical Venom in Wasp Spiders (Argiope bruennichi).

Spiders use venom to subdue their prey, but little is known about the diversity of venoms in different spider families. Given the limited data available for orb-weaver spiders (Araneidae), we selected the wasp spider Argiope bruennichi for detailed analysis. Our strategy combined a transcriptomics pipeline based on multiple assemblies with a dual proteomics workflow involving parallel mass spectrometry techniques and electrophoretic profiling. We found that the remarkably simple venom of A. bruennichi has an atypical composition compared to other spider venoms, prominently featuring members of the cysteine-rich secretory protein, antigen 5 and pathogenesis-related protein 1 (CAP) superfamily and other, mostly high-molecular-weight proteins. We also detected a subset of potentially novel toxins similar to neuropeptides. We discuss the potential function of these proteins in the context of the unique hunting behavior of wasp spiders, which rely mostly on silk to trap their prey. We propose that the simplicity of the venom evolved to solve an economic dilemma between two competing yet metabolically expensive weapon systems. This study emphasizes the importance of cutting-edge methods to encompass the lineages of smaller venomous species that have yet to be characterized in detail, allowing us to understand the biology of their venom systems and to mine this prolific resource for translational research.

Chemosensory Cell-Derived Acetylcholine Drives Tracheal Mucociliary Clearance in Response to Virulence-Associated Formyl Peptides.

Mucociliary clearance through coordinated ciliary beating is a major innate defense removing pathogens from the lower airways, but the pathogen sensing and downstream signaling mechanisms remain unclear. We identified virulence-associated formylated bacterial peptides that potently stimulated ciliary-driven transport in the mouse trachea. This innate response was independent of formyl peptide and taste receptors but depended on key taste transduction genes. Tracheal cholinergic chemosensory cells expressed these genes, and genetic ablation of these cells abrogated peptide-driven stimulation of mucociliary clearance. Trpm5-deficient mice were more susceptible to infection with a natural pathogen, and formylated bacterial peptides were detected in patients with chronic obstructive pulmonary disease. Optogenetics and peptide stimulation revealed that ciliary beating was driven by paracrine cholinergic signaling from chemosensory to ciliated cells operating through muscarinic M3 receptors independently of nerves. We provide a cellular and molecular framework that defines how tracheal chemosensory cells integrate chemosensation with innate defense.

Bacterial MgrB peptide activates chemoreceptor Fpr3 in mouse accessory olfactory system and drives avoidance behaviour.

Innate immune chemoreceptors of the formyl peptide receptor (Fpr) family are expressed by vomeronasal sensory neurons (VSNs) in the accessory olfactory system. Their biological function and coding mechanisms remain unknown. We show that mouse Fpr3 (Fpr-rs1) recognizes the core peptide motif f-MKKFRW that is predominantly present in the signal sequence of the bacterial protein MgrB, a highly conserved regulator of virulence and antibiotic resistance in Enterobacteriaceae. MgrB peptide can be produced and secreted by bacteria, and is selectively recognized by a subset of VSNs. Exposure to the peptide also stimulates VSNs in freely behaving mice and drives innate avoidance. Our data shows that Fpr3 is required for neuronal detection and avoidance of peptides derived from a conserved master virulence regulator of enteric bacteria.

Alpha-1 Antitrypsin Inhibits ATP-Mediated Release of Interleukin-1ß via CD36 and Nicotinic Acetylcholine Receptors.

While interleukin (IL)-1ß is a potent pro-inflammatory cytokine involved in host defense, high levels can cause life-threatening sterile inflammation including systemic inflammatory response syndrome. Hence, the control of IL-1ß secretion is of outstanding biomedical importance. In response to a first inflammatory stimulus such as lipopolysaccharide, pro-IL-1ß is synthesized as a cytoplasmic inactive pro-form. Extracellular ATP originating from injured cells is a prototypical second signal for inflammasome-dependent maturation and release of IL-1ß. The human anti-protease alpha-1 antitrypsin (AAT) and IL-1ß regulate each other via mechanisms that are only partially understood. Here, we demonstrate that physiological concentrations of AAT efficiently inhibit ATP-induced release of IL-1ß from primary human blood mononuclear cells, monocytic U937 cells, and rat lung tissue, whereas ATP-independent IL-1ß release is not impaired. Both, native and oxidized AAT are active, suggesting that the inhibition of IL-1ß release is independent of the anti-elastase activity of AAT. Signaling of AAT in monocytic cells involves the lipid scavenger receptor CD36, calcium-independent phospholipase A2ß, and the release of a small soluble mediator. This mediator leads to the activation of nicotinic acetylcholine receptors, which efficiently inhibit ATP-induced P2X7 receptor activation and inflammasome assembly. We suggest that AAT controls ATP-induced IL-1ß release from human mononuclear blood cells by a novel triple-membrane-passing signaling pathway. This pathway may have clinical implications for the prevention of sterile pulmonary and systemic inflammation.

The Eukaryotic Elongation Factor 1 Alpha (eEF1α) from the Parasite Leishmania infantum Is Modified with the Immunomodulatory Substituent Phosphorylcholine (PC).

Proteins and glycolipids have been found to be decorated with phosphorylcholine (PC) both in protozoa and nematodes that parasitize humans and animals. PC epitopes can provoke various effects on immune cells leading to an immunomodulation of the host's immune system that allows long-term persistence of the parasites. So far, only a limited number of PC-modified proteins, mainly from nematodes, have been identified. Infections caused by Leishmania spp. (e.g., L. infantum in southern Europe) affect about 12 million people worldwide and are characterized by a wide spectrum of clinical forms in humans, ranging from cutaneous to fatal visceral leishmaniasis. To establish and maintain the infection, these protozoa are dependent on the secretion of effector molecules into the host for modulating their immune system. In this project, we analyzed the PC modification of L. infantum promastigotes by 2D-gel based proteomics. Western blot analysis with the PC-specific antibody TEPC-15 revealed one PC-substituted protein in this organism, identified as eEF1α. We could demonstrate that the binding of eEF1α to one of its downstream effectors is dependent on its PC-modification. In this study we provide evidence that in this parasite the modification of eEF1α with PC may be essential for its function as an important virulence factor.

Time course of Tau toxicity and pharmacologic prevention in a cell model of Tauopathy.

The aggregation of Tau protein is a hallmark of neurodegenerative diseases including Alzheimer's disease. Previously, we generated a cell model of tauopathy based on the 4-repeat domain with the FTDP-17 mutation ΔK280 (Tau4RDΔK) which is expressed in a regulatable fashion (tet-on). The deletion variant ΔK280 is highly amyloidogenic and forms fibrous aggregates in neuroblastoma N2a cells staining with the reporter dye Thioflavin S. The aggregation of Tau4RDΔK is toxic, contrary to wildtype or anti-aggregant variants of the protein. Using a novel approach for monitoring in situ Tau aggregation and toxicity by combination of microscopic analysis with FACS and biochemical analysis of cells enabled the dissection of the aggregating species which cause a time-dependent increase of toxicity. The dominant initiating step is the dimerization of Tau4RDΔK which leads to further aggregation and induces a strong increase in reactive oxygen species (ROS) and cytoplasmic Ca2+ which damage the membranes and cause cell death. Tau-based treatments using Tau aggregation inhibitors reduce both soluble oligomeric and fully aggregated Tau species and decrease their toxicity.

The PCome of Ascaris suum as a model system for intestinal nematodes: identification of phosphorylcholine-substituted proteins and first characterization of the PC-epitope structures.

In multicellular parasites (e.g., nematodes and protozoa), proteins and glycolipids have been found to be decorated with phosphorylcholine (PC). PC can provoke various effects on immune cells leading to an immunomodulation of the host's immune system. This immunomodulation allows long-term persistence but also prevents severe pathology due to downregulation of cellular immune responses. PC-containing antigens have been found to interfere with key proliferative signaling pathways in B and T cells, development of dendritic cells and macrophages, and mast cell degranulation. These effects contribute to the observed modulated cytokine levels and impairment of lymphocyte proliferation. In contrast to glycosphingolipids, little is known about the PC-epitopes of proteins. So far, only a limited number of PC-modified proteins from nematodes have been identified. In this project, PC-substituted proteins and glycolipids in Ascaris suum have been localized by immunohistochemistry in specific tissues of the body wall, intestine, and reproductive tract. Subsequently, we investigated the PCome of A. suum by 2D gel-based proteomics and detection by Western blotting using the PC-specific antibody TEPC-15. By peptide-mass-fingerprint matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), we could identify 59 PC-substituted proteins, which are in involved multiple cellular processes. In addition to membrane proteins like vitellogenin-6, we found proteins with structural (e.g., tubulins) and metabolic (e.g., pyruvate dehydrogenase) functions or which can act in the defense against the host's immune response (e.g., serpins). Initial characterization of the PC-epitopes revealed a predominant linkage of PC to the proteins via N-glycans. Our data form the basis for more detailed investigations of the PC-epitope structures as a prerequisite for comprehensive understanding of the molecular mechanisms of immunomodulation.

Phosphocholine-Modified Macromolecules and Canonical Nicotinic Agonists Inhibit ATP-Induced IL-1ß Release.

IL-1ß is a potent proinflammatory cytokine of the innate immune system that is involved in host defense against infection. However, increased production of IL-1ß plays a pathogenic role in various inflammatory diseases, such as rheumatoid arthritis, gout, sepsis, stroke, and transplant rejection. To prevent detrimental collateral damage, IL-1ß release is tightly controlled and typically requires two consecutive danger signals. LPS from Gram-negative bacteria is a prototypical first signal inducing pro-IL-1ß synthesis, whereas extracellular ATP is a typical second signal sensed by the ATP receptor P2X7 that triggers activation of the NLRP3-containing inflammasome, proteolytic cleavage of pro-IL-1ß by caspase-1, and release of mature IL-1ß. Mechanisms controlling IL-1ß release, even in the presence of both danger signals, are needed to protect from collateral damage and are of therapeutic interest. In this article, we show that acetylcholine, choline, phosphocholine, phosphocholine-modified LPS from Haemophilus influenzae, and phosphocholine-modified protein efficiently inhibit ATP-mediated IL-1ß release in human and rat monocytes via nicotinic acetylcholine receptors containing subunits α7, α9, and/or α10. Of note, we identify receptors for phosphocholine-modified macromolecules that are synthesized by microbes and eukaryotic parasites and are well-known modulators of the immune system. Our data suggest that an endogenous anti-inflammatory cholinergic control mechanism effectively controls ATP-mediated release of IL-1ß and that the same mechanism is used by symbionts and misused by parasites to evade innate immune responses of the host.

Detection and site localization of phosphorylcholine-modified peptides by NanoLC-ESI-MS/MS using precursor ion scanning and multiple reaction monitoring experiments.

Phosphorylcholine (PC)-modified biomolecules like lipopolysaccharides, glycosphingolipids, and (glyco)proteins are widespread, highly relevant antigens of parasites, since this small hapten shows potent immunomodulatory capacity, which allows the establishment of long-lasting infections of the host. Especially for PC-modified proteins, structural data is rare because of the zwitterionic nature of the PC substituent, resulting in low sensitivities and unusual but characteristic fragmentation patterns. We have developed a targeted mass spectrometric approach using hybrid triple quadrupole/linear ion trap (QTRAP) mass spectrometry coupled to nanoflow chromatography for the sensitive detection of PC-modified peptides from complex proteolytic digests, and the localization of the PC-modification within the peptide backbone. In a first step, proteolytic digests are screened using precursor ion scanning for the marker ions of choline (m/z 104.1) and phosphorylcholine (m/z 184.1) to establish the presence of PC-modified peptides. Potential PC-modified precursors are then subjected to a second analysis using multiple reaction monitoring (MRM)-triggered product ion spectra for the identification and site localization of the modified peptides. The approach was first established using synthetic PC-modified synthetic peptides and PC-modified model digests. Following the optimization of key parameters, we then successfully applied the method to the detection of PC-peptides in the background of a proteolytic digest of a whole proteome. This methodological invention will greatly facilitate the detection of PC-substituted biomolecules and their structural analysis.

Oligomer formation of tau protein hyperphosphorylated in cells.

Abnormal phosphorylation ("hyperphosphorylation") and aggregation of Tau protein are hallmarks of Alzheimer disease and other tauopathies, but their causative connection is still a matter of debate. Tau with Alzheimer-like phosphorylation is also present in hibernating animals, mitosis, or during embryonic development, without leading to pathophysiology or neurodegeneration. Thus, the role of phosphorylation and the distinction between physiological and pathological phosphorylation needs to be further refined. So far, the systematic investigation of highly phosphorylated Tau was difficult because a reliable method of preparing reproducible quantities was not available. Here, we generated full-length Tau (2N4R) in Sf9 cells in a well defined phosphorylation state containing up to ∼20 phosphates as judged by mass spectrometry and Western blotting with phospho-specific antibodies. Despite the high concentration in living Sf9 cells (estimated ∼230 µm) and high phosphorylation, the protein was not aggregated. However, after purification, the highly phosphorylated protein readily formed oligomers, whereas fibrils were observed only rarely. Exposure of mature primary neuronal cultures to oligomeric phospho-Tau caused reduction of spine density on dendrites but did not change the overall cell viability.

Microtubule affinity-regulating kinase 2 (MARK2) turns on phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) at Thr-313, a mutation site in Parkinson disease: effects on mitochondrial transport.

The kinase MARK2/Par-1 plays key roles in several cell processes, including neurodegeneration such as Alzheimer disease by phosphorylating tau and detaching it from microtubules. In search of interaction partners of MARK2, we identified phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1), which is important for the survival of neurons and whose mutations are linked to familial Parkinson disease (PD). MARK2 phosphorylated and activated the cleaved form of PINK1 (ΔN-PINK1; amino acids 156-581). Thr-313 was the primary phosphorylation site, a residue mutated to a non-phosphorylatable form (T313M) in a frequent variant of PD. Mutation of Thr-313 to Met or Glu in PINK1 showed toxic effects with abnormal mitochondrial distribution in neurons. MARK2 and PINK1 were found to colocalize with mitochondria and regulate their transport. ΔN-PINK1 promoted anterograde transport and increased the fraction of stationary mitochondria, whereas full-length PINK1 promoted retrograde transport. In both cases, MARK2 enhanced the effects. The results identify MARK2 as an upstream regulator of PINK1 and ΔN-PINK1 and provide insights into the regulation of mitochondrial trafficking in neurons and neurodegeneration in PD.