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The nervous system monitors the environment to maintain homeostasis, which can be affected by stressful conditions. Using mammalian models of chronic stress, we previously observed altered brain levels of GPM6A, a protein involved in neuronal morphology. However, GPM6A's role in systemic stress responses remains unresolved. The nematode Caenorhabditis elegans expresses a GPM6A ortholog, the neuronal membrane glycoprotein 1 (NMGP-1). Because of the shared features between nematode and mammalian nervous systems and the vast genetic tools available in C. elegans, we used the worm to elucidate the role of GPM6A in the stress response. We first identified nmgp-1 expression in different amphid and phasmid neurons. To understand the nmgp-1 role, we characterized the behavior of nmgp-1(RNAi) animals and two nmgp-1 mutant alleles. Compared to control animals, mutant and RNAi-treated worms exhibited increased recovery time from the stress-resistant dauer stage, altered SDS chemosensation and reduced egg-laying rate resulting in egg retention (bag-of-worms phenotype). Silencing of nmgp-1 expression induced morphological abnormalities in the ASJ sensory neurons, partly responsible for dauer exit. These results indicate that nmgp-1 is required for neuronal morphology and for behaviors associated with chemosensation. Finally, we propose nmgp-1 mutants as a tool to screen drugs for human nervous system pathologies., (© 2021 International Society for Neurochemistry.)

Nowadays, great efforts are made to gain insight into the molecular mechanisms that underlie structural neuronal plasticity. Moreover, the identification of signaling pathways involved in the development of psychiatric disorders aids the screening of possible therapeutic targets. Genetic variations or alterations in GPM6A expression are linked to neurological disorders such as schizophrenia, depression, and Alzheimer's disease. GPM6A encodes the neuronal surface glycoprotein M6a that promotes filopodia/spine, dendrite, and synapse formation by unknown mechanisms. A substantial body of evidence suggests that the extracellular loops of M6a command its function. However, the proteins that associate with them and that modulate neuronal plasticity have not been determined yet. To address this question, we generated a chimera protein that only contains the extracellular loops of M6a and performed a co-immunoprecipitation with rat hippocampus samples followed by TMT/MS. Here, we report 72 proteins, which are good candidates to interact with M6a's extracellular loops and modify its function. Gene ontology (GO) analysis showed that 63% of the potential M6a's interactor proteins belong to the category "synapse," at both sides of the synaptic cleft, "neuron projections" (51%) and "presynapse" (49%). In this sense, we showed that endogenous M6a interacts with piccolo, synaptic vesicle protein 2B, and synapsin 1 in mature cultured hippocampal neurons. Interestingly, about 28% of the proteins left were related to the "myelin sheath" annotation, suggesting that M6a could interact with proteins at the surface of oligodendrocytes. Indeed, we demonstrated the ( cis and trans ) interaction between M6a and proteolipid protein (PLP) in neuroblastoma N2a cells. Finally, the 72 proteins were subjected to disease-associated genes and variants screening by DisGeNET. Apart from the diseases that have already been associated with M6a, most of the proteins are also involved in "autistic disorder," "epilepsy," and "seizures" increasing the spectrum of disorders in which M6a could play a role. Data are available via ProteomeXchange with identifier PXD017347 ., (Copyright © 2020 Aparicio, Formoso, León, Frasch and Scorticati.)

The trans-sialidase protein expressed by Trypanosoma cruzi is an important enzyme in the life cycle of this human pathogenic parasite and is considered a promising target for the development of new drug treatments against Chagas' disease. Here we describe α-amino phosphonates as a novel class of inhibitor of T. cruzi trans-sialidase. Molecular modelling studies were initially used to predict the active-site binding affinities for a series of amino phosphonates, which were subsequently synthesised and their IC 50 s determined in vitro. The measured inhibitory activities show some correlation with the predictions from molecular modelling, with 1-napthyl derivatives found to be the most potent inhibitors having IC 50 s in the low micromolar range. Interestingly, kinetic analysis of the mode of inhibition demonstrated that the α-aminophosphonates tested here operate in a non-competitive manner., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Neuronal membrane glycoprotein M6a (Gpm6a) is a protein with four transmembrane regions and the N- and the C-ends facing the cytosol. It functions in processes of neuronal development, outgrowth of neurites, and formation of filopodia, spines, and synapsis. Molecular mechanisms by which Gpm6a acts in these processes are not fully comprehended. Structural similarities of Gpm6a with tetraspanins led us to hypothesize that, similarly to tetraspanins, the cytoplasmic tails function as connections with cytoskeletal and/or signaling proteins. Here, we demonstrate that the C- but not the N-terminal cytosolic end of Gpm6a is required for the formation of filopodia by Gpm6a in cultured neurons from rat hippocampus and in neuroblastoma cells N2a. Further immunofluorescence microcopy and flow cytometry analysis show that deletion of neither the N- nor the C-terminal intracellular domains interferes with the recognition of Gpm6a by the function-blocking antibody directed against the extracellular part of Gpm6a. Expression levels of both truncation mutants were not affected but we observed decrease in the amount of both truncated proteins on cell surface suggesting that the incapacity of the Gpm6a lacking C-terminus to induce filopodium formation is not due to the lower amount of Gpm6a on cell surface. Following colocalization assays shows that deletion of the C- but not the N-terminus diminishes the association of Gpm6a with clathrin implying involvement of clathrin-mediated trafficking events. Next, using comprehensive alanine scanning mutagenesis of the C-terminus we identify K250, K255, and E258 as the key residues for the formation of filopodia by Gpm6a. Substitution of these charged residues with alanine also diminishes the amount of Gpm6a on cell surface and in case of K255 and E258 leads to the lower amount of total expressed protein. Subsequent bioinformatic analysis of Gpm6a amino acid sequence reveals that highly conserved and functional residues cluster preferentially within the C- and not within the N-terminus and that K250, K255, and E258 are predicted as part of sorting signals of transmembrane proteins. Altogether, our results provide evidence that filopodium outgrowth induced by Gpm6a requires functionally critical residues within the C-terminal cytoplasmic tail.

Trypanosomes, protozoan parasites of medical importance, essentially rely on post-transcriptional mechanisms to regulate gene expression in insect vectors and vertebrate hosts. RNA binding proteins (RBPs) that associate to the 3'-UTR of mature mRNAs are thought to orchestrate master developmental programs for these processes to happen. Yet, the molecular mechanisms by which differentiation occurs remain largely unexplored in these human pathogens. Here, we show that ectopic inducible expression of the RBP TcUBP1 promotes the beginning of the differentiation process from non-infective epimastigotes to infective metacyclic trypomastigotes in Trypanosoma cruzi. In early-log epimastigotes TcUBP1 promoted a drop-like phenotype, which is characterized by the presence of metacyclogenesis hallmarks, namely repositioning of the kinetoplast, the expression of an infective-stage virulence factor such as trans-sialidase, increased resistance to lysis by human complement and growth arrest. Furthermore, TcUBP1-ectopic expression in non-infective late-log epimastigotes promoted full development into metacyclic trypomastigotes. TcUBP1-derived metacyclic trypomastigotes were infective in cultured cells, and developed normally into amastigotes in the cytoplasm. By artificial in vivo tethering of TcUBP1 to the 3' untranslated region of a reporter mRNA we were able to determine that translation of the reporter was reduced by 8-fold, while its mRNA abundance was not significantly compromised. Inducible ectopic expression of TcUBP1 confirmed its role as a translational repressor, revealing significant reduction in the translation rate of multiple proteins, a reduction of polysomes, and promoting the formation of mRNA granules. Expression of TcUBP1 truncated forms revealed the requirement of both N and C-terminal glutamine-rich low complexity sequences for the development of the drop-like phenotype in early-log epimastigotes. We propose that a rise in TcUBP1 levels, in synchrony with nutritional deficiency, can promote the differentiation of T. cruzi epimastigotes into infective metacyclic trypomastigotes., Competing Interests: The authors have declared that no competing interests exist.

Membrane neuronal glycoprotein M6a is highly expressed in the brain and contributes to neural plasticity promoting neurite growth and spine and synapse formation. We have previously showed that chronic stressors alter hippocampal M6a mRNA levels in rodents and tree shrews. We now show that M6a glycoprotein can be detected in mouse blood. M6a is a transmembrane glycoprotein and, as such, unlikely to be free in blood. Here we demonstrate that, in blood, M6a is transported in extracellular vesicles (EVs). It is also shown that M6a-containing EVs are delivered from cultured primary neurons as well as from M6a-transfected COS-7 cells. Released EVs containing M6a can be incorporated into COS-7 cells changing its phenotype through formation of membrane protrusions. Thus, M6a-containing EVs might contribute to maintain cellular plasticity. M6a presence in blood was used to monitor stress effects. Chronic restraint stress modulated M6a protein level in a sex dependent manner. Analysis of individual animals indicated that M6a level variations depend on the stressor applied. The response to stressors in blood makes M6a amenable to further studies in the stress disorder field.

Neuronal glycoprotein M6a belongs to the tetraspan proteolipid protein (PLP) family. Mutations in GPM6A gene have been related to mental disorders like schizophrenia, bipolar disorders and claustrophobia. M6a is expressed mainly in neuronal cells of the central nervous system and it has been extensively related to neuronal plasticity. M6a induces neuritogenesis and axon/filopodium outgrowth; however its mechanism of action is still unresolved. We recently reported that the integrity of the transmembrane domains (TMDs) 2 and 4 are critical for M6a filopodia induction. There is also experimental data suggesting that M6a might be involved in synaptogenesis. In this regard, we have previously determined that M6a is involved in filopodia motility, a process that is described in the first step of the filopodial model for synaptogenesis. In this work we analyzed the possible involvement of M6a in synaptogenesis and spinogenesis, and evaluated the effect of two non-synonymous SNPs present in the coding region of TMD2-GPM6A in these processes. The results showed that endogenous M6a colocalized with both, pre-synaptic (synaptophysin) and post-synaptic (NMDA-R1), markers along of neuronal soma and dendrites. M6a-overexpressing neurons displayed an increased number of synaptophysin and NMDA-R1 puncta and, also, an increased number of colocalization puncta between both markers. Conversely, the number of synaptic puncta markers in neurons expressing nsSNP variants was similar to those of control neurons. Overexpression of M6a is accompanied by an increase in spine density, particularly in mature spines, as compared with neurons expressing mGFP or GPM6A nsSNP variants. Taken together, these results suggest that M6a contributes positively to spine and, likely, synapse formation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The p21-activated kinases (PAKs) of group I are the main effectors for the small Rho GTPases, critically involved in neurodevelopment, plasticity and maturation of the nervous system. Moreover, the neuronal complexity controlled by PAK1/PAK3 signaling determines the postnatal brain size and synaptic properties. Stress induces alterations at the level of structural and functional synaptic plasticity accompanied by reductions in size and activity of the hippocampus and the prefrontal cortex (PFC). These abnormalities are likely to contribute to the pathology of depression and, in part, reflect impaired cytoskeleton remodeling pointing to the role of Rho GTPase signaling. Thus, the present study assessed the expression of the group I PAKs and their activators in the brain of depressed subjects. Using quantitative polymerase chain reaction (qPCR), mRNA levels and coexpression of the group I PAKs: PAK1, PAK2, and PAK3 as well as of their activators: RAC1, CDC42 and ARHGEF7 were examined in postmortem samples from the PFC (n=25) and the hippocampus (n=23) of subjects with depression and compared to control subjects (PFC n=24; hippocampus n=21). Results demonstrated that mRNA levels of PAK1 and PAK3, are significantly reduced in the brain of depressed subjects, with PAK1 being reduced in the PFC and PAK3 in the hippocampus. No differences were observed for the ubiquitously expressed PAK2. Following analysis of gene coexpression demonstrated disruption of coordinated gene expression in the brain of subjects with depression. Abnormalities in mRNA expression of PAK1 and PAK3 as well as their altered coexpression patterns were detected in the brain of subjects with depression., Competing Interests: The authors report no biomedical financial interests or potential conflicts of interest. Dr. Pandey reports grants from USA: National Institute of Health, during the conduct of the study., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

Stress-responsive neuronal membrane glycoprotein M6a (Gpm6a) functions in neurite extension, filopodium and spine formation and synaptogenesis. The mechanisms of Gpm6a action in these processes are incompletely understood. Previously, we identified the actin regulator coronin-1a (Coro1a) as a putative Gpm6a interacting partner. Here, we used co-immunoprecipitation assays with the anti-Coro1a antibody to show that Coro1a associates with Gpm6a in rat hippocampal neurons. By immunofluorescence microscopy, we demonstrated that in hippocampal neurons Coro1a localizes in F-actin-enriched regions and some of Coro1a spots co-localize with Gpm6a labeling. Notably, the over-expression of a dominant-negative form of Coro1a as well as its down-regulation by siRNA interfered with Gpm6a-induced filopodium formation. Coro1a is known to regulate the plasma membrane translocation and activation of small GTPase Rac1. We show that Coro1a co-immunoprecipitates with Rac1 together with Gpm6a. Pharmacological inhibition of Rac1 resulted in a significant decrease in filopodium formation by Gpm6a. The same was observed upon the co-expression of Gpm6a with the inactive GDP-bound form of Rac1. In this case, the elevated membrane recruitment of GDP-bound Rac1 was detected as well. Moreover, the kinase activity of the p21-activated kinase 1 (Pak1), a main downstream effector of Rac1 that acts downstream of Coro1a, was required for Gpm6a-induced filopodium formation. Taken together, our results provide evidence that a signaling pathway including Coro1a, Rac1, and Pak1 facilitates Gpm6a-induced filopodium formation. Formation of filopodia by membrane glycoprotein M6a (Gpm6a) requires actin regulator coronin-1a (Coro1a), known to regulate plasma membrane localization and activation of Rac1 and its downstream effector Pak1. Coro1a associates with Gpm6a. Blockage of Coro1a, Rac1, or Pak1 interferes with Gpm6a-induced filopodium formation. Moreover, Gpm6a facilitates Rac1 membrane recruitment. Altogether, a mechanistic insight into the process of Gpm6a-induced neuronal filopodium formation is provided., (© 2016 International Society for Neurochemistry.)

Regulation of gene expression in trypanosomatid parasitic protozoa is mainly achieved posttranscriptionally. RNA-binding proteins (RBPs) associate to 3' untranslated regions in mRNAs through dedicated domains such as the RNA recognition motif (RRM). Trypanosoma cruzi UBP1 (TcUBP1) is an RRM-type RBP involved in stabilization/degradation of mRNAs. TcUBP1 uses its RRM to associate with cytoplasmic mRNA and to mRNA granules under starvation stress. Here, we show that under starvation stress, TcUBP1 is tightly associated with condensed cytoplasmic mRNA granules. Conversely, under high nutrient/low density-growing conditions, TcUBP1 ribonucleoprotein (RNP) complexes are lax and permeable to mRNA degradation and disassembly. After dissociating from mRNA, TcUBP1 can be phosphorylated only in unstressed parasites. We have identified TcP22, the ortholog of mammalian P32/C1QBP, as an interactor of TcUBP1 RRM. Overexpression of TcP22 decreased the number of TcUBP1 granules in starved parasites in vivo. Endogenous TcUBP1 RNP complexes could be dissociated in vitro by addition of recombinant TcP22, a condition stimulating TcUBP1 phosphorylation. Biochemical and in silico analysis revealed that TcP22 interacts with the RNA-binding surface of TcUBP1 RRM. We propose a model for the decondensation of TcUBP1 RNP complexes in T. cruzi through direct interaction with TcP22 and phosphorylation., (© 2015 John Wiley & Sons Ltd.)

Membrane glycoprotein M6a, which belongs to the tetraspan proteolipid protein family, promotes structural plasticity in neurons and cell lines by unknown mechanisms. This glycoprotein is encoded by Gpm6a, a stress-regulated gene. The hippocampus of animals chronically stressed by either psychosocial or physical stressors shows decreased M6a expression. Stressed Gpm6a-null mice develop a claustrophobia-like phenotype. In humans, de novo duplication of GPM6A results in learning/behavioral abnormalities, and two single-nucleotide polymorphisms (SNPs) in the non-coding region are linked to mood disorders. Here, we studied M6a dimerization in neuronal membranes and its functional relevance. We showed that the self-interaction of M6a transmembrane domains (TMDs) might be driving M6a dimerization, which is required to induce filopodia formation. Glycine mutants located in TMD2 and TMD4 of M6a affected its dimerization, thus preventing M6a-induced filopodia formation in neurons. In silico analysis of three non-synonymous SNPs located in the coding region of TMDs suggested that these mutations induce protein instability. Indeed, these SNPs prevented M6a from being functional in neurons, owing to decreased stability, dimerization or improper folding. Interestingly, SNP3 (W141R), which caused endoplasmic reticulum retention, is equivalent to that mutated in PLP1, W161L, which causes demyelinating Pelizaeus-Merzbacher disease. In this work we analyzed the functional contribution of transmembrane domains (TMDs) of the neuronal membrane glycoprotein M6a. We determined that certain glycines present in TMD2 and TMD4 are critical for filopodia induction in neurons. In addition, three nsSNPs located in the coding region of TMD2 and TMD3 of GPM6A impair M6a function by affecting its stability, folding and dimer formation., (© 2015 International Society for Neurochemistry.)

Background: Expression of the neuronal membrane glycoprotein M6a (GPM6A), the proteolipid protein (PLP/DM20) family member, is downregulated in the hippocampus of chronically stressed animals. Its neuroplastic function involves a role in neurite formation, filopodium outgrowth and synaptogenesis through an unknown mechanism. Disruptions in neuroplasticity mechanisms have been shown to play a significant part in the etiology of depression. Thus, the current investigation examined whether GPM6A expression is also altered in human depressed brain., Methods: Expression levels and coexpression patterns of GPM6A, GPM6B, and PLP1 (two other members of PLP/DM20 family) as well as of the neuroplasticity-related genes identified to associate with GPM6A were determined using quantitative polymerase chain reaction (qPCR) in postmortem samples from the hippocampus (n = 18) and the prefrontal cortex (PFC) (n = 25) of depressed suicide victims and compared with control subjects (hippocampus n = 18; PFC n = 25). Neuroplasticity-related proteins that form complexes with GPM6A were identified by coimmunoprecipitation technique followed by mass spectrometry., Results: Results indicated transcriptional downregulation of GPM6A and GPM6B in the hippocampus of depressed suicides. The expression level of calcium/calmodulin-dependent protein kinase II alpha (CAMK2A) and coronin1A (CORO1A) was also significantly decreased. Subsequent analysis of coexpression patterns demonstrated coordinated gene expression in the hippocampus and in the PFC indicating that the function of these genes might be coregulated in the human brain. However, in the brain of depressed suicides this coordinated response was disrupted., Conclusions: Disruption of coordinated gene expression as well as abnormalities in GPM6A and GPM6B expression and expression of the components of GPM6A complexes were detected in the brain of depressed suicides., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15 mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15 mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼ threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Neuronal glycoprotein M6a is involved in neuronal plasticity, promoting neurite and filopodia outgrowth and, likely, synaptogenesis. Polymorphisms in the human M6a gene GPM6A have recently been associated with mental illnesses such as schizophrenia, bipolar disorders, and claustrophobia. Nevertheless, the molecular bases underlying these observations remain unknown. We have previously documented that, to induce filopodia formation, M6a depends on the association of membrane lipid microdomains and the activation of Src and mitogen-activated protein kinase kinases. Here, in silico analysis of the phosphorylation of tyrosine 251 (Y251) at the C-terminus of M6a showed that it could be a target of Src kinases. We examined whether phosphorylation of M6a at Y251 affects neurite and filopodia outgrowth and the targets involved in its signal propagation. This work provides evidence that the Src kinase family and the phosphatidylinositide 3-kinase (PI3K), but not Ras, participate in M6a signal cascade leading to neurite/filopodia outgrowth in hippocampal neurons and murine neuroblastoma N2a cells. Phosphorylation of M6a at Y251 is essential only for neurite outgrowth by the PI3K/AKT-mediated pathway and, moreover, rescues the inhibition caused by selective Src inhibitor and external M6a monoclonal antibody treatment. Thus, we suggest that phosphorylation of M6a at Y251 is critical for a specific stage of neuronal development and triggers redundant signaling pathways leading to neurite extension., (© 2014 Wiley Periodicals, Inc.)

Prenatal stress (PS) exerts strong impact on fetal brain development and on adult offspring brain functions. Previous work demonstrated that chronic stress alters the mRNA expression of GPM6A, a neuronal glycoprotein involved in filopodium extension. In this work, we analyzed the effect of PS on gpm6a expression and the epigenetic mechanisms involved. Pregnant Wistar rats received restraint stress during the last week of gestation. Male offspring were sacrificed on postnatal days 28 and 60. Hippocampus and prefrontal cortex samples were analyzed for gene expression (qPCR for mRNAs and microRNAs), methylation status (bisulfite conversion) and protein levels. Hippocampal neurons in culture were used to analyze microRNA overexpression effects. Prenatal stress induced changes in gpm6a levels in both tissues and at both ages analyzed, indicating a persistent effect. Two CpG islands in the gpm6a gene were identified. Variations in the methylation pattern at three specific CpGs were found in hippocampus, but not in PFC samples from PS offspring. microRNAs predicted to target gpm6a were identified in silico. qPCR measurements showed that PS modified the expression of several microRNAs in both tissues, being microRNA-133b the most significantly altered. Further studies overexpressing this microRNA in neuronal cultures showed a reduction in gmp6a mRNA and protein level. Moreover filopodium density was also reduced, suggesting that GPM6A function was affected. Gestational stress affected gpm6a gene expression in offspring likely through changes in methylation status and in posttranscriptional regulation by microRNAs. Thus, our findings propose gpm6a as a novel target for epigenetic regulation during prenatal stress.

In eukaryotic cells, a group of messenger ribonucleic acids (mRNAs) encoding functionally interrelated proteins together with the trans-acting factors that coordinately modulate their expression is termed a post-transcriptional regulon, due to their partial analogy to a prokaryotic polycistron. This mRNA clustering is organized by sequence-specific RNA-binding proteins (RBPs) that bind cis-regulatory elements in the noncoding regions of genes, and mediates the synchronized control of their fate. These recognition motifs are often characterized by conserved sequences and/or RNA structures, and it is likely that various classes of cis-elements remain undiscovered. Current evidence suggests that RNA regulons govern gene expression in trypanosomes, unicellular parasites which mainly use post-transcriptional mechanisms to control protein synthesis. In this study, we used motif discovery tools to test whether groups of functionally related trypanosomatid genes contain a common cis-regulatory element. We obtained conserved structured RNA motifs statistically enriched in the noncoding region of 38 out of 53 groups of metabolically related transcripts in comparison with a random control. These motifs have a hairpin loop structure, a preferred sense orientation and are located in close proximity to the open reading frames. We found that 15 out of these 38 groups represent unique motifs in which most 3'-UTR signature elements were group-specific. Two extensively studied Trypanosoma cruzi RBPs, TcUBP1 and TcRBP3 were found associated with a few candidate RNA regulons. Interestingly, 13 motifs showed a strong correlation with clusters of developmentally co-expressed genes and six RNA elements were enriched in gene clusters affected after hyperosmotic stress. Here we report a systematic genome-wide in silico screen to search for novel RNA-binding sites in transcripts, and describe an organized network of several coordinately regulated cohorts of mRNAs in T. cruzi. Moreover, we found that structured RNA elements are also conserved in other human pathogens. These results support a model of regulation of gene expression by multiple post-transcriptional regulons in trypanosomes.

The trans-sialidase of Trypanosoma cruzi (TcTS) catalyzes the transfer of sialic acid from host glycoconjugates to terminal β-galactopyranosides in the mucins of the parasite. During infection, the enzyme is actively shed by the parasite to the bloodstream inducing hematological alterations. Lactitol prevents cell apoptosis caused by the TcTS, although it is rapidly eliminated from the circulatory system. Linear polyethyleneglycol (PEG) conjugates of lactose analogs were prepared but their clearance from blood was still quite fast. With the aim of improving their circulating half-lives in vivo, we now synthesized covalent conjugates of eight-arm PEG. The star-shape of these conjugates allows an increase in the molecular weight together with the loading of the active sugar. Two approaches were used for PEGylation of disaccharide derivatives containing β-D-Galp as the non-reducing unit. (1) Amide formation between benzyl β-D-galactopyranosyl-(1→6)-2-amino-2-deoxy-α-D-glucopyranoside and a succinimide-activated PEG. (2) Conjugation of lactobionolactone with amino end-functionalized PEG. Two 8-arm PEG derivatives (20 and 40 kDa) were used for each sugar. Substitution of all arms was proved by (1)H nuclear magnetic resonance (NMR) spectroscopy. The bioavailability of the conjugates in mice plasma was considerably improved with respect to the 5 kDa linear PEG conjugates retaining their inhibitory properties.

Background: Members of the proteolipid protein family, including the four-transmembrane glycoprotein M6a, are involved in neuronal plasticity in mammals. Results from our group previously demonstrated that M6, the only proteolipid protein expressed in Drosophila, localizes to the cell membrane in follicle cells. M6 loss triggers female sterility, which suggests a role for M6 in follicular cell remodeling. These results were the basis of the present study, which focused on the function and requirements of M6 in the fly nervous system., Results: The present study identified two novel, tissue-regulated M6 isoforms with variable N- and C- termini, and showed that M6 is the functional fly ortholog of Gpm6a. In the adult brain, the protein was localized to several neuropils, such as the optic lobe, the central complex, and the mushroom bodies. Interestingly, although reduced M6 levels triggered a mild rough-eye phenotype, hypomorphic M6 mutants exhibited a defective response to light., Conclusions: Based on its ability to induce filopodium formation we propose that M6 is key in cell remodeling processes underlying visual system function. These results bring further insight into the role of M6/M6a in biological processes involving neuronal plasticity and behavior in flies and mammals.

Trypanosoma cruzi, the agent of Chagas disease, does not seem to control gene expression through regulation of transcription initiation and makes use of post-transcriptional mechanisms. We report here a 43-nt U-rich RNA element located in the 3'-untranslated region (3'-UTR) of a large number of T. cruzi mRNAs that is important for mRNA abundance in the intracellular amastigote stage of the parasite. Whole genome scan analysis, differential display RT-PCR, Northern blot, and RT-PCR analyses were used to determine the transcript levels of more than 900 U-rich-containing mRNAs of large gene families as well as single and low copy number genes. Our results indicate that the 43-nt U-rich mRNA element is preferentially present in amastigotes. The cis-element of a protein kinase 3'-UTR but not its mutated version promoted the expression of the green fluorescent protein reporter gene in amastigotes. The regulatory cis-element, but not its mutated version, was also shown to interact with the trypanosome-specific RNA-binding protein (RBP) TcUBP1 but not with other related RBPs. Co-immunoprecipitation experiments of TcUBP1-containing ribonucleoprotein complexes formed in vivo validated the interaction with representative endogenous RNAs having the element. These results suggest that this 43-nt U-rich element together with other yet unidentified sequences might be involved in the modulation of abundance and/or translation of subsets of transcripts in the amastigote stage.

Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi and is one of the most important endemic problems in Latin America. Lately, it has also become a health concern in the United States and Europe. Currently, a diagnosis of Chagas' disease and the screening of blood supplies for antiparasite antibodies are achieved by conventional serological tests that show substantial variation in the reproducibility and reliability of their results. In addition, the specificity of these assays is curtailed by antigenic cross-reactivity with sera from patients affected by other endemic diseases, such as leishmaniasis. Here we used a highly sensitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) to evaluate a recombinant protein core of a mucin-like molecule (termed trypomastigote small surface antigen [TSSA]) for the detection of specific serum antibodies in a broad panel of human sera. The same samples were evaluated by CL-ELISA using as the antigen either a mixture of native T. cruzi trypomastigote mucins or an epimastigote extract and, for further comparison, by conventional serologic tests, such as an indirect hemagglutination assay and indirect immunofluorescence assay. TSSA showed ∼87% sensitivity among the seropositive Chagasic panel, a value which was increased up to >98% when only parasitologically positive samples were considered. More importantly, TSSA showed a significant increase in specificity (97.4%) compared to those of currently used assays, which averaged 80 to 90%. Overall, our data demonstrate that recombinant TSSA may be a useful antigen for the immunodiagnosis of Chagas' disease.

A neuronal integral membrane glycoprotein M6a has been suggested to be involved in a number of biological processes, including neuronal remodeling and differentiation, trafficking of mu-opioid receptors, and Ca(2+) transportation. Moreover, pathological situations such as chronic stress in animals and depression in humans have been associated with alterations in M6a sequence and expression. The mechanism of action of M6a is essentially unknown. In this work, we analyze the relevance of M6a distribution in plasma membrane, namely its lipid microdomain targeting, for its biological function in filopodia formation. We demonstrate that M6a is localized in membrane microdomains compatible with lipid rafts in cultured rat hippocampal neurons. Removal of cholesterol from neuronal membranes with methyl-β-cyclodextrin decreases M6a-induced filopodia formation, an effect that is reversed by the addition of cholesterol. Inhibition of Src kinases and MAPK prevents filopodia formation in M6a-over-expressing neurons. Src-deficient SYF cells over-expressing M6a fail to promote filopodia formation. Taken together, our findings reveal that the association of M6a with lipid rafts is important for its role in filopodia formation and Src and MAPK kinases participate in M6a signal propagation., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Trypanosomatids are protozoan micro-organisms that cause serious health problems in humans and domestic animals. In addition to their medical relevance, these pathogens have novel biological structures and processes. From nuclear DNA transcription to mRNA translation, trypanosomes use unusual mechanisms to control gene expression. For example, transcription by RNAPII (RNA polymerase II) is polycistronic, and only a few transcription initiation sites have been identified so far. The sequences present in the polycistronic units code for proteins having unrelated functions, that is, not involved in a similar metabolic pathway. Owing to these biological constraints, these micro-organisms regulate gene expression mostly by post-transcriptional events. Consequently, the function of proteins that recognize RNA elements preferentially at the 3' UTR (untranslated region) of transcripts is central. It was recently shown that mRNP (messenger ribonucleoprotein) complexes are organized within post-transcriptional operons to co-ordinately regulate gene expression of functionally linked transcripts. In the present chapter we will focus on particular characteristics of gene expression in the so-called TriTryp parasites: Trypanosoma cruzi, Trypanosoma brucei and Leishmania major.

We had previously shown that the transmembrane glycoprotein M6a, a member of the proteolipid protein (PLP) family, regulates neurite/filopodium outgrowth, hence, M6a might be involved in neuronal remodeling and differentiation. In this work we focused on M6, the only PLP family member present in Drosophila, and ortholog to M6a. Unexpectedly, we found that decreased expression of M6 leads to female sterility. M6 is expressed in the membrane of the follicular epithelium in ovarioles throughout oogenesis. Phenotypes triggered by M6 downregulation in hypomorphic mutants included egg collapse and egg permeability, thus suggesting M6 involvement in eggshell biosynthesis. In addition, RNAi-mediated M6 knockdown targeted specifically to follicle cells induced an arrest of egg chamber development, revealing that M6 is essential in oogenesis. Interestingly, M6-associated phenotypes evidenced abnormal changes of the follicle cell shape and disrupted follicular epithelium in mid- and late-stage egg chambers. Therefore, we propose that M6 plays a role in follicular epithelium maintenance involving membrane cell remodeling during oogenesis in Drosophila.

Trypanosoma cruzi, the agent of Chagas disease, expresses a unique enzyme, the trans-sialidase (TcTS) involved in the transfer of sialic acid from host glycoconjugates to mucins of the parasite. The enzyme is shed to the medium and may affect the immune system of the host. We have previously described that lactose derivatives effectively inhibited the transfer of sialic acid to N-acetyllactosamine. Lactitol also prevented the apoptosis caused by the TcTS, although it is rapidly eliminated from the circulatory system. In this paper we report covalent conjugation of polyethylene glycol (PEG) with lactose, lactobionolactone and benzyl beta-D-galactopyranosyl-(1-->6)-2-amino-2-deoxy-alpha-D-glucopyranoside (1) with the hope to improve the bioavailability, though retaining their inhibitory properties. Different conjugation methods have been used and the behavior of the PEGylated products in the TcTS reaction was studied.

Chronic stress causes morphological alterations in the hippocampus of rodents and tree shrews, including atrophy of CA3 dendrites and loss of synapses. The molecular mechanisms underlying these structural changes remain largely unknown. We have previously identified M6a as a stress responsive gene and shown that M6a is involved in filopodium/spine outgrowth and, likely, synapse formation. M6a belongs to the proteolipid protein (PLP) family, all of their members having four transmembrane domains that allow their localization at the plasma membrane. In the present work, we analyzed other members of this family, the closely related M6b as well as PLP and its splice variant DM20. We found that chronic restraint stress in mice reduces M6b and DM20, but not PLP, mRNA levels in the hippocampus. In addition, M6b and DM20, but again not PLP, induce filopodium formation in primary cultures of hippocampal neurons. Several M6b protein isoforms were studied, all of them having similar effects except for the one lacking the transmembrane domains. Our results reveal a conserved cellular function and a stress-mediated regulation among members of the proteolipid protein family, suggesting an involvement of proteolipid proteins in the stress response., ((c) 2009 Wiley-Liss, Inc.)

In eukaryotic cells, a regulated import and export of factors is required to fulfill the requirements of precise gene expression. Post-transcriptional regulation of gene expression has proven to provide ubiquitous control, as well as a quick response to environmental changes when required. RNA-binding proteins (RBP) are involved in the several steps at which mRNA biogenesis, stability, translation and decay is exerted. The most characterized RBPs contain single or multiple copies of an RNA Recognition Motif (RRM). Here, we concentrate on RRMs mediating protein nuclear import by virtue of its ability to interact with proteins, besides interacting with nucleic acids. The consensus on how RRM-protein interactions take place is non-existent, and so is the involvement of the RRM as a nuclear localization signal (NLS). Within the cases examined, the single RRM from a trypanosome RBP behaves as a structural NLS, alternating nuclear import and RNA-binding.

M6a is a neuronal membrane glycoprotein whose expression diminishes during chronic stress. M6a overexpression in rat primary hippocampal neurons induces the formation of filopodial protrusions that could be spine precursors. As the filopodium and spine motility has been associated with synaptogenesis, we analysed the motility of M6a-induced protrusions by time-lapse imaging. Our data demonstrate that the motile protrusions formed by the neurons overexpressing M6a were more abundant and moved faster than those formed in control cells. When different putative M6a phosphorylation sites were mutated, the neurons transfected with a mutant lacking intracellular phosphorylation sites bore filopodia, but these protrusions did not move as fast as those formed by cells overexpressing wild-type M6a. This suggests a role for M6a phosphorylation state in filopodium motility. Furthermore, we show that M6a-induced protrusions could be stabilized upon contact with presynaptic region. The motility of filopodia contacting or not neurites overexpressing synaptophysin was analysed. We show that the protrusions that apparently contacted synaptophysin-labeled cells exhibited less motility. The behavior of filopodia from M6a-overexpressing cells and control cells was alike. Thus, M6a-induced protrusions may be spine precursors that move to reach presynaptic membrane. We suggest that M6a is a key molecule for spine formation during development.

RNA-binding proteins (RBPs) and RNA metabolism are considered to be important for modulating gene expression in trypanosomes, because these protozoan parasites mainly rely on post-transcriptional mechanisms to regulate protein levels. Previously, we have identified TcUBP1, a single RNA recognition motif (RRM)-type RBP from Trypanosoma cruzi. TcUBP1 is a cytoplasmic protein with roles in stabilization/degradation of mRNAs and in the protection of transcripts through their recruitment into cytoplasmic granules. We now show that TcUBP1, and the closely related protein TcUBP2, can be found in small amounts in the nucleus under normal conditions, and are able to accumulate in the nucleus under arsenite stress. The kinetics of nuclear accumulation, and export to the cytoplasm, are consistent with the shuttling of TcUBP1 between the nucleus and the cytoplasm. The sequence required for TcUBP1 nuclear accumulation was narrowed to the RRM, and point mutations affecting RNA binding abolished nuclear import. This RRM was also shown to be efficiently exported from the nucleus in unstressed parasites, a property that relied on the binding to RNA. TcUBP1 nuclear accumulation was dependent on active transcription, and colocalized with transcripts in the nucleus, suggesting nuclear binding of the mRNA. We propose that TcUBP1 could be linking the mRNA metabolism at both sides of the nuclear pore complex, using the RRM as a nuclear localization signal, and being exported as a cargo on mRNA.

Gpm6a was identified as a stress-responsive gene in the hippocampal formation. This gene is down-regulated in the hippocampus of both socially and physically stressed animals, and this effect can be reversed by antidepressant treatment. Previously we showed that the stress-regulated protein M6a is a key modulator for neurite outgrowth and filopodium/spine formation. In the present work, mutational analysis was used to characterize the action of M6a at the molecular level. We show that four cysteines 162, 174, 192, and 202 within EC2 are functionally crucial sites. The presence of cysteines 162 and 202 is essential for the efficient cell surface expression of the M6a protein. In contrast, cysteines 174 and 192, which form a disulfide bridge as shown by biochemical analysis, are not required for the efficient surface expression of M6a. Their mutation to alanine does not interfere with the localization of M6a to filopodial protrusions in primary hippocampal neurons. The neurons expressing C174A and/or C192A mutants display decreased filopodia number. In non-permeabilized cells, these mutant proteins are not recognized by a function-blocking monoclonal antibody directed to M6a. Moreover, neurons in contact with axons expressing C174A/C192A mutant display significantly lower density of presynaptic clusters over their dendrites. Taken together, this study demonstrates that cysteines in the EC2 domain are critical for the role of M6a in filopodium outgrowth and synaptogenesis.

trans-Sialidase from Trypanosoma cruzi (TcTS) has emerged as a potential drug target for treatment of Chagas disease. Here, we report the results of virtual screening for the discovery of novel TcTS inhibitors, which targeted both the sialic acid and sialic acid acceptor sites of this enzyme. A library prepared from the Evotec database of commercially available compounds was screened using the molecular docking program GOLD, following the application of drug-likeness filters. Twenty-three compounds selected from the top-scoring ligands were purchased and assayed using a fluorimetric assay. Novel inhibitor scaffolds, with IC(50) values in the submillimolar range were discovered. The 3-benzothiazol-2-yl-4-phenyl-but-3-enoic acid scaffold was studied in more detail, and TcTS inhibition was confirmed by an alternative sialic acid transfer assay. Attempts to obtain crystal structures of these compounds with TcTS proved unsuccessful but provided evidence of ligand binding at the active site.

Background: Trypanosomes mostly control gene expression by post-transcriptional events such as modulation of mRNA stability and translational efficiency. These mechanisms involve RNA-binding proteins (RBPs), which associate with transcripts to form messenger ribonucleoprotein (mRNP) complexes., Results: In this study, we report the identification of mRNA targets for Trypanosoma cruzi U-rich RBP 1 (TcUBP1) and T. cruzi RBP 3 (TcRBP3), two phylogenetically conserved proteins among Kinetoplastids. Co-immunoprecipitated RBP-associated RNAs were extracted from mRNP complexes and binding of RBPs to several targets was confirmed by independent experimental assays. Analysis of target transcript sequences allowed the identification of different signature RNA motifs for each protein. Cis-elements for RBP binding have a stem-loop structure of 30-35 bases and are more frequently represented in the 3'-untranslated region (UTR) of mRNAs. Insertion of the correctly folded RNA elements to a non-specific mRNA rendered it into a target transcript, whereas substitution of the RNA elements abolished RBP interaction. In addition, RBPs competed for RNA-binding sites in accordance with the distribution of different and overlapping motifs in the 3'-UTRs of common mRNAs., Conclusion: Functionally related transcripts were preferentially associated with a given RBP; TcUBP1 targets were enriched in genes encoding proteins involved in metabolism, whereas ribosomal protein-encoding transcripts were the largest group within TcRBP3 targets. Together, these results suggest coordinated control of different mRNA subsets at the post-transcriptional level by specific RBPs.

The trans-sialidase from Trypanosoma cruzi catalyzes the transfer of a sialic acid moiety from sialylated donor substrates to the terminal galactose moiety of lactose and lactoside acceptors to yield alpha-(2,3)-sialyllactose or its derivatives with net retention of anomeric configuration. Through kinetic analyses in which the concentrations of two different donor aryl alpha-sialoside substrates and the acceptor substrate lactose were independently varied, we have demonstrated that this enzyme follows a ping-pong bi-bi kinetic mechanism. This is supported for both the native enzyme and a mutant (D59A) in which the putative acid/base catalyst has been replaced by the demonstration of the half-reaction in which a sialyl-enzyme intermediate is formed. Mass spectrometric analysis of the protein directly demonstrates the formation of a covalent intermediate, while the observation of release of a full equivalent of p-nitrophenol by the mutant in a pre-steady state burst provides further support. The active site nucleophile is confirmed to be Tyr342 by trapping of the sialyl-enzyme intermediate using the D59A mutant and sequencing of the purified peptic peptide. The role of D59 as the acid/base catalyst is confirmed by chemical rescue studies in which activity is restored to the D59A mutant by azide and a sialyl azide product is formed.

The sialic acid present in the protective surface mucin coat of Trypanosoma cruzi is added by a membrane anchored trans-sialidase (TcTS), a modified sialidase that is expressed from a large gene family. In this work, we analyzed single domain camelid antibodies produced against trans-sialidase. Llamas were immunized with a recombinant trans-sialidase and inhibitory single-domain antibody fragments were obtained by phage display selection, taking advantage of a screening strategy using an inhibition test instead of the classic binding assay. Four single domain antibodies displaying strong trans-sialidase inhibition activity against the recombinant enzyme were identified. They share the same complementarity-determining region 3 length (17 residues) and have very similar sequences. This result indicates that they likely derived from a unique clone. Probably there is only one structural solution for tight binding inhibitory antibodies against the TcTS used for immunization. To our surprise, this single domain antibody that inhibits the recombinant TcTS, failed to inhibit the enzymatic activity present in parasite extracts. Analysis of individual recombinant trans-sialidases showed that enzymes expressed from different genes were inhibited to different extents (from 8 to 98%) by the llama antibodies. Amino acid changes at key positions are likely to be responsible for the differences in inhibition found among the recombinant enzymes. These results suggest that the presence of a large and diverse trans-sialidase family might be required to prevent the inhibitory response against this essential enzyme and might thus constitute a novel strategy of T. cruzi to evade the host immune system.

In the African trypanosome Trypanosoma brucei nearly all control of gene expression is posttranscriptional; sequences in the 3'-untranslated regions of mRNAs determine the steady-state mRNA levels by regulation of RNA turnover. Here we investigate the roles of two related proteins, TbUBP1 and TbUBP2, containing a single RNA recognition motif, in trypanosome gene expression. TbUBP1 and TbUBP2 are in the cytoplasm and nucleus, comprise ca. 0.1% of the total protein, and are not associated with polysomes or RNA degradation enzymes. Overexpression of TbUBP2 upregulated the levels of several mRNAs potentially involved in cell division, including the CFB1 mRNA, which encodes a protein with a cyclin F-box domain. CFB1 regulation was mediated by the 3'-untranslated region and involved stabilization of the mRNA. Depletion of TbUBP2 and TbUBP1 inhibited growth and downregulated expression of the cyclin F box protein gene CFB2; trans splicing was unaffected. The results of pull-down assays indicated that all tested mRNAs were bound to TbUBP2 or TbUBP1, with some preference for CFB1. We suggest that TbUBP1 and TbUBP2 may be relatively nonspecific RNA-binding proteins and that specific effects of overexpression or depletion could depend on competition between various different proteins for RNA binding.

Trypanosomes are outstanding examples of the importance of mRNA metabolism in the regulation of gene expression, as these unicellular eukaryotes mostly control protein synthesis by post-transcriptional mechanisms. Here, we show that mRNA metabolism in these organisms involves recruitment of mRNAs and proteins to microscopically visible ribonucleoprotein granules in the cytoplasm. These structures engage transcripts that are being translated and protect mRNAs from degradation. Analysis of the protein composition of trypanosomal mRNA granules indicated that they contain orthologous proteins to those present in P bodies and stress granules from metazoan organisms. Formation of mRNA granules was observed after carbon-source deprivation of parasites in axenic culture. More important, mRNA granules are formed naturally in trypanosomes present in the intestinal tract of the insect vector. We suggest that trypanosomes make use of mRNA granules for transient transcript protection as a strategy to cope with periods of starvation that they have to face during their complex life cycles.

Trypanosomes are unique eukaryotic cells, in that they virtually lack mechanisms to control gene expression at the transcriptional level. These microorganisms mostly control protein synthesis by posttranscriptional regulation processes, like mRNA stabilization and degradation. Transcription in these cells is polycistronic. Tens to hundreds of protein-coding genes of unrelated function are arrayed in long clusters on the same DNA strand. Polycistrons are cotranscriptionally processed by trans-splicing at the 5' end and polyadenylation at the 3' end, generating monocistronic units ready for degradation or translation. In this work, we show that some trans-splicing/polyadenylation sites may be skipped during normal polycistronic processing. As a consequence, dicistronic units or monocistronic transcripts having long 3' UTRs are produced. Interestingly, these unspliced transcripts can be processed into mature mRNAs by the conventional trans-splicing/polyadenylation events leading to translation. To our knowledge, this is a previously undescribed mRNA maturation by trans-splicing uncoupled from transcription. We identified an RNA-recognition motif-type protein, homologous to the mammalian polypyrimidine tract-binding protein, interacting with one of the partially processed RNAs analyzed here that might be involved in exon skipping. We propose that splice-site skipping might be part of a posttranscriptional mechanism to regulate gene expression in trypanosomes, through the generation of premature nontranslatable RNA molecules.

The procyclic stage of Trypanosoma brucei in the insect vector expresses a surface-bound trans-sialidase (TbTS) that transfers sialic acid from glycoconjugates in the environment to glycosylphosphatidylinositol-anchored proteins on its surface membrane. RNA interference against TbTS abolished trans-sialidase activity in procyclic cells but did not diminish sialidase activity, suggesting the presence of a separate sialidase enzyme for hydrolyzing sialic acid. A search of the T. brucei genome sequence revealed seven other putative genes encoding proteins with varying similarity to TbTS. RNA interference directed against one of these proteins, TbSA C, greatly decreased the sialidase activity but had no effect on trans-sialidase activity. The deduced amino acid sequence of TbSA C shares only 40% identity with TbTS but conserves most of the relevant residues required for catalysis. However, the sialidase has a tryptophan substitution for a tyrosine at position 170 that is crucial in binding the terminal galactose that accepts the transferred sialic acid. When this same tryptophan substitution in the sialidase was placed into the recombinant trans-sialidase, the mutant enzyme lost almost all of its trans-sialidase activity and increased its sialidase activity, further confirming that the gene and protein identified correspond to the parasite sialidase. Thus, in contrast to all other trypanosomes analyzed to date that express either a trans-sialidase or a sialidase but not both, T. brucei expresses these two enzymatic activities in two separate proteins. These results suggest that African trypanosomes could regulate the amount of critical sialic acid residues on their surface by modulating differential expression of each of these enzymes.

Polysialic acid (PSA) attached to NCAM is involved in cell-cell interactions participating in structural and functional plasticity of neuronal circuits. Two polysialyltransferases, ST8SiaII/STX and ST8SiaIV/PST, polysialylate NCAM. We previously suggested that ST8SiaII/STX is the key enzyme for polysialylation in hippocampus. Here, polysialyltransferase mRNA interference experiments showed that, knock down of ST8SiaIV/PST transcripts did not affect PSA expression, but PSA was almost absent from neuronal surfaces when ST8SiaII/STX mRNA was interfered. Non-polysialylated neurons bore a similar number of neurites per cell than polysialylated neurons. However, non-polysialylated processes were shorter and a lower density of synaptophysin clusters accompanied this reduced neuritic growth. Therefore, ST8SiaII/STX expression is essential to allow a correct neuritic development at initial stages of hippocampus ontogeny.

The trans-sialidase, a modified sialidase that transfers sialyl residues among macromolecules, is a unique enzymatic activity expressed by some parasitic trypanosomes being essential for their survival in the mammalian host and/or in the insect vector. The enzyme from Trypanosoma cruzi, the agent of Chagas disease, is found in blood and able to act far from the infection site by inducing apoptosis in cells from the immune system. A central and still unsolved question is whether trans-sialidase-mediated addition or removal of sialic acid to/from host acceptor molecules is the event associated with the apoptosis induced by the enzyme. Here we show that lactitol, a competitive inhibitor that precluded the transference of the sialyl residue to endogenous acceptors but not the hydrolase activity of the enzyme, prevented ex vivo and in vivo the apoptosis caused by the trans-sialidase. By lectin histochemistry, the transference of sialyl residue to the cell surface was demonstrated in vivo and found associated with the apoptosis induction. The sialylation of the CD43 mucin, a key molecule involved in trans-sialidase-apoptotic process, was readily detected and also prevented by lactitol on thymocytes. Therefore, lesions induced by trans-sialidase on the immune system are due to the sialylation of endogenous acceptor molecules.

The surface of the protozoan parasite Trypanosoma cruzi is covered in mucins, which contribute to parasite protection and to the establishment of a persistent infection. Their importance is highlighted by the fact that the approximately 850 mucin-encoding genes comprise approximately 1% of the parasite genome and approximately 6% of all predicted T. cruzi genes. The coordinate expression of a large repertoire of mucins containing variable regions in the mammal-dwelling stages of the T. cruzi life cycle suggests a possible strategy to thwart the host immune response. Here, we discuss the expression profiling of T. cruzi mucins, the mechanisms leading to the acquisition of mucin diversity and the possible consequences of a mosaic surface coat in the interplay between parasite and host.

The surface of Trypanosoma cruzi is covered by different groups of mucins that are differentially expressed during the parasite life cycle. We have previously identified the major mucins from the bloodstream trypomastigote stage. Here, we present additional evidence that together with our previous observations allows for the identification of a second mucin group also expressed in the mammal-dwelling stages, but predominant in the intracellular amastigote. These mucins are encoded by many genes, are mostly composed of tandem repeats and are highly conserved except for an exposed hypervariable (HV) N-terminal peptide. Antibodies against HV-peptides are restricted to approximately 50% of the chronically infected human population, are monospecific (i.e. directed towards a single HV), and display low-avidity. In contrast, immunization with a single HV-peptide triggers high-avidity, cross-reacting humoral responses against multiple HV sequences, but not against other T. cruzi surface antigens. The diversity present in the HV regions and the characteristics of the antibody response against them suggest a role of these molecules in eluding and/or modulating the mammalian host immune system.

Background: Chronic stress has significant effects on hippocampal structure and function. We have previously identified nerve growth factor (NGF), membrane glycoprotein 6a (M6a), the guanine nucleotide binding protein (G protein) alpha q polypeptide (GNAQ), and CDC-like kinase 1 (CLK-1) as genes regulated by psychosocial stress and clomipramine treatment in the hippocampus of tree shrews. These genes encode proteins involved in neurite outgrowth., Methods: To analyze whether regulation of the above-mentioned genes is conserved between different species, stressors, and antidepressant drugs, we subjected mice to repeated restraint stress and tianeptine treatment and measured hippocampal messenger RNA (mRNA) levels by real time reverse transcription polymerase chain reaction (RT-PCR)., Results: Chronically stressed mice displayed a reduction in transcript levels for NGF, M6a, GNAQ, and CLK-1. In addition, other genes implicated in neuronal plasticity, such as brain-derived neurotrophic factor (BDNF), cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), protein kinase C (PKC), neural cell adhesion molecule (NCAM), and synapsin I were downregulated in stressed mice. Tianeptine treatment reversed the stress effects for the genes analyzed. Alterations in gene expression were dependent on the duration of the stress treatment and, in some cases, were only observed in male mice., Conclusions: These results suggest that genes involved in neurite remodeling are one of the main targets for regulation by chronic stress. The finding that this regulation is conserved in different stress models and antidepressant treatments highlights the biological relevance of the genes analyzed and suggests that they might be involved in stress-related disorders.

RNA-binding proteins are important in many aspects of RNA processing, function, and destruction. One class of such proteins contains the RNA recognition motif (RRM), which consists of about 90 amino acid residues, including the canonical RNP1 octapeptide: (K/R)G(F/Y)(G/A)FVX(F/Y). We used a variety of homology searches to classify all of the RRM proteins of the three kinetoplastids Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. All three organisms have similar sets of RRM-containing protein orthologues, suggesting common posttranscriptional processing and regulatory pathways. Of the 75 RRM proteins identified in T. brucei, only 13 had clear homologues in other eukaryotes, although 8 more could be given putative functional assignments. A comparison with the 18 RRM proteins of the obligate intracellular parasite Encephalitozoon cuniculi revealed just 3 RRM proteins which appear to be conserved at the primary sequence level throughout eukaryotic evolution: poly(A) binding protein, the rRNA-processing protein MRD1, and the nuclear cap binding protein.

Neuronal remodeling is a fundamental process by which the brain responds to environmental influences, e.g., during stress. In the hippocampus, chronic stress causes retraction of dendrites in CA3 pyramidal neurons. We have recently identified the glycoprotein M6a as a stress-responsive gene in the hippocampal formation. This gene is down-regulated in the hippocampus of both socially and physically stressed animals, and this effect can be reversed by antidepressant treatment. In the present work, we analyzed the biological function of the M6a protein. Immunohistochemistry showed that the M6a protein is abundant in all hippocampal subregions, and subcellular analysis in primary hippocampal neurons revealed its presence in membrane protrusions (filopodia/spines). Transfection experiments revealed that M6a overexpression induces neurite formation and increases filopodia density in hippocampal neurons. M6a knockdown with small interference RNA methodology showed that M6a low-expressing neurons display decreased filopodia number and a lower density of synaptophysin clusters. Taken together, our findings indicate that M6a plays an important role in neurite/filopodium outgrowth and synapse formation. Therefore, reduced M6a expression might be responsible for the morphological alterations found in the hippocampus of chronically stressed animals. Potential mechanisms that might explain the biological effects of M6a are discussed.

Whole-genome sequencing of the protozoan pathogen Trypanosoma cruzi revealed that the diploid genome contains a predicted 22,570 proteins encoded by genes, of which 12,570 represent allelic pairs. Over 50% of the genome consists of repeated sequences, such as retrotransposons and genes for large families of surface molecules, which include trans-sialidases, mucins, gp63s, and a large novel family (>1300 copies) of mucin-associated surface protein (MASP) genes. Analyses of the T. cruzi, T. brucei, and Leishmania major (Tritryp) genomes imply differences from other eukaryotes in DNA repair and initiation of replication and reflect their unusual mitochondrial DNA. Although the Tritryp lack several classes of signaling molecules, their kinomes contain a large and diverse set of protein kinases and phosphatases; their size and diversity imply previously unknown interactions and regulatory processes, which may be targets for intervention.

Leishmania species cause a spectrum of human diseases in tropical and subtropical regions of the world. We have sequenced the 36 chromosomes of the 32.8-megabase haploid genome of Leishmania major (Friedlin strain) and predict 911 RNA genes, 39 pseudogenes, and 8272 protein-coding genes, of which 36% can be ascribed a putative function. These include genes involved in host-pathogen interactions, such as proteolytic enzymes, and extensive machinery for synthesis of complex surface glycoconjugates. The organization of protein-coding genes into long, strand-specific, polycistronic clusters and lack of general transcription factors in the L. major, Trypanosoma brucei, and Trypanosoma cruzi (Tritryp) genomes suggest that the mechanisms regulating RNA polymerase II-directed transcription are distinct from those operating in other eukaryotes, although the trypanosomatids appear capable of chromatin remodeling. Abundant RNA-binding proteins are encoded in the Tritryp genomes, consistent with active posttranscriptional regulation of gene expression.

TcUBP1 is a trypanosome cytoplasmic RNA-binding protein containing a single, conserved RNA-recognition motif (RRM) domain involved in selective destabilization of U-rich mRNAs such as the Trypanosoma cruzi small mucin gene family mRNA, TcSMUG. TcUBP1 binds specific transcripts in vivo and co-localizes in the perinuclear part of the cell with components of the mRNA-stability determinant pathway such as poly(A)-binding protein 1 (PABP1) and TcUBP2, a closely related RRM-containing protein. In TcUBP proteins, the RRM domain is flanked by N-terminal Gln-rich and C-terminal Gly-Gln-rich extensions, which are involved in protein-protein interactions. In this work, we determined the solution structure of the TcUBP1 RRM domain by nuclear magnetic resonance (NMR) spectroscopy. The domain has a characteristic betaalphabetabetaalphabeta fold, consisting of a beta sheet composed of four antiparallel betastrands and two alpha helices packed against one face of the beta sheet. A unique aspect of TcUBP1 is the participation of a beta hairpin (beta4-beta5) in the beta sheet, resulting in an enlarged RNA-binding surface. Detailed analysis of the TcUBP1 interaction with a short single-stranded RNA derived from the 3' UTR of TcSMUG was carried out by titration experiments using both NMR spectroscopy and isothermal titration calorimetry. This analysis revealed that amino acids located within the beta hairpin (beta4-beta5) contribute to complex formation. This enlarged protein-RNA interface could compensate for the lack of additional RNA-binding domains in TcUBP1, as observed in many other RRM-containing proteins. The structure of TcUBP1 reveals new aspects of single RRM-RNA interactions and insight into how N- and C-terminal extensions can contribute to RNA binding.

Trypanosoma cruzi, the agent of Chagas disease, expresses a modified sialidase, the trans-sialidase, which transfers sialic acid from host glycoconjugates to beta-galactose present in parasite mucins. Another American trypanosome, Trypanosoma rangeli, expresses a homologous protein that has sialidase activity but is devoid of transglycosidase activity. Based on the recently determined structures of T.rangeli sialidase (TrSA) and T.cruzi trans-sialidase (TcTS), we have now constructed mutants of TrSA with the aim of studying the relevant residues in transfer activity. Five mutations, Met96-Val, Ala98-Pro, Ser120-Tyr, Gly249-Tyr and Gln284-Pro, were enough to obtain a sialidase mutant (TrSA(5mut)) with trans-sialidase activity; and a sixth mutation increased the activity to about 10% that of wild-type TcTS. The crystal structure of TrSA(5mut) revealed the formation of a trans-sialidase-like binding site for the acceptor galactose, primarily defined by the phenol group of Tyr120 and the indole ring of Trp313, which adopts a new conformation, similar to that in TcTS, induced by the Gln284-Pro mutation. The transition state analogue 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA), which inhibits sialidases but is a poor inhibitor of trans-sialidase, was used to probe the active site conformation of mutant enzymes. The results show that the presence of a sugar acceptor binding-site, the fine-tuning of protein-substrate interactions and the flexibility of crucial active site residues are all important to achieve transglycosidase activity from the TrSA sialidase scaffold.