Molecular phenotype of zebrafish ovarian follicle by serial analysis of gene expression and proteomic profiling, and comparison with the transcriptomes of other animals

BACKGROUND: The ability of an oocyte to develop into a viable embryo depends on the accumulation of specific maternal information and molecules, such as RNAs and proteins. A serial analysis of gene expression (SAGE) was carried out in parallel with proteomic analysis on fully-grown ovarian follicles from zebrafish (Danio rerio). The data obtained were compared with ovary/follicle/egg molecular phenotypes of other animals, published or available in public sequence databases. RESULTS: Sequencing of 27,486 SAGE tags identified 11,399 different ones, including 3,329 tags with an occurrence superior to one. Fifty-eight genes were expressed at over 0.15% of the total population and represented 17.34% of the mRNA population identified. The three most expressed transcripts were a rhamnose-binding lectin, beta-actin 2, and a transcribed locus similar to the H2B histone family. Comparison with the large-scale expressed sequence tags sequencing approach revealed highly expressed transcripts that were not previously known to be expressed at high levels in fish ovaries, like the short-sized polarized metallothionein 2 transcript. A higher sensitivity for the detection of transcripts with a characterized maternal genetic contribution was also demonstrated compared to large-scale sequencing of cDNA libraries. Ferritin heavy polypeptide 1, heat shock protein 90-beta, lactate dehydrogenase B4, beta-actin isoforms, tubulin beta 2, ATP synthase subunit 9, together with 40 S ribosomal protein S27a, were common highly-expressed transcripts of vertebrate ovary/unfertilized egg. Comparison of transcriptome and proteome data revealed that transcript levels provide little predictive value with respect to the extent of protein abundance. All the proteins identified by proteomic analysis of fully-grown zebrafish follicles had at least one transcript counterpart, with two exceptions: eosinophil chemotactic cytokine and nothepsin. CONCLUSION: This study provides a complete sequence data set of maternal mRNA stored in zebrafish germ cells at the end of oogenesis. This catalogue contains highly-expressed transcripts that are part of a vertebrate ovarian expressed gene signature. Comparison of transcriptome and proteome data identified downregulated transcripts or proteins potentially incorporated in the oocyte by endocytosis. The molecular phenotype described provides groundwork for future experimental approaches aimed at identifying functionally important stored maternal transcripts and proteins involved in oogenesis and early stages of embryo development

Natural HLA-B*2705 protein ligands with glutamine as anchor motif: implications for HLA-B27 association with spondyloarthropathy

The presentation of short viral peptide antigens by human leukocyte antigen (HLA) class I molecules on cell surfaces is a key step in the activation of cytotoxic T lymphocytes, which mediate the killing of pathogen-infected cells or initiate autoimmune tissue damage. HLA-B27 is a well known class I molecule that is used to study both facets of the cellular immune response. Using mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of HLA-B*2705(+) cells, we identified 200 naturally processed HLA-B*2705 ligands. Our analyses revealed that a change in the position (P) 2 anchor motif was detected in the 3% of HLA-B*2705 ligands identified. B*2705 class I molecules were able to bind these six GlnP2 peptides, which showed significant homology to pathogenic bacterial sequences, with a broad range of affinities. One of these ligands was able to bind with distinct conformations to HLA-B27 subtypes differentially associated with ankylosing spondylitis. These conformational differences could be sufficient to initiate autoimmune damage in patients with ankylosing spondylitis-associated subtypes. Therefore, these kinds of peptides (short, with GlnP2, and similar low affinity to all HLA-B27 subtypes tested but with unlike conformations in differentially ankylosing spondylitis-associated subtypes) must not be excluded from future researches involving potential arthritogenic peptides.This work was supported by grants from the Programa Ramón y Cajal and the Ministerio de Ciencia e Innovación (to D. L.) and from the Israel Science Foundation (ISF 916/05) (to A. A.).N

Numerous proteins with unique characteristics are degraded by the 26S proteasome following monoubiquitination

The "canonical" proteasomal degradation signal is a substrateanchored polyubiquitin chain. However, a handful of proteins were shown to be targeted following monoubiquitination. In this study, we established-in both human and yeast cells-a systematic approach for the identification of monoubiquitination-dependent proteasomal substrates. The cellular wild-type polymerizable ubiquitin was replaced with ubiquitin that cannot form chains. Using proteomic analysis, we screened for substrates that are nevertheless degraded under these conditions compared with those that are stabilized, and therefore require polyubiquitination for their degradation. For randomly sampled representative substrates, we confirmed that their cellular stability is in agreement with our screening prediction. Importantly, the two groups display unique features: monoubiquitinated substrates are smaller than the polyubiquitinated ones, are enriched in specific pathways, and, in humans, are structurally less disordered. We suggest that monoubiquitination-dependent degradation is more widespread than assumed previously, and plays key roles in various cellular processes

Immunoproteomic analysis of a Chikungunya poxvirus-based vaccine reveals high HLA class II immunoprevalence

BACKGROUND: Efficient adaptive antiviral cellular and humoral immune responses require previous recognition of viral antigenic peptides bound to human leukocyte antigen (HLA) class I and II molecules, which are exposed on the surface of infected and antigen presenting cells, respectively. The HLA-restricted immune response to Chikungunya virus (CHIKV), a mosquito-borne Alphavirus of the Togaviridae family responsible for severe chronic polyarthralgia and polyarthritis, is largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a high-throughput mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of human cells infected with a vaccinia virus (VACV) recombinant expressing CHIKV structural proteins was carried out. Twelve viral ligands from the CHIKV polyprotein naturally presented by different HLA-A, -B, and -C class I, and HLA-DR and -DP class II molecules were identified. CONCLUSIONS/SIGNIFICANCE: The immunoprevalence of the HLA class II but not the HLA class I-restricted cellular immune response against the CHIKV structural polyprotein was greater than that against the VACV vector itself. In addition, most of the CHIKV HLA class I and II ligands detected by mass spectrometry are not conserved compared to its closely related O'nyong-nyong virus. These findings have clear implications for analysis of both cytotoxic and helper immune responses against CHIKV as well as for the future studies focused in the exacerbated T helper response linked to chronic musculoskeletal disorders in CHIKV patients.This work was supported by the Spanish Ministry of Economy grants SAF2014-58052 and “Acción Estratégica en Salud” 2018 to DL, SAF-2013-45232-R and SAF-2017-88089-R to ME, and by Israel Science Foundation, grant No. 1435/16 to AA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Trade-off between transcriptome plasticity and genome evolution in cephalopods

Author Posting. © The Author(s), 2017. This is the author's version of the work. It is posted here by permission of Cell Press for personal use, not for redistribution. The definitive version was published in Cell 169 (2017): 191-202, doi:10.1016/j.cell.2017.03.025.RNA editing, a post-transcriptional process, allows the diversification of proteomes beyond the genomic blueprint; however it is infrequently used among animals. Recent reports suggesting increased levels of RNA editing in squids thus raise the question of their nature and effects in these organisms. We here show that RNA editing is particularly common in behaviorally sophisticated coleoid cephalopods, with tens of thousands of evolutionarily conserved sites. Editing is enriched in the nervous system affecting molecules pertinent for excitability and neuronal morphology. The genomic sequence flanking editing sites is highly conserved, suggesting that the process confers a selective advantage. Due to the large number of sites, the surrounding conservation greatly reduces the number of mutations and genomic polymorphisms in protein coding regions. This trade-off between genome evolution and transcriptome plasticity highlights the importance of RNA recoding as a strategy for diversifying proteins, particularly those associated with neural function.NLB was supported by a post-doctoral scholarship from the Center for Nanoscience and Nanotechnology, Tel-Aviv University. The research of RU is supported by the Israel Science Foundation (772/13). The research of EYL was supported by the European Research Council (311257) and the Israel Science Foundation (1380/14). The research of JJCR was supported by the National Institutes of Health [1R0111223855, 1R01NS64259], the National Science Foundation (HRD- 1137725), and the Frank R. Lillie and Laura and Arthur Colwin Research Fellowships from the Marine Biological Laboratory in Woods Hole. The work of JJCR and EE was supported by grant No 094/2013 from the United States-Israel Binational Science Foundation (BSF).2018-04-0

Acrylamide acute neurotoxicity in adult zebrafish

Un articulo indexadoAcute exposure to acrylamide (ACR), a type-2 alkene, may lead to a ataxia, skeletal muscles weakness and numbness of the extremities in human and laboratory animals. In the present manuscript, ACR acute neurotoxicity has been characterized in adult zebrafish, a vertebrate model increasingly used in human neuropharmacology and toxicology research. At behavioral level, ACR-treated animals exhibited “depression-like” phenotype comorbid with anxiety behavior. At transcriptional level, ACR induced down-regulation of regeneration-associated genes and up-regulation of oligodendrocytes and reactive astrocytes markers, altering also the expression of genes involved in the presynaptic vesicle cycling. ACR induced also significant changes in zebrafish brain proteome and formed adducts with selected cysteine residues of specific proteins, some of them essential for the presynaptic function. Finally, the metabolomics analysis shows a depletion in the monoamine neurotransmitters, consistent with the comorbid depression and anxiety disorder, in the brain of the exposed fish.Conacy

Pro-inflammatory Cytokines Alter the Immunopeptidome Landscape by Modulation of HLA-B Expression

Antigen presentation on HLA molecules is a major mechanism by which the immune system monitors self and non-self-recognition. Importantly, HLA-I presentation has gained much attention through its role in eliciting anti-tumor immunity. Several determinants controlling the peptides presented on HLA have been uncovered, mainly through the study of model substrates and large-scale immunopeptidome analyses. These determinants include the relative abundances of proteins in the cell, the stability or turnover rate of these proteins and the binding affinities of a given peptide to the HLA haplotypes found in a cell. However, the regulatory principles involved in selection and regulation of specific antigens in response to tumor pro-inflammatory signals remain largely unknown. Here, we chose to examine the effect that TNFα and IFNγ stimulation may exert on the immunopeptidome landscape of lung cancer cells. We show that the expression of many of the proteins involved in the class I antigen presentation pathway are changed by pro-inflammatory cytokines. Further, we could show that increased expression of the HLA-B allomorph drives a significant change in HLA-bound antigens, independently of the significant changes observed in the cellular proteome. Finally, we observed increased HLA-B levels in correlation with tumor infiltration across the TCGA lung cancer cohorts. Taken together, our results suggest that the immunopeptidome landscape should be examined in the context of anti-tumor immunity whereby signals in the microenvironment may be critical in shaping and modulating this important aspect of host-tumor interactions

Therapeutic potential of N-acetylcysteine in acrylamide acute neurotoxicity in adult zebrafish

Two essential key events in acrylamide (ACR) acute neurotoxicity are the formation of adducts with nucleophilic sulfhydryl groups on cysteine residues of selected proteins in the synaptic terminals and the depletion of the glutathione (GSx) stores in neural tissue. The use of N-acetylcysteine (NAC) has been recently proposed as a potential antidote against ACR neurotoxicity, as this chemical is not only a well-known precursor of the reduced form of glutathione (GSH), but also is an scavenger of soft electrophiles such as ACR. In this study, the suitability of 0.3 and 0.75 mM NAC to protect against the neurotoxic effect of 0.75 mM ACR has been tested in vivo in adult zebrafish. NAC provided only a mild to negligible protection against the changes induced by ACR in the motor function, behavior, transcriptome and proteome. The permeability of NAC to cross blood-brain barrier (BBB) was assessed, as well as the ACR-scavenging activity and the gamma-glutamyl-cysteine ligase (γ-GCL) and acylase I activities. The results show that ACR not only depletes GSx levels but also inhibits it synthesis from NAC/cysteine, having a dramatic effect over the glutathione system. Moreover, results indicate a very low NAC uptake to the brain, probably by a combination of low BBB permeability and high deacylation of NAC during the intestinal absorption. These results strongly suggest that the use of NAC is not indicated in ACR acute neurotoxicity treatment.This work was supported by the NATO SfP project MD.SFPP 984777 (D.R.) and the Spanish Government (CTM2017-83242-R; D.R.). M.F acknowledges financial support from the Beatriu de Pinós programme (Grant No. 2016 BP 00233) provided by the Secretariat of Universities and Research department of the Ministry for Business and Knowledge, Catalonia Government. Mention of specific products or trade names does not indicate endorsement by the US federal government.Peer reviewe

The viral transcription group determines the HLA class I cellular immune response against human respiratory syncytial virus

The cytotoxic T-lymphocyte-mediated killing of virus-infected cells requires previous recognition of short viral antigenic peptides bound to human leukocyte antigen class I molecules that are exposed on the surface of infected cells. The cytotoxic T-lymphocyte response is critical for the clearance of human respiratory syncytial virus infection. In this study, naturally processed viral human leukocyte antigen class I ligands were identified with mass spectrometry analysis of complex human leukocyte antigen-bound peptide pools isolated from large amounts of human respiratory syncytial virus-infected cells. Acute antiviral T-cell response characterization showed that viral transcription determines both the immunoprevalence and immunodominance of the human leukocyte antigen class I response to human respiratory syncytial virus. These findings have clear implications for antiviral vaccine design.This work was supported by the Spanish Ministry of Economy grants BIO2011-25636 to D.L. and to A. A. from the ISF 916/05. The funding agencies had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. We have no conflicting financial interestsS