Papers by Michalis Aivaliotis
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Journal of Cellular Physiology
Schizophrenia is a debilitating disorder with complex and unclarified etiological factors. Sex di... more Schizophrenia is a debilitating disorder with complex and unclarified etiological factors. Sex differences have been observed in humans but animal models have only focused on male subjects. In this study, we report the establishment of the neurodevelopmental MAM model of schizophrenia in mice and compare the schizotypic-like characteristics and cognitive function in both sexes. Pregnant mice were injected with 26mg/kg(i.p.) of Methylazoxy-methanol acetate (MAM) or saline (5ml/kg) on gestational day (GD) 16 (MAM-16) or 17 (MAM-17). Behavioral, histological and electrophysiological and mass spectrometry-based comparative proteomic techniques were employed to assess the schizotypic-like characteristics and cognitive function of adult male and female offspring (MAM- or saline-treated). Female MAM-16, but not MAM-17 treated mice exhibited enhanced hyperlocomotion after acute administration of the NMDA receptor antagonist, MK-801, compared to saline treated mice. Male MAM-16, but not MAM-...
Nucleic acids research, Jan 17, 2017
Profiling of proteome dynamics is crucial for understanding cellular behavior in response to intr... more Profiling of proteome dynamics is crucial for understanding cellular behavior in response to intrinsic and extrinsic stimuli and maintenance of homeostasis. Over the last 20 years, mass spectrometry (MS) has emerged as the most powerful tool for large-scale identification and characterization of proteins. Bottom-up proteomics, the most common MS-based proteomics approach, has always been challenging in terms of data management, processing, analysis and visualization, with modern instruments capable of producing several gigabytes of data out of a single experiment. Here, we present ProteoSign, a freely available web application, dedicated in allowing users to perform proteomics differential expression/abundance analysis in a user-friendly and self-explanatory way. Although several non-commercial standalone tools have been developed for post-quantification statistical analysis of proteomics data, most of them are not end-user appealing as they often require very stringent installation...
Nature Cell Biology, 2017
Journal of the American Chemical Society, 2017
The full extent of proline (Pro) hydroxylation has yet to be established, as it is largely unexpl... more The full extent of proline (Pro) hydroxylation has yet to be established, as it is largely unexplored in bacteria. We describe here a so far unknown Pro hydroxylation activity which occurs in active sites of polysaccharide deacetylases (PDAs) from bacterial pathogens, modifying the protein backbone at the Cα atom of a Pro residue to produce 2-hydroxyproline (2-Hyp). This process modifies with high specificity a conserved Pro, shares with the deacetylation reaction the same active site and one catalytic residue, and utilizes molecular oxygen as source for the hydroxyl group oxygen of 2-Hyp. By providing additional hydrogen-bonding capacity, the Pro→2-Hyp conversion alters the active site and enhances significantly deacetylase activity, probably by creating a more favorable environment for transition-state stabilization. Our results classify this process as an active-site "maturation", which is highly atypical in being a protein backbone-modifying activity, rather than a side-chain-modifying one.
Scientific Reports, 2017
Accumulating evidence during the last decades revealed that androgen can exert membrane initiated... more Accumulating evidence during the last decades revealed that androgen can exert membrane initiated actions that involve signaling via specific kinases and the modulation of significant cellular processes, important for prostate cancer cell growth and metastasis. Results of the present work clearly show that androgens can specifically act at the membrane level via the GPCR oxoeicosanoid receptor 1 (OXER1) in prostate cancer cells. In fact, OXER1 expression parallels that of membrane androgen binding in prostate cancer cell lines and tumor specimens, while in silico docking simulation of OXER1 showed that testosterone could bind to OXER1 within the same grove as 5-OxoETE, the natural ligand of OXER1. Interestingly, testosterone antagonizes the effects of 5-oxoETE on specific signaling pathways and rapid effects such as actin cytoskeleton reorganization that ultimately can modulate cell migration and metastasis. These findings verify that membrane-acting androgens exert specific effects through an antagonistic interaction with OXER1. Additionally, this interaction between androgen and OXER1, which is an arachidonic acid metabolite receptor expressed in prostate cancer, provides a novel link between steroid and lipid actions and renders OXER1 as new player in the disease. These findings should be taken into account in the design of novel therapeutic approaches in prostate cancer. Prostate cancer cells are highly dependent for their growth on testosterone (at least at the initial stages of the disease), with chemical castration by the administration of anti-androgen, being the primary line of treatment 1. However, after a rather short time period (18-36 months) castration resistance develops and prostate cancer cells can grow independently of androgens. Hence, it seems (as recently shown by van der Sluis and his colleagues 2) that, even at this stage, prostate cancer cells still dependent on hormones for migration, invasion and ultimately metastasis. Indeed, testosterone has been shown to induce migration and invasion of prostate cancer cells 2 and serum testosterone levels to be correlated with a high-grade pathology and Gleason score 3. These findings strongly designate testosterone as an important player in prostate cancer, with its mechanism of action requiring thorough investigation. Androgen actions are classically mediated via intracellular androgen receptors (AR) that belong to the nuclear receptor superfamily. AR dimerizes and translocates to the nucleus after androgen binding, affecting gene expression. However, during the last fifteen years, a large amount of evidence points out an alternative mode of androgen action, that is initiated at the cell membrane, involves rapid signaling via specific kinases and modulates a significant number of cellular processes 4. Previous work demonstrated that membrane androgen sites are present in a number of physiological (T lymphocytes, macrophages, spermocytes, sperm, osteoblasts) 5-7 , and cancer cells (prostate, breast, colon) 8-10. In prostate and breast cancer cell lines, we have shown that membrane-acting androgens induce rapid cytoskeletal changes, resulting in the modulation of the adhesive and migratory capacity
Journal of Chromatography B, 2016
Mass spectrometry-based quantitative proteomics specifically applied to comprehend the pathogenes... more Mass spectrometry-based quantitative proteomics specifically applied to comprehend the pathogenesis of lymphoma has incremental value in deciphering the heterogeneity in complex deregulated molecular mechanisms/pathways of the lymphoma entities, implementing the current diagnostic and therapeutic strategies. Essential global, targeted and functional differential proteomics analyses although still evolving, have been successfully implemented to shed light on lymphoma pathogenesis to discover and explore the role of potential lymphoma biomarkers and drug targets. This review aims to outline and appraise the present status of MS-based quantitative proteomic approaches in lymphoma research, introducing the current state-of-the-art MS-based proteomic technologies, the opportunities they offer in biological discovery in human lymphomas and the related limitation issues arising from sample preparation to data evaluation. It is a synopsis containing information obtained from recent research articles, reviews and public proteomics repositories (PRIDE). We hope that this review article will aid, assimilate and assess all the information aiming to accelerate the development and validation of diagnostic, prognostic or therapeutic targets for an improved and empowered clinical proteomics application in lymphomas in the nearby future.
mBio, Jan 31, 2018
Many plant-pathogenic bacteria of considerable economic importance rely on type III secretion sys... more Many plant-pathogenic bacteria of considerable economic importance rely on type III secretion systems (T3SSs) of the Hrc-Hrp 1 family to subvert their plant hosts. T3SS gene expression is regulated through the HrpG and HrpV proteins, while secretion is controlled by the gatekeeper HrpJ. A link between the two mechanisms was so far unknown. Here, we show that a mechanistic coupling exists between the expression and secretion cascades through the direct binding of the HrpG/HrpV heterodimer, acting as a T3SS chaperone, to HrpJ. The ternary complex is docked to the cytoplasmic side of the inner bacterial membrane and orchestrates intermediate substrate secretion, without affecting early substrate secretion. The anchoring of the ternary complex to the membranes potentially keeps HrpG/HrpV away from DNA. In their multiple roles as transcriptional regulators and gatekeeper chaperones, HrpV/HrpG provide along with HrpJ potentially attractive targets for antibacterial strategies. On the basi...
Molecular & Cellular Proteomics, 2013
Biological membranes are essential for cell viability. Their functional characteristics strongly ... more Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content which consists of transmembrane (integral) as well as peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. While transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools and carry no discernible membrane targeting signals. Here we experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multi-disciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding sub-cellular localization, using literature searches, manual curation and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies, a previously unsuspected, ~ 19% of the basic E. coli BL21(DE3) proteome and the detected peripheral inner membrane proteome ~ 25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communications with biological membrane surfaces that we are only beginning to decipher. Papanastasiou et al. E.coli peripherome 4
Immunology, 2014
Soluble MHCII (sMHCII) molecules are present in body fluids of healthy individuals and are consid... more Soluble MHCII (sMHCII) molecules are present in body fluids of healthy individuals and are considered to be involved in the maintenance of self tolerance, and are also related to various diseases. Their concentration increases during in vivo antigen-specific tolerogenic stimulation and it was recently shown that exosome-mediated tolerance is MHCII dependent. At the cellular level, sMHCII proteins compete with membrane MHCII for T-cell receptor binding on CD4(+) T cells. Immunoaffinity purification techniques isolated sMHCII antigens from the serum of human serum albumin (HSA) -tolerant mice as a single highly glycosylated protein of ~ 60,000 molecular weight, specifically interacting with anti-class II antibodies in Western blotting and ELISA. Mass spectroscopy showed that these sMHCII proteins were loaded with the tolerogenic peptide as well as multiple self peptides. At the cellular level, sMHCII suppressed antigen-specific, and to a lesser degree antigen-non-specific, spleen cell proliferation and induced CD25 in naive T cells. In T cells activated by antigen-seeded macrophages, sMHCII decreased CD28 and increased CTLA-4 protein expression, while decreasing interleukin-2 and increasing interleukin-10 production. In this case, sMHCII proteins were shown to decrease ZAP-70 and LAT phosphorylation. The results presented here for the first time provide evidence for the role of sMHCII proteins in immune response suppression and maintenance of tolerance, revealing novel regulatory mechanisms for immune system manipulation.
PROTEOMICS, 2015
Biological membranes define cells and cellular compartments and are essential in regulating bidir... more Biological membranes define cells and cellular compartments and are essential in regulating bidirectional flow of chemicals and signals. Characterizing their protein content therefore is required to determine their function, nevertheless, the comprehensive determination of membrane-embedded sub-proteomes remains challenging. Here, we experimentally characterized the inner membrane proteome (IMP) of the model organism E. coli BL21(DE3). We took advantage of the recent extensive re-annotation of the theoretical E. coli IMP regarding the sub-cellular localization of all its proteins. Using surface proteolysis of IMVs with variable chemical treatments followed by nanoLC-MS/MS analysis, we experimentally identified ∼45% of the expressed IMP in wild type E. coli BL21(DE3) with 242 proteins reported here for the first time. Using modified label-free approaches we quantified 220 IM proteins. Finally, we compared protein levels between wild type cells and those over-synthesizing the membrane-embedded translocation channel SecYEG proteins. We propose that this proteomics pipeline will be generally applicable to the determination of IMP from other bacteria. This article is protected by copyright. All rights reserved.
Immunology, 2014
Soluble MHCII (sMHCII) molecules are present in body fluids of healthy individuals and are consid... more Soluble MHCII (sMHCII) molecules are present in body fluids of healthy individuals and are considered to be involved in the maintenance of self tolerance, and are also related to various diseases. Their concentration increases during in vivo antigen-specific tolerogenic stimulation and it was recently shown that exosome-mediated tolerance is MHCII dependent. At the cellular level, sMHCII proteins compete with membrane MHCII for T-cell receptor binding on CD4(+) T cells. Immunoaffinity purification techniques isolated sMHCII antigens from the serum of human serum albumin (HSA) -tolerant mice as a single highly glycosylated protein of ~ 60,000 molecular weight, specifically interacting with anti-class II antibodies in Western blotting and ELISA. Mass spectroscopy showed that these sMHCII proteins were loaded with the tolerogenic peptide as well as multiple self peptides. At the cellular level, sMHCII suppressed antigen-specific, and to a lesser degree antigen-non-specific, spleen cell proliferation and induced CD25 in naive T cells. In T cells activated by antigen-seeded macrophages, sMHCII decreased CD28 and increased CTLA-4 protein expression, while decreasing interleukin-2 and increasing interleukin-10 production. In this case, sMHCII proteins were shown to decrease ZAP-70 and LAT phosphorylation. The results presented here for the first time provide evidence for the role of sMHCII proteins in immune response suppression and maintenance of tolerance, revealing novel regulatory mechanisms for immune system manipulation.
Journal of Proteome Research, 2007
Characterization of protein N-terminal peptides supports the quality assessment of data derived f... more Characterization of protein N-terminal peptides supports the quality assessment of data derived from genomic sequences (e.g., the correct assignment of start codons) and hints to in vivo N-terminal modifications such as N-terminal acetylation and removal of the initiator methionine. The current work represents the first large-scale identification of N-terminal peptides from prokaryotes, of the two halophilic euryarchaeota Halobacterium salinarum and Natronomonas pharaonis. Two methods were used that specifically allow the characterization of protein N-terminal peptides: combined fractional diagonal chromatography (COFRADIC) and strong cation exchange chromatography (SCX), both known to enrich for N-terminally blocked peptides. In addition to these specific methods, N-terminal peptide identifications were extracted from our previous genome-wide proteomic data. Combining all data, 606 N-terminal peptides from Hbt. salinarum and 328 from Nmn. pharaonis were reliably identified. These results constitute the largest available dataset holding identified and characterized protein N-termini for prokaryotes (archaea and bacteria). They allowed the validation/improvement of start codon assignments as automatic gene finders tend to misassign start codons for GC-rich genomes. In addition, the dataset allowed unravelling N-terminal protein maturation in archaea, showing that 60% of the proteins undergo methionine cleavage and thatsin contrast to current knowledgesN R -acetylation is common in the archaeal domain of life with 13-18% of the proteins being N R -acetylated. The protein sets described in this paper are available by FTP and might be used as reference sets to test the performance of new gene finders.
PROTEOMICS, 2015
Biological membranes define cells and cellular compartments and are essential in regulating bidir... more Biological membranes define cells and cellular compartments and are essential in regulating bidirectional flow of chemicals and signals. Characterizing their protein content therefore is required to determine their function, nevertheless, the comprehensive determination of membrane-embedded sub-proteomes remains challenging. Here, we experimentally characterized the inner membrane proteome (IMP) of the model organism E. coli BL21(DE3). We took advantage of the recent extensive re-annotation of the theoretical E. coli IMP regarding the sub-cellular localization of all its proteins. Using surface proteolysis of IMVs with variable chemical treatments followed by nanoLC-MS/MS analysis, we experimentally identified ∼45% of the expressed IMP in wild type E. coli BL21(DE3) with 242 proteins reported here for the first time. Using modified label-free approaches we quantified 220 IM proteins. Finally, we compared protein levels between wild type cells and those over-synthesizing the membrane-embedded translocation channel SecYEG proteins. We propose that this proteomics pipeline will be generally applicable to the determination of IMP from other bacteria. This article is protected by copyright. All rights reserved.
The FASEB Journal, 2015
The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor protein is re... more The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor protein is regulated by various mechanisms that are not fully understood. This includes regulation by Tyr phosphorylation by a mechanism that remains elusive. Here, we show that focal adhesion kinase (FAK) phosphorylates PTEN in vitro, in cell-free systems and in cells. Furthermore, by mass spectrometry, we identified Tyr336 on PTEN as being phosphorylated by FAK. Tyr336 phosphorylation increased phosphatase activity, protein-lipid interaction, and protein stability of PTEN. In cells, including primary mouse macrophages and human cancer cell lines, FAK was found to be negatively regulated by p110δ phosphoinositide-3 kinase (PI3K), whereas the activation of FAK was positively regulated by RhoA-associated kinase (ROCK). Indeed, the phosphorylation of FAK was unexpectedly increased in macrophages derived from mice expressing kinase-dead p110δ. Pharmacologic inactivation of RhoA/ROCK reduced the phosphorylation of FAK to normal levels in cells with genetically inactivated p110δ. Likewise, pharmacologic inactivation of FAK reduced the phosphorylation of PTEN in cells expressing kinase-dead p110δ and restored the functional defects of p110δ inactivation, including Akt phosphorylation and cell proliferation. This work identifies FAK as a target of p110δ PI3K that links RhoA with PTEN and establishes for the first time that PTEN is a substrate of FAK-mediated Tyr phosphorylation.-Tzenaki, N., Aivaliotis, M., Papakonstanti, E. A. Focal adhesion kinase phosphorylates the phosphatase and tensin homolog deleted on chromosome 10 under the control of p110δ phosphoinositide-3 kinase.
Preferred Presentation Method: Oral or Poster Introduction: The type III secretion system (T3SS) ... more Preferred Presentation Method: Oral or Poster Introduction: The type III secretion system (T3SS) is a specialized bacterial protein secretory pathway that plays an essential role in the pathogenesis of Gram-negative bacteria (e.g. Enteropathogenic E. coli, EPEC) [1]. It is encoded by the Locus of Enterocyte Effacement (LEE), and injects effector proteins into the host cell, modulating key cellular processes [2]. The precise mechanisms of T3SS remain poorly understood. Methods: Cytosolic protein complexes were isolated from wt and selected deletion mutant and fractionated by Native polyacrylamide gel electrophoresis (N-PAGE) and size-exclusion chromatography (SEC). In a targeted approach, His-tagged T3SS-related proteins were used for the isolation of protein complexes which were fractionated by N-PAGE and SEC. The secretome was collected and concentrated by acid-mediated precipitation. Complexome and secretome were analyzed by " bottom-up " proteomics. Protein identificati...
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Papers by Michalis Aivaliotis