Mariana Fioramonte

Proteomic analysis of the reproductive tract fluids from tropically-adapted Santa Ines rams ☆

The present study is focused on the proteome of reproductive tract fluids from tropically-adapted Santa Ines rams. Seminal plasma, cauda epididymal (CEF) and vesicular gland fluid (VGF) proteins were analyzed by 2-D electrophoresis and mass spectrometry. Seminal plasma maps contained 302 ± 16 spots, within the 4-7 pH range. From these maps, 73 spots were identified, corresponding to 41 proteins. Ram Seminal Vesicle Proteins (RSVP) 14 and 22kDa and bodhesins 1 and 2 represented the most abundant seminal components. Other seminal proteins included clusterin, angiotensin-converting enzyme, matrix metalloproteinase-2, tissue-inhibitor of metalloproteinase-2, plasma glutamate carboxypeptidase, albumin, lactoferrin, alpha enolase, peroxiredoxin, leucine aminopeptidase, β-galactosidase, among others. Later, seminal plasma gels were run within narrow pH intervals (3.9-5.1; 4.7-5.9; 5.5-6.7), allowing the additional identification of 21 proteins not detected in 4-7 pH maps. Major proteins of CEF and VGF were albumin and transferrin, and RSVPs, respectively. Western blots confirmed that RSVPs were mainly present in VGF while bodhesins, in VGF and CEF. Based on RT-PCR, RSVP and bodhesin genes were primarily expressed in the vesicular glands. In summary, the reproductive tract fluids of Brazilian hairy rams contain several categories of proteins, with potential roles in sperm protection, capacitation, acrosome reaction and sperm-oocyte interaction.

The Biginelli reaction under batch and continuous flow conditions: catalysis, mechanism and antitumoral activity

Two novel coordination polymers (CPs) have been synthesized, characterized and successfully applied as robust heterogeneous catalysts for the Biginelli multicomponent reaction to obtain 3,4-dihydropyrimidin-2(1H)-one or thione (DHPMs) derivatives. The reaction was initially developed using both CPs and the Zn-based material showed much better catalytic activity. After the reaction optimization under batch conditions, a continuous flow protocol was developed and applied with impressive results. Four bioactive DHPMs were successfully synthesized with high yields. The mechanism of the transformation was also investigated by electrospray (tandem) mass spectrometry (ESI-MS(/MS)) analyses. Online monitoring of the reaction indicated under the developed conditions that the iminium mechanism is preferred over the enamine- and Knoevenagel-based mechanisms. Nine DHPMs had their antitumoral activities evaluated against MCF-7 (human breast cancer cells), A549 (human alveolar basal epithelial cells) and Caco-2 (human epithelial colorectal cells) cancer cell lineages. Fibroblasts (healthy cells) were not affected by the tested DHPMs showing an excellent selectivity for tumour cells. Three DHPMs returned impressive results, being capable of inhibiting tumour cell proliferation in 72 h.

FERM domain interaction with myosin negatively regulates FAK in cardiomyocyte hypertrophy

Focal adhesion kinase (FAK) regulates cellular processes that affect several aspects of development and disease. The FAK N-terminal FERM (4.1 protein-ezrin-radixin-moesin homology) domain, a compact clover-leaf structure, binds partner proteins and mediates intramolecular regulatory interactions. Combined chemical cross-linking coupled to MS, small-angle X-ray scattering, computational docking and mutational analyses showed that the FAK FERM domain has a molecular cleft (~998 Å(2)) that interacts with sarcomeric myosin, resulting in FAK inhibition. Accordingly, mutations in a unique short amino acid sequence of the FERM myosin cleft, FP-1, impaired the interaction with myosin and enhanced FAK activity in cardiomyocytes. An FP-1 decoy peptide selectively inhibited myosin interaction and increased FAK activity, promoting cardiomyocyte hypertrophy through activation of the AKT-mammalian target of rapamycin pathway. Our findings uncover an inhibitory interaction between the FAK FERM domain and sarcomeric myosin that presents potential opportunities to modulate the cardiac hypertrophic response through changes in FAK activity.

Analysis of secondary structure in proteins by chemical cross-linking coupled to MS

Chemical cross‐linking is an attractive technique for the study of the structure of protein complexes due to its low sample consumption and short analysis time. Furthermore, distance constraints obtained from the identification of cross‐linked peptides by MS can be used to construct and validate protein models. If a sufficient number of distance constraints are obtained, then determining the secondary structure of a protein can allow inference of the proteins fold. In this work, we show how the distance constraints obtained from cross‐linking experiments can identify secondary structures within the protein sequence. Molecular modeling of alpha helices and beta sheets reveals that each secondary structure presents different cross‐linking possibilities due to the topological distances between reactive residues. Cross‐linking experiments performed with amine reactive cross‐linkers with model alpha helix containing proteins corroborated the molecular modeling predictions. The cross‐linking patterns established here can be extended to other cross‐linkers with known lengths for the determination of secondary structures in proteins.

[des-Arg(1)]-Proctolin: A novel NEP-like enzyme inhibitor identified in Tityus serrulatus venom

The scorpion Tityus serrulatus venom comprises a complex mixture of molecules that paralyzes and kills preys, especially insects. However, venom components also interact with molecules in humans, causing clinic envenomation. This cross-interaction may result from homologous molecular targets in mammalians and insects, such as (NEP)-like enzymes. In face of these similarities, we searched for peptides in Tityus serrulatus venom using human NEP as a screening tool. We found a NEP-inhibiting peptide with the primary sequence YLPT, which is very similar to that of the insect neuropeptide proctolin (RYLPT). Thus, we named the new peptide [des-Arg(1)]-proctolin. Comparative NEP activity assays using natural substrates demonstrated that [des-Arg(1)]-proctolin has high specificity for NEP and better inhibitory activity than proctolin. To test the initial hypothesis that molecular homologies allow Tityus serrulatus venom to act on both mammal and insect targets, we investigated the presence of a NEP-like in cockroaches, the main scorpion prey, that could be likewise inhibited by [des-Arg(1)]-proctolin. Indeed, we detected a possible NEP-like in a homogenate of cockroach heads whose activity was blocked by thiorphan and also by [des-Arg(1)]-proctolin. Western blot analysis using a human NEP monoclonal antibody suggested a NEP-like enzyme in the homogenate of cockroach heads. Our study describes for the first time a proctolin-like peptide, named [des-Arg(1)]-proctolin, isolated from Tityus serrulatus venom. The tetrapeptide inhibits human NEP activity and a NEP-like activity in a cockroach head homogenate, thus it may play a role in human envenomation as well as in the paralysis and death of scorpion preys.

The SEB-1 Transcription Factor Binds to the STRE Motif in Neurospora crassa and Regulates a Variety of Cellular Processes Including the Stress Response and Reserve Carbohydrate Metabolism

When exposed to stress conditions, all cells induce mechanisms resulting in an attempt to adapt to stress that involve proteins which, once activated, trigger cell responses by modulating specific signaling pathways. In this work, using a combination of pulldown assays and mass spectrometry analyses, we identified the Neurospora crassa SEB-1 transcription factor that binds to the Stress Response Element (STRE) under heat stress. Orthologs of SEB-1 have been functionally characterized in a few filamentous fungi as being involved in stress responses; however, the molecular mechanisms mediated by this transcription factor may not be conserved. Here, we provide evidences for the involvement of N. crassa SEB-1 in multiple cellular processes, including response to heat, as well as osmotic and oxidative stress. The Δseb-1 strain displayed reduced growth under these conditions, and genes encoding stress-responsive proteins were differentially regulated in the Δseb-1 strain grown under the same conditions. In addition, the SEB-1-GFP protein translocated from the cytosol to the nucleus under heat, osmotic, and oxidative stress conditions. SEB-1 also regulates the metabolism of the reserve carbohydrates glycogen and trehalose under heat stress, suggesting an interconnection between metabolism control and this environmental condition. We demonstrated that SEB-1 binds in vivo to the promoters of genes encoding glycogen metabolism enzymes and regulates their expression. A genome-wide transcriptional profile of the Δseb-1 strain under heat stress was determined by RNA-seq, and a broad range of cellular processes was identified that suggests a role for SEB-1 as a protein interconnecting these mechanisms.

Insights into scorpion venom peptides: Alternative processing of β-KTx propeptide from Tityus serrulatus venom results in a new naturally occurring thimet oligopeptidase inhibitor

Most functions attributed to Tityus serrulatus venom (TsV) are related to active molecules on ion-channels; however, here we describe a new pentapeptide that was discovered through enzymatic assay selection using EP24.15. The primary structure analysis revealed the sequence KEXXG (X means Ile or Leu), similar to the sequence present in the β-KTX propeptide described from the venom of Tityus spp. We confirmed through HPLC analysis that KEILG is the peptide present in TsV, but that KELLG also inhibits EP24.15 although through different mechanisms.

XPlex: An Effective, Multiplex Cross-Linking Chemistry for Acidic Residues

Cross-linking/Mass spectrometry (XLMS) is a consolidated technique for structural characterization of proteins and protein complexes. Despite its success, the cross-linking chemistry currently used is mostly based on N-hydroxysuccinimide (NHS) esters, which react primarily with lysine residues. One way to expand the current applicability of XLMS into several new areas is to increase the number of cross-links obtainable for a target protein. We introduce a multiplex chemistry (denoted XPlex) that targets Asp, Glu, Lys, and Ser residues. XPlex can generate significantly more cross-links with reactions occurring at lower temperatures and enables targeting proteins that are not possible with NHS ester-based cross-linkers. We demonstrate the effectiveness of our approach in model proteins as well as a target Lys-poor protein, SalBIII. Identification of XPlex spectra requires a search engine capable of simultaneously considering multiple cross-linkers on the same run; to achieve this, we updated the SIM-XL search algorithm with a search mode tailored toward XPlex. In summary, we present a complete chemistry/computational solution for significantly increasing the number of possible distance constraints by mass spectrometry experiments, and thus, we are convinced that XPlex poses as a real complementary approach for structural proteomics studies.

Heteropolyacid-Containing Ionic Liquid-Catalyzed Multicomponent Synthesis of Bridgehead Nitrogen Heterocycles: Mechanisms and Mitochondrial Staining

The current manuscript describes the use of a heteropolyacid-containing task-specific ionic liquid, supported in imidazolium-based ionic liquids, as the catalyst for an efficient multicomponent synthesis of hexahydroimidazo[1,2-α]pyridine derivatives. The reactions conditions were fully optimized, and the bridgehead nitrogen heterocycle derivatives could be obtained in just 1 h exclusively as a single isomer ( trans). Single crystal X-ray analysis confirmed the trans derivative as the only isomer. The mechanism of the reaction was investigated by ESI(+)-MS(/MS), and the use of the elegant charge-tag strategy allowed a catalytic cycle to be proposed for the current transformation and revealed the possibility of at least two reaction pathways. One derivative bearing a coumarin scaffold was synthesized, and its fluorescent properties allowed it to be tested as a probe for live-cell imaging experiments with a preference for mitochondria.

32.3 MAGNIFYING THE PROTEOME OF SCHIZOPHRENIA BRAINS

Abstract Background In the post-genomic era, proteomics has emerged as a powerful tool to unravel biomarker candidates and to understand human diseases from the molecular point of view. In the last decade, our group mapped the proteome of several post-mortem brain regions and the cerebrospinal fluid from schizophrenia patients and controls to help deciphering schizophrenia’s pathobiology. These results led us to more recently focus on subproteomes and protein interaction analysis. Methods Using ion mobility-enhanced, data-independent acquisitions and 2D-nano UPLC fractionation, we investigated the mitochondrial, nuclear and cytosolic subproteomes of the dentate nucleus and caudate nucleus as well as the protein interactome of potential protein targets to schizophrenia. Results As previously observed, have found recurrently the differential expression energy associated proteins as well as signaling pathways associated to glutamatergic dysfunction. Proteins associated to translation machinery were also found differentially expressed, implicating spliceosome dysfunction to schizophrenia. In order to understanding this better, we investigated the protein interactome of hnRNPs by co-immunoprecipitation in the disease context. Discussion Proteomics findings may provide an integrated picture of schizophrenia′s pathobiology and the identification of proteins associated to energy, signalling and translational pathways may trace back the origins of schizophrenia.