Abundance of intrinsic disorder in SV-IV, a multifunctional androgen-dependent protein secreted from rat seminal vesicle

The potent immunomodulatory, anti-inflammatory and procoagulant properties of the
protein no. 4 secreted from the rat seminal vesicle epithelium (SV-IV) have been
previously found to be modulated by a supramolecular monomer-trimer equilibrium.
More structural details that integrate experimental data into a predictive framework
have recently been reported. Unfortunately, homology modelling and fold-recognition
strategies were not successful in creating a theoretical model of the structural
organization of SV-IV. It was inferred that the global structure of SV-IV is not similar
to any protein of known three-dimensional structure. Reversing the classical approach
to the sequence-structure-function paradigm, in this paper we report on novel
information obtained by comparing physicochemical parameters of SV-IV with two
datasets made of intrinsically unfolded and ideally globular proteins. In addition, we
have analysed the SV-IV sequence by several publicly available disorder-oriented
predictors. Overall, disorder predictions and a re-examination of existing experimental
data strongly suggest that SV-IV needs large plasticity to efficiently interact with the
different targets that characterize its multifaceted biological function and should be
therefore better classified as an intrinsically disordered protein

Challenges and frontiers of computational modelling of biomolecular recognition

Biomolecular recognition including binding of small molecules, peptides and proteins to their target receptors plays a key role in cellular function and has been targeted for therapeutic drug design. However, the high flexibility of biomolecules and slow binding and dissociation processes have presented challenges for computational modelling. Here, we review the challenges and computational approaches developed to characterize biomolecular binding, including molecular docking, molecular dynamics simulations (especially enhanced sampling) and machine learning. Further improvements are still needed in order to accurately and efficiently characterise binding structures, mechanisms, thermodynamics and kinetics of biomolecules in the future

Investigating the roles of neuropeptides in the development of the sea urchin, Strongylocentrotus purpuratus

Neuropeptides are ancient signalling molecules that bind to receptors on target cells to induce a variety of growth, metabolic, reproductive and behavioural responses. At least 30 neuropeptide signalling systems were present in the common ancestor of bilaterians, and therefore neuropeptide signalling systems offer a good opportunity to characterise neuronal cell types and help determine the evolution of neuronal cell types and nervous systems. 38 neuropeptide precursor (NP) genes have so far been identified in the invertebrate deuterostome, the purple sea urchin, Strongylocentrotus purpuratus, but their function is largely unknown. Here I molecularly characterise and investigate the roles of NP signalling systems in the S. purpuratus embryo and larva. I first present the spatio-temporal data for NP genes and G-protein coupled receptors (GPCRs) genes during the development of S. purpuratus. QPCR and transcriptome data have revealed that almost all of these NP genes are expressed in the late larval stage, when cells differentiate. ISH data of these NP genes has revealed at least nine distinct populations of peptidergic neurons and showed that the sea urchin larval nervous system is more neurochemically complex than previously thought. Interestingly, some NP genes are also expressed in the pre-gastrula phase of embryogenesis, before cells differentiate. These early expressing NP genes are expressed in undifferentiated ectodermal and endodermal cells, suggesting that these signalling molecules may have a novel developmental role as well as a neuronal role. I also optimised CRISPR/Cas9 genome-editing in the sea urchin and used CRISPR/Cas9 and morpholino oligonucleotides to perturb the expression of an NP gene Sp-Thyrotropin releasing hormone (Sp-TRH) and its proven receptor. Here I show that the TRH neuropeptide signalling system acts as a local ectodermal cue, to regulate larval skeleton growth through VEGF and FGF signalling. This is the first study to investigate the roles of TRH signalling in a marine invertebrate larvae, which together with previous studies in adults reveals that the TRH signalling system is an ancient regulator of growth

Neurofly 2008 abstracts : the 12th European Drosophila neurobiology conference 6-10 September 2008 Wuerzburg, Germany

This volume consists of a collection of conference abstracts

Structural characterization of protein-DNA response element ternary complex

openI fattori di trascrizione sono elementi fondamentali alla base di diversi processi biologici, tra cui la regolazione del ciclo cellulare e del differenziamento cellulare durante lo sviluppo, il controllo della risposta immunitaria e il mantenimento dell’omeostasi cellulare. La loro capacità di regolare la trascrizione di specifici geni permette alla cellula di rispondere a diversi stimoli esterni. Sono elementi dinamici che intervengono alla fine della cascata di trasduzione quando, attivati da diverse reazioni chimiche, legano il DNA e ne permettono la trascrizione. L‘interazione con il DNA avviene per la maggior parte dei casi in collaborazione con altri fattori di trascrizione o numerosi cofattori. Alterazioni di questo meccanismo portano all‘insorgenza di molteplici patologie tra cui il cancro. Per questo motivo, l’intricata rete in cui partecipano i fattori di trascrizione con i cofattori e le interazioni che creano è un campo della ricerca attivo. In questa tesi, due fattori di trascrizione, in particolare TEAD1 e FOXO4 sono stati analizzati per ottenere informazioni riguardo cambi conformazionali che avvengono nell’interazione con il DNA e nell’interazione proteina-proteina in assenza o in presenza del DNA. Basandosi su precedenti evidenze di una possibile interazione tra TEAD1 e FOXO4, è stato deciso di svolgere l’esperimento coniugando una reazione di cross-linking con l’analisi LC-MS. Le due proteine sono state analizzate in condizioni differenti utilizzando DSA cross-linker marcato con isotopi nella sua versione leggera e pesante. Conoscendo la posizione degli aminoacidi coinvolti nel legame con il cross-linking agent e sapendo che il cross-linking agent ha una lunghezza definita che determina la distanza tra i gruppi funzionali che lega, è stato possibile ottenere informazioni su come TEAD e FOX04 riarrangiano la loro conformazione Oltretutto, DSA cross-linker ha permesso, essendo marcato con isotopi, la quantificazione dei cambiamenti conformazionali avvenuti nelle proteine nelle condizioni studiate e le dinamiche di interazione delle proteine. Sono stati caratterizzati i cambi conformazionali delle proteine nella sola presenza del DNA. Nel caso di TEAD1, 7 cross-links sono stati individuati e tra questi, tre in particolare indicano che TEAD cambia conformazione nell‘interazione con il DNA. Al contrario, per FOXO4 i tre cross-links individuati rimangono quantitativamente invariati anche quando il fattore di trascrizione lega il DNA Inoltre, quando TEAD1 e FOXO4 sono in soluzione insieme, nell’aggiunta del DNA `e stato notata un’interazione tra le due proteine per formare un complesso ternario TEAD1-FOXO4-DNA. La presenza significativa del cross-link ha confermato quest’interazione.Transcription factors are key elements of several biological processes such as control of cell cycle progression, differentiation of cells during development, immune response, or maintenance of intracellular metabolic balance. Their ability to regulate the transcription of specific sets of genes makes the cells responsive to diverse stimuli. They are dynamic elements participating in the last step of a signalling cascade with the ability to bind the DNA cooperating with other transcription factors and with numerous cofactors. They are elements of interest since dysregulation of the transcription leads to many diseases including cancer. For this reason, the complicated network of transcription factors and cofactors and their interactions is an active research field. In this work, two transcription factors TEAD and FOXO4 were analysed to retrieve some information about their structural change in conformation when they are interacting with the DNA response element and when they are in a mixture together either with or without the DNA. Based on previous evidence of a possible interaction between TEAD and FOXO4, the experiment was set on the use of isotopic cross-linkers conjugated with LC-MS analysis. The two proteins were studied in different conditions in the presence of the isotopically labelled DSA cross-linking agents. The positions of the cross-linked amino acids combined with the known cross-linking agent length that imposes a distance constraint between functional groups within a protein or a complex gave information on the 3D structure of TEAD, FOXO4 and their complex. Moreover, the isotopically labelled DSA cross-linker allowed the quantification of conformational changes of proteins under different conditions and the protein interaction dynamics. It was identified how the proteins change conformation when they are in the only presence of DNA. In the case of TEAD, 7 cross-links were depicted and among them, 3 highlighted a change in the structure of the protein in the presence of the DNA. On the contrary, for FOXO4 3 cross-links were identified which are slightly changing when the transcription factor interacts with the DNA. Furthermore, when TEAD and FOXO4 are mixed in the presence of the DNA, it was noticed that they interact and change conformation to adjust themselves in the formation of a ternary complex TEAD-FOXO4-DNA and a significant presence of a cross-link confirmed an interaction of TEAD and FOXO4 to form the complex with the DNA

Role of neuronal IGF-1R signaling for the pathogenesis of Alzheimer's disease

Post mortem investigations of brains from patients with AD revealed a markedly down regulated expression IGF-1R, and insulin receptor substrate (IRS) proteins, and these changes progress with severity of neurodegeneration. A common feature in neurons from AD patients is a downregulation of IGF-1R. To investigate the role of neuronal IGF-1R signaling in AD neuron-specific IGF-1R (nIGF-1R-/-) deficient mice were generated. These mice were crossed with mice expressing the Swedish mutation of human APP695 harbouring the double mutation Lys670→; Asn, Met671→; Leu which was found in a Swedish family with early-onset AD (APPsw, Tg2576 mice). nIGF-1R-/- mice were generated using the cre-loxP-system under the control of the neuron-specific synapsin-1 promoter and crossed them into the Tg2576 background. The offsprings of these mice (WT, Tg2576, nIGF-1R-/-, nIGF-1R-/-Tg2576) were analysed at two different time points. Kaplan-Meier analysis, amyloid accumulation as well as metabolic and somatic factors of the offspring were investigated during an observation period of 60 weeks. Western blot analysis of isolated hippocampi displayed a 40% reduced IGF-1R expression in nIGF-1R-/- and nIGF-1R-/-Tg2576 compared to WT and Tg2576 animals, whereas other brain regions e.g. cortex or cerebellum did not show significant IGF-1R deletion. Thus, conditional IGF-1R deletion using Cre recombinase expression under the control of the synapsin-1 promoter leads to a hippocampus-specific downregulation of IGF-1R. Further analysis of Cre recombinase expression in a lacZ reporter mouse strain revealed a Cre recombinase activity driven by the synapsin-1 promoter in the dentus gyrus and the CA3 region of the hippocampus. Kaplan-Meier-analysis revealed a 60% mortality of Tg2576 mice after 60 weeks of observation. In contrast nIGF-1R-/-Tg2576 were protected against premature mortality of Tg2576 mice (p&≤0.02; Tg2576 vs. nIGF-1R-/-Tg2576). Isolated hippocampi of 28 and 60 weeks old nIGF-1R-/-Tg2576 animals showed a 50% reduced Aβ1-40 and Aβ1-42 accumulation compared to Tg2576. Additionally, APP α- and β-C-terminal fragments were reduced in hippocampi of nIGF-1R-/-Tg2576 compared Tg2576 mice due to a modification of α- and β-secretases activity. In addition Aβ plaque burden was reduced in Tg2576 animals with neuronal IGF-1R deletion. Taken together the results of the present thesis demonstrate that decreased neuronal IGF-1R signaling predominantly in the hippocampus protects against APPsw induced mortality. Moreover IGF-1R mediated signals influence APP processing due to a modification of α- and β-secretases leading to reduced Aβ accumulation and amyloid plaque burden. Thus, downregulation of IGF-1R observed in neurons of patients suffering from Alzheimer's disease is most likely a compensatory phenomenon to decrease amyloid accumulation and prolong survival

Brain-derived neurotrophic factor in megakaryocytes

The biosynthesis of endogenous BDNF has thus far been examined in neurons where it is expressed at very low levels, in an activity-dependent fashion. In humans, BDNF has long been known to accumulate in circulating platelets, at levels far higher than in the brain. During the process of blood coagulation, BDNF is released from platelets which has led to its extensive use as a readily accessible biomarker, under the assumption that serum levels may somehow reflect brain levels. To identify the cellular origin of BDNF in platelets, we established primary cultures of megakaryocytes, the progenitors of platelets, and found that human and rat megakaryocytes express the BDNF gene. Surprisingly, the pattern of mRNA transcripts is similar to neurons. In the presence of thapsigargin and of external calcium, the levels of the mRNA species leading to efficient BDNF translation rapidly increase. Under these conditions, pro-BDNF, the obligatory precursor of biologically active BDNF, becomes readily detectable. Megakaryocytes store BDNF in α-granules, with more than 80% of them also containing platelet factor 4. By contrast, BDNF is undetectable in mouse megakaryocytes, in line with the absence of BDNF in mouse serum. These findings suggest that alterations of BDNF levels in human serum as reported in studies dealing with depression or physical exercise may primarily reflect changes occurring in megakaryocytes and platelets, including the ability of the latter to retain and release BDNF

A novel genome-wide approach to identify in vivo targets of Notch signalling

The Notch signalling pathway regulates many developmental processes in metazoan embryos and adults such as cell proliferation, stem cell maintenance, cell fate specification and apoptosis. Despite the importance of this pathway, few targets have been identified, with the Hes (Hairy and Enhancer-of-split) protein family being the best-characterised group of downstream effectors. I have established transgenic mice carrying Biotin Acceptor Peptide (BAP)- tagged versions of Notch1. The tagged protein is fully functional and is biotinylated after crossing to mice expressing the biotinylase from E. coli. Biotinylation was confirmed in a range of different tissues. However, streptavidin chromatin pull-down (bioChIP) experiments from these tissues showed no significant enrichment of known Notch1 target sequences. A possible explanation could be the indirect and transient nature of the interaction between Notch, its DNA binding partner CSL and the promoter of the target gene. A transgenic mouse line expressing a BAP-tagged version of the transcription factor Hes7, a downstream effector of Notch signalling and key regulator of somitogenesis, was similarly generated. Although the tagged Hes7 protein is functional and gets biotinylated in cell culture assays, the transgenic mice exhibit a severe somite/skeletal phenotype indicating that the tagged allele is hypomorphic. A detailed analysis of the phenotype revealed differential axial requirements for Hes7

The Role of Hedgehog Acyltransferase & Heparan Sulphate Proteoglycans in Human Sonic Hedgehog Signalling

Hedgehog proteins (Hh) are morphogens and major mediators in many developmental processes. Hh signalling is significant for many aspects of embryonic development, whereas dysregulation of this pathway is associated with several types of cancers. Hh proteins require dual lipidation and Heparan Sulfate Proteoglycans (HSPGs) for their proper distribution and signalling activity. My first aim was to study the role of HSPGs in human (h) Sonic Hedgehog (Shh) signalling and clarify the biological function of hShh/HSPGs complexes in hShh signalling, by investigating the interaction between human hShh and HSPGs. I used DNA mutagenesis and heparin affinity chromatography to determine key residues in hShh involved in heparin binding (K37/38 and K178). The activity of these mutants was tested by detecting induced Alkaline Phosphatase activity in C3H10T1/2 cells and hShh-inducible gene expression in PANC1 human pancreatic carcinoma cells. I examined the biological function of mutated hShhs (K37/38S, K178S and K37/38/178S) that cannot interact with heparin efficiently and showed that they had reduced signalling activity compared to wild type hShh and a control mutation (K74S). Also, I showed that mutant hShh proteins mediate reduced proliferation and invasion of PANC1 cells following hShh RNAi knockdown (KD), and this correlated with reduced Shh multimeric complex formation. Structurally, Shh proteins are unusual in being dually lipid-modified to be fully active. During the post-translational modifications of Shh, N-terminal palmitoylation is facilitated by the product (Hhat) of the hedgehog acyltransferase gene. I have carried out a thorough analysis of Hhat in PANC1 cells. First, I characterised an antibody prepared in the lab to hHhat. I confirmed the specificity of the antibody by immunoblotting using a self-constructed hHhat-EGFP clone, and a control mGup1-EGFP clone. By subcellular fractionation and Western blotting I found Hhat to be a membrane protein. In addition, I used the hHhat antibody to determine the intracellular localisation of hHhat in PANC1 cells by confocal microscopy and showed that hHhat localised in ER mainly but not in Golgi apparatus. I confirmed this using the hHhat-EGFP clone for fluorescence microscopy in transfected cells. To illuminate the biological function of palmitoylation of hShh in production of active hShh and in the formation of hShh multimeric complex I optimised hHhat RNAi knockdown (KD) in PANC1 cells and confirmed this by a cell-based palmitoylation assay. Using semi-quantitative RT-PCR and immunoblot analyses, I showed that hHhat KD caused decreased signalling through the Shh pathway due to reduced production of active hShh. In addition, I investigated the effect of the addition of palmitate to hShh on its association with cells by comparing hHhat KD cells with control cells. Immunoblotting suggests that palmitoylation of Shh improves its ability to associate to cell membranes. Using hHhat KD, gel filtration of high molecular weight complexes of hShh and immunoblotting of hShh I characterised the role of palmitoylation of hShh in multimeric complex formation. Lastly, I investigated the effect of hHhat KD on PANC1 proliferation and invasion, showing that it represses PANC1 proliferation and invasion. These studies provide a firm basis for understanding the functional roles of hShh palmitoylation and its interactions with HSPGs, and provide proof-of-principle for targeting these aspects of hShh biology in tumour cell therapeutics, specifically in the pancreatic carcinoma context