PtdIns4P-mediated electrostatic forces influence S-acylation of peripheral proteins at the Golgi complex

Protein S-acylation is a reversible post-translational modification involving the addition of fatty acids to cysteines and is catalyzed by transmembrane protein acyltransferases (PATs) mainly expressed at the Golgi complex. In case of soluble proteins, S-acylation confers stable membrane attachment. Myristoylation or farnesylation of many soluble proteins constitutes the initial transient membrane adsorption step prior to S-acylation. However, some S-acylated soluble proteins, such as the neuronal growth-associated protein Growth-associated protein-43 (GAP-43), lack the hydrophobic modifications required for this initial membrane interaction. The signals for GAP-43 S-acylation are confined to the first 13 amino acids, including the S-acylatable cysteines 3 and 4 embedded in a hydrophobic region, followed by a cluster of basic amino acids. We found that mutation of critical basic amino acids drastically reduced membrane interaction and hence S-acylation of GAP-43. Interestingly, acute depletion of phosphatidylinositol 4-phosphate (PtdIns4P) at the Golgi complex reduced GAP-43 membrane binding, highlighting a new, pivotal role for this anionic lipid and supporting the idea that basic amino acid residues are involved in the electrostatic interactions between GAP-43 and membranes of the Golgi complex where they are S-acylated.Fil: Chumpen Ramirez, Sabrina Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Astrada, Micaela R.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Daniotti, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentin

An update on transcriptional and post-translational regulation of brain voltage-gated sodium channels

Voltage-gated sodium channels are essential proteins in brain physiology, as they generate the sodium currents that initiate neuronal action potentials. Voltage-gated sodium channels expression, localisation and function are regulated by a range of transcriptional and post-translational mechanisms. Here, we review our understanding of regulation of brain voltage-gated sodium channels, in particular SCN1A (Naᵥ1.1), SCN2A (Naᵥ1.2), SCN3A (Naᵥ1.3) and SCN8A (Naᵥ1.6), by transcription factors, by alternative splicing, and by post-translational modifications. Our focus is strongly centred on recent research lines, and newly generated knowledge

The computational analysis of post-translational modifications

The post-translational modification (PTMs) of proteins presents a means to increase the proteome size and diversity of an organism through the inclusion of structural elements not encoded at the sequence-level alone. Their erroneous inclusion or exclusion has been linked to a variety of diseases and disorders thus their characterisation has the potential to present viable drug targets. The proliferation of newer high-throughput methods, such as mass spectrometry, to identify such modifications has led to a rapid increase in the number of databases and tools to display and analyse such vast amounts of data effectively. This study covers the development of one such tool; PTM Browser, and the construction of the underlying database that it is based upon. This new database was initially seeded with annotations from the Swiss-Prot and Phospho.ELM resources. The initial database of PTMs was then expanded to include a large repertoire of previously unannotated proteins for a selection of topical species (e.g. Danio rerio and Tetraodon nigroviridis). Orthologue assignments have also been added to the database – to allow for queries to be performed regarding the conservation of modifications between homologous proteins. The PTM Browser tool allows for a full exploration of this new database of PTMs – with a special focus on allowing users to identify modifications that are both shared between and are specific to particular species. This tool is freely available for non-commercial use at the following URL: http://www.ptmbrowser.org. An analysis is presented on the conservation of modifications between members of the tumour suppressor family, p53, using this new tool. This tool has also been used to analysis the conservation of modifications between super-kingdoms and Eukaryote species

Drug Discovery

Natural products are a constant source of potentially active compounds for the treatment of various disorders. The Middle East and tropical regions are believed to have the richest supplies of natural products in the world. Plant derived secondary metabolites have been used by humans to treat acute infections, health disorders and chronic illness for tens of thousands of years. Only during the last 100 years have natural products been largely replaced by synthetic drugs. Estimates of 200 000 natural products in plant species have been revised upward as mass spectrometry techniques have developed. For developing countries the identification and use of endogenous medicinal plants as cures against cancers has become attractive. Books on drug discovery will play vital role in the new era of disease treatment using natural products

Palmitoylation of BK channels

Palmitoylation is a post-translational modification that has been implicated in the control of multiple proteins, including ion channels. S-Palmitoylation is a lipophilic modification that involves the attachment of palmitate through a thioester linkage to a cysteine residue in a target protein. By increasing the hydrophobicity of the target region, palmitoylation can promote membrane targeting. Here, palmitoylation is shown to play an important role in regulating large conductance calcium- and voltage- activated (BK) potassium channels. The STREX splice variant of the BK channel contains a 58 amino acid insert at the splice site C2 within the intracellular C-terminal RCK1-RCK2 linker that confers increased calcium sensitivity to the channel and determines PKA inhibition of channel activity. The cysteine rich STREX domain was predicted to be palmitoylated, and using an imaging assay STREX was shown to act as a membrane targeting domain through palmitoylation of a di-cysteine motif (C645:C645). A membrane potential assay and electrophysiological analysis demonstrates that palmitoylation at the C645:C646 site in STREX is important in mediating the increased calcium sensitive properties inherent to the STREX channel. Palmitoylation is also shown to modulate PKA channel inhibition. The stability of palmitoylation can often be reliant on the local environment within the protein. Generally in most proteins; lipidated regions, basic domains or transmembrane domains are found adjacent to a palmitoylation site. In STREX, a polybasic domain composed of 11 basic residues just upstream from the C645:C646 palmitoylation site, functions to control the palmitoylation status of the STREX insert. A site directed mutagenesis approach to disrupt the polybasic domain revealed an important role in controlling membrane targeting of the STREX C-terminus, mediating the increased calcium sensitivity inherent to STREX channels and controlling the palmitoylation status of the C645:C646 palmitoylation site using multiple techniques involving electrophysiology, fluorescent imaging and biochemical assays. Further to this, using imaging to examine the membrane association of fluorescently tagged C-terminal proteins, phosphorylation is shown to function as a physiological electrostatic switch to regulate the polybasic region in controlling palmitoylation of the STREX insert. Finally, an additional palmitoylation site that is constitutively expressed in all BK channels was identified to be located in the S0-S1 linker (C53:C54:C56). Mutation of the C53:C54:C56 palmitoylation site in the S0-S1 linker was shown to abolish all palmitoylation in BK channels that did not contain the STREX insert. Palmitoylation allows the S0-S1 linker to associate with the plasma membrane however the mutated de-palmitoylated channels did not affect channel conductance or the calcium/voltage sensitivity of the channel. Palmitoylation of the S0-S1 linker was shown to be a critical determinant of cell surface expression of BK channels, as steady state surface expression levels were reduced by ~55% in the C53:C54:C56 mutant. STREX channels that could not be palmitoylated in the S0-S1 linker also showed decreased surface expression even through STREX insert palmitoylation was unaffected. Palmitoylation is rapidly emerging as an important post-translational mechanism to control ion channel behaviour. This work reveals that palmitoylation of the BK channel can control channel function of the STREX splice variant channel and can regulate cell surface expression in all other channel variants. Palmitoylation appears to be functionally independent at these two distinct sites expressed within the same channel protein

The cellular functions of mammalian type II phosphatidylinositol 4-kinases

Type II phosphatidylinositol 4-kinases (PI4KIIs), PI4KIIα and PI4KIIβ, both catalyse phosphatidylinositol 4-phosphate (PI(4)P) synthesis and are implicated in the control of trafficking from the trans-Golgi network (TGN) and endosomal membranes. It has been suggested that these closely related isoforms perform redundant roles. This study addresses the issue of functional overlap, by studying the location of the PI(4)P pools synthesised by each isoform, the associated membrane trafficking routes and the functional consequences of loss of these PI4P pools. The TGN localisations of PI4KIIα and PI4KIIβ could be distinguished by co-immunostaining with TGN markers syntaxin 6 and TGN46, indicating that the isoforms localise to separate TGN domains. In addition, depletion of PI4KII isoforms using small interfering RNA (siRNA) had differential effects on TGN pools of PI(4)P, with PI4KIIα loss significantly affecting a syntaxin 6 positive PI(4)P pool while PI4KIIβ depletion altered a TGN46 positive pool; thus indicating the synthesis of metabolically separate PI(4)P pools by these two isoforms. Depletion of either PI4KII isoform also impaired post-TGN traffic of cation independent mannose 6-phosphate receptor (CI-M6PR) and the endo-lysosomal traffic and degradation of the EGF receptor which is suggestive of overlapping roles for both isoforms in post-TGN traffic. PI4KII gene silencing also had differential effects on the actin cytoskeleton. Loss of PI4KIIα led to increased stress fibre formation while PI4KIIβ depletion induced the formation of functional invadopodia containing membrane type I matrix metalloproteinase (MT1-MMP). This was accompanied by decreased colocalization of MT1-MMP with the endosomal markers Rab5 and Rab7 that control lysosomal trafficking and regulate surface levels of MT1-MMP. However, MT1-MMP showed increased colocalisation with Rab8, which mediates exocytic trafficking and pro-invasive activity of MT1-MMP. In addition, depletion of PI4KIIβ conferred a migratory phenotype on minimally invasive HeLa and MCF-7 cell lines. This cell phenotype was substantiated by oncogenomic database analyses showing that loss of PI4KIIβ expression was a risk factor for numerous human carcinomas

Protein S‐palmitoylation: advances and challenges in studying a therapeutically important lipid modification

The lipid post‐translational modification S‐palmitoylation is a vast developing field, with the modification itself and the enzymes that catalyse the reversible reaction implicated in a number of diseases. In this review we discuss the past and recent advances in the experiment tools used in this field, including pharmacological tools, animal models and techniques to understand how palmitoylation controls protein localisation and function. Additionally, we discuss the obstacles to overcome in order to advance the field, particularly to the point at which modulating palmitoylation may be achieved as a therapeutic strategy

Protein-protein interactions in GCR1 signalling in arabidopsis thaliana

The G-protein coupled receptors (GPCRs) are seven-transmembrane receptors that transduce signals from the cell surface to intracellular effectors. There are more than 1000 GPCRs in metazoans, while no GPCR has been definitively identified in plants. The most promising plant GPCR candidate, Arabidopsis G-protein coupled receptor 1 (GCR1), physically couples to the G-protein < subunit GPA1 and is involved in cell cycle regulation, blue light and phytohormone responses, but its signalling network remains largely unknown. This project aimed to achieve a better understanding of GCR1 signalling by identifying its interactors using a novel yeast two hybrid system – the Ras Recruitment System (RRS). Screening of an Arabidopsis cDNA library using a bait comprising intracellular loop 1 (i1) and 2 (i2) of GCR1 resulted in the isolation of 20 potential interactors. Extensive reconfirmation screening demonstrated that three of these interactors: Thioredoxin h3 (TRX3), Thioredoxin h4 (TRX4) and a DHHC type zinc finger family protein (zf-DHHC1) interact specifically with both i1 and i2 of GCR1. This was supported by the reverse RRS (rRRS) and 6xHis-pull-down assays. It is speculated that TRX3 and TRX4, which can reduce disulfide bridges of target proteins and act as powerful antioxidants, may regulate GCR1-mediated signalling events in response to oxidative stress. Alternatively, they may modulate GCR1 targeting or signalling through their chaperone activities. zf-DHHC1 has a predicted membrane topography that is shared by most DHHC domain-containing palmitoyl acyl transferases. It may modify GCR1 activity through palmitoylation of the two cysteines located at the cytoplasmic end of the first transmembrane domain. Together, these findings contribute to the growing understanding of the GCR1 signalling network, and provide valuable starting points for further investigation.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Novel chemical proteomics approaches to study N-myristoylation and N-terminal methionine excision

N-terminal modifications may constitute the first modifications any protein acquires and can modulate protein function by altering protein stability and 3D structure, promoting or interfering with protein-protein interactions and regulating membrane targeting, localisation or secretion. Here, two interlinked N-terminal protein modifications have been studied: initiator methionine (iMet) excision and co-translational N-myristoylation. N-myristoylation involves de attachment of myristic acid, a 14-carbon saturated fatty acid, onto exposed N-terminal glycines of protein substrates. In humans, this reaction is catalysed by N-myristoyltransferases 1 and 2. The two NMT isoforms share a high degree of sequence and structural similarity, which has hindered the development of isoform-specific inhibitors to date and prevented the dissection of isoform- specific substrate pools. Using CRISPR/Cas9 in combination with a previously described metabolic labelling approach and whole proteome profiling, NMT1, and not NMT2, was defined as the main enzyme responsible for N-myristoylation of proteins in the cancer cell. In addition, a novel method was designed to accurately assess on-target activity of NMT inhibitors and the fate of N-myristoylated substrates across the whole proteome upon NMT inhibition. This new approach relies on post-lysis labelling of exposed N-terminal glycines by S. aureus sortase A (SrtA) and no longer relying on metabolic labelling, it can be applied to any type of biological sample. Methionine aminopeptidases (MetAPs) catalyse initiator methionine (iMet) removal from nascent proteins and are essential to maintain healthy proteome dynamics by priming other N-terminal modifications such as N-acetylation or N-myristoylation and modulating protein localisation and stability. MetAP2 has been explored by pharmaceutical companies for decades for the treatment of cancer and obesity. However, the links between MetAP2 inhibition and phenotypic effects are still poorly understood. Here, a novel chemical proteomics workflow is proposed to elucidate the substrates of MetAP2 systematically and uncover the missing link between MetAP2 inhibition and phenotype. This new strategy is based on metabolic labelling of cells with the methionine analogue azidohomoalanine (AHA) and in combination with specific pharmacological inhibition of MetAP2, allowed identification of >70 substrates of MetAP2, 94% of which were unknown until reported here. Together, this work provides fundamental insights into the biological role and importance of N-myristoylation and iMet excision in cancer and shapes the path for future steps in the use of NMT and MetAP2 inhibitors for the treatment of human disease.Open Acces