Correlation of calpain sensitivity, Bradford assay instability, and electrophoretic mobility in phosphomimetic mutants of GlyT2 N-terminus.

The glycine transporter GlyT2 plays an important role in glycine-inhibitory neurotransmission of the hindbrain and spinal cord. Its special feature is the extended N-terminus, which contains a large number of potentially phosphorylated serine and threonine residues. Due to its unstructured nature, it is difficult to address the changes introduced by potential phosphorylation. Here, we used relatively simple methods such as calpain sensitivity, Bradford instability, and SDS electrophoretic mobility shift to investigate the effect of multiple phosphomimetic mutations versus neutral mutations on GlyT2N properties. The replacement of several serines or threonines with neutral alanines did not have a significant effect on the studied GlyT2N properties. Replacement of the same residues with phosphomimetic aspartate resulted in significant alterations in calpain cleavage patterns, Bradford instability, and SDS gel protein mobility. Interestingly, a correlation between the relative intensity of the measured effects was observed, indicating that they all reflect similar structural changes introduced by potential phosphorylation in vivo. Results indicate that a potential single or multiple phosphorylation significantly alters the proteomic properties of the glycine transporter GlyT2 N-terminus. Assays can be helpful in the first screening of structurally significant and possibly phosphorylated residues in the N-terminus of GlyT2.

Manganese- and zinc-coordinated interaction of Schistosoma japonicum glutathione S-transferase with neurotransmitter transporters GlyT1 and GAT3 in vitro.

Glutathione S-transferases (GSTs) are a family of multifunctional isoenzymes involved in the neutralization of toxic compounds, drug resistance and several other cellular functions. The glutathione S-transferase enzyme of Schistosoma japonicum (SjGST-26) plays a role in human schistosomiasis and is also a frequently used fusion partner in mammalian and bacterial expression and pull-down systems. GSTs seem not to be naturally associated with metal ions. Exceptionally, in vitro, metal binding sites have been previously described in some schistosome GSTs; however, their possible physiological role is unclear. Molecules of several neurotransmitter transporters also contain a regulatory zinc binding site, which affects their transport cycle. Here we show that among several metals, manganese and zinc are able to induce a specific protein interaction of SjGST-26 with the glycine transporter GlyT1 and the GABA transporter GAT3 in vitro. The results suggest that metal-binding sites on SjGST-26 and neurotransmitter transporters might function in metal-coordinated interactions with other metalloproteins. Our results additionally indicate that the presence of metal ions in SjGST-26-based GST protein pull-down assays may lead to a false-positive interaction if the potential interacting target is the metalloprotein.

PDZ interaction of the GABA transporter GAT1 with the syntenin-1 in Neuro-2a cells.

The GABA transporter GAT1 regulates brain inhibitory neurotransmission and it is considered a potential therapeutic target for the treatment of wide spectrum of neurological diseases including epilepsy, stroke and autism. Syntenin-1 binds to syntaxin 1A, which is known to regulate the plasma membrane insertion of several neurotransmitter transporters. Previously, a direct interaction of syntenin-1 with the glycine transporter GlyT2 was reported. Here, we show that the GABA transporter GAT1 also directly interacts with syntenin-1, involving both unidentified protein interaction interface and the GAT1 C-terminal PDZ binding motif interacting mainly with syntenin-1 PDZ domain 1. The PDZ interaction was eliminated by the mutation of GAT1 isoleucine 599 and tyrosine 598 located in PDZ positions 0 and -1, respectively. This indicates an unconventional PDZ interaction and possible regulation of the transporter PDZ motif via tyrosine phosphorylation. Whole syntenin-1 protein fused to GST protein and immobilised on glutathione resin coprecipitated intact GAT1 transporter from an extract of GAT1 transfected neuroblastoma N2a cells. This coprecipitation was inhibited by tyrosine phosphatases inhibitor pervanadate. The fluorescence tagged GAT1 and syntenin-1 colocalized upon coexpression in N2a cells. The above results show that syntenin-1 might be, in addition to GlyT2, directly involved in the trafficking of GAT1 transporter.

A Significant Difference in Core PDZ Interactivity of SARS-CoV, SARS-CoV2 and MERS-CoV Protein E Peptide PDZ Motifs In Vitro.

Small structural E protein of coronaviruses uses its C-terminal PDZ motif to compromise the cellular PDZ interactome. In this work we compared core PDZ interactivity of small (seven amino acids) peptide PDZ motifs, originating from the envelope proteins of recently transmitted coronaviruses SARS-CoV, SARS-CoV2, and MERS-CoV. As the interaction targets we used 23 domains of the largest PDZ proteins MUPP1/MPDZ and PATJ/INAD. Results revealed exceptional affinity and interaction promiscuity of MERS-CoV PDZ motif in vitro, suggesting an increased probability of potential PDZ targets in vivo. We hypothesize that together with its known ability to enter the cells from both apical and basolateral sites, this might further contribute to its elevated disruption of cellular PDZ pathways and higher virulence.

Phosphorylation of Serine 157 Protects the Rat Glycine Transporter GlyT2 from Calpain Cleavage.

The N-terminal region of the rat glycine transporter 2 (rGlyT2, SLC6A5) is cleaved by calpain protease in vitro, which raises the question of its protection against calpain in vivo. Here, we used a phosphomimetic and orthogonal phosphoserine translation approach to investigate the possible role of phosphorylation in the protection of two calpain cleavage sites, M156/S157 and G164/T165, previously identified in the N-terminus region of the rat GlyT2. Replacement of serine 157 with phosphomimetic aspartate or with orthogonal phosphoserine blocked both calpain cleavage sites and caused an electrophoretic mobility shift of rGlyT2N fusion proteins. Both effects can be reversed by dephosphorylation, suggesting that phosphorylation might induce structural changes in the rGlyT2 N-terminus, preventing the accessibility of the M156/S157 and G164/T165 cleavage sites to calpain in vivo. In comparison with the wild type, the phosphomimetic mutation S157D increased the total immunoreactivity of the transporter expressed in neuroblastoma cells, suggesting that serine 157 phosphorylation or phosphorylation-regulated calpain cleavage might contribute to the turnover of the glycine transporter GlyT2.

Comparison of SynCAM1/CADM1 PDZ interactions with MUPP1 using mammalian and bacterial pull-down systems.

BACKGROUND: Synaptic cell adhesion molecule 1 (SynCAM1) also known as cell adhesion molecule 1 (CADM1) is a transmembrane cell adhesion protein that operates in a variety of physiological and pathological cellular contexts, and its interaction with the PDZ signalling protein MUPP1 have been previously implicated in autism spectrum disorder (ASD). METHODS: We used in vitro pull-down systems based on the bacterial and mammalian extracts to study SynCAM1/CADM1 PDZ interactions with MUPP1 at various conditions. RESULTS: So far, the investigated interaction of SynCAM1/CADM1 with MUPP1 has been mostly attributed to an unspecified region of MUPP1 PDZ domains 1-5 or exclusively to domain 2, using a yeast two-hybrid system. We also confirmed the single interaction of native synaptosomal CADM1 with PDZ domain 2. However, in this work, using recombinant proteins overexpressed in bacteria, we found an in vitro pull-down conditions in which all first five domains and, to a much lesser extent, MUPP1 domains 7 and 11 significantly interacted with the whole C-terminal domain of SynCAM1/CADM1. These PDZ interactions were confirmed by a pull-down assay using the last seven amino acids of the SynCAM1/CADM1 PDZ motif and using two fusion partners. Multiple interactions were additionally replicated using the continuous N-terminal MUPP1 protein fragment, which included first five PDZ domains, containing either intact or mutated domain 2. CONCLUSIONS: We hypothesize that multiple interactions might exist in vivo, representing transient low-affinity interactions or alternative binding sites on MUPP1 when domain 2 is occupied or occluded by the interaction with other ligands. This newly identified interactions extend the potential genetic mutations, possibly affecting SynCAM1/CADM1/MUPP1 function. Possible reasons for the absence of some of the identified CADM1 PDZ interactions in mammalian extracts are discussed.

Correction to: Phosphomimetic Mutation of Glycine Transporter GlyT1 C-Terminal PDZ Binding Motif Inhibits its Interactions with PSD95.

The original version of this article unfortunately contained two mistakes.

Phosphomimetic Mutation of Glycine Transporter GlyT1 C-Terminal PDZ Binding Motif Inhibits its Interactions with PSD95.

Even though the abundance of GlyT1 in postsynaptic aspects of forebrain neurons is low, its previously reported interaction with postsynaptic density protein PSD95 represents a prototype of interaction, which might uncover the binding mechanism and regulation of the GlyT1 C-terminal PDZ motif. We used a phosphomimetic approach to mimic the potential phosphorylation of GlyT1 C-terminal serines 640, 643, 644, and 649 of the mouse GlyT1b subtype (mGlyT1b) (Uniprot P28571-2) and its effect on GlyT1-PSD95 PDZ interaction. Among them, only phosphomimetic mutation of serine 649 to aspartate, which resides in the minimal PDZ motif -SRI, significantly eliminated the interaction of the GlyT1 C-terminus with PSD95 PDZ domain 2. The effect was observed with recombinant fusion proteins as well as with GlyT1 and PSD95 expressed in tissue culture. Results indicate that phosphorylation of mouse GlyT1b serine 649 and equivalent serines of GlyT1a and GlyT1c subtypes might regulate the PDZ interaction of the GlyT1 C-terminal PDZ binding motif.

Specific glycine to alanine mutation eliminates dynamic interaction of polymeric GlyT1a N-terminus with Coomassie Brilliant Blue G-250.

We previously found that multimeric GlyT1aN16 protein exhibits increased diffusion in a polyacrylamide gel and shows an unusual time-dependent absorbance rearrangement, as revealed by the Bradford assay. Here, we find that glycine to alanine mutation eliminates the absorbance shift, but not the altered diffusion properties of GlyT1aN16, indicating that these two phenomena are not interconnected. The absorbance shift is apparent with both native and urea-denatured GlyT1aN16, suggesting that the effect is either not dependent on protein structure, or the required structure is restored very quickly following denaturant removal. In the far-UV spectra, circular dichroism (CD) curves for both wild-type and mutated GlyT1aN16 are under the zero line, indicating largely unstructured character. However, a significant shift of the mutant CD curve suggests possible microstructural heterogeneity. Deconvolution of the CD data indicates a potential 3-fold increase in isolated helical content, which would inhibit an absorbance shift, as we demonstrated previously. These results suggest that, in addition to protein quantification, Coomassie Brilliant Blue G-250 can be used to reveal certain properties of the secondary structure of proteins.

A Dynamic Interaction of Coomassie Dye with the Glycine Transporters N-termini.

Coomassie Brilliant Blue interacts with proteins and even though the interactions exhibit variation due to the amino acid content, reported dye interactions with individual proteins appear to be relatively stable. Here we report an atypical dynamic interaction of glycine transporters 1 and 2 N-termini with Coomassie dye, resulting in intramolecular interference with their Bradford assay. These proteins exhibit classic protein-Coomassie G-250 complex with absorption maximum at 595 nm, which within minutes starts to decrease and parallel increase of absorbance shoulders above 300 and 700 nm is observed. Interestingly, these effects are eliminated upon fusion of glycine transporters N-termini with glutathione S-transferase protein or by the presence of glutathione S-transferase or bovine serum albumin in the same solution. Circular dichroism data revealed largely unstructured character of glycine transporters N-termini, which suggests that dynamic properties of these protein- Coomassie complexes might be a signature of high flexibility and protein disorder. The assay might potentially reveal similar domains in other proteins and help to associate them with particular functions.

The elution of certain protein affinity tags with millimolar concentrations of diclofenac.

Diclofenac (2-[(2, 6-dichlorophenyl)amino] benzeneacetic acid) is a sparingly soluble, nonsteroidal anti-inflammatory drug therapeutically acting at low micromolar concentrations. In pH range from 8 to 11, its aqueous solubility can be increased up to 200 times by the presence of counter ions such as sodium. Our protein interaction studies revealed that a millimolar concentration of sodium diclofenac is able to elute glutathione S-transferase (GST), cellulose binding protein (CBD), and maltose binding protein (MBP) but not histidine-tagged or PDZ-tagged proteins from their affinity resins. The elution efficiency of diclofenac is comparable with the eluting agents normally used at similar concentrations. Native gel electrophoresis of sodium diclofenac-treated proteins showed that the interaction is non-covalent and non-denaturing. These results suggest that sodium diclofenac, in addition to its pharmaceutical applications, can also be exploited as a lead for the development of new proteomics reagents.

Structural insights into the benzophenanthridines binding to human glycine transporter GlyT1.

We previously identified cysteine 475 as a key residue for the inhibitory action of sanguinarine on the human glycine transporter GlyT1c. To define potential benzophenanthridine binding pocket more closely, we created a structural homology model of GlyT1 and also mutated several amino acids in the vicinity of cysteine 475. Even though this area contains the molecular determinants of the glycine and sodium permeation pathways, and several mutations resulted in an inactive transporter, we found that the mutation of a polar aromatic tyrosine 370 to purely aromatic phenylalanine, but not to an aliphatic leucine, significantly increased the sensitivity of GlyT1 to both sanguinarine and chelerythrine. The reduction of sanguinarine to dihydrosanguinarine completely eliminated the alkaloid's inhibitory potency. Both these results suggest that aromaticity is important in the interaction of benzophenanthridines with GlyT1. Even though tyrosine 370 is part of the conformationaly highly flexible glycine binding site, and is accesible during the transport process from both intra and extracellular sites, the cytoplasmic location of the second alkaloid sensitive residue, cysteine 475, suggests that the benzophenanthridines might attack the area of the GlyT1 intracellular gates.

Using a collection of MUPP1 domains to investigate the similarities of neurotransmitter transporters C-terminal PDZ motifs.

A ubiquitous feature of neurotransmitter transporters is the presence of short C-terminal PDZ binding motifs acting as important trafficking elements. Depending on their very C-terminal sequences, PDZ binding motifs are usually divided into at least three groups; however this classification has recently been questioned. To introduce a 3D aspect into transporter's PDZ motif similarities, we compared their interactions with the natural collection of all 13 PDZ domains of the largest PDZ binding protein MUPP1. The GABA, glycine and serotonin transporters showed unique binding preferences scattered over one or several MUPP1 domains. On the contrary, the dopamine and norepinephrine transporter PDZ motifs did not show any significant affinity to MUPP1 domains. Interestingly, despite their terminal sequence diversity all three GABA transporter PDZ motifs interacted with MUPP1 domain 7. These results indicate that similarities in binding schemes of individual transporter groups might exist. Results also suggest the existence of variable PDZ binding modes, allowing several transporters to interact with identical PDZ domains and potentially share interaction partners in vivo.

Calcium dependent interaction of calmodulin with the GlyT1 C-terminus.

The cytoplasmic regions of neurotransmitter transporters play an important role in their trafficking. This process is, to a high extent, tuned by calcium and calcium binding proteins, but the exact molecular connection are still not fully understood. In this work we found that the C-terminal region of the mouse glycine transporter GlyT1b is able to specifically interact with calmodulin in the presence of calcium. We found that several GlyT1 C-terminal mutations, including those in the ER retention signal, either eliminate or increase calmodulin interaction in vitro. In tissue-culture-expressed GlyT1 at least two of these mutations altered the sensitivity of GlyT1 surface expression and glycine uptake to calmodulin antagonists. These results suggest the possible involvement of calmodulin or calmodulin-like interactions in the regulation of GlyT1C-mediated transporter trafficking.

Effect of phosphomimetic mutations on the C-terminal sensitivity of glycine transporter GlyT1 to calpain.

Previously, we found that the C-terminus of the glycine transporter GlyT1 loses the most of its epitopes during pathological calcium increase in rodent synaptosomes but that the more internal epitopes are spared. We also found that epitope immunoreactivity likely decreases via both phosphorylation and calpain-mediated proteolysis. Here we show that the predicted phosphomimetic mutation S605D fully blocks the adjacent (T602/T603) internal calpain cleavage in the mouse GlyT1b C-terminal fusion protein, but that the neutral S605A mutation does not. Consistent with this, the phophomimetic mutation, but not the neutral mutation, significantly protected the internal GlyT1 C-terminal antiGlyT1C603-626 epitopes against calpain when introduced into tissue culture expressed GlyT1b. Because similar effects can be obtained using phosphatase inhibitors, it may be that phosphorylation of S605 protects the GlyT1 C-terminal sequences from calpain cleavage in vivo.

Expression and purification of recombinant calpain-derived N-terminal peptides from glycine transporter GlyT2.

Glycine transporter GlyT2 contains an extended N-terminal domain which is about three times longer than the N-termini of its closest family members. We previously found that this domain could be separated from the transporter by proteolysis with calpain resulting in the generation of at least two GlyT2N derived peptides. In this work we analyzed the properties of these peptides using bio-informatics, by expressing them in mammalian cell lines and by overexpressing them in bacteria. When expressed in mammalian cell lines, these peptides show widespread localization in the cytoplasm. Their unusually high number of alanine, proline and glycine residues suggests that they posses significant disorder and conformational flexibility, which is supported by their thermal resistivity. Making use of these phenomena, we developed a simple purification method for obtaining pure recombinant GlyT2N derived calpain fragments without using an affinity tag. This procedure can be used to obtain peptide fragments in large amounts for structural, interaction studies or for determining their potential biological activity.

Molecular basis for differential glycine transporters sensitivity to sanguinarine.

We previously demonstrated that glycine transporters GlyT1 and GlyT2 are differentially affected by toxic benzophenanthridine alkaloids. Using a combination of homology modeling, knowledge of the sensitivity of sanguinarine to sulfhydryl reagents and site directed mutagenesis we show here that the increased sensitivity of human GlyT1c to sanguinarine is abolished by the mutation of only cysteine 475. Inhibition requires the membrane permeable alkaloid alkanolamine, which is consistent with the intracellular location of the targeted cysteine.

Differential effect of the benzophenanthridine alkaloids sanguinarine and chelerythrine on glycine transporters.

Glycine transporter inhibitors modulate the transmission of pain signals. Since it is well known that extracts from medicinal plants such as Chelidonium majus exhibit analgesic properties, we investigated the effects of alkaloids typically present in this plant on glycine transporters. We found that chelerythrine and sanguinarine selectively inhibit the glycine transporter GlyT1 with comparable potency in the low micromolar range while berberine shows no inhibition at all. At this concentration both alkaloids only minimally affected transport of the closely related glycine transporter GlyT2, suggesting that the effect is not mediated by the inhibitory activity of these alkaloids on the Na(+)/K(+) ATPase. GlyT1 inhibition was time-dependent, noncompetitive and increased with glycine concentration. While chelerythrine inhibition was reversible, the effect of sanguinarine was resistant to wash out. These results suggest that benzophenanthridine alkaloids interact with glycine transporters and at low micromolar range selectively target glycine transporter GlyT1.

Calcium dependent modification of distal C-terminal sequences of glycine transporter GlyT1.

Glycine transporter GlyT1 plays important role in maintaining accurate glycine concentration in local brain microenvironment. Transporting efficiency of GlyT1 is strongly affected by the state of its distal C-terminus, which regulates transporter trafficking and cellular surface density. Using selected range of antibody epitopes against C-terminal region of GlyT1 we investigated its changes during calcium overload, the ubiquitous phenomena of several brain pathologies. We show that immunoreactivity against the last 12 amino acids of GlyT1C-terminal region exhibits robust calcium dependent decline, while the immunoreactivity of closely located region shows relatively small changes. Process is fully blocked by calcium chelation and inhibited by cysteine proteases inhibitors as well as inhibitors of protein kinase C. Distal GlyT1C-terminal end contains PDZ binding motif responsible for GlyT1 interaction with trafficking and clustering proteins. Its removal/modification could be part of the mechanism changing glycine homeostasis during physiological/pathological conditions characterized by elevated calcium.

Modification of the cytosolic regions of GABA transporter GAT1 by calpain.

Cytosolic regions of sodium dependent neurotransmitter transporters regulate their surface density and transporting function by interconnecting themselves with intracellular signaling pathways. Here we show that calpain activation in rat brain synaptosomes leads to cleavage of both N- and C-terminal regions of GABA transporter GAT1. In the C-terminal region, calpain removes a short segment of amino acids involved in binding of GAT1 to a high-density PDZ anchoring matrix. Using a protein pull-down assay, we found that C-terminal truncation of GAT1 results in modification of its interacting proteome in vitro. Results indicate that calpain activation/inhibition in GABAergic terminals may influence the scaffolding and surface expression of GABA transporter GAT1 under normal conditions or imbalance GAT1-mediated GABAergic transmission under pathological states.