Benita Sjögren

Heat Stabilization of the Tissue Proteome: A New Technology for Improved Proteomics

After tissue or body fluid sampling, proteases and other protein-modifying enzymes can rapidly change composition of the proteome. As a direct consequence, analytical results will reflect a mix of in vivo proteome and ex vivo degradation products. Vital information about the presampling state may be destroyed or distorted, leading to variation between samples and incorrect conclusions. Sample stabilization and standardization of sample handling can reduce or eliminate this problem. Here, a novel tissue stabilization system which utilizes a combination of heat and pressure under vacuum was used to stop degradation in mouse brain tissue immediately after sampling. It was found by biochemical assays that enzymatic activity was reduced to background levels in stabilized samples. Western blot analysis confirmed that post-translational phosphorylations of analyzed proteins were stable and conserved for up to 2 h at room temperature and that peptide extracts were devoid of abundant protein degradation fragments. The combination of reduced complexity and proteolytic inactivation enabled mass spectrometric identification of several neuropeptides and endogenous peptides including modified species at higher levels compared to nonstabilized samples. The tissue stabilizing system ensures reproducible and rapid inactivation of enzymes. Therefore, the system provides a powerful improvement to proteomics by greatly reducing the complexity and dynamic range of the proteome in tissue samples and enables enhanced possibilities for discovery and analysis of clinically relevant protein/peptide biomarkers.

Coupling surface plasmon resonance to mass spectrometry to discover novel protein–protein interactions

The elucidation of protein–protein interaction networks is a crucial task in the postgenomic era. In this protocol, we describe our approach to discover protein–protein interactions using the surface plasmon resonance technique coupled to mass spectrometry (MS). A peptide or a protein is immobilized on a sensor chip and then exposed to brain extracts injected through the surface of the chip by a microfluidic system. The interactions between the immobilized ligand and the extracts can be monitored in real time. Proteins interacting with the peptide/protein are recovered, trypsinated and identified using MS. The data obtained are searched against a sequence database using the Mascot 2.1 software. Control experiments using blank sensor chips and/or randomized peptides are carried out to exclude nonspecific interactors. The protocol can be carried out in <3 days. Other methods, such as yeast two-hybrid systems or pull-down approaches followed by MS, are widely used to screen protein–protein interactions. However, as the yeast two-hybrid system requires protein interactions in the nucleus of yeast, proteins that are abundant in other compartments may not be detected. Pull-down approaches based on immunoprecipitation can be used to study endogenous proteins but they require specific antibodies. The protocol presented here does not require the specific labeling or modification of proteins.

5-HT1A and 5-HT7 receptor crosstalk in the regulation of emotional memory: implications for effects of selective serotonin reuptake inhibitors

This study utilized pharmacological manipulations to analyze the role of direct and indirect activation of 5-HT(7) receptors (5-HT(7)Rs) in passive avoidance learning by assessing emotional memory in male C57BL/6J mice. Additionally, 5-HT(7)R binding affinity and 5-HT(7)R-mediated protein phosphorylation of downstream signaling targets were determined. Elevation of 5-HT by the selective serotonin reuptake inhibitor (SSRI) fluoxetine had no effect by itself, but facilitated emotional memory performance when combined with the 5-HT(1A)R antagonist NAD-299. This facilitation was blocked by the selective 5-HT(7)R antagonist SB269970, revealing excitatory effects of the SSRI via 5-HT(7)Rs. The enhanced memory retention by NAD-299 was blocked by SB269970, indicating that reduced activation of 5-HT(1A)Rs results in enhanced 5-HT stimulation of 5-HT(7)Rs. The putative 5-HT(7)R agonists LP-44 when administered systemically and AS19 when administered both systemically and into the dorsal hippocampus failed to facilitate memory. This finding is consistent with the low efficacy of LP-44 and AS19 to stimulate protein phosphorylation of 5-HT(7)R-activated signaling cascades. In contrast, increasing doses of the dual 5-HT(1A)R/5-HT(7)R agonist 8-OH-DPAT impaired memory, while co-administration with NAD-299 facilitated of emotional memory in a dose-dependent manner. This facilitation was blocked by SB269970 indicating 5-HT(7)R activation by 8-OH-DPAT. Dorsohippocampal infusion of 8-OH-DPAT impaired passive avoidance retention through hippocampal 5-HT(1A)R activation, while 5-HT(7)Rs appear to facilitate memory processes in a broader cortico-limbic network and not the hippocampus alone.

Regulation of serotonin receptor function in the nervous system by lipid rafts and adaptor proteins.

5-HT is a phylogenetically conserved monoaminergic neurotransmitter which is crucial for a number of physiological processes and is dysregulated in several disease states including depression, anxiety and schizophrenia. 5-HT neurons in the central nervous system are localized in the raphe nuclei and project to a wide range of target areas. 5-HT exerts its functions through 14 subtypes of 5-HT receptors. The tertiary structures of seven transmembrane 5-HT receptors contain several important features, including cholesterol consensus motifs, prominent intracellular loops and free C-termini. Alterations of cholesterol levels affect binding of ligands to 5-HT receptors and cholesterol-enriched microdomains in the cell membrane, termed lipid rafts, regulate 5-HT receptor internalization and signaling. The intracellular loops and the C-termini of 5-HT receptors provide binding sites for interacting adaptor proteins. Adaptor proteins affect internalization, desensitization as well as G-protein dependent and independent signaling via 5-HT receptors. We will here briefly review recent progress on the role of lipid rafts and adaptor proteins in the regulation of localization, trafficking, signaling and ligand bias of 5-HT receptors.

Cholesterol reduction attenuates 5-HT1A receptor-mediated signaling in human primary neuronal cultures

Lipid rafts regulate functions of various G protein-coupled receptors and signaling proteins. We show that human primary neuronal cultures contain high levels of 5-HT1A receptors. Stimulation with the 5-HT1A/7 receptor agonist, 8-OH-DPAT, reduced P-T185/Y187-ERK2. This reduction could be blocked by the 5-HT1A receptor antagonist, WAY100635. Pretreatment with the cholesterol sequestering agent, methyl-β-cyclodextrin, before adding 8-OH-DPAT, significantly counteracted the inhibitory influence of 8-OH-DPAT on P-T185/Y187-ERK2 and P-S133-CREB. These data indicate that reduction of cholesterol levels significantly influence signaling via 5-HT1A receptors in intact neurons.

Caveolin-1 affects serotonin binding and cell surface levels of human 5-HT7(a) receptors

Studies using HeLa cells expressing 5‐HT7 receptors showed that detergent resistant membranous lipid rafts purified by sucrose gradient centrifugation contained 5‐HT7 receptors and caveolin‐1. Caveolin‐1 siRNA‐mediated knockdown or serotonin (5‐HT) treatment caused significant reductions of maximum [3H]5‐HT binding to 5‐HT7 receptors and total immunoreactivity of membranous 5‐HT7 receptors in intact cells. Co‐treatment with 5‐HT, caveolin‐1 siRNA and methyl‐β‐cyclodextrin had no additive effects on reducing maximum binding of [3H]5‐HT to 5‐HT7 receptors. 5‐HT‐mediated reduction of [3H]5‐HT binding to 5‐HT7 receptors was counteracted by genistein, but not by sucrose. Caveolin‐1, specifically localized in cholesterol‐enriched lipid rafts, appears to regulate constitutive and agonist‐stimulated cell surface levels of 5‐HT7 receptors via a clathrin‐independent mechanism.

Identification of Protein‐Protein Interactions by Surface Plasmon Resonance followed by Mass Spectrometry

Elucidation of the function and meaning of the protein networks can be useful in the understanding of many pathological processes and the identification of new therapeutic targets. This unit describes an approach to discover protein-protein interactions by coupling surface plasmon resonance to mass spectrometry. Briefly, a protein is covalently bound to a sensor chip, which is then exposed to brain extracts injected over the surface via a microfluidic system. This allows the monitoring in real-time of the interactions between the immobilized ligand and the extracts. Interacting proteins from the extracts are then recovered, trypsinized, and identified using mass spectrometry. The data obtained are searched against a sequence database using the Mascot software. To exclude nonspecific interactors, control experiments using blank sensor chips, and/or randomized peptides, are performed. The protocol presented here does not require specific labeling or modification of proteins and can be performed in <4 days.

Use of surface plasmon resonance coupled with mass spectrometry reveals an interaction between the voltage-gated sodium channel type X alpha-subunit and caveolin-1.

The combination of surface plasmon resonance and mass spectrometry is emerging as a sensitive tool for the elucidation of protein-protein interactions. With the use of surface plasmon resonance-mass spectrometry, peptides, and brain extracts, we now report a novel interaction between the voltage-gated sodium channel type X alpha-subunit and caveolin-1, the central protein controlling caveolae formation. Surface plasmon resonance binding analyses show that this interaction involves amino acids 85-103 of voltage-gated sodium channel type X alpha-subunit and amino acids 81-100 of caveolin-1, a known scaffolding domain of caveolin-1. It is anticipated that the surface plasmon resonance-mass spectrometry approach utilized in this study will be important for the elucidation of protein-protein network analysis in native tissues including the brain.