Alicia Lundby

Proteomic Analysis of Lysine Acetylation Sites in Rat Tissues Reveals Organ Specificity and Subcellular Patterns

SUMMARY Lysine acetylation is a major posttranslational modification involved in a broad array of physiological functions. Here, we provide an organ-wide map of lysine acetylation sites from 16 rat tissues analyzed by high-resolution tandem mass spectrometry. We quantify 15,474 modification sites on 4,541 proteins and provide the data set as a web-based database. We demonstrate that lysine acetylation displays site-specific sequence motifs that diverge between cellular compartments, with a significant fraction of nuclear sites conforming to the consensus motifs G-AcK and AcK-P. Our data set reveals that the subcellular acetylation distribution is tissue-type dependent and that acetylation targets tissue-specific pathways involved in fundamental physiological processes. We compare lysine acetylation patterns for rat as well as human skeletal muscle biopsies and demonstrate its general involvement in muscle contraction. Furthermore, we illustrate that acetylation of fructose-bisphosphate aldolase and glycerol-3-phosphate dehydrogenase serves as a cellular mechanism to switch off enzymatic activity.

Quantitative maps of protein phosphorylation sites across 14 different rat organs and tissues

Deregulated cellular signalling is a common hallmark of disease, and delineating tissue phosphoproteomes is key to unravelling the underlying mechanisms. Here we present the broadest tissue catalogue of phosphoproteins to date, covering 31,480 phosphorylation sites on 7,280 proteins quantified across 14 rat organs and tissues. We provide the data set as an easily accessible resource via a web-based database, the CPR PTM Resource. A major fraction of the presented phosphorylation sites are tissue-specific and modulate protein interaction networks that are essential for the function of individual organs. For skeletal muscle, we find that phosphotyrosines are over-represented, which is mainly due to proteins involved in glycogenolysis and muscle contraction, a finding we validate in human skeletal muscle biopsies. Tyrosine phosphorylation is involved in both skeletal and cardiac muscle contraction, whereas glycogenolytic enzymes are tyrosine phosphorylated in skeletal muscle but not in the liver. The presented phosphoproteomic method is simple and rapid, making it applicable for screening of diseased tissue samples.

Engineering of a Genetically Encodable Fluorescent Voltage Sensor Exploiting Fast Ci-VSP Voltage-Sensing Movements

Ci-VSP contains a voltage-sensing domain (VSD) homologous to that of voltage-gated potassium channels. Using charge displacement (‘gating’ current) measurements we show that voltage-sensing movements of this VSD can occur within 1 ms in mammalian membranes. Our analysis lead to development of a genetically encodable fluorescent protein voltage sensor (VSFP) in which the fast, voltage-dependent conformational changes of the Ci-VSP voltage sensor are transduced to similarly fast fluorescence read-outs.

In Vivo Phosphoproteomics Analysis Reveals the Cardiac Targets of β-Adrenergic Receptor Signaling

Analysis of phosphorylated proteins from the hearts of mice given drugs targeting β-adrenergic receptors may aid in treating heart disease. Getting to the Heart of Signaling Patients with high blood pressure and other heart-related conditions routinely take inhibitors of β-adrenergic receptors (βARs) to prevent cardiac dysfunction. βAR signaling leads to the increased contractility of cardiomyocytes, among other effects; however, the number of downstream targets of βARs is unclear. Lundby et al. treated mice with combinations of specific β1AR and β2AR agonists and antagonists to activate each receptor isoform individually before harvesting the hearts and subjecting them to phosphoproteomics analysis. The authors identified previously uncharacterized peptides and sites phosphorylated in response to β1AR signaling, as well as characterized the activation of a potassium channel important for increasing heart rate. This in vivo approach provides insights into βAR signaling pathways that may help in understanding how heart diseases develop and how they may be treated. β-Blockers are widely used to prevent cardiac arrhythmias and to treat hypertension by inhibiting β-adrenergic receptors (βARs) and thus decreasing contractility and heart rate. βARs initiate phosphorylation-dependent signaling cascades, but only a small number of the target proteins are known. We used quantitative in vivo phosphoproteomics to identify 670 site-specific phosphorylation changes in murine hearts in response to acute treatment with specific βAR agonists. The residues adjacent to the regulated phosphorylation sites exhibited a sequence-specific preference (R-X-X-pS/T), and integrative analysis of sequence motifs and interaction networks suggested that the kinases AMPK (adenosine 5′-monophosphate–activated protein kinase), Akt, and mTOR (mammalian target of rapamycin) mediate βAR signaling, in addition to the well-established pathways mediated by PKA (cyclic adenosine monophosphate–dependent protein kinase) and CaMKII (calcium/calmodulin-dependent protein kinase type II). We found specific regulation of phosphorylation sites on six ion channels and transporters that mediate increased ion fluxes at higher heart rates, and we showed that phosphorylation of one of these, Ser92 of the potassium channel KV7.1, increased current amplitude. Our data set represents a quantitative analysis of phosphorylated proteins regulated in vivo upon stimulation of seven-transmembrane receptors, and our findings reveal previously unknown phosphorylation sites that regulate myocardial contractility, suggesting new potential targets for the treatment of heart disease and hypertension.

KCNE3 Mutation V17M Identified in a Patient with Lone Atrial Fibrillation

Background: Atrial fibrillation (AF) is the most common cardiac rhythm disorder with a lifetime risk for development of 25 % for people aged 40 or older [1]. In this study we aim for the functional assessment of a mutation in KCNE3 identified in a proband with early-onset lone AF. Methods: Screening of genomic DNA from the proband led to identification of a KCNE3 V17M missense mutation. We heterologously expressed the accessory channel subunit in Xenopus laevis oocytes together with its known interacting potassium channel α-subunits. Further, we applied RT-PCR on human total RNA from left and right atria and ventricle. Results: Electrophysiological recordings revealed an increased activity of Kv4.3/KCNE3 and Kv11.1/KCNE3 generated currents by the mutation, thereby conferring susceptibility of mutation carriers to faster cardiac action potential repolarization and thus vulnerability to re-entrant wavelets in the atria and thereby AF. Conclusion: Here we report a novel mutation in KCNE3 identified in a proband with early-onset lone AF possibly leading to gain-of-function of several cardiac currents. We suggest abnormalities in the KCNE3 gene as a potential genetic risk factor for initiation and/or maintenance of AF.

Differential effects of the transient outward K+ current activator NS5806 in the canine left ventricle

To examine the electrophysiological and molecular properties of the transient outward current (I(to)) in canine left ventricle using a novel I(to) activator, NS5806, I(to) was measured in isolated epicardial (Epi), midmyocardial (Mid) and endocardial (Endo) cells using whole-cell patch-clamp techniques. NS5806 activation of K(v)4.3 current was also studied in CHO-K1 cells and Xenopus laevis oocytes. In CHO-K1 cells co-transfected with K(v)4.3 and KChIP2, NS5806 (10 microM) caused a 35% increase in current amplitude and a marked slowing of current decay with tau increasing from 7.0+/-0.4 to 10.2+/-0.3 ms. In the absence of KChIP2, current decay was unaffected by NS5806. In ventricular myocytes, NS5806 increased I(to) density by 80%, 82%, and 16% in Epi, Mid, and Endo myocytes, respectively (at +40 mV) and shifted steady-state inactivation to negative potentials. NS5806 also significantly slowed decay of I(to), increasing total charge to 227%, 192% and 83% of control in Epi, Mid and Endo cells, respectively (+40 mV, p<0.05). Quantification of K(v)4.3 and KChIP2 mRNA in the 3 ventricular cell types revealed that levels of K(v)4.3 message was uniform but those of KChIP2 were significantly greater in Epi and Mid cells. The KChIP2 gradient was confirmed at the protein level by Western blot. Our results suggest that NS5806 augments I(to) by increasing current density and slowing decay and that both depend on the presence of KChIP2. I(to) and its augmentation by NS5806 are greatest in Epi and Mid cells because KChIP2 levels are highest in these cell types.

Effect of the Ito activator NS5806 on cloned Kv4 channels depends on the accessory protein KChIP2

The compound NS5806 increases the transient outward current (Ito) in canine ventricular cardiomyocytes and slows current decay. In human and canine ventricle, Ito is thought to be mediated by KV4.3 and various ancillary proteins, yet, the exact subunit composition of Ito channels is still debated. Here we characterize the effect of NS5806 on heterologously expressed putative Ito channel subunits and other potassium channels.

GeLCMS for In-Depth Protein Characterization and Advanced Analysis of Proteomes

In recent years the array of mass spectrometry (MS) applications to address questions in molecular and cellular biology has greatly expanded and continues to grow. Modern mass spectrometers allow for identification, characterization, as well as quantification of protein compositions and their modifications in complex biological samples. Prior to MS analysis any biological sample needs to be properly prepared for the experiment. Here we present a protocol that combines pre-separation of proteins by 1D gel electrophoresis followed by analysis of in situ digested protein products by tandem mass spectrometry (MS/MS). All steps of the sample preparation are explained in detail, and the procedure is compatible with downstream analysis on any mass spectrometer available. With minor adjustments the protocol can be used with 2D gels as well. The protocol provided can be applied to analyze specific proteins of particular interest as well as entire proteomes. If SILAC-labeled protein samples are mixed prior to gel separation, the protein content of the sample can furthermore be accurately quantified.

Annotation of loci from genome-wide association studies using tissue-specific quantitative interaction proteomics

Genome-wide association studies (GWAS) have identified thousands of loci associated with complex traits, but it is challenging to pinpoint causal genes in these loci and to exploit subtle association signals. We used tissue-specific quantitative interaction proteomics to map a network of five genes involved in the Mendelian disorder long QT syndrome (LQTS). We integrated the LQTS network with GWAS loci from the corresponding common complex trait, QT-interval variation, to identify candidate genes that were subsequently confirmed in Xenopus laevis oocytes and zebrafish. We used the LQTS protein network to filter weak GWAS signals by identifying single-nucleotide polymorphisms (SNPs) in proximity to genes in the network supported by strong proteomic evidence. Three SNPs passing this filter reached genome-wide significance after replication genotyping. Overall, we present a general strategy to propose candidates in GWAS loci for functional studies and to systematically filter subtle association signals using tissue-specific quantitative interaction proteomics.

Structural basis for KV7.1–KCNEx interactions in the IKs channel complex

The cardiac I(Ks) current is involved in action potential repolarization, where its primary function is to limit action potential prolongation during sympathetic stimulation. The I(Ks) channel is mainly composed of K(V)7.1 ion channels associated with KCNE1 auxiliary subunits. The availability of KCNE1 solution structure by nuclear magnetic resonance spectroscopy in conjunction with biochemical assays addressing K(V)7.1-KCNE1 residue interactions has provided new insights into the structural basis for K(V)7.1 modulation by KCNE1. Recent evidence further suggests that KCNE2 may associate with the K(V)7.1-KCNE1 channel complex and modulate its current amplitude. Here we review recent studies in this area and discuss potential roles for multiple KCNE(x) subunits in I(Ks) generation and modulation as well as the clinical relevance of the new information.