1. Intracellular Ca(2+) overload has been associated with established atrial arrhythmogenesis. The present experiments went on to correlate acute initiation of atrial arrhythmogenesis in Langendorff-perfused mouse hearts with changes in Ca(2+) homeostasis in isolated atrial myocytes following pharmacological procedures that modified the storage or release of sarcoplasmic reticular (SR) Ca(2+) or inhibited entry of extracellular Ca(2+). 2. Caffeine (1 mmol/L) elicited diastolic Ca(2+) waves in regularly stimulated atrial myocytes immediately following addition. This was followed by a decline in the amplitude of the evoked transients and the disappearance of such diastolic events, suggesting partial SR Ca(2+) depletion. 3. Cyclopiazonic acid (CPA; 0.15 micromol/L) produced more gradual reductions in evoked Ca(2+) transients and abolished diastolic Ca(2+) events produced by the further addition of caffeine. 4. Nifedipine (0.5 micromol/L) produced immediate reductions in evoked Ca(2+) transients. Further addition of caffeine produced an immediate increase followed by a decline in the amplitude of the evoked Ca(2+) transients, without eliciting diastolic Ca(2+) events. 5. These findings correlated with changes in spontaneous and provoked atrial arrhythmogenecity in mouse isolated Langendorf-perfused hearts. Thus, caffeine was pro-arrhythmogenic immediately following but not > 5 min after application and both CPA and nifedipine pretreatment inhibited such arrhythmogenesis. 6. Together, these findings relate acute atrial arrhythmogenesis in intact hearts to diastolic Ca(2+) events in atrial myocytes that, in turn, depend upon a finite SR Ca(2+) store and diastolic Ca(2+) release following Ca(2+)-induced Ca(2+) release initiated by the entry of extracellular Ca(2+).

Recent studies have reported that human mutations in Nav1.5 predispose to early age onset atrial arrhythmia. The present experiments accordingly assess atrial arrhythmogenicity in aging Scn5a+/KPQ mice modeling long QT3 syndrome in relationship to cardiac Na(+) channel, Nav1.5, expression. Atrial electrophysiological properties in isolated Langendorff-perfused hearts from 3- and 12-month-old wild type (WT), and Scn5a+/KPQ mice were assessed using programmed electrical stimulation and their Nav1.5 expression assessed by Western blot. Cardiac conduction properties were assessed electrocardiographically in intact anesthetized animals. Monophasic action potential recordings demonstrated increased atrial arrhythmogenicity specifically in aged Scn5a+/DeltaKPQ hearts. These showed greater action potential duration/refractory period ratios but lower atrial Nav1.5 expression levels than aged WT mice. Atrial Nav1.5 levels were higher in young Scn5a+/DeltaKPQ than young WT. These levels increased with age in WT but not Scn5a+/DeltaKPQ. Both young and aged Scn5a+/DeltaKPQ mice showed lower heart rates and longer PR intervals than their WT counterparts. Young Scn5a+/DeltaKPQ mice showed longer QT and QTc intervals than young WT. Aged Scn5a+/DeltaKPQ showed longer QRS durations than aged WT. PR intervals were prolonged and QT intervals were shortened in young relative to aged WT. In contrast, ECG parameters were similar between young and aged Scn5a+/DeltaKPQ. Aged murine Scn5a+/DeltaKPQ hearts thus exhibit an increased atrial arrhythmogenicity. The differing Nav1.5 expression and electrocardiographic indicators of slowed cardiac conduction between Scn5a+/DeltaKPQ and WT, which show further variations associated with aging, may contribute toward atrial arrhythmia in aged Scn5a+/DeltaKPQ hearts.

Voltage-gated sodium (Na) channels contribute to the regulation of cellular excitability due to their role in the generation and propagation of action potentials. They are composed of a pore-forming alpha subunit and are modulated by at least two of four distinct beta subunits (beta1-4). Recent studies have implicated a role for the intracellular domain of beta subunits in modulating Na channel gating and trafficking. In beta3, the intracellular domain contains a serine residue at position 161 that is replaced by an alanine in beta1. In this study, we have probed the functional importance of beta3S161 for modulating Na channel gating. Wild-type beta3 and point mutations beta3S161A or beta3S161E were individually co-expressed in HEK 293 cells stably expressing human Na(v)1.2. WTbeta3 expression increased Na current density, shifted steady-state inactivation in a depolarized direction, and accelerated the kinetics of recovery from inactivation of the Na current. Analogous effects were observed with beta3S161E co-expression. In contrast, beta3S161A abolished the shifts in steady-state inactivation and recovery from inactivation of the Na current, but did increase Na current density. Immunocytochemistry and Western blot experiments demonstrate membrane expression of WTbeta3, beta3S161E, and beta3S161A, suggesting that the differences in Na channel gating were not due to disruptions in beta subunit trafficking. These studies suggest that modification of beta3S161 may be important in modulating Na-channel gating.

Clathrin and clathrin-dependent events are evolutionary conserved although it is believed that there are differences in the requirement for clathrin in yeast and higher vertebrates. Clathrin is a long-lived protein and thus, with clathrin knockdowns only long-term consequences of clathrin depletion can be studied. Here, we characterize the first vertebrate temperature-sensitive clathrin heavy chain mutant as a tool to investigate responses to rapid clathrin inactivation in higher eukaryotes. Although we created this mutant using a clathrin cryo-electron microscopy model and a yeast temperature-sensitive mutant as a guide, the resulting temperature-sensitive clathrin showed an altered phenotype compared to the corresponding yeast temperature-sensitive clathrin. First, it seemed to form stable triskelions at the non-permissive temperature although endocytosis was impaired under these conditions. Secondly, as a likely consequence of the stable triskelions at the non-permissive temperature, clathrin also localized correctly to its target membranes. Thirdly, we did not observe missorting of the lysosomal enzyme beta-glucuronidase which could indicate that the temperature-sensitive clathrin is still operating at the non-permissive temperature at the Golgi or, that, like in yeast, more than one TGN trafficking pathway exists. Fourthly, in contrast to yeast, actin does not appear to actively compensate in general endocytosis. Thus, there seem to be differences between vertebrates and yeast which can be studied in further detail with this newly created tool.

According to the Arg/N-end rule pathway, proteins with basic N-termini are targeted for degradation by the Arabidopsis thaliana E3 ligase, PROTEOLYSIS6 (PRT6). Proteins can also become PRT6 substrates following post-translational arginylation by arginyltransferases ATE1 and 2. Here, we undertook a quantitative proteomics study of Arg/N-end rule mutants, ate1/2 and prt6, to investigate the impact of this pathway on the root proteome. Tandem mass tag labelling identified a small number of proteins with increased abundance in the mutants, some of which represent downstream targets of transcription factors known to be N-end rule substrates. Isolation of N-terminal peptides using terminal amine isotope labelling of samples (TAILS) combined with triple dimethyl labelling identified 1465 unique N-termini. Stabilising residues were over-represented among the free neo-N-termini, but destabilising residues were not markedly enriched in N-end rule mutants. The majority of free neo-N-termini were revealed following cleavage of organellar targeting signals, thus compartmentation may account in part for the presence of destabilising residues in the wild-type N-terminome. Our data suggest that PRT6 does not have a marked impact on the global proteome of Arabidopsis roots and is likely involved in the controlled degradation of relatively few regulatory proteins. All MS data have been deposited in the ProteomeXchange with identifier PXD001719 (http://proteomecentral.proteomexchange.org/dataset/PXD001719).

Pull-down assays can identify members of protein complexes but suffer from co-isolation of contaminants. The problem is particularly acute when the specifically interacting partners are of low-abundance and/or bind transiently with low affinity. To differentiate true interacting partners from contaminants, we have combined SILAC labelling with a proteomic method called "Interactomes by Parallel Affinity Capture" (iPAC). In our method, a cell-line stably expressing a doubly tagged target endogenous protein and its tag-less control cell-line are differentially SILAC labelled. Lysates from the two cell-lines are mixed and the tagged protein is independently purified for MS analysis using multiple affinity resins in parallel. This allows the quantitative identification of tagged proteins and their binding partners. SILAC-iPAC provides a rigorous and sensitive approach that can discriminate between genuine binding partners and contaminants, even when the contaminants in the pull-down are in large excess. We employed our method to examine the interacting partners of phosphatidyl inositol 5-phosphate 4-kinase 2β subunit (PI5P4K2β) and the Fanconi anaemia core complex in the chicken pre-B cell-line DT40. We confirmed known components of these two complexes, and we have identified new potential binding partners. Combining the iPAC approach with SILAC labelling provides a sensitive and fully quantitative method for the discrimination of specific interactions under conditions where low signal to noise ratios are unavoidable. In addition, our work provides the first characterisation of the most abundant proteins within the DT40 proteome and the non-specific DT40 'beadomes' (non-specific proteins binding to beads) for common epitope tags. Given the importance and widespread use of the DT40 cell-line, these will be important resources for the cell biology and immunology communities. Biological significance SILAC-iPAC provides an improved method for the analysis of low-affinity and/or low abundance protein-protein interactions. We use it to clarify two examples where the nature of the protein complexes are known, or are currently unclear. The method is simple and quantitative and will be applicable to many problems in cell and molecular biology. We also report the first chicken beadomes.

Experimental spatial proteomics, i.e. the high-throughput assignment of proteins to sub-cellular compartments based on quantitative proteomics data, promises to shed new light on many biological processes given adequate computational tools.

α-Klotho has emerged as a powerful regulator of the aging process. To date, the expression profile of α-Klotho in human tissues is unknown, and its existence in some human tissue types is subject to much controversy.

RNA-binding proteins (RBPs) have essential roles in determining the fate of RNA from synthesis to decay and have been studied on a protein-by-protein basis, or computationally based on a number of well-characterised RNA-binding domains. Recently, high-throughput methods enabled the capture of mammalian RNA-binding proteomes. To gain insight into the role of Arabidopsis thaliana RBPs at the systems level, we have employed interactome capture techniques using cells from different ecotypes grown in cultures and leaves. In vivo UV-crosslinking of RNA to RBPs, oligo(dT) capture and mass spectrometry yielded 1,145 different proteins including 550 RBPs that either belong to the functional category 'RNA-binding', have known RNA-binding domains or have orthologs identified in mammals, C. elegans, or S. cerevisiae in addition to 595 novel candidate RBPs. We noted specific subsets of RBPs in cultured cells and leaves and a comparison of Arabidopsis, mammalian, C. elegans, and S. cerevisiae RBPs reveals a common set of proteins with a role in intermediate metabolism, as well as distinct differences suggesting that RBPs are also species and tissue specific. This study provides a foundation for studies that will advance our understanding of the biological significance of RBPs in plant developmental and stimulus specific responses.

Brugada syndrome (BrS) is associated with ventricular tachycardia originating particularly in the right ventricle (RV). We explore electrophysiological features predisposing to such arrhythmic tendency and their possible RV localization in a heterozygotic Scn5a+/- murine model. Na(v)1.5 mRNA and protein expression were lower in Scn5a+/- than wild-type (WT), with a further reduction in the RV compared with the left ventricle (LV). RVs showed higher expression levels of K(v)4.2, K(v)4.3 and KChIP2 in both Scn5a+/- and WT. Action potential upstroke velocity and maximum Na(+) current (I(Na)) density were correspondingly decreased in Scn5a+/-, with a further reduction in the RV. The voltage dependence of inactivation was shifted to more negative values in Scn5a+/-. These findings are predictive of a localized depolarization abnormality leading to slowed conduction. Persistent Na(+) current (I(pNa)) density was decreased in a similar pattern to I(Na). RV transient outward current (I(to)) density was greater than LV in both WT and Scn5a+/-, and had larger time constants of inactivation. These findings were also consistent with the observation that AP durations were smallest in the RV of Scn5a+/-, fulfilling predictions of an increased heterogeneity of repolarization as an additional possible electrophysiological mechanism for arrhythmogenesis in BrS.

Alternans and arrhythmogenicity were studied in hypokalaemic (3.0 mM K(+)) Langendorff-perfused murine hearts paced at high rates. Epicardial and endocardial monophasic action potentials were recorded and durations quantified at 90% repolarization. Alternans and arrhythmia occurred in hypokalaemic, but not normokalaemic (5.2 mM K(+)) hearts (P<0.01): this was prevented by treatment with lidocaine (10 microM, P<0.01). Fourier analysis then confirmed transition from monomorphic to polymorphic waveforms for the first time in the murine heart. Alternans and arrhythmia were associated with increases in the slopes of restitution curves, obtained for the first time in the murine heart, while the anti-arrhythmic effect of lidocaine was associated with decreased slopes. Thus, hypokalaemia significantly increased (P<0.05) maximal gradients (from 0.55+/-0.14 to 2.35+/-0.67 in the epicardium and from 0.67+/-0.13 to 1.87 +/-0.28 in the endocardium) and critical diastolic intervals (DIs) at which gradients equalled unity (from -2.14+/-0.52 ms to 50.93+/-14.45 ms in the epicardium and from 8.14+/-1.49 ms to 44.64+/-5 ms in the endocardium). While treatment of normokalaemic hearts with lidocaine had no significant effect (P>0.05) on either maximal gradients (0.78+/-0.27 in the epicardium and 0.83+/-0.45 in the endocardium) or critical DIs (6.06+/-2.10 ms and 7.04+/-3.82 ms in the endocardium), treatment of hypokalaemic hearts with lidocaine reduced (P<0.05) both these parameters (1.05+/-0.30 in the epicardium and 0.89+/-0.36 in the endocardium and 30.38+/-8.88 ms in the epicardium and 31.65+/-4.78 ms in the endocardium, respectively). We thus demonstrate that alternans contributes a dynamic component to arrhythmic substrate during hypokalaemia, that restitution may furnish an underlying mechanism and that these phenomena are abolished by lidocaine, both recapitulating and clarifying clinical findings.

Emiliania huxleyi virus 86 (EhV-86) is the type species of the genus Coccolithovirus within the family Phycodnaviridae. The fully sequenced 407,339 bp genome is predicted to encode 473 protein coding sequences (CDSs) and is the largest Phycodnaviridae sequenced to date. The majority of EhV-86 CDSs exhibit no similarity to proteins in the public databases.

Flecainide suppresses cardiac tachyarrhythmias including paroxysmal atrial fibrillation, supraventricular tachycardia and arrhythmic long QT syndromes (LQTS), as well as the Ca2+ -mediated, catecholaminergic polymorphic ventricular tachycardia (CPVT). However, flecainide can also exert pro-arrhythmic effects most notably following myocardial infarction and when used to diagnose Brugada syndrome (BrS). These divergent actions result from its physiological and pharmacological actions at multiple, interacting levels of cellular organization. These were studied in murine genetic models with modified Nav channel or intracellular ryanodine receptor (RyR2)-Ca2+ channel function. Flecainide accesses its transmembrane Nav 1.5 channel binding site during activated, open, states producing a use-dependent antagonism. Closing either activation or inactivation gates traps flecainide within the pore. An early peak INa related to activation of Nav channels followed by rapid de-activation, drives action potential (AP) upstrokes and their propagation. This is diminished in pro-arrhythmic conditions reflecting loss of function of Nav 1.5 channels, such as BrS, accordingly exacerbated by flecainide challenge. Contrastingly, pro-arrhythmic effects attributed to prolonged AP recovery by abnormal late INaL following gain-of-function modifications of Nav 1.5 channels in LQTS3 are reduced by flecainide. Anti-arrhythmic effects of flecainide that reduce triggering in CPVT models mediated by sarcoplasmic reticular Ca2+ release could arise from its primary actions on Nav channels indirectly decreasing [Ca2+ ]i through a reduced [Na+ ]i and/or direct open-state RyR2-Ca2+ channel antagonism. The consequent [Ca2+ ]i alterations could also modify AP propagation velocity and therefore arrhythmic substrate through its actions on Nav 1.5 channel function. This is consistent with the paradoxical differences between flecainide actions upon Na+ currents, AP conduction and arrhythmogenesis under circumstances of normal and increased RyR2 function.

Abiotic stresses are considered the most deleterious factor affecting growth and development of plants worldwide. Such stresses are largely unavoidable and trigger adaptive responses affecting different cellular processes and target different compartments. Shotgun proteomic and mass spectrometry-based approaches offer an opportunity to elucidate the response of the proteome to abiotic stresses. In this study, the severe drought or water-deficit response in Arabidopsis thaliana was mimicked by treating cell suspension callus with 40% polyethylene glycol for 10 and 30 min. Resulting data demonstrated that 310 proteins were differentially expressed in response to this treatment with a strict ±2.0-fold change. Over-representation was observed in the gene ontology categories of 'ribosome' and its related functions as well as 'oxidative phosphorylation', indicating both structural and functional drought responses at the cellular level. Proteins in the category 'endocytosis' also show significant enrichment and this is consistent with increased active transport and recycling of membrane proteins in response to abiotic stress. This is supported by the particularly pronounced enrichment in proteins of the endosomal sorting complexes that are required for membrane remodelling. Taken together, the findings point to rapid and complex physiological and structural changes essential for survival in response to sudden severe drought stress.

Ageing and age-related bioenergetic conditions including obesity, diabetes mellitus and heart failure constitute clinical ventricular arrhythmic risk factors.

Ciprofloxacin is one of the most widely-used antibiotics, and has proven especially effective at controlling infections associated with the opportunistic human pathogen, Pseudomonas aeruginosa. In this work, we show that sub-inhibitory concentrations of ciprofloxacin induce discrete changes in the intracellular proteome. Central metabolism and cell envelope-associated functions are particularly affected. In spite of the low magnitude of the intracellular proteomic changes, we found that sub-lethal concentrations of ciprofloxacin had substantial effects on motility and exoprotein secretion. Crucially, the proteomic and phenotypic modulations that we observed were absolutely dependent upon the presence of wild-type GyrA; an isogenic strain of P. aeruginosa carrying a ciprofloxacin-insensitive form of GyrA (a T83→I mutant) did not display ciprofloxacin-dependent changes unless complemented with wild-type gyrA in trans. These results show that the diverse effects of sub-inhibitory ciprofloxacin on the cell are routed through its primary target in the cell, DNA gyrase.

Exercising horses uniquely accommodate 7-8-fold increases in heart rate (HR). The present experiments for the first time analysed the related adaptations in action potential (AP) restitution properties recorded by in vivo telemetric electrocardiography from Thoroughbred horses. The horses were subjected to a period of acceleration from walk to canter. The QRS durations, and QT and TQ intervals yielded AP conduction velocities, AP durations (APDs) and diastolic intervals respectively. From these, indices of active, λ = QT/(QRS duration), and resting, λ0 = TQ/(QRS duration), AP wavelengths were calculated. Critical values of QT and TQ intervals, and of λ and λ0 at which plots of these respective pairs of functions showed unity slope, were obtained. These were reduced by 38.9±2.7% and 86.2±1.8%, and 34.1±3.3% and 85.9±1.2%, relative to their resting values respectively. The changes in λ were attributable to falls in QT interval rather than QRS duration. These findings both suggested large differences between the corresponding critical (129.1±10.8 or 117.4±5.6 bpm respectively) and baseline HRs (32.9±2.1 (n = 7) bpm). These restitution analyses thus separately identified concordant parameters whose adaptations ensure the wide range of HRs over which electrophysiological activation takes place in an absence of heart block or arrhythmias in equine hearts. Since the horse is amenable to this in vivo electrophysiological analysis and displays a unique wide range of heart rates, it could be a novel cardiac electrophysiology animal model for the study of sudden cardiac death in human athletes.

Mutations in the cardiac ryanodine receptor Ca2+ release channel (RyR2) can cause deadly ventricular arrhythmias and atrial fibrillation (AF). The RyR2-P2328S mutation produces catecholaminergic polymorphic ventricular tachycardia (CPVT) and AF in hearts from homozygous RyR2P2328S/P2328S (denoted RyR2S/S) mice. We have now examined P2328S RyR2 channels from RyR2S/S hearts. The activity of wild-type (WT) and P2328S RyR2 channels was similar at a cytoplasmic [Ca2+] of 1 mM, but P2328S RyR2 was significantly more active than WT at a cytoplasmic [Ca2+] of 1 µM. This was associated with a >10-fold shift in the half maximal activation concentration (AC50) for Ca2+ activation, from ∼3.5 µM Ca2+ in WT RyR2 to ∼320 nM in P2328S channels and an unexpected >1000-fold shift in the half maximal inhibitory concentration (IC50) for inactivation from ∼50 mM in WT channels to ≤7 μM in P2328S channels, which is into systolic [Ca2+] levels. Unexpectedly, the shift in Ca2+ activation was not associated with changes in sub-conductance activity, S2806 or S2814 phosphorylation or the level of FKBP12 (also known as FKBP1A) bound to the channels. The changes in channel activity seen with the P2328S mutation correlate with altered Ca2+ homeostasis in myocytes from RyR2S/S mice and the CPVT and AF phenotypes.This article has an associated First Person interview with the first author of the paper.

Cyanobacteria are complex prokaryotes, incorporating a Gram-negative cell wall and internal thylakoid membranes (TMs). However, localization of proteins within cyanobacterial cells is poorly understood. Using subcellular fractionation and quantitative proteomics, we produced an extensive subcellular proteome map of an entire cyanobacterial cell, identifying ∼67% of proteins in Synechocystis sp. PCC 6803, ∼1000 more than previous studies. Assigned to six specific subcellular regions were 1,712 proteins. Proteins involved in energy conversion localized to TMs. The majority of transporters, with the exception of a TM-localized copper importer, resided in the plasma membrane (PM). Most metabolic enzymes were soluble, although numerous pathways terminated in the TM (notably those involved in peptidoglycan monomer, NADP+, heme, lipid, and carotenoid biosynthesis) or PM (specifically, those catalyzing lipopolysaccharide, molybdopterin, FAD, and phylloquinol biosynthesis). We also identified the proteins involved in the TM and PM electron transport chains. The majority of ribosomal proteins and enzymes synthesizing the storage compound polyhydroxybuyrate formed distinct clusters within the data, suggesting similar subcellular distributions to one another, as expected for proteins operating within multicomponent structures. Moreover, heterogeneity within membrane regions was observed, indicating further cellular complexity. Cyanobacterial TM protein localization was conserved in Arabidopsis (Arabidopsis thaliana) chloroplasts, suggesting similar proteome organization in more developed photosynthetic organisms. Successful application of this technique in Synechocystis suggests it could be applied to mapping the proteomes of other cyanobacteria and single-celled organisms. The organization of the cyanobacterial cell revealed here substantially aids our understanding of these environmentally and biotechnologically important organisms.

Uncharacterized and unannotated open-reading frames, which we refer to as novel open reading frames (nORFs), may sometimes encode peptides that remain unexplored for novel therapeutic opportunities. To our knowledge, no systematic identification and characterization of transcripts encoding nORFs or their translation products in cancer, or in any other physiological process has been performed. We use our curated nORFs database (nORFs.org), together with RNA-Seq data from The Cancer Genome Atlas (TCGA) and Genotype-Expression (GTEx) consortiums, to identify transcripts containing nORFs that are expressed frequently in cancer or matched normal tissue across 22 cancer types. We show nORFs are subject to extensive dysregulation at the transcript level in cancer tissue and that a small subset of nORFs are associated with overall patient survival, suggesting that nORFs may have prognostic value. We also show that nORF products can form protein-like structures with post-translational modifications. Finally, we perform in silico screening for inhibitors against nORF-encoded proteins that are disrupted in stomach and esophageal cancer, showing that they can potentially be targeted by inhibitors. We hope this work will guide and motivate future studies that perform in-depth characterization of nORF functions in cancer and other diseases.