Identification of the -1 translational frameshift sites using a liquid chromatography-tandem mass spectrometric approach

Translational frameshifting, a ubiquitous mechanism used to produce alternative proteins for different biological purposes, appears in a variety of genes in probably all organisms. In the past, the combinational use of sophisticated expression vectors, specific endopeptidases, and Edman degradation has been the main approach for identification of the translational frameshift sites. Although Edman degradation is highly reliable, it is also time-consuming and costly. In this article, we report a new liquid chromatography-tandem mass spectrometric (LC-MS/MS) approach for identifying the -1 translational frameshift sites. The approach consists of three steps: (i) LC-MS/MS analysis of the protein digests, (ii) primary data analysis using the known mRNA sequence, and (iii) advanced data analysis using a new database containing distinct mRNA sequences with single insertion at particular positions. We first validated our approach by analyzing the previously documented slippery sequence, A4G, from IS3. With this approach, we further determined whether the TTTTTTG (T6G) sequence of IS1372 from Streptomyces lividans had the -1 translational frameshifting potential. The identified amino acid sequence of the transframe peptide indicated that the -1 frameshifting occurred at the T6G motif, as predicted previously. The results on IS3 (A4G) and IS1372 (T6G) suggested that this approach is effective for the translational frameshifting studies.     

Regulation of cardiac sarcoplasmic reticulum Ca release by luminal [Ca] and altered gating assessed with a mathematical model

Cardiac excitation-contraction coupling is initialized by the release of Ca from the sarcoplasmic reticulum (SR) in response to a sudden increase in local cytosolic [Ca] ([Ca]i) within the junctional cleft. We have tested the hypothesis that functional ryanodine receptor (RyR) regulation plays a major role in the regulation of myocyte Ca. A mathematical model with unique characteristics was used to simulate Ca homeostasis. Specifically, the model was designed to accurately represent the SR [Ca]-dependence of release from a variety of experimentally produced data sets. The simulated data for altered RyR Ca sensitivity demonstrated a regulatory feedback loop that resulted in the same release at lower [Ca]SR. This suggests that the primary role of myocyte RyR regulation may be to decrease SR [Ca] without decreasing the size of the [Ca]i transient. The model results suggest that this action moderates the increased SR [Ca] observed with adrenergic stimulation and may keep the [Ca]SR below the threshold for delayed afterdepolarizations and arrhythmia. However, increased Ca affinity of the RyR increased the probability of delayed afterdepolarizations when heart failure was simulated. We conclude that RyR regulation may play a role in preventing arrhythmias in healthy myocytes but that the same regulation may have the opposite effect in chronic heart failure.     

Dynamic alterations in myoplasmic Ca2+ in malignant hyperthermia and central core disease

Ca2+ ions play a pivotal role in a wide array of cellular processes ranging from fertilization to cell death. In skeletal muscle, a mechanical interaction between plasma membrane dihydropyridine receptors (DHPRs, L-type Ca2+ channels) and Ca2+ release channels (ryanodine receptors, RyR1s) of the sarcoplasmic reticulum orchestrates a complex, bi-directional Ca2+ signaling process that converts electrical impulses in the sarcolemma into myoplasmic Ca2+ transients during excitation-contraction coupling. Mutations in the genes that encode the two proteins that coordinate this electrochemical conversion process (the DHPR and RyR1) result in a variety of skeletal muscle disorders including malignant hyperthermia (MH), central core disease (CCD), multiminicore disease, nemaline rod myopathy, and hypokalemic periodic paralysis. Although RyR1 and DHPR disease mutations are thought to alter excitability and Ca2+ homeostasis in skeletal muscle, only recently has research begun to probe the molecular mechanisms by which these genetic defects lead to distinct clinical and histopathological manifestations. This review focuses on recent advances in determining the impact of MH and CCD mutations in RyR1 on muscle Ca2+ signaling and how these effects contribute to disease-specific aspects of these disorders.     

Voltage-dependent Ca2+ fluxes in skeletal myotubes determined using a removal model analysis

The purpose of this study was to quantify the Ca2+ fluxes underlying Ca2+ transients and their voltage dependence in myotubes by using the "removal model fit" approach. Myotubes obtained from the mouse C2C12 muscle cell line were voltage-clamped and loaded with a solution containing the fluorescent indicator dye fura-2 (200 microM) and a high concentration of EGTA (15 mM). Ca2+ inward currents and intracellular ratiometric fluorescence transients were recorded in parallel. The decaying phases of Ca2+-dependent fluorescence signals after repolarization were fitted by theoretical curves obtained from a model that included the indicator dye, a slow Ca2+ buffer (to represent EGTA), and a sequestration mechanism as Ca2+ removal components. For each cell, the rate constants of slow buffer and transport and the off rate constant of fura-2 were determined in the fit. The resulting characterization of the removal properties was used to extract the Ca2+ input fluxes from the measured Ca2+ transients during depolarizing pulses. In most experiments, intracellular Ca2+ release dominated the Ca2+ input flux. In these experiments, the Ca2+ flux was characterized by an initial peak followed by a lower tonic phase. The voltage dependence of peak and tonic phase could be described by sigmoidal curves that reached half-maximal activation at -16 and -20 mV, respectively, compared with -2 mV for the activation of Ca2+ conductance. The ratio of the peak to tonic phase (flux ratio) showed a gradual increase with voltage as in rat muscle fibers indicating the similarity to EC coupling in mature mammalian muscle. In a subgroup of myotubes exhibiting small fluorescence signals and in cells treated with 30 microM of the SERCA pump inhibitor cyclopiazonic acid (CPA) and 10 mM caffeine, the calculated Ca2+ input flux closely resembled the L-type Ca2+ current, consistent with the absence of SR Ca2+ release under these conditions and in support of a valid determination of the time course of myoplasmic Ca2+ input flux based on the optical indicator measurements.     

Regulation of sodium channel function by bilayer elasticity: the importance of hydrophobic coupling. Effects of Micelle-forming amphiphiles and cholesterol

Membrane proteins are regulated by the lipid bilayer composition. Specific lipid-protein interactions rarely are involved, which suggests that the regulation is due to changes in some general bilayer property (or properties). The hydrophobic coupling between a membrane-spanning protein and the surrounding bilayer means that protein conformational changes may be associated with a reversible, local bilayer deformation. Lipid bilayers are elastic bodies, and the energetic cost of the bilayer deformation contributes to the total energetic cost of the protein conformational change. The energetics and kinetics of the protein conformational changes therefore will be regulated by the bilayer elasticity, which is determined by the lipid composition. This hydrophobic coupling mechanism has been studied extensively in gramicidin channels, where the channel-bilayer hydrophobic interactions link a "conformational" change (the monomer<-->dimer transition) to an elastic bilayer deformation. Gramicidin channels thus are regulated by the lipid bilayer elastic properties (thickness, monolayer equilibrium curvature, and compression and bending moduli). To investigate whether this hydrophobic coupling mechanism could be a general mechanism regulating membrane protein function, we examined whether voltage-dependent skeletal-muscle sodium channels, expressed in HEK293 cells, are regulated by bilayer elasticity, as monitored using gramicidin A (gA) channels. Nonphysiological amphiphiles (beta-octyl-glucoside, Genapol X-100, Triton X-100, and reduced Triton X-100) that make lipid bilayers less "stiff", as measured using gA channels, shift the voltage dependence of sodium channel inactivation toward more hyperpolarized potentials. At low amphiphile concentration, the magnitude of the shift is linearly correlated to the change in gA channel lifetime. Cholesterol-depletion, which also reduces bilayer stiffness, causes a similar shift in sodium channel inactivation. These results provide strong support for the notion that bilayer-protein hydrophobic coupling allows the bilayer elastic properties to regulate membrane protein function.     

De novo folding of membrane proteins: an exploration of the structure and NMR properties of the fd coat protein

De novo folding simulations of the major pVIII coat protein from filamentous fd bacteriophage, using a newly developed implicit membrane generalized Born model and replica-exchange molecular dynamics, are presented and discussed. The quality of the predicted structures, judged by comparison of the root-mean-square deviations of a room temperature ensemble of conformations from the replica-exchange simulations and experimental structures from both solid-state NMR in lipid bilayers and solution-phase NMR on the protein in micelles, was quite good, reinforcing the general quality of the folding simulations. The transmembrane helical segment of the protein was well defined in comparison with experiment and the amphipathic helical fragment remained at the membrane/aqueous phase boundary while undergoing significant conformational flexibility due to the loop connecting the two helical segments of the protein. Additional comparisons of computed solid-state NMR properties, the 15N chemical shift and 15N-1H dipolar coupling constants, showed semi-quantitative agreement with the corresponding measurements. These findings suggest an emerging potential for the de novo investigation of integral membrane peptides and proteins and a mechanism to assist experimental approaches to the characterization and structure determination of these important systems.     

Location of divergent region 2 on the three-dimensional structure of cardiac muscle ryanodine receptor/calcium release channel

Ryanodine receptors (RyRs) are a family of calcium release channels found on intracellular calcium-handing organelles. Molecular cloning studies have identified three different RyR isoforms, which are 66-70% identical in amino acid sequence. In mammals, the three isoforms are encoded by three separate genes located on different chromosomes. The major variations among the isoforms occur in three regions, known as divergent regions 1, 2, and 3 (DR1, DR2, and DR3). In the present study, a modified RyR2 (cardiac isoform) cDNA was constructed, into which was inserted a green fluorescent protein (GFP)-encoding cDNA within DR2, specifically after amino acid residue Thr1366 (RyR2(T1366-GFP)). HEK293 cells expressing RyR2(T1366-GFP) cDNAs showed caffeine-sensitive and ryanodine-sensitive calcium release, demonstrating that RyR2(T1366-GFP) forms functional calcium release channels. Cells expressing RyR2(T1366-GFP) were identified readily by the characteristic fluorescence of GFP, indicating that the overall structure of the inserted GFP was retained. Cryo-electron microscopy (cryo-EM) of purified RyR2(T1366-GFP) showed structurally intact receptors, and a three-dimensional reconstruction was obtained by single-particle image processing. The location of the inserted GFP was obtained by comparing this three-dimensional reconstruction to one obtained for wild-type RyR2. The inserted GFP and, consequently Thr1366 within DR2, was mapped on the three-dimensional structure of RyR2 to domain 6, one of the characteristic cytoplasmic domains that form part of the multi-domain "clamp" regions of RyR2. The three-dimensional location of DR2 suggests that it plays roles in the RyR conformational changes that occur during channel gating, and possibly in RyR's interaction with the dihydropyridine receptor in excitation-contraction coupling. This study further demonstrates the feasibility and reliability of the GFP insertion/cryo-EM approach for correlating RyR's amino acid sequence with its three-dimensional structure, thereby enhancing our understanding of the structural basis of RyR function.     

The Drosophila fragile X-related gene regulates axoneme differentiation during spermatogenesis

Macroorchidism (i.e., enlarged testicles) and mental retardation are the two hallmark symptoms of Fragile X syndrome (FraX). The disease is caused by loss of fragile X mental retardation protein (FMRP), an RNA-binding translational regulator. We previously established a FraX model in Drosophila, showing that the fly FMRP homologue, dFXR, acts as a negative translational regulator of microtubule-associated Futsch to control stability of the microtubule cytoskeleton during nervous system development. Here, we investigate dFXR function in the testes. Male dfxr null mutants have the enlarged testes characteristic of the disease and are nearly sterile (>90% reduced male fecundity). dFXR protein is highly enriched in Drosophila testes, particularly in spermatogenic cells during the early stages of spermatogenesis. Cytological analyses reveal that spermatogenesis is arrested specifically in late-stage spermatid differentiation following individualization. Ultrastructurally, dfxr mutants lose specifically the central pair microtubules in the sperm tail axoneme. The frequency of central pair microtubule loss becomes progressively greater as spermatogenesis progresses, suggesting that dFXR regulates microtubule stability. Proteomic analyses reveal that chaperones Hsp60B-, Hsp68-, Hsp90-related protein TRAP1, and other proteins have altered expression in dfxr mutant testes. Taken together with our previous nervous system results, these data suggest a common model in which dFXR regulates microtubule stability in both synaptogenesis in the nervous system and spermatogenesis in the testes. The characterization of dfxr function in the testes paves the way to genetic screens for modifiers of dfxr-induced male sterility, as a means to efficiently dissect FMRP-mediated mechanisms.     

Nanogold-plasmon-resonance-based glucose sensing

Noble metal nanoparticles are well known for their strong interactions with light through the resonant excitations of the collective oscillations of the conduction electrons on the particles, the so-called surface plasmon resonances. The close proximity of two nanoparticles is known to result in a red-shifted resonance wavelength peak, due to near-field coupling. We have subsequently employed this phenomenon and developed a new approach to glucose sensing, which is based on the aggregation and disassociation of 20-nm gold particles and the changes in plasmon absorption induced by the presence of glucose. High-molecular-weight dextran-coated nanoparticles are aggregated with concanavalin A (Con A), which results in a significant shift and broadening of the gold plasmon absorption. The addition of glucose competitively binds to Con A, reducing gold nanoparticle aggregation and therefore the plasmon absorption when monitored at a near-red arbitrary wavelength. We have optimized our plasmonic-type glucose nanosensors with regard to particle stability, pH effects, the dynamic range for glucose sensing, and the observation wavelength to be compatible with clinical glucose requirements and measurements. In addition, by modifying the amount of dextran or Con A used in nanoparticle fabrication, we can to some extent tune the glucose response range, which means that a single sensing platform could potentially be used to monitor microM --> mM glucose levels in many physiological fluids, such as tears, blood, and urine, where the glucose concentrations are significantly different.     

Topological studies on the twin-arginine translocase component TatC

The twin-arginine translocation (Tat) system can translocate folded proteins across biological membranes. Among the known Tat-system components in Escherichia coli, TatC is the only protein with multiple trans-membrane domains. TatC is important for translocon interactions with Tat substrates. The knowledge of its membrane topology is therefore crucial for the understanding of substrate binding and translocon function. Recently, based on active PhoA reporter fusions to the second predicted cytoplasmic loop of TatC, a topology with four trans-membrane domains has been suggested, calling in silico predictions of six trans-membrane domains into question. Here we report studies with translational fusions of TatC to the topological marker enzymes PhoA and LacZ which provide strong evidence for a six-trans-membrane domain topology. The stop transfer capacity of the fourth trans-membrane domain was found to be strongly influenced by the succeeding cytoplasmic domain. The presence of linker sequences at PhoA-fusion sites of the cytoplasmic domain induced PhoA leakage. In the case of one tested fusion (S185-PhoA), the stop-transfer efficiency was already low due to the negative charge in the center of the fourth trans-membrane domain (E170). The results point to the importance of cytoplasmic loops for the stabilization of stop-transfer sequences and revoke evidence for only four trans-membrane domains of TatC.