Skeletal Myosin Regulatory Light Chain Research Articles

Measuring quantitative proteomic distance between Spanish beef breeds

Estimates of quantitative proteomic distance between populations have not been reported to date. Here, quantitative proteomic distances between three Spanish bovine breeds (Asturiana de los Valles, AV; Retinta, RE; and Rubia Gallega, RG) were estimated from two-dimensional electrophoresis profiles of meat samples of longissimus thoracis muscle at 2 h post-mortem. Statistically significant distances were detected between AV/RG and the most genetically different RE breed, using the novel QD measure of quantitative proteomic distance. In total, 18 differentially abundant myofibrillar and sarcoplasmic proteins/isoforms contributing to proteomic distances between breeds were confidently identified by tandem mass spectrometry. The fast skeletal myosin regulatory light chain 2 followed by other five interacting proteins exhibited the most pronounced relative change between breeds. In addition, most differentially represented proteins could be associated with variations in meat tenderness. Therefore, they could be candidate biomarkers for molecular breeding programs and authentication of the three Spanish beef breeds.

Effects of IVS6-1 Mutation in MYl2 Associated with Cardioskeletal Myopathy and Early Cardiac Death of Infants

The homozygous appearance of intronic mutation (IVS6-1) in MYL2 encoding myosin ventricular/slow skeletal myosin regulatory light chain (RLC) was recently linked to the development of slow skeletal muscle fiber type I hypotrophy. The affected infants died between 4 and 6 months of age due to dilated or hypertrophic cardiomyopathy. In this report we have investigated, the molecular mechanism and functional consequences associated with IVS6-1 using recombinant human cardiac IVS6-1-RLC reconstituted in RLC-depleted porcine cardiac muscle preparations. The results were compared with those obtained for recombinant human cardiac WT-RLC. Despite the ability of the mutant to become phosphorylated, IVS6-1 displayed a significantly lower Vmax of the actin-activated myosin ATPase activity compared with WT (0.18±0.2 s-1 vs. 0.25±0.02 s-1; n=3), indicating a slower myosin cross-bridge turnover rate of the mutant. Likewise, stopped flow kinetics of the ATP induced dissociation of the acto-myosin complex showed a significantly reduced slope of the kobs-[MgATP] relationship for IVS6-1 reconstituted myosin (4.3±0.02×105 M-1 s-1, n=5), depicting slower second-order MgATP binding rates compared with WT (5.3±0.02×105 M-1 s-1, n=5). Steady-state fluorescence binding experiments of mutant vs. WT reconstituted myosin to pyrene labeled F-actin under rigor conditions demonstrated significantly reduced binding affinity of IVS6-1 (Kd=16.7±2.8 nM, n=3) compared with WT (Kd=4.6±1.3 nM, n=3). In skinned porcine cardiac muscles, a significant decrease in maximal force was observed for IVS6-1 reconstituted fibers compared with WT (27.1±1.0 kN/m2 vs. 34.9±2.4 kN/m2, n=6). The Ca2+ sensitivity and cooperativity were significantly different in IVS6-1 (pCa50=5.66±0.01, nH=2.9±0.15) vs. WT (pCa50=5.48±0.01, nH=2.2±0.14). In conclusion, we demonstrate that IVS6-1 may lead to cardiac dysfunction by disrupting the acto-myosin interaction (steady-state and kinetics) and compromising the ability of the mutant myosin to develop contractile force.

Tackling proteome changes in the longissimus thoracis bovine muscle in response to pre-slaughter stress

Pre-slaughter stress has adverse effects on meat quality that can lead to the occurrence of Dark Firm Dry (DFD) meat in cattle. This study explores the previously uncharacterized proteome changes linked to pre-slaughter stress in the longissimus thoracis (LT) bovine muscle. Differential proteome profiles of DFD and normal (non-DFD) LT meat samples from male calves of the Rubia Gallega breed were assessed by 2-DE coupled to MS analysis (LC-MS/MS and MALDI TOF/TOF MS). A total of seven structural-contractile proteins (three different myosin light chain isoforms, two fast skeletal myosin light chain 2 isoforms, troponin C type 2 and cofilin-2) and three metabolism enzymes (triosephosphate isomerase, ATP synthase and beta-galactoside alpha-2,6-sialyltransferase) were found to have statistically significant differential abundance in sample groups. In addition, 2-DE in combination with the phosphoprotein-specific fluorescent dye Pro-Q DPS revealed that highly phosphorylated fast skeletal myosin regulatory light chain 2 isoforms underwent the most intense relative change in muscle conversion to DFD meat. Therefore, they appear to be the most sensitive biomarkers of stress just prior to slaughter in Rubia Gallega. Overall, these findings will facilitate a more integrative understanding of the biochemical processes associated with stress in cattle muscle and their effects in meat quality. Biological significancePre-slaughter stress is a crucial factor in meat production. Animals destined for slaughter are stressed by a variety of endogenous and exogenous factors that negatively affect the complex post-mortem biochemical events underlying the conversion of muscle into meat. The study of the muscle proteome has a great relevance for understanding the molecular mechanisms associated with stress. However, there is no information available on the molecular changes linked to pre-slaughter stress in cattle on the proteome scale. Our study led to the identification of a number of candidate proteins associated with the response to pre-slaughter stress in the LT bovine muscle of the Rubia Gallega breed. The functions of those significantly changed proteins have a clear biological relationship with stress response. These findings contribute to a deeper insight into the molecular pathways that respond to stress in cattle.

Myosin regulatory light chain phosphorylation inhibits shortening velocities of skeletal muscle fibers in the presence of the myosin inhibitor blebbistatin

Phosphorylation of skeletal myosin regulatory light chain (RLC) occurs in fatigue and may play a role in the inhibition of shortening velocities observed in vivo. Forces and shortening velocities were measured in permeabilized rabbit psoas fibers with either phosphorylated or dephosphorylated RLCs and in the presence or absence of the myosin inhibitor blebbistatin. Addition of 20 microM blebbistatin decreased tensions by approximately 80% in fibers, independent of phosphorylation. In blebbistatin maximal shortening velocities (V(max)) at 30 degrees C, were decreased by 45% (3.2 +/- 0.34 vs. 5.8 +/- 0.18 lengths/s) in phosphorylated fibers but were not inhibited in dephosphorylated fibers (6.0 +/- 0.30 vs. 5.4 +/- 0.30). In the presence of 20 microM blebbistatin, K(m) for V(max) as a function of [ATP] was lower for phosphorylated fibers than for dephosphorylated fibers (50 +/- 20 vs. 330 +/- 84 microM) indicating that the apparent binding of ATP is stronger in these fibers. Phosphorylation of RLC in situ during fiber preparation or by addition of myosin light chain kinase yielded similar data. RLC phosphorylation inhibited velocity in blebbistatin at both 30 and 10 degrees C, unlike previous reports where RLC phosphorylation only affected shortening velocities at higher temperatures.

A point mutation in the regulatory light chain reduces the step size of skeletal muscle myosin.

Current evidence favors the theory that, when the globular motor domain of myosin attaches to actin, the light chain binding domain or "lever arm" rotates, and thereby generates movement of actin filaments. Myosin is uniquely designed for such a role in that a long alpha-helix (approximately 9 nm) extending from the C terminus of the catalytic core is stabilized by two calmodulin-like molecules, the regulatory light chain (RLC) and the essential light chain (ELC). Here, we introduce a single-point mutation into the skeletal myosin RLC, which results in a large (approximately 50%) reduction in actin filament velocity (V(actin)) without any loss in actin-activated MgATPase activity. Single-molecule analysis of myosin by optical trapping showed a comparable 2-fold reduction in unitary displacement or step size (d), without a significant change in the duration of the strongly attached state (tau(on)) after the power stroke. Assuming that V(actin) approximately d/tau(on), we can account for the change in velocity primarily by a change in the step size of the lever arm without incurring any change in the kinetic properties of the mutant myosin. These results suggest that a principal role for the many light chain isoforms in the myosin II class may be to modulate the flexural rigidity of the light chain binding domain to maximize tension development and movement during muscle contraction.

Preparation and incorporation of probe-labeled apoA-I for fluorescence resonance energy transfer studies of rHDL

Apolipoprotein A-I (apoA-I), the major constituent of HDL, plays an essential role in regulating cholesterol metabolism, acting as the physiological activator of lecithin: cholesterol acyltransferase, which converts cholesterol to cholesterol ester. Thiol-reactive fluorescent probes attached to cysteine-containing apoA-I mutants are currently being used to investigate the "LCAT active" conformation of lipid-bound apoA-I. Herein, we report new methodologies allowing rapid expression, fluorescent labeling, and recombinant HDL (rHDL) preparation for use in apoA-I in fluorescence resonance energy transfer (FRET) studies. Cysteine-containing mutant forms of human apoA-I were cloned into the pTYB12 vector containing a T7 promoter, a modified self-splicing protein element (intein), and a small affinity tag [chitin binding domain (CBD)]. The fusion proteins were expressed in Escherichia coli, isolated from cell lysates, and bound to a chitin-affinity column. Release of mature human apoA-I was initiated by the addition of DTT, which induced self-cleavage at the COOH terminus of the intein - CBD fusion protein. ApoA-I was further purified by Q-sepharose and then used for fluorescent probe labeling. Discoidal rHDL were then prepared with donor and/or acceptor labeled apoA-I and characterized with respect to their size, composition and ability to activate LCAT.

Proximity Relationships between Engineered Cysteine Residues in Chicken Skeletal Myosin Regulatory Light Chain:: A RESONANCE ENERGY TRANSFER STUDY

Resonance energy transfer was used to measure the distances between pairs of cysteines, Cys2 and Cys155 and Cys73 and Cys155, in recombinant chicken skeletal myosin regulatory light chains in the free and bound states. The fluorescent and nonfluorescent probes N-iodoacetyl-N'-(5-sulfo-1-naphthyl) ethylenediamine and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide were used as the donor and the acceptor, respectively. The distance between Cys2 and Cys155 was measured to be 35 and 30 A in the absence and presence of myosin heavy chain, respectively, suggesting a slightly more compact structure for the light chain in the bound state. The distance between Cys73 and Cys155 measured in a similar manner was 31 and 30 A in the free and bound states, respectively; this latter value is in good agreement with that derived from crystallographic structures. For heavy chain-bound light chains, no measurable distance changes were detected with the binding of ATP or actin. These results show that no gross structural changes occur within the regulatory light chain during the contraction cycle, but that resonance energy transfer between other sites could be used to monitor potential changes in the myosin head upon the binding of nucleotides and actin.