scholarly journals Lysine acetylation of F-actin decreases tropomyosin-based inhibition of actomyosin activity

2020 ◽  
Vol 295 (46) ◽  
pp. 15527-15539
Author(s):  
William Schmidt ◽  
Aditi Madan ◽  
D. Brian Foster ◽  
Anthony Cammarato
Keyword(s):  
Electrostatic Interactions ◽  
Striated Muscle ◽  
Force Production ◽  
Lysine Acetylation ◽  
Post Translational Modification ◽  
Thin Filaments ◽  
Thin Filament Regulation ◽  
Labeling Approach ◽  
Positively Charged

Recent proteomics studies of vertebrate striated muscle have identified lysine acetylation at several sites on actin. Acetylation is a reversible post-translational modification that neutralizes lysine's positive charge. Positively charged residues on actin, particularly Lys326 and Lys328, are predicted to form critical electrostatic interactions with tropomyosin (Tpm) that promote its binding to filamentous (F)-actin and bias Tpm to an azimuthal location where it impedes myosin attachment. The troponin (Tn) complex also influences Tpm's position along F-actin as a function of Ca2+ to regulate exposure of myosin-binding sites and, thus, myosin cross-bridge recruitment and force production. Interestingly, Lys326 and Lys328 are among the documented acetylated residues. Using an acetic anhydride-based labeling approach, we showed that excessive, nonspecific actin acetylation did not disrupt characteristic F-actin–Tpm binding. However, it significantly reduced Tpm-mediated inhibition of myosin attachment, as reflected by increased F-actin–Tpm motility that persisted in the presence of Tn and submaximal Ca2+. Furthermore, decreasing the extent of chemical acetylation, to presumptively target highly reactive Lys326 and Lys328, also resulted in less inhibited F-actin–Tpm, implying that modifying only these residues influences Tpm's location and, potentially, thin filament regulation. To unequivocally determine the residue-specific consequences of acetylation on Tn–Tpm–based regulation of actomyosin activity, we assessed the effects of K326Q and K328Q acetyl (Ac)-mimetic actin on Ca2+-dependent, in vitro motility parameters of reconstituted thin filaments (RTFs). Incorporation of K328Q actin significantly enhanced Ca2+ sensitivity of RTF activation relative to control. Together, our findings suggest that actin acetylation, especially Lys328, modulates muscle contraction via disrupting inhibitory Tpm positioning.

scholarly journals Effects of cardiomyopathy-linked mutations K15N and R21H in tropomyosin on thin-filament regulation and pointed-end dynamics

2019 ◽  
Vol 30 (2) ◽  
pp. 268-281 ◽  
Author(s):  
Thu Ly ◽  
Christopher T. Pappas ◽  
Dylan Johnson ◽  
William Schlecht ◽  
Mert Colpan ◽  
...  
Keyword(s):  
Thin Filament ◽  
Structural Changes ◽  
Striated Muscle ◽  
In Vitro Assays ◽  
Missense Mutations ◽  
Thin Filaments ◽  
Thin Filament Regulation ◽  
Pointed End ◽  
Kinetics Of

Missense mutations K15N and R21H in striated muscle tropomyosin are linked to dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), respectively. Tropomyosin, together with the troponin complex, regulates muscle contraction and, along with tropomodulin and leiomodin, controls the uniform thin-filament lengths crucial for normal sarcomere structure and function. We used Förster resonance energy transfer to study effects of the tropomyosin mutations on the structure and kinetics of the cardiac troponin core domain associated with the Ca2+-dependent regulation of cardiac thin filaments. We found that the K15N mutation desensitizes thin filaments to Ca2+ and slows the kinetics of structural changes in troponin induced by Ca2+ dissociation from troponin, while the R21H mutation has almost no effect on these parameters. Expression of the K15N mutant in cardiomyocytes decreases leiomodin’s thin-filament pointed-end assembly but does not affect tropomodulin’s assembly at the pointed end. Our in vitro assays show that the R21H mutation causes a twofold decrease in tropomyosin’s affinity for F-actin and affects leiomodin’s function. We suggest that the K15N mutation causes DCM by altering Ca2+-dependent thin-filament regulation and that one of the possible HCM-causing mechanisms by the R21H mutation is through alteration of leiomodin’s function.

scholarly journals Analysis of myofibrillar structure and assembly using fluorescently labeled contractile proteins.

1984 ◽  
Vol 98 (3) ◽  
pp. 825-833 ◽  
Author(s):  
J W Sanger ◽  
B Mittal ◽  
J M Sanger
Keyword(s):  
Actin Filaments ◽  
Striated Muscle ◽  
Contractile Proteins ◽  
Actin Binding ◽  
Thin Filaments ◽  
In Vitro System ◽  
Self Association ◽  
Cross Bridges

To study how contractile proteins become organized into sarcomeric units in striated muscle, we have exposed glycerinated myofibrils to fluorescently labeled actin, alpha-actinin, and tropomyosin. In this in vitro system, alpha-actinin bound to the Z-bands and the binding could not be saturated by prior addition of excess unlabeled alpha-actinin. Conditions known to prevent self-association of alpha-actinin, however, blocked the binding of fluorescently labeled alpha-actinin to Z-bands. When tropomyosin was removed from the myofibrils, alpha-actinin then added to the thin filaments as well as the Z-bands. Actin bound in a doublet pattern to the regions of the myosin filaments where there were free cross-bridges i.e., in that part of the A-band free of interdigitating native thin filaments but not in the center of the A-band which lacks cross-bridges. In the presence of 0.1-0.2 mM ATP, no actin binding occurred. When unlabeled alpha-actinin was added first to myofibrils and then labeled actin was added fluorescence occurred not in a doublet pattern but along the entire length of the myofibril. Tropomyosin did not bind to myofibrils unless the existing tropomyosin was first removed, in which case it added to the thin filaments in the l-band. Tropomyosin did bind, however, to the exogenously added tropomyosin-free actin that localizes as a doublet in the A-band. These results indicate that the alpha-actinin present in Z-bands of myofibrils is fully complexed with actin, but can bind exogenous alpha-actinin and, if actin is added subsequently, the exogenous alpha-actinin in the Z-band will bind the newly formed fluorescent actin filaments. Myofibrillar actin filaments did not increase in length when G-actin was present under polymerizing conditions, nor did they bind any added tropomyosin. These observations are discussed in terms of the structure and in vivo assembly of myofibrils.

scholarly journals Conserved tau microtubule-binding repeat histidines confer pH-dependent tau-microtubule association

2018 ◽  
Author(s):  
Rabab A. Charafeddine ◽  
Wilian A. Cortopassi ◽  
Parnian Lak ◽  
Matthew P. Jacobson ◽  
Diane L. Barber ◽  
...  
Keyword(s):  
Intracellular Ph ◽  
Electrostatic Interactions ◽  
Cell Model ◽  
Interaction Strength ◽  
Microtubule Binding ◽  
Intrinsically Disordered ◽  
C Terminus ◽  
Decreasing Ph ◽  
Positively Charged

ABSTRACTTau, a member of the MAP2/tau family of microtubule-associated proteins, functions to stabilize and organize axonal microtubules in healthy neurons. In contrast, tau dissociates from microtubules and forms neurotoxic extracellular aggregates in neurodegenerative tauopathies. MAP2/tau family proteins are characterized by three to five conserved, intrinsically disordered repeat regions that mediate electrostatic interactions with the microtubule surface. We use molecular dynamics, microtubule-binding experiments and live cell microscopy to show that highly conserved histidine residues near the C terminus of each MT-binding repeat are pH sensors that can modulate tau-MT interaction strength within the physiological intracellular pH range. At lower pH, these histidines are positively charged and form cation-π interactions with phenylalanine residues in a hydrophobic cleft between adjacent tubulin dimers. At higher pH, tau deprotonation decreases microtubule-binding both in vitro and in cells. However, electrostatic and hydrophobic characteristics of histidine are required for tau-MT-binding as substitution with constitutively positively charged, non-aromatic lysine or uncharged alanine greatly reduces or abolishes tau-MT binding. Consistent with these findings, tau-MT binding is reduced in a cancer cell model with increased intracellular pH but is rapidly rescued by decreasing pH to normal levels. Thus, these data add a new dimension to the intracellular regulation of tau activity and could be relevant in normal and pathological conditions.

Preparation and In Vitro Release Study of Quaternized Chitosan Nanoparticles

2014 ◽  
Vol 1053 ◽  
pp. 466-472
Author(s):  
Yan Wen ◽  
Xiao Yang Zhang ◽  
Li Sheng ◽  
Xi Jun Lian
Keyword(s):  
Electrostatic Interactions ◽  
Fourier Transforms ◽  
Sodium Tripolyphosphate ◽  
Drug Carrier ◽  
Chitosan Nanoparticles ◽  
Buffer Solution ◽  
Solution Ph ◽  
Substitution Degree ◽  
Positively Charged

In order to exploit a novel drug carrier for 5-fluorocrai (5-Fu) with improved entrapment efficiency (EE) and loading capacity (LC) determined by UV/Vis spectrophotometer, N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC) nanoparticles were prepared via the ionic gelation based on the electrostatic interactions between positively charged HTCC and negatively charged sodium tripolyphosphate (TPP). The in vitro release behavior of 5-Fu molecules encapsulated within HTCC nanoparticles took a controlled manner in PBS buffer solution (pH 7.4), initiating from a rapid release within 2 hours following a slower sustained release. A higher substitution degree of quaternary ammonium group (DQ) along HTCC chain generated a stronger electrostatic interaction between positively charged HTCC and negatively charged 5-Fu as a result of a higher positively charge density, and contributed to the controlled release of 5-Fu from HTCC nonaparticles. The formation mechanism of HTCC nanoparticles and 5-Fu-loaded HTCC nanoparticles was probed based on the results of fourier transforms infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and size and zeta potential analysis.

scholarly journals Heparin modifies the immunogenicity of positively charged proteins

Blood ◽  
2010 ◽  
Vol 116 (26) ◽  
pp. 6046-6053 ◽  
Author(s):  
Shalini L. Chudasama ◽  
Benjamin Espinasse ◽  
Fred Hwang ◽  
Rui Qi ◽  
Manali Joglekar ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Cell Activation ◽  
High Titer ◽  
Interleukin 12 ◽  
Dendritic Cell Activation ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Positively Charged

Abstract The immune response in heparin-induced thrombocytopenia is initiated by and directed to large multimolecular complexes of platelet factor 4 (PF4) and heparin (H). We have previously shown that PF4:H multimolecular complexes assemble through electrostatic interactions and, once formed, are highly immunogenic in vivo. Based on these observations, we hypothesized that other positively charged proteins would exhibit similar biologic interactions with H. To test this hypothesis, we selected 2 unrelated positively charged proteins, protamine (PRT) and lysozyme, and studied H-dependent interactions using in vitro and in vivo techniques. Our studies indicate that PRT/H and lysozyme/H, like PF4/H, show H-dependent binding over a range of H concentrations and that formation of complexes occurs at distinct stoichiometric ratios. We show that protein/H complexes are capable of eliciting high-titer antigen-specific antibodies in a murine immunization model and that PRT/H antibodies occur in patients undergoing cardiopulmonary bypass surgery. Finally, our studies indicate that protein/H complexes, but not uncomplexed protein, directly activate dendritic cells in vitro leading to interleukin-12 release. Taken together, these studies indicate that H significantly alters the biophysical and biologic properties of positively charged compounds through formation of multimolecular complexes that lead to dendritic cell activation and trigger immune responses in vivo.

scholarly journals Dual Interactions of Amphiphilic Gelatin Copolymer and Nanocurcumin Improving the Delivery Efficiency of the Nanogels

Polymers ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 814 ◽  
Author(s):  
Dinh Trung Nguyen ◽  
Van Thoai Dinh ◽  
Le Hang Dang ◽  
Dang Nam Nguyen ◽  
Bach Long Giang ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
In Vitro Study ◽  
Pluronic F127 ◽  
Spherical Form ◽  
Hierarchical Assembly ◽  
Transmission Electron ◽  
New Process ◽  
Complex Micelles ◽  
Positively Charged

Herein, a new process to manufacture multicore micelles nanoparticles reinforced with co-assembly via hydrophobic interaction and electrostatic interaction under the help of ultrasonication was developed. The precise co-assembly between negative/hydrophobic drug and positive charged amphiphilic copolymer based pluronic platform allows the formation of complex micelles structures as the multicore motif with predefined functions. In this study, curcumin was selected as a drug model while positively charged copolymer was based on a pluronic-conjugated gelatin with different hydrophobicity length of Pluronic F87 and Pluronic F127. Under impact of dual hydrophobic and electrostatic interactions, the nCur-encapsulated core–shell micelles formed ranging from 40 nm to 70 nm and 40–100 nm by transmission electron microscopy (TEM) and Dynamic Light Scattering (DLS), respectively. It is found that the structures emerged depended on the relative lengths of the hydrophobic blocks in pluronic. Regarding g2(τ) behavior from DLS measurement, the nanogels showed a high stability in spherical form. Surprisingly, the release profiles showed a sustainable behavior of Cur from this system for drug delivery approaches. In vitro study exhibited that nCur-encapsulated complex micelles increased inhibitory activity against cancer cells growth with IC50 is 4.02 ± 0.11 mg/L (10.92 ± 0.3 µM) which is higher than of free curcumin at 9.40 ± 0.17 mg/L (25.54 ± 0.18 µM). The results obtained can provide the new method to generate the hierarchical assembly of copolymers with incorporated loading with the same property.

scholarly journals Acetylation of glucosyltransferases regulates Streptococcus mutans biofilm formation and virulence

2021 ◽  
Vol 17 (12) ◽  
pp. e1010134
Author(s):  
Qizhao Ma ◽  
Yangyang Pan ◽  
Yang Chen ◽  
Shuxing Yu ◽  
Jun Huang ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Extracellular Polysaccharides ◽  
Lysine Acetylation ◽  
Post Translational Modification ◽  
Caries Model ◽  
Acetyl Group ◽  
Important Virulence Factor ◽  
Protein Acetyltransferase

Lysine acetylation is a frequently occurring post-translational modification (PTM), emerging as an important metabolic regulatory mechanism in prokaryotes. This process is achieved enzymatically by the protein acetyltransferase (KAT) to specifically transfer the acetyl group, or non-enzymatically by direct intermediates (acetyl phosphate or acetyl-CoA). Although lysine acetylation modification of glucosyltransferases (Gtfs), the important virulence factor in Streptococcus mutans, was reported in our previous study, the KAT has not been identified. Here, we believe that the KAT ActG can acetylate Gtfs in the enzymatic mechanism. By overexpressing 15 KATs in S. mutans, the synthesized water-insoluble extracellular polysaccharides (EPS) and biofilm biomass were measured, and KAT (actG) was identified. The in-frame deletion mutant of actG was constructed to validate the function of actG. The results showed that actG could negatively regulate the water-insoluble EPS synthesis and biofilm formation. We used mass spectrometry (MS) to identify GtfB and GtfC as the possible substrates of ActG. This was also demonstrated by in vitro acetylation assays, indicating that ActG could increase the acetylation levels of GtfB and GtfC enzymatically and decrease their activities. We further found that the expression level of actG in part explained the virulence differences in clinically isolated strains. Moreover, overexpression of actG in S. mutans attenuated its cariogenicity in the rat caries model. Taken together, our study demonstrated that the KAT ActG could induce the acetylation of GtfB and GtfC enzymatically in S. mutans, providing insights into the function of lysine acetylation in bacterial virulence and pathogenicity.

scholarly journals Assembly of different isoforms of actin and tropomyosin into the skeletal tropomyosin-enriched microfilaments during differentiation of muscle cells in vitro.

1986 ◽  
Vol 103 (6) ◽  
pp. 2173-2183 ◽  
Author(s):  
J J Lin ◽  
J L Lin
Keyword(s):  
Monoclonal Antibody ◽  
Striated Muscle ◽  
Chicken Embryo Fibroblast ◽  
Muscle Cells ◽  
Electron Microscopic ◽  
Thin Filaments ◽  
Microscopic Studies ◽  
The One ◽  
Muscle Isoforms

We have used a monoclonal antibody (CL2) directed against striated muscle isoforms of tropomyosin to selectively isolate a class of microfilaments (skeletal tropomyosin-enriched microfilaments) from differentiating muscle cells. This class of microfilaments differed from the one (tropomyosin-enriched microfilaments) isolated from the same cells by a monoclonal antibody (LCK16) recognizing all isoforms of muscle and nonmuscle tropomyosin. In myoblasts, the skeletal tropomyosin-enriched microfilaments had a higher content of alpha-actin and phosphorylated isoforms of tropomyosin as compared with the tropomyosin-enriched microfilaments. Moreover, besides muscle isoforms of actin and tropomyosin, significant amounts of nonmuscle isoforms of actin and tropomyosin were found in the skeletal tropomyosin-enriched microfilaments of myoblasts and myotubes. These results suggest that different isoforms of actin and tropomyosin can assemble into the same set of microfilaments, presumably pre-existing microfilaments, to form the skeletal tropomyosin-enriched microfilaments, which will eventually become the thin filaments of myofibrils. Therefore, the skeletal tropomyosin-enriched microfilaments detected here may represent an intermediate class of microfilaments formed during thin filament maturation. Electron microscopic studies of the isolated microfilaments from myoblasts and myotubes showed periodic localization of tropomyosin molecules along the microfilaments. The tropomyosin periodicity in the microfilaments of myoblasts and myotubes was 35 and 37 nm, respectively, whereas the nonmuscle tropomyosin along chicken embryo fibroblast microfilaments had a 34-nm repeat.

scholarly journals The interaction of tropomodulin with tropomyosin stabilizes thin filaments in cardiac myocytes

2003 ◽  
Vol 162 (6) ◽  
pp. 1057-1068 ◽  
Author(s):  
Ryan E. Mudry ◽  
Cynthia N. Perry ◽  
Meredith Richards ◽  
Velia M. Fowler ◽  
Carol C. Gregorio
Keyword(s):  
Cardiac Myocytes ◽  
Actin Filament ◽  
Thin Filament ◽  
Fluorescent Protein ◽  
Striated Muscle ◽  
Thin Filaments ◽  
Multifunctional Protein ◽  
Capping Protein ◽  
Green Fluorescent

Actin (thin) filament length regulation and stability are essential for striated muscle function. To determine the role of the actin filament pointed end capping protein, tropomodulin1 (Tmod1), with tropomyosin, we generated monoclonal antibodies (mAb17 and mAb8) against Tmod1 that specifically disrupted its interaction with tropomyosin in vitro. Microinjection of mAb17 or mAb8 into chick cardiac myocytes caused a dramatic loss of the thin filaments, as revealed by immunofluorescence deconvolution microscopy. Real-time imaging of live myocytes expressing green fluorescent protein–α-tropomyosin and microinjected with mAb17 revealed that the thin filaments depolymerized from their pointed ends. In a thin filament reconstitution assay, stabilization of the filaments before the addition of mAb17 prevented the loss of thin filaments. These studies indicate that the interaction of Tmod1 with tropomyosin is critical for thin filament stability. These data, together with previous studies, indicate that Tmod1 is a multifunctional protein: its actin filament capping activity prevents thin filament elongation, whereas its interaction with tropomyosin prevents thin filament depolymerization.

Characteristics of the Interaction between Thrombin Exosite1 and the Sequence 269-297 of Platelet Glycoprotein Ibα

1998 ◽  
Vol 80 (08) ◽  
pp. 310-315 ◽  
Author(s):  
Marie-Christine Bouton ◽  
Christophe Thurieau ◽  
Marie-Claude Guillin ◽  
Martine Jandrot-Perrus
Keyword(s):  
Platelet Activation ◽  
Electrostatic Interactions ◽  
Platelet Glycoprotein ◽  
Thrombin Receptor ◽  
Central Position ◽  
Amidolytic Activity ◽  
N Terminus ◽  
Hydrolysis Of ◽  
Chemical Cross Linking ◽  
Positively Charged

SummaryThe interaction between GPIb and thrombin promotes platelet activation elicited via the hydrolysis of the thrombin receptor and involves structures located on the segment 238-290 within the N-terminal domain of GPIbα and the positively charged exosite 1 on thrombin. We have investigated the ability of peptides derived from the 269-287 sequence of GPIbα to interact with thrombin. Three peptides were synthesized, including Ibα 269-287 and two scrambled peptides R1 and R2 which are comparable to Ibα 269-287 with regards to their content and distribution of anionic residues. However, R2 differs from both Ibα 269-287 and R1 by the shifting of one proline from a central position to the N-terminus. By chemical cross-linking, we observed the formation of a complex between 125I-Ibα 269-287 and α-thrombin that was inhibited by hirudin, the C-terminal peptide of hirudin, sodium pyrophosphate but not by heparin. The complex did not form when γ-thrombin was substituted for α-thrombin. Ibα 269-287 produced only slight changes in thrombin amidolytic activity and inhibited thrombin binding to fibrin. R1 and R2 also formed complexes with α-thrombin, modified slightly its catalytic activity and inhibited its binding to fibrin. Peptides Ibα 269-287 and R1 inhibited platelet aggregation and secretion induced by low thrombin concentrations whereas R2 was without effect. Our results indicate that Ibα 269-287 interacts with thrombin exosite 1 via mainly electrostatic interactions, which explains why the scrambled peptides also interact with exosite 1. Nevertheless, the lack of effect of R2 on thrombin-induced platelet activation suggests that proline 280 is important for thrombin interaction with GPIb.

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