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The Dark Side of Actin: Cardiac Actin Variants Highlight the Role of Allostery in Disease Development

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The Dark Side of Actin: Cardiac Actin Variants Highlight the Role of Allostery in Disease Development Archives of Biochemistry and Biophysics 695 (2020) 108624 Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi The Dark Side of Actin: Cardiac actin variants highlight the role of allostery in disease development Grace Zi Teng, John F. Dawson * Department of Molecular & Cellular Biology and Centre for Cardiovascular Investigations, University of Guelph, Guelph, ON, N1G 2W1, Canada ARTICLE INFO ABSTRACT Keywords: Mutations in the α-cardiac actin ACTC1 gene cause dilated or hypertrophic cardiomyopathy. These diseases are Actin the result of changes in protein interactions between ACTC protein and force-generating β-myosin or the calcium- Allostery dependent cardiac-tropomyosin (cTm) and cardiac troponin (cTn) regulatory complex, altering the overall Cardiomyopathy contractile force. The T126I and S271F ACTC variants possess amino acid substitutions on the other side of actin Calcium sensitivity pathway relative to the myosin or regulatory protein binding sites on what we call the “dark side” of actin. The T126I Altered force pathway change results in hyposensitivity to calcium, in accordance with the calcium sensitivity pathway of cardiomy­ opathy development while the S271F change alters the maximum in vitro motility sliding speed, reflecting a change in maximum force. These results demonstrate the role of actin allostery in the cardiac disease development. 1. Introduction (2008) [9]. The effects of the S271F and T126I variants on ACTC and their interactions with myosin have not been determined. For the past decade, cardiovascular diseases (CVD) have been the Allostery is a key aspect of the function of actin and is thought to be a number one cause of death worldwide [1]. A significantportion of CVD major contributor to the extreme conservation of the actin protein is due to heritable gene mutations [2]. Cardiomyopathy is the most sequence throughout eukaryotes [10,11]. Within the filament,subunits commonly inherited CVD [3,4] with hypertrophic cardiomyopathy communicate with each other through structural changes dictated by (HCM) and dilated cardiomyopathy (DCM) being two main forms. HCM their nucleotide state. Those allosteric changes are sensed or influenced is characterized by thick ventricular and septal walls, decreasing the by actin binding protein interactions. volume of the left chamber [5]. In contrast, DCM is characterized by thin Disease-related actin variants have been linked to a pathogenic helix and weak ventricular walls, resulting in enlarged ventricles [6]. that extends from residues K113 to T126 of actin molecules [12]. For Mutations in genes encoding sarcomere proteins can lead to HCM or example, K118 N γ-cytoplasmic actin (ACTG) is linked to early-onset DCM. The human ACTC1 gene encoding α-cardiac actin (ACTC) is one of deafness [13], and E117K β-actin (ACTB) was identified in patients the few genes where mutations are linked to both HCM and DCM [7]. with severe cases of Baraitser-Winter syndrome [14]. However, the During heart contractions, the interactions between filamentouscardiac position of the amino acid changes on the actin molecule are far actin and β-myosin are regulated by cardiac-tropomyosin (cTm) and removed from sites of major actin binding protein interactions, sug­ cardiac troponin (cTn) complex in a calcium dependent manner. gesting that allosteric changes are responsible for functional alterations. To date, 18 missense mutations in the ACTC1 gene have been iden­ Both the T126I and S271F ACTC amino acid substitutions are located tifiedby genetic screening of HCM and DCM patients. In genetic testing on the back side of the conventional figureof actin distal to the myosin on patients diagnosed with DCM by Lakdawala et al. (2014), one patient or regulatory protein binding sites or what we call the “dark side” of was identifiedwith a cytosine to thymine transition at nucleotide 383 of actin (Fig. 1). The T126I change is located in the pathogenic helix, while ACTC1 that resulted in the T126I substitution mutation [8]. The S271F recent structural studies showed that the hydrophobic plug of actin is ACTC variant was identified in a patient with HCM by Olivotto et al. part of a tripartite interaction involving residues E270, R39 and D286 of Abbreviations: HCM, hypertrophic cardiomyopathy; ACTC, cardiac actin; RTF, regulated thin filament;IVM, in vitro motility; cTn, cardiac troponin; cTm, cardiac tropomyosin; HMM, heavy meromyosin. * Corresponding author. Department of Molecular & Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada. E-mail address: [email protected] (J.F. Dawson). https://doi.org/10.1016/j.abb.2020.108624 Received 30 June 2020; Received in revised form 24 September 2020; Accepted 5 October 2020 Available online 10 October 2020 0003-9861/© 2020 Elsevier Inc. All rights reserved. G.Z. Teng and J.F. Dawson Archives of Biochemistry and Biophysics 695 (2020) 108624 two neighbouring protomers in the actin filament. During actomyosin glycerol, 1.3 M β-mercaptoethanol (BME), and 0.1% bromophenol blue) interactions, residue E270 is repositioned due to allosteric effects [15]. It [18]. Gels were run for 1 h at 150 mV with a 1 × running buffer (25 mM is hypothesized that the substitution of serine with phenylalanine at Tris, 250 mM glycine, and 0.1% SDS), stained with Coomassie blue, and residue 271 would impact this allosteric change and alter actomyosin destained in 40% methanol and 10% acetic acid. interactions [3]. Models of CVD development with ACTC variants include changes in 2.2. Protein purification the intrinsic properties of the actin molecule or changes in contractility due to what we call the calcium sensitivity pathway, where hypersen­ As described by Teng et al.(2019) [19], recombinant human ACTC sitivity to calcium in the sarcomere leads to HCM and hyposensitivity proteins were purified from Sf9 cells infected with baculovirus and results in DCM. These hypotheses are not mutually exclusive, since full-length myosin and heavy meromyosin (HMM) were isolated from changes in the intrinsic properties of actin can impact the F-actin con­ rabbit soleus muscle. formations accessible to different actin binding proteins (ABPs), Regulatory proteins cardiac tropomyosin and cardiac troponin changing their affinities for different F-actin conformations [16,17]. complex were purifiedfrom bovine cardiac ether powder adapted from Therefore, to test these hypotheses, we biochemically characterized the the protocols of Greaser and Gergely (1971) [20] and Adelstein and S271F and T126I ACTC variants, examining the protein stability, acto­ Tobacman (1986) [21]. myosin interactions and troponin/tropomyosin (Tn/Tm)-dependent calcium sensitivity of the two variants. The T126I ACTC variant dis­ 2.3. Actin intrinsic properties assays played calcium hyposensitivity, aligning with changes in the calcium sensitivity pathway. Alternatively, S271F ACTC variant did not exhibit Thermal Shift Assay –0.4 mg/ml of recombinant ACTC protein stored major changes in calcium sensitivity but had slower IVM filamentsliding in G buffer (2 mM Tris pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP, 0.5 mM beta- speeds, suggesting altered force production. Our work supports the mercaptoethanol, 0.002% NaN3) was mixed with 10 × SYPRO Orange model that altered force development at physiological calcium levels are Dye in a 9:1 ratio. The sample was aliquoted into RT-PCR 96 Well Plates the molecular cause of cardiomyopathies, whether the result of changed in triplicate. The StepOne Plus Software 2.3 was used to measure fluo­ ◦ calcium sensitivity or the maximum force possible in the system. The rescence with readings starting at 4 C and increasing one degree every ◦ location of the T126I and S271F ACTC changes further reinforces the minute until reaching 100 C. role of actin allostery in the regulation of force and the development of Polymerization Assay – The fluorescence of 5 μM sample containing disease. 85% recombinant ACTC protein and 15% pyrene-labeled α-skeletal actin was measured using Cary Eclipse Fluorescence Spectrophotometer with 2. Materials and methods an excitation wavelength of 347 nm and an emission wavelength at 407 nm. The baseline intensity was measured for 30 min, then the poly­ 2.1. Reagents and basic protocols merization reaction was initiated with the addition of 10 × polymeri­ zation buffer (finalconcentration 25 mM Tris, pH 8.0, 50 mM KCl, 1 mM Unless stated otherwise, all reagents were from ThermoFisher Sci­ EGTA, 2 mM MgCl2 and 0.1 mM ATP). The reaction ran for 400 min with entificor Sigma–Aldrich. Spodoptera frugiperda (Sf9) cells were cultured intensity measurements every 5 min. in I-Max media (Wisent Bioproducts, Toronto, ON) supplemented with 1% Penicillin/Streptomycin (PenStrep) mix were obtained from Gibco 2.4. Actomyosin activity assay (Life Technologies, Mississauga, ON). The concentration of recombinant ACTC protein was determined Unregulated Actin Activated Myosin ATPase Assay – Unregulated with the Bradford colourmetric assay using Protein Assay Dye Reagent myosin ATPase activity was measured with an molybdate-based col­ (Bio-Rad, Hercules, CA). Additionally, polyacrylamide gels with 10% ourimeteric Pi release assay from Trybus (2000) and adapted to a 96- resolving gels and 5% stacking gel were used. Samples were mixed in a well format as described [22]. Samples of 15 μM recombinant ACTC 1:1 ratio with 2 × Laemmli buffer (50 mM Tris (pH 6.8), 2% SDS, 10% protein were polymerized with 10 × AB buffer (75 mM KCl, 10 mM Fig. 1. The Dark Side of Actin. Shown is the structure of three actin subunits (A1, A2, and A3) from Mentes et al., 2018 [15] (PDB 6C1D) from the perspective of the customary front (left), side (middle), and “dark side” (left) of the filament.T126 (blue) is located in subdomain 1 on the outer surface of the filament,while S271 (red) is in the hydrophobic plug located between subdomain 3 and 4 of the large domain of actin in the interior of the filament.At left, the A2 subunit of one F-actin strand ◦ is rotated 180 away from subunits A1 and A3 of the opposite strand, revealing the location of S271 on A2 and its site of interaction between subunits A1 and A3 (dotted circles).
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