A novel method for preparation of HAMLET-like protein complexes

Biochimie 93 (2011) 1495e1501

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Research paper

A novel method for preparation of HAMLET-like protein complexes Sergei E. Permyakov a, b, *, Ekaterina L. Knyazeva a, *, Marina V. Leonteva a, b, Roman S. Fadeev c, Aleksei V. Chekanov c, Andrei P. Zhadan a, Anders P. Håkansson d, Vladimir S. Akatov c, Eugene A. Permyakov a, b a

Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia Department of Biomedical Engineering, Pushchino State University, Pushchino, Moscow Region 142290, Russia c Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia d Department of Microbiology and Immunology, University at Buffalo, Buffalo, NY 14214, USA b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 March 2011 Accepted 4 May 2011 Available online 11 May 2011

Some natural proteins induce tumor-selective apoptosis. a-Lactalbumin (a-LA), a milk calcium-binding protein, is converted into an antitumor form, called HAMLET/BAMLET, via partial unfolding and association with oleic acid (OA). Besides triggering multiple cell death mechanisms in tumor cells, HAMLET exhibits bactericidal activity against Streptococcus pneumoniae. The existing methods for preparation of active complexes of a-LA with OA employ neutral pH solutions, which greatly limit water solubility of OA. Therefore these methods suffer from low scalability and/or heterogeneity of the resulting a-LA e OA samples. In this study we present a novel method for preparation of a-LA e OA complexes using alkaline conditions that favor aqueous solubility of OA. The unbound OA is removed by precipitation under acidic conditions. The resulting sample, bLAeOA-45, bears 11 OA molecules and exhibits physico-chemical properties similar to those of BAMLET. Cytotoxic activities of bLAeOA-45 against human epidermoid larynx carcinoma and S. pneumoniae D39 cells are close to those of HAMLET. Treatment of S. pneumoniae with bLAeOA-45 or HAMLET induces depolarization and rupture of the membrane. The cells are markedly rescued from death upon pretreatment with an inhibitor of Ca2þ transport. Hence, the activation mechanisms of S. pneumoniae death are analogous for these two complexes. The developed express method for preparation of active a-LA e OA complex is high-throughput and suited for development of other protein complexes with low-molecular-weight amphiphilic substances possessing valuable cytotoxic properties. Ó 2011 Elsevier Masson SAS. All rights reserved.

Keywords: a-lactalbumin Oleic acid HAMLET Cytotoxicity Bactericidal activity

1. Introduction Despite prominent achievements in development of novel anticancer therapies, the need for new anticancer drug candidates Abbreviations: OA, oleic acid (C18:1:9cis); CMC, critical micelle concentration;

a-LA, a-lactalbumin; intact a-LA, a-lactalbumin isolated from milk; hLA, human a-lactalbumin; bLA, bovine a-lactalbumin; HAMLET(BAMLET), complex of human(bovine) a-lactalbumin with oleic acid, prepared as described in [1,2]; bLAeOA-45, complex of bovine a-lactalbumin with oleic acid, prepared as described in Materials and methods; reference bLA, sample of bovine a-lactalbumin

subjected to procedures described for preparation of bLAeOA-45, skipping addition of oleic acid; MS, mass spectrometry; CD, circular dichroism; lmax, maximum position of fluorescence emission spectrum; T1/2, mid-transition temperature; HEp2, human epidermoid larynx carcinoma cells; CFUs, colony-forming units. * Corresponding authors. Institute for Biological Instrumentation of the Russian Academy of Sciences, Institutskaya 7, Pushchino, Moscow Region 142290, Russia. Tel.: þ7 4967 73 41 35; fax: þ7 4967 33 05 22. E-mail addresses: [email protected] (S.E. Permyakov), elknyazeva@rambler. ru (E.L. Knyazeva). 0300-9084/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2011.05.002

is still urgent. One of the most intriguing classes of substances with antitumor therapeutic potential is a set of natural proteins, selectively inducing apoptosis in tumor cells: apoptin, E4orf4, NS1, mda7, TRAIL, HAMLET and ELOA (for reviews, see [3,4]). Some of them have shown positive results in pre-clinical and clinical studies (mda-7, TRAIL and HAMLET). Aside from direct use of these proteins for therapeutic purposes, they provide valuable information on molecular mechanisms used to defeat the biochemical machinery of malignant cells. In this respect, HAMLET is of special interest due to its ability to attack several critical organelles (mitochondrion, proteasome and nucleosome), activating different death pathways in tumor cells (for reviews, see [4,5]). HAMLET represents a complex between a small calcium-binding protein from human milk, a-lactalbumin (a-LA; for review, see [6]), and oleic acid (OA) [1]. In the lactating mammary gland, a-LA serves as a regulatory subunit of the lactose synthase enzyme complex [7]. The binding of OA to a-LA following a specific procedure converts it into a form (HAMLET, Human Alpha-lactalbumin Made LEthal to

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Tumor cells), which induces death of tumor cells and undifferentiated cells, but spares healthy and mature cells in cell culture and tissue [1,8e12]. Besides its tumoricidal activity, this complex also exhibits bactericidal activity against antibiotic-sensitive and resistant strains of Streptococcus pneumoniae [13,14]. Although a-LA is able to bind other fatty acids [15], C18:1:9cis (OA) or C18:1:11cis (vaccenic acid) is required to form an a-LA complex with pronounced antitumor activity [16]. Meanwhile, human a-LA can be replaced by bovine, equine, porcine or caprine a-LA without loss of the complex activity [2]. Moreover, binding of OA to Ca2þ-binding equine lysozyme also gives rise to tumoricidal activity, analogous to HAMLET [17]. This fact and similar evidences discussed by Spolaore et al. [18] indicate that the cytotoxic effect of HAMLET is mostly due to OA, which is cytotoxic by itself [19e21], while the proteinaceous component of the complex mainly serves as a carrier of the poorly water soluble OA [22e24]. Nevertheless, there are evidences that a-LA within HAMLET directly affects some of key cellular processes [25], implying that both components of the complex are important for its cytotoxicity. The binding of OA to a-LA requires partial unfolding of the protein [1], which can be achieved via Ca2þ depletion [26] or thermal denaturation. The existing procedures for preparing tumoricidal aLA e OA complexes differ in the procedure of application of OA, in the method of a-LA unfolding, and in the OA concentration used. The original method employs chromatography of Ca2þ-free a-LA on a DEAE-TrisacrylÒ M column preconditioned with OA followed by elution of the complex by high salt concentration [1]. Other methods are based upon direct binding of dissolved OA by Ca2þ-depleted [21,27] or thermally denatured [28] a-LA. Only the method by Knyazeva et al. takes into account low water solubility of OA at neutral pH via use of OA concentrations below its effective critical micelle concentration (CMC). The absence of such a control of CMC values in other methods is fraught with formation of extended phases of OA [22e24], making the a-LA e OA system highly heterogeneous. The heterogeneity of this system is documented in the work by Spolaore et al. [18]. Possibly this circumstance partly rationalizes the fact that different oligomeric states of a-LA and various OA to a-LA molar ratios were reported for active complexes prepared using different approaches (for review, see [18]). The CMC values of OA do not exceed 70 mM under optimized solution conditions used in the method by Knyazeva et al. [21]. The low water solubility of OA at neutral pH limits scalability of this method. In the present work we present a novel method for preparation of biologically active a-LA e OA complexes based on the use of alkaline solvent conditions, greatly favoring aqueous solubility of OA, thereby allowing us to overcome the scalability limitation of the previous method. The developed low-cost express method enables preparation under laboratory conditions of quantities of active sample suitable for animal testing and pre-clinical studies. Furthermore, the use of extreme alkaline conditions enables the extension of this method to other proteins, thus opening up wide opportunities for preparation of protein complexes with fatty acids or other low-molecular-weight amphiphilic substances with valuable cytotoxic properties. The proposed method of dissolving of otherwise hardly water soluble cytotoxic substances into the hydrophobic interior of natural proteins can be exploited for cell delivery of these substances.

Human a-lactalbumin (hLA) was isolated from milk as described [29,30]. Ca2þ-depleted bovine a-lactalbumin (bLA) and oleic acid (C18:1:9cis) were from SigmaeAldrich Co. Chemistry grade 96% (v/v) ethanol was treated with K2MnO4 and activated coal, followed by double distillation; the absence of organic impurities was confirmed spectrophotometrically. Stock solution of OA was prepared by sonication of an ethanol suspension of OA for 3 min, and stored at room temperature. HAMLET and BAMLET were prepared using the original procedure [1,2]. Concentrations of a-LA were evaluated using extinction coefficients A1% 1 cm;280 of 20.1 and 18.2 for bLA/BAMLET and hLA/HAMLET, respectively [31,32]. Ultra-grade H3BO3 was bought from Merck. Ultra-grade ammonium bicarbonate, CaCl2 standard solution, DEAE-TrisacrylÒ M, DMEM culture medium, crystal violet, Ruthenium Red and propidium iodide were from SigmaeAldrich Co. Fetal bovine serum was from HyClone. Todd-Hewitt medium and Bacto-Yeast extract were from BD Biosciences. DiBAC4(3) was from Invitrogen. EDTA standard solution and dialysis tubing were from Thermo Fisher Scientific Inc. Water with conductivity of 18.2 MU cm was used. HPLC grade acetonitrile was from Panreac. 2.2. Estimation of the effective critical micelle concentration of OA Estimation of the effective critical micelle concentration of OA was performed as previously described [21]. The appearance of OA micelles was monitored photometrically at 335 nm. The lowest estimate of OA binding capacity of a-LA, nmax, was found from the dependence of effective CMC of OA upon protein concentration, [P] (CMC0 is CMC value of free OA):

CMC ¼ CMC0 þ nmax $½P

(1)

2.3. Preparation of bLAeOA-45 state 6 ml of 0.6 mM Ca2þ-free bLA at pH 12.0 (5 mM KOH, 1 mM EDTA) was titrated at 45  C by 12 ml aliquots of 1 M OA solution in 96% ethanol up to the effective CMC of OA, 22 mM. The unbound OA was separated via acidification of the solution down to pH 2.0 and centrifugation at 12,000g for 30 min at 40  C. The free from unbound OA water phase was collected, and its pH was adjusted by KOH to w5.5. The solution was triply dialyzed against 100-fold volume of distilled water, freeze-dried and stored at 18  C. Bovine a-lactalbumin, subjected to the described procedure, but skipping addition of OA, will be referred to as “reference bLA”. The integrity of bLAeOA-45 and reference bLA was confirmed by SDS-PAGE. 2.4. Quantitation of the content of OA in protein samples

2. Materials and methods

Quantitation of the content of OA in protein samples was performed using LC/MS system LCMS-2010EV (Shimadzu Co.), ESI probe coupled to a single quadrupole detector. Sample of 0.1e1 mM a-LA in 50:50 (v/v) mixture of 20 mM ammonium bicarbonate pH 8.5 (buffer A) and acetonitrile was applied to Shimadzu Silica 100 2.5 mm RP 4,4  50 mm column at 40  C. OA was eluted with buffer A:acetonitrile 20:80 (v/v) at 50 ml/min flow rate and infused into MS detector (interface 4 kV, negative mode, SIM mode 281  0.5 u). The chromatogram was integrated and quantitated using a calibration curve. The calibration was carried out using 0.01e100 mM OA in 96% ethanol.

2.1. Materials

2.5. Secondary structure content in a-LA

Human epidermoid larynx carcinoma (HEp-2) cells were from the Russian Cell Culture Collection (Institute of Cytology, Russian Academy of Sciences, St. Petersburg).

Secondary structure content in a-LA was determined from farUV circular dichroism (CD) spectra using the CDPro software package [33] as previously described [21].

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2.6. Thermal stability of a-LA

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S. pneumoniae D39 [34] was stored in glycerol stocks at 80  C. Frozen stocks were used to seed Todd-Hewitt medium containing 0.5% Bacto-Yeast Extract. In late logarithmic growth phase the bacteria were harvested by centrifugation at 1500g for 2 min and suspended in PBS buffer (30 mM Na2HPO4, 10 mM KH2PO4, 120 mM NaCl, pH 7.4) to an appropriate concentration for experiments. The effect of a-LA samples on bacterial viability was assessed by plating 10-fold dilutions of treated and untreated bacteria on blood agar and counting of viable colony-forming units (CFUs) after overnight growth at 37  C. The effect of Ruthenium Red was assessed via addition of protein complexes after 15 min pretreatment of the cells with the inhibitor. The data were analysed using two-tailed Student’s t-test.

strength) the respective CMC values reported for pH 8.3 under similar solution conditions [21]. The maximal CMC value of (2.1  0.6) mM is reached in the absence of metal cations at 20  C. On the contrary, calcium ions (1 mM CaCl2) lower the CMC of OA down to 4 mM, regardless of temperature. Similar values (3e4 mM) were previously observed at pH 8.3 [21]. Since calcium ions drastically reduce water solubility of OA, they were avoided in further optimization steps. It is further assumed that 1 mM EDTA is added. Examination of the dependence of effective CMC of OA upon bLA concentration at pH 12 and 45  C (Fig. 1) shows that OA binding capacity of bLA, nmax (see Equation [1]), grows at bLA concentrations above 0.5 mM, which reflects loading of the low-affinity sites of the protein (OA binding constants ca 103 M1). The use of bLA concentrations above 0.6e1 mM would require EDTA concentrations above 1 mM for proper depletion of contaminating Ca2þ ions. Since this high EDTA concentration may result in a binding of EDTA to bLA [37], bLA concentration was restricted to 0.6 mM. Optimization of the process of a-LA e OA interaction via selection of ionic strength and temperature conditions ensuring maximal OA binding capacity of bLA (Table 1) shows that the most favorable solution conditions are achieved in the absence of metal cations at 45  C. The corresponding nmax value of 34 is about twofold higher than the maximal nmax estimate for pH 8.3 [21]. The fact that negatively charged OA molecules efficiently associate with almost fully deprotonated protein at pH 12.0 (bLA contains one Arg residue) suggests a crucial role of the hydrophobic effect in stabilization of the bLA e OA complex. Thus, the process of bLA association with OA can be regarded as dissolution of the hydrophobic moiety of OA in the non-polar interior of the protein. This mechanism seems to enable the binding of multiple OA molecules to bLA (Table 1).

2.10. Measurement of bacterial outer membrane potential and integrity of the membrane

3.2. Preparation of bLAeOA-45 sample

Thermal stability of a-LA was estimated spectrofluorimetrically as previously described [21]. 2.7. Calcium affinity of a-LA Calcium affinity of a-LA was estimated from spectrofluorimetric Ca2þ-titration of the Ca2þ-depleted protein using the cooperative metal binding scheme as previously described [21]. 2.8. Viability of human larynx carcinoma (HEp-2) cells Viability of human larynx carcinoma (HEp-2) cells was estimated using crystal violet assay as previously described [21]. 2.9. Bactericidal assay

S. pneumoniae D39 were pelleted by centrifugation at 1500g and washed twice by resuspension in PBS buffer (pH 7.4) after centrifugation. The bacterial pellet was resuspended in half of the initial PBS volume and energized with 50 mM glucose for 15 min at 37  C. Propidium iodide (40 mg/ml) and DiBAC4(3) (0.5 mM) were added to the energized bacteria and 100 ml of bacterial suspension was mixed with 100 ml of PBS alone or PBS containing Ruthenium Red in each well of a 96-well microtiter plate (Falcon, BD Biosciences) (final concentrations are 25 mM glucose, 20 mg/ml propidium iodide and 0.25 mM DiBAC4(3)). The plate was placed into a Synergy 2 microplate reader (BioTek Instruments, Inc.) at 37  C and fluorescence was read for 40 min before addition of a-LA complexes to allow the dyes to equilibrate. DiBAC4(3) fluorescence (485/20 nm excitation, 520/25 nm emission) and propidium iodide fluorescence (485/20 nm excitation, 590/35 nm emission) were then read for 1 h to monitor changes in membrane potential and integrity, respectively.

The 0.6 mM solution of bLA was titrated by OA up to its effective CMC value under optimized solution conditions: pH 12.0, 5 mM KOH, 1 mM EDTA, 45  C. The intrinsically cytotoxic [19e21] unbound OA was removed using the procedure employing low solubility of fatty acids under acidic conditions. Acidification of the solution down to pH 2.0 induces protonation of carboxylates of OA,

3. Results and discussion 3.1. Optimization of conditions for formation of a-LA e OA complexes The optimal conditions should ensure maximal OA binding capacity of a-LA. This requirement entails the need for fulfillment of two conditions: a-LA unfolding, previously shown to be critical for association of OA [1], and high solubility of OA. Both conditions are fully satisfied under extreme alkaline conditions of pH above 12 [35,36]. The CMCs of Ca2þ-free OA (1 mM EDTA) estimated at pH 12.0 exceed 2e105-fold (depending upon temperature and ionic

Fig. 1. The dependence of effective CMC of OA upon bLA concentration in the absence of Ca2þ (1 mM EDTA) at pH 12.0 (5 mM KOH) and 45  C, in the absence/presence of 150 mM KCl (open/filled circles). The lines represent linear fits of the initial regions of the experimental curves.

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Table 1 The dependence of OA binding capacity of bLA (nmax) at pH 12.0 (5 mM KOH) upon calcium concentration, ionic strength and temperature. Protein concentration was 0.6 mM. Temperature

20  C 45  C

1 mM EDTA

1 mM CaCl2 nmax, 150 mM KCl

nmax, no salt

nmax, 150 mM KCl

0 0

18  1 34  5

26  2 22  3

resulting in its precipitation. The precipitates were removed via centrifugation. The resulting sample was adjusted to pH w5.5, dialyzed against water and freeze-dried. The bLA sample subjected to the same procedures, but without addition of OA, will be referred to as “reference bLA”. 3.3. Physico-chemical characterization of bLAeOA-45 complex The number of OA molecules per a-LA molecule in bLAeOA-45 sample estimated by LC/MS is 11, which is comparable to the content of OA in an LAeOA-45 sample of human a-LA [21]. Thus, about one third of the initially bound OA molecules are retained in bLAeOA-45 sample. Notably, examination of the samples of other proteins binding OA only non-specifically, which were subjected to the same procedures used for preparation of bLAeOA-45 sample, have shown for some of them at least an order of magnitude lower OA content. This fact confirms efficacy of the acidic method of OA removal and indicates that bLAeOA-45 sample retains OA molecules due to their specific binding. The CD estimates of a-helical content for Ca2þ-depleted forms of different bLA states at 5  C (pH 8.3) are shown in Table 2. Reference bLA exhibits a-helical content lowered by (4.6  0.7)%, evidencing some structural perturbations caused by the procedures used for preparation of bLAeOA-45 sample. At the same time, the bLAeOA45 state has the same a-helical content as intact protein. Hence, modification of bLA with OA improves its a-helicity. An analogous effect is observed for BAMLET (Table 2) and hLA e OA complexes [21]. This effect is also observed at 45  C. Spectrofluorimetric measurement of thermal unfolding of the Ca2þ-free bLAeOA-45 shows that its thermal stability is equivalent to that of the BAMLET state, being lowered by about 8  C with respect to both intact and reference bLA samples (Table 2). This effect is in agreement with the previous data, evidencing a decrease in stability of apo-a-LA upon association with OA [21,38]. The lowered stabilities of the Ca2þ-free bLA complexes with OA are in line with red shifts (9e12 nm) of their fluorescence emission spectra (Table 2), which indicate higher accessibility of emitting tryptophans to the solvent. A similar, but less pronounced, effect (7 nm red shift) is observed for reference bLA. Calcium-binding (1 mM CaCl2) lowers this effect to 5 nm. The structural perturbations in reference bLA affect protein’s secondary structure and the environment of emitting tryptophans, but do not change thermal stability of bLA. Calcium binding (1 mM CaCl2) renders all bLA states studied equally stable (mid-transition temperatures of 61e62  C). Despite

that, fluorescence spectrum of bLAeOA-45 is red-shifted by 11 nm in comparison with intact bLA and BAMLET. Hence, in the case of bLAeOA-45, the association with OA increases solvent accessibility of emitting tryptophans of the Ca2þ-bound protein, without affecting its thermal stability. Spectrofluorimetric measurements of calcium affinity of various bLA states at 45  C show that association of OA does not affect protein calcium affinity, but decreases cooperativity of Ca2þbinding by bLAeOA-45 (Table 2). Since calcium affinity data for bLAeOA-45 and reference bLA are indistinguishable, the modification of the protein by OA does not affect calcium affinity of bLA. Overall, our data show that conformational and physicochemical properties of bLAeOA-45 state are similar to those of BAMLET. 3.4. Cytotoxic effects of bLAeOA-45 upon HEp-2 and S. pneumoniae D39 cells The concentration dependencies of the cytotoxicites of bLAeOA45, HAMLET and respective control samples on human larynx carcinoma (HEp-2) cells in vitro are shown in Fig. 2. Intact and reference bLA at concentrations up to 1 mM were shown to be nontoxic. Oleic acid exerts toxic effect on HEp-2 cells only at concentrations above 300 mM. The IC50 values for bLAeOA-45 (about 20 mM) and HAMLET (40 mM) differ insignificantly. The comparison of bactericidal activities of bLAeOA-45 and HAMLET against S. pneumoniae D39 is shown in Table 3. In the range of protein concentrations of 50e150 mg/ml (3.5e10.6 mM) bLAeOA-45 reduces viability of the bacteria by 2.4e6.2 orders of magnitude, which is close to the bactericidal activity of HAMLET. Intact a-LA at a concentration of 150 mg/ml does not exert any bactericidal activity. To evaluate the specific contribution of OA to the bactericidal activity, we exposed bacteria to a concentration of OA, equivalent to the content of OA in 10.6 mM of bLAeOA-45 sample. An OA concentration of 106 mM exhibits a bactericidal activity of 6.3  0.2 orders of magnitude, which is similar to activity for 10.6 mM of bLAeOA-45. Hence, the cytotoxic effect of bLAeOA45 sample could be ascribed primarily to the toxic action of OA. The same conclusion follows from the cytotoxicity data presented in Fig. 2 and the previous data on the hLA e OA complexes [21]. These facts favor the view that the proteinaceous component of the LA e OA complexes mainly serves as a carrier of the otherwise poorly soluble cytotoxic component, oleic acid. Summarizing, bLAeOA-45 and HAMLET samples are equally effective with respect to both HEp-2 and S. pneumoniae D39 cells. 3.5. The initial stages of bLAeOA-45 action upon S. pneumoniae D39 cells Pretreatment of S. pneumoniae D39 cells with 30 mM Ruthenium Red, an inhibitor of Ca2þ transport, significantly prevents the bLAe OA-45einduced death of the cells, indicated by a reduction of logdeath from 3.1  0.5 to 1.48  0.17 (protein concentration is 75 mg/ ml), which suggests that Ca2þ transport is directly involved in the cytotoxic action of this complex. This is analogous to the 51% log-

Table 2 Physico-chemical properties of various states of bLA: a-helical content, maximum position of fluorescence emission spectrum (lmax) and mid-transition temperature (T1/2) for Ca2þ-depleted protein (1 mM EDTA), equilibrium Ca2þ association constant (K) and Hill coefficient (n) at 45  C. Buffer conditions: pH 8.3, 20 mM H3BO3eKOH, 150 mM NaCl. Protein state

a (5  C), %

Intact bLA Reference bLA bLAeOA-45 BAMLET

31.0 26.4 31.0 38.4

   

0.5 0.5 0.5 0.5

lmax (10  C), nm 324.6 331.4 336.2 333.6

   

0.1 0.1 0.1 0.1

K, M1

T1/2,  C 37.4 38.1 30.0 29.5

   

0.8 1.0 0.7 1.4

(1.1 (2.0 (2.0 (1.2

   

n 0.2) 0.3) 0.3) 0.2)

   

106 106 106 106

0.95 0.65 0.68 0.98

   

0.02 0.08 0.04 0.01

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Fig. 2. Concentration dependences of cytotoxicites against human larynx carcinoma cells evaluated using the crystal violet assay for bLAeOA-45 (open circles), HAMLET (open triangles), OA (filled triangles), reference bLA (filled circles) and intact bLA (open squares).

reduction recently shown for HAMLET [14]. Similar results are observed for bLAeOA-45einduced death of S. pneumoniae D39 cells upon their pretreatment with other inhibitors of Ca2þ transport: 100 mM of tetrandrine or dichlorobenzamil (reduction of log-death to 1.3  0.6 and 1.2  1.2, respectively). The involvement of Ca2þ transport in the initial stages of cytotoxic action of hLA e OA complexes was previously shown on plasma membrane of Chara coralline [39]. To assess the effects of bLAeOA-45 and HAMLET on bacterial plasma membrane the depolarization and integrity of the membranes were monitored using the fluorescence dyes DiBAC4(3) and propidium iodide, respectively. DiBAC4(3) is known to enter depolarized cells where it binds to intracellular proteins or membranes, which causes an enhancement in its fluorescence [40]. Propidium iodide is unable to penetrate into intact cells, but binds to cellular DNA in damaged cells, resulting in appearance of red fluorescence [41]. Addition of bLAeOA-45 or HAMLET to pneumococci results in a depolarization of the bacterial membrane within 2 min after the addition, as monitored by DiBAC4(3) fluorescence (Fig. 3A). The kinetics of disturbance of integrity of the membranes detected by fluorescence of propidium iodide (Fig. 3B) suggests that the membrane depolarization leads to membrane damage. Both these effects were greatly inhibited by Ruthenium Red, suggesting a role of calcium in the activation of depolarization and cell death. Intact a-LA does not cause depolarization or rupture of bacterial membrane. These effects are in accord with the previous data on plasmalemma of C. coralline [39]. Taken together, the experimental data demonstrate a high degree of similarity in the initiation of bacterial death induced by bLAeOA-45 and HAMLET.

Table 3 The bactericidal activities of bLAeOA-45 and HAMLET against S. pneumoniae D39. Protein concentration,

mg/ml 50 75 100 150

Log (control CFUs)/CFUs HAMLET 3.6 4.8 6.0 6.5

   

0.9 0.8 1.1 1.3

bLAeOA-45 2.4 3.1 6.0 6.2

   

0.3 0.5 1.1 0.7

Fig. 3. The kinetics of the response of S. pneumoniae D39 cells to bLAeOA-45 (50e150 mg/ml, solid curves) or HAMLET (100 mg/ml, dotted curves) fluorimetrically monitored using 0.25 mM DiBAC4(3) (A) or 20 mg/ml propidium iodide (B). The results represent a ratio of fluorescence intensities for cells treated and untreated by a-LA sample. bLAeOA-45/HAMLET was added 40 min after the dyes.

4. Conclusions The use of extreme alkaline solution for preparation of the biologically active bLA e OA complex enables us to achieve both aLA unfolding and higher water solubility of OA. The latter factor facilitates saturation of bLA with OA. The bLA e OA complex was formed under optimized conditions ensuring maximal OA binding capacity of bLA. Notably, the OA concentration was kept below its effective CMC value, which ensures the absence of extended phases of OA and the absence of sample heterogeneity related to them. Furthermore, the sample was subjected to removal of the unbound OA via solution acidification, centrifugation and dialysis. The resulting complex, bLAeOA-45, contains about 11 molecules of OA per bLA molecule, and possesses secondary structure, thermal stability and calcium affinity closely resembling those of the BAMLET state. The cytotoxic activities of bLAeOA-45 with respect to HEp-2 and S. pneumoniae D39 cells were shown to be equivalent to those for HAMLET. Importantly, the initial stages of the cytotoxic effect of bLAeOA-45 against S. pneumoniae cells are very similar to those for HAMLET: depolarization of the bacterial membrane followed by its rupture. Both effects are dependent upon Ca2þ

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transport. Overall, the bLAeOA-45 sample is in many ways equivalent to HAMLET/BAMLET. The developed low-cost express method for preparation of the HAMLET-like complex lacks the scalability limitation of the previous method and is well suited for preparation under regular laboratory conditions of quantities of the active complex necessary to conduct animal testing and pre-clinical studies. The shown crucial role of hydrophobic effect in stabilization of the a-LA e OA complex opens up the possibility to prepare similar complexes of alkaline-denatured proteins with fatty acids and other low-molecular-weight amphiphilic substances, possessing cytotoxic properties with respect to malignant cells. The possibility of dissolving cytotoxic hydrophobic substances in the non-polar interior of natural proteins opens up a new avenue for cell delivery of these substances. Further efforts aimed at development of the complexes of this kind are required. Conflict of interests We confirm that there is no conflict of interest in this manuscript.

[11]

[12] [13]

[14]

[15]

[16]

[17]

[18]

Author contributions [19]

Sergei E. Permyakov was involved in experiment design, data treatment and analysis, and manuscript preparation. Ekaterina L. Knyazeva was involved in data collection, data treatment and analysis, manuscript preparation. Marina V. Leonteva, Roman S. Fadeev, Aleksei V. Chekanov and Andrei P. Zhadan were involved in data collection, data treatment and analysis. Anders P. Håkansson was involved in experiment design, data collection, data treatment and analysis, and manuscript preparation. Vladimir S. Akatov was involved in experiment design. Eugene A. Permyakov was involved in experiment design, and manuscript preparation.

[20] [21]

[22]

[23]

[24]

Acknowledgements This work was supported by grant to P.E.A. from the Program of the Russian Academy of Sciences «Molecular and Cellular Biology», grant to K.E.L. from the Dynasty Foundation, and grants to A.P.H. from the Bill and Melinda Gates Foundation (Grant 53085), the JR Oishei Foundation, and The American Lung Association (Grant RG123721-N).

[25]

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