Triacylglycerol mimetics regulate membrane interactions of glycogen branching enzyme: implications for therapy
2017; Elsevier BV; Volume: 58; Issue: 8 Linguagem: Inglês
10.1194/jlr.m075531
ISSN1539-7262
AutoresRafael Álvarez, Jesús Casas, David J. López, Maitane Ibarguren, Ariadna Suari-Rivera, Silvia Terés, Francisca Guardiola-Serrano, Alexander Lossos, Xavier Busquets, Or Kakhlon, Pablo V. Escribá,
Tópico(s)Lysosomal Storage Disorders Research
ResumoAdult polyglucosan body disease (APBD) is a neurological disorder characterized by adult-onset neurogenic bladder, spasticity, weakness, and sensory loss. The disease is caused by aberrant glycogen branching enzyme (GBE) (GBE1Y329S) yielding less branched, globular, and soluble glycogen, which tends to aggregate. We explore here whether, despite being a soluble enzyme, GBE1 activity is regulated by protein-membrane interactions. Because soluble proteins can contact a wide variety of cell membranes, we investigated the interactions of purified WT and GBE1Y329S proteins with different types of model membranes (liposomes). Interestingly, both triheptanoin and some triacylglycerol mimetics (TGMs) we have designed (TGM0 and TGM5) markedly enhance GBE1Y329S activity, possibly enough for reversing APBD symptoms. We show that the GBE1Y329S mutation exposes a hydrophobic amino acid stretch, which can either stabilize and enhance or alternatively, reduce the enzyme activity via alteration of protein-membrane interactions. Additionally, we found that WT, but not Y329S, GBE1 activity is modulated by Ca2+ and phosphatidylserine, probably associated with GBE1-mediated regulation of energy consumption and storage. The thermal stabilization and increase in GBE1Y329S activity induced by TGM5 and its omega-3 oil structure suggest that this molecule has a considerable therapeutic potential for treating APBD. Adult polyglucosan body disease (APBD) is a neurological disorder characterized by adult-onset neurogenic bladder, spasticity, weakness, and sensory loss. The disease is caused by aberrant glycogen branching enzyme (GBE) (GBE1Y329S) yielding less branched, globular, and soluble glycogen, which tends to aggregate. We explore here whether, despite being a soluble enzyme, GBE1 activity is regulated by protein-membrane interactions. Because soluble proteins can contact a wide variety of cell membranes, we investigated the interactions of purified WT and GBE1Y329S proteins with different types of model membranes (liposomes). Interestingly, both triheptanoin and some triacylglycerol mimetics (TGMs) we have designed (TGM0 and TGM5) markedly enhance GBE1Y329S activity, possibly enough for reversing APBD symptoms. We show that the GBE1Y329S mutation exposes a hydrophobic amino acid stretch, which can either stabilize and enhance or alternatively, reduce the enzyme activity via alteration of protein-membrane interactions. Additionally, we found that WT, but not Y329S, GBE1 activity is modulated by Ca2+ and phosphatidylserine, probably associated with GBE1-mediated regulation of energy consumption and storage. The thermal stabilization and increase in GBE1Y329S activity induced by TGM5 and its omega-3 oil structure suggest that this molecule has a considerable therapeutic potential for treating APBD. The glycogen branching enzyme (GBE)1 (EC 2.4.1.18) catalyzes two reactions. In amylase-type hydrolysis, GBE1 cleaves an α-1,4-linked segment of six or more glucose units, every 8–14 glucose residues from the nonreducing end of a glucan chain. In the transglucosylation reaction, it transfers the cleaved oligosaccharide, via an α-1,6-glucosidic linkage, to the C6 hydroxyl group of a glucose acceptor unit within the same chain (intramolecular) or onto a different neighboring chain [intermolecular (1.Froese D.S. Michaeli A. McCorvie T.J. Krojer T. Sasi M. Melaev E. Goldblum A. Zatsepin M. Lossos A. Alvarez R. et al.Structural basis of glycogen branching enzyme deficiency and pharmacologic rescue by rational peptide design.Hum. Mol. Genet. 2015; 24: 5667-5676Crossref PubMed Scopus (39) Google Scholar)]. The resulting glycogen is a highly branched molecule whose spherical and soluble structure favors its cytoplasmic stability and metabolism (2.Meléndez R. Meléndez-Hevia E. Canela E.I. The fractal structure of glycogen: a clever solution to optimize cell metabolism.Biophys. J. 1999; 77: 1327-1332Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). A mutant GBE1 enzyme may affect the structure of glycogen, reducing its cytoplasmic solubility and hindering its bio-availability due to the formation of aggregates called polyglucosans. Different mutations partially or completely inactivate GBE1, and the severity of the pathophysiological symptoms can vary as a function of the mutation (3.Moses S.W. Parvari R. The variable presentations of glycogen storage disease type IV: a review of clinical, enzymatic and molecular studies.Curr. Mol. Med. 2002; 2: 177-188Crossref PubMed Scopus (142) Google Scholar, 6.Dainese L. Monin M.L. Demeret S. Brochier G. Froissart R. Spraul A. Schiffmann R. Seilhean D. Mochel F. Abnormal glycogen in astrocytes is sufficient to cause adult polyglucosan body disease.Gene. 2013; 515: 376-379Crossref PubMed Scopus (15) Google Scholar). Adult polyglucosan body disease (APBD) is a rare neurodegenerative disease that most often affects adults from the Ashkenazi Jewish community afflicted by the substitution of tyrosine-329 by a serine residue, which impairs GBE1 activity. Inactivation of GBE1 and the activation of glycogen synthase (GYS) leads to the generation of polyglucosan bodies (amylopectin-like polysaccharides with fewer branch points), which are harmful to neurons and glial cells. These aggregates may cause different phenotypic alterations, such as neurogenic bladder, partial motor dysfunction in the extremities, sensorial dysfunction in the lower part of the body, and possibly cognitive impairment. The advanced state of the disease is characterized by difficulty in walking, impaired balance, and progressive weakness, sometimes accompanied by early death (4.Lossos A. Meiner Z. Barash V. Soffer D. Schlesinger I. Abramsky O. Argov Z. Shpitzen S. Meiner V. Adult polyglucosan body disease in Ashkenazi Jewish patients carrying the Tyr329Ser mutation in the glycogen-branching enzyme gene.Ann. Neurol. 1998; 44: 867-872Crossref PubMed Scopus (97) Google Scholar, 5.Hussain A. Armistead J. Gushulak L. Kruck C. Pind S. Triggs-Raine B. Natowicz M.R. The adult polyglucosan body disease mutation GBE1 c.1076A>C occurs at high frequency in persons of Ashkenazi Jewish background.Biochem. Biophys. Res. Commun. 2012; 426: 286-288Crossref PubMed Scopus (7) Google Scholar). Triheptanoin (TH; a triacylglycerol with three heptanoic acid moieties) is currently under investigation as a possible treatment for APBD [ClinicalTrials.gov identifier NCT00947960 (7.Roe C.R. Bottiglieri T. Wallace M. Arning E. Martin A. Adult polyglucosan body disease (APBD): anaplerotic diet therapy (triheptanoin) and demonstration of defective methylation pathways.Mol. Genet. Metab. 2010; 101: 246-252Crossref PubMed Scopus (36) Google Scholar, 8.Roe C.R. Sweetman L. Roe D.S. David F. Brunengraber H. Treatment of cardiomyopathy and rhabdomyolysis in long-chain fat oxidation disorders using an anaplerotic odd-chain triglyceride.J. Clin. Invest. 2002; 110: 259-269Crossref PubMed Scopus (225) Google Scholar)]. However, little is known about the molecular events underlying the effects of TH and its possible clinical efficacy is still unclear. Thus, this drug remains far from achieving marketing approval from the major regulatory agencies (e.g., European Medicines Agency, Food and Drug Administration, etc.). Most processes in the cell occur in, at, or around membranes (9.Escribá P.V. González-Ros J.M. Goñi F.M. Kinnunen P.K. Vigh L. Sánchez-Magraner L. Fernández A.M. Busquets X. Horváth I. Barceló-Coblijn G. Membranes: a meeting point for lipids, proteins and therapies.J. Cell. Mol. Med. 2008; 12: 829-875Crossref PubMed Scopus (279) Google Scholar). The high density and the variety of cell membranes in the cytoplasm, with different compositions and physicochemical properties [i.e.: the plasma membrane; the mitochondrial, nuclear, lysosomal, endosomal, peroxisomal, Golgi, and vesicular trafficking membranes; the endoplasmic reticulum (ER); and other types of vesicular and organellar membranes in different cell types] favors the interactions of soluble proteins with different lipid bilayers. Because the p.Y329S GBE1 mutation causes partial misfolding that may expose internal hydrophobic regions of the enzyme to the aqueous milieu, the activity of this enzyme may be modulated by GBE1-membrane interactions. Such interactions would be influenced by changes in membrane lipid composition (1.Froese D.S. Michaeli A. McCorvie T.J. Krojer T. Sasi M. Melaev E. Goldblum A. Zatsepin M. Lossos A. Alvarez R. et al.Structural basis of glycogen branching enzyme deficiency and pharmacologic rescue by rational peptide design.Hum. Mol. Genet. 2015; 24: 5667-5676Crossref PubMed Scopus (39) Google Scholar). Indeed, the therapeutic effects of the lipid TH suggest that membrane lipids may play a relevant role in the etiology, pathophysiology, and therapy of APBD. As such, the present study was designed to investigate the interactions of WT and p.Y329S-mutated GBE1 with model membranes, so as to study the molecular bases underlying APBD. In addition, we set out to design and investigate molecules that could regulate this interaction, enhancing GBE1 activity, in the hope of defining possible therapeutic tools for the treatment of this condition. The pFastBac 1 plasmid and SYPRO-Orange were purchased from Invitrogen (Barcelona, Spain), EcoRI and NotI were obtained from Fermentas (Madrid, Spain), while shrimp alkaline phosphatase was obtained from USB Corporation (Staufen, Germany). Agarose D-1 was obtained from Conda Laboratories (Barcelona, Spain) and Grace's medium was from GIBCO (Madrid, Spain). Penicillin and streptomycin were purchased from PAA (Pasching, Austria) and β-mercaptoethanol from Acros Organics (Madrid, Spain). Purified commercial GBE1 protein and the anti-GBE1 antibody were supplied by OriGene Technologies, Inc. (Rockville, MD). Due to the altered membrane binding and the potential loss of activity of this commercial GBE1 (supplemental Fig. S1), we produced our own WT and GBE1Y329S enzymes (see below). The α-D-[14C(U)]glucose-1-phosphate dipotassium salt was purchased from PerkinElmer (Waltham, MA) and α-D-glucose-1-phosphate was purchased from Alfa Aesar (Karlsruhe, Germany). IRDye 800CW-linked donkey anti-mouse IgG was provided by Li-Cor Biosciences (Madrid, Spain), while egg phosphatidylcholine (PC), liver phosphatidylethanolamine (PE), egg SM, brain phosphatidylserine (PS), brain phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], and cholesterol (Cho) were all obtained from Avanti Polar Lipids (Alabaster, AL). DTT, HEPES, phosphorylase-a, Tris-HCl, proteinase inhibitors, and all other reagents were purchased from Sigma-Aldrich (Madrid, Spain). TH and the triacylglycerol mimetics (TGMs) were produced by BEGA Pharmaceuticals (Palma de Mallorca, Spain). The TGMs used here are triacylglycerols with three identical hydroxylated fatty acid molecules, such that: TGM0 is the hydroxylated analog of TH and it contains three 2-hydroxy-heptanoic acid moieties; TGM1 has three 2-hydroxy-oleic acid moieties; TGM2 has 2-hydroxy-linoleic acid moieties; TGM3A has 2-hydroxy-α-linolenic acid moieties; TGM3G has 2-hydroxy-γ-linolenic acid moieties; TGM4 has 2-hydroxy-arachidonic acid; TGM5 has 2-hydroxy-eicosapentaenoic acid moieties; and finally, TGM6 has 2-hydroxy-docosahexaenoic acid moieties. The number in the TGM abbreviation corresponds to the number of double bonds in the fatty acid molecule. The cDNA encoding the recombinant human GBE1 protein (GenBank accession number BC012098) was kindly provided in the pOTB7 expression vector (1.8 kb) by Dr. Hasan O. Akman (Columbia University, NY). This cDNA was subcloned into the pFastBac-1 vector, adding a C-terminal 6xHis-tag by PCR amplification with the Pfu DNA polymerase (Thermo Scientific, Schwerte, Germany) and using primers with the appropriate sequence (Fig. 1, Table 1). After an initial incubation at 94°C for 5 min, the PCR mixture was subjected to 35 cycles (denaturation at 94°C for 45 s, annealing at 65°C for 45 s, and elongation at 72°C for 2 min), followed by a final 10 min elongation step at 72°C. Human GBE1Y329S (Fig. 1) was generated from the recombinant pFastBac-1 plasmid by PCR amplification using primers that contained the selected mutation (Table 1). As such, overlap-extension PCR was carried out in two consecutive reactions using a high-fidelity DNA polymerase (Exact Polymerase; 5 Prime Co., Hilden, Germany). In the first reaction, an initial 2 min incubation at 94°C was followed by 32 thermal cycles (denaturation at 94°C for 45 s, annealing at 68°C for 45 s, and elongation at 72°C for 2 min) and a final 10 min elongation step at 72°C. In this first PCR, two independent reactions were set up, using the "GBE1 forward" and "internal reverse" primers in one of the reactions, and in the second reaction the "internal forward" and "GBE1 reverse" primers. The products of both of these reactions were combined for a second amplification process under the same conditions without the addition of further reagents. The amplicon resulting from this last reaction contained the modifications shown in Fig. 1. Subsequent reactions of enzymatic digestion and ligation, as well as bacterial transformation and purification of the resultant recombinant constructs were done as described elsewhere (10.Böttcher C.J.F. Van Gent C.M. Pries C. A rapid and sensitive sub-micro phosphorus determination.Anal. Chim. Acta. 1961; 24: 203-204Crossref Scopus (851) Google Scholar).TABLE 1PCR primers used for WT and GBE1Y329S amplificationGBE1 forward5′-ATCGAATTCATGGCGGCTCCGATGACTCCCGCGGCT-3′aEcoRI restriction enzyme site underlined.GBE1 reverse5′-CTGGCGGCCGCTCAATGATGATGATGATGATGATTCGGCAGATCCACATTCTGAAGGATGAG-3′bNotI restriction enzyme site underlined.Internal forward GBE1Y329S5′-GTTTGCCTCCTCCAGCTGGGAAGTTTTAAGATTCCTTCTGTCAAACATAA-3′Internal reverse GBE1Y329S5′-TTATGTTTGACAGAAGGAATCTTAAAACTTCCCAGCTGGAGGAGGCAAAC-3′a EcoRI restriction enzyme site underlined.b NotI restriction enzyme site underlined. Open table in a new tab Recombinant GBE1 proteins (WT and Y329S) were produced in Sf9 cells cultured in suspension in Grace's medium supplemented with 10% FCS (v/v), penicillin (100 units/ml), and streptomycin (100 μg/ml). The proteins were purified as described elsewhere with slight modifications (11.Álvarez R. López D.J. Casas J. Lladó V. Higuera M. Nagy T. Barceló M. Busquets X. Escribá P.V. G protein-membrane interactions I: galphai1 myristoyl and palmitoyl modifications in protein-lipid interactions and its implications in membrane microdomain localization.Biochim. Biophys. Acta. 2015; 1851: 1511-1520Crossref PubMed Scopus (20) Google Scholar). Briefly, the GBE1 proteins (Fig. 1) were overexpressed and purified from the cytosolic fraction of infected Sf9 cells after harvesting the cells by centrifugation and resuspending them in 6 ml of ice-cold 20 mM HEPES buffer (pH 8.0) containing β-mercaptoethanol (10 mM), NaCl (100 mM), and proteinase inhibitors (lysis buffer). A final wash of the nickel-nitrilotriacetic acid (Ni-NTA) affinity column was done with 12 ml of HEPES buffer (20 mM, pH 8.0), containing MgCl2 (0.5 mM), β-mercaptoethanol (10 mM), NaCl (100 mM), leupeptin (0.5 μg/ml), and imidazole (15 mM) at 30°C. Finally, the GBE1 proteins were eluted with HEPES buffer (20 mM, pH 8.0) containing β-mercaptoethanol (10 mM), NaCl (100 mM), and MgCl2 (1 mM, elution buffer) and supplemented with imidazole in a step gradient (20, 40, 80, 120, 240, and 300 mM). The purified protein was desalted, filtered, concentrated with Amicon Ultra-15 (30 kDa) and stored at −80°C. The WT protein produced had good activity, whereas the activity of GBE1Y329S was always less than that of the WT form. In all cases, the activity of GBE1 increased in the presence of PC membranes. Model membranes (liposomes) were prepared from 10 mM stock solutions of natural lipids (PC, PE, PS, Cho, and SM) and synthetic triacylglycerols by mixing the appropriate volumes of each in glass vials. The solvent (chloroform:methanol, 2:1, v/v) was then removed under argon and the lipid film was subjected to vacuum for 2 h to remove traces of solvent. Subsequently, the lipid films were resuspended by vigorous vortexing in binding buffer [20 mM HEPES buffer, 100 mM KCl, and 0.1 mM EDTA (pH 7.4)] to a final concentration of 3 mM (lipid phosphorus). These multilamellar vesicles were submitted to 10 freeze/thaw cycles (−196°C/42°C) and, subsequently, the different lipid emulsions were passed 11 times through a 400 nm pore polycarbonate membrane to generate large unilamellar vesicles (LUVs) using a mini-extruder (Avanti Polar Lipids). The final concentration of the liposomes was determined as described elsewhere (10.Böttcher C.J.F. Van Gent C.M. Pries C. A rapid and sensitive sub-micro phosphorus determination.Anal. Chim. Acta. 1961; 24: 203-204Crossref Scopus (851) Google Scholar). LUVs were prepared in the presence of calcium following an identical protocol except that 2 mM CaCl2 was added to the binding buffer. For activity assays, unilamellar vesicles of PC and PC:TGM (4:1) were prepared in activity buffer (20 mM MES buffer, pH 6.3) containing 54 mM glucose-1-phosphate and 2.2 mM AMP. Liposomes (1 mM) were incubated for 1 h at 25°C in the presence of 100 ng of purified GBE1 in a total volume of 300 μl binding buffer. Membrane-bound GBE1 was then differentiated from unbound GBE1 by centrifugation at 90,000 g for 1 h at 25°C. Finally, the membrane pellets were resuspended in 36 μl of 80 mM Tris-HCl buffer (pH 6.8) containing 4% SDS, and combined with 4 μl of 10× electrophoresis loading buffer [120 mM Tris-HCl (pH 6.8), 1.43 M β-mercaptoethanol, 2% SDS, and 50% glycerol]. In binding assays with calcium, 2 mM CaCl2 was added to the binding buffer. Binding of WT and mutant GBE1 proteins to model membranes was quantified as described elsewhere (11.Álvarez R. López D.J. Casas J. Lladó V. Higuera M. Nagy T. Barceló M. Busquets X. Escribá P.V. G protein-membrane interactions I: galphai1 myristoyl and palmitoyl modifications in protein-lipid interactions and its implications in membrane microdomain localization.Biochim. Biophys. Acta. 2015; 1851: 1511-1520Crossref PubMed Scopus (20) Google Scholar). Briefly, proteins present in the membrane pellets were fractionated on 9% polyacrylamide gels and then transferred to nitrocellulose membranes. The membranes were then incubated for 1 h at room temperature with gentle rocking in blocking solution containing PBS and 5% (w/v) nonfat dry milk. After blocking, the membranes were probed overnight at 4°C with anti-GBE1 (diluted 1:750 in fresh blocking solution containing 0.1% Tween 20), washed with PBS, and then incubated at room temperature for 1 h with IRDye 800CW-linked donkey anti-mouse IgG (diluted 1:4,000 in blocking solution containing 0.1% Tween 20 and 0.01% SDS). Antibody binding was detected by near infrared fluorescence using an Odyssey near infrared radiation detection system (LI-COR Biosciences). GBE1 protein was quantified using standard curves [i.e., a plot of the GBE1 protein loaded against the integrated optical density (IOD) of the immunoreactive bands], consisting of four points of different protein content prepared from a commercial GBE1 protein batch (OriGene). This commercial GBE1 protein was used only for quantification purposes. A linear relationship between the amount of protein loaded onto the gel and the IOD was found over the entire range of protein used. The amount of GBE1 protein in the experimental samples was obtained by interpolation of the IOD value from these samples in the standard curve. Thus, the amount of GBE1 protein bound to LUVs was expressed as the proportion of protein in the pellet fraction with respect to the total quantity of GBE1 initially used in the binding experiment (100 ng). Model membrane suspensions (34 μl containing 6 mM total lipid) and 4 μl of GBE1 (10 ng) were incubated for 1 h at 25°C with shaking at 300 rpm. Enzyme activity was then analyzed at 37°C by determining the glucose-1-phosphate incorporation into glycogen in the absence of the exogenous glycogen primer, as described elsewhere (12.Bruno C. Servidei S. Shanske S. Karpati G. Carpenter S. McKee D. Barohn R.J. Hirano M. Rifai Z. DiMauro S. Glycogen branching enzyme deficiency in adult polyglucosan body disease.Ann. Neurol. 1993; 33: 88-93Crossref PubMed Scopus (94) Google Scholar). Briefly, 139 μl of activity buffer, 2 μl of [14C]glucose-1-P (0.02 mCi/ml), and 25 μl of boiled rat muscle 10% homogenate were added to each sample, and a 50 μl aliquot of the reaction mixture was analyzed as the baseline (time point 0). Subsequently, 5 μl of rabbit muscle phosphorylase-a (22 μg/μl) were added to the reaction medium and 50 μl aliquots were taken after 30 and 60 min. In assays with calcium, 2 mM CaCl2 were added to the activity buffer. All these samples were then spotted onto filter paper, dried for 3 min, and washed twice with 66% ethanol and once with acetone for 5 min. Finally, the filter papers were dried and 4 ml of scintillation liquid were added to the filter paper in each bottle. The radioactivity was measured by a scintillation counter, measuring the total radioactivity in nonboiled samples and the nonspecific incorporation in preboiled samples. Specific activity was determined as the difference between the total and nonspecific radioactivity. Melting temperatures (Tms) for WT and GBE1Y329S proteins were determined in strip-tubes using a StepOnePlus RT-PCR thermal cycler (Life Technologies Corporation). Each sample (20 μl) consisted of purified GBE1 protein (0.5 μM) in 20 mM HEPES (pH 8.0), 100 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, and 5× SYPRO-Orange (1:1,000 dilution from the original stock). The effects of PC membranes (LUVs) were analyzed in the presence or absence of 20 mol% of the different triacylglycerols, TH, TGM0, or TGM5. A concentration of 12.5 μM of total lipid was used in these assays with model membranes. Fluorescence intensities were measured from 25°C to 95°C with a ramp rate of 1°C/min. The Tm was determined by plotting intensity as a function of temperature and fitting the curve to the Boltzman equation (13.Niesen F.H. Berglund H. Vedadi M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability.Nat. Protoc. 2007; 2: 2212-2221Crossref PubMed Scopus (1702) Google Scholar, 14.Froese D.S. Healy S. McDonald M. Kochan G. Oppermann U. Niesen F.H. Gravel R.A. Thermolability of mutant MMACHC protein in the vitamin B12-responsive cblC disorder.Mol. Genet. Metab. 2010; 100: 29-36Crossref PubMed Scopus (45) Google Scholar). Relative fluorescence (RF) was calculated considering the relative increases of fluorescence at each temperature in the following way: RFi = 1 − [(Fmax − Fi)/(Fmax − Fmin)], where Fi is the fluorescence detected at the temperature considered i, Fmax is the maximum fluorescence intensity detected, and Fmin is the minimum fluorescence intensity in the temperature range of increasing emission intensities. The raw data were analyzed using the DSF Analysis 3.0 software (ftp://ftp.sgc.ox.ac.uk/pub/biophysics). The thermal behavior of the model membranes was studied by differential scanning calorimetry (DSC) on a TA Instruments (New Castle, DE) 2920 calorimeter. Briefly, 15 mg 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) were mixed with 1 mol% of each of the different natural and synthetic triacylglycerides indicated previously, each of which was dissolved in chloroform:methanol (2:1, by volume). The lipid mixtures were dried under an argon flux and solvent traces were removed under vacuum for at least 3 h at room temperature before hydration. Multilamellar lipid vesicles were formed by resuspending the lipid film in 10 mM HEPES, 100 mM KCl, and 1 mM EDTA (pH 7.4), following eight freeze/thaw cycles (−196°C/40°C). The mixture was loaded in aluminum hermetic pans and it was subjected to five consecutive scans between −10°C and 80°C at a scan rate of 1°C or 2°C/min. The transition enthalpy and transition temperature values shown here corresponded to the means of all the scans measured and they were obtained using the software provided by the manufacturer (TA Universal Analysis). The effect of TH and of TGMs 0, 1, and 5 on GBE1 activity was tested in peripheral blood mononuclear cells (PBMCs) collected from a healthy donor and APBD patients homozygous for the p.Y329S mutation, and isolated by centrifugation on Ficoll density gradient, as described previously (4.Lossos A. Meiner Z. Barash V. Soffer D. Schlesinger I. Abramsky O. Argov Z. Shpitzen S. Meiner V. Adult polyglucosan body disease in Ashkenazi Jewish patients carrying the Tyr329Ser mutation in the glycogen-branching enzyme gene.Ann. Neurol. 1998; 44: 867-872Crossref PubMed Scopus (97) Google Scholar). Briefly, whole blood was diluted 1:1 with PBS, added to the top of a Ficoll gradient (Axis-Shield, Oslo, Norway) and centrifuged at 800 g for 20 min at room temperature. The interphase between the plasma and Ficoll-erythrocyte fractions containing the PBMCs was collected, washed three times with PBS, and the cells were suspended at 2 × 106 cells/ml in PBS supplemented with 2% FBS. TH and the TGMs (300 μM) were then added to the PBMCs, shaken overnight at 37°C, harvested, and assayed for GBE1 activity, as described previously (12.Bruno C. Servidei S. Shanske S. Karpati G. Carpenter S. McKee D. Barohn R.J. Hirano M. Rifai Z. DiMauro S. Glycogen branching enzyme deficiency in adult polyglucosan body disease.Ann. Neurol. 1993; 33: 88-93Crossref PubMed Scopus (94) Google Scholar). This protocol was approved by the Hadassah-Hebrew University Medical Center Institutional Review Board according to The Code of Ethics of the World Medical Association (Declaration of Helsinki). Hydrophobicity profiles were generated using the Kyte-Doolittle method [available at the website http://web.expasy.org/protscale (15.Kyte J. Doolittle R.F. A simple method for displaying the hydropathic character of a protein.J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17169) Google Scholar)], using an amino acid window size of 19 and a relative weight for window edges of 100%. Exposure of amino acids on the surface was calculated considering the molar fraction (percent) of 3,220 accessible residues (16.Janin J. Surface and inside volumes in globular proteins.Nature. 1979; 277: 491-492Crossref PubMed Scopus (597) Google Scholar), with a window size of 19 and a relative weight for window edges of 100%. The 3D structure of GBE1 was generated using the coordinates from the Molecular Modeling Database (identifier #131789) and the Protein Data Bank [identifier #4BZY; (1.Froese D.S. Michaeli A. McCorvie T.J. Krojer T. Sasi M. Melaev E. Goldblum A. Zatsepin M. Lossos A. Alvarez R. et al.Structural basis of glycogen branching enzyme deficiency and pharmacologic rescue by rational peptide design.Hum. Mol. Genet. 2015; 24: 5667-5676Crossref PubMed Scopus (39) Google Scholar)]. Certain amino acids were highlighted using the website application. Origin software was used for the data and statistical analysis. Unless otherwise indicated, the results are expressed as the mean ± SEM from the number of experiments indicated (n). To determine statistical significance, an ANOVA or an unpaired two-sample t-test was used when appropriate. Differences were considered statistically significant at P < 0.05. Recombinant human WT GBE1 protein and its mutant counterpart (GBE1Y329S) were purified by affinity chromatography (Fig. 2) and their interactions with the most representative membrane lipids were analyzed. There is considerable evidence that the p.Y329S mutation introduces important structural changes in GBE1 (1.Froese D.S. Michaeli A. McCorvie T.J. Krojer T. Sasi M. Melaev E. Goldblum A. Zatsepin M. Lossos A. Alvarez R. et al.Structural basis of glycogen branching enzyme deficiency and pharmacologic rescue by rational peptide design.Hum. Mol. Genet. 2015; 24: 5667-5676Crossref PubMed Scopus (39) Google Scholar) and, indeed, this mutant protein had a different Ni-NTA affinity elution profile (Fig. 2). In addition, more mutant GBE1Y329S bound to LUVs (Fig. 3), suggesting that the internal hydrophobic region flanking Y329S could be exposed at the protein's surface and become involved in membrane lipid interactions, along with other domains. In fact, the p.Y329S mutation drastically altered the binding of GBE1 to PC, PC:PE, PC:PS, PC:PE:Cho:SM, and PC:PE:PS:Cho:SM membranes (Fig. 3, supplemental Fig. S1).Fig. 3WT GBE1 and GBE1Y329S binding to model membranes. Model membranes of PC, PC:PE (2:3 molar ratio), PC:PS (3:2), PC:PE:PS (3:4:3), PC:PE:Cho:SM (1:1:1:1), and PC:PE:PS:Cho:SM (2.3:2:2.6:2.3:0.8) were used. The bars indicate the proportion (mean ± SEM) of WT and GBE1Y329S protein bound to the model membranes resembling different membrane types or membrane microdomains. Representative immunoblots of each binding experiment are shown in the same order as in the graph. The data represent the mean ± SEM of from three to four independent experiments. *P < 0.05; **P < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT) TH is currently undergoing clinical trials to investigate its efficacy to treat APBD and other conditions (ClinicalTrials.gov identifiers #NCT00947960 and #NCT01993186). For this reason, we designed and synthesized a number of TH analogs (TGMs) in order to obtain potentially improved therapeutic tools. We then investigated the effects of TH and the aforementioned TGMs on the membrane interactions and activity of GBE1 (WT and Y329S). TH and its closest analog, TGM0, were seen to induce a significant concentration-dependent reduction in
Referência(s)