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• W2898073575 abstract "Lipoprotein glomerulopathy (LPG) is a rare renal disease, characterized by lipoprotein thrombi in glomerular capillaries. A series of apoE mutations have been associated with LPG development. We previously showed that three mutants based on apoE3 sequence, in which an arginine was substituted by proline, are thermodynamically destabilized and aggregation-prone. To examine whether other LPG-associated apoE3 mutations induce similar effects, we characterized three nonproline LPG-associated apoE3 mutations, namely, R25C (apoEKyoto), R114C (apoETsukuba), and A152D (apoELasVegas). All three apoE3 variants are found to have significantly reduced helical content and to be thermodynamically destabilized, both in lipid-free and lipoprotein-associated form, and to expose a larger portion of hydrophobic surface to the solvent compared with WT apoE3. Furthermore, all three apoE3 variants are aggregation-prone, as shown by dynamic light-scattering measurements and by their enhanced capacity to bind the amyloid probe thioflavin T. Overall, our data suggest that the LPG-associated apoE3 mutations R25C, R114C, and A152D induce protein misfolding, which may contribute to protein aggregation in glomerular capillaries. The similar effects of both LPG-associated proline and nonproline mutations on apoE3 structure suggest that the thermodynamic destabilization and enhanced aggregation of apoE3 may constitute a common underlying mechanism behind the pathogenesis of LPG. Lipoprotein glomerulopathy (LPG) is a rare renal disease, characterized by lipoprotein thrombi in glomerular capillaries. A series of apoE mutations have been associated with LPG development. We previously showed that three mutants based on apoE3 sequence, in which an arginine was substituted by proline, are thermodynamically destabilized and aggregation-prone. To examine whether other LPG-associated apoE3 mutations induce similar effects, we characterized three nonproline LPG-associated apoE3 mutations, namely, R25C (apoEKyoto), R114C (apoETsukuba), and A152D (apoELasVegas). All three apoE3 variants are found to have significantly reduced helical content and to be thermodynamically destabilized, both in lipid-free and lipoprotein-associated form, and to expose a larger portion of hydrophobic surface to the solvent compared with WT apoE3. Furthermore, all three apoE3 variants are aggregation-prone, as shown by dynamic light-scattering measurements and by their enhanced capacity to bind the amyloid probe thioflavin T. Overall, our data suggest that the LPG-associated apoE3 mutations R25C, R114C, and A152D induce protein misfolding, which may contribute to protein aggregation in glomerular capillaries. The similar effects of both LPG-associated proline and nonproline mutations on apoE3 structure suggest that the thermodynamic destabilization and enhanced aggregation of apoE3 may constitute a common underlying mechanism behind the pathogenesis of LPG. Lipoprotein glomerulopathy (LPG) is a rare renal disease, characterized by distinctive lipoprotein thrombi in the glomerular capillaries, often abnormal lipid profile, proteinuria, and progressive kidney failure (1.Saito T. Matsunaga A. Ito K. Nakashima H. Topics in lipoprotein glomerulopathy: an overview.Clin. Exp. Nephrol. 2014; 18: 214-217Crossref PubMed Scopus (16) Google Scholar, 2.Tsimihodimos V. Elisaf M. Lipoprotein glomerulopathy.Curr. Opin. Lipidol. 2011; 22: 262-269Crossref PubMed Scopus (23) Google Scholar). The relapse of LPG following kidney transplantation in LPG patients (3.Mourad G. Cristol J.P. Turc-Baron C. Djamali A. Lipoprotein glomerulopathy: a new apolipoprotein-E-related disease that recurs after renal transplantation.Transplant. Proc. 1997; 29: 2376Crossref PubMed Scopus (11) Google Scholar, 4.Miyata T. Sugiyama S. Nangaku M. Suzuki D. Uragami K. Inagi R. Sakai H. Kurokawa K. Apolipoprotein E2/E5 variants in lipoprotein glomerulopathy recurred in transplanted kidney.J. Am. Soc. Nephrol. 1999; 10: 1590-1595Crossref PubMed Google Scholar) has suggested that factors outside the kidney should be crucial for the disease pathogenesis. One such factor has been proposed to be mutated apoE, because LPG has been related with inherited mutations within the apoE gene that act in a dominant way but with incomplete penetrance (1.Saito T. Matsunaga A. Ito K. Nakashima H. Topics in lipoprotein glomerulopathy: an overview.Clin. Exp. Nephrol. 2014; 18: 214-217Crossref PubMed Scopus (16) Google Scholar, 2.Tsimihodimos V. Elisaf M. Lipoprotein glomerulopathy.Curr. Opin. Lipidol. 2011; 22: 262-269Crossref PubMed Scopus (23) Google Scholar, 5.Matsunaga A. Saito T. Apolipoprotein E mutations: a comparison between lipoprotein glomerulopathy and type III hyperlipoproteinemia.Clin. Exp. Nephrol. 2014; 18: 220-224Crossref PubMed Scopus (18) Google Scholar, 6.Stratikos E. Chroni A. A possible structural basis behind the pathogenic role of apolipoprotein E hereditary mutations associated with lipoprotein glomerulopathy.Clin. Exp. Nephrol. 2014; 18: 225-229Crossref PubMed Scopus (5) Google Scholar). ApoE is a major protein component of the lipoprotein transport system and plays critical roles in dyslipidemia and atherosclerosis (7.Zannis V.I. Kypreos K.E. Chroni A. Kardassis D. Zanni E.E. Lipoproteins and atherogenesis.Molecular Mechanisms of Atherosclerosis. 2004; : 111-174Google Scholar, 8.Huang Y. Mahley R.W. Apolipoprotein E: structure and function in lipid metabolism, neurobiology, and Alzheimer's diseases.Neurobiol. Dis. 2014; 72: 3-12Crossref PubMed Scopus (370) Google Scholar). Human apoE has three common isoforms, the apoE2, apoE3, and apoE4, each differing in the amino acid positions 112 and 158 (9.Zannis V.I. Breslow J.L. Human very low density lipoprotein apolipoprotein E isoprotein polymorphism is explained by genetic variation and posttranslational modification.Biochemistry. 1981; 20: 1033-1041Crossref PubMed Scopus (384) Google Scholar). The polymorphic background and mutations in the apoE gene have been linked with the pathogenesis of several diseases related to lipid metabolism, such as type III hyperlipoproteinemia, atherosclerosis, diabetic dyslipidemia, and LPG, as well as of neurodegenerative disorders, such as Alzheimer's disease (5.Matsunaga A. Saito T. Apolipoprotein E mutations: a comparison between lipoprotein glomerulopathy and type III hyperlipoproteinemia.Clin. Exp. Nephrol. 2014; 18: 220-224Crossref PubMed Scopus (18) Google Scholar, 6.Stratikos E. Chroni A. A possible structural basis behind the pathogenic role of apolipoprotein E hereditary mutations associated with lipoprotein glomerulopathy.Clin. Exp. Nephrol. 2014; 18: 225-229Crossref PubMed Scopus (5) Google Scholar, 8.Huang Y. Mahley R.W. Apolipoprotein E: structure and function in lipid metabolism, neurobiology, and Alzheimer's diseases.Neurobiol. Dis. 2014; 72: 3-12Crossref PubMed Scopus (370) Google Scholar, 10.Mahley R.W. Weisgraber K.H. Huang Y. Apolipoprotein E: structure determines function, from atherosclerosis to Alzheimer's disease to AIDS.J. Lipid Res. 2009; 50: S183-S188Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 11.Johnson L.A. Arbones-Mainar J.M. Fox R.G. Pendse A.A. Altenburg M.K. Kim H.S. Maeda N. Apolipoprotein E4 exaggerates diabetic dyslipidemia and atherosclerosis in mice lacking the LDL receptor.Diabetes. 2011; 60: 2285-2294Crossref PubMed Scopus (24) Google Scholar). ApoE is highly helical with labile tertiary structure (12.Morrow J.A. Hatters D.M. Lu B. Hochtl P. Oberg K.A. Rupp B. Weisgraber K.H. Apolipoprotein E4 forms a molten globule. A potential basis for its association with disease.J. Biol. Chem. 2002; 277: 50380-50385Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar) that can undergo significant conformational changes during its physiological functions that include lipid binding, protein-protein interactions, and other processes (10.Mahley R.W. Weisgraber K.H. Huang Y. Apolipoprotein E: structure determines function, from atherosclerosis to Alzheimer's disease to AIDS.J. Lipid Res. 2009; 50: S183-S188Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 13.Hatters D.M. Peters-Libeu C.A. Weisgraber K.H. Apolipoprotein E structure: insights into function.Trends Biochem. Sci. 2006; 31: 445-454Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 14.Chen J. Li Q. Wang J. Topology of human apolipoprotein E3 uniquely regulates its diverse biological functions.Proc. Natl. Acad. Sci. USA. 2011; 108: 14813-14818Crossref PubMed Scopus (169) Google Scholar, 15.Frieden C. Wang H. Ho C.M.W. A mechanism for lipid binding to apoE and the role of intrinsically disordered regions coupled to domain-domain interactions.Proc. Natl. Acad. Sci. USA. 2017; 114: 6292-6297Crossref PubMed Scopus (51) Google Scholar, 16.Chetty P.S. Mayne L. Lund-Katz S. Englander S.W. Phillips M.C. Helical structure, stability, and dynamics in human apolipoprotein E3 and E4 by hydrogen exchange and mass spectrometry.Proc. Natl. Acad. Sci. USA. 2017; 114: 968-973Crossref PubMed Scopus (26) Google Scholar). Lipid-free apoE is folded into two structural domains, an N-terminal and a carboxyl-terminal, connected with a hinge region (10.Mahley R.W. Weisgraber K.H. Huang Y. Apolipoprotein E: structure determines function, from atherosclerosis to Alzheimer's disease to AIDS.J. Lipid Res. 2009; 50: S183-S188Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 13.Hatters D.M. Peters-Libeu C.A. Weisgraber K.H. Apolipoprotein E structure: insights into function.Trends Biochem. Sci. 2006; 31: 445-454Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 14.Chen J. Li Q. Wang J. Topology of human apolipoprotein E3 uniquely regulates its diverse biological functions.Proc. Natl. Acad. Sci. USA. 2011; 108: 14813-14818Crossref PubMed Scopus (169) Google Scholar, 15.Frieden C. Wang H. Ho C.M.W. A mechanism for lipid binding to apoE and the role of intrinsically disordered regions coupled to domain-domain interactions.Proc. Natl. Acad. Sci. USA. 2017; 114: 6292-6297Crossref PubMed Scopus (51) Google Scholar, 16.Chetty P.S. Mayne L. Lund-Katz S. Englander S.W. Phillips M.C. Helical structure, stability, and dynamics in human apolipoprotein E3 and E4 by hydrogen exchange and mass spectrometry.Proc. Natl. Acad. Sci. USA. 2017; 114: 968-973Crossref PubMed Scopus (26) Google Scholar). Crystal structure analysis of the N-terminal domain of apoE showed that this domain folds as a four-helix bundle of amphipathic α-helices (17.Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Three-dimensional structure of the LDL receptor-binding domain of human apolipoprotein E.Science. 1991; 252: 1817-1822Crossref PubMed Scopus (599) Google Scholar). NMR analysis of full-length apoE also showed a four-helix bundle in the N-terminal domain, as well as two helices in the hinge region and three helices in the carboxyl-terminal domain (14.Chen J. Li Q. Wang J. Topology of human apolipoprotein E3 uniquely regulates its diverse biological functions.Proc. Natl. Acad. Sci. USA. 2011; 108: 14813-14818Crossref PubMed Scopus (169) Google Scholar). Interactions between the amino- and carboxyl-terminal regions of apoE are proposed to affect the overall folding of the protein. Additionally, it has been shown that apoE contains intrinsically disordered regions and smaller flexible regions that surround structured helices, affecting the conformation and functional properties of apoE (14.Chen J. Li Q. Wang J. Topology of human apolipoprotein E3 uniquely regulates its diverse biological functions.Proc. Natl. Acad. Sci. USA. 2011; 108: 14813-14818Crossref PubMed Scopus (169) Google Scholar, 15.Frieden C. Wang H. Ho C.M.W. A mechanism for lipid binding to apoE and the role of intrinsically disordered regions coupled to domain-domain interactions.Proc. Natl. Acad. Sci. USA. 2017; 114: 6292-6297Crossref PubMed Scopus (51) Google Scholar, 16.Chetty P.S. Mayne L. Lund-Katz S. Englander S.W. Phillips M.C. Helical structure, stability, and dynamics in human apolipoprotein E3 and E4 by hydrogen exchange and mass spectrometry.Proc. Natl. Acad. Sci. USA. 2017; 114: 968-973Crossref PubMed Scopus (26) Google Scholar). Several biophysical studies have shown that apoE displays low thermodynamic stability and significant conformational plasticity, and allelic differences as well as single point mutations have been shown to affect protein conformation and to hinder physiological function (13.Hatters D.M. Peters-Libeu C.A. Weisgraber K.H. Apolipoprotein E structure: insights into function.Trends Biochem. Sci. 2006; 31: 445-454Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 15.Frieden C. Wang H. Ho C.M.W. A mechanism for lipid binding to apoE and the role of intrinsically disordered regions coupled to domain-domain interactions.Proc. Natl. Acad. Sci. USA. 2017; 114: 6292-6297Crossref PubMed Scopus (51) Google Scholar, 16.Chetty P.S. Mayne L. Lund-Katz S. Englander S.W. Phillips M.C. Helical structure, stability, and dynamics in human apolipoprotein E3 and E4 by hydrogen exchange and mass spectrometry.Proc. Natl. Acad. Sci. USA. 2017; 114: 968-973Crossref PubMed Scopus (26) Google Scholar, 18.Georgiadou D. Stamatakis K. Efthimiadou E.K. Kordas G. Gantz D. Chroni A. Stratikos E. Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathy.J. Lipid Res. 2013; 54: 164-176Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 19.Argyri L. Dafnis I. Theodossiou T.A. Gantz D. Stratikos E. Chroni A. Molecular basis for increased risk for late-onset Alzheimer disease due to the naturally occurring L28P mutation in apolipoprotein E4.J. Biol. Chem. 2014; 289: 12931-12945Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 20.Dafnis I. Argyri L. Sagnou M. Tzinia A. Tsilibary E.C. Stratikos E. Chroni A. The ability of apolipoprotein E fragments to promote intraneuronal accumulation of amyloid beta peptide 42 is both isoform and size-specific.Sci. Rep. 2016; 6: 30654Crossref PubMed Scopus (28) Google Scholar). Furthermore, apoE has been shown to be prone to aggregation and to form oligomeric structures in solution (21.Gau B. Garai K. Frieden C. Gross M.L. Mass spectrometry-based protein footprinting characterizes the structures of oligomeric apolipoprotein E2, E3, and E4.Biochemistry. 2011; 50: 8117-8126Crossref PubMed Scopus (53) Google Scholar, 22.Huang R.Y. Garai K. Frieden C. Gross M.L. Hydrogen/deuterium exchange and electron-transfer dissociation mass spectrometry determine the interface and dynamics of apolipoprotein E oligomerization.Biochemistry. 2011; 50: 9273-9282Crossref PubMed Scopus (68) Google Scholar). The majority of LPG-associated apoE mutations are located in the helical N-terminal domain of the molecule (5.Matsunaga A. Saito T. Apolipoprotein E mutations: a comparison between lipoprotein glomerulopathy and type III hyperlipoproteinemia.Clin. Exp. Nephrol. 2014; 18: 220-224Crossref PubMed Scopus (18) Google Scholar, 6.Stratikos E. Chroni A. A possible structural basis behind the pathogenic role of apolipoprotein E hereditary mutations associated with lipoprotein glomerulopathy.Clin. Exp. Nephrol. 2014; 18: 225-229Crossref PubMed Scopus (5) Google Scholar). Most LPG patients are heterozygous for the apoE mutations and have elevated plasma apoE levels (5.Matsunaga A. Saito T. Apolipoprotein E mutations: a comparison between lipoprotein glomerulopathy and type III hyperlipoproteinemia.Clin. Exp. Nephrol. 2014; 18: 220-224Crossref PubMed Scopus (18) Google Scholar, 6.Stratikos E. Chroni A. A possible structural basis behind the pathogenic role of apolipoprotein E hereditary mutations associated with lipoprotein glomerulopathy.Clin. Exp. Nephrol. 2014; 18: 225-229Crossref PubMed Scopus (5) Google Scholar, 23.Saito T. Ishigaki Y. Oikawa S. Yamamoto T.T. Role of apolipoprotein E variants in lipoprotein glomerulopathy and other renal lipidoses.Clin. Exp. Nephrol. 2001; 5: 201-208Crossref Scopus (17) Google Scholar, 24.Hu Z. Huang S. Wu Y. Liu Y. Liu X. Su D. Tao Y. Fu P. Zhang X. Peng Z. et al.Hereditary features, treatment, and prognosis of the lipoprotein glomerulopathy in patients with the APOE Kyoto mutation.Kidney Int. 2014; 85: 416-424Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 25.Xin Z. Zhihong L. Shijun L. Jinfeng Z. Huiping C. Caihong Z. Daxi J. Leishi L. Successful treatment of patients with lipoprotein glomerulopathy by protein A immunoadsorption: a pilot study.Nephrol. Dial. Transplant. 2009; 24: 864-869Crossref PubMed Scopus (22) Google Scholar). Initially, apoE mutations were discovered in patients from East Asian countries, and it was believed that the disease was restricted in those areas, but several LPG cases and apoE mutations have been reported in patients of Caucasian ancestry in Europe and the United States (1.Saito T. Matsunaga A. Ito K. Nakashima H. Topics in lipoprotein glomerulopathy: an overview.Clin. Exp. Nephrol. 2014; 18: 214-217Crossref PubMed Scopus (16) Google Scholar). The direct relation of apoE mutations with LPG has been supported by gene transfer studies of apoESendai (R145P), the second more frequent apoE mutant detected in LPG patients, to apoE-deficient mice that lead to lipoprotein depositions in the glomerulus of the kidney (26.Ishigaki Y. Oikawa S. Suzuki T. Usui S. Magoori K. Kim D.H. Suzuki H. Sasaki J. Sasano H. Okazaki M. et al.Virus-mediated transduction of apolipoprotein E (ApoE)-sendai develops lipoprotein glomerulopathy in ApoE-deficient mice.J. Biol. Chem. 2000; 275: 31269-31273Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). These findings suggested that apoE dysfunction may be an etiological cause of LPG, although the exact mechanism remains elusive. In an effort to gain mechanistic insight linking the presence of apoE mutations and the development of LPG, we previously examined the effects of three Arg to Pro substitutions at positions 145, 147, and 158 of the apoE3 sequence on structural and conformational integrity of protein. We showed that apoE3 variants carrying these mutations [R145P (apoESendai), R147P (apoEChicago), and R158P (apoEOsaka or apoEKurashiki)] displayed major thermodynamic destabilization, structural perturbations, and increased hydrophobic surface exposure to the solvent and were aggregation prone (18.Georgiadou D. Stamatakis K. Efthimiadou E.K. Kordas G. Gantz D. Chroni A. Stratikos E. Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathy.J. Lipid Res. 2013; 54: 164-176Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). These findings suggested that the folding defects and aggregation propensity of LPG-associated apoE mutations may constitute a component of the disease pathogenic mechanism. Because, however, proline reduces the helical content of apoE3, it is possible that these destabilization effects are limited to the proline mutations and not to other known LPG-associated apoE3 mutants. To test the generality of this mechanism, we set forth to examine whether substitutions of apoE3 amino acids with residues that are, in principle, compatible with helical secondary structure may also lead to perturbations in apoE structure and function. We therefore evaluated the effect of three other LPG-associated mutations based on apoE3 sequence, namely, R25C (apoEKyoto) (1.Saito T. Matsunaga A. Ito K. Nakashima H. Topics in lipoprotein glomerulopathy: an overview.Clin. Exp. Nephrol. 2014; 18: 214-217Crossref PubMed Scopus (16) Google Scholar, 27.Matsunaga A. Sasaki J. Komatsu T. Kanatsu K. Tsuji E. Moriyama K. Koga T. Arakawa K. Oikawa S. Saito T. et al.A novel apolipoprotein E mutation, E2 (Arg25Cys), in lipoprotein glomerulopathy.Kidney Int. 1999; 56: 421-427Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), which has been reported in various parts of the world and is the most common apoE mutation in LPG (1.Saito T. Matsunaga A. Ito K. Nakashima H. Topics in lipoprotein glomerulopathy: an overview.Clin. Exp. Nephrol. 2014; 18: 214-217Crossref PubMed Scopus (16) Google Scholar), R114C (apoETsukuba) (28.Hagiwara M. Yamagata K. Matsunaga T. Arakawa Y. Usui J. Shimizu Y. Aita K. Nagata M. Koyama A. Zhang B. et al.A novel apolipoprotein E mutation, ApoE Tsukuba (Arg 114 Cys), in lipoprotein glomerulopathy.Nephrol. Dial. Transplant. 2008; 23: 381-384Crossref PubMed Scopus (24) Google Scholar), and A152D (apoELasVegas) (29.Bomback A.S. Song H. D'Agati V.D. Cohen S.D. Neal A. Appel G.B. Rovin B.H. A new apolipoprotein E mutation, apoE Las Vegas, in a European-American with lipoprotein glomerulopathy.Nephrol. Dial. Transplant. 2010; 25: 3442-3446Crossref PubMed Scopus (22) Google Scholar), on the structural, thermodynamic, and aggregation properties of the protein. Here, we demonstrate that the three LPG-associated apoE3 mutants, R25C (apoEKyoto), R114C (apoETsukuba), and A152D (apoELasVegas), display structural and thermodynamic perturbations both in lipid-free and lipoprotein-associated forms and expose a larger portion of hydrophobic surface to the solvent as compared with WT apoE3, as it was previously observed for R145P (apoESendai), R147P (apoEChicago), and R158P (apoEOsaka or apoEKurashiki) mutants (18.Georgiadou D. Stamatakis K. Efthimiadou E.K. Kordas G. Gantz D. Chroni A. Stratikos E. Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathy.J. Lipid Res. 2013; 54: 164-176Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). In addition, the three apoE3 mutants are aggregation prone, similarly to LPG-associated apoE3 mutants studied previously (18.Georgiadou D. Stamatakis K. Efthimiadou E.K. Kordas G. Gantz D. Chroni A. Stratikos E. Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathy.J. Lipid Res. 2013; 54: 164-176Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Overall, our data indicate the presence of common themes in the dysfunction of LPG-associated apoE3 mutants that are related to structural and thermodynamic perturbations of protein, pointing toward a unifying mechanism contributing to LPG pathogenesis. The pET32-E33C vector containing a Trx tag, a 6× His-tag, and a 3C-protease site at the fusion junction with the human cDNA for full-length apoE3 has been described previously (18.Georgiadou D. Stamatakis K. Efthimiadou E.K. Kordas G. Gantz D. Chroni A. Stratikos E. Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathy.J. Lipid Res. 2013; 54: 164-176Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The R25C, R114C, and A152D mutations were introduced into the gene of apoE3 by site-directed mutagenesis, by using the QuikChange II XL site-directed mutagenesis kit (Agilent, Santa Clara, CA), according to the manufacturer's instructions. The sequences of primers used for mutagenesis were: R25C: 5′-GTG GCA GAG CGG CCA GTG CTG GGA ACT GGC ACT GG-3′ and 5′-CCA GTG CCA GTT CCC AGC ACT GGC CGC TCT GCC AC-3′ R114C: 5′-GGA GGA CGT GTG CGG CTG CCT GGT GCA GTA CCG CG-3′ and 5′-CGC GGT ACT GCA CCA GGC AGC CGC ACA CGT CCT CC-3′ and A152D: 5′-GCG GCT CCT CCG CGA TGA CGA TGA CCT GCA GAA GC-3′ and 5′-GCT TCT GCA GGT CAT CGT CAT CGC GGA GGA GCC GC-3′. Successful mutagenesis was confirmed by DNA sequencing. The expression and purification of WT apoE3, as well as of mutant apoE3 forms R145P and R158P, was carried out as described previously (18.Georgiadou D. Stamatakis K. Efthimiadou E.K. Kordas G. Gantz D. Chroni A. Stratikos E. Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathy.J. Lipid Res. 2013; 54: 164-176Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The production of mutant apoE3 forms R25C, R114C, and A152D was performed using the same protocol. Briefly, BL21-Gold (DE3) cells (Stratagene, Cedar Creek, TX) were transformed with the vectors, and the recombinant proteins expression was induced with isopropyl-β-d-thiogalactopyranoside. All Trx-fused proteins were expressed soluble and purified by Ni-nitrilotriacetic acid (Ni-NTA) resin (Thermo Scientific, Rockford, IL) affinity chromatography following elution by increasing concentrations of imidazole (18.Georgiadou D. Stamatakis K. Efthimiadou E.K. Kordas G. Gantz D. Chroni A. Stratikos E. Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathy.J. Lipid Res. 2013; 54: 164-176Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The Trx-tag was subsequently cleaved from apoE by His-tagged 3C protease, prepared using the pET-24/His-3C vector kindly provided by Dr. Arie Geerlof (EMBL, Heidelberg, Germany), and the released apoE was isolated by a second Ni-NTA resin affinity chromatography step in the flow-through (18.Georgiadou D. Stamatakis K. Efthimiadou E.K. Kordas G. Gantz D. Chroni A. Stratikos E. Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathy.J. Lipid Res. 2013; 54: 164-176Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). After purification, each apoE form was extensively dialyzed against 5 mM NH4HCO3, lyophilized, and stored at −80°C. Before analyses, the lyophilized proteins were dissolved in 6 M guanidine hydrochloride (GndHCl) in 50 mM sodium phosphate buffer, pH 7.4, containing 1 mM DTT and refolded by extensive dialysis against the same buffer (50 mM sodium phosphate buffer, pH 7.4, and 1 mM DTT). The samples were then centrifuged at 12.000 g for 20 min, at 4°C, to remove any precipitated protein. The refolded proteins were approximately 98% pure, as estimated by SDS-PAGE. All analyses were performed on freshly refolded proteins. Reconstituted discoidal lipoprotein particles containing WT or mutant apoE3 forms were prepared, using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC):cholesterol:apoE:sodium cholate in 50 mM sodium phosphate buffer (pH 7.4) and 1 mM DTT at a molar ratio of 100:10:1:100, as described (18.Georgiadou D. Stamatakis K. Efthimiadou E.K. Kordas G. Gantz D. Chroni A. Stratikos E. Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathy.J. Lipid Res. 2013; 54: 164-176Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 30.Dafnis I. Metso J. Zannis V.I. Jauhiainen M. Chroni A. Influence of isoforms and carboxyl-terminal truncations on the capacity of apolipoprotein E to associate with and activate phospholipid transfer protein.Biochemistry. 2015; 54: 5856-5866Crossref PubMed Scopus (6) Google Scholar). All lipoprotein samples were prepared using the same phospholipid-cholesterol suspension, and the procedure was performed in parallel. Particles were stored at 4°C under N2 to prevent lipid oxidation. Far-UV circular dichroism (CD) spectra were recorded from 190 to 260 nm at 20°C with a Jasco 715 spectropolarimeter, and the spectra were collected as described before (18.Georgiadou D. Stamatakis K. Efthimiadou E.K. Kordas G. Gantz D. Chroni A. Stratikos E. Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathy.J. Lipid Res. 2013; 54: 164-176Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 30.Dafnis I. Metso J. Zannis V.I. Jauhiainen M. Chroni A. Influence of isoforms and carboxyl-terminal truncations on the capacity of apolipoprotein E to associate with and activate phospholipid transfer protein.Biochemistry. 2015; 54: 5856-5866Crossref PubMed Scopus (6) Google Scholar). The concentration of lipid-free WT and mutant apoE3 forms was 0.08–0.1 mg/ml in 50 mM sodium phosphate buffer (pH 7.4) and 1 mM DTT and of the protein component (apoE3 forms) of lipoprotein particles was 0.1 mg/ml in 50 mM sodium phosphate buffer (pH 7.4). Helical content was calculated using the molecular ellipticity at 222 nm (31.Morrisett J.D. David J.S. Pownall H.J. Gotto Jr., A.M. Interaction of an apolipoprotein (apoLP-alanine) with phosphatidylcholine.Biochemistry. 1973; 12: 1290-1299Crossref PubMed Scopus (250) Google Scholar) by the equation: For thermal denaturation analysis of lipid-free apoE3 forms, the change in molar ellipticity at 222 nm was monitored while varying the temperature from 20°C to 80°C at a rate of 1°C/min. The thermal denaturation curve was fitted to a Boltzmann simple sigmoidal model using the GraphPad Prism™ software (GraphPad Software Inc., La Jolla, CA). The apparent melting temperature Tm was determined by the sigmoidal fit as midpoint of the thermal transition. The relative enthalpy change was calculated as described previously (18.Georgiadou D. Stamatakis K. Efthimiadou E.K. Kordas G. Gantz D. Chroni A. Stratikos E. Thermodynamic and structural destabilization of apoE3 by hereditary mutations associated with the development of lipoprotein glomerulopathy.J. Lipid Res. 2013; 54: 164-176Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 32.Gorshkova I.N. Liadaki K. Gursky O. Atkinson D. Zannis V.I. Probing the lipid-free structure and stability of apolipoprotein A-I by mutation.Biochemistry. 2000; 39: 15910-15919Crossref PubMed Scopus (45) Google Scholar). For thermal denaturation analysis of lipoprotein particles, all measuring parameters were identical to that of the lipid-free apoE3 forms, with the exception of the temperature range that varied from 20°C to 100°C." @default.
• W2898073575 created "2018-10-26" @default.
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