Biochmical et Biophysica Acta - General Subjects
Differences in solution dynamics between lens β-crystallin homodimers and heterodimers probed by hydrogen–deuterium exchange and deamidation
Publication date: January 2016
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part B
Author(s): Kirsten J. Lampi, Matthew R. Murray, Matthew P. Peterson, Bryce S. Eng, Eileen Yue, Alice R. Clark, Elisar Barbar, Larry L. David
Background Lens transparency is due to the ordered arrangement of the major structural proteins, called crystallins. βB2 crystallin in the lens of the eye readily forms dimers with other β-crystallin subunits, but the resulting heterodimer structures are not known and were investigated in this study. Methods Structures of βA3 and βB2 crystallin homodimers and the βA3/βB2 crystallin heterodimers were probed by measuring changes in solvent accessibility using hydrogen–deuterium exchange with mass spectrometry. We further mimicked deamidation in βB2 and probed the effect on the βA3/βB2 heterodimer. Results were confirmed with chemical crosslinking and NMR. Results Both βA3 and βB2 had significantly decreased deuterium levels in the heterodimer compared to their respective homodimers, suggesting that they had less solvent accessibility and were more compact in the heterodimer. The compact structure of βB2 was supported by the identification of chemical crosslinks between lysines in βB2 within the heterodimer that were inconsistent with βB2's extended homodimeric structure. The compact structure of βA3 was supported by an overall decrease in mobility of βA3 in the heterodimer detected by NMR. In βB2, peptides 70–84 and 121–134 were exposed in the homodimer, but buried in the heterodimer with ≥50% decreases in deuterium levels. Homologous peptides in βA3, 97–109 and 134–149, had 25–50% decreases in deuterium levels in the heterodimer. These peptides are probable sites of interaction between βB2 and βA3 and are located at the predicted interface between subunits with bent linkers. Deamidation at Q184 in βB2 at this predicted interface led to a less compact βB2 in the heterodimer. The more compact structure of the βA3/βB2 heterodimer was also more heat stable than either of the homodimers. Conclusions The major structural proteins in the lens, the β-crystallins, are not static, but dynamic in solution, with differences in accessibility between the homo-and hetero-dimers. This structural flexibility, particularly of βB2, may facilitate formation of different size higher-ordered structures found in the transparent lens. General significance Understanding complex hetero-oligomer interactions between β-crystallins in normal lens and how these interactions change during aging is fundamental to understanding the cause of cataracts. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Graphical abstract
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part B
Author(s): Kirsten J. Lampi, Matthew R. Murray, Matthew P. Peterson, Bryce S. Eng, Eileen Yue, Alice R. Clark, Elisar Barbar, Larry L. David
Background Lens transparency is due to the ordered arrangement of the major structural proteins, called crystallins. βB2 crystallin in the lens of the eye readily forms dimers with other β-crystallin subunits, but the resulting heterodimer structures are not known and were investigated in this study. Methods Structures of βA3 and βB2 crystallin homodimers and the βA3/βB2 crystallin heterodimers were probed by measuring changes in solvent accessibility using hydrogen–deuterium exchange with mass spectrometry. We further mimicked deamidation in βB2 and probed the effect on the βA3/βB2 heterodimer. Results were confirmed with chemical crosslinking and NMR. Results Both βA3 and βB2 had significantly decreased deuterium levels in the heterodimer compared to their respective homodimers, suggesting that they had less solvent accessibility and were more compact in the heterodimer. The compact structure of βB2 was supported by the identification of chemical crosslinks between lysines in βB2 within the heterodimer that were inconsistent with βB2's extended homodimeric structure. The compact structure of βA3 was supported by an overall decrease in mobility of βA3 in the heterodimer detected by NMR. In βB2, peptides 70–84 and 121–134 were exposed in the homodimer, but buried in the heterodimer with ≥50% decreases in deuterium levels. Homologous peptides in βA3, 97–109 and 134–149, had 25–50% decreases in deuterium levels in the heterodimer. These peptides are probable sites of interaction between βB2 and βA3 and are located at the predicted interface between subunits with bent linkers. Deamidation at Q184 in βB2 at this predicted interface led to a less compact βB2 in the heterodimer. The more compact structure of the βA3/βB2 heterodimer was also more heat stable than either of the homodimers. Conclusions The major structural proteins in the lens, the β-crystallins, are not static, but dynamic in solution, with differences in accessibility between the homo-and hetero-dimers. This structural flexibility, particularly of βB2, may facilitate formation of different size higher-ordered structures found in the transparent lens. General significance Understanding complex hetero-oligomer interactions between β-crystallins in normal lens and how these interactions change during aging is fundamental to understanding the cause of cataracts. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Graphical abstract
Deamidation of N76 in human γS-crystallin promotes dimer formation
Publication date: January 2016
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part B
Author(s): Nicholas J. Ray, Damien Hall, John A. Carver
Background Cataract formation is often attributed to the build-up of post-translational modifications in the crystallin proteins of the eye lens. One such modification, the deamidation of N76 in human γS-crystallin to D76, is highly correlated with age-related cataract (Hooi et al. Invest. Ophthalmol. Vis. Sci. 53 (2012) 3554–3561). In the current work, this modification has been extensively characterised in vitro. Methods Biophysical characterisation was performed on wild type and N76D γS-crystallins using turbidity measurements to monitor aggregation, intrinsic fluorescence and circular dichroism spectroscopy to determine the folded state and NMR spectroscopy for identifying local changes in structure. Protein mass was determined using SEC–MALLS and analytical ultracentrifugation methods. Results Relative to the wild type protein, deamidation at N76 in γS-crystallin causes an increase in the thermal stability and resistance to thermally induced aggregation alongside a decrease in stability to denaturants, a propensity to aggregate rapidly once destabilised and a tendency to form a dimer. We ascribe the apparent increase in thermal stability upon deamidation to the formation of dimer which prevents the unfolding of the inherently less stable monomer. Conclusions Deamidation causes a decrease in stability of γS-crystallin but this is offset by an increased tendency for dimer formation. General significance Deamidation at N76 in human γS-crystallin likely has a combinatorial effect with other post-translational crystallin modifications to induce age-related cataract. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part B
Author(s): Nicholas J. Ray, Damien Hall, John A. Carver
Background Cataract formation is often attributed to the build-up of post-translational modifications in the crystallin proteins of the eye lens. One such modification, the deamidation of N76 in human γS-crystallin to D76, is highly correlated with age-related cataract (Hooi et al. Invest. Ophthalmol. Vis. Sci. 53 (2012) 3554–3561). In the current work, this modification has been extensively characterised in vitro. Methods Biophysical characterisation was performed on wild type and N76D γS-crystallins using turbidity measurements to monitor aggregation, intrinsic fluorescence and circular dichroism spectroscopy to determine the folded state and NMR spectroscopy for identifying local changes in structure. Protein mass was determined using SEC–MALLS and analytical ultracentrifugation methods. Results Relative to the wild type protein, deamidation at N76 in γS-crystallin causes an increase in the thermal stability and resistance to thermally induced aggregation alongside a decrease in stability to denaturants, a propensity to aggregate rapidly once destabilised and a tendency to form a dimer. We ascribe the apparent increase in thermal stability upon deamidation to the formation of dimer which prevents the unfolding of the inherently less stable monomer. Conclusions Deamidation causes a decrease in stability of γS-crystallin but this is offset by an increased tendency for dimer formation. General significance Deamidation at N76 in human γS-crystallin likely has a combinatorial effect with other post-translational crystallin modifications to induce age-related cataract. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Increased hydrophobic surface exposure in the cataract-related G18V variant of human γS-crystallin
Publication date: January 2016
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part B
Author(s): Domarin Khago, Eric K. Wong, Carolyn N. Kingsley, J. Alfredo Freites, Douglas J. Tobias, Rachel W. Martin
Background The objective of this study was to determine whether the cataract-related G18V variant of human γS-crystallin has increased exposure of hydrophobic residues that could explain its aggregation propensity and/or recognition by αB-crystallin. Methods We used an ANS fluorescence assay and NMR chemical shift perturbation to experimentally probe exposed hydrophobic surfaces. These results were compared to flexible docking simulations of ANS molecules to the proteins, starting with the solution-state NMR structures of γS-WT and γS-G18V. Results γS-G18V exhibits increased ANS fluorescence, suggesting increased exposed hydrophobic surface area. The specific residues involved in ANS binding were mapped by NMR chemical shift perturbation assays, revealing ANS binding sites in γS-G18V that are not present in γS-WT. Molecular docking predicts three binding sites that are specific to γS-G18V corresponding to the exposure of a hydrophobic cavity located at the interdomain interface, as well as two hydrophobic patches near a disordered loop containing solvent-exposed cysteines, all but one of which is buried in γS-WT. Conclusions Although both proteins display non-specific binding, more residues are involved in ANS binding to γS-G18V, and the affected residues are localized in the N-terminal domain and the nearby interdomain interface, proximal to the mutation site. General significance Characterization of changes in exposed hydrophobic surface area between wild-type and variant proteins can help elucidate the mechanisms of aggregation propensity and chaperone recognition, presented here in the context of cataract formation. Experimental data and simulations provide complementary views of the interactions between proteins and the small molecule probes commonly used to study aggregation. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Graphical abstract
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part B
Author(s): Domarin Khago, Eric K. Wong, Carolyn N. Kingsley, J. Alfredo Freites, Douglas J. Tobias, Rachel W. Martin
Background The objective of this study was to determine whether the cataract-related G18V variant of human γS-crystallin has increased exposure of hydrophobic residues that could explain its aggregation propensity and/or recognition by αB-crystallin. Methods We used an ANS fluorescence assay and NMR chemical shift perturbation to experimentally probe exposed hydrophobic surfaces. These results were compared to flexible docking simulations of ANS molecules to the proteins, starting with the solution-state NMR structures of γS-WT and γS-G18V. Results γS-G18V exhibits increased ANS fluorescence, suggesting increased exposed hydrophobic surface area. The specific residues involved in ANS binding were mapped by NMR chemical shift perturbation assays, revealing ANS binding sites in γS-G18V that are not present in γS-WT. Molecular docking predicts three binding sites that are specific to γS-G18V corresponding to the exposure of a hydrophobic cavity located at the interdomain interface, as well as two hydrophobic patches near a disordered loop containing solvent-exposed cysteines, all but one of which is buried in γS-WT. Conclusions Although both proteins display non-specific binding, more residues are involved in ANS binding to γS-G18V, and the affected residues are localized in the N-terminal domain and the nearby interdomain interface, proximal to the mutation site. General significance Characterization of changes in exposed hydrophobic surface area between wild-type and variant proteins can help elucidate the mechanisms of aggregation propensity and chaperone recognition, presented here in the context of cataract formation. Experimental data and simulations provide complementary views of the interactions between proteins and the small molecule probes commonly used to study aggregation. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Graphical abstract
Gamma crystallins of the human eye lens
Publication date: January 2016
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part B
Author(s): Venkata Pulla Rao Vendra, Ismail Khan, Sushil Chandani, Anbukkarasi Muniyandi, Dorairajan Balasubramanian
Background Protein crystallins co me in three types (α, β and γ) and are found predominantly in the eye, and particularly in the lens, where they are packed into a compact, plastic, elastic, and transparent globule of proper refractive power range that aids in focusing incoming light on to the retina. Of these, the γ-crystallins are found largely in the nuclear region of the lens at very high concentrations (>400mg/ml). The connection between their structure and inter-molecular interactions and lens transparency is an issue of particular interest. Scope of review We review the origin and phylogeny of the gamma crystallins, their special structure involving the use of Greek key supersecondary structural motif, and how they aid in offering the appropriate refractive index gradient, intermolecular short range attractive interactions (aiding in packing them into a transparent ball), the role that several of the constituent amino acid residues play in this process, the thermodynamic and kinetic stability and how even single point mutations can upset this delicate balance and lead to intermolecular aggregation, forming light-scattering particles which compromise transparency. We cite several examples of this, and illustrate this by cloning, expressing, isolating and comparing the properties of the mutant protein S39C of human γS-crystallin (associated with congenital cataract-microcornea), with those of the wild type molecule. In addition, we note that human γ-crystallins are also present in other parts of the eye (e.g., retina), where their functions are yet to be understood. Major conclusions There are several ‘crucial’ residues in and around the Greek key motifs which are essential to maintain the compact architecture of the crystallin molecules. We find that a mutation that replaces even one of these residues can lead to reduction in solubility, formation of light-scattering particles and loss of transparency in the molecular assembly. General significance Such a molecular understanding of the process helps us construct the continuum of genotype–molecular structural phenotype–clinical (pathological) phenotype. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part B
Author(s): Venkata Pulla Rao Vendra, Ismail Khan, Sushil Chandani, Anbukkarasi Muniyandi, Dorairajan Balasubramanian
Background Protein crystallins co me in three types (α, β and γ) and are found predominantly in the eye, and particularly in the lens, where they are packed into a compact, plastic, elastic, and transparent globule of proper refractive power range that aids in focusing incoming light on to the retina. Of these, the γ-crystallins are found largely in the nuclear region of the lens at very high concentrations (>400mg/ml). The connection between their structure and inter-molecular interactions and lens transparency is an issue of particular interest. Scope of review We review the origin and phylogeny of the gamma crystallins, their special structure involving the use of Greek key supersecondary structural motif, and how they aid in offering the appropriate refractive index gradient, intermolecular short range attractive interactions (aiding in packing them into a transparent ball), the role that several of the constituent amino acid residues play in this process, the thermodynamic and kinetic stability and how even single point mutations can upset this delicate balance and lead to intermolecular aggregation, forming light-scattering particles which compromise transparency. We cite several examples of this, and illustrate this by cloning, expressing, isolating and comparing the properties of the mutant protein S39C of human γS-crystallin (associated with congenital cataract-microcornea), with those of the wild type molecule. In addition, we note that human γ-crystallins are also present in other parts of the eye (e.g., retina), where their functions are yet to be understood. Major conclusions There are several ‘crucial’ residues in and around the Greek key motifs which are essential to maintain the compact architecture of the crystallin molecules. We find that a mutation that replaces even one of these residues can lead to reduction in solubility, formation of light-scattering particles and loss of transparency in the molecular assembly. General significance Such a molecular understanding of the process helps us construct the continuum of genotype–molecular structural phenotype–clinical (pathological) phenotype. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Editorial Board
Publication date: December 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 12
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 12
Beyond water homeostasis: Diverse functional roles of mammalian aquaporins
Publication date: December 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 12
Author(s): Philip Kitchen, Rebecca E. Day, Mootaz M. Salman, Matthew T. Conner, Roslyn M. Bill, Alex C. Conner
Background Aquaporin (AQP) water channels are best known as passive transporters of water that are vital for water homeostasis. Scope of review AQP knockout studies in whole animals and cultured cells, along with naturally occurring human mutations suggest that the transport of neutral solutes through AQPs has important physiological roles. Emerging biophysical evidence suggests that AQPs may also facilitate gas (CO2) and cation transport. AQPs may be involved in cell signalling for volume regulation and controlling the subcellular localization of other proteins by forming macromolecular complexes. This review examines the evidence for these diverse functions of AQPs as well their physiological relevance. Major conclusions As well as being crucial for water homeostasis, AQPs are involved in physiologically important transport of molecules other than water, regulation of surface expression of other membrane proteins, cell adhesion, and signalling in cell volume regulation. General significance Elucidating the full range of functional roles of AQPs beyond the passive conduction of water will improve our understanding of mammalian physiology in health and disease. The functional variety of AQPs makes them an exciting drug target and could provide routes to a range of novel therapies.
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 12
Author(s): Philip Kitchen, Rebecca E. Day, Mootaz M. Salman, Matthew T. Conner, Roslyn M. Bill, Alex C. Conner
Background Aquaporin (AQP) water channels are best known as passive transporters of water that are vital for water homeostasis. Scope of review AQP knockout studies in whole animals and cultured cells, along with naturally occurring human mutations suggest that the transport of neutral solutes through AQPs has important physiological roles. Emerging biophysical evidence suggests that AQPs may also facilitate gas (CO2) and cation transport. AQPs may be involved in cell signalling for volume regulation and controlling the subcellular localization of other proteins by forming macromolecular complexes. This review examines the evidence for these diverse functions of AQPs as well their physiological relevance. Major conclusions As well as being crucial for water homeostasis, AQPs are involved in physiologically important transport of molecules other than water, regulation of surface expression of other membrane proteins, cell adhesion, and signalling in cell volume regulation. General significance Elucidating the full range of functional roles of AQPs beyond the passive conduction of water will improve our understanding of mammalian physiology in health and disease. The functional variety of AQPs makes them an exciting drug target and could provide routes to a range of novel therapies.
Endostatin's emerging roles in angiogenesis, lymphangiogenesis, disease, and clinical applications
Publication date: December 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 12
Author(s): Amit Walia, Jessica F. Yang, Yu-hui Huang, Mark I. Rosenblatt, Jin-Hong Chang, Dimitri T. Azar
Background Angiogenesis is the process of neovascularization from pre-existing vasculature and is involved in various physiological and pathological processes. Inhibitors of angiogenesis, administered either as individual drugs or in combination with other chemotherapy, have been shown to benefit patients with various cancers. Endostatin, a 20-kDa C-terminal fragment of type XVIII collagen, is one of the most potent inhibitors of angiogenesis. Scope of review We discuss the biology behind endostatin in the context of its endogenous production, the various receptors to which it binds, and the mechanisms by which it acts. We focus on its inhibitory role in angiogenesis, lymphangiogenesis, and cancer metastasis. We also present emerging clinical applications for endostatin and its potential as a therapeutic agent in the form a short peptide. Major conclusions The delicate balance between pro- and anti-angiogenic factors can be modulated to result in physiological wound healing or pathological tumor metastasis. Research in the last decade has emphasized an emerging clinical potential for endostatin as a biomarker and as a therapeutic short peptide. Moreover, elevated or depressed endostatin levels in diseased states may help explain the pathophysiological mechanisms of the particular disease. General significance Endostatin was once sought after as the ‘be all and end all’ for cancer treatment; however, research throughout the last decade has made it apparent that endostatin's effects are complex and involve multiple mechanisms. A better understanding of newly discovered mechanisms and clinical applications still has the potential to lead to future advances in the use of endostatin in the clinic.
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 12
Author(s): Amit Walia, Jessica F. Yang, Yu-hui Huang, Mark I. Rosenblatt, Jin-Hong Chang, Dimitri T. Azar
Background Angiogenesis is the process of neovascularization from pre-existing vasculature and is involved in various physiological and pathological processes. Inhibitors of angiogenesis, administered either as individual drugs or in combination with other chemotherapy, have been shown to benefit patients with various cancers. Endostatin, a 20-kDa C-terminal fragment of type XVIII collagen, is one of the most potent inhibitors of angiogenesis. Scope of review We discuss the biology behind endostatin in the context of its endogenous production, the various receptors to which it binds, and the mechanisms by which it acts. We focus on its inhibitory role in angiogenesis, lymphangiogenesis, and cancer metastasis. We also present emerging clinical applications for endostatin and its potential as a therapeutic agent in the form a short peptide. Major conclusions The delicate balance between pro- and anti-angiogenic factors can be modulated to result in physiological wound healing or pathological tumor metastasis. Research in the last decade has emphasized an emerging clinical potential for endostatin as a biomarker and as a therapeutic short peptide. Moreover, elevated or depressed endostatin levels in diseased states may help explain the pathophysiological mechanisms of the particular disease. General significance Endostatin was once sought after as the ‘be all and end all’ for cancer treatment; however, research throughout the last decade has made it apparent that endostatin's effects are complex and involve multiple mechanisms. A better understanding of newly discovered mechanisms and clinical applications still has the potential to lead to future advances in the use of endostatin in the clinic.
Nitric oxide and the thioredoxin system: a complex interplay in redox regulation
Publication date: December 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 12
Author(s): Moran Benhar
Background The free radical nitric oxide (NO) and the thiol oxidoreductase thioredoxin (Trx) play essential roles in cellular redox regulation. Recent biochemical and cellular studies have revealed a complex thiol-dependent crosstalk between NO and Trx that modulates multiple redox-dependent pathways. Scope of review This review aims to discuss recent progress, as well as the remaining questions, regarding the interaction and cross regulation between NO and Trx in cellular function and dysfunction. Major conclusions The importance and ubiquity of NO-mediated S-nitrosylation of protein thiols as a signaling mechanism is increasingly recognized as is the central role of Trx in regulating S-nitrosylation processes. By denitrosylating diverse protein substrates, Trx plays an active role in attenuating NO signaling as well as in ameliorating nitrosative stress. Yet, at the same time, Trx can also support the activity of NO synthases, thus promoting NO production and its downstream effects. Finally, NO can reciprocally modulate the redox activity of Trx and Trx reductase. General significance Further elucidation of the crosstalk between NO and Trx will be important for an improved understanding of the effects of reactive oxygen and nitrogen species on cellular signaling and function.
Graphical abstract
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 12
Author(s): Moran Benhar
Background The free radical nitric oxide (NO) and the thiol oxidoreductase thioredoxin (Trx) play essential roles in cellular redox regulation. Recent biochemical and cellular studies have revealed a complex thiol-dependent crosstalk between NO and Trx that modulates multiple redox-dependent pathways. Scope of review This review aims to discuss recent progress, as well as the remaining questions, regarding the interaction and cross regulation between NO and Trx in cellular function and dysfunction. Major conclusions The importance and ubiquity of NO-mediated S-nitrosylation of protein thiols as a signaling mechanism is increasingly recognized as is the central role of Trx in regulating S-nitrosylation processes. By denitrosylating diverse protein substrates, Trx plays an active role in attenuating NO signaling as well as in ameliorating nitrosative stress. Yet, at the same time, Trx can also support the activity of NO synthases, thus promoting NO production and its downstream effects. Finally, NO can reciprocally modulate the redox activity of Trx and Trx reductase. General significance Further elucidation of the crosstalk between NO and Trx will be important for an improved understanding of the effects of reactive oxygen and nitrogen species on cellular signaling and function.
Graphical abstract
The S100 proteins in epidermis: Topology and function
Publication date: December 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 12
Author(s): Wiesława Leśniak, Agnieszka Graczyk-Jarzynka
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 12
Author(s): Wiesława Leśniak, Agnieszka Graczyk-Jarzynka
Lupeol derivative mitigates Echis carinatus venom-induced tissue destruction by neutralizing venom toxins and protecting collagen and angiogenic receptors on inflammatory cells
Publication date: December 2015
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 12
Author(s): G.D. Katkar, Rachana D. Sharma, G.J. Vishalakshi, S.K. Naveenkumar, Gaurav Madhur, R.M. Thushara, T. Narender, K.S. Girish, K. Kemparaju
Background Echis carinatus bite is a serious threat in South-Asian countries including India, as it causes highest number of deaths and terrifying long-term tissue destruction at the bitten site. Although venom metalloproteinases and hyaluronidases are the suggested key players, studies on the effect of venom on polymorphonuclear cells, peripheral blood mononuclear cells and platelets, and their role in long-term tissue destruction are still in infancy. While, the effect of venom on collagen receptors, integrin α2β1/GP VI/DDR1 and CX3CR1 chemokine receptor present on these cells is an untouched area. Methods Lupeol, lupeol acetate, its synthetic derivatives 2–8 were screened for inhibition of E. carinatus venom induced-hemorrhage in mouse model where compound 8 was found to be the most potent. Further, compound 8 efficiently neutralized venom induced hemorrhage, edema, dermonecrosis, myonecrosis, myotoxicity, pro-coagulant, oxidative stress, inflammatory cytokines and cleavage of collagen and CX3CR1 receptors on inflammatory cells in in vivo, in silico, ex vivo and in vitro studies. Conclusions This study for the first time demonstrated the cleavage of collagen receptors and the receptor for angiogenesis and wound healing by the venom and its inhibition by compound 8, as these are important for firm adhesion of inflammatory cells at the damaged site to resolve inflammation and promote tissue repair. General significance This study provides a lead in venom pharmacology, wherein, compound 8 could be a therapeutic agent for the better management of viper venom-induced long-term tissue destruction.
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1850, Issue 12
Author(s): G.D. Katkar, Rachana D. Sharma, G.J. Vishalakshi, S.K. Naveenkumar, Gaurav Madhur, R.M. Thushara, T. Narender, K.S. Girish, K. Kemparaju
Background Echis carinatus bite is a serious threat in South-Asian countries including India, as it causes highest number of deaths and terrifying long-term tissue destruction at the bitten site. Although venom metalloproteinases and hyaluronidases are the suggested key players, studies on the effect of venom on polymorphonuclear cells, peripheral blood mononuclear cells and platelets, and their role in long-term tissue destruction are still in infancy. While, the effect of venom on collagen receptors, integrin α2β1/GP VI/DDR1 and CX3CR1 chemokine receptor present on these cells is an untouched area. Methods Lupeol, lupeol acetate, its synthetic derivatives 2–8 were screened for inhibition of E. carinatus venom induced-hemorrhage in mouse model where compound 8 was found to be the most potent. Further, compound 8 efficiently neutralized venom induced hemorrhage, edema, dermonecrosis, myonecrosis, myotoxicity, pro-coagulant, oxidative stress, inflammatory cytokines and cleavage of collagen and CX3CR1 receptors on inflammatory cells in in vivo, in silico, ex vivo and in vitro studies. Conclusions This study for the first time demonstrated the cleavage of collagen receptors and the receptor for angiogenesis and wound healing by the venom and its inhibition by compound 8, as these are important for firm adhesion of inflammatory cells at the damaged site to resolve inflammation and promote tissue repair. General significance This study provides a lead in venom pharmacology, wherein, compound 8 could be a therapeutic agent for the better management of viper venom-induced long-term tissue destruction.
Basic leucine zipper (bZIP) transcription factors involved in abiotic stresses: A molecular model of a wheat bZIP factor and implications of its structure in function
Publication date: January 2016
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part A
Author(s): Pradeep Sornaraj, Sukanya Luang, Sergiy Lopato, Maria Hrmova
Background Basic leucine zipper (bZIP) genes encode transcription factors (TFs) that control important biochemical and physiological processes in plants and all other eukaryotic organisms. Scope of review Here we present (i) the homo-dimeric structural model of bZIP consisting of basic leucine zipper and DNA binding regions, in complex with the synthetic Abscisic Acid-Responsive Element (ABREsyn); (ii) discuss homo- and hetero-dimerisation patterns of bZIP TFs; (iii) summarise the current progress in understanding the molecular mechanisms of function of bZIP TFs, including features determining the specificity of their binding to DNA cis-elements, and (iv) review information on interaction partners of bZIPs during plant development and stress response, as well as on types and roles of post-translational modifications, and regulatory aspects of protein-degradation mediated turn-over. Finally, we (v) recapitulate on the recent advances regarding functional roles of bZIP factors in major agricultural crops, and discuss the potential significance of bZIP-based genetic engineering in improving crop yield and tolerance to abiotic stresses. Major conclusions An accurate analysis and understanding of roles of plant bZIP TFs in different biological processes requires the knowledge of interacting partners, time and location of expression in plant organs, and the information on mechanisms of homo- and hetero-dimerisation of bZIP TFs. General significance Studies on molecular mechanisms of plant bZIP TFs at the atomic levels will provide novel insights into the regulatory processes during plant development, and responses to abiotic and biotic stresses.
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part A
Author(s): Pradeep Sornaraj, Sukanya Luang, Sergiy Lopato, Maria Hrmova
Background Basic leucine zipper (bZIP) genes encode transcription factors (TFs) that control important biochemical and physiological processes in plants and all other eukaryotic organisms. Scope of review Here we present (i) the homo-dimeric structural model of bZIP consisting of basic leucine zipper and DNA binding regions, in complex with the synthetic Abscisic Acid-Responsive Element (ABREsyn); (ii) discuss homo- and hetero-dimerisation patterns of bZIP TFs; (iii) summarise the current progress in understanding the molecular mechanisms of function of bZIP TFs, including features determining the specificity of their binding to DNA cis-elements, and (iv) review information on interaction partners of bZIPs during plant development and stress response, as well as on types and roles of post-translational modifications, and regulatory aspects of protein-degradation mediated turn-over. Finally, we (v) recapitulate on the recent advances regarding functional roles of bZIP factors in major agricultural crops, and discuss the potential significance of bZIP-based genetic engineering in improving crop yield and tolerance to abiotic stresses. Major conclusions An accurate analysis and understanding of roles of plant bZIP TFs in different biological processes requires the knowledge of interacting partners, time and location of expression in plant organs, and the information on mechanisms of homo- and hetero-dimerisation of bZIP TFs. General significance Studies on molecular mechanisms of plant bZIP TFs at the atomic levels will provide novel insights into the regulatory processes during plant development, and responses to abiotic and biotic stresses.
CacyBP/SIP — Structure and variety of functions
Publication date: January 2016
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part A
Author(s): Agnieszka M. Topolska-Woś, Walter J. Chazin, Anna Filipek
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part A
Author(s): Agnieszka M. Topolska-Woś, Walter J. Chazin, Anna Filipek
Effects of cytosine methylation on DNA morphology: An atomic force microscopy study
Publication date: January 2016
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part A
Author(s): V. Cassina, M. Manghi, D. Salerno, A. Tempestini, V. Iadarola, L. Nardo, S. Brioschi, F. Mantegazza
Methylation is one of the most important epigenetic mechanisms in eukaryotes. As a consequence of cytosine methylation, the binding of proteins that are implicated in transcription to gene promoters is severely hindered, which results in gene regulation and, eventually, gene silencing. To date, the mechanisms by which methylation biases the binding affinities of proteins to DNA are not fully understood; however, it has been proposed that changes in double-strand conformations, such as stretching, bending, and over-twisting, as well as local variations in DNA stiffness/flexibility may play a role. The present work investigates, at the single molecule level, the morphological consequences of DNA methylation in vitro. By tracking the atomic force microscopy images of single DNA molecules, we characterize DNA conformations pertaining to two different degrees of methylation. In particular, we observe that methylation induces no relevant variations in DNA contour lengths, but produces measurable incremental changes in persistence lengths. Furthermore, we observe that for the methylated chains, the statistical distribution of angles along the DNA coordinate length is characterized by a double exponential decay, in agreement with what is predicted for polyelectrolytes. The results reported herein support the claim that the biological consequences of the methylation process, specifically difficulties in protein-DNA binding, are at least partially due to DNA conformation modifications.
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part A
Author(s): V. Cassina, M. Manghi, D. Salerno, A. Tempestini, V. Iadarola, L. Nardo, S. Brioschi, F. Mantegazza
Methylation is one of the most important epigenetic mechanisms in eukaryotes. As a consequence of cytosine methylation, the binding of proteins that are implicated in transcription to gene promoters is severely hindered, which results in gene regulation and, eventually, gene silencing. To date, the mechanisms by which methylation biases the binding affinities of proteins to DNA are not fully understood; however, it has been proposed that changes in double-strand conformations, such as stretching, bending, and over-twisting, as well as local variations in DNA stiffness/flexibility may play a role. The present work investigates, at the single molecule level, the morphological consequences of DNA methylation in vitro. By tracking the atomic force microscopy images of single DNA molecules, we characterize DNA conformations pertaining to two different degrees of methylation. In particular, we observe that methylation induces no relevant variations in DNA contour lengths, but produces measurable incremental changes in persistence lengths. Furthermore, we observe that for the methylated chains, the statistical distribution of angles along the DNA coordinate length is characterized by a double exponential decay, in agreement with what is predicted for polyelectrolytes. The results reported herein support the claim that the biological consequences of the methylation process, specifically difficulties in protein-DNA binding, are at least partially due to DNA conformation modifications.
Cellular response of human neuroblastoma cells to α-synuclein fibrils, the main constituent of Lewy bodies
Publication date: January 2016
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part A
Author(s): Laura Pieri, Philippe Chafey, Morgane Le Gall, Guilhem Clary, Ronald Melki, Virginie Redeker
Graphical abstract
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part A
Author(s): Laura Pieri, Philippe Chafey, Morgane Le Gall, Guilhem Clary, Ronald Melki, Virginie Redeker
Graphical abstract
The mitochondrial carnitine/acylcarnitine carrier is regulated by hydrogen sulfide via interaction with C136 and C155
Publication date: January 2016
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part A
Author(s): Nicola Giangregorio, Annamaria Tonazzi, Lara Console, Imma Lorusso, Annalisa De Palma, Cesare Indiveri
Background The carnitine/acylcarnitine carrier (CAC or CACT) mediates transport of acylcarnitines into mitochondria for the β-oxidation. CAC possesses Cys residues which respond to redox changes undergoing to SH/disulfide interconversion. Methods The effect of H2S has been investigated on the [3H]carnitine/carnitine antiport catalyzed by recombinant or native CAC reconstituted in proteoliposomes. Site-directed mutagenesis was employed for identifying Cys reacting with H2S. Results H2S led to transport inhibition, which was dependent on concentration, pH and time of incubation. Best inhibition with IC50 of 0.70μM was observed at physiological pH after 30–60min incubation. At longer times of incubation, inhibition was reversed. After oxidation of the carrier by O2, transport activity was rescued by H2S indicating that the inhibition/activation depends on the initial redox state of the protein. The observed effects were more efficient on the native rat liver transporter than on the recombinant protein. Only the protein containing both C136 and C155 responded to the reagent as the WT. While reduced responses were observed in the mutants containing C136 or C155. Multi-alignment of known mitochondrial carriers, highlighted that only the CAC possesses both Cys residues. This correlates well with the absence of effects of H2S on carriers which does not contain the Cys couple. Conclusions Altogether, these data demonstrate that H2S regulates the CAC by inhibiting or activating transport on the basis of the redox state of the protein. General significance CAC represents a specific target of H2S among mitochondrial carriers in agreement with the presence of a reactive Cys couple.
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part A
Author(s): Nicola Giangregorio, Annamaria Tonazzi, Lara Console, Imma Lorusso, Annalisa De Palma, Cesare Indiveri
Background The carnitine/acylcarnitine carrier (CAC or CACT) mediates transport of acylcarnitines into mitochondria for the β-oxidation. CAC possesses Cys residues which respond to redox changes undergoing to SH/disulfide interconversion. Methods The effect of H2S has been investigated on the [3H]carnitine/carnitine antiport catalyzed by recombinant or native CAC reconstituted in proteoliposomes. Site-directed mutagenesis was employed for identifying Cys reacting with H2S. Results H2S led to transport inhibition, which was dependent on concentration, pH and time of incubation. Best inhibition with IC50 of 0.70μM was observed at physiological pH after 30–60min incubation. At longer times of incubation, inhibition was reversed. After oxidation of the carrier by O2, transport activity was rescued by H2S indicating that the inhibition/activation depends on the initial redox state of the protein. The observed effects were more efficient on the native rat liver transporter than on the recombinant protein. Only the protein containing both C136 and C155 responded to the reagent as the WT. While reduced responses were observed in the mutants containing C136 or C155. Multi-alignment of known mitochondrial carriers, highlighted that only the CAC possesses both Cys residues. This correlates well with the absence of effects of H2S on carriers which does not contain the Cys couple. Conclusions Altogether, these data demonstrate that H2S regulates the CAC by inhibiting or activating transport on the basis of the redox state of the protein. General significance CAC represents a specific target of H2S among mitochondrial carriers in agreement with the presence of a reactive Cys couple.
CdTe quantum dots as fluorescent probes to study transferrin receptors in glioblastoma cells
Publication date: January 2016
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part A
Author(s): Paulo E. Cabral Filho, Ana L.C. Cardoso, Maria I.A. Pereira, Ana P.M. Ramos, Fernando Hallwas, M. Margarida C.A. Castro, Carlos F.G.C. Geraldes, Beate S. Santos, Maria C. Pedroso de Lima, Giovannia A.L. Pereira, Adriana Fontes
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part A
Author(s): Paulo E. Cabral Filho, Ana L.C. Cardoso, Maria I.A. Pereira, Ana P.M. Ramos, Fernando Hallwas, M. Margarida C.A. Castro, Carlos F.G.C. Geraldes, Beate S. Santos, Maria C. Pedroso de Lima, Giovannia A.L. Pereira, Adriana Fontes
Alpha-lipoic acid supplementation protects enzymes from damage by nitrosative and oxidative stress
Publication date: January 2016
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part A
Author(s): Sylvia Hiller, Robert DeKroon, Eric D. Hamlett, Longquan Xu, Cristina Osorio, Jennifer Robinette, Witold Winnik, Stephen Simington, Nobuyo Maeda, Oscar Alzate, Xianwen Yi
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part A
Author(s): Sylvia Hiller, Robert DeKroon, Eric D. Hamlett, Longquan Xu, Cristina Osorio, Jennifer Robinette, Witold Winnik, Stephen Simington, Nobuyo Maeda, Oscar Alzate, Xianwen Yi
Identification of a neuropeptide precursor protein that gives rise to a “cocktail” of peptides that bind Cu(II) and generate metal-linked dimers
Publication date: January 2016
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part A
Author(s): Christopher E. Jones, Meet Zandawala, Dean C. Semmens, Sarah Anderson, Graeme R. Hanson, Daniel A. Janies, Maurice R. Elphick
Background Neuropeptides with an Amino Terminal Cu(II), Ni(II) Binding (ATCUN) motif (H2N-xxH) bind Cu(II)/Ni(II) ions. Here we report the novel discovery of a neuropeptide precursor that gives rise to a “cocktail” of peptides that bind Cu(II)/Ni(II) and form ternary complexes — the L-type SALMFamide precursor in the starfish Asterias rubens. Methods Echinoderm transcriptome sequence data were analysed to identify transcripts encoding precursors of SALMFamide-type neuropeptides. The sequence of the L-type SALMFamide precursor in the starfish Asterias rubens was confirmed by cDNA sequencing and peptides derived from this precursor (e.g. AYHSALPF-NH2, GYHSGLPF-NH2 and LHSALPF-NH2) were synthesized. The ability of these peptides to bind metals was investigated using UV/Vis, NMR, circular dichroism and EPR spectroscopy. Results AYHSALPF-NH2 and GYHSGLPF-NH2 bind Cu(II) and Ni(II) and generate metal-linked dimers to form ternary complexes with LHSALPF-NH2. Investigation of the evolutionary history of the histidine residue that confers these properties revealed that it can be traced to the common ancestor of echinoderms, which is estimated to have lived ~500million years ago. However, L-type precursors comprising multiple SALMFamides with the histidine residue forming an ATCUN motif appears to be a feature that has evolved uniquely in starfish (Asteroidea). General Significance The discovery of a SALMFamide-type neuropeptide precursor protein that gives rise to a “cocktail” of peptides that bind metal ions and generate metal-linked dimers provides a new insight on ATCUN motif-containing neuropeptides. This property of L-type SALMFamides in the Asteroidea may be associated with a role in regulation of the unusual extra-oral feeding behaviour of starfish.
Graphical abstract
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part A
Author(s): Christopher E. Jones, Meet Zandawala, Dean C. Semmens, Sarah Anderson, Graeme R. Hanson, Daniel A. Janies, Maurice R. Elphick
Background Neuropeptides with an Amino Terminal Cu(II), Ni(II) Binding (ATCUN) motif (H2N-xxH) bind Cu(II)/Ni(II) ions. Here we report the novel discovery of a neuropeptide precursor that gives rise to a “cocktail” of peptides that bind Cu(II)/Ni(II) and form ternary complexes — the L-type SALMFamide precursor in the starfish Asterias rubens. Methods Echinoderm transcriptome sequence data were analysed to identify transcripts encoding precursors of SALMFamide-type neuropeptides. The sequence of the L-type SALMFamide precursor in the starfish Asterias rubens was confirmed by cDNA sequencing and peptides derived from this precursor (e.g. AYHSALPF-NH2, GYHSGLPF-NH2 and LHSALPF-NH2) were synthesized. The ability of these peptides to bind metals was investigated using UV/Vis, NMR, circular dichroism and EPR spectroscopy. Results AYHSALPF-NH2 and GYHSGLPF-NH2 bind Cu(II) and Ni(II) and generate metal-linked dimers to form ternary complexes with LHSALPF-NH2. Investigation of the evolutionary history of the histidine residue that confers these properties revealed that it can be traced to the common ancestor of echinoderms, which is estimated to have lived ~500million years ago. However, L-type precursors comprising multiple SALMFamides with the histidine residue forming an ATCUN motif appears to be a feature that has evolved uniquely in starfish (Asteroidea). General Significance The discovery of a SALMFamide-type neuropeptide precursor protein that gives rise to a “cocktail” of peptides that bind metal ions and generate metal-linked dimers provides a new insight on ATCUN motif-containing neuropeptides. This property of L-type SALMFamides in the Asteroidea may be associated with a role in regulation of the unusual extra-oral feeding behaviour of starfish.
Graphical abstract
Ligand tunnels in T. brucei and human CYP51: Insights for parasite-specific drug design
Publication date: January 2016
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part A
Author(s): Xiaofeng Yu, Prajwal Nandekar, Ghulam Mustafa, Vlad Cojocaru, Galina I. Lepesheva, Rebecca C. Wade
Background Cytochrome P450 sterol 14α-demethylase (CYP51) is an essential enzyme for sterol biosynthesis and a target for anti-parasitic drug design. However, the design of parasite-specific drugs that inhibit parasitic CYP51 without severe side effects remains challenging. The active site of CYP51 is situated in the interior of the protein. Here, we characterize the potential ligand egress routes and mechanisms in Trypanosoma brucei and human CYP51 enzymes. Methods We performed Random Acceleration Molecular Dynamics simulations of the egress of four different ligands from the active site of models of soluble and membrane-bound T. brucei CYP51 and of soluble human CYP51. Results In the simulations, tunnel 2f, which leads to the membrane, was found to be the predominant ligand egress tunnel for all the ligands studied. Tunnels S, 1 and W, which lead to the cytosol, were also used in T. brucei CYP51, whereas tunnel 1 was the only other tunnel used significantly in human CYP51. The common tunnels found previously in other CYPs were barely used. The ligand egress times were shorter for human than T. brucei CYP51, suggesting lower barriers to ligand passage. Two gating residues, F105 and M460, in T. brucei CYP51 that modulate the opening of tunnels 2f and S were identified. Conclusions Although the main egress tunnel was the same, differences in the tunnel-lining residues, ligand passage and tunnel usage were found between T. brucei and human CYP51s. General Significance The results provide a basis for the design of selective anti-parasitic agents targeting the ligand tunnels.
Graphical abstract
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part A
Author(s): Xiaofeng Yu, Prajwal Nandekar, Ghulam Mustafa, Vlad Cojocaru, Galina I. Lepesheva, Rebecca C. Wade
Background Cytochrome P450 sterol 14α-demethylase (CYP51) is an essential enzyme for sterol biosynthesis and a target for anti-parasitic drug design. However, the design of parasite-specific drugs that inhibit parasitic CYP51 without severe side effects remains challenging. The active site of CYP51 is situated in the interior of the protein. Here, we characterize the potential ligand egress routes and mechanisms in Trypanosoma brucei and human CYP51 enzymes. Methods We performed Random Acceleration Molecular Dynamics simulations of the egress of four different ligands from the active site of models of soluble and membrane-bound T. brucei CYP51 and of soluble human CYP51. Results In the simulations, tunnel 2f, which leads to the membrane, was found to be the predominant ligand egress tunnel for all the ligands studied. Tunnels S, 1 and W, which lead to the cytosol, were also used in T. brucei CYP51, whereas tunnel 1 was the only other tunnel used significantly in human CYP51. The common tunnels found previously in other CYPs were barely used. The ligand egress times were shorter for human than T. brucei CYP51, suggesting lower barriers to ligand passage. Two gating residues, F105 and M460, in T. brucei CYP51 that modulate the opening of tunnels 2f and S were identified. Conclusions Although the main egress tunnel was the same, differences in the tunnel-lining residues, ligand passage and tunnel usage were found between T. brucei and human CYP51s. General Significance The results provide a basis for the design of selective anti-parasitic agents targeting the ligand tunnels.
Graphical abstract
De novo lipogenesis in Atlantic salmon adipocytes
Publication date: January 2016
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part A
Author(s): Marta Bou, Marijana Todorčević, Jacob Torgersen, Stanko Škugor, Isabel Navarro, Bente Ruyter
Background Carnivorous teleost fish utilize glucose poorly, and the reason for this is not known. It is possible that the capacity of adipocytes to synthesize lipids from carbohydrate precursors through a process known as “de novo lipogenesis” (DNL) is one of the factors that contributes to glucose intolerance in Atlantic salmon. Methods Primary adipocytes from Atlantic salmon differentiated in vitro were incubated with radiolabelled glucose in order to explore the capacity of salmon adipocytes to synthesize and deposit lipids from glucose through DNL. The lipid-storage capacity of adipocytes incubated with glucose was compared with that of cells incubated with the fatty acid palmitic acid. Quantitative PCR and immunohistochemistry were used to assess changes of genes and proteins involved in glucose and lipid transport and metabolism. Results Less than 0.1% of the radiolabelled glucose was metabolized to the fatty acids 16:0 and the stearoyl-CoA desaturase products 16:1 and 18:1 by DNL, whereas approximately 40% was converted to glycerol to form the triacylglycerol backbone of lipids. Transcriptional analysis indicated that adipocytes ensure the availability of necessary cofactors and other substrates for lipid synthesis and storage from glycolysis, the pentose phosphate pathway and glyceroneogenesis. Conclusions We have shown for the first time that the DNL pathway is active in fish adipocytes. The capacity of the pathway to convert glucose into cellular lipids for storage is relatively low. General significance The limited capacity of adipocytes to utilize glucose as a substrate for lipid deposition may contribute to glucose intolerance in salmonids.
Source:Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1860, Issue 1, Part A
Author(s): Marta Bou, Marijana Todorčević, Jacob Torgersen, Stanko Škugor, Isabel Navarro, Bente Ruyter
Background Carnivorous teleost fish utilize glucose poorly, and the reason for this is not known. It is possible that the capacity of adipocytes to synthesize lipids from carbohydrate precursors through a process known as “de novo lipogenesis” (DNL) is one of the factors that contributes to glucose intolerance in Atlantic salmon. Methods Primary adipocytes from Atlantic salmon differentiated in vitro were incubated with radiolabelled glucose in order to explore the capacity of salmon adipocytes to synthesize and deposit lipids from glucose through DNL. The lipid-storage capacity of adipocytes incubated with glucose was compared with that of cells incubated with the fatty acid palmitic acid. Quantitative PCR and immunohistochemistry were used to assess changes of genes and proteins involved in glucose and lipid transport and metabolism. Results Less than 0.1% of the radiolabelled glucose was metabolized to the fatty acids 16:0 and the stearoyl-CoA desaturase products 16:1 and 18:1 by DNL, whereas approximately 40% was converted to glycerol to form the triacylglycerol backbone of lipids. Transcriptional analysis indicated that adipocytes ensure the availability of necessary cofactors and other substrates for lipid synthesis and storage from glycolysis, the pentose phosphate pathway and glyceroneogenesis. Conclusions We have shown for the first time that the DNL pathway is active in fish adipocytes. The capacity of the pathway to convert glucose into cellular lipids for storage is relatively low. General significance The limited capacity of adipocytes to utilize glucose as a substrate for lipid deposition may contribute to glucose intolerance in salmonids.