Fabrizio Chiti - Academia.edu (original) (raw)
Papers by Fabrizio Chiti
European Journal of Medicinal Chemistry, 2016
Amyloid or similar protein aggregates are the hallmarks of many disorders, including Alzheimer's,... more Amyloid or similar protein aggregates are the hallmarks of many disorders, including Alzheimer's, Parkinson's, Huntington's diseases and amyloidoses. The inhibition of the formation of these aberrant species by small molecules is a promising strategy for disease treatment. However, at present, all such diseases lack an appropriate therapeutic approach based on small molecules. In this work we have evaluated five bis(indolyl)phenylmethane derivatives to reduce amyloid fibril formation by hen egg white lysozyme (HEWL) and its associated cytotoxicity. HEWL is a widely used model system to study the fundamentals of amyloid fibril formation and is heterologous to human lysozyme, which forms amyloid fibrils in a familial form of systemic amyloidosis. HEWL aggregation was tested in the presence and absence of the five compounds, under conditions in which the protein is partially unfolded. To this purpose, various techniques were used, including Congo red and Thioflavin T binding assays, atomic force microscopy, Fourier-Transform Infrared spectroscopy and cell-based cytotoxicity assays, such as the MTT reduction test and the trypan blue test. It was found that all compounds inhibited the formation of amyloid fibrils and their associated toxicity, diverging the aggregation process towards the formation of large, morphologically amorphous, unstructured, nontoxic aggregates, thus resembling class I molecules defined previously. In addition, the five compounds also appeared to disaggregate pre-formed fibrils of HEWL, which categorizes them into class IA. The half maximal inhibitory concentration (IC50) was found to be ca 12.3 ± 1.0 mM for the forefather compound.
Iris Lindberg,1 James Shorter,2 R. Luke Wiseman,3 Fabrizio Chiti,4 Chad A. Dickey,5 and XPamela J... more Iris Lindberg,1 James Shorter,2 R. Luke Wiseman,3 Fabrizio Chiti,4 Chad A. Dickey,5 and XPamela J. McLean6 1University of Maryland School of Medicine, Baltimore, Maryland 21201, 2University of Pennsylvania, Philadelphia, Pennsylvania 19104, 3The Scripps Research Institute, La Jolla, California 92037, 4University of Florence, 50121 Florence, Italy, 5University of South Florida, Tampa, Florida 33620, and 6Mayo Clinic, Jacksonville, Florida 32224
ACS Chemical Neuroscience
The molecular composition of the plasma membrane plays a key role in mediating the susceptibility... more The molecular composition of the plasma membrane plays a key role in mediating the susceptibility of cells to perturbations induced by toxic molecules. The pharmacological regulation of the properties of the cell membrane has therefore the potential to enhance cellular resilience to a wide variety of chemical and biological compounds. In this study, we investigate the ability of claramine, a blood−brain barrier permeable small molecule in the aminosterol class, to neutralize the toxicity of acute biological threat agents, including melittin from honeybee venom and α-hemolysin from Staphylococcus aureus. Our results show that claramine neutralizes the toxicity of these pore-forming agents by preventing their interactions with cell membranes without perturbing their structures in a detectable manner. We thus demonstrate that the exogenous administration of an aminosterol can tune the properties of lipid membranes and protect cells from diverse biotoxins, including not just misfolded protein oligomers as previously shown but also biological protein-based toxins. Our results indicate that the investigation of regulators of the physicochemical properties of cell membranes offers novel opportunities to develop countermeasures against an extensive set of cytotoxic effects associated with cell membrane disruption.
ACS Chemical Neuroscience, 2019
Cellular and Molecular Life Sciences
Alzheimer's disease is characterized by the accumulation in the brain of the amyloid β (Aβ) p... more Alzheimer's disease is characterized by the accumulation in the brain of the amyloid β (Aβ) peptide in the form of senile plaques. According to the amyloid hypothesis, the aggregation process of Aβ also generates smaller soluble misfolded oligomers that contribute to disease progression. One of the mechanisms of Aβ oligomer cytotoxicity is the aberrant interaction of these species with the phospholipid bilayer of cell membranes, with a consequent increase in cytosolic Ca2+ levels, flowing from the extracellular space, and production of reactive oxygen species (ROS). Here we investigated the relationship between the increase in Ca2+ and ROS levels immediately after the exposure to misfolded protein oligomers, asking whether they are simultaneous or instead one precedes the other. Using Aβ42-derived diffusible ligands (ADDLs) and type A HypF-N model oligomers (OAs), we followed the kinetics of ROS production and Ca2+ influx in human neuroblastoma SH-SY5Y cells and rat primary cort...
Molecules
TAR DNA-binding protein 43 (TDP-43) forms intraneuronal cytoplasmic inclusions associated with am... more TAR DNA-binding protein 43 (TDP-43) forms intraneuronal cytoplasmic inclusions associated with amyotrophic lateral sclerosis and ubiquitin-positive frontotemporal lobar degeneration. Its N-terminal domain (NTD) can dimerise/oligomerise with the head-to-tail arrangement, which is essential for function but also favours liquid-liquid phase separation and inclusion formation of full-length TDP-43. Using various biophysical approaches, we identified an alternative conformational state of NTD in the presence of Sulfobetaine 3-10 (SB3-10), with higher content of α-helical structure and tryptophan solvent exposure. NMR shows a highly mobile structure, with partially folded regions and β-sheet content decrease, with a concomitant increase of α-helical structure. It is monomeric and reverts to native oligomeric NTD upon SB3-10 dilution. The equilibrium GdnHCl-induced denaturation shows a cooperative folding and a somewhat lower conformational stability. When the aggregation processes were co...
Scientific Reports, 2020
Studies on the amyloidogenic N-terminal domain of the E. coli HypF protein (HypF-N) have contribu... more Studies on the amyloidogenic N-terminal domain of the E. coli HypF protein (HypF-N) have contributed significantly to a detailed understanding of the pathogenic mechanisms in neurodegenerative diseases characterised by the formation of misfolded oligomers, by proteins such as amyloid-β, α-synuclein and tau. Given that both cell membranes and mitochondria are increasingly recognised as key targets of oligomer toxicity, we investigated the damaging effects of aggregates of HypF-N on mitochondrial membranes. Essentially, we found that HypF-N oligomers characterised by high surface hydrophobicity (type A) were able to trigger a robust permeabilisation of mito-mimetic liposomes possessing cardiolipin-rich membranes and dysfunction of isolated mitochondria, as demonstrated by a combination of mitochondrial shrinking, lowering of mitochondrial membrane potential and cytochrome c release. Furthermore, using single-channel electrophysiology recordings we obtained evidence that the type A agg...
The Journal of biological chemistry, Jan 29, 2018
A set of missense mutations in the gene encoding profilin-1 has been linked to the onset of famil... more A set of missense mutations in the gene encoding profilin-1 has been linked to the onset of familial forms of ALS (fALS), also known as Lou Gehrig's disease. The pathogenic potential of these mutations is linked to the formation of intracellular inclusions of the mutant proteins and correlates with the mutation-induced destabilization of its native, fully folded state. However, the mechanism by which these mutations promote misfolding and self-assembly is yet unclear. Here, using temperature-jump and stopped-flow kinetic measurements, we show that, during refolding, WT profilin-1 transiently populates a partially folded (PF) state endowed with hydrophobic clusters exposed to the solvent and with no detectable secondary structure. We observed that this conformational state is marginally stable at neutral pH but becomes significantly populated at mildly acidic pH. Interestingly, the fALS-associated mutations did not cause a change in the refolding mechanism of profilin-1, but indu...
Biological Chemistry, 2016
Profilin-1 is a small protein involved in actin-mediated cytoskeleton rearrangement. Recently, mu... more Profilin-1 is a small protein involved in actin-mediated cytoskeleton rearrangement. Recently, mutations of profilin-1 have been associated with familial amyotrophic lateral sclerosis. It was previously reported that pathogenic mutations of profilin-1 increase the aggregation propensity of this protein, leaving its function unaffected. However, it is not clear if the mutations act by decreasing the conformational stability or by promoting structural perturbations of the folded state of this protein. In this work we have purified three novel profilin-1 mutants that were recently discovered and have investigated their conformational stability, structural features and aggregation behaviourin vitro. Analysis of the data obtained with the three novel variants, and a global statistical analysis with all profilin-1 mutants so far characterised, indicate significant correlations between aggregation propensity and structural perturbations of the folded state, rather than its conformational s...
Scientific Reports, 2019
We have studied the intrinsic fluorescence spectra of a monomeric variant of human transthyretin ... more We have studied the intrinsic fluorescence spectra of a monomeric variant of human transthyretin (M-ttR), a protein involved in the transport of the thyroid hormone and retinol and associated with various forms of amyloidosis, extending our analysis to the second order derivative of the spectra. This procedure allowed to identify three peaks readily assigned to Trp41, as the three peaks were also visible in a mutant lacking the other tryptophan (Trp79) and had similar FRET efficiency values with an acceptor molecule positioned at position 10. The wavelength values of the three peaks and their susceptibility to acrylamide quenching revealed that the three corresponding conformers experience different solvent-exposure, polarity of the environment and flexibility. We could monitor the three peaks individually in urea-unfolding and pH-unfolding curves. This revealed changes in the distribution of the corresponding conformers, indicating conformational changes and alterations of the dynamics of the microenvironment that surrounds the associated tryptophan residue in such transitions, but also native-like conformers of such residues in unfolded states. We also found that the amyloidogenic state adopted by M-TTR at mildly low pH has a structural and dynamical microenvironment surrounding Trp41 indistinguishable from that of the fully folded and soluble state at neutral pH.
Scientific Reports, 2019
We have studied the intrinsic fluorescence spectra of a monomeric variant of human transthyretin ... more We have studied the intrinsic fluorescence spectra of a monomeric variant of human transthyretin (M-ttR), a protein involved in the transport of the thyroid hormone and retinol and associated with various forms of amyloidosis, extending our analysis to the second order derivative of the spectra. This procedure allowed to identify three peaks readily assigned to Trp41, as the three peaks were also visible in a mutant lacking the other tryptophan (Trp79) and had similar FRET efficiency values with an acceptor molecule positioned at position 10. The wavelength values of the three peaks and their susceptibility to acrylamide quenching revealed that the three corresponding conformers experience different solvent-exposure, polarity of the environment and flexibility. We could monitor the three peaks individually in urea-unfolding and pH-unfolding curves. This revealed changes in the distribution of the corresponding conformers, indicating conformational changes and alterations of the dynamics of the microenvironment that surrounds the associated tryptophan residue in such transitions, but also native-like conformers of such residues in unfolded states. We also found that the amyloidogenic state adopted by M-TTR at mildly low pH has a structural and dynamical microenvironment surrounding Trp41 indistinguishable from that of the fully folded and soluble state at neutral pH.
Journal of Biological Chemistry, 2019
Novel imaging techniques with ever-increasing resolution are invaluable tools for the study of pr... more Novel imaging techniques with ever-increasing resolution are invaluable tools for the study of protein deposition, as they allow the self-assembly of proteins to be directly investigated in living cells. For the first time, the acceleration in Aβ42 aggregation induced by the Arctic mutation was monitored in cells, revealing a number of distinct morphologies that form sequentially. This approach will help discriminate the impacts of mutations on amyloid protein processing, Aβ aggregation propensity, and other mechanistic outcomes.
Frontiers in Molecular Neuroscience, 2017
Chaperones have long been recognized to play well defined functions such as to: (i) assist protei... more Chaperones have long been recognized to play well defined functions such as to: (i) assist protein folding and promote formation and maintenance of multisubunit complexes; (ii) mediate protein degradation; (iii) inhibit protein aggregation; and (iv) promote disassembly of undesired aberrant protein aggregates. In addition to these well-established functions, it is increasingly clear that chaperones can also interact with aberrant protein aggregates, such as pre-fibrillar oligomers and fibrils, and inhibit their toxicity commonly associated with neurodegenerative diseases without promoting their disassembly. In particular, the evidence collected so far in different labs, exploiting different experimental approaches and using different chaperones and client aggregated proteins, indicates the existence of two distinct mechanisms of action mediated by the chaperones to neutralize the toxicity of aberrant proteins oligomers: (i) direct binding of the chaperones to the hydrophobic patches exposed on the oligomer/fibril surface, with resulting shielding or masking of the moieties responsible for the aberrant interactions with cellular targets; (ii) chaperone-mediated conversion of aberrant protein aggregates into large and more innocuous species, resulting in a decrease of their surface-to-volume ratio and diffusibility and in deposits more easily manageable by clearance mechanisms, such as autophagy. In this review article we will describe the in vitro and in vivo evidence supporting both mechanisms and how this results in a suppression of the detrimental effects caused by protein misfolded aggregates.
Journal of Biological Chemistry, 2019
Novel imaging techniques with ever-increasing resolution are invaluable tools for the study of pr... more Novel imaging techniques with ever-increasing resolution are invaluable tools for the study of protein deposition, as they allow the self-assembly of proteins to be directly investigated in living cells. For the first time, the acceleration in Aβ42 aggregation induced by the Arctic mutation was monitored in cells, revealing a number of distinct morphologies that form sequentially. This approach will help discriminate the impacts of mutations on amyloid protein processing, Aβ aggregation propensity, and other mechanistic outcomes.
Frontiers in Molecular Neuroscience, 2017
Chaperones have long been recognized to play well defined functions such as to: (i) assist protei... more Chaperones have long been recognized to play well defined functions such as to: (i) assist protein folding and promote formation and maintenance of multisubunit complexes; (ii) mediate protein degradation; (iii) inhibit protein aggregation; and (iv) promote disassembly of undesired aberrant protein aggregates. In addition to these well-established functions, it is increasingly clear that chaperones can also interact with aberrant protein aggregates, such as pre-fibrillar oligomers and fibrils, and inhibit their toxicity commonly associated with neurodegenerative diseases without promoting their disassembly. In particular, the evidence collected so far in different labs, exploiting different experimental approaches and using different chaperones and client aggregated proteins, indicates the existence of two distinct mechanisms of action mediated by the chaperones to neutralize the toxicity of aberrant proteins oligomers: (i) direct binding of the chaperones to the hydrophobic patches exposed on the oligomer/fibril surface, with resulting shielding or masking of the moieties responsible for the aberrant interactions with cellular targets; (ii) chaperone-mediated conversion of aberrant protein aggregates into large and more innocuous species, resulting in a decrease of their surface-to-volume ratio and diffusibility and in deposits more easily manageable by clearance mechanisms, such as autophagy. In this review article we will describe the in vitro and in vivo evidence supporting both mechanisms and how this results in a suppression of the detrimental effects caused by protein misfolded aggregates.
The Journal of biological chemistry, Jan 21, 2016
Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin positive... more Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-U) are two clinically distinct neurodegenerative conditions sharing a similar histopathology characterized by the nuclear clearance of TDP-43 and its associated deposition into cytoplasmic inclusions in different areas of the central nervous system. Given the concomitant occurrence of TDP-43 nuclear depletion and cytoplasmic accumulation, it has been proposed that TDP-43 proteinopathies originate from either a loss-of-function (LOF) mechanism, a gain-of-function (GOF) process, or both. We have addressed this issue by transfecting murine NSC34 and N2a cells with siRNA for endogenous murine TDP-43 and with human recombinant TDP-43 inclusion bodies (IBs). These two strategies allowed the depletion of nuclear TDP-43 and the accumulation of cytoplasmic TDP-43 aggregates to occur separately and independently. Endogenous and exogenous TDP-43 were monitored and quantified using...
The Journal of biological chemistry, Jan 21, 2016
Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin positive... more Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-U) are two clinically distinct neurodegenerative conditions sharing a similar histopathology characterized by the nuclear clearance of TDP-43 and its associated deposition into cytoplasmic inclusions in different areas of the central nervous system. Given the concomitant occurrence of TDP-43 nuclear depletion and cytoplasmic accumulation, it has been proposed that TDP-43 proteinopathies originate from either a loss-of-function (LOF) mechanism, a gain-of-function (GOF) process, or both. We have addressed this issue by transfecting murine NSC34 and N2a cells with siRNA for endogenous murine TDP-43 and with human recombinant TDP-43 inclusion bodies (IBs). These two strategies allowed the depletion of nuclear TDP-43 and the accumulation of cytoplasmic TDP-43 aggregates to occur separately and independently. Endogenous and exogenous TDP-43 were monitored and quantified using...
Protein Engineering Design and Selection, 2011
Acta Crystallographica Section A Foundations of Crystallography, 2005
European Journal of Medicinal Chemistry, 2016
Amyloid or similar protein aggregates are the hallmarks of many disorders, including Alzheimer's,... more Amyloid or similar protein aggregates are the hallmarks of many disorders, including Alzheimer's, Parkinson's, Huntington's diseases and amyloidoses. The inhibition of the formation of these aberrant species by small molecules is a promising strategy for disease treatment. However, at present, all such diseases lack an appropriate therapeutic approach based on small molecules. In this work we have evaluated five bis(indolyl)phenylmethane derivatives to reduce amyloid fibril formation by hen egg white lysozyme (HEWL) and its associated cytotoxicity. HEWL is a widely used model system to study the fundamentals of amyloid fibril formation and is heterologous to human lysozyme, which forms amyloid fibrils in a familial form of systemic amyloidosis. HEWL aggregation was tested in the presence and absence of the five compounds, under conditions in which the protein is partially unfolded. To this purpose, various techniques were used, including Congo red and Thioflavin T binding assays, atomic force microscopy, Fourier-Transform Infrared spectroscopy and cell-based cytotoxicity assays, such as the MTT reduction test and the trypan blue test. It was found that all compounds inhibited the formation of amyloid fibrils and their associated toxicity, diverging the aggregation process towards the formation of large, morphologically amorphous, unstructured, nontoxic aggregates, thus resembling class I molecules defined previously. In addition, the five compounds also appeared to disaggregate pre-formed fibrils of HEWL, which categorizes them into class IA. The half maximal inhibitory concentration (IC50) was found to be ca 12.3 ± 1.0 mM for the forefather compound.
Iris Lindberg,1 James Shorter,2 R. Luke Wiseman,3 Fabrizio Chiti,4 Chad A. Dickey,5 and XPamela J... more Iris Lindberg,1 James Shorter,2 R. Luke Wiseman,3 Fabrizio Chiti,4 Chad A. Dickey,5 and XPamela J. McLean6 1University of Maryland School of Medicine, Baltimore, Maryland 21201, 2University of Pennsylvania, Philadelphia, Pennsylvania 19104, 3The Scripps Research Institute, La Jolla, California 92037, 4University of Florence, 50121 Florence, Italy, 5University of South Florida, Tampa, Florida 33620, and 6Mayo Clinic, Jacksonville, Florida 32224
ACS Chemical Neuroscience
The molecular composition of the plasma membrane plays a key role in mediating the susceptibility... more The molecular composition of the plasma membrane plays a key role in mediating the susceptibility of cells to perturbations induced by toxic molecules. The pharmacological regulation of the properties of the cell membrane has therefore the potential to enhance cellular resilience to a wide variety of chemical and biological compounds. In this study, we investigate the ability of claramine, a blood−brain barrier permeable small molecule in the aminosterol class, to neutralize the toxicity of acute biological threat agents, including melittin from honeybee venom and α-hemolysin from Staphylococcus aureus. Our results show that claramine neutralizes the toxicity of these pore-forming agents by preventing their interactions with cell membranes without perturbing their structures in a detectable manner. We thus demonstrate that the exogenous administration of an aminosterol can tune the properties of lipid membranes and protect cells from diverse biotoxins, including not just misfolded protein oligomers as previously shown but also biological protein-based toxins. Our results indicate that the investigation of regulators of the physicochemical properties of cell membranes offers novel opportunities to develop countermeasures against an extensive set of cytotoxic effects associated with cell membrane disruption.
ACS Chemical Neuroscience, 2019
Cellular and Molecular Life Sciences
Alzheimer's disease is characterized by the accumulation in the brain of the amyloid β (Aβ) p... more Alzheimer's disease is characterized by the accumulation in the brain of the amyloid β (Aβ) peptide in the form of senile plaques. According to the amyloid hypothesis, the aggregation process of Aβ also generates smaller soluble misfolded oligomers that contribute to disease progression. One of the mechanisms of Aβ oligomer cytotoxicity is the aberrant interaction of these species with the phospholipid bilayer of cell membranes, with a consequent increase in cytosolic Ca2+ levels, flowing from the extracellular space, and production of reactive oxygen species (ROS). Here we investigated the relationship between the increase in Ca2+ and ROS levels immediately after the exposure to misfolded protein oligomers, asking whether they are simultaneous or instead one precedes the other. Using Aβ42-derived diffusible ligands (ADDLs) and type A HypF-N model oligomers (OAs), we followed the kinetics of ROS production and Ca2+ influx in human neuroblastoma SH-SY5Y cells and rat primary cort...
Molecules
TAR DNA-binding protein 43 (TDP-43) forms intraneuronal cytoplasmic inclusions associated with am... more TAR DNA-binding protein 43 (TDP-43) forms intraneuronal cytoplasmic inclusions associated with amyotrophic lateral sclerosis and ubiquitin-positive frontotemporal lobar degeneration. Its N-terminal domain (NTD) can dimerise/oligomerise with the head-to-tail arrangement, which is essential for function but also favours liquid-liquid phase separation and inclusion formation of full-length TDP-43. Using various biophysical approaches, we identified an alternative conformational state of NTD in the presence of Sulfobetaine 3-10 (SB3-10), with higher content of α-helical structure and tryptophan solvent exposure. NMR shows a highly mobile structure, with partially folded regions and β-sheet content decrease, with a concomitant increase of α-helical structure. It is monomeric and reverts to native oligomeric NTD upon SB3-10 dilution. The equilibrium GdnHCl-induced denaturation shows a cooperative folding and a somewhat lower conformational stability. When the aggregation processes were co...
Scientific Reports, 2020
Studies on the amyloidogenic N-terminal domain of the E. coli HypF protein (HypF-N) have contribu... more Studies on the amyloidogenic N-terminal domain of the E. coli HypF protein (HypF-N) have contributed significantly to a detailed understanding of the pathogenic mechanisms in neurodegenerative diseases characterised by the formation of misfolded oligomers, by proteins such as amyloid-β, α-synuclein and tau. Given that both cell membranes and mitochondria are increasingly recognised as key targets of oligomer toxicity, we investigated the damaging effects of aggregates of HypF-N on mitochondrial membranes. Essentially, we found that HypF-N oligomers characterised by high surface hydrophobicity (type A) were able to trigger a robust permeabilisation of mito-mimetic liposomes possessing cardiolipin-rich membranes and dysfunction of isolated mitochondria, as demonstrated by a combination of mitochondrial shrinking, lowering of mitochondrial membrane potential and cytochrome c release. Furthermore, using single-channel electrophysiology recordings we obtained evidence that the type A agg...
The Journal of biological chemistry, Jan 29, 2018
A set of missense mutations in the gene encoding profilin-1 has been linked to the onset of famil... more A set of missense mutations in the gene encoding profilin-1 has been linked to the onset of familial forms of ALS (fALS), also known as Lou Gehrig's disease. The pathogenic potential of these mutations is linked to the formation of intracellular inclusions of the mutant proteins and correlates with the mutation-induced destabilization of its native, fully folded state. However, the mechanism by which these mutations promote misfolding and self-assembly is yet unclear. Here, using temperature-jump and stopped-flow kinetic measurements, we show that, during refolding, WT profilin-1 transiently populates a partially folded (PF) state endowed with hydrophobic clusters exposed to the solvent and with no detectable secondary structure. We observed that this conformational state is marginally stable at neutral pH but becomes significantly populated at mildly acidic pH. Interestingly, the fALS-associated mutations did not cause a change in the refolding mechanism of profilin-1, but indu...
Biological Chemistry, 2016
Profilin-1 is a small protein involved in actin-mediated cytoskeleton rearrangement. Recently, mu... more Profilin-1 is a small protein involved in actin-mediated cytoskeleton rearrangement. Recently, mutations of profilin-1 have been associated with familial amyotrophic lateral sclerosis. It was previously reported that pathogenic mutations of profilin-1 increase the aggregation propensity of this protein, leaving its function unaffected. However, it is not clear if the mutations act by decreasing the conformational stability or by promoting structural perturbations of the folded state of this protein. In this work we have purified three novel profilin-1 mutants that were recently discovered and have investigated their conformational stability, structural features and aggregation behaviourin vitro. Analysis of the data obtained with the three novel variants, and a global statistical analysis with all profilin-1 mutants so far characterised, indicate significant correlations between aggregation propensity and structural perturbations of the folded state, rather than its conformational s...
Scientific Reports, 2019
We have studied the intrinsic fluorescence spectra of a monomeric variant of human transthyretin ... more We have studied the intrinsic fluorescence spectra of a monomeric variant of human transthyretin (M-ttR), a protein involved in the transport of the thyroid hormone and retinol and associated with various forms of amyloidosis, extending our analysis to the second order derivative of the spectra. This procedure allowed to identify three peaks readily assigned to Trp41, as the three peaks were also visible in a mutant lacking the other tryptophan (Trp79) and had similar FRET efficiency values with an acceptor molecule positioned at position 10. The wavelength values of the three peaks and their susceptibility to acrylamide quenching revealed that the three corresponding conformers experience different solvent-exposure, polarity of the environment and flexibility. We could monitor the three peaks individually in urea-unfolding and pH-unfolding curves. This revealed changes in the distribution of the corresponding conformers, indicating conformational changes and alterations of the dynamics of the microenvironment that surrounds the associated tryptophan residue in such transitions, but also native-like conformers of such residues in unfolded states. We also found that the amyloidogenic state adopted by M-TTR at mildly low pH has a structural and dynamical microenvironment surrounding Trp41 indistinguishable from that of the fully folded and soluble state at neutral pH.
Scientific Reports, 2019
We have studied the intrinsic fluorescence spectra of a monomeric variant of human transthyretin ... more We have studied the intrinsic fluorescence spectra of a monomeric variant of human transthyretin (M-ttR), a protein involved in the transport of the thyroid hormone and retinol and associated with various forms of amyloidosis, extending our analysis to the second order derivative of the spectra. This procedure allowed to identify three peaks readily assigned to Trp41, as the three peaks were also visible in a mutant lacking the other tryptophan (Trp79) and had similar FRET efficiency values with an acceptor molecule positioned at position 10. The wavelength values of the three peaks and their susceptibility to acrylamide quenching revealed that the three corresponding conformers experience different solvent-exposure, polarity of the environment and flexibility. We could monitor the three peaks individually in urea-unfolding and pH-unfolding curves. This revealed changes in the distribution of the corresponding conformers, indicating conformational changes and alterations of the dynamics of the microenvironment that surrounds the associated tryptophan residue in such transitions, but also native-like conformers of such residues in unfolded states. We also found that the amyloidogenic state adopted by M-TTR at mildly low pH has a structural and dynamical microenvironment surrounding Trp41 indistinguishable from that of the fully folded and soluble state at neutral pH.
Journal of Biological Chemistry, 2019
Novel imaging techniques with ever-increasing resolution are invaluable tools for the study of pr... more Novel imaging techniques with ever-increasing resolution are invaluable tools for the study of protein deposition, as they allow the self-assembly of proteins to be directly investigated in living cells. For the first time, the acceleration in Aβ42 aggregation induced by the Arctic mutation was monitored in cells, revealing a number of distinct morphologies that form sequentially. This approach will help discriminate the impacts of mutations on amyloid protein processing, Aβ aggregation propensity, and other mechanistic outcomes.
Frontiers in Molecular Neuroscience, 2017
Chaperones have long been recognized to play well defined functions such as to: (i) assist protei... more Chaperones have long been recognized to play well defined functions such as to: (i) assist protein folding and promote formation and maintenance of multisubunit complexes; (ii) mediate protein degradation; (iii) inhibit protein aggregation; and (iv) promote disassembly of undesired aberrant protein aggregates. In addition to these well-established functions, it is increasingly clear that chaperones can also interact with aberrant protein aggregates, such as pre-fibrillar oligomers and fibrils, and inhibit their toxicity commonly associated with neurodegenerative diseases without promoting their disassembly. In particular, the evidence collected so far in different labs, exploiting different experimental approaches and using different chaperones and client aggregated proteins, indicates the existence of two distinct mechanisms of action mediated by the chaperones to neutralize the toxicity of aberrant proteins oligomers: (i) direct binding of the chaperones to the hydrophobic patches exposed on the oligomer/fibril surface, with resulting shielding or masking of the moieties responsible for the aberrant interactions with cellular targets; (ii) chaperone-mediated conversion of aberrant protein aggregates into large and more innocuous species, resulting in a decrease of their surface-to-volume ratio and diffusibility and in deposits more easily manageable by clearance mechanisms, such as autophagy. In this review article we will describe the in vitro and in vivo evidence supporting both mechanisms and how this results in a suppression of the detrimental effects caused by protein misfolded aggregates.
Journal of Biological Chemistry, 2019
Novel imaging techniques with ever-increasing resolution are invaluable tools for the study of pr... more Novel imaging techniques with ever-increasing resolution are invaluable tools for the study of protein deposition, as they allow the self-assembly of proteins to be directly investigated in living cells. For the first time, the acceleration in Aβ42 aggregation induced by the Arctic mutation was monitored in cells, revealing a number of distinct morphologies that form sequentially. This approach will help discriminate the impacts of mutations on amyloid protein processing, Aβ aggregation propensity, and other mechanistic outcomes.
Frontiers in Molecular Neuroscience, 2017
Chaperones have long been recognized to play well defined functions such as to: (i) assist protei... more Chaperones have long been recognized to play well defined functions such as to: (i) assist protein folding and promote formation and maintenance of multisubunit complexes; (ii) mediate protein degradation; (iii) inhibit protein aggregation; and (iv) promote disassembly of undesired aberrant protein aggregates. In addition to these well-established functions, it is increasingly clear that chaperones can also interact with aberrant protein aggregates, such as pre-fibrillar oligomers and fibrils, and inhibit their toxicity commonly associated with neurodegenerative diseases without promoting their disassembly. In particular, the evidence collected so far in different labs, exploiting different experimental approaches and using different chaperones and client aggregated proteins, indicates the existence of two distinct mechanisms of action mediated by the chaperones to neutralize the toxicity of aberrant proteins oligomers: (i) direct binding of the chaperones to the hydrophobic patches exposed on the oligomer/fibril surface, with resulting shielding or masking of the moieties responsible for the aberrant interactions with cellular targets; (ii) chaperone-mediated conversion of aberrant protein aggregates into large and more innocuous species, resulting in a decrease of their surface-to-volume ratio and diffusibility and in deposits more easily manageable by clearance mechanisms, such as autophagy. In this review article we will describe the in vitro and in vivo evidence supporting both mechanisms and how this results in a suppression of the detrimental effects caused by protein misfolded aggregates.
The Journal of biological chemistry, Jan 21, 2016
Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin positive... more Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-U) are two clinically distinct neurodegenerative conditions sharing a similar histopathology characterized by the nuclear clearance of TDP-43 and its associated deposition into cytoplasmic inclusions in different areas of the central nervous system. Given the concomitant occurrence of TDP-43 nuclear depletion and cytoplasmic accumulation, it has been proposed that TDP-43 proteinopathies originate from either a loss-of-function (LOF) mechanism, a gain-of-function (GOF) process, or both. We have addressed this issue by transfecting murine NSC34 and N2a cells with siRNA for endogenous murine TDP-43 and with human recombinant TDP-43 inclusion bodies (IBs). These two strategies allowed the depletion of nuclear TDP-43 and the accumulation of cytoplasmic TDP-43 aggregates to occur separately and independently. Endogenous and exogenous TDP-43 were monitored and quantified using...
The Journal of biological chemistry, Jan 21, 2016
Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin positive... more Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-U) are two clinically distinct neurodegenerative conditions sharing a similar histopathology characterized by the nuclear clearance of TDP-43 and its associated deposition into cytoplasmic inclusions in different areas of the central nervous system. Given the concomitant occurrence of TDP-43 nuclear depletion and cytoplasmic accumulation, it has been proposed that TDP-43 proteinopathies originate from either a loss-of-function (LOF) mechanism, a gain-of-function (GOF) process, or both. We have addressed this issue by transfecting murine NSC34 and N2a cells with siRNA for endogenous murine TDP-43 and with human recombinant TDP-43 inclusion bodies (IBs). These two strategies allowed the depletion of nuclear TDP-43 and the accumulation of cytoplasmic TDP-43 aggregates to occur separately and independently. Endogenous and exogenous TDP-43 were monitored and quantified using...
Protein Engineering Design and Selection, 2011
Acta Crystallographica Section A Foundations of Crystallography, 2005