The plasma protein transthyretin (TTR) is linked to human amyloidosis. Dissociation of its native... more The plasma protein transthyretin (TTR) is linked to human amyloidosis. Dissociation of its native tetrameric assembly is a rate-limiting step in the conversion from a native structure into a pathological amyloidogenic fold. Binding of small molecule ligands within the thyroxine binding site of TTR can stabilize the tetrameric integrity and is a potential therapeutic approach. However, through the characterization of nine different tetramer-stabilizing ligands we found that unspecific binding to plasma components might significantly compromise ligand efficacy. Surprisingly the binding strength between a particular ligand and TTR does not correlate well with its selectivity in plasma. However, through analysis of the thermodynamic signature using isothermal titration calorimetry we discovered a better correlation between selectivity and the enthalpic component of the interaction. This is of specific interest in the quest for more efficient TTR stabilizers, but a high selectivity is an...
Pro-inflammatory S100A9 protein is increasingly recognized as an important contributor to inflamm... more Pro-inflammatory S100A9 protein is increasingly recognized as an important contributor to inflammation-related neurodegeneration. Here, we provide insights into S100A9 specific mechanisms of action in Alzheimer’s disease (AD). Due to its inherent amyloidogenicity S100A9 contributes to amyloid plaque formation together with Aβ. In traumatic brain injury (TBI) S100A9 itself rapidly forms amyloid plaques, which were reactive with oligomer-specific antibodies, but not with Aβ and amyloid fibrillar antibodies. They may serve as precursor-plaques for AD, implicating TBI as an AD risk factor. S100A9 was observed in some hippocampal and cortical neurons in TBI, AD and non-demented aging. In vitro S100A9 forms neurotoxic linear and annular amyloids resembling Aβ protofilaments. S100A9 amyloid cytotoxicity and native S100A9 pro-inflammatory signaling can be mitigated by its co-aggregation with Aβ, which results in a variety of micron-scale amyloid complexes. NMR and molecular docking demonstrated transient interactions between native S100A9 and Aβ. Thus, abundantly present in AD brain pro-inflammatory S100A9, possessing also intrinsic amyloidogenic properties and ability to modulate Aβ aggregation, can serve as a link between the AD amyloid and neuroinflammatory cascades and as a prospective therapeutic target.
Amyloid formation is inherent property of proteins which under certain circumstances can become a... more Amyloid formation is inherent property of proteins which under certain circumstances can become a pathologic feature of a group of diseases called amyloidosis. There are about 30 known human amyloidosis and more than 27 identified proteins involved in these ...
... reference ». Author: Gharibyan, Anna (Umeå University, Faculty of Medicine, Department of Med... more ... reference ». Author: Gharibyan, Anna (Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics). ... Subject category: Neurosciences Cell and Molecular Biology. Research subject: biomedicinsk laboratorievetenskap. In thesis: Gharibyan, Anna. ...
... reference ». Author: Gharibyan, Anna (Umeå University, Faculty of Medicine, Department of Med... more ... reference ». Author: Gharibyan, Anna (Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics). ... Cardiac and Cardiovascular Systems Cell and Molecular Biology. Research subject: biomedicinsk laboratorievetenskap. In thesis: Gharibyan, Anna ...
Dynorphins are endogenous opioid peptide products of the prodynorphin gene. An extensive literatu... more Dynorphins are endogenous opioid peptide products of the prodynorphin gene. An extensive literature suggests that dynorphins have deleterious effects on CNS injury outcome. We thus examined whether a deficiency of dynorphin would protect against tissue damage after spinal cord injury (SCI), and if individual cell types would be specifically affected. Wild-type and prodynorphin−/− mice received a moderate contusion injury at 10th thoracic vertebrae (T10). Caspase-3 activity at the injury site was significantly decreased in tissue homogenates from prodynorphin−/− mice after 4 h. We examined frozen sections at 4 h post-injury by immunostaining for active caspase-3. At 3–4 mm rostral or caudal to the injury, >90% of all neurons, astrocytes and oligodendrocytes expressed active caspase-3 in both wild-type and knockout mice. At 6–7 mm, there were fewer caspase-3+ oligodendrocytes and astrocytes than at 3–4 mm. Importantly, caspase-3 activation was significantly lower in prodynorphin−/− oligodendrocytes and astrocytes, as compared with wild-type mice. In contrast, while caspase-3 expression in neurons also declined with further distance from the injury, there was no effect of genotype. Radioimmunoassay showed that dynorphin A(1-17) was regionally increased in wild-type injured versus sham-injured tissues, although levels of the prodynorphin processing product Arg6-Leu-enkephalin were unchanged. Our results indicate that dynorphin peptides affect the extent of post-injury caspase-3 activation, and that glia are especially sensitive to these effects. By promoting caspase-3 activation, dynorphin peptides likely increase the probability of glial apoptosis after SCI. While normally beneficial, our findings suggest that prodynorphin or its peptide products become maladaptive following SCI and contribute to secondary injury.
In the current study we investigated the molecular mechanisms of cytotoxicity of amyloid oligomer... more In the current study we investigated the molecular mechanisms of cytotoxicity of amyloid oligomers of horse milk lysozyme. We have shown that lysozyme forms soluble amyloid oligomers and protofibrils during incubation at pH 2.0 and 4.5 and 57 degrees C. These structures bind the amyloid-specific dyes thioflavin T and Congo Red, and their morphology and size were analyzed by atomic force microscopy. Monomeric lysozyme and its fibrils did not affect the viability of three cell types used in our experiments including primary murine neurons and fibroblasts, as well as neuroblastoma cell line IMR-32. However, soluble amyloid oligomers of lysozyme caused death of all these cell types, as estimated by flow-cytometry counting dead cells stained with ethidium bromide. The primary cell cultures appeared to be more sensitive to amyloid than neuroblastoma cell line IMR-32. Amyloid cytotoxicity depends on the size of oligomeric particles: samples containing 20-mers formed at pH 4.5 were more toxic than tetramers and octamers present in the solution at pH 2.0. Soluble amyloid oligomers can self-assemble into doughnut-like structures; however, no correlation was observed between the amount of the doughnut-like structures in the sample and its cytotoxicity. The fact that the intermediate oligomers of such an abundant protein as lysozyme display cytotoxicity confirms a hypothesis that cytotoxicity is a common feature of protein amyloid. Inhibition of intermediate oligomer formation is crucial in preventing amyloid pathogeneses.
The plasma protein transthyretin (TTR) is linked to human amyloidosis. Dissociation of its native... more The plasma protein transthyretin (TTR) is linked to human amyloidosis. Dissociation of its native tetrameric assembly is a rate-limiting step in the conversion from a native structure into a pathological amyloidogenic fold. Binding of small molecule ligands within the thyroxine binding site of TTR can stabilize the tetrameric integrity and is a potential therapeutic approach. However, through the characterization of nine different tetramer-stabilizing ligands we found that unspecific binding to plasma components might significantly compromise ligand efficacy. Surprisingly the binding strength between a particular ligand and TTR does not correlate well with its selectivity in plasma. However, through analysis of the thermodynamic signature using isothermal titration calorimetry we discovered a better correlation between selectivity and the enthalpic component of the interaction. This is of specific interest in the quest for more efficient TTR stabilizers, but a high selectivity is an...
Pro-inflammatory S100A9 protein is increasingly recognized as an important contributor to inflamm... more Pro-inflammatory S100A9 protein is increasingly recognized as an important contributor to inflammation-related neurodegeneration. Here, we provide insights into S100A9 specific mechanisms of action in Alzheimer’s disease (AD). Due to its inherent amyloidogenicity S100A9 contributes to amyloid plaque formation together with Aβ. In traumatic brain injury (TBI) S100A9 itself rapidly forms amyloid plaques, which were reactive with oligomer-specific antibodies, but not with Aβ and amyloid fibrillar antibodies. They may serve as precursor-plaques for AD, implicating TBI as an AD risk factor. S100A9 was observed in some hippocampal and cortical neurons in TBI, AD and non-demented aging. In vitro S100A9 forms neurotoxic linear and annular amyloids resembling Aβ protofilaments. S100A9 amyloid cytotoxicity and native S100A9 pro-inflammatory signaling can be mitigated by its co-aggregation with Aβ, which results in a variety of micron-scale amyloid complexes. NMR and molecular docking demonstrated transient interactions between native S100A9 and Aβ. Thus, abundantly present in AD brain pro-inflammatory S100A9, possessing also intrinsic amyloidogenic properties and ability to modulate Aβ aggregation, can serve as a link between the AD amyloid and neuroinflammatory cascades and as a prospective therapeutic target.
Amyloid formation is inherent property of proteins which under certain circumstances can become a... more Amyloid formation is inherent property of proteins which under certain circumstances can become a pathologic feature of a group of diseases called amyloidosis. There are about 30 known human amyloidosis and more than 27 identified proteins involved in these ...
... reference ». Author: Gharibyan, Anna (Umeå University, Faculty of Medicine, Department of Med... more ... reference ». Author: Gharibyan, Anna (Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics). ... Subject category: Neurosciences Cell and Molecular Biology. Research subject: biomedicinsk laboratorievetenskap. In thesis: Gharibyan, Anna. ...
... reference ». Author: Gharibyan, Anna (Umeå University, Faculty of Medicine, Department of Med... more ... reference ». Author: Gharibyan, Anna (Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics). ... Cardiac and Cardiovascular Systems Cell and Molecular Biology. Research subject: biomedicinsk laboratorievetenskap. In thesis: Gharibyan, Anna ...
Dynorphins are endogenous opioid peptide products of the prodynorphin gene. An extensive literatu... more Dynorphins are endogenous opioid peptide products of the prodynorphin gene. An extensive literature suggests that dynorphins have deleterious effects on CNS injury outcome. We thus examined whether a deficiency of dynorphin would protect against tissue damage after spinal cord injury (SCI), and if individual cell types would be specifically affected. Wild-type and prodynorphin−/− mice received a moderate contusion injury at 10th thoracic vertebrae (T10). Caspase-3 activity at the injury site was significantly decreased in tissue homogenates from prodynorphin−/− mice after 4 h. We examined frozen sections at 4 h post-injury by immunostaining for active caspase-3. At 3–4 mm rostral or caudal to the injury, >90% of all neurons, astrocytes and oligodendrocytes expressed active caspase-3 in both wild-type and knockout mice. At 6–7 mm, there were fewer caspase-3+ oligodendrocytes and astrocytes than at 3–4 mm. Importantly, caspase-3 activation was significantly lower in prodynorphin−/− oligodendrocytes and astrocytes, as compared with wild-type mice. In contrast, while caspase-3 expression in neurons also declined with further distance from the injury, there was no effect of genotype. Radioimmunoassay showed that dynorphin A(1-17) was regionally increased in wild-type injured versus sham-injured tissues, although levels of the prodynorphin processing product Arg6-Leu-enkephalin were unchanged. Our results indicate that dynorphin peptides affect the extent of post-injury caspase-3 activation, and that glia are especially sensitive to these effects. By promoting caspase-3 activation, dynorphin peptides likely increase the probability of glial apoptosis after SCI. While normally beneficial, our findings suggest that prodynorphin or its peptide products become maladaptive following SCI and contribute to secondary injury.
In the current study we investigated the molecular mechanisms of cytotoxicity of amyloid oligomer... more In the current study we investigated the molecular mechanisms of cytotoxicity of amyloid oligomers of horse milk lysozyme. We have shown that lysozyme forms soluble amyloid oligomers and protofibrils during incubation at pH 2.0 and 4.5 and 57 degrees C. These structures bind the amyloid-specific dyes thioflavin T and Congo Red, and their morphology and size were analyzed by atomic force microscopy. Monomeric lysozyme and its fibrils did not affect the viability of three cell types used in our experiments including primary murine neurons and fibroblasts, as well as neuroblastoma cell line IMR-32. However, soluble amyloid oligomers of lysozyme caused death of all these cell types, as estimated by flow-cytometry counting dead cells stained with ethidium bromide. The primary cell cultures appeared to be more sensitive to amyloid than neuroblastoma cell line IMR-32. Amyloid cytotoxicity depends on the size of oligomeric particles: samples containing 20-mers formed at pH 4.5 were more toxic than tetramers and octamers present in the solution at pH 2.0. Soluble amyloid oligomers can self-assemble into doughnut-like structures; however, no correlation was observed between the amount of the doughnut-like structures in the sample and its cytotoxicity. The fact that the intermediate oligomers of such an abundant protein as lysozyme display cytotoxicity confirms a hypothesis that cytotoxicity is a common feature of protein amyloid. Inhibition of intermediate oligomer formation is crucial in preventing amyloid pathogeneses.
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