Dataset Information

Characterization of ?-domains in C-terminal fragments of TDP-43 by scanning tunneling microscopy.

ABSTRACT: The TAR DNA-binding protein 43 (TDP-43) has been identified as a critical player in a range of neurodegenerative diseases, including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Recent discoveries demonstrate the important role of carboxyl-terminal fragments of TDP-43 in its proteinopathy. Herein, we report the characterization of ?-domains in the C-terminal fragments of TDP-43 using scanning tunneling microscopy (STM). Careful comparison of the wild-type TDP-43 (Wt) and the three mutant TDP-43 peptides: an ALS-related mutant peptide: phosphorylated A315T mutant TDP-43 (A315T(p)) and two model peptides: A315T mutant TDP-43 (A315T), A315E mutant TDP-43 (A315E) reveals that A315T(p) has a longer core region of the ?-domain than Wt. A315E possesses the longest core region of the ?-domain and A315T(p) mutant TDP-43 has the second longest core region of the ?-domain. The core regions of the ?-domains for A315T and Wt TDP-43 have the same length. This observation provides a supportive evidence of a higher tendency in beta-sheet formation of A315T(p) containing TDP-43 fragment, and structural mechanism for the higher cytotoxicity and accelerated fibril formation of the A315T(p) mutation-containing TDP-43 peptide as compared with Wt TDP-43.

SUBMITTER: Xu M

PROVIDER: S-EPMC3787119 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Characterization of β-domains in C-terminal fragments of TDP-43 by scanning tunneling microscopy.

Xu Meng M Zhu Li L Liu Jianghong J Yang Yanlian Y Wu Jane Y JY Wang Chen C

Journal of structural biology 20121105 1

The TAR DNA-binding protein 43 (TDP-43) has been identified as a critical player in a range of neurodegenerative diseases, including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Recent discoveries demonstrate the important role of carboxyl-terminal fragments of TDP-43 in its proteinopathy. Herein, we report the characterization of β-domains in the C-terminal fragments of TDP-43 using scanning tunneling microscopy (STM). Careful comparison of the wild-type TDP ...[more]

PMID: 23138004

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Project description:The pathological hallmark of amyotrophic lateral sclerosis (ALS) is the presence of cytoplasmic inclusions, containing C-terminal fragments of the protein TDP-43. Here, we tested the hypothesis that highly sensitive mass spectrometry with parallel reaction monitoring (MS-PRM) can generate a high-resolution map of pathological TDP-43 peptide ratios to form the basis for quantitation of abnormal C-terminal TDP-43 fragment enrichment. Human cortex and spinal cord, microscopically staged for the presence of p-TDP-43, p-tau, alpha-synuclein, and beta-amyloid pathology, were biochemically fractionated and analyzed by immunoblot and MS for the detection of full-length and truncated (disease-specific) TDP-43 peptides. This informed the synthesis of heavy isotope-labeled peptides for absolute quantification of TDP-43 by MS-PRM across 16 ALS, 8 Parkinson's, 8 Alzheimer's disease, and 8 aged control cases. We confirmed by immunoblot the previously described enrichment of pathological C-terminal fragments in ALS-TDP urea fractions. Subsequent MS analysis resolved specific TDP-43 N- and C-terminal peptides, including a novel N-terminal truncation site-specific peptide. Absolute quantification of peptides by MS-PRM showed an increased C:N-terminal TDP-43 peptide ratio in ALS-TDP brain compared to normal and disease controls. A C:N-terminal ratio >1.5 discriminated ALS from controls with a sensitivity of 100% (CI 79.6-100) and specificity of 100% (CI 68-100), and from Parkinson's and Alzheimer's disease with a sensitivity of 93% (CI 70-100) and specificity of 100% (CI 68-100). N-terminal truncation site-specific peptides were increased in ALS in line with C-terminal fragment enrichment, but were also found in a proportion of Alzheimer cases with normal C:N-terminal ratio but coexistent limbic TDP-43 neuropathological changes. In conclusion this is a novel, sensitive, and specific method to quantify the enrichment of pathological TDP-43 fragments in human brain, which could form the basis for an antibody-free assay. Our methodology has the potential to help clarify if specific pathological TDP-43 peptide signatures are associated with primary or secondary TDP-43 proteinopathies.

Project description:The pathological hallmark of amyotrophic lateral sclerosis (ALS) is the presence of cytoplasmic inclusions, containing C-terminal fragments of the protein TDP-43. Here, we tested the hypothesis that highly sensitive mass spectrometry with parallel reaction monitoring (MS-PRM) can generate a high-resolution map of pathological TDP-43 peptide ratios to form the basis for quantitation of abnormal C-terminal TDP-43 fragment enrichment. Human cortex and spinal cord, microscopically staged for the presence of phosphoTDP-43, p-tau, alpha-synuclein and beta-amyloid pathology, were biochemically fractionated and analysed by immunoblot and MS for detection of full-length and truncated (disease-specific) TDP-43 peptides. This informed synthesis of heavy isotope-labelled peptides for the absolute quantification of TDP-43 by MS-PRM across 16 ALS, 8 Parkinson’s and 8 Alzheimer’s disease and 8 aged control cases. We confirmed by immunoblot the previously described enrichment of pathological C-terminal fragments in ALS-TDP urea fractions. Subsequent MS analysis resolved specific TDP-43 N- and C-terminal peptides, including a novel N-terminal truncation site-specific peptide. Absolute quantification of peptides by MS-PRM showed an increased C:N-terminal TDP-43 peptide ratio in ALS-TDP brain compared to normal and disease controls. A C:N-terminal ratio >1.5 discriminated ALS from controls with a sensitivity of 100% (CI 79.6-100) and specificity of 100% (CI 68-100), and from Parkinson’s and Alzheimer’s disease with a sensitivity of 93% (CI 70-100) and specificity of 100% (CI 68-100). N-terminal Truncation site-specific peptides were increased in ALS in line with C-terminal fragment enrichment, but also found in a proportion of Alzheimer cases with normal C:N-terminal ratio but coexistent TDP-43 pathology. In conclusion this is a novel, sensitive and specific method to quantify the enrichment of pathological TDP-43 fragments in human brain, which could form the basis for an antibody-free assay. Our methodology has the potential to help clarify if specific pathological TDP-43 peptide signatures are associated with primary or secondary TDP-43 proteinopathies.

Project description:Many of the molecular mechanisms underlying the pathological aggregation of proteins observed in neurodegenerative diseases are still not fully understood. Among the aggregate-associated diseases, Amyotrophic Lateral Sclerosis (ALS) is of relevant importance. In fact, although understanding the processes that cause the disease is still an open challenge, its relationship with protein aggregation is widely known. In particular, human TDP-43, an RNA/DNA binding protein, is a major component of the pathological cytoplasmic inclusions observed in ALS patients. Indeed, the deposition of the phosphorylated full-length TDP-43 in spinal cord cells has been widely studied. Moreover, it has also been shown that the brain cortex presents an accumulation of phosphorylated C-terminal fragments (CTFs). Even if it is debated whether the aggregation of CTFs represents a primary cause of ALS, it is a hallmark of TDP-43 related neurodegeneration in the brain. Here, we investigate the CTFs aggregation process, providing a computational model of interaction based on the evaluation of shape complementarity at the molecular interfaces. To this end, extensive Molecular Dynamics (MD) simulations were conducted for different types of protein fragments, with the aim of exploring the equilibrium conformations. Adopting a newly developed approach based on Zernike polynomials, able to find complementary regions in the molecular surface, we sampled a large set of solvent-exposed portions of CTFs structures as obtained from MD simulations. Our analysis proposes and assesses a set of possible association mechanisms between the CTFs, which could drive the aggregation process of the CTFs. To further evaluate the structural details of such associations, we perform molecular docking and additional MD simulations to propose possible complexes and assess their stability, focusing on complexes whose interacting regions are both characterized by a high shape complementarity and involve β3 and β5 strands at their interfaces.

Dataset Information

Characterization of ?-domains in C-terminal fragments of TDP-43 by scanning tunneling microscopy.

Publications

Characterization of β-domains in C-terminal fragments of TDP-43 by scanning tunneling microscopy.

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