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Mohammad A Alsenaidy - One of the best experts on this subject based on the ideXlab platform.

  • sodium louroyl sarcosinate Sarkosyl modulate amyloid fibril formation in hen egg white lysozyme hewl at alkaline ph a molecular insight study
    Journal of Biomolecular Structure & Dynamics, 2018
    Co-Authors: Javed Masood Khan, Mohammad A Alsenaidy, Mohd Shahnawaz Khan, Anwar Ahmed, Priyankar Sen, Mohammad Oves, Nasser Abdulatif Alshabib, Rizwan Hasan Khan
    Abstract:

    Amyloid fibril formation is responsible for several neurodegenerative diseases and are formed when native proteins misfold and stick together with different interactive forces. In the present study, we have determined the mode of interaction of the anionic surfactant Sarkosyl with hen egg white lysozyme (HEWL) [EC No. 3.2.1.17] at two pHs (9.0 and 13.0) and investigated its impact on fibrillogenesis. Our data suggested that Sarkosyl is promoting amyloid fibril formation in HEWL at the concentration range between 0.9 and 3.0 mM and no amyloid fibril formation was observed in the concentration range of 3.0-20.0 mM at pH 9.0. The results were confirmed by several biophysical and computational techniques, such as turbidity measurement, dynamic light scattering, Raleigh scattering, ThT fluorescence, intrinsic fluorescence, far-UV CD and atomic force microscopy. Sarkosyl was unable to induce aggregation in HEWL at pH 13.0 as confirmed by turbidity and RLS measurements. HEWL forms larger amyloid fibrils in the presence of 1.6 mM of Sarkosyl. The spectroscopic, microscopic and molecular docking data suggest that the negatively charged carboxylate group and 12-carbon hydrophobic tail of Sarkosyl stimulate amyloid fibril formation in HEWL via electrostatic and hydrophobic interaction. This study leads to new insight into the process of suppression of fibrillogenesis in HEWL which can be prevented by designing ligands that can retard the electrostatic and hydrophobic interaction between Sarkosyl and HEWL.

  • biophysical evaluation of amyloid fibril formation in bovine cytochrome c by sodium lauroyl sarcosinate Sarkosyl in acidic conditions
    Journal of Molecular Liquids, 2017
    Co-Authors: Mohammad A Alsenaidy
    Abstract:

    Abstract Surfactant-induced amyloid fibril formation in protein has a lot of association in the laboratory and industrial application. Sodium lauroyl sarcosinate (Sarkosyl) is used to solubilize protein aggregates during protein purification, however, its amyloid fibril induction properties are very less understood. The aim of the current work is to examine the role of Sarkosyl on the amyloid fibrillation of bovine cytochrome c protein at pH 3.5. We have used various spectroscopic (turbidity, RLS, ThT and far-UV CD) and microscopic (TEM) techniques to characterize the fibrillation of cytochrome c at low pH. We have demonstrated that Sarkosyl in the concentrations range 0.5 to 10.0 mM is inducing large size aggregates in cytochrome c with fibrillar morphology. At concentrations of ≤ 0.5 mM, Sarkosyl was not able to induce amyloid-like aggregates in cytochrome c at the same pH. The α-helical content of cytochrome c is transformed into β-sheet structure (single minima at 218 nm) in the presence of ≥ 0.5 mM of Sarkosyl. The amyloid fibril formation potency is dependent on the concentrations of Sarkosyl and solution pH. We believe the results of this work will not only help to explain the molecular mechanism of amyloid fibril formation, but also give us an insight on rationally designing potential anti-amyloidogenic molecules.

Shu Hui Yen - One of the best experts on this subject based on the ideXlab platform.

  • Homocysteine Increases Tau Phosphorylation, Truncation and Oligomerization
    MDPI AG, 2018
    Co-Authors: Norimichi Shirafuji, Shu Hui Yen, Tadanori Hamano, Nicholas M. Kanaan, Hirotaka Yoshida, Kouji Hayashi, Masamichi Ikawa, Osamu Yamamura, Masaru Kuriyama, Yasunari Nakamoto
    Abstract:

    Increased plasma homocysteinemia is considered a risk factor of dementia, including Alzheimer’s disease (AD) and vascular dementia. However, the reason elevated plasma homocysteinemia increases the risk of dementia remains unknown. A pathological hallmark of AD is neurofibrillary tangles (NFTs) that consist of pathologically phosphorylated tau proteins. The effect of homocysteine (Hcy) on tau aggregation was explored using human neuroblastoma M1C cells that constitutively express human wild-type tau (4R0N) under the control of a tetracycline off system, primary mouse cultured neurons, and by inducing hyperhomocysteinemia in a mouse model of tauopathy (HHCy mice). A wide range of Hcy concentrations (10–1000 µM) increased total tau and phosphorylated tau protein levels. Hcy activated glycogen synthase kinase 3, and cyclin dependent kinase 5, major tau phosphokinases, and inactivated protein phosphatase 2A, a main tau phosphatase. Hcy exhibited cytotoxic effects associated with enhanced activation of caspase. Truncation of tau in the C-terminus, the cleavage site of caspase 3 (i.e., D421, detected by the TauC3 antibody) was also increased. Total tau, phosphorylated tau, as well as C-terminal cleaved tau were increased in the Sarkosyl insoluble tau fraction. Hcy also increased the level of tau oligomers, as indicated by the tau oligomer complex 1 (TOC1) antibody that specifically identifies oligomeric tau species, in the tris insoluble, Sarkosyl soluble fraction. The levels of TOC1-positive oligomeric tau were increased in brain lysates from HHCy mice, and treating HHCy mice with S-adenosylmethionine, an intermediate of Hcy, reduced the levels of oligomeric tau to control levels. These observations suggest that Hcy increases the levels of phosphorylated tau as well as truncated tau species via caspase 3 activation, and enhanced tau oligomerization and aggregation

  • Original Article Concentration-dependent Effects of Proteasomal Inhibition on tau Processing in a Cellular Model of
    2015
    Co-Authors: Tadanori Hamano, Tania F Gendron, Shu Hui Yen
    Abstract:

    Abstract: Tauopathies are characterized by accumulation of filamentous tau aggregates. These aggregates can be recapitulated in transfectant M1C overproducing wild-type human brain tau 4R0N via the tetracycline off (TetOff) inducible expression mechanism. To determine the contribution of proteasomes to tau degradation and aggregation, we exposed M1C cells to epoxomicin (Epx; 2-50 nM) or MG132 (0.5 μM) on the 3rd or 4th day of a 5-day TetOff induction and demonstrated a reduction of proteasomal activity. Cultures treated with 2 nM Exp showed accumulation of full-length tau without affecting ubiquitin and β-catenin immunoblotting profiles. In contrast, cells treated with 10, 50 nM Epx or MG132 displayed changes in ubiquitin or β-catenin immunoblotting profiles and extensive tau degradation/truncation. The increase of tau degradation/truncation was accompanied with accumulation of oligomers and Sarkosyl-insoluble aggregates of tau, augmented thioflavin-binding and activation of caspases and calpains. Truncated, oligomeric and Sarkosyl-insoluble tau derivatives appeared with caspase-specific cleavage and their production was diminished when pretreated with a pan-caspase inhibitor. The results demonstrate (i) a dose-dependent, opposite effect of proteasome inhibition on tau processing, (ii) the participation of proteasome-dependent, ubiquitination-independent mechanisms in tau degradation and aggregation, and (iii) the promotion of tau aggregation by caspase-mediated tau degradation/truncation

  • concentration dependent effects of proteasomal inhibition on tau processing in a cellular model of tauopathy
    International Journal of Clinical and Experimental Pathology, 2009
    Co-Authors: Tadanori Hamano, Tania F Gendron, Shu Hui Yen
    Abstract:

    Tauopathies are characterized by accumulation of filamentous tau aggregates. These aggregates can be recapitulated in transfectant M1C overproducing wild-type human brain tau 4R0N via the tetracycline off (TetOff) inducible expression mechanism. To determine the contribution of proteasomes to tau degradation and aggregation, we exposed M1C cells to epoxomicin (Epx; 2-50 nM) or MG132 (0.5 μM) on the 3rd or 4th day of a 5-day TetOff induction and demonstrated a reduction of proteasomal activity. Cultures treated with 2 nM Exp showed accumulation of full-length tau without affecting ubiquitin and β-catenin immunoblotting profiles. In contrast, cells treated with 10, 50 nM Epx or MG132 displayed changes in ubiquitin or β-catenin immunoblotting profiles and extensive tau degradation/truncation. The increase of tau degradation/truncation was accompanied with accumulation of oligomers and Sarkosyl-insoluble aggregates of tau, augmented thioflavin-binding and activation of caspases and calpains. Truncated, oligomeric and Sarkosyl-insoluble tau derivatives appeared with caspase-specific cleavage and their production was diminished when pretreated with a pan-caspase inhibitor. The results demonstrate (i) a dose-dependent, opposite effect of proteasome inhibition on tau processing, (ii) the participation of proteasome-dependent, ubiquitination-independent mechanisms in tau degradation and aggregation, and (iii) the promotion of tau aggregation by caspase-mediated tau degradation/truncation.

  • phosphorylated p38mapk specific antibodies cross react with Sarkosyl insoluble hyperphosphorylated tau proteins
    Journal of Neurochemistry, 2004
    Co-Authors: Naruhiko Sahara, Jada Lewis, Eileen Mcgowan, Dennis W Dickson, Mike Hutton, Irving E Vega, Takashi Ishizawa, Shu Hui Yen
    Abstract:

    Neurofibrillary tangles (NFT) accumulated in Alzheimer's diseases and related disorders contain hyperphosphorylated tau and display immunoreactivity for active forms of various kinases. To understand the role of p38MAPK (mitogen-activated protein kinase) in NFT formation, we have studied a transgenic (Tg) mouse model of tauopathy, JNPL3, that expresses P301L mutant tau, and bigenic mice, TAPP, generated by cross-breeding of JNPL3 with Tg2576 mice. Age-matched non-Tg mice (NTg), wild-type human tau Tg mice (JN25), and Tg2576 mice were used as controls. Phosphorylated p38MAPK (active form) immunoreactivity was consistently located in NFT and granulovaculolar degeneration in JNPL3 and TAPP mice older than 5 months of age. Unphosphorylated/total-p38MAPK was not detectable in spinal cord and brain sections from 2- to 11-month-old mice, even though JNPL3 mice, but not controls had an age-dependent increase of total-p38MAPK by western blotting. Spinal cord/brain extracts from mice and human with tauopathy were demonstrated to have insignificant amount of active-p38MAPK. However, they contained antiactive-p38MAPK cross-reactive proteins insoluble in Sarkosyl and similar to phosphorylated tau in size. Consistently, antiactive-p38MAPK immunoprecipitates displayed tau immunoreactivity, but not total-p38MAPK, and antitau immunoprecipitates displayed active-p38MAPK immunoreactivity. Together, the results indicate that the cross-reactivity of antiactive-p38MAPK antibody with phosphorylated tau is responsible for the immunolabeling of tau-positive inclusion.

  • assembly of tau in transgenic animals expressing p301l tau alteration of phosphorylation and solubility
    Journal of Neurochemistry, 2002
    Co-Authors: Naruhiko Sahara, Jada Lewis, Michael Deture, Eileen Mcgowan, Dennis W Dickson, Mike Hutton, Shu Hui Yen
    Abstract:

    Transgenic mice (JNPL3), which develop neurofibrillary degeneration and express four-repeat human tau with P301L missense mutation, were characterized biochemically to determine whether the development of aggregated tau from soluble tau involves an intermediate stage. Homogenates from mice of different ages were separated into buffer-soluble (S1), Sarkosyl- and salt-extractable (S2) and Sarkosyl-insoluble pellet (P3) fractions, and analyzed for human tau distribution, phosphorylation and filament formation. S1 and S2 fractions contained 50-60-kDa tau whereas the S2 fraction also had 64-kDa tau. The level of tau in the P3 fraction increased in an age-dependent manner and correlated positively with the soluble tau concentration. The P3 fraction from 2.5-6.5-month-old mice contained 64- and 50-60-kDa tau, whereas that from 8.5-month and older transgenic animals contained mostly 64-kDa and higher molecular weight tau. The S2 and P3 fractions contained comparable amounts of 64-kDa tau. The 64-kDa tau was predominantly human, and phosphorylated at multiple sites: Thr181, Ser202/Thr205, Thr212, Thr231, Ser262, Ser396/Ser404, Ser409 and Ser422. Most of these sites were phosphorylated to a lesser extent in S2 than in P3 fractions. Tau polymers were detected in P3 fractions from 3-month and older female JNPL3 mice, but not in non-transgenic controls. The results suggest that tau in S2 represents an intermediate from which insoluble tau is derived, and that phosphorylation may play a role in filament formation and/or stabilization.

Maurizio Gatti - One of the best experts on this subject based on the ideXlab platform.

  • the kl 3 loop of the y chromosome of drosophila melanogaster binds a tektin like protein
    Genetics, 1993
    Co-Authors: C Pisano, Silvia Bonaccorsi, Maurizio Gatti
    Abstract:

    Primary spermatocyte nuclei of Drosophila melanogaster exhibit three giant lampbrush-like loops formed by the kl-5, kl-3 and ks-1 Y-chromosome fertility factors. These structures contain and abundantly transcribe highly repetitive, simple sequence DNAs and accumulate large amounts of non-Y-encoded proteins. By immunizing mice with the 53-kD fraction (enriched in beta 2-tubulin) excised from a sodium dodecyl sulfate-polyacrylamide gel loaded with Drosophila testis proteins we raised a polyclonal antibody, designated as T53-1, which decorates the kl-3 loop and the sperm flagellum. Two dimensional immunoblot analysis showed that the T53-1 antibody reacts with a single protein of about 53 kD, different from the tubulins and present both in X/Y and X/O males. Moreover, the antigen recognized by the T53-1 antibody proved to be testis-specific because it was detected in testes and seminal vesicles but not in other male tissues or in females. The characteristics of the protein recognized by the T53-1 antibody suggested that it might be a member of a class of axonemal proteins, the tektins, known to form Sarkosyl-urea insoluble filaments in the wall of flagellar microtubules. Purification of the Sarkosyl-urea insoluble fraction of D. melanogaster sperm revealed that it contains four polypeptides having molecular masses ranging from 51 to 57 kD. One of these polypeptides reacts strongly with the T53-1 antibody but none of them reacts with antitubulin antibodies. These results indicate that the kl-3 loop binds a non-Y encoded, testis-specific, tektin-like protein which is a constituent of the sperm flagellum. This finding supports the hypothesis that the Y loops fulfill a protein-binding function required for the proper assembly of the axoneme components.

Nobuyuki Nukina - One of the best experts on this subject based on the ideXlab platform.

  • a seeding reaction recapitulates intracellular formation of Sarkosyl insoluble transactivation response element tar dna binding protein 43 inclusions
    Journal of Biological Chemistry, 2011
    Co-Authors: Yoshiaki Furukawa, Kumi Kaneko, Shoji Watanabe, Koji Yamanaka, Nobuyuki Nukina
    Abstract:

    The transactivation response element (TAR) DNA-binding protein-43 (TDP-43) is a nuclear protein that normally regulates transcription and splicing. Abnormal accumulation of insoluble inclusions containing TDP-43 has been recently reported in the affected tissues of amyotrophic lateral sclerosis (ALS) patients. Here, we show that intracellular aggregation of TDP-43 can be triggered by transduction of fibrillar aggregates prepared from in vitro functional TDP-43. Sarkosyl is found to be incapable of solubilizing those intracellularly seeded aggregates of TDP-43, which is consistent with the observation that TDP-43 inclusions in ALS patients are Sarkosyl-insoluble. In addition, intracellular seeding in our cell models reproduces ubiquitination of TDP-43 aggregates, which is another prominent feature of TDP-43 inclusions in ALS patients. Although it has been so far difficult to initiate disease-associated changes of TDP-43 using cultured cell models, we propose that a seeding reaction is a key to construct a model to monitor TDP-43 pathologies.

John T Lis - One of the best experts on this subject based on the ideXlab platform.

  • Direct cloning of DNA that interacts in vivo with a specific protein: application to RNA polymerase II and sites of pausing in Drosophila
    Nucleic acids research, 1998
    Co-Authors: Adam Law, Kazunori Hirayoshi, Thomas O'brien, John T Lis
    Abstract:

    A new method is described for cloning DNA sequences occupied by a specific protein on chromatin in vivo. The approach uses UV cross-linking to couple proteins covalently to DNA and the resulting complexes are then purified under stringent conditions. Particular adducts are immunoprocipitated with antibody to the protein of interest. The resulting DNA (iDNA) is amplified by PCR, cloned and characterized. The model system used was RNA polymerase II (Pol II), whose density on particular DNAs under various conditions is well documented. Pol II can exist in several states on DNA. While Pol II can simply be bound to DNA, the bulk of DNA-associated Pol II is transcriptionally engaged in either the transcribing or paused states. Paused Pol IIs that have previously been characterized are found at promoters and have the distinctive property that their transcription in isolated nuclei is stimulated by Sarkosyl or high salt. Here we isolate and sequence DNAs that cross-link to Pol II molecules. We identify by nuclear run-on assays those DNAs that have Pol II engaged in transcription. Twenty one percent of the iDNA clones that have detectable transcriptionally engaged Pol II appear to be paused, in that they display Sarkosyl-stimulated trancription in a nuclear run-on transcription assay. At least some of these map to the 54-ends of genes. These results suggest that transcriptional pausing of Pol II is a general phenomenon in vivo.