Brain Lysates (brain + lysate)

Distribution by Scientific Domains


Selected Abstracts


Identification of calreticulin as a ligand of GABARAP by phage display screening of a peptide library

FEBS JOURNAL, Issue 21 2007
Jeannine Mohrlüder
4-Aminobutyrate type A (GABAA) receptor-associated protein (GABARAP) is a ubiquitin-like modifier implicated in the intracellular trafficking of GABAA receptors, and belongs to a family of proteins involved in intracellular vesicular transport processes, such as autophagy and intra-Golgi transport. In this article, it is demonstrated that calreticulin is a high affinity ligand of GABARAP. Calreticulin, although best known for its functions as a Ca2+ -dependent chaperone and a Ca2+ -buffering protein in the endoplasmic reticulum, is also localized to the cytosol and exerts a variety of extra-endoplasmic reticulum functions. By phage display screening of a randomized peptide library, peptides that specifically bind GABARAP were identified. Their amino acid sequences allowed us to identify calreticulin as a potential GABARAP binding protein. GABARAP binding to calreticulin was confirmed by pull-down experiments with brain lysate and colocalization studies in N2a cells. Calreticulin and GABARAP interact with a dissociation constant Kd = 64 nm and a mean lifetime of the complex of 20 min. Thus, the interaction between GABARAP and calreticulin is the strongest so far reported for each protein. [source]


Oligophrenin-1, a Rho GTPase-activating protein (RhoGAP) involved in X-linked mental retardation, is expressed in the enteric nervous system

THE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 2 2003
Junhua Xiao
Abstract Oligophrenin-1 is a RhoGTPase-activating protein (RhoGAP) that is involved in the regulation of shape changes in dendritic spines, and outgrowth of axons and dendrites in the brain. These changes in neuronal morphology are central to the mechanisms of plasticity, learning, and memory. Although the enteric nervous system also exhibits long-term changes in neuronal function, the expression and involvement of oligophrenin-1 has not previously been investigated. We show by RT-PCR analysis that oligophrenin-1 mRNA is expressed in the myenteric plexus (MP) of the guinea pig ileum. Sequencing of RT-PCR products showed that guinea pig oligophrenin-1 mRNA is 98% and 87% homologous to human and mouse oligophrenin-1, respectively, except that a 42 bp sequence is absent from the guinea pig mRNA. This 42 bp sequence codes for a sequence of 14 amino acids located near the carboxy-terminal end of the RhoGAP domain in the human sequence. An antibody that recognizes human oligophrenin-1 identified a 91 kDa protein band in rat and mouse brain lysates and in guinea pig sciatic nerve, and a 36 kDa protein band in both purified enteric ganglion cell and brain lysate from guinea pig. Oligophrenin-1 is localized specifically to neurons and varicose axons in the MPs and submucosal plexuses (SMPs) of the guinea pig and rat, but is not detectable in glial cells, smooth muscle, or other cell types. These findings indicate that oligophrenin-1 is expressed in the enteric nervous system, where it may regulate morphological changes in axons and dendrites, and thus modulate neuronal connectivity. Anat Rec Part A 273A:671,676, 2003. © 2003 Wiley-Liss, Inc. [source]


Novel brain 14-3-3 interacting proteins involved in neurodegenerative disease

FEBS JOURNAL, Issue 16 2005
Shaun Mackie
We isolated two novel 14-3-3 binding proteins using 14-3-3 , as bait in a yeast two-hybrid screen of a human brain cDNA library. One of these encoded the C-terminus of a neural specific armadillo-repeat protein, ,-catenin (neural plakophilin-related arm-repeat protein or neurojungin). ,-Catenin from brain lysates was retained on a 14-3-3 affinity column. Mutation of serine 1072 in the human protein and serine 1094 in the equivalent site in the mouse homologue (in a consensus binding motif for 14-3-3) abolished 14-3-3 binding to ,-catenin in vitro and in transfected cells. ,-catenin binds to presenilin-1, encoded by the gene most commonly mutated in familial Alzheimer's disease. The other clone was identified as the insulin receptor tyrosine kinase substrate protein of 53 kDa (IRSp53). Human IRSp53 interacts with the gene product implicated in dentatorubral-pallidoluysian atrophy, an autosomal recessive disorder associated with glutamine repeat expansion of atrophin-1. [source]


Ca2+ - and thromboxane-dependent distribution of MaxiK channels in cultured astrocytes: From microtubules to the plasma membrane

GLIA, Issue 12 2009
J. W. Ou
Abstract Large-conductance, voltage- and Ca2+ -activated K+ channels (MaxiK) are broadly expressed ion channels minimally assembled by four pore-forming ,-subunits (MaxiK,) and typically observed as plasma membrane proteins in various cell types. In murine astrocyte primary cultures, we show that MaxiK, is predominantly confined to the microtubule network. Distinct microtubule distribution of MaxiK, was visualized by three independent labeling approaches: (1) MaxiK,-specific antibodies, (2) expressed EGFP-labeled MaxiK,, and (3) fluorophore-conjugated iberiotoxin, a specific MaxiK pore-blocker. This MaxiK, association with microtubules was further confirmed by in vitro His-tag pulldown, co-immunoprecipitation from brain lysates, and microtubule depolymerization experiments. Changes in intracellular Ca2+ elicited by general pharmacological agents, caffeine or thapsigargin, resulted in increased MaxiK, labeling at the plasma membrane. More notably, U46619, an analog of thromboxane A2 (TXA2), which triggers Ca2+ -release pathways and whose levels increase during cerebral hemorrhage/trauma, also elicits a similar increase in MaxiK, surface labeling. Whole-cell patch clamp recordings of U46619-stimulated cells develop a ,3-fold increase in current amplitude indicating that TXA2 stimulation results in the recruitment of additional, functional MaxiK channels to the surface membrane. While microtubules are largely absent in mature astrocytes, immunohistochemistry results in brain slices show that cortical astrocytes in the newborn mouse (P1) exhibit a robust expression of microtubules that significantly colocalize with MaxiK,. The results of this study provide the novel insight that suggests that Ca2+ released from intracellular stores may play a key role in regulating the traffic of intracellular, microtubule-associated MaxiK, stores to the plasma membrane of developing murine astrocytes. © 2009 Wiley-Liss, Inc. [source]


Sequestosome 1/p62 shuttles polyubiquitinated tau for proteasomal degradation

JOURNAL OF NEUROCHEMISTRY, Issue 1 2005
Jeganathan Ramesh Babu
Abstract Inclusions isolated from several neurodegenerative diseases, including Alzheimer's disease (AD), are characterized by ubiquitin-positive proteinaceous aggregates. Employing confocal and immunoelectron microscopy, we find that the ubiquitin-associating protein sequestosome1/p62, co-localizes to aggregates isolated from AD but not control brain, along with the E3 ubiquitin ligase, TRAF6. This interaction could be recapitulated by co-transfection in HEK293 cells. Employing both in vitro and in vivo approaches, tau was found to be a substrate of the TRAF6, possessing lysine 63 polyubiquitin chains. Moreover, tau recovered from brain of TRAF6 knockout mice, compared with wild type, was not ubiquitinated. Tau degradation took place through the ubiquitin,proteasome pathway and was dependent upon either the K63-polyubiquitin chains or upon p62. In brain lysates of p62 knockout mice, tau fails to co-interact with Rpt1, a proteasomal subunit, thereby indicating a requirement for p62 shuttling of tau to the proteasome. Our results demonstrate that p62 interacts with K63-polyubiquitinated tau through its UBA domain and serves a novel role in regulating tau proteasomal degradation. We propose a model whereby either a decline in p62 expression or a decrease in proteasome activity may contribute to accumulation of insoluble/aggregated K63-polyubiquitinated tau. [source]


Oligophrenin-1, a Rho GTPase-activating protein (RhoGAP) involved in X-linked mental retardation, is expressed in the enteric nervous system

THE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 2 2003
Junhua Xiao
Abstract Oligophrenin-1 is a RhoGTPase-activating protein (RhoGAP) that is involved in the regulation of shape changes in dendritic spines, and outgrowth of axons and dendrites in the brain. These changes in neuronal morphology are central to the mechanisms of plasticity, learning, and memory. Although the enteric nervous system also exhibits long-term changes in neuronal function, the expression and involvement of oligophrenin-1 has not previously been investigated. We show by RT-PCR analysis that oligophrenin-1 mRNA is expressed in the myenteric plexus (MP) of the guinea pig ileum. Sequencing of RT-PCR products showed that guinea pig oligophrenin-1 mRNA is 98% and 87% homologous to human and mouse oligophrenin-1, respectively, except that a 42 bp sequence is absent from the guinea pig mRNA. This 42 bp sequence codes for a sequence of 14 amino acids located near the carboxy-terminal end of the RhoGAP domain in the human sequence. An antibody that recognizes human oligophrenin-1 identified a 91 kDa protein band in rat and mouse brain lysates and in guinea pig sciatic nerve, and a 36 kDa protein band in both purified enteric ganglion cell and brain lysate from guinea pig. Oligophrenin-1 is localized specifically to neurons and varicose axons in the MPs and submucosal plexuses (SMPs) of the guinea pig and rat, but is not detectable in glial cells, smooth muscle, or other cell types. These findings indicate that oligophrenin-1 is expressed in the enteric nervous system, where it may regulate morphological changes in axons and dendrites, and thus modulate neuronal connectivity. Anat Rec Part A 273A:671,676, 2003. © 2003 Wiley-Liss, Inc. [source]