scholarly journals The Early Onset Dystonia Protein TorsinA Interacts with Kinesin Light Chain 1

2004 ◽  
Vol 279 (19) ◽  
pp. 19882-19892 ◽  
Author(s):  
Christoph Kamm ◽  
Heather Boston ◽  
Jeffrey Hewett ◽  
Jeremy Wilbur ◽  
David P. Corey ◽  
...  
Keyword(s):  
Rat Brain ◽  
Light Chain ◽  
Cortical Neurons ◽  
Early Onset ◽  
Subcellular Fractionation ◽  
Wild Type ◽  
Anterograde Transport ◽  
Glutamic Acid Residue ◽  
Carboxyl Terminal

Early onset dystonia is a movement disorder caused by loss of a glutamic acid residue (Glu302/303) in the carboxyl-terminal portion of the AAA+protein, torsinA. We identified the light chain subunit (KLC1) of kinesin-I as an interacting partner for torsinA, with binding occurring between the tetratricopeptide repeat domain of KLC1 and the carboxyl-terminal region of torsinA. Coimmunoprecipitation analysis demonstrated that wild-type torsinA and kinesin-I form a complexin vivo. In cultured cortical neurons, both proteins co-localized along processes with enrichment at growth cones. Wild-type torsinA expressed in CAD cells co-localized with endogenous KLC1 at the distal end of processes, whereas mutant torsinA remained confined to the cell body. Subcellular fractionation of adult rat brain revealed torsinA and KLC associated with cofractionating membranes, and both proteins were co-immunoprecipitated after cross-linking cytoplasmically oriented proteins on isolated rat brain membranes. These studies suggest that wild-type torsinA undergoes anterograde transport along microtubules mediated by kinesin and may act as a molecular chaperone regulating kinesin activity and/or cargo binding.

scholarly journals Domain structure and functional analysis of the carboxyl-terminal polyacidic sequence of the RAD6 protein of Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (3) ◽  
pp. 1179-1185 ◽  
Author(s):  
A Morrison ◽  
E J Miller ◽  
L Prakash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Proteinase K ◽  
Intrinsic Activity ◽  
Structural Domain ◽  
Globular Domain ◽  
Wild Type ◽  
Calculated Mass ◽  
Stokes Radius ◽  
Carboxyl Terminal

The RAD6 gene of Saccharomyces cerevisiae, which is required for normal tolerance of DNA damage and for sporulation, encodes a 172-residue protein whose 23 carboxyl-terminal residues are almost all acidic. We show that this polyacidic sequence appends to RAD6 protein as a polyanionic tail and that its function in vivo does not require stoichiometry of length. RAD6 protein was purified to near homogeneity from a yeast strain carrying a RAD6 overproducing plasmid. Approximately the first 150 residues of RAD6 protein composed a structural domain that was resistant to proteinase K and had a Stokes radius typical of a globular protein of its calculated mass. The carboxyl-terminal polyacidic sequence was sensitive to proteinase K, and it endowed RAD6 protein with an aberrantly large Stokes radius that indicates an asymmetric shape. We deduce that RAD6 protein is monomeric and comprises a globular domain with a freely extending polyacidic tail. We tested the phenotypic effects of partial or complete deletion of the polyacidic sequence, demonstrating the presence of the shortened proteins in the cell by using antibody to RAD6 protein. Removal of the entire polyacidic sequence severely reduced sporulation but only slightly affected survival after UV irradiation or UV-induced mutagenesis. Strains with deletions of all but the first 4 or 15 residues of the polyacidic sequence were phenotypically almost wild type or wild type, respectively. We conclude that the intrinsic activity of RAD6 protein resides in the globular domain, that the polyacidic sequence has a stimulatory or modifying role evident primarily in sporulation, and that only a short section apparently functions as effectively as the entire polyacidic sequence.

Phenotypic Correction of Adamts13-Deficient Mice by Early Intra-Amniotic Gene Transfer of Lentiviral Vector Encoding ADAMTS13 Genes.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 197-197
Author(s):  
Masami Niiya ◽  
Masayuki Endo ◽  
Philip W. Zoltick ◽  
Nidal E. Muvarak ◽  
David G. Motto ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Proteolytic Activity ◽  
Lentiviral Vector ◽  
Wild Type ◽  
Occlusion Time ◽  
Carboxyl Terminal ◽  
Human Wild Type

Abstract ADAMTS13, a member of A Disintegrin and Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS) family, is mainly synthesized in the hepatic stellate cells, endothelial cells and megakaryocytes or platelets. It controls the sizes of von Willebrand factor (VWF) multimers by cleaving VWF at the Tyr1605-Met1606 bond. Genetic deficiency of plasma ADAMTS13 activity results in hereditary thrombotic thrombocytopenic purpura (TTP), also named Upshaw-Schülman syndrome. To develop a potential gene therapy approach and to determine the domains of ADAMTS13 required for recognition and cleavage of VWF in vivo, a self-inactivating lentiviral vector encoding human wild-type ADAMTS13 or variant truncated after the spacer domain (construct MDTCS) was administrated by intra-amniotic injection on embryonic day 8. Direct stereomicroscopy and immunofluorescent microscopic analysis revealed that the green fluorescent protein (GFP) reporter, ADAMTS13 and MDTCS were predominantly expressed in the heart, kidneys and skin. The synthesized ADAMTS13 and truncated variant were detectable in mouse plasma by immunoprecipitation and Western blot, as well as by proteolytic cleavage of FRETS-VWF73 substrate. The levels of proteolytic activity in plasma of mice expressing ADAMTS13 and MDTCS were 5 ± 7% and 60 ± 70%, respectively using normal human plasma as a standard, and this proteolytic activity persisted for at least 24 weeks in Adamts13−/−mice and 42 weeks in wild-type mice tested (the duration of observation). The mice expressing both recombinant ADAMTS13 and MDTCS showed a significantly decreased ratio of plasma VWF collagen-binding activity to antigen and a reduction in VWF multimer sizes as compared to those in the controls. Moreover, the mice expressing ADAMTS13 and MDTCS showed a significant prolongation of ferric chloride-induced carotid arterial occlusion time (9.0 ± 0.6 and 25.2 ± 3.2 min, respectively) as compared to the Adamts13−/− mice expressing GFP alone (5.6 ± 0.5 min) (p<0.01). The ferric chloride-induced carotid occlusion time in Adamts13−/− mice expressing ADAMTS13 was almost identical to that in wild type mice with same genetic background (C56BL/6) (8.0 ± 0.2 min) (p>0.05). The data demonstrate the correction of the prothrombotic phenotype in Adamts13−/−mice by gene transfer to the fetus by viral vectors encoding human wild type ADAMTS13 and the carboxyl terminal truncated variant (MDTCS), supporting the feasibility of developing a gene therapy based treatment for hereditary TTP. The discrepancy in the proteolytic activity of MDTCS between in vitro (Zhang P et al. Blood, 2007 in press) and in vivo in the present study suggests the potential cofactors in murine circulation that may rescue the defective proteolytic activity of the carboxyl-terminal truncated ADAMTS13 protease seen in vitro.

scholarly journals Sequence and functional analysis of the positively acting regulatory gene amdR from Aspergillus nidulans.

1990 ◽  
Vol 10 (6) ◽  
pp. 3194-3203 ◽  
Author(s):  
A Andrianopoulos ◽  
M J Hynes
Keyword(s):  
Dna Binding ◽  
Aspergillus Nidulans ◽  
Nuclear Localization ◽  
Regulatory Gene ◽  
Transcription Activation ◽  
Binding Motif ◽  
Wild Type ◽  
Amino Terminal ◽  
Carboxyl Terminal

The positively acting regulatory gene amdR of Aspergillus nidulans coordinately regulates the expression of five structural genes involved in the catabolism of certain amides (amdS), omega amino acids (gatA and gabA), and lactams (lamA and lamB) in the presence of omega amino acid inducers. Analysis of the amdR gene showed that it contains three small introns, heterogeneous 5' and 3' transcription sites, and multiple AUG codons prior to the major AUG initiator. The predicted amdR protein sequence has a cysteine-rich "zinc finger" DNA-binding motif at the amino-terminal end, four putative acidic transcription activation motifs in the carboxyl-terminal half, and two sequences homologous to the simian virus 40 large T antigen nuclear localization motif. These nuclear localization sequences overlap the cysteine-rich DNA-binding motif. A series of 5', 3', and internal deletions were examined in vivo for transcription activator function and showed that the amdR product contains at least two activation regions in the carboxyl-terminal half. Each of these activator amdR product contains at least two activation regions in the carboxyl-terminal half. Each of these activator regions may function independently, but both are required for wild-type levels of transcription activation. A number of the amdR deletion products were found to compete with the wild-type amdR product in vivo. Development of a rapid method for the localization of amdR mutations is presented, and using this technique, we localized and sequenced the mutation in the semiconstitutive amdR6c allele. The amdR6c missense mutation occurs in the middle of the gene, and it is suggested that it results in an altered protein which activates gene expression efficiently in the absence of an inducer.

Three-dimensional in vivo analysis of Dictyostelium mounds reveals directional sorting of prestalk cells and defines a role for the myosin II regulatory light chain in prestalk cell sorting and tip protrusion

Development ◽  
2000 ◽  
Vol 127 (12) ◽  
pp. 2715-2728 ◽  
Author(s):  
P.A. Clow ◽  
T. Chen ◽  
R.L. Chisholm ◽  
J.G. McNally
Keyword(s):  
Long Range ◽  
Light Chain ◽  
Cell Sorting ◽  
Myosin Ii ◽  
Regulatory Light Chain ◽  
Upward Movement ◽  
Wild Type ◽  
Vertical Column ◽  
Initial Cluster

During cell sorting in Dictyostelium, we observed that GFP-tagged prestalk cells (ecmAO-expressing cells) moved independently and directionally to form a cluster. This is consistent with a chemotaxis model for cell sorting (and not differential adhesion) in which a long-range signal attracts many of the prestalk cells to the site of cluster formation. Surprisingly, the ecmAO prestalk cluster that we observed was initially found at a random location within the mound of this Ax3 strain, defining an intermediate sorting stage not widely reported in Dictyostelium. The cluster then moved en masse to the top of the mound to produce the classic, apical pattern of ecmAO prestalk cells. Migration of the cluster was also directional, suggesting the presence of another long-range guidance cue. Once at the mound apex, the cluster continued moving upward leading to protrusion of the mound's tip. To investigate the role of the cluster in tip protrusion, we examined ecmAO prestalk-cell sorting in a myosin II regulatory light chain (RLC) null in which tips fail to form. In RLC-null mounds, ecmAO prestalk cells formed an initial cluster that began to move to the mound apex, but then arrested as a vertical column that extended from the mound's apex to its base. Mixing experiments with wild-type cells demonstrated that the RLC-null ecmAO prestalk-cell defect is cell autonomous. These observations define a specific mechanism for myosin's function in tip formation, namely a mechanical role in the upward movement of the ecmAO prestalk cluster. The wild-type data demonstrate that cell sorting can occur in two steps, suggesting that, in this Ax3 strain, spatially and temporally distinct cues may guide prestalk cells first to an initial cluster and then later to the tip.

scholarly journals Protein Kinase C Phosphorylation of a γ-Protocadherin C-terminal Lipid Binding Domain Regulates Focal Adhesion Kinase Inhibition and Dendrite Arborization

2015 ◽  
Vol 290 (34) ◽  
pp. 20674-20686 ◽  
Author(s):  
Austin B. Keeler ◽  
Dietmar Schreiner ◽  
Joshua A. Weiner
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Cortical Neurons ◽  
Lipid Binding ◽  
Binding Motif ◽  
Wild Type ◽  
Western Blots ◽  
Phospholipid Binding ◽  
C Terminus

The γ-protocadherins (γ-Pcdhs) are a family of 22 adhesion molecules with multiple critical developmental functions, including the proper formation of dendritic arbors by forebrain neurons. The γ-Pcdhs bind to and inhibit focal adhesion kinase (FAK) via a constant C-terminal cytoplasmic domain shared by all 22 proteins. In cortical neurons lacking the γ-Pcdhs, aberrantly high activity of FAK and of PKC disrupts dendrite arborization. Little is known, however, about how γ-Pcdh function is regulated by other factors. Here we show that PKC phosphorylates a serine residue situated within a phospholipid binding motif at the shared γ-Pcdh C terminus. Western blots using a novel phospho-specific antibody against this site suggest that a portion of γ-Pcdh proteins is phosphorylated in the cortex in vivo. We find that PKC phosphorylation disrupts both phospholipid binding and the γ-Pcdh inhibition of (but not binding to) FAK. Introduction of a non-phosphorylatable (S922A) γ-Pcdh construct into wild-type cortical neurons significantly increases dendrite arborization. This same S922A construct can also rescue dendrite arborization defects in γ-Pcdh null neurons cell autonomously. Consistent with these data, introduction of a phosphomimetic (S/D) γ-Pcdh construct or treatment with a PKC activator reduces dendrite arborization in wild-type cortical neurons. Together, these data identify a novel mechanism through which γ-Pcdh control of a signaling pathway important for dendrite arborization is regulated.

scholarly journals Murine Cytomegalovirus Open Reading Frame M27 Plays an Important Role in Growth and Virulence in Mice

2001 ◽  
Vol 75 (4) ◽  
pp. 1697-1707 ◽  
Author(s):  
Gerardo Abenes ◽  
Manfred Lee ◽  
Erik Haghjoo ◽  
Tuong Tong ◽  
Xiaoyan Zhan ◽  
...  
Keyword(s):  
Scid Mice ◽  
Open Reading Frame ◽  
Murine Cytomegalovirus ◽  
Wild Type Virus ◽  
Wild Type ◽  
Reading Frame ◽  
Viral Virulence ◽  
Carboxyl Terminal

ABSTRACT Using a Tn3-based transposon mutagenesis approach, we have generated a pool of murine cytomegalovirus (MCMV) mutants. In this study, one of the mutants, RvM27, which contained the transposon sequence at open reading frame M27, was characterized both in tissue culture and in immunocompetent BALB/c mice and immunodeficient SCID mice. Our results suggest that the M27 carboxyl-terminal sequence is dispensable for viral replication in vitro. Compared to the wild-type strain and a rescued virus that restored the M27 region, RvM27 was attenuated in growth in both BALB/c and SCID mice that were intraperitoneally infected with the viruses. Specifically, the titers of RvM27 in the salivary glands, lungs, spleens, livers, and kidneys of the infected SCID mice at 21 days postinfection were 50- to 500-fold lower than those of the wild-type virus and the rescued virus. Moreover, the virulence of the mutant virus appeared to be attenuated, because no deaths occurred among SCID mice infected with RvM27 for up to 37 days postinfection, while all the animals infected with the wild-type and rescued viruses died within 27 days postinfection. Our observations provide the first direct evidence to suggest that a disruption of M27 expression results in reduced viral growth and attenuated viral virulence in vivo in infected animals. Moreover, these results suggest that M27 is a viral determinant required for optimal MCMV growth and virulence in vivo and provide insight into the functions of the M27 homologues found in other animal and human CMVs as well as in other betaherpesviruses.

scholarly journals c-Jun NH2-terminal Kinase (JNK)-interacting Protein-3 (JIP3) Regulates Neuronal Axon Elongation in a Kinesin- and JNK-dependent Manner

2013 ◽  
Vol 288 (20) ◽  
pp. 14531-14543 ◽  
Author(s):  
Tao Sun ◽  
Nuo Yu ◽  
Lu-Kai Zhai ◽  
Na Li ◽  
Chao Zhang ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Neuronal Development ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Anterograde Transport ◽  
Interacting Protein ◽  
Axon Elongation

The development of neuronal polarity is essential for the establishment of the accurate patterning of neuronal circuits in the brain. However, little is known about the underlying molecular mechanisms that control rapid axon elongation during neuronal development. Here, we report that c-Jun NH2-terminal kinase (JNK)-interacting protein-3 (JIP3) is highly expressed at axon tips during the critical period for axon development. Using gain- and loss-of-function approaches, immunofluorescence analysis, and in utero electroporation, we find that JIP3 can enhance axon elongation in primary hippocampal neurons and cortical neurons in vivo. We further demonstrate that JIP3 promotes axon elongation in a kinesin- and JNK-dependent manner using several deletion mutants of JIP3. Next, we demonstrate that the successful transportation of JIP3 to axon tips by kinesin is a prerequisite for enhancing JNK phosphorylation in this area and therefore promotes axon elongation, constituting a novel mechanism for coupling JIP3 anterograde transport with JNK signaling at the distal axons and axon elongation. Finally, our immunofluorescence data suggest that the activation of JNK at axon tips facilitates axon elongation by modulating cofilin activity and actin filament dynamics. These findings may have important implications for our understanding of neuronal axon elongation during development.

scholarly journals Uptake and metabolic fate of [HisA8,HisB4,GluB10,HisB27]insulin in rat liver in vivo

1998 ◽  
Vol 332 (2) ◽  
pp. 421-430 ◽  
Author(s):  
François AUTHIER ◽  
Gianni M. Di GUGLIELMO ◽  
Gillian M. DANIELSEN ◽  
John J. M. BERGERON
Keyword(s):  
Insulin Receptor ◽  
Subcellular Fractionation ◽  
Wild Type ◽  
Binding Assays ◽  
Insulin Degrading Enzyme ◽  
Direct Binding ◽  
Temporal Interaction ◽  
Kinetics Of Uptake

Receptor-mediated endocytosis and subsequent endosomal proteolysis of [125I]TyrA14-[HisA8,HisB4,GluB10,HisB27]insulin ([125I]TyrA14-H2 analogue), an insulin analogue exhibiting a high affinity for the insulin receptor, has been studied in liver parenchymal cells by quantitative subcellular fractionation and compared with that of wild-type [125I]TyrA14-insulin. Whereas the kinetics of uptake of the H2 analogue by liver was not different from that of insulin, the H2 analogue radioactivity after the 2 min peak declined significantly more slowly. A significant retention of the H2 analogue compared with insulin in both plasma membrane and endosomal fractions was observed and corresponded to decreased processing and dissociation of the H2 analogue. Cell-free endosomes preloaded in vivo with radiolabelled ligands and incubated in vitro processed insulin and extraluminally released insulin intermediates at a 2–3-fold higher rate than the H2 analogue. In vitro proteolysis of both non-radiolabelled and monoiodinated molecules by endosomal lysates showed a decreased response to the endosomal proteolytic machinery for the H2 analogue. However, in cross-linking and competition studies the H2 analogue exhibited an affinity for insulin-degrading enzyme identical with that of wild-type insulin. Brij-35-permeabilized endosomes revealed a 2-fold higher rate of dissociation of insulin from internalized receptors compared with the H2 analogue. After the administration of a saturating dose of both ligands, a rapid and reversible ligand-induced translocation of insulin receptor was observed, but without receptor loss. The H2 analogue induced a higher receptor concentration and tyrosine autophosphorylation of the receptor β subunit in endosomes. Moreover, a prolonged temporal interaction of the in vivo injected H2 analogue with receptor was observed by direct binding assays performed on freshly prepared subcellular fractions. These results indicate that endosomal proteolysis for the H2 analogue is slowed as a result of an increased residence time of the analogue on the insulin receptor and a low affinity of endosomal acidic insulinase for the dissociated H2 molecule.

Domain structure and functional analysis of the carboxyl-terminal polyacidic sequence of the RAD6 protein of Saccharomyces cerevisiae

1988 ◽  
Vol 8 (3) ◽  
pp. 1179-1185 ◽  
Author(s):  
A Morrison ◽  
E J Miller ◽  
L Prakash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Proteinase K ◽  
Intrinsic Activity ◽  
Structural Domain ◽  
Globular Domain ◽  
Wild Type ◽  
Calculated Mass ◽  
Stokes Radius ◽  
Carboxyl Terminal

The RAD6 gene of Saccharomyces cerevisiae, which is required for normal tolerance of DNA damage and for sporulation, encodes a 172-residue protein whose 23 carboxyl-terminal residues are almost all acidic. We show that this polyacidic sequence appends to RAD6 protein as a polyanionic tail and that its function in vivo does not require stoichiometry of length. RAD6 protein was purified to near homogeneity from a yeast strain carrying a RAD6 overproducing plasmid. Approximately the first 150 residues of RAD6 protein composed a structural domain that was resistant to proteinase K and had a Stokes radius typical of a globular protein of its calculated mass. The carboxyl-terminal polyacidic sequence was sensitive to proteinase K, and it endowed RAD6 protein with an aberrantly large Stokes radius that indicates an asymmetric shape. We deduce that RAD6 protein is monomeric and comprises a globular domain with a freely extending polyacidic tail. We tested the phenotypic effects of partial or complete deletion of the polyacidic sequence, demonstrating the presence of the shortened proteins in the cell by using antibody to RAD6 protein. Removal of the entire polyacidic sequence severely reduced sporulation but only slightly affected survival after UV irradiation or UV-induced mutagenesis. Strains with deletions of all but the first 4 or 15 residues of the polyacidic sequence were phenotypically almost wild type or wild type, respectively. We conclude that the intrinsic activity of RAD6 protein resides in the globular domain, that the polyacidic sequence has a stimulatory or modifying role evident primarily in sporulation, and that only a short section apparently functions as effectively as the entire polyacidic sequence.

scholarly journals Ste6p Mutants Defective in Exit from the Endoplasmic Reticulum (ER) Reveal Aspects of an ER Quality Control Pathway inSaccharomyces cerevisiae

1998 ◽  
Vol 9 (10) ◽  
pp. 2767-2784 ◽  
Author(s):  
Diego Loayza ◽  
Amy Tam ◽  
Walter K. Schmidt ◽  
Susan Michaelis
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Subcellular Fractionation ◽  
Metabolic Labeling ◽  
Wild Type ◽  
Er Quality Control ◽  
Ubiquitin Proteasome ◽  
Mutant Forms ◽  
Er Membrane

We are studying the intracellular trafficking of the multispanning membrane protein Ste6p, the a-factor transporter inSaccharomyces cerevisiae and a member of the ATP-binding cassette superfamily of proteins. In the present study, we have used Ste6p as model for studying the process of endoplasmic reticulum (ER) quality control, about which relatively little is known in yeast. We have identified three mutant forms of Ste6p that are aberrantly ER retained, as determined by immunofluorescence and subcellular fractionation. By pulse-chase metabolic labeling, we demonstrate that these mutants define two distinct classes. The single member of Class I, Ste6–166p, is highly unstable. We show that its degradation involves the ubiquitin–proteasome system, as indicated by its in vivo stabilization in certain ubiquitin–proteasome mutants or when cells are treated with the proteasome inhibitor drug MG132. The two Class II mutant proteins, Ste6–13p and Ste6–90p, are hyperstable relative to wild-type Ste6p and accumulate in the ER membrane. This represents the first report of a single protein in yeast for which distinct mutant forms can be channeled to different outcomes by the ER quality control system. We propose that these two classes of ER-retained Ste6p mutants may define distinct checkpoint steps in a linear pathway of ER quality control in yeast. In addition, a screen for high-copy suppressors of the mating defect of one of the ER-retained ste6 mutants has identified a proteasome subunit, Hrd2p/p97, previously implicated in the regulated degradation of wild-type hydroxymethylglutaryl-CoA reductase in the ER membrane.

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