scholarly journals Targeting of synaptotagmin to neurite terminals in neuronally differentiated PC12 cells

2000 ◽  
Vol 113 (8) ◽  
pp. 1389-1404
Author(s):  
P.A. Krasnov ◽  
G. Enikolopov
Keyword(s):  
Amino Acids ◽  
Pc12 Cells ◽  
Neuronal Cell ◽  
Structural Element ◽  
Alanine Scanning Mutagenesis ◽  
Mutant Proteins ◽  
Pheochromocytoma Pc12 ◽  
Carboxy Terminal ◽  
Related Proteins ◽  
Point Mutagenesis

We have investigated structural elements that determine the accumulation of synaptotagmin, a major synaptic vesicle protein, in neurite terminals of neuronally differentiated neuroendocrine pheochromocytoma PC12 cells. We performed extensive deletion and point mutagenesis of rat synaptotagmin II, expressed mutant proteins in PC12 cells differentiated by nerve growth factor (NGF) and monitored their intracellular distribution by immunofluorescence. We found a structural element located at the carboxy-terminal domain of the synaptotagmin molecule, which is necessary for its accumulation at the terminal. Using alanine-scanning mutagenesis, we have identified two amino acids in this element, tryptophan W405 and leucine L408, that are critical for correct targeting of synaptotagmin II to neurite terminals. Changing either one of them to alanine prevents the accumulation of the protein at the terminals. These amino acids are evolutionarily conserved throughout the entire synaptotagmin family and also among synaptotagmin-related proteins, suggesting that different synaptotagmins may have similar mechanisms of targeting to neuronal cell terminals.

scholarly journals Negative Regulation of DNA Replication by the Retinoblastoma Protein Is Mediated by Its Association with MCM7

1998 ◽  
Vol 18 (5) ◽  
pp. 2748-2757 ◽  
Author(s):  
Jacqueline M. Sterner ◽  
Susan Dew-Knight ◽  
Christine Musahl ◽  
Sally Kornbluth ◽  
Jonathan M. Horowitz
Keyword(s):  
Amino Acids ◽  
Dna Replication ◽  
Protein Complexes ◽  
Replication Assay ◽  
Amino Terminal ◽  
Licensing Factor ◽  
Human Proteins ◽  
Carboxy Terminal ◽  
Related Proteins

ABSTRACT A yeast two-hybrid screen was employed to identify human proteins that specifically bind the amino-terminal 400 amino acids of the retinoblastoma (Rb) protein. Two independent cDNAs resulting from this screen were found to encode the carboxy-terminal 137 amino acids of MCM7, a member of a family of proteins that comprise replication licensing factor. Full-length Rb and MCM7 form protein complexes in vitro, and the amino termini of two Rb-related proteins, p107 and p130, also bind MCM7. Protein complexes between Rb and MCM7 were also detected in anti-Rb immunoprecipitates prepared from human cells. The amino-termini of Rb and p130 strongly inhibited DNA replication in an MCM7-dependent fashion in a Xenopus in vitro DNA replication assay system. These data provide the first evidence that Rb and Rb-related proteins can directly regulate DNA replication and that components of licensing factor are targets of the products of tumor suppressor genes.

scholarly journals Targeting of P-selectin to two regulated secretory organelles in PC12 cells.

1996 ◽  
Vol 134 (5) ◽  
pp. 1229-1240 ◽  
Author(s):  
J P Norcott ◽  
R Solari ◽  
D F Cutler
Keyword(s):  
Amino Acids ◽  
Pc12 Cells ◽  
Transient Expression ◽  
Subcellular Fractionation ◽  
Cytoplasmic Domain ◽  
Dense Core ◽  
The Other ◽  
Lysosomal Targeting ◽  
Dense Core Granules ◽  
Carboxy Terminal

Targeting of P-selectin to the regulated secretory organelles (RSOs) of phaeochromocytoma PC12 cells has been investigated. By expressing from cDNA a chimera composed of HRP and P-selectin, and then following HRP activity through subcellular fractionation, we have discovered that P-selectin contains signals that target HRP to the synaptic-like microvesicles (SLMV) as well as the dense-core granules (DCGs) of these cells. Mutagenesis of the chimera followed by transient expression in PC12 cells shows that at least two different sequences within the carboxy-terminal cytoplasmic tail of P-selectin are necessary, but that neither is sufficient for trafficking to the SLMV. One of these sequences is centred on the 10 amino acids of the membrane-proximal C1 exon that is also implicated in lysosomal targeting. The other sequence needed for trafficking to the SLMV includes the last four amino acids of the protein. The same series of mutations have a different effect on DCG targeting, showing that traffic to the two different RSOs depends on different features within the cytoplasmic domain of P-selectin.

scholarly journals High Glucose-Induced PC12 Cell Death by Increasing Glutamate Production and Decreasing Methyl Group Metabolism

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Minjiang Chen ◽  
Hong Zheng ◽  
Tingting Wei ◽  
Dan Wang ◽  
Huanhuan Xia ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Death ◽  
Pc12 Cells ◽  
High Glucose ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Creatine Phosphate ◽  
Metabolic Changes ◽  
Extracellular Medium ◽  
Methyl Group

Objective. High glucose- (HG-) induced neuronal cell death is responsible for the development of diabetic neuropathy. However, the effect of HG on metabolism in neuronal cells is still unclear.Materials and Methods. The neural-crest derived PC12 cells were cultured for 72 h in the HG (75 mM) or control (25 mM) groups. We used NMR-based metabolomics to examine both intracellular and extracellular metabolic changes in HG-treated PC12 cells.Results. We found that the reduction in intracellular lactate may be due to excreting more lactate into the extracellular medium under HG condition. HG also induced the changes of other energy-related metabolites, such as an increased succinate and creatine phosphate. Our results also reveal that the synthesis of glutamate from the branched-chain amino acids (isoleucine and valine) may be enhanced under HG. Increased levels of intracellular alanine, phenylalanine, myoinositol, and choline were observed in HG-treated PC12 cells. In addition, HG-induced decreases in intracellular dimethylamine, dimethylglycine, and 3-methylhistidine may indicate a downregulation of methyl group metabolism.Conclusions. Our metabolomic results suggest that HG-induced neuronal cell death may be attributed to a series of metabolic changes, involving energy metabolism, amino acids metabolism, osmoregulation and membrane metabolism, and methyl group metabolism.

scholarly journals Mutational Analysis of RsbT, an Activator of the Bacillus subtilis Stress Response Transcription Factor, σB

2004 ◽  
Vol 186 (9) ◽  
pp. 2789-2797 ◽  
Author(s):  
Robyn L. Woodbury ◽  
Tingqiu Luo ◽  
Lindsay Grant ◽  
W. G. Haldenwang
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Tertiary Structure ◽  
Mutational Analysis ◽  
Physical Stress ◽  
Negative Regulator ◽  
Alanine Scanning Mutagenesis ◽  
Charged Amino Acids ◽  
Carboxy Terminal ◽  
Stress Pathway

ABSTRACT σB, the stress-activated σ factor of Bacillus subtilis, requires the RsbT protein as an essential positive regulator of its physical stress pathway. Stress triggers RsbT to both inactivate the principal negative regulator of the physical stress pathway (RsbS) by phosphorylation and activate a phosphatase (RsbU) required for σB induction. Neither the regions of RsbT that are involved in responding to stress signaling nor those required for downstream events have been established. We used alanine scanning mutagenesis to examine the contributions of RsbT's charged amino acids to the protein's stability and activities. Eleven of eighteen rsbT mutations blocked σB induction by stress. The carboxy terminus of RsbT proved to be particularly important for accumulation in Bacillus subtilis. Four of the five most carboxy-terminal mutations yielded rsbT alleles whose products were undetectable in B. subtilis extracts. Charged amino acids in the central region of RsbT were less critical, with four of the five substitutions in this region having no measurable effect on RsbT accumulation or activity. Only when the substitutions extended into a region of kinase homology was σB induction affected. Six other RsbT variants, although present at levels adequate for activity, failed to activate σB and displayed significant changes in their ability to interact with RsbT's normal binding partners in a yeast dihybrid assay. These changes either dramatically altered the proteins' tertiary structure without affecting their stability or defined regions of RsbT that are involved in multiple interactions.

Toxicity and Metabolism of the Chloral-Derived Mammalian Alkaloid 1-Trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo) in PC12 Cells

2006 ◽  
Vol 61 (7-8) ◽  
pp. 601-610 ◽  
Author(s):  
Gerhard Bringmann ◽  
Doris Feineis ◽  
Miriam Münchbach ◽  
Ralf God ◽  
Karl Peters ◽  
...  
Keyword(s):  
Pc12 Cells ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Reductive Dehalogenation ◽  
Ring Plane ◽  
Cytotoxic Potential ◽  
X Ray ◽  
Pheochromocytoma Pc12 ◽  
Dose Dependent

Chloral-derived β-carbolines, which are structurally similar to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 5), are discussed to contribute to neuronal cell death in idiopathic Parkinson’s disease. The cytotoxicity of 1-trichloromethyl- 1,2,3,4-tetrahydro-β-carboline (TaClo, 4) to neuronal-like clonal pheochromocytoma PC12 cells was examined by the determination of lactate dehydrogenase (LDH) release. After incubation for 48 h, 4 showed a strong dose-dependent cytotoxic activity towards PC12 cells with an ED50 value of 230 μᴍ. In PC12 cells reductive dehalogenation of 4 was observed giving rise to the formation of 1-dichloromethyl-1,2,3,4-tetrahydro-β-carboline (6) as a main TaClo metabolite exhibiting a cytotoxic potential comparable to that of TaClo. An X-ray structure analysis, performed for the trifluoroacetyl derivative of 6, revealed the N-substituent of such a highly chlorinated agent to be dramatically pushed out of the β-carboline ring ‘plane’ due to the high steric demand of the huge dichloromethyl group at C(1).

scholarly journals Analysis of progressive deletions of the transmembrane and cytoplasmic domains of influenza hemagglutinin.

1986 ◽  
Vol 103 (4) ◽  
pp. 1193-1204 ◽  
Author(s):  
C Doyle ◽  
J Sambrook ◽  
M J Gething
Keyword(s):  
Amino Acids ◽  
Dna Sequences ◽  
Intracellular Transport ◽  
Hydrophobic Domain ◽  
Mutant Proteins ◽  
Influenza Hemagglutinin ◽  
Influenza Virus Hemagglutinin ◽  
Hydrophobic Amino Acids ◽  
Carboxy Terminal ◽  
Mutant Genes

Site-directed oligonucleotide mutagenesis has been used to introduce chain termination codons into the cloned DNA sequences encoding the carboxy-terminal transmembrane (27 amino acids) and cytoplasmic (10 amino acids) domains of influenza virus hemagglutinin (HA). Four mutant genes were constructed which express truncated forms of HA that lack the cytoplasmic domain and terminate at amino acids 9, 14, 17, or 27 of the wild-type hydrophobic domain. Analysis of the biosynthesis and intracellular transport of these mutants shows that the cytoplasmic tail is not needed for the efficient transport of HA to the cell surface; the stop-transfer sequences are located in the hydrophobic domain; 17 hydrophobic amino acids are sufficient to anchor HA stably in the membrane; and mutant proteins with truncated hydrophobic domains show drastic alterations in transport, membrane association, and stability.

scholarly journals Protective Effect of Ferulic Acid against Hydrogen Peroxide Induced Apoptosis in PC12 Cells

Molecules ◽  
2020 ◽  
Vol 26 (1) ◽  
pp. 90
Author(s):  
Hironao Nakayama ◽  
Masako Nakahara ◽  
Erina Matsugi ◽  
Midori Soda ◽  
Tomoka Hattori ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Ferulic Acid ◽  
Pc12 Cells ◽  
Pc12 Cell ◽  
Cell Injury ◽  
Biological Effects ◽  
Extracellular Signal ◽  
Pheochromocytoma Pc12 ◽  
Induced Apoptosis ◽  
Related Proteins

Ferulic Acid (FA) is a highly abundant phenolic phytochemical which is present in plant tissues. FA has biological effects on physiological and pathological processes due to its anti-apoptotic and anti-oxidative properties, however, the detailed mechanism(s) of function is poorly understood. We have identified FA as a molecule that inhibits apoptosis induced by hydrogen peroxide (H2O2) or actinomycin D (ActD) in rat pheochromocytoma, PC12 cell. We also found that FA reduces H2O2-induced reactive oxygen species (ROS) production in PC12 cell, thereby acting as an anti-oxidant. Then, we analyzed FA-mediated signaling responses in rat pheochromocytoma, PC12 cells using antibody arrays for phosphokinase and apoptosis related proteins. This FA signaling pathway in PC12 cells includes inactivation of pro-apoptotic proteins, SMAC/Diablo and Bad. In addition, FA attenuates the cell injury by H2O2 through the inhibition of phosphorylation of the extracellular signal-regulated kinase (ERK). Importantly, we find that FA restores expression levels of brain-derived neurotrophic factor (BDNF), a key neuroprotective effector, in H2O2-treated PC12 cells. As a possible mechanism, FA increases BDNF by regulating microRNA-10b expression following H2O2 stimulation. Taken together, FA has broad biological effects as a neuroprotective modulator to regulate the expression of phosphokinases, apoptosis-related proteins and microRNAs against oxidative stress in PC12 cells.

scholarly journals Assembly and routing of von Willebrand factor variants: the requirements for disulfide-linked dimerization reside within the carboxy-terminal 151 amino acids.

1991 ◽  
Vol 113 (1) ◽  
pp. 195-205 ◽  
Author(s):  
J Voorberg ◽  
R Fontijn ◽  
J Calafat ◽  
H Janssen ◽  
J A van Mourik ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Von Willebrand Factor ◽  
Amino Acid Residues ◽  
Mutant Proteins ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Von Willebrand ◽  
Willebrand Factor

The precursor protein of von Willebrand factor (pro-vWF) consists of four different repeated domains, denoted D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2, followed by a carboxy-terminal region of 151 amino acids without obvious internal homology. Previously, we have shown the requirement of the domains D1, D2, D', and D3 of pro-vWF in the assembly of pro-vWF dimers into multimers. Here, we define the domains of vWF involved in dimerization, using deletion mutants of full-length vWF cDNA transiently expressed in monkey kidney COS-1 cells. It is shown that only the carboxy-terminal 151 amino acid residues of vWF are required for dimerization. In addition, by analyzing a construct, encoding only the carboxy-terminal 151 amino acids of vWF, we find that the formation of dimers is an event independent of other domains present on pro-vWF, such as the domains C1 and C2 previously suggested to be involved in dimerization. Furthermore, it is shown that a deletion mutant of vWF, lacking the carboxy-terminal 151 amino acid residues and thus unable to dimerize, is proteolytically degraded in the ER. In contrast, a mutant protein, composed only of the carboxy-terminal 151 amino acids of vWF, and able to dimerize, is transported from the ER in a similar fashion as wild-type vWF. The role of the ER in the assembly of vWF is discussed with regard to the data presented in this paper on the intracellular fate of several vWF mutant proteins.

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