scholarly journals The protein bcl-2 alpha does not require membrane attachment, but two conserved domains to suppress apoptosis.

1994 ◽  
Vol 126 (4) ◽  
pp. 1059-1068 ◽  
Author(s):  
C Borner ◽  
I Martinou ◽  
C Mattmann ◽  
M Irmler ◽  
E Schaerer ◽  
...  
Keyword(s):  
Sympathetic Neurons ◽  
Point Mutations ◽  
Cell Types ◽  
In Vitro Transcription ◽  
Site Directed Mutagenesis ◽  
Full Activity ◽  
Membrane Attachment ◽  
Hydrophobic Tail ◽  
Critical Regions

Bcl-2 is a mitochondrial- and perinuclear-associated protein that prolongs the lifespan of a variety of cell types by interfering with programmed cell death (apoptosis). Bcl-2 seems to function in an antioxidant pathway, and it is believed that membrane attachment mediated by a COOH-terminal hydrophobic tail is required for its full activity. To identify critical regions in bcl-2 alpha for subcellular localization, activity, and/or interaction with other proteins, we created, by site-directed mutagenesis, various deletion, truncation, and point mutations. We show here that membrane attachment is not required for the survival activity of bcl-2 alpha. A truncation mutant of bcl-2 alpha lacking the last 33 amino acids (T3.1) including the hydrophobic COOH terminus shows full activity in blocking apoptosis of nerve growth factor-deprived sympathetic neurons or TNF-alpha-treated L929 fibroblasts. Confocal microscopy reveals that the T3 mutant departs into the extremities of neurites in neurons and filopodias in fibroblasts. Consistently, T3 is predominantly detected in the soluble fraction by Western blotting, and is not inserted into microsomes after in vitro transcription/translation. We further provide evidence for motifs (S-N and S-II) at the NH2 and COOH terminus of bcl-2, which are crucial for its activity.

Domains of the rat rDNA promoter must be aligned stereospecifically

1992 ◽  
Vol 12 (3) ◽  
pp. 1266-1275
Author(s):  
W Q Xie ◽  
L I Rothblum
Keyword(s):  
Protein Complexes ◽  
Point Mutations ◽  
In Vitro Transcription ◽  
Promoter Elements ◽  
Repeat Distance ◽  
Deletion Mutations ◽  
The Core ◽  
In Vivo Experiments

Efficient transcription from the rat rDNA promoter results from an undefined interaction between the core (CPE) and upstream (UPE) promoter elements or the protein complexes which form on them. These interactions were demonstrated by the behavior of promoters that contained either linker-scanning or deletion mutations of the UPE in combination with point mutations of the CPE (bidomain mutants). In vivo transcription experiments using point mutations within the CPE (G----A mutation at either -16 or -7) demonstrated that the CPE may in fact consist of two domains. Whereas both of these mutants were rescued by the addition of UBF to in vitro transcription reactions, the CPE mutant -7A/G was inactive in vivo. Experiments with these bidomain mutants demonstrated that the UPE was required for the rescue of the CPE mutants. We also examined the hypothesis that this interaction might require a stereospecific alignment of the promoter elements. Our results indicate that the promoter consists of several domains with differing responses to mutations that alter the distance between, or within, the promoter elements. For example, the insertion or deletion of half-multiples of the helical repeat distance between -167 and -147 had no significant effect on transcription. On the other hand, some sites were sensitive to deletions of any size but not to insertions of up to 20 bp. The analyses of two sites yielded results suggesting that they lay between domains of the promoter that must be on the same side of the DNA helix for promoter activity. The first of these sites mapped between -106 and -95.(ABSTRACT TRUNCATED AT 250 WORDS)

Architecture of the maize mitochondrial atp1 promoter as determined by linker-scanning and point mutagenesis

1993 ◽  
Vol 13 (12) ◽  
pp. 7232-7238
Author(s):  
W D Rapp ◽  
D S Lupold ◽  
S Mack ◽  
D B Stern
Keyword(s):  
Promoter Region ◽  
Consensus Sequence ◽  
Point Mutations ◽  
In Vitro Transcription ◽  
Wild Type ◽  
Transcription Start ◽  
Transcription System ◽  
Mitochondrial Promoters ◽  
Point Mutagenesis

Plant mitochondrial promoters are poorly conserved but generally share a loose consensus sequence spanning approximately 17 nucleotides. Using a homologous in vitro transcription system, we have previously shown that an 11-nucleotide sequence within this region comprises at least part of the maize mitochondrial atp1 promoter (W. Rapp and D. Stern, EMBO J. 11:1065-1073, 1992). We have extended this finding by using a series of linker-scanning and point mutations to define the atp1 promoter in detail. Our results show that mutations at positions -12 to +5, relative to the major transcription start site, can decrease initiation rates to between < 10 and 40% of wild-type levels. Some mutations, scattered throughout this region, have lesser effects or no effect. Taken together, our data suggest a model in which the atp1 promoter consists of a central domain extending from -7 to +5 and an upstream domain of 1 to 3 bp that is centered around -11 to -12. Because many mutations within this promoter region are tolerated in vitro, the maize atp1 promoter is distinct from the highly conserved yeast mitochondrial promoters.

scholarly journals The Hox cooperativity motif of the chimeric oncoprotein E2a-Pbx1 is necessary and sufficient for oncogenesis.

1997 ◽  
Vol 17 (1) ◽  
pp. 81-88 ◽  
Author(s):  
C P Chang ◽  
I de Vivo ◽  
M L Cleary
Keyword(s):  
Dna Binding ◽  
Single Point ◽  
Point Mutations ◽  
Cell Types ◽  
Site Directed Mutagenesis ◽  
Binding Assays ◽  
Cellular Assays ◽  
Potential Contact ◽  
Carboxy Terminal ◽  
Necessary And Sufficient

E2a-Pbx1 chimeric oncoproteins result from fusion of the E2A and PBX1 genes at the sites of t(1;19) chromosomal translocations in a subset acute lymphoblastic leukemias. Experimentally, E2a-Pbx1 transforms a variety of cell types, including fibroblasts, myeloid progenitors, and lymphoblasts. Structure-function studies have shown that contributions from both E2a and Pbx1 are necessary for oncogenesis, but the Pbx1 homeodomain is dispensable and the required portion of Pbx1 has not been delineated. In this study, we used deletional and site-directed mutagenesis to identify portions of Pbx1 necessary for oncogenic and transcriptional activities of E2a-Pbx1. These studies defined a motif (named the Hox cooperativity motif [HCM]) carboxy terminal to the Pbx homeodomain that is required for cooperative DNA binding, cellular transcriptional activity, and the oncogenic potential of E2a-Pbx1. The HCM is highly conserved throughout the Pbx/exd subfamily of divergent homeodomain proteins and functions in DNA-binding assays as a potential contact site for Hox dimerization. E2a-Pbx1 proteins with interstitial deletion or single-point mutations in the HCM could neither activate transcription in cellular assays nor transform NIH 3T3 cells. An E2a-Pbx1 mutant containing 50 amino acids of Pbx1b spanning the HCM but lacking the homeodomain was capable of inducing fibroblast transformation. Thus, the HCM is a necessary and sufficient contribution of Pbx1 for oncogenesis induced by E2a-Pbx1 and accounts for its homeodomain-independent transforming properties. Since subtle alterations of the Pbx HCM result in complete abrogation of transforming activity whereas the homeodomain is entirely dispensable, we conclude that interactions mediated by the HCM are more important for transformation by E2a-Pbx1 than interactions with cognate Pbx DNA sites.

Dissection of functional domains in Bcl-2α by site-directed mutagenesis

1994 ◽  
Vol 72 (11-12) ◽  
pp. 463-469 ◽  
Author(s):  
Christoph Borner ◽  
Reynald Olivier ◽  
Isabelle Martinou ◽  
Chantal Mattmann ◽  
Jurg Tschopp ◽  
...  
Keyword(s):  
Cell Survival ◽  
Protein Structures ◽  
Cell Types ◽  
Site Directed Mutagenesis ◽  
Functional Domains ◽  
Directed Mutagenesis ◽  
Membrane Attachment ◽  
Alternatively Spliced ◽  
Membrane Bound ◽  
Related Proteins

Bcl-2α is a mitochondrial or perinuclear-associated oncoprotein that prolongs the life span of a variety of cell types by interfering with programmed cell death. How Bcl-2 confers cell survival is unknown, although antioxidant and antiprotease functions have been proposed. In addition, protein structures of Bcl-2 that are crucial for its survival activity are still ill-defined. Bcl-2 can occur as Bcl-2α or Bcl-2β, two alternatively spliced forms which solely differ in their carboxyl termini. The finding that Bcl-2α is active and membrane bound, but Bcl-2β is inactive and cytosolic, indicates that the carboxyl terminus contributes to the survival activity of Bcl-2. This region contains two subdomains, a domain X with unknown function and a hydrophobic stretch reported to mediate membrane assocation of Bcl-2α. Recently Bcl-2-related proteins have been identified. These include Bax that heterodimerizes with Bcl-2 and, when overxpressed, counteracts Bcl-2. Bax contains two highly conserved regions of sequence homology with Bcl-2, referred to as Bcl-2 homology 1 and 2 (BH1 and BH2) domains. Site-directed mutagenesis studies have revealed that both domains are not only novel dimerization motifs for the interaction of Bax with Bcl-2 but also crucial for the survival activity of Bcl-2. Interestingly, the C-terminal end of BH2 encompasses the Bcl-2α/β splice site, as well as part of domain X in Bcl-2α. To better define the role of domain X and the hydrophobic C-terminal stretch of Bcl-2α for its survival activity, we created various deletion and truncation mutations in these regions by site-directed mutagenesis. We show here that membrane attachment and therefore the hydrophobic stretch is not required for the survival activity of Bcl-2, but part of domain X appears to be indispensable.Key words: apoptosis, Bcl-2, mutagenesis, cell survival, functional domains.

scholarly journals Sequences and factors required for the F9 embryonal carcinoma stem cell E1a-like activity.

1991 ◽  
Vol 11 (11) ◽  
pp. 5534-5540 ◽  
Author(s):  
E J Murray ◽  
D Stott ◽  
P W Rigby
Keyword(s):  
Stem Cells ◽  
Embryonal Carcinoma ◽  
Point Mutations ◽  
Cell Types ◽  
In Vitro Mutagenesis ◽  
Sequence Element ◽  
Cell Extracts ◽  
Transcription In Vitro ◽  
Efficient Expression

F9 embryonal carcinoma (EC) stem cells contain an E1a-like activity that is absent from differentiated derivatives. We have previously characterized proteins present in F9 EC cell extracts that bind to the E1a-dependent E2A promoter and have shown that two of them, TF68 and DRTF1, are required for efficient transcription in vitro (N. B. La Thangue, B. Thimmapaya, and P. W. J. Rigby, Nucleic Acids Res. 18:2929-2938, 1990). We now show that the E1a-like activity is detectable in transient transfection assays. Deletion mutations show that a distal sequence element, which includes the ATF/CREB consensus, is required for expression in both cell types, although it does not mediate the down-regulation of promoter activity that accompanies differentiation. A series of point mutations generated by in vitro mutagenesis confirm this and show that sequences around -60 are necessary for efficient expression in stem cells but not in differentiated derivatives. These sequences bind DRTF1, the activity of which is strongly down-regulated during differentiation. Surprisingly, mutations in a previously uncharacterized region of the promoter restore activity to a promoter carrying the -60 mutation and lead to the formation of a new DNA-protein complex.

scholarly journals The ubiquitous octamer-binding protein(s) is sufficient for transcription of immunoglobulin genes.

1990 ◽  
Vol 10 (3) ◽  
pp. 982-990 ◽  
Author(s):  
D G Johnson ◽  
L Carayannopoulos ◽  
J D Capra ◽  
P W Tucker ◽  
J H Hanke
Keyword(s):  
B Cell ◽  
Protein S ◽  
Cell Types ◽  
In Vitro Transcription ◽  
Immunoglobulin Gene ◽  
Immunoglobulin Genes ◽  
Specific Expression ◽  
Specific Regulation

All immunoglobulin genes contain a conserved octanucleotide promoter element, ATGCAAAT, which has been shown to be required for their normal B-cell-specific transcription. Proteins that bind this octamer have been purified, and cDNAs encoding octamer-binding proteins have been cloned. Some of these proteins (referred to as OTF-2) are lymphoid specific, whereas at least one other, and possibly more (referred to as OTF-1), is found ubiquitously in all cell types. The exact role of these different proteins in directing the tissue-specific expression of immunoglobulin genes is unclear. We have identified two human pre-B-cell lines that contain extremely low levels of OTF-2 yet still express high levels of steady-state immunoglobulin heavy-chain mRNA in vivo and efficiently transcribe an immunoglobulin gene in vitro. Addition of a highly enriched preparation of OTF-1 made from one of these pre-B cells or from HeLa cells specifically stimulated in vitro transcription of an immunoglobulin gene. Furthermore, OFT-1 appeared to have approximately the same transactivation ability as OTF-2 when normalized for binding activity. These results suggest that OTF-1, without OTF-2, is sufficient for transcription of immunoglobulin genes and that OTF-2 alone is not responsible for the B-cell-specific regulation of immunoglobulin gene expression.

Binding of the transcription factor EBP-80 mediates the methylation response of an intracisternal A-particle long terminal repeat promoter

1991 ◽  
Vol 11 (1) ◽  
pp. 117-125
Author(s):  
M Falzon ◽  
E L Kuff
Keyword(s):  
Promoter Activity ◽  
In Vitro Transcription ◽  
Site Directed Mutagenesis ◽  
Long Terminal Repeats ◽  
Transcription System ◽  
A Cell ◽  
Induced Changes ◽  
Intracisternal A Particle

Intracisternal A-particle (IAP) expression in mouse cells has been correlated with hypomethylation of HhaI and HpaII sites in proviral long terminal repeats (LTRs). In a previous study, in vitro methylation of three HhaI sites in the U3 region of the LTR from the cloned genomic IAP element, MIA14, was shown to inhibit promoter activity in vivo. In this study, we found by site-directed mutagenesis that the two more downstream HhaI sites within this LTR were responsible for the methylation effects on promoter activity in vivo; methylation of the other (5') HhaI site, which lies within a putative SP1 binding domain, did not affect promoter activity. Methylation of the HhaI sites also inhibited promoter activity of the LTR in a cell-free transcription system. Exonuclease III footprinting demonstrated methylation-induced changes in protein binding over the region encompassing the downstream HhaI site, designated the Enh2 domain. The protein that interacts specifically with this domain, EBP-80, was characterized in a previous study (M. Falzon and E. L. Kuff, J. Biol. Chem. 264:21915-21922, 1989). We show here that the presence of methylcytosine in the HhaI site within the Enh2 domain inhibited binding of EBP-80 in vitro. The methylated MIA14 LTR construct was much less responsive to added EBP-80 in an in vitro transcription system than was the unmethylated construct. These data suggest that CpG methylation within the Enh2 domain may exert its effect on transcription in vivo by altering the interaction between EBP-80 and its cognate DNA sequence.

Sequences and factors required for the F9 embryonal carcinoma stem cell E1a-like activity

1991 ◽  
Vol 11 (11) ◽  
pp. 5534-5540
Author(s):  
E J Murray ◽  
D Stott ◽  
P W Rigby
Keyword(s):  
Stem Cells ◽  
Embryonal Carcinoma ◽  
Point Mutations ◽  
Cell Types ◽  
In Vitro Mutagenesis ◽  
Sequence Element ◽  
Cell Extracts ◽  
Transcription In Vitro ◽  
Efficient Expression

F9 embryonal carcinoma (EC) stem cells contain an E1a-like activity that is absent from differentiated derivatives. We have previously characterized proteins present in F9 EC cell extracts that bind to the E1a-dependent E2A promoter and have shown that two of them, TF68 and DRTF1, are required for efficient transcription in vitro (N. B. La Thangue, B. Thimmapaya, and P. W. J. Rigby, Nucleic Acids Res. 18:2929-2938, 1990). We now show that the E1a-like activity is detectable in transient transfection assays. Deletion mutations show that a distal sequence element, which includes the ATF/CREB consensus, is required for expression in both cell types, although it does not mediate the down-regulation of promoter activity that accompanies differentiation. A series of point mutations generated by in vitro mutagenesis confirm this and show that sequences around -60 are necessary for efficient expression in stem cells but not in differentiated derivatives. These sequences bind DRTF1, the activity of which is strongly down-regulated during differentiation. Surprisingly, mutations in a previously uncharacterized region of the promoter restore activity to a promoter carrying the -60 mutation and lead to the formation of a new DNA-protein complex.

Modeling Sympathetic Hyperactivity in Alzheimer’s Related Bone Loss

2021 ◽  
pp. 1-12
Author(s):  
Robert A. Culibrk ◽  
Ahmad S. Arabiyat ◽  
Carisa A. DeKalb ◽  
Mariah S. Hahn
Keyword(s):  
Bone Loss ◽  
Sympathetic Neurons ◽  
Cell Types ◽  
Collagen Type I ◽  
Type I ◽  
Mineral Density ◽  
Necrosis Factor Alpha ◽  
Glycol Diacrylate ◽  
Tnf Α

Background: A significant subset of patients with Alzheimer’s disease (AD) exhibit low bone mineral density and are therefore more fracture-prone, relative to their similarly aged neurotypical counterparts. In addition to chronic immune hyperactivity, behavioral dysregulation of effector peripheral sympathetic neurons—which densely innervate bone and potently modulate bone remodeling—is implicated in this pathological bone reformation. Objective: Thus, there exists a pressing need for a robust in vitro model which allows interrogation of the paracrine interactions between the putative mediators of AD-related osteopenia: sympathetic neurons (SNs) and mesenchymal stem cells (MSCs). Methods: Toward this end, activated SN-like PC12 cells and bone marrow derived MSCs were cultured in poly(ethylene glycol) diacrylate (PEGDA) hydrogels in the presence or absence of the AD-relevant inflammatory cytokine tumor necrosis factor alpha (TNF-α) under mono- and co-culture conditions. Results: PC12s and MSCs exposed separately to TNF-α displayed increased expression of pro-inflammatory mediators and decreased osteopontin (OPN), respectively. These data indicate that TNF-α was capable of inducing a dysregulated state in both cell types consistent with AD. Co-culture of TNF-α-activated PC12s and MSCs further exacerbated pathological behaviors in both cell types. Specifically, PC12s displayed increased secretion of interleukin 6 relative to TNF-α stimulated monoculture controls. Similarly, MSCs demonstrated a further reduction in osteogenic capacity relative to TNF-α stimulated monoculture controls, as illustrated by a significant decrease in OPN and collagen type I alpha I chain. Conclusion: Taken together, these data may indicate that dysregulated sympathetic activity may contribute to AD-related bone loss.

scholarly journals Selective SUMO modification of cAMP-specific phosphodiesterase-4D5 (PDE4D5) regulates the functional consequences of phosphorylation by PKA and ERK

2010 ◽  
Vol 428 (1) ◽  
pp. 55-65 ◽  
Author(s):  
Xiang Li ◽  
Suryakiran Vadrevu ◽  
Allan Dunlop ◽  
Jon Day ◽  
Noopur Advant ◽  
...  
Keyword(s):  
Cell Types ◽  
Site Directed Mutagenesis ◽  
Peptide Arrays ◽  
Post Translational Modification ◽  
Sumo Modification ◽  
Erk Phosphorylation ◽  
Extracellular Signal ◽  
Catalytic Unit ◽  
Mitogen Activated Protein

Enzymes from the PDE (phosphodiesterase) 4 cAMP-specific PDE family are crucial for the maintenance of compartmentalized cAMP responses in many cell types. Regulation of PDE activity can be achieved via post-translational modification such as phosphorylation by ERK (extracellular-signal-regulated kinase) MAPKs (mitogen-activated protein kinases) and PKA (protein kinase A). In the present paper, we report for the first time that PDE4 isoforms from the PDE4A and PDE4D subfamilies can be selectively modified by SUMO (small ubiquitin-related modifier). We have identified a single SUMO site within a consensus tetrapeptide motif, ΨKXE (where Ψ represents a hydrophobic residue), which lies in the catalytic unit of these enzymes. SUMO modification of PDE4 at this site was observed upon overexpression of the SUMO E3 ligase PIASy [protein inhibitor of activated STAT (signal transducer and activator of transcription) Y] in HEK (human embryonic kidney)-293 cells and we identify PIASy as a novel binding partner for long PDE4 isoforms. Site-directed mutagenesis of the acceptor lysine residue ablated conjugation of PDE4 with SUMO, suggesting the presence of a single SUMO site in the first subdomain of the conserved PDE4 catalytic unit. This observation was supported by both cell-free in vitro SUMOylation assays and analysis of SUMOylated spot-immobilized peptide arrays. SUMO modification of long PDE4 isoforms serves to augment their activation by PKA phosphorylation and repress their inhibition by ERK phosphorylation. Following ligation of β-adrenergic receptors, SUMOylation of PDE4 isoforms sufficiently amplified PKA-stimulated PDE4 activity to reduce markedly the PKA phosphorylation status of the β2-adrenergic receptor. These results highlight a new means whereby cells might achieve the selective regulation of the activity of cAMP-specific PDE4 enyzmes.

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