scholarly journals The processing pathway of prelamin A

1994 ◽  
Vol 107 (1) ◽  
pp. 61-67 ◽  
Author(s):  
M. Sinensky ◽  
K. Fantle ◽  
M. Trujillo ◽  
T. McLain ◽  
A. Kupfer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Line ◽  
Genetic Background ◽  
Initial Step ◽  
Carboxyl Terminus ◽  
Lamin A ◽  
Prelamin A ◽  
Carboxyl Terminal ◽  
Prenylated Proteins

The conversion of mammalian prelamin A to mature lamin A proceeds through the removal of 18 amino acids from the carboxyl terminus. The initial step in this processing is the isoprenylation of a CAAX box cysteine. This proteolytic event is distinctive for prelamin A among the known prenylated mammalian proteins. Since the carboxyl terminus of prelamin A is removed during maturation, it is not obvious that this protein would undergo the two reactions subsequent to prenylation observed in other CAAX box proteins--the endoproteolytic removal of the carboxyl-terminal 3 amino acids and the subsequent methylation of the now carboxyl-terminal cysteine. To characterize the maturation of prelamin A further, we have developed a CHO-K1 cell line that possesses a dexamethasone-inducible human prelamin A against a genetic background of high mevalonate uptake. Utilizing this cell line in association with antibodies specific to the transgenic prelamin A, we have been able to demonstrate directly in vivo that prelamin A undergoes farnesylation and carboxymethylation prior to conversion to lamin A, as is the case for other prenylated proteins. We have demonstrated previously that in the absence of isoprenylation, conversion of prelamin A to lamin A is blocked, but that unprocessed prelamin A is transported to the nucleus where it can still undergo maturation. Consistent with the implications of these prior studies, we now demonstrate the presence of both subunits of farnesyl-protein transferase in the nucleus.

Features of the pp60v-src carboxyl terminus that are required for transformation

1986 ◽  
Vol 6 (8) ◽  
pp. 2807-2819
Author(s):  
P Yaciuk ◽  
D Shalloway
Keyword(s):  
Amino Acids ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Cellular Proteins ◽  
Carboxyl Terminus ◽  
Deletion Mutants ◽  
Carboxyl Terminal ◽  
Protein Instability ◽  
Wide Family

Analysis of the biological and biochemical activities of pp60recombinant-src proteins encoded by 12 carboxyl-terminal mutants showed that a wide family of alternate src carboxyl termini permit complete transforming and kinase activities. src proteins having carboxyl termini which are up to 10 amino acids longer than that of pp60c-src (17 amino acids longer than that of pp60v-src) still permit transformation. Transformation-positive mutations preserve leucine-516, a residue which is highly conserved in protein-tyrosine kinase sequences; removal causes in vivo protein instability. Successive deletion mutants show that this residue is at the boundary of a region required for kinase activity. pp60src which is truncated just outside this point still transforms cells and binds both pp50 and pp90 cellular proteins.

scholarly journals Features of the pp60v-src carboxyl terminus that are required for transformation.

1986 ◽  
Vol 6 (8) ◽  
pp. 2807-2819 ◽  
Author(s):  
P Yaciuk ◽  
D Shalloway
Keyword(s):  
Amino Acids ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Cellular Proteins ◽  
Carboxyl Terminus ◽  
Deletion Mutants ◽  
Carboxyl Terminal ◽  
Protein Instability ◽  
Wide Family

Analysis of the biological and biochemical activities of pp60recombinant-src proteins encoded by 12 carboxyl-terminal mutants showed that a wide family of alternate src carboxyl termini permit complete transforming and kinase activities. src proteins having carboxyl termini which are up to 10 amino acids longer than that of pp60c-src (17 amino acids longer than that of pp60v-src) still permit transformation. Transformation-positive mutations preserve leucine-516, a residue which is highly conserved in protein-tyrosine kinase sequences; removal causes in vivo protein instability. Successive deletion mutants show that this residue is at the boundary of a region required for kinase activity. pp60src which is truncated just outside this point still transforms cells and binds both pp50 and pp90 cellular proteins.

scholarly journals The carboxyl terminus of the human cytomegalovirus UL37 immediate-early glycoprotein is conserved in primary strains and is important for transactivation

2001 ◽  
Vol 82 (7) ◽  
pp. 1569-1579 ◽  
Author(s):  
Wail A. Hayajneh ◽  
Despina G. Contopoulos-Ioannidis ◽  
Marci M. Lesperance ◽  
Ana M. Venegas ◽  
Anamaris M. Colberg-Poley
Keyword(s):  
Amino Acids ◽  
Human Cytomegalovirus ◽  
Immediate Early ◽  
Carboxyl Terminus ◽  
Wild Type ◽  
Reading Frame ◽  
Cytosolic Domains ◽  
Carboxyl Terminal

The human cytomegalovirus (HCMV) UL37 exon 3 (UL37x3) open reading frame (ORF) encodes the carboxyl termini of two immediate-early glycoproteins (gpUL37 and gpUL37M). UL37x3 homologous sequences are not required for mouse cytomegalovirus (MCMV) growth in vitro; yet, they are important for MCMV growth and pathogenesis in vivo. Similarly, UL37x3 sequences are dispensable for HCMV growth in culture, but their requirement for HCMV growth in vivo is not known. To determine this requirement, we directly sequenced the complete UL37x3 gene in multiple HCMV primary strains. A total of 63 of the 310 amino acids in the UL37x3 ORF differ non-conservatively in one or more HCMV primary strains. The HCMV UL37x3 genetic diversity is non-random: the N-glycosylation (46/186 aa) and basic (9/15 aa) domains have the highest proportion of non-conservative variant amino acids. Nonetheless, most (15/17 signals) of the N-glycosylation signals are retained in all HCMV primary strains. Moreover, new N-glycosylation signals are encoded by 5/20 primary strains. In sharp contrast, the UL37x3 transmembrane (TM) ORF completely lacks diversity in all 20 HCMV sequenced primary strains, and only 1 of 28 cytosolic tail residues differs non-conservatively. To test the functional significance of the conserved carboxyl terminus, gpUL37 mutants lacking the TM and/or cytosolic tail were tested for transactivating activity. The gpUL37 carboxyl-terminal mutants are partially defective in hsp70 promoter transactivation even though they trafficked similarly to the wild-type protein into the endoplasmic reticulum and to mitochondria. From these results, we conclude that N-glycosylated gpUL37, particularly its TM and cytosolic domains, is important for HCMV growth in humans.

Lamin A precursor is localized to intranuclear foci

1995 ◽  
Vol 108 (1) ◽  
pp. 273-285 ◽  
Author(s):  
A.M. Sasseville ◽  
Y. Raymond
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Nuclear Lamina ◽  
Subcellular Location ◽  
Cysteine Residue ◽  
Carboxyl Terminus ◽  
Nuclear Space ◽  
Lamin A ◽  
Carboxyl Terminal ◽  
Processing Steps

Lamin A is synthesized in the cytoplasm as a precursor bearing a carboxyl-terminal CaaX box or isoprenylation signal. This precursor is post-translationally processed through multiple steps: isoprenylation with a farnesyl residue on the cysteine of the CaaX box, proteolytic removal of the last three amino acids, carboxymethylation of the cysteine residue and, finally, proteolytic removal of 15 amino acids from the carboxyl terminus. This last step gives rise to mature lamin A from which the isoprenylated terminus has been removed. Isoprenylation is a prerequisite for all other steps of processing. The subcellular location of these processing steps for lamin A is still a matter of debate. We have produced an antibody specific to the 18 amino acid carboxyl terminus of the lamin A precursor that does not recognize mature lamin A. This antibody detects intranuclear foci by immunofluorescence. Larger amounts of lamin A precursor were accumulated by treating cells with mevinolin (MVN), an inhibitor of isoprenoid synthesis. In MVN-treated cells, the lamin A precursor accumulated most strikingly in the peripheral nuclear lamina where it was assembled, while intranuclear foci were maintained. The addition of an excess of mevalonate (MVA), which restores isoprenylation activity, to MVN-treated cells led to a progressive disappearance of the lamin A precursor from the peripheral lamina. This process was completed after 4 hours of MVA treatment, after which the lamin A precursor was restricted to intranuclear foci. We conclude from these results that the non-isoprenylated lamin A precursor appears competent for assembly into the peripheral nuclear lamina, and that all the processing steps leading to mature lamin A can occur within the nuclear space.

scholarly journals Extensive mutagenesis of a transcriptional activation domain identifies single hydrophobic and acidic amino acids important for activation in vivo.

1997 ◽  
Vol 17 (1) ◽  
pp. 115-122 ◽  
Author(s):  
M B Sainz ◽  
S A Goff ◽  
V L Chandler
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transcriptional Activation ◽  
Carboxyl Terminus ◽  
Wild Type ◽  
Activation Domain ◽  
Hydrophobic Residues ◽  
Transcriptional Activation Domain ◽  
Acidic Amino Acids

C1 is a transcriptional activator of genes encoding biosynthetic enzymes of the maize anthocyanin pigment pathway. C1 has an amino terminus homologous to Myb DNA-binding domains and an acidic carboxyl terminus that is a transcriptional activation domain in maize and yeast cells. To identify amino acids critical for transcriptional activation, an extensive random mutagenesis of the C1 carboxyl terminus was done. The C1 activation domain is remarkably tolerant of amino acid substitutions, as changes at 34 residues had little or no effect on transcriptional activity. These changes include introduction of helix-incompatible amino acids throughout the C1 activation domain and alteration of most single acidic amino acids, suggesting that a previously postulated amphipathic alpha-helix is not required for activation. Substitutions at two positions revealed amino acids important for transcriptional activation. Replacement of leucine 253 with a proline or glutamine resulted in approximately 10% of wild-type transcriptional activation. Leucine 253 is in a region of C1 in which several hydrophobic residues align with residues important for transcriptional activation by the herpes simplex virus VP16 protein. However, changes at all other hydrophobic residues in C1 indicate that none are critical for C1 transcriptional activation. The other important amino acid in C1 is aspartate 262, as a change to valine resulted in only 24% of wild-type transcriptional activation. Comparison of our C1 results with those from VP16 reveal substantial differences in which amino acids are required for transcriptional activation in vivo by these two acidic activation domains.

scholarly journals RhoB prenylation is driven by the three carboxyl-terminal amino acids of the protein: Evidenced in vivo by an anti-farnesyl cysteine antibody

2000 ◽  
Vol 97 (21) ◽  
pp. 11626-11631 ◽  
Author(s):  
R. Baron ◽  
E. Fourcade ◽  
I. Lajoie-Mazenc ◽  
C. Allal ◽  
B. Couderc ◽  
...  
Keyword(s):  
Amino Acids ◽  
Terminal Amino ◽  
Carboxyl Terminal

scholarly journals The C Terminus of Substrates Is Critical but Not Sufficient for Their Degradation by the Pseudomonas aeruginosa CtpA Protease

2020 ◽  
Vol 202 (16) ◽  
Author(s):  
Sammi Chung ◽  
Andrew J. Darwin
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Cell Wall ◽  
Outer Membrane ◽  
Content Type ◽  
C Terminus ◽  
Outer Membrane Lipoprotein ◽  
Membrane Lipoprotein ◽  
Carboxyl Terminal

ABSTRACT Bacterial carboxyl-terminal processing proteases (CTPs) are widely conserved and have been linked to important processes, including signal transduction, cell wall metabolism, and virulence. However, the features that target proteins for CTP-dependent cleavage are unclear. Studies of the Escherichia coli CTP Prc suggested that it cleaves proteins with nonpolar and/or structurally unconstrained C termini, but it is not clear if this applies broadly. Pseudomonas aeruginosa has a divergent CTP, CtpA, which is required for virulence. CtpA works in complex with the outer membrane lipoprotein LbcA to degrade cell wall hydrolases. In this study, we investigated if the C termini of two nonhomologous CtpA substrates are important for their degradation. We determined that these substrates have extended C termini compared to those of their closest E. coli homologs. Removing 7 amino acids from these extensions was sufficient to reduce their degradation by CtpA both in vivo and in vitro. Degradation of one truncated substrate was restored by adding the C terminus from the other but not by adding an unrelated sequence. However, modification of the C termini of nonsubstrates, by adding the C-terminal amino acids from a substrate, did not cause their degradation by CtpA. Therefore, the C termini of CtpA substrates are required but not sufficient for their efficient degradation. Although C-terminal truncated substrates were protected from degradation, they still associated with the LbcA-CtpA complex in vivo. Therefore, degradation of a protein by CtpA requires a C terminus-independent interaction with the LbcA-CtpA complex, followed by C terminus-dependent degradation, perhaps because CtpA normally initiates cleavage at a C-terminal site. IMPORTANCE Carboxyl-terminal processing proteases (CTPs) are found in all three domains of life, but exactly how they work is poorly understood, including how they recognize substrates. Bacterial CTPs have been associated with virulence, including CtpA of Pseudomonas aeruginosa, which works in complex with the outer membrane lipoprotein LbcA to degrade potentially dangerous peptidoglycan hydrolases. We report an important advance by revealing that efficient degradation by CtpA requires at least two separable phenomena and that one of them depends on information encoded in the substrate C terminus. A C terminus-independent association with the LbcA-CtpA complex is followed by C terminus-dependent cleavage by CtpA. Increased understanding of how CTPs target proteins is significant, due to their links to virulence, peptidoglycan remodeling, and other important processes.

scholarly journals Carboxyl-Terminal Extensions beyond the Conserved Pentapeptide Reduce Rates of Chemoreceptor Adaptational Modification

2005 ◽  
Vol 187 (15) ◽  
pp. 5115-5121 ◽  
Author(s):  
Wing-Cheung Lai ◽  
Gerald L. Hazelbauer
Keyword(s):  
Sensory System ◽  
Bacterial Chemotaxis ◽  
Covalent Modification ◽  
High Abundance ◽  
Carboxyl Terminus ◽  
Carboxyl Terminal ◽  
Catalyzed Reactions ◽  
Chemosensory Systems

ABSTRACT Sensory adaptation in bacterial chemotaxis is mediated by covalent modification of chemoreceptors. Specific glutamyl residues are methylated and demethylated in reactions catalyzed by methyltransferase CheR and methylesterase CheB. In the well-characterized chemosensory systems of Escherichia coli and Salmonella spp., efficient modification by either enzyme is dependent on a conserved pentapeptide sequence, NWETF or NWESF, present at the extreme carboxyl terminus of high-abundance chemoreceptors. To what extent is position at the extreme carboxyl terminus important for pentapeptide-mediated enhancement of adaptational modification? Is this position equally important for enhancement of both enzyme activities? To address these questions, we created forms of high-abundance receptor Tsr or Tar carrying one, six, or eight additional amino acids extending beyond the pentapeptide at their carboxyl termini and assayed methylation, demethylation, deamidation, and ability to mediate chemotaxis. In vitro and in vivo, all three carboxyl-terminal extensions reduced pentapeptide-mediated enhancement of rates of adaptational modification. CheB-catalyzed reactions were more affected than CheR-catalyzed reactions. Effects were less severe for the complete sensory system in vivo than for the minimal system of receptor and modification enzymes in vitro. Notably, extended receptors mediated chemotaxis as efficiently as wild-type receptors, providing a striking example of robustness in chemotactic systems. This could reflect compensatory reductions of rates for both modification reactions, mitigation of effects of slower reactions by the intertwined circuitry of signaling and adaptation, or tolerance of a range of reactions rates for adaptational modification. No matter what the mechanism, the observations provide a challenging test for mathematical models of chemotaxis.

In Vitro Labeling Strategies for Identifying Primary Neural Tissue and a Neuronal Cell Line after Transplantation in the Cns

1993 ◽  
Vol 2 (2) ◽  
pp. 131-149 ◽  
Author(s):  
Stephen M. Onifer ◽  
Linda A. White ◽  
Scott R. Whittemore ◽  
Vicky R. Holets
Keyword(s):  
Escherichia Coli ◽  
Cell Line ◽  
Neuronal Cell ◽  
Fluorescein Isothiocyanate ◽  
Initial Step ◽  
Host Cells ◽  
Lacz Gene ◽  
Raphe Neurons

Potential labels for identifying embryonic raphe neurons and a clonal, neuronally differentiating, raphe-derived cell line, RN33B, in CNS transplantation studies were tested by first characterizing the labels in vitro. The labels that were tested included 4,6-diamidino-2-phenylindole hydrochloride, 1,1′-dioctadecyl-3,3,3′-tetramethylindocarbocyanine perchlorate, the Escherichia coli lacZ gene, Fast Blue, Fluoro-Gold, fluorescein-conjugated latex microspheres, fluorescein isothiocyanate-conjugated or nonconjugated Phaseolus vulgaris leucoagglutinin, methyl o-(6-amino-3′-imino-3H-xanthen-9-yl) benzoate monohydrochloride, or tetanus toxin C fragment. Subsequently, the optimal in vitro labels for embryonic raphe neurons and for RN33B cells were characterized in vivo after CNS transplantation. In vitro, 1,1-dioctadecyl-3,3,3′-tetramethylindocarbocyanine perchlorate (DiI) optimally labeled embryonic neurons. The Escherichia coli lacZ gene optimally labeled RN33B cells. Most labels were rapidly diluted in cultures of embryonic astrocytes and proliferating RN33B cells. Some labels were toxic and were often retained in cellular debris. In vivo, DiI was visualized in transplanted, DiI-labeled raphe neurons, but not in astrocytes up to 1 mo posttransplant. DiI-labeled host cells were seen after transplantation of lysed, DiI-labeled cells. β-Galactosidase was visualized in transplanted, Escherichia coli lacZ gene-labeled RN33B cells after 15 days in vivo. No β-galactosidase was seen in host cells after transplantation of lysed, lacZ-labeled RN33B cells. The results demonstrate that labels for use in CNS transplantation studies should be optimized for the specific population of donor cells under study, with the initial step being characterization in vitro followed by in vivo analysis. Appropriate controls for false-positive labeling of host cells should always be assessed.

scholarly journals Rce1 deficiency accelerates the development of K-RAS–induced myeloproliferative disease

Blood ◽  
2006 ◽  
Vol 109 (2) ◽  
pp. 763-768 ◽  
Author(s):  
Annika M. Wahlstrom ◽  
Briony A. Cutts ◽  
Christin Karlsson ◽  
Karin M. E. Andersson ◽  
Meng Liu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Converting Enzyme ◽  
Myeloproliferative Disease ◽  
Ras Proteins ◽  
Carboxyl Terminal ◽  
Caax Motif

Abstract The RAS proteins undergo farnesylation of a carboxyl-terminal cysteine (the “C” of the carboxyl-terminal CaaX motif). After farnesylation, the 3 amino acids downstream from the farnesyl cysteine (the -aaX of the CaaX motif) are released by RAS-converting enzyme 1 (RCE1). We previously showed that inactivation of Rce1 in mouse fibroblasts mislocalizes RAS proteins away from the plasma membrane and inhibits RAS transformation. Therefore, we hypothesized that the inactivation of Rce1 might inhibit RAS transformation in vivo. To test this hypothesis, we used Cre/loxP recombination techniques to simultaneously inactivate Rce1 and activate a latent oncogenic K-RAS allele in hematopoietic cells in mice. Normally, activation of the oncogenic K-RAS allele in hematopoietic cells leads to rapidly progressing and lethal myeloproliferative disease. Contrary to our hypothesis, the inactivation of Rce1 actually increased peripheral leukocytosis, increased the release of immature hematopoietic cells into the circulation and the infiltration of cells into liver and spleen, and caused mice to die more rapidly. Moreover, in the absence of Rce1, splenocytes and bone marrow cells expressing oncogenic K-RAS yielded more and larger colonies when grown in methylcellulose. We conclude that the inactivation of Rce1 worsens the myeloproliferative disease caused by oncogenic K-RAS.

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