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.

Mutagenesis of the borage Δ6 fatty acid desaturase

2000 ◽  
Vol 28 (6) ◽  
pp. 636-638 ◽  
Author(s):  
O. Sayanova ◽  
F. Beaudoin ◽  
B. Libisch ◽  
P. Shewry ◽  
J. Napier
Keyword(s):  
Fatty Acid ◽  
Consensus Sequence ◽  
Fatty Acid Desaturase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
The Third ◽  
Membrane Bound ◽  
Fusion Domain ◽  
Δ6 Desaturase

The consensus sequence of the third histidine box of a range of Δ5, Δ6, Δ8 and sphingolipid desaturases differs from that of the membrane-bound non-fusion Δ12 and Δ15 desaturases in the presence of glutamine instead of histidine. We have used site-directed mutagenesis to determine the importance of glutamine and other residues of the third histidine box and created a chimaeric enzyme to determine the ability of the Cyt b5 fusion domain from the plant sphingolipid desaturase to substitute for the endogenous domain of the Δ6 desaturase.

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.

scholarly journals Novel 3,6-Dihydroxypicolinic Acid Decarboxylase Mediated Picolinic Acid Catabolism inAlcaligenes faecalisJQ135

2018 ◽  
Author(s):  
Qiu Jiguo ◽  
Zhang Yanting ◽  
Yao Shigang ◽  
Ren Hao ◽  
Qian Meng ◽  
...  
Keyword(s):  
Aromatic Compounds ◽  
Picolinic Acid ◽  
Degradation Pathway ◽  
Site Directed Mutagenesis ◽  
Acid Decarboxylase ◽  
Directed Mutagenesis ◽  
Deficient Mutant ◽  
Carbon And Nitrogen ◽  
Genetic Deletion ◽  
Related Proteins

AbstractAlcaligenesfaecalisstrain JQ135 utilizes picolinic acid (PA) as sole carbon and nitrogen source for growth. In this study, we screened a 6-hydroxypicolinic acid (6HPA) degradation-deficient mutant through random transposon mutagenesis. The mutant hydroxylated 6HPA into an intermediate, identified as 3,6-dihydroxypicolinic acid (3,6DHPA) with no further degradation. A novel decarboxylase PicC was identified that was found to be responsible for the decarboxylation of 3,6DHPA to 2.5-dihydroxypyridine. Although, PicC belonged to amidohydrolase_2 family, it shows low similarity (<45%) when compared to other reported amidohydrolase_2 family decarboxylases. Moreover, PicC was found to form a monophyletic group in the phylogenetic tree constructed using PicC and related proteins. Further, the genetic deletion and complementation results demonstrated thatpicCwas essential for PA degradation. The PicC was Zn2+-dependent non-oxidative decarboxylase that can specifically catalyze the irreversible decarboxylation of 3,6DHPA to 2.5-dihydroxypyridine. TheKmandkcattowards 3,6DHPA were observed to be 13.44 μM and 4.77 s-1, respectively. Site-directed mutagenesis showed that His163 and His216 were essential for PicC activity.ImportancePicolinic acid is a natural toxic pyridine derived from L-tryptophan metabolism and some aromatic compounds in mammalian and microbial cells. Microorganisms can degrade and utilize picolinic acid for their growth, and thus, a microbial degradation pathway of picolinic acid has been proposed. Picolinic acid is converted into 6-hydroxypicolinic acid, 3,6-dihydroxypicolinic acid, and 2,5-dihydroxypyridine in turn. However, there was no physiological and genetic validation for this pathway. This study demonstrated that 3,6DHPA was an intermediate in PA catabolism process and further identified and characterized a novel amidohydrolase_2 family decarboxylase PicC. It was also shown that PicC could catalyze the decarboxylation process of 3,6-dihydroxypicolinic acid into 2,5-dihydroxypyridine. This study provides a basis for understanding PA degradation pathway and the underlying molecular mechanism.

Fibroblasts expressing mouse c locus tyrosinase produce an authentic enzyme and synthesize phaeomelanin

1993 ◽  
Vol 104 (2) ◽  
pp. 467-475 ◽  
Author(s):  
A.J. Winder ◽  
A. Wittbjer ◽  
E. Rosengren ◽  
H. Rorsman
Keyword(s):  
Cell Types ◽  
Tyrosinase Activity ◽  
Melanin Synthesis ◽  
Post Translational Modification ◽  
Heterologous Promoter ◽  
Dopachrome Tautomerase ◽  
Membrane Bound ◽  
Related Proteins ◽  
Melanocytic Differentiation ◽  
Melanoma Cell Lines

Recent advances in the study of the molecular biology of mouse pigmentation have led to the discovery of a family of proteins involved in the control of melanin synthesis. It has been confirmed that the product of the mouse c (albino) locus is the key melanogenic enzyme tyrosinase, but study of its function and regulation have been hampered by the presence of closely related proteins within melanin-synthesising cells. To overcome these problems, we have established lines of mouse fibroblasts expressing the c locus mouse tyrosinase. Here we describe characterisation of the tyrosinase synthesised by these cells and demonstrate considerable similarity between the expressed tyrosinase and the native enzyme. The expressed tyrosinase is proteolytically cleaved to produce membrane-bound and soluble forms of the expected molecular mass and is rich in N-linked carbohydrate, suggesting that melanocytic differentiation is not a prerequisite for post-translational modification of the protein. The expressed enzyme has tyrosinase activity, but not catalase or dopachrome tautomerase activity, confirming that it is an authentic tyrosinase. Transfected fibroblasts expressing tyrosinase are shown to share several physiological characteristics with melanoma cell lines, including increased pigmentation and tyrosinase activity in response to increased cell density. Since tyrosinase is expressed under a heterologous promoter, these shared characteristics probably reflect translational or post-translational controls that operate in both non-melanocytic and melanocytic cell types. We demonstrate that pigmented fibroblasts contain the melanin synthesis intermediates 5-S-cysteinyldopa and 5-S-glutathionyl-dopa, and produce a phaeomelanin-like pigment, but do not contain detectable eumelanin. Expression of tyrosine is therefore sufficient for the synthesis of a form of melanin pigment in fibroblasts.

A model system for studying membrane biogenesis. Overexpression of cytochrome b5 in yeast results in marked proliferation of the intracellular membrane

1993 ◽  
Vol 106 (1) ◽  
pp. 249-259 ◽  
Author(s):  
G. Vergeres ◽  
T.S. Yen ◽  
J. Aggeler ◽  
J. Lausier ◽  
L. Waskell
Keyword(s):  
Cytochrome B5 ◽  
Alpha Helix ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Membrane Biogenesis ◽  
Wild Type ◽  
Membrane Anchor ◽  
Type Protein ◽  
Wild Type Protein ◽  
Membrane Bound

Cytochrome b5 is an amphipathic microsomal protein that is anchored to the endoplasmic reticulum by a single hydrophobic transmembrane alpha-helix located near the carboxyl terminus of the protein. In yeast, cytochrome b5 provides electrons for fatty acid desaturation and ergosterol biosynthesis. High level expression of cytochrome b5 in Saccharomyces cerevisiae was achieved using the yeast metallothionein promoter and a synthetic cytochrome b5 gene. In order to accommodate the markedly increased amount of the membrane-bound cytochrome b5, the yeast cell proliferated its nuclear membrane. As many as 20 pairs of stacked membranes could be observed to partially encircle the nucleus. This morphological arrangement of membrane around the nucleus is known as a karmella. In an effort to understand which part of the cytochrome b5 molecule, i.e. the membrane anchor or the soluble heme domain, which is competent in electron transfer, provided the signal for the de novo membrane biogenesis, a series of studies, including site-directed mutagenesis, was undertaken. The results of these experiments demonstrated that the inactive hemedeficient apo form of the membrane-bound protein stimulates membrane proliferation to the same extent as the holo wild-type protein, whereas cytosolic forms of cytochrome b5 did not induce membrane synthesis. These data demonstrate that membrane proliferation is a consequence of the cell's ability to monitor the level of membrane proteins and to compensate for alterations in these levels rather than the result of the ability of the extra cytochrome b5 to catalyze synthesis of extra lipid that had to be accommodated in new membrane. Site-directed mutagenesis studies of the membrane binding domain of cytochrome b5 provided additional clues about the nature of the signal for membrane proliferation. Replacement of the membrane anchor by a non-physiological nonsense sequence of 22 leucines gave rise to a mutant protein that triggered membrane biosynthesis. The conclusion from these experiments is clear; the signal for membrane proliferation does not reside in some specific amino acid sequence but instead in the hydrophobic properties of the proliferant. Interestingly, these membranes are somewhat diminished in quantity and have a slightly altered morphology compared to those induced by the wild-type protein. It was also observed that disruption of the putative alpha helix of the membrane anchor by an Ala116Pro mutation, which gives rise to two sequential prolines at positions 115 and 116 results in a protein with diminished capacity to induce membrane formation.(ABSTRACT TRUNCATED AT 400 WORDS)

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