scholarly journals A novel N-terminal motif for palmitoylation of G-protein α subunits

1993 ◽  
Vol 291 (2) ◽  
pp. 349-353 ◽  
Author(s):  
M Parenti ◽  
M A Viganó ◽  
C M H Newman ◽  
G Milligan ◽  
A I Magee
Keyword(s):  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Hybrid Cells ◽  
Protein Synthesis Inhibitors ◽  
Cos Cells ◽  
Metabolic Labelling ◽  
And Function ◽  
Α Subunits ◽  
Caax Motif ◽  
Novel Protein

We have examined the post-translational processing of G alpha subunits expressed endogenously in rat PC12 and NG108-15 rat/mouse hybrid cells, and after transfection of cDNA expression constructs into COS cells. Thioester-linked palmitoylation of alpha o, alpha s, alpha q/alpha 11 and alpha 12 has been detected by metabolic labelling with [3H]palmitate and immunoprecipitation. Palmitoylation of alpha o occurs post-translationally in cells treated with protein-synthesis inhibitors, suggesting possible dynamic acylation. Palmitoylation of the C-terminal CAAX motif has been excluded. Site-directed mutagenesis of alpha o has been used to implicate the site of modification as a cysteine residue next to the N-terminal myristoylated glycine, in a novel protein-lipid modification motif Met-Gly-Cys. The non-palmitoylated alpha o mutant is still myristoylated but shows reduced membrane binding, suggesting that reversible palmitoylation may regulate G alpha localization and function.

scholarly journals Molecular and Pharmacological Characterization of the Interaction between Human Geranylgeranyltransferase Type I and Ras-Related Protein Rap1B

2021 ◽  
Vol 22 (5) ◽  
pp. 2501
Author(s):  
Sonja Hinz ◽  
Dominik Jung ◽  
Dorota Hauert ◽  
Hagen S. Bachmann
Keyword(s):  
Protein Function ◽  
Tumor Development ◽  
Cysteine Residue ◽  
Type I ◽  
Pharmacological Characterization ◽  
Anti Cancer ◽  
And Function ◽  
Caax Motif ◽  
Important Drug

Geranylgeranyltransferase type-I (GGTase-I) represents an important drug target since it contributes to the function of many proteins that are involved in tumor development and metastasis. This led to the development of GGTase-I inhibitors as anti-cancer drugs blocking the protein function and membrane association of e.g., Rap subfamilies that are involved in cell differentiation and cell growth. In the present study, we developed a new NanoBiT assay to monitor the interaction of human GGTase-I and its substrate Rap1B. Different Rap1B prenylation-deficient mutants (C181G, C181S, and ΔCQLL) were designed and investigated for their interaction with GGTase-I. While the Rap1B mutants C181G and C181S still exhibited interaction with human GGTase-I, mutant ΔCQLL, lacking the entire CAAX motif (defined by a cysteine residue, two aliphatic residues, and the C-terminal residue), showed reduced interaction. Moreover, a specific, peptidomimetic and competitive CAAX inhibitor was able to block the interaction of Rap1B with GGTase-I. Furthermore, activation of both Gαs-coupled human adenosine receptors, A2A (A2AAR) and A2B (A2BAR), increased the interaction between GGTase-I and Rap1B, probably representing a way to modulate prenylation and function of Rap1B. Thus, A2AAR and A2BAR antagonists might be promising candidates for therapeutic intervention for different types of cancer that overexpress Rap1B. Finally, the NanoBiT assay provides a tool to investigate the pharmacology of GGTase-I inhibitors.

scholarly journals Characterization of iduronate sulphatase mutants affecting N-glycosylation sites and the cysteine-84 residue

1997 ◽  
Vol 326 (1) ◽  
pp. 243-247 ◽  
Author(s):  
Gilles MILLAT ◽  
Roseline FROISSART ◽  
Irène MAIRE ◽  
Dominique BOZON
Keyword(s):  
Catalytic Activity ◽  
Amino Acid Position ◽  
Cysteine Residue ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Lysosomal Storage ◽  
Post Translational Modification ◽  
Cos Cells ◽  
Conserved Sequence ◽  
Glycosylation Sites

Iduronate sulphatase (IDS) is responsible for mucopolysaccharidosis type II, a rare recessive X-linked lysosomal storage disease. The aim of this work was to evaluate the functional importance of each N-glycosylation site, and of the cysteine-84 residue. IDS mutant cDNAs, lacking one of the eight potential N-glycosylation sites, were expressed in COS cells. Although each of the potential sites was used, none of the eight glycosylation sites appeared to be essential for lysosomal targeting. Another important sulphatase co- or post-translational modification for generating catalytic activity involves the conversion of a cysteine residue surrounded by a conserved sequence C-X-P-S-R into a 2-amino-3-oxopropionic acid residue [Schmidt, Selmer, Ingendoh and von Figura (1995) Cell 82, 271–278]. This conserved cysteine, located at amino acid position 84 in IDS, was replaced either by an alanine (C84A) or by a threonine (C84T) using site-directed mutagenesis. C84A and C84T mutant cDNAs were expressed either in COS cells or in human lymphoblastoid cells deleted for the IDS gene. C84A had a drastic effect both for IDS processing and for catalytic activity. The C84T mutation produced a small amount of mature forms but also abolished enzyme activity, confirming that the cysteine residue at position 84 is required for IDS activity.

scholarly journals Palmitoylation of multiple Src-family kinases at a homologous N-terminal motif

1994 ◽  
Vol 303 (3) ◽  
pp. 749-753 ◽  
Author(s):  
M Koegl ◽  
P Zlatkine ◽  
S C Ley ◽  
S A Courtneidge ◽  
A I Magee
Keyword(s):  
Tyrosine Kinases ◽  
Recombinant Baculovirus ◽  
Fatty Acid Analysis ◽  
Site Directed Mutagenesis ◽  
Protein Tyrosine Kinases ◽  
Protein Tyrosine ◽  
Cos Cells ◽  
Cysteine Motif ◽  
Lipid Fatty Acid ◽  
And Function

We have recently identified a novel N-terminal cysteine-containing motif which specifies the palmitoylation of several G-protein alpha-subunits [Parenti, Viganó, Newman, Milligan and Magee (1993) Biochem. J. 291, 349-353]. A related motif occurs at the N-terminus of members of the Src family of protein tyrosine kinases except for Src itself and Blk. We have investigated whether the Src, Fyn, Yes and Lck gene products are palmitoylated. Src was not labelled with [3H]palmitate when endogenously expressed in COS cells. In contrast, endogenous Yes immunoprecipitated from COS cells was palmitoylated. Fyn was palmitoylated in insect cells infected with a recombinant baculovirus and the palmitoylation was independent of protein synthesis, suggesting a dynamic turnover of this lipid. Fatty acid analysis indicated that most of the label was incorporated as palmitate. Lck was palmitoylated when expressed by transfection in COS cells. All of these protein tyrosine kinases were also detectably myristoylated in each of the systems tested. Experiments performed with mutants of Lck expressed by transfection in COS cells indicated that cysteines at positions 3 and 5 were both palmitoylation sites and that myristoylation was required for palmitoylation. To confirm that palmitoylation was occurring on cysteines in the N-terminal region of Fyn, site-directed mutagenesis was used to replace the cysteines at positions 3 and 6 with alanine. The resulting protein was not palmitoylated but was still myristoylated when expressed in COS cells. A glycine to alanine mutant at position 2 was also not palmitoylated, showing that myristoylation is a prerequisite for palmitoylation. Our data indicate that Src family members containing the N-terminal cysteine motif are indeed palmitoylated. By analogy with Ras, it is possible that palmitoylation may play an important role in the localization and function of Src family protein tyrosine kinases.

scholarly journals New insights into structure and function of bis-phosphinic acid derivatives and implications for CFTR modulation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sara Bitam ◽  
Ahmad Elbahnsi ◽  
Geordie Creste ◽  
Iwona Pranke ◽  
Benoit Chevalier ◽  
...  
Keyword(s):  
Phosphinic Acid ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Transmembrane Conductance Regulator ◽  
Intracellular Loop ◽  
Respiratory Cells ◽  
Cftr Protein ◽  
Dynamics Simulations ◽  
And Function ◽  
Molecular Mode

AbstractC407 is a compound that corrects the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein carrying the p.Phe508del (F508del) mutation. We investigated the corrector effect of c407 and its derivatives on F508del-CFTR protein. Molecular docking and dynamics simulations combined with site-directed mutagenesis suggested that c407 stabilizes the F508del-Nucleotide Binding Domain 1 (NBD1) during the co-translational folding process by occupying the position of the p.Phe1068 side chain located at the fourth intracellular loop (ICL4). After CFTR domains assembly, c407 occupies the position of the missing p.Phe508 side chain. C407 alone or in combination with the F508del-CFTR corrector VX-809, increased CFTR activity in cell lines but not in primary respiratory cells carrying the F508del mutation. A structure-based approach resulted in the synthesis of an extended c407 analog G1, designed to improve the interaction with ICL4. G1 significantly increased CFTR activity and response to VX-809 in primary nasal cells of F508del homozygous patients. Our data demonstrate that in-silico optimized c407 derivative G1 acts by a mechanism different from the reference VX-809 corrector and provide insights into its possible molecular mode of action. These results pave the way for novel strategies aiming to optimize the flawed ICL4–NBD1 interface.

Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function

1997 ◽  
Vol 75 (6) ◽  
pp. 687-696 ◽  
Author(s):  
Tamo Fukamizo ◽  
Ryszard Brzezinski
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Binding ◽  
Structure And Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Streptomyces Sp ◽  
Binding Cleft ◽  
And Function

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.

scholarly journals Site-Directed Mutagenesis of the Heterotrimeric Killer Toxin Zymocin Identifies Residues Required for Early Steps in Toxin Action

2014 ◽  
Vol 80 (20) ◽  
pp. 6549-6559 ◽  
Author(s):  
Sabrina Wemhoff ◽  
Roland Klassen ◽  
Friedhelm Meinhardt
Keyword(s):  
Kluyveromyces Lactis ◽  
Killer Toxin ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Target Cells ◽  
Directed Mutagenesis ◽  
Content Type ◽  
Protein Toxin ◽  
Assisted Delivery

ABSTRACTZymocin is aKluyveromyces lactisprotein toxin composed of αβγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αβ-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activityin vitrothat might be important during γ import.Saccharomyces cerevisiaestrains carrying k1-derived hybrid elements deficient in either αβ (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α's chitinase domain and the sole cysteine residue of β (Cys250). Since βγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of β C250 and γ C231 in zymocin assembly. To test the capability of αβ to carry alternative cargos, the heterologous ACNase fromPichia acaciae(P. acaciaeOrf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αβ's capability to deliver other cargo proteins into target cells.

scholarly journals Acetylcholine Receptor Gating at Extracellular Transmembrane Domain Interface: the “Pre-M1” Linker

2007 ◽  
Vol 130 (6) ◽  
pp. 559-568 ◽  
Author(s):  
Prasad Purohit ◽  
Anthony Auerbach
Keyword(s):  
Acetylcholine Receptors ◽  
State Energy ◽  
Transmembrane Domain ◽  
Salt Bridge ◽  
Side Chain ◽  
Coupling Energy ◽  
And Function ◽  
Loop 2 ◽  
Α Subunits ◽  
Α1 Subunit

Charged residues in the β10–M1 linker region (“pre-M1”) are important in the expression and function of neuromuscular acetylcholine receptors (AChRs). The perturbation of a salt bridge between pre-M1 residue R209 and loop 2 residue E45 has been proposed as being a principle event in the AChR gating conformational “wave.” We examined the effects of mutations to all five residues in pre-M1 (positions M207–P211) plus E45 in loop 2 in the mouse α1-subunit. M207, Q208, and P211 mutants caused small (approximately threefold) changes in the gating equilibrium constant (Keq), but the changes for R209, L210, and E45 were larger. Of 19 different side chain substitutions at R209 on the wild-type background, only Q, K, and H generated functional channels, with the largest change in Keq (67-fold) from R209Q. Various R209 mutants were functional on different E45 backgrounds: H, Q, and K (E45A), H, A, N, and Q (E45R), and K, A, and N (E45L). Φ values for R209 (on the E45A background), L210, and E45 were 0.74, 0.35, and 0.80, respectively. Φ values for R209 on the wt and three other backgrounds could not be estimated because of scatter. The average coupling energy between 209/45 side chains (six different pairs) was only −0.33 kcal/mol (for both α subunits, combined). Pre-M1 residues are important for expression of functional channels and participate in gating, but the relatively modest changes in closed- vs. open-state energy caused mutations, the weak coupling energy between these residues and the functional activity of several unmatched-charge pairs are not consistent with the perturbation of a salt bridge between R209 and E45 playing the principle role in gating.

scholarly journals N-terminal fatty acylation of the α-subunit of the G-protein Gi1: only the myristoylated protein is a substrate for palmitoylation

1994 ◽  
Vol 303 (3) ◽  
pp. 697-700 ◽  
Author(s):  
F Galbiati ◽  
F Guzzi ◽  
A I Magee ◽  
G Milligan ◽  
M Parenti
Keyword(s):  
Palmitic Acid ◽  
G Protein ◽  
Fatty Acyl ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Α Subunit ◽  
Metabolic Labelling ◽  
Fatty Acylation ◽  
Absolute Requirement ◽  
Cdna Construct

The alpha-subunit of the G-protein Gi1 carries two fatty acyl moieties covalently bound to its N-terminal region: myristic acid is linked to glycine-2 and palmitic acid is linked to cysteine-3. Using site-directed mutagenesis on a cDNA construct of alpha i1 we have generated an alpha i1-G2A mutant, carrying alanine instead of glycine at position 2, and alpha i1-C3S mutant, in which serine replaced cysteine-3 and a double mutant with both substitutions (alpha i1-G2A/C3S). These constructs were individually expressed by transfection in Cos-7 cells, and incorporation of fatty acids into the various mutants was compared with wild-type alpha i1 monitoring metabolic labelling with [3H]palmitate or [3H]myristate. The disruption of the palmitoylation site in alpha i1-C3S did not influence myristoylation, whereas prevention of myristoylation in alpha i1-G2A also abolished palmitoylation. Co-translational myristoylation is thus an absolute requirement for alpha i1 to be post-translationally palmitoylated. The non-palmitoylated alpha i1-C3S showed reduced membrane binding to the same extent as the non-myristoylated/non-palmitoylated alpha i1-G2A and alpha i1-G2A/C3S mutants, indicating that the attachment of palmitic acid is necessary for proper interaction with the membrane.

scholarly journals The CaaX motif is required for isoprenylation, carboxyl methylation, and nuclear membrane association of lamin B2.

1991 ◽  
Vol 113 (1) ◽  
pp. 13-23 ◽  
Author(s):  
G T Kitten ◽  
E A Nigg
Keyword(s):  
Nuclear Membrane ◽  
Stop Codon ◽  
Mevalonic Acid ◽  
Cysteine Residue ◽  
Inner Nuclear Membrane ◽  
Nuclear Lamins ◽  
Carboxyl Methylation ◽  
Reticulocyte Lysates ◽  
Caax Motif

Recent evidence suggests that the conserved COOH-terminal CaaX motif of nuclear lamins may play a role in targeting newly synthesized proteins to the nuclear envelope. We have shown previously that in rabbit reticulocyte lysates the cysteine residue of the CaaX motif of chicken lamin B2 is necessary for incorporation of a derivative of mevalonic acid, the precursor of isoprenoids. Here we have analyzed the properties of normal and mutated forms of chicken lamin B2 stably expressed in mouse L cells. Mutation of the cysteine residue of the CaaX motif to alanine or introduction of a stop codon immediately after the cysteine residue was found to abolish both isoprenylation and carboxyl methylation of transfected lamin B2. Concomitantly, although nuclear import of the mutant lamin B2 proteins was preserved, their association with the inner nuclear membrane was severely impaired. From these results we conclude that the COOH-terminal CaaX motif is required for isoprenylation and carboxyl methylation of lamins in vivo, and that these modifications are important for association of B-type lamins with the nucleoplasmic surface of the inner nuclear membrane.

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