A novel N-terminal motif for palmitoylation of G-protein alpha subunits.
Ontology highlight
ABSTRACT:
SUBMITTER: Parenti M
PROVIDER: S-EPMC1132531 | biostudies-other | 1993 Apr
REPOSITORIES: biostudies-other
Similar Datasets
Project description: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.
| S-EPMC1137610 | biostudies-other
Project description:Modification of NMDA receptor function and trafficking contributes to the regulation of synaptic transmission and is important for several forms of synaptic plasticity. Here, we report that NMDA receptor subunits NR2A and NR2B have two distinct clusters of palmitoylation sites in their C-terminal region. Palmitoylation within the first cluster on a membrane-proximal region increases tyrosine phosphorylation of tyrosine-based internalization motifs by Src family protein tyrosine kinases, leading to enhanced stable surface expression of NMDA receptors. In addition, palmitoylation of these sites regulates constitutive internalization of the NMDA receptor in developing neurons. In marked contrast, palmitoylation of the second cluster in the middle of C terminus by distinct palmitoyl transferases causes receptors to accumulate in the Golgi apparatus and reduces receptor surface expression. These data suggest that regulated palmitoylation of NR2 subunits differentially modulates receptor trafficking and might be important for NMDA-receptor-dependent synaptic plasticity.
| S-EPMC2788208 | biostudies-literature
Project description:Proteolytic processing of amyloid precursor protein (APP) by beta- and gamma-secretases generates beta-amyloid (Abeta) peptides, which accumulate in the brains of individuals affected by Alzheimer disease. Detergent-resistant membrane microdomains (DRM) rich in cholesterol and sphingolipid, termed lipid rafts, have been implicated in Abeta production. Previously, we and others reported that the four integral subunits of the gamma-secretase associate with DRM. In this study we investigated the mechanisms underlying DRM association of gamma-secretase subunits. We report that in cultured cells and in brain the gamma-secretase subunits nicastrin and APH-1 undergo S-palmitoylation, the post-translational covalent attachment of the long chain fatty acid palmitate common in lipid raft-associated proteins. By mutagenesis we show that nicastrin is S-palmitoylated at Cys(689), and APH-1 is S-palmitoylated at Cys(182) and Cys(245). S-Palmitoylation-defective nicastrin and APH-1 form stable gamma-secretase complexes when expressed in knock-out fibroblasts lacking wild type subunits, suggesting that S-palmitoylation is not essential for gamma-secretase assembly. Nevertheless, fractionation studies show that S-palmitoylation contributes to DRM association of nicastrin and APH-1. Moreover, pulse-chase analyses reveal that S-palmitoylation is important for nascent polypeptide stability of both proteins. Co-expression of S-palmitoylation-deficient nicastrin and APH-1 in cultured cells neither affects Abeta40, Abeta42, and AICD production, nor intramembrane processing of Notch and N-cadherin. Our findings suggest that S-palmitoylation plays a role in stability and raft localization of nicastrin and APH-1, but does not directly modulate gamma-secretase processing of APP and other substrates.
| S-EPMC2615504 | biostudies-literature
Project description: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.
| S-EPMC1137601 | biostudies-other
Project description:BackgroundSmall secreted proteins (SSPs) are employed by plant pathogenic fungi as essential strategic tools for their successful colonization. SSPs are often species-specific and so far only a few widely phylogenetically distributed SSPs have been identified.ResultsA novel fungal SSP family consisting of 107 members was identified in the poplar tree fungal pathogen Marssonina brunnea, which accounts for over 17% of its secretome. We named these proteins IGY proteins (IGYPs) based on the conserved three amino acids at the N-terminus. In spite of overall low sequence similarity among IGYPs; they showed conserved N- and C-terminal motifs and a unified gene structure. By RT-PCR-seq, we analyzed the IGYP gene models and validated their expressions as active genes during infection. IGYP homologues were also found in 25 other Dikarya fungal species, all of which shared conserved motifs and the same gene structure. Furthermore, 18 IGYPs from 11 fungi also shared similar genomic contexts. Real-time RT-PCR showed that 8 MbIGYPs were highly expressed in the biotrophic stage. Interestingly, transient assay of 12 MbIGYPs showed that the MbIGYP13 protein induced cell death in resistant poplar clones.ConclusionsIn total, 154 IGYPs in 26 fungi of the Dikarya subkingdom were discovered. Gene structure and genomic context analyses indicated that IGYPs originated from a common ancestor. In M. brunnea, the expansion of highly divergent MbIGYPs possibly is associated with plant-pathogen arms race.
| S-EPMC4367982 | biostudies-literature
Project description:Essentially all proteins known to fold kinetically in a two-state manner have their N- and C-terminal secondary structural elements in contact, and the terminal elements often dock as part of the experimentally measurable initial folding step. Conversely, all N-C no-contact proteins studied so far fold by non-two-state kinetics. By comparison, about half of the single domain proteins in the Protein Data Bank have their N- and C-terminal elements in contact, more than expected on a random probability basis but not nearly enough to account for the bias in protein folding. Possible reasons for this bias relate to the mechanisms for initial protein folding, native state stability, and final turnover.
| S-EPMC545867 | biostudies-other
Project description:Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) are central to the signaling processes of multicellular organisms. We have explored the diversity of the G protein subunits in mammals and found evidence for a large family of genes that encode the alpha subunits. Amino acid sequence comparisons show that the different alpha subunits fall into at least three classes. These classes have been conserved in animals separated by considerable evolutionary distances; they are present in mammals, Drosophila, and nematodes. We have now obtained cDNA clones encoding two murine alpha subunits, G alpha 12 and G alpha 13, that define a fourth class. The translation products are predicted to have molecular masses of 44 kDa and to be insensitive to ADP-ribosylation by pertussis toxin. They share 67% amino acid sequence identity with each other and less than 45% identity with other alpha subunits. Their transcripts can be detected in every tissue examined, although the relative levels of the G alpha 13 message appear somewhat variable.
| S-EPMC51921 | biostudies-other
Project description:Mycobacteria regulate their energy (ATP) levels to sustain their survival even in stringent living conditions. Recent studies have shown that mycobacteria not only slow down their respiratory rate but also block ATP hydrolysis of the F-ATP synthase (α3:β3:γ:δ:ε:a:b:b':c9) to maintain ATP homeostasis in situations not amenable for growth. The mycobacteria-specific α C-terminus (α533-545) has unraveled to be the major regulative of latent ATP hydrolysis. Its deletion stimulates ATPase activity while reducing ATP synthesis. In one of the six rotational states of F-ATP synthase, α533-545 has been visualized to dock deep into subunit γ, thereby blocking rotation of γ within the engine. The functional role(s) of this C-terminus in the other rotational states are not clarified yet and are being still pursued in structural studies. Based on the interaction pattern of the docked α533-545 region with subunit γ, we attempted to study the druggability of the α533-545 motif. In this direction, our computational work has led to the development of an eight-featured α533-545 peptide pharmacophore, followed by database screening, molecular docking, and pose selection, resulting in eleven hit molecules. ATP synthesis inhibition assays using recombinant ATP synthase as well as mycobacterial inverted membrane vesicles show that one of the hits, AlMF1, inhibited the mycobacterial F-ATP synthase in a micromolar range. The successful targeting of the α533-545-γ interaction motif demonstrates the potential to develop inhibitors targeting the α site to interrupt rotary coupling with ATP synthesis.
| S-EPMC8698299 | biostudies-literature
Project description:Compared with other mammalian species, humans are particularly susceptible to tau-mediated neurodegenerative disorders. Differential interactions of the tau protein with other proteins are critical for mediating tau's physiological functions as well as tau-associated pathological processes. Primate tau harbors an 11-amino acid-long motif in its N-terminal region (residues 18-28), which is not present in non-primate species and whose function is unknown. Here, we used deletion mutagenesis to remove this sequence region from the longest human tau isoform, followed by glutathione S-transferase (GST) pulldown assays paired with isobaric tags for relative and absolute quantitation (iTRAQ) multiplex labeling, a quantitative method to measure protein abundance by mass spectrometry. Using this method, we found that the primate-specific N-terminal tau motif differentially mediates interactions with neuronal proteins. Among these binding partners are proteins involved in synaptic transmission (synapsin-1 and synaptotagmin-1) and signaling proteins of the 14-3-3 family. Furthermore, we identified an interaction of tau with a member of the annexin family (annexin A5) that was linked to the 11-residue motif. These results suggest that primate Tau has evolved specific residues that differentially regulate protein-protein interactions compared with tau proteins from other non-primate mammalian species. Our findings provide in vitro insights into tau's interactions with other proteins that may be relevant to human disease.
| S-EPMC5846163 | biostudies-literature
Project description:The N-terminal approximately 440 aa of integrin alpha subunits contain seven sequence repeats. These are predicted here to fold into a beta-propeller domain. A homologous domain from the enzyme phosphatidylinositol phospholipase D is predicted to have the same fold. The domains contain seven four-stranded beta-sheets arranged in a torus around a pseudosymmetry axis. The trimeric G-protein beta subunit (G beta) appears to be the most closely related beta-propeller. Integrin ligands and a putative Mg2+ ion are predicted to bind to the upper face of the beta-propeller. This face binds substrates in beta-propeller enzymes and is used by the G protein beta subunit to bind the G protein alpha subunit. The integrin alpha subunit I domain, which is structurally homologous to the G protein alpha subunit, is tethered to the top of the beta-propeller domain by a hinge that may allow movement of the domains relative to one another. The Ca2+-binding motifs in integrin alpha subunits are on the lower face of the beta-propeller.
| S-EPMC19237 | biostudies-literature