scholarly journals Myristoylation alone is sufficient for PKA catalytic subunits to fractionally associate with the plasma membrane to regulate neuronal functions

2020 ◽  
Author(s):  
Wei-Hong Xiong ◽  
Maozhen Qin ◽  
Haining Zhong
Keyword(s):  
Plasma Membrane ◽  
Ampa Receptors ◽  
Fluorescent Proteins ◽  
Membrane Association ◽  
Post Translational Modification ◽  
Functional Roles ◽  
Membrane Affinity ◽  
Neuronal Dendrites ◽  
Neuronal Synapses ◽  
Protein Functions

AbstractMyristoylation is a post-translational modification that plays diverse functional roles in many protein species. The myristate moiety is considered insufficient for protein-membrane associations unless additional membrane-affinity motifs, such as a stretch of positively charged residues, are present. Here, we report that the electrically neutral N-terminal fragment of the protein kinase A catalytic subunit (PKA-C), in which myristoylation is the only functional motif, is sufficient for membrane association. This myristoylation can associate a fraction of PKA-C molecules or fluorescent proteins (FPs) to the plasma membrane in neuronal dendrites. The net neutral charge of PKA-C is evolutionally conserved, even though its membrane affinity can be readily tuned by changing charges near the myristoylation site. The observed membrane association, while moderate, is sufficient to concentrate PKA activity at the membrane by nearly 20-fold, and is required for PKA regulation of AMPA receptors at neuronal synapses. Our results indicate that myristoylation alone may be sufficient to drive functionally significant membrane association in the absence of assisting motifs. This provides a revised foundation for the understanding of how myristoylation regulates protein functions.

scholarly journals Myristoylation alone is sufficient for PKA catalytic subunits to associate with the plasma membrane to regulate neuronal functions

2021 ◽  
Vol 118 (15) ◽  
pp. e2021658118
Author(s):  
Wei-Hong Xiong ◽  
Maozhen Qin ◽  
Haining Zhong
Keyword(s):  
Plasma Membrane ◽  
Ampa Receptors ◽  
Fluorescent Proteins ◽  
Membrane Association ◽  
Conceptual Base ◽  
Functional Roles ◽  
Membrane Affinity ◽  
Neuronal Dendrites ◽  
Neuronal Synapses ◽  
Protein Functions

Myristoylation is a posttranslational modification that plays diverse functional roles in many protein species. The myristate moiety is considered insufficient for protein–membrane associations unless additional membrane-affinity motifs, such as a stretch of positively charged residues, are present. Here, we report that the electrically neutral N-terminal fragment of the protein kinase A catalytic subunit (PKA-C), in which myristoylation is the only functional motif, is sufficient for membrane association. This myristoylation can associate a fraction of PKA-C molecules or fluorescent proteins (FPs) to the plasma membrane in neuronal dendrites. The net neutral charge of the PKA-C N terminus is evolutionally conserved, even though its membrane affinity can be readily tuned by changing charges near the myristoylation site. The observed membrane association, while moderate, is sufficient to concentrate PKA activity at the membrane by nearly 20-fold and is required for PKA regulation of AMPA receptors at neuronal synapses. Our results indicate that myristoylation may be sufficient to drive functionally significant membrane association in the absence of canonical assisting motifs. This provides a revised conceptual base for the understanding of how myristoylation regulates protein functions.

Annexins sense changes in intracellular pH during hypoxia

2007 ◽  
Vol 409 (1) ◽  
pp. 65-75 ◽  
Author(s):  
Katia Monastyrskaya ◽  
Fabian Tschumi ◽  
Eduard B. Babiychuk ◽  
Deborah Stroka ◽  
Annette Draeger
Keyword(s):  
Plasma Membrane ◽  
Intracellular Ph ◽  
Fluorescent Proteins ◽  
Annexin A2 ◽  
Transport Systems ◽  
Physiological Parameter ◽  
Membrane Association ◽  
Ph Dependent

The pHi (intracellular pH) is an important physiological parameter which is altered during hypoxia and ischaemia, pathological conditions accompanied by a dramatic decrease in pHi. Sensors of pHi include ion transport systems which control intracellular Ca2+ gradients and link changes in pHi to functions as diverse as proliferation and apoptosis. The annexins are a protein family characterized by Ca2+-dependent interactions with cellular membranes. Additionally, in vitro evidence points to the existence of pH-dependent, Ca2+-independent membrane association of several annexins. We show that hypoxia promotes the interaction of the recombinant annexin A2–S100A10 (p11) and annexin A6 with the plasma membrane. We have investigated in vivo the influence of the pHi on the membrane association of human annexins A1, A2, A4, A5 and A6 tagged with fluorescent proteins, and characterized this interaction for endogenous annexins present in smooth muscle and HEK (human embryonic kidney)-293 cells biochemically and by immunofluorescence microscopy. Our results show that annexin A6 and the heterotetramer A2–S100A10 (but not annexins A1, A4 and A5) interact independently of Ca2+ with the plasma membrane at pH 6.2 and 6.6. The dimerization of annexin A2 within the annexin A2–S100A10 complex is essential for the pH-dependent membrane interaction at this pH range. The pH-induced membrane binding of annexins A6 and A2–S100A10 might have consequences for their functions as membrane organizers and channel modulators.

Towards Computational Models of Identifying Protein Ubiquitination Sites

2019 ◽  
Vol 20 (5) ◽  
pp. 565-578 ◽  
Author(s):  
Lidong Wang ◽  
Ruijun Zhang
Keyword(s):  
Computational Methods ◽  
Computational Models ◽  
Feature Representation ◽  
Biological Sequence ◽  
Post Translational Modification ◽  
Test Dataset ◽  
Protein Ubiquitination ◽  
Protein Functions ◽  
Independent Test Dataset ◽  
Benchmark Datasets

Ubiquitination is an important post-translational modification (PTM) process for the regulation of protein functions, which is associated with cancer, cardiovascular and other diseases. Recent initiatives have focused on the detection of potential ubiquitination sites with the aid of physicochemical test approaches in conjunction with the application of computational methods. The identification of ubiquitination sites using laboratory tests is especially susceptible to the temporality and reversibility of the ubiquitination processes, and is also costly and time-consuming. It has been demonstrated that computational methods are effective in extracting potential rules or inferences from biological sequence collections. Up to the present, the computational strategy has been one of the critical research approaches that have been applied for the identification of ubiquitination sites, and currently, there are numerous state-of-the-art computational methods that have been developed from machine learning and statistical analysis to undertake such work. In the present study, the construction of benchmark datasets is summarized, together with feature representation methods, feature selection approaches and the classifiers involved in several previous publications. In an attempt to explore pertinent development trends for the identification of ubiquitination sites, an independent test dataset was constructed and the predicting results obtained from five prediction tools are reported here, together with some related discussions.

scholarly journals Contribution of the Cytoplasmic Determinants of Vpu to the Expansion of Virus-Containing Compartments in HIV-1-Infected Macrophages

2019 ◽  
Vol 93 (11) ◽  
Author(s):  
Olivier Leymarie ◽  
Leslie Lepont ◽  
Margaux Versapuech ◽  
Delphine Judith ◽  
Sophie Abelanet ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Viral Particle ◽  
Transmembrane Domain ◽  
Immune Surveillance ◽  
Restriction Factor ◽  
Viral Particles ◽  
Protein Functions ◽  
Specific Accumulation ◽  
Model Cell ◽  
Hiv 1

ABSTRACTHIV-1 infection of macrophages leads to the sequestration of newly formed viruses in intracellular plasma membrane-connected structures termed virus-containing compartments (VCCs), where virions remain infectious and hidden from immune surveillance. The cellular restriction factor bone marrow stromal cell antigen 2 (BST2), which prevents HIV-1 dissemination by tethering budding viral particles at the plasma membrane, can be found in VCCs. The HIV-1 accessory protein Vpu counteracts the restriction factor BST2 by downregulating its expression and removing it from viral budding sites. Numerous studies described these Vpu countermeasures in CD4+T cells or model cell lines, but the interplay between Vpu and BST2 in VCC formation and HIV-1 production in macrophages is less explored. Here, we show that Vpu expression in HIV-1-infected macrophages enhances viral release. This effect is related to Vpu’s ability to circumvent BST2 antiviral activity. We show that in absence of Vpu, BST2 is enriched in VCCs and colocalizes with capsid p24, whereas Vpu expression significantly reduces the presence of BST2 in these compartments. Furthermore, our data reveal that BST2 is dispensable for the formation of VCCs and that Vpu expression impacts the volume of these compartments. This Vpu activity partly depends on BST2 expression and requires the integrity of the Vpu transmembrane domain, the dileucine-like motif E59XXXLV64and phosphoserines 52 and 56 of Vpu. Altogether, these results highlight that Vpu controls the volume of VCCs and promotes HIV-1 release from infected macrophages.IMPORTANCEHIV-1 infection of macrophages leads to the sequestration of newly formed viruses in virus-containing compartments (VCCs), where virions remain infectious and hidden from immune surveillance. The restriction factor BST2, which prevents HIV-1 dissemination by tethering budding viral particles, can be found in VCCs. The HIV-1 Vpu protein counteracts BST2. This study explores the interplay between Vpu and BST2 in the viral protein functions on HIV-1 release and viral particle sequestration in VCCs in macrophages. The results show that Vpu controls the volume of VCCs and favors viral particle release. These Vpu functions partly depend on Vpu’s ability to antagonize BST2. This study highlights that the transmembrane domain of Vpu and two motifs of the Vpu cytoplasmic domain are required for these functions. These motifs were notably involved in the control of the volume of VCCs by Vpu but were dispensable for the prevention of the specific accumulation of BST2 in these structures.

scholarly journals Post translational modifications of milk proteins in geographically diverse goat breeds

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
P. K. Rout ◽  
M. Verma
Keyword(s):  
Protein Function ◽  
Whey Proteins ◽  
Milk Proteins ◽  
Goat Milk ◽  
Peptide Sequence ◽  
Cow Milk ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Functional Roles ◽  
Dpp Iv

AbstractGoat milk is a source of nutrition in difficult areas and has lesser allerginicity than cow milk. It is leading in the area for nutraceutical formulation and drug development using goat mammary gland as a bioreactor. Post translational modifications of a protein regulate protein function, biological activity, stabilization and interactions. The protein variants of goat milk from 10 breeds were studied for the post translational modifications by combining highly sensitive 2DE and Q-Exactive LC-MS/MS. Here we observed high levels of post translational modifications in 201 peptides of 120 goat milk proteins. The phosphosites observed for CSN2, CSN1S1, CSN1S2, CSN3 were 11P, 13P, 17P and 6P, respectively in 105 casein phosphopeptides. Whey proteins BLG and LALBA showed 19 and 4 phosphosites respectively. Post translational modification was observed in 45 low abundant non-casein milk proteins mainly associated with signal transduction, immune system, developmental biology and metabolism pathways. Pasp is reported for the first time in 47 sites. The rare conserved peptide sequence of (SSSEE) was observed in αS1 and αS2 casein. The functional roles of identified phosphopeptides included anti-microbial, DPP-IV inhibitory, anti-inflammatory and ACE inhibitory. This is first report from tropics, investigating post translational modifications in casein and non-casein goat milk proteins and studies their interactions.

scholarly journals Insulin Stimulates Membrane Fusion and GLUT4 Accumulation in Clathrin Coats on Adipocyte Plasma Membranes

2007 ◽  
Vol 27 (9) ◽  
pp. 3456-3469 ◽  
Author(s):  
Shaohui Huang ◽  
Larry M. Lifshitz ◽  
Christine Jones ◽  
Karl D. Bellve ◽  
Clive Standley ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Insulin Signaling ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Tirf Microscopy ◽  
Adipocyte Plasma Membranes ◽  
Kinetics Of

ABSTRACT Total internal reflection fluorescence (TIRF) microscopy reveals highly mobile structures containing enhanced green fluorescent protein-tagged glucose transporter 4 (GLUT4) within a zone about 100 nm beneath the plasma membrane of 3T3-L1 adipocytes. We developed a computer program (Fusion Assistant) that enables direct analysis of the docking/fusion kinetics of hundreds of exocytic fusion events. Insulin stimulation increases the fusion frequency of exocytic GLUT4 vesicles by ∼4-fold, increasing GLUT4 content in the plasma membrane. Remarkably, insulin signaling modulates the kinetics of the fusion process, decreasing the vesicle tethering/docking duration prior to membrane fusion. In contrast, the kinetics of GLUT4 molecules spreading out in the plasma membrane from exocytic fusion sites is unchanged by insulin. As GLUT4 accumulates in the plasma membrane, it is also immobilized in punctate structures on the cell surface. A previous report suggested these structures are exocytic fusion sites (Lizunov et al., J. Cell Biol. 169:481-489, 2005). However, two-color TIRF microscopy using fluorescent proteins fused to clathrin light chain or GLUT4 reveals these structures are clathrin-coated patches. Taken together, these data show that insulin signaling accelerates the transition from docking of GLUT4-containing vesicles to their fusion with the plasma membrane and promotes GLUT4 accumulation in clathrin-based endocytic structures on the plasma membrane.

scholarly journals Tracking the Fate of Genetically Distinct Vesicular Stomatitis Virus Matrix Proteins Highlights the Role for Late Domains in Assembly

2015 ◽  
Vol 89 (23) ◽  
pp. 11750-11760 ◽  
Author(s):  
Timothy K. Soh ◽  
Sean P. J. Whelan
Keyword(s):  
Plasma Membrane ◽  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Fluorescent Proteins ◽  
Endosomal Sorting ◽  
Escrt Machinery ◽  
Viral Particles ◽  
The Matrix ◽  
Vesicular Stomatitis ◽  
Late Domains

ABSTRACTVesicular stomatitis virus (VSV) assembly requires condensation of the viral ribonucleoprotein (RNP) core with the matrix protein (M) during budding from the plasma membrane. The RNP core comprises the negative-sense genomic RNA completely coated by the nucleocapsid protein (N) and associated by a phosphoprotein (P) with the large polymerase protein (L). To study the assembly of single viral particles, we tagged M and P with fluorescent proteins. We selected from a library of viruses with insertions in the M gene a replication-competent virus containing a fluorescent M and combined that with our previously described virus containing fluorescent P. Virus particles containing those fusions maintained the same bullet shape appearance as wild-type VSV but had a modest increase in particle length, reflecting the increased genome size. Imaging of the released particles revealed a variation in the amount of M and P assembled into the virions, consistent with a flexible packaging mechanism. We used the recombinants to further study the importance of the late domains in M, which serve to recruit the endosomal sorting complex required for transport (ESCRT) machinery during budding. Mutations in late domains resulted in the accumulation of virions that failed to pinch off from the plasma membrane. Imaging of single virions released from cells that were coinfected with M tagged with enhanced green fluorescent protein and M tagged with mCherry variants in which the late domains of one virus were inactivated by mutation showed a strong bias against the incorporation of the late-domain mutant into the released virions. In contrast, the intracellular expression and membrane association of the two variants were unaltered. These studies provide new tools for imaging particle assembly and enhance our resolution of existing models for assembly of VSV.IMPORTANCEAssembly of vesicular stomatitis virus (VSV) particles requires the separate trafficking of the viral replication machinery, a matrix protein (M) and a glycoprotein, to the plasma membrane. The matrix protein contains a motif termed a “late domain” that engages the host endosomal sorting complex required for transport (ESCRT) machinery to facilitate the release of viral particles. Inactivation of the late domains through mutation results in the accumulation of virions arrested at the point of release. In the study described here, we developed new tools to study VSV assembly by fusing fluorescent proteins to M and to a constituent of the replication machinery, the phosphoprotein (P). We used those tools to show that the late domains of M are required for efficient incorporation into viral particles and that the particles contain a variable quantity of M and P.

scholarly journals Individual Palmitoyl Residues Serve Distinct Roles in H-Ras Trafficking, Microlocalization, and Signaling

2005 ◽  
Vol 25 (15) ◽  
pp. 6722-6733 ◽  
Author(s):  
Sandrine Roy ◽  
Sarah Plowman ◽  
Barak Rotblat ◽  
Ian A. Prior ◽  
Cornelia Muncke ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Lipid Rafts ◽  
Lipid Bilayer ◽  
Spatial Organization ◽  
Cysteine Residues ◽  
Wild Type ◽  
Membrane Affinity ◽  
Lateral Segregation

ABSTRACT H-ras is anchored to the plasma membrane by two palmitoylated cysteine residues, Cys181 and Cys184, operating in concert with a C-terminal S-farnesyl cysteine carboxymethylester. Here we demonstrate that the two palmitates serve distinct biological roles. Monopalmitoylation of Cys181 is required and sufficient for efficient trafficking of H-ras to the plasma membrane, whereas monopalmitoylation of Cys184 does not permit efficient trafficking beyond the Golgi apparatus. However, once at the plasma membrane, monopalmitoylation of Cys184 supports correct GTP-regulated lateral segregation of H-ras between cholesterol-dependent and cholesterol-independent microdomains. In contrast, monopalmitoylation of Cys181 dramatically reverses H-ras lateral segregation, driving GTP-loaded H-ras into cholesterol-dependent microdomains. Intriguingly, the Cys181 monopalmitoylated H-ras anchor emulates the GTP-regulated microdomain interactions of N-ras. These results identify N-ras as the Ras isoform that normally signals from lipid rafts but also reveal that spacing between palmitate and prenyl groups influences anchor interactions with the lipid bilayer. This concept is further supported by the different plasma membrane affinities of the monopalmitoylated anchors: Cys181-palmitate is equivalent to the dually palmitoylated wild-type anchor, whereas Cys184-palmitate is weaker. Thus, membrane affinity of a palmitoylated anchor is a function both of the hydrophobicity of the lipid moieties and their spatial organization. Finally we show that the plasma membrane affinity of monopalmitoylated anchors is absolutely dependent on cholesterol, identifying a new role for cholesterol in promoting interactions with the raft and nonraft plasma membrane.

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