1,720,967 research outputs found
The role of protein-protein interactions in the intracellular traffic of the potassium channels TASK-1 and TASK-3
No full textThe intracellular transport of membrane proteins is controlled by trafficking signals: Short peptide motifs that mediate the contact with COPI, COPII or various clathrin-associated coat proteins. In addition, many membrane proteins interact with accessory proteins that are involved in the sorting of these proteins to different intracellular compartments. In the K-2P channels, TASK-1 and TASK-3, the influence of protein-protein interactions on sorting decisions has been studied in some detail. Both TASK paralogues interact with the adaptor protein 14-3-3; TASK-1 interacts, in addition, with the adaptor protein p11 (S100A10) and the endosomal SNARE protein syntaxin-8. The role of these interacting proteins in controlling the intracellular traffic of the channels and the underlying molecular mechanisms are summarised in this review. In the case of 14-3-3, the interacting protein masks a retention signal in the C-terminus of the channel; in the case of p11, the interacting protein carries a retention signal that localises the channel to the endoplasmic reticulum; and in the case of syntaxin-8, the interacting protein carries an endocytosis signal that complements an endocytosis signal of the channel. These examples illustrate some of the mechanisms by which interacting proteins may determine the itinerary of a membrane protein within a cell and suggest that the intracellular traffic of membrane proteins may be adapted to the specific functions of that protein by multiple protein-protein interactions
Signal recognition initiates reorganization of the presequence translocase during protein import.
Get PDFThe mitochondrial presequence translocase interacts with presequence-containing precursors at the intermembrane space (IMS) side of the inner membrane to mediate their translocation into the matrix. Little is known as too how these matrix-targeting signals activate the translocase in order to initiate precursor transport. Therefore, we analysed how signal recognition by the presequence translocase initiates reorganization among Tim-proteins during import. Our analyses revealed that the presequence receptor Tim50 interacts with Tim21 in a signal-sensitive manner in a process that involves the IMS-domain of the Tim23 channel. The signal-driven release of Tim21 from Tim50 promotes recruitment of Pam17 and thus triggers formation of the motor-associated form of the TIM23 complex required for matrix transport
A GTPase-induced switch in phospholipid affinity of collybistin contributes to synaptic gephyrin clustering
Get PDFSynaptic transmission between neurons relies on the exact spatial organization of postsynaptic transmitter receptors, which are recruited and positioned by dedicated scaffolding and regulatory proteins. At GABAergic synapses, the regulatory protein collybistin (Cb, also known as ARHGEF9) interacts with small GTPases, cell adhesion proteins and phosphoinositides to recruit the scaffolding protein gephyrin and GABAA receptors to nascent synapses. We dissected the interaction of Cb with the small Rho-like GTPase TC10 (also known as RhoQ) and phospholipids. Our data define a protein– lipid interaction network that controls the clustering of gephyrin at synapses. Within this network, TC10 and monophosphorylated phosphoinositides, particulary phosphatidylinositol 3-phosphate (PI3P), provide a coincidence detection platform that allows the accumulation and activation of Cb in endomembranes. Upon activation, TC10 induces a phospholipid affinity switch in Cb, which allows Cb to specifically interact with phosphoinositide species present at the plasma membrane. We propose that this GTPase- based regulatory switch mechanism represents an important step in the process of tethering of Cb-dependent scaffolds and receptors at nascent postsynapses
Discovery and Characterization of an ALFA-Tag-Specific Affinity Resin Optimized for Protein Purification at Low Temperatures in Physiological Buffer
No full textEpitope tags are widely employed as tools to detect, purify and manipulate proteins in various experimental systems. We recently introduced the ALFA-tag together with two ALFA-specific single-domain antibodies (sdAbs), NbALFA and NbALFAPE, featuring high or intermediate affinity, respectively. Together, the ALFA system can be employed for a broad range of applications in microscopy, cell biology and biochemistry requiring either extraordinarily stable binding or mild competitive elution at room temperature. In order to further enhance the versatility of the ALFA system, we, here, aimed at developing an sdAb optimized for efficient elution at low temperatures. To achieve this, we followed a stringent selection scheme tailored to the specific application. We found candidates combining a fast capture of ALFA-tagged proteins with an efficient competitive elution at 4 °C in physiological buffer. Importantly, by employing a structure-guided semisynthetic library based on well-characterized NbALFA variants, the high specificity and consistent binding of proteins harboring ALFA-tags at either terminus could be maintained. ALFA SelectorCE, a resin presenting the cold-elutable NbALFACE, is an ideal tool for the one-step purification of sensitive protein complexes or temperature-labile enzymes. We believe that the general approach followed during the selection and screening can be transferred to other challenging sdAb discovery projects
The SUMO1-E67 interacting loop peptide is an allosteric inhibitor of the dipeptidyl peptidases 8 and 9.
Get PDFThe intracellular peptidases dipeptidyl peptidase (DPP) 8 and DPP9 are involved in multiple cellular pathways including antigen maturation, cellular homeostasis, energy metabolism, and cell viability. Previously we showed that the small ubiquitin-like protein modifier SUMO1 interacts with an armlike structure in DPP9, leading to allosteric activation of the peptidase. Here we demonstrate that the E67-interacting loop (EIL) peptide, which corresponds to the interaction surface of SUMO1 with DPP9, acts as a noncompetitive inhibitor of DPP9. Moreover, by analyzing the sensitivity of DPP9 arm mutants to the EIL peptide, we mapped specific residues in the arm that are important for inhibition by the EIL, suggesting that the peptide acts as an allosteric inhibitor of DPP9. By modifying the EIL peptide, we constructed peptide variants with more than a 1,000-fold selectivity toward DPP8 (147 nm) and DPP9 (170 nm) over DPPIV (200 μm). Furthermore, application of these peptides to cells leads to a clear inhibition of cellular prolyl peptidase activity. Importantly, in line with previous publications, inhibition of DPP9 with these novel allosteric peptide inhibitors leads to an increase in EGF-mediated phosphorylation of Akt. This work highlights the potential use of peptides that mimic interaction surfaces for modulating enzyme activity
A dual phosphorylation switch controls 14-3-3-dependent cell surface expression of TASK-1.
Get PDFThe transport of the K+ channels TASK-1 and TASK-3 (also known as KCNK3 and KCNK9, respectively) to the cell surface is controlled by the binding of 14-3-3 proteins to a trafficking control region at the extreme C-terminus of the channels. The current model proposes that phosphorylation-dependent binding of 14-3-3 sterically masks a COPI-binding motif. However, the direct effects of phosphorylation on COPI binding and on the binding parameters of 14-3-3 isoforms are still unknown. We find that phosphorylation of the trafficking control region prevents COPI binding even in the absence of 14-3-3, and we present a quantitative analysis of the binding of all human 14-3-3 isoforms to the trafficking control regions of TASK-1 and TASK-3. Surprisingly, the affinities of 14-3-3 proteins for TASK-1 are two orders of magnitude lower than for TASK-3. Furthermore, we find that phosphorylation of a second serine residue in the C-terminus of TASK-1 inhibits 14-3-3 binding. Thus, phosphorylation of the trafficking control region can stimulate or inhibit transport of TASK-1 to the cell surface depending on the target serine residue. Our findings indicate that control of TASK-1 trafficking by COPI, kinases, phosphatases and 14-3-3 proteins is highly dynamic
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Quantitative analysis of protein-protein interactions governing TASK-1/TASK-3 intracellular transport
Get PDFThe transport of the K+-channels TASK-1 and TASK-3 to the cell surface is
regulated by protein-protein interactions with either the COPI vesicle coat, or
members of the phosphoadaptor protein family 14-3-3. Interactions are
mediated via a trafficking control region present at the distal C-terminus of
either K+-channel. This trafficking control region comprises a polybasic ER
retention and retrieval motif and an adjacent mode III 14-3-3 binding motif.
Phosphorylation of a conserved serine residue, as part of the mode III 14-3-3
binding motif, is followed by the recruitment of 14-3-3, thereby releasing the
channel from ER retention by sterically preventing the COPI vesicle coat from
binding to the overlapping ER retention and retrieval motif. Following
phosphorylation and 14-3-3 binding, the channel is transported forward to the
cell surface. In this thesis I determined the binding parameters of all seven
human 14-3-3 isoforms to the trafficking control regions of TASK-1 and TASK-
3. Furthermore, I investigated the direct effect of phosphorylation of the TASK-
1 and TASK-3 C-terminus on COPI binding. I observed distinctly different
binding parameters between individual 14-3-3 isoforms and different channel
C-termini demonstrating that 14-3-3 isoforms bind the same substrate in an
isoform specific manner. Surprisingly, the binding affinities determined for
TASK-1 were approximately two orders of magnitude lower than the binding
affinities determined for TASK-3. I explain these differences by small, but
physiologically relevant, amino acid sequence differences within the trafficking
control regions of TASK-1 and TASK-3. While TASK-3 presents a second
lysine residue that allows for high affinity binding of 14-3-3 proteins to this
trafficking control region, TASK-1 presents a second serine residue that upon
phosphorylation inhibits 14-3-3 binding. I further correlate my in vitro
observations with reporter protein assays performed in vivo (COS7),
assessing the relative cell surface expression of TASK-derived reporter
proteins. My findings indicate that the control of TASK-1 protein trafficking is
highly dynamic, modulated by COPI, 14-3-3, kinases and phosphatases.
Binding experiments performed with the yeast COPI vesicle coat and
phosphorylated or unphosphorylated constructs comprising the distal C-terminus of TASK-1 and TASK-3 (the last 15 amino acids) demonstrate that
the phosphorylation of these trafficking control regions is sufficient to interfere
with COPI binding, in absence of 14-3-3. In summary, my findings contribute
substantially to the quantitative understanding of events governing the
intracellular transport of TASK-1 and TASK-3
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