PDZ结构域介导的PIP脂质信号转导

PDZ结构域是调控蛋白质/蛋白质相互作用的一类重要结构域,能特异结合蛋白质C末端一段有规律的氨基酸序列。含有PDZ结构域的支架蛋白能够组装成超大的蛋白质复合体来调控细胞内的信号转导通路。最新研究表明,PDZ结构域还能与PIP脂质直接相互作用,从而参与调控PIP脂质信号通路。将综合最新研究进展,阐明PDZ结构域与PIP脂质的作用方式,以及对相关PIP脂质信号转导的调控过程。     

Divalent cation-dependent formation of electrostatic PIP2 clusters in lipid monolayers

Polyphosphoinositides are among the most highly charged molecules in the cell membrane, and the most common polyphosphoinositide, phosphatidylinositol-4,5-bisphosphate (PIP₂), is involved in many mechanical and biochemical processes in the cell membrane. Divalent cations such as calcium can cause clustering of the polyanionic PIP₂, but the origin and strength of the effective attractions leading to clustering has been unclear. In addition, the question of whether the ion-mediated attractions could be strong enough to alter the mechanical properties of the membrane, to our knowledge, has not been addressed. We study phase separation in mixed monolayers of neutral and highly negatively charged lipids, induced by the addition of divalent positively charged counterions, both experimentally and numerically. We find good agreement between experiments on mixtures of PIP₂ and 1-stearoyl-2-oleoyl phosphatidylcholine and simulations of a simplified model in which only the essential electrostatic interactions are retained. In addition, we find numerically that under certain conditions the effective attractions can rigidify the resulting clusters. Our results support an interpretation of PIP₂ clustering as governed primarily by electrostatic interactions. At physiological pH, the simulations suggest that the effective attractions are strong enough to give nearly pure clusters of PIP₂ even at small overall concentrations of PIP₂.     

Assembly of Trp1 in a signaling complex associated with caveolin-scaffolding lipid raft domains

Trp1 has been proposed as a component of the store-operated Ca(2+) entry (SOC) channel. However, neither the molecular mechanism of SOC nor the role of Trp in this process is yet understood. We have examined possible molecular interactions involved in the regulation of SOC and Trp1 and report here for the first time that Trp1 is assembled in signaling complex associated with caveolin-scaffolding lipid raft domains. Endogenous hTrp1 and caveolin-1 were present in low density fractions of Triton X-100-extracted human submandibular gland cell membranes. Depletion of plasma membrane cholesterol increased Triton X-100 solubility of Trp1 and inhibited carbachol-stimulated Ca(2+) signaling. Importantly, thapsigargin stimulated Ca(2+) influx, but not internal Ca(2+) release, and inositol 1,4,5-triphosphate (IP(3))-stimulated I(soc) were also attenuated. Furthermore, both anti-Trp1 and anti-caveolin-1 antibodies co-immunoprecipitated hTrp1, caveolin-1, Galpha(q/11), and IP(3) receptor-type 3 (IP(3)R3). These results demonstrate that caveolar microdomains provide a scaffold for (i) assembly of key Ca(2+) signaling proteins into a complex and (ii) coordination of the molecular interactions leading to the activation of SOC. Importantly, we have shown that Trp1 is also localized in this microdomain where it interacts with one or more components of this complex, including IP(3)R3. This finding is potentially important in elucidating the physiological function of Trp.     

Simulations of Kindlin-2 PIP binding domains reveal protonation-dependent membrane binding modes

Kindlin-2, a member of the Kindlin family of peripheral membrane proteins, is important for integrin activation and stabilization of epidermal growth factor receptor. It associates with the cytoplasmic face of the plasma membrane via dedicated phosphatidylinositol phosphate binding domains located in the N-terminal F0 and Pleckstrin Homology domains. These domains have binding affinity for phosphatidylinositol 4,5-bisphosphate and, to a greater degree, phosphatidylinositol 3,4,5-trisphosphate. The biological significance of the differential binding of these phosphatidylinositol phosphates to Kindlin-2 and the mechanism by which they activate Kindlin-2 are not well understood. Recently, ssNMR identified the predominant protonation states of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate near physiological pH in the presence of anionic lipids. Here, we perform atomistic simulation of the bound state of the Pleckstrin Homology and F0 domains of Kindlin-2 at membranes containing phosphatidylinositol 4,5-bisphosphate/phosphatidylinositol 3,4,5-trisphosphate with differing protonation states. This computational approach demonstrates that these two phosphatidylinositol phosphates differently modulate Kindlin-2 subdomain binding in a protonation-state-dependent manner. We speculate these variations in binding mode provide a mechanism for intracellular pH and Ca²⁺ influx to control the membrane binding behavior and activity of Kindlin-2.     

The PIP2 binding mode of the C2 domains of rabphilin-3A

Phosphatidylinositol-4,5-bisphosphate (PIP2) is a key player in the neurotransmitter release process. Rabphilin-3A is a neuronal C2 domain tandem containing protein that is involved in this process. Both its C2 domains (C2A and C2B) are able to bind PIP2. The investigation of the interactions of the two C2 domains with the PIP2 headgroup IP3 (inositol-1,4,5-trisphosphate) by NMR showed that a well-defined binding site can be described on the concave surface of each domain. The binding modes of the two domains are different. The binding of IP3 to the C2A domain is strongly enhanced by Ca(2+) and is characterized by a K(D) of 55 microM in the presence of a saturating concentration of Ca(2+) (5 mM). Reciprocally, the binding of IP3 increases the apparent Ca(2+)-binding affinity of the C2A domain in agreement with a Target-Activated Messenger Affinity (TAMA) mechanism. The C2B domain binds IP3 in a Ca(2+)-independent fashion with low affinity. These different PIP2 headgroup recognition modes suggest that PIP2 is a target of the C2A domain of rabphilin-3A while this phospholipid is an effector of the C2B domain.     

RUN domains: a new family of domains involved in Ras-like GTPase signaling

RUN domains are present in several proteins that are linked particularly to the functions of GTPases in the Rap and Rab families. They could hence play an important role in multiple Ras-like GTPase signaling pathways.     

Cutting edge: association of phospholipase C-gamma 2 Src homology 2 domains with BLNK is critical for B cell antigen receptor signaling.

To explore the mechanism(s) by which phospholipase C (PLC)-gamma 2 participates in B cell Ag receptor (BCR) signaling, we have studied the function of PLC-gamma 2 mutants in B cells deficient in PLC-gamma 2. Mutation of the N-terminal Src homology 2 domain [SH2(N)] resulted in the complete loss of inositol 1,4, 5-trisphosphate generation upon BCR engagement. A possible explanation for the SH2(N) requirement was provided by findings that this mutation abrogates the association of PLC-gamma 2 with an adaptor protein BLNK. Moreover, expression of a membrane-associated form (CD16/PLC-gamma 2) with SH2(N) mutation required coligation of BCR and CD16 for inositol 1,4,5-trisphosphate generation. Together, our results suggest a central role for the SH2(N) domain in directing PLC-gamma 2 into the close proximity of BCR signaling complex by its association with BLNK, whereby PLC-gamma 2 becomes tyrosine phosphorylated and thereby activated.     

Signaling through C2 domains: More than one lipid target

C2 domains are membrane-binding modules that share a common overall fold: a single compact Greek-key motif organized as an eight-stranded anti-parallel β-sandwich consisting of a pair of four-stranded β-sheets. A myriad of studies have demonstrated that in spite of sharing the common structural β-sandwich core, slight variations in the residues located in the interconnecting loops confer C2 domains with functional abilities to respond to different Ca^2 + concentrations and lipids, and to signal through protein–protein interactions as well. This review summarizes the main structural and functional findings on Ca^2 + and lipid interactions by C2 domains, including the discovery of the phosphoinositide-binding site located in the β3–β4 strands. The wide variety of functions, together with the different Ca^2 + and lipid affinities of these domains, converts this superfamily into a crucial player in many functions in the cell and more to be discovered. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

Angiopoietin 2 signaling plays a critical role in neural crest cell migration

Background

Collective neural crest cell migration is critical to the form and function of the vertebrate face and neck, distributing bone, cartilage, and nerve cells into peripheral targets that are intimately linked with head vasculature. The vasculature and neural crest structures are ultimately linked, but when and how these patterns develop in the early embryo are not well understood.

Results

Using in vivo imaging and sophisticated cell behavior analyses, we show that quail cranial neural crest and endothelial cells share common migratory paths, sort out in a dynamic multistep process, and display multiple types of motion. To better understand the underlying molecular signals, we examined the role of angiopoietin 2 (Ang2), which we found expressed in migrating cranial neural crest cells. Overexpression of Ang2 causes neural crest cells to be more exploratory as displayed by invasion of off-target locations, the widening of migratory streams into prohibitive zones, and differences in cell motility type. The enhanced exploratory phenotype correlates with increased phosphorylated focal adhesion kinase activity in migrating neural crest cells. In contrast, loss of Ang2 function reduces neural crest cell exploration. In both gain and loss of function of Ang2, we found disruptions to the timing and interplay between cranial neural crest and endothelial cells.

Conclusions

Together, these data demonstrate a role for Ang2 in maintaining collective cranial neural crest cell migration and suggest interdependence with endothelial cell migration during vertebrate head patterning.

     

Phosphatidic acid: a multifunctional stress signaling lipid in plants

Phosphatidic acid (PA) has only recently been identified as an important signaling molecule in both plants and animals. Nonetheless, it already promises to rival the importance of the classic second messengers Ca(2+) and cAMP. In plants, its formation is triggered in response to various biotic and abiotic stress factors, including pathogen infection, drought, salinity, wounding and cold. In general, PA signal production is fast (minutes) and transient. Recently, our understanding of the role of PA formation in stress responses as a result of phospholipases C and D activity has greatly increased. Moreover, the first protein targets of PA have been identified. Based on this recent work, potential mechanisms by which PA provokes downstream effects are emerging.     

Membrane effects of ethanol: bulk lipid versus lipid domains

It has been well-established that ethanol fluidizes the bulk lipid of membranes and that this effect may alter cell function and be involved in ethanol sensitivity and tolerance. This hypothesis has been supported in several studies, however, there is also a considerable amount of data that do not support such an explanation, e.g., direct effect of ethanol on proteins, other membrane acting drugs, temperature effects, effects of ethanol on aged membranes and inconsistent effects of chronic ethanol consumption on lipid content. This review examined the bulk membrane fluidization hypothesis in light of those data and proposed a modification of the bulk membrane hypothesis that is based on recent data that show that ethanol and other alcohols have a specific effect on the structural properties of different membrane domains. This specific effect of ethanol is discussed within the context of how changes in fluidity of domains may alter membrane function.     

Microbial rhamnolipid production: a critical re-evaluation of published data and suggested future publication criteria

High production cost and potential pathogenicity of Pseudomonas aeruginosa, commonly used for rhamnolipid synthesis, have led to extensive research for safer producing strains and cost-effective production methods. This has resulted in numerous research publications claiming new non-pathogenic producing strains and novel production techniques many of which are unfortunately without proper characterisation of product and/or producing strain/s. Genes responsible for rhamnolipid production have only been confirmed in P. aeruginosa, Burkholderia thailandensis and Burkholderia pseudomallei. Comparing yields in different publications is also generally unreliable especially when different methodologies were used for rhamnolipid quantification. After reviewing the literature in this area, we strongly feel that numerous research outputs have insufficient evidence to support claims of rhamnolipid-producing strains and/or yields. We therefore recommend that standards should be set for reporting new rhamnolipid-producing strains and production yields. These should include (1) molecular and bioinformatic tools to fully characterise new microbial isolates and confirm the presence of the rhamnolipid rhl genes for all bacterial strains, (2) using gravimetric methods to quantify crude yields and (3) use of a calibrated method (high-performance liquid chromatography or ultra-performance liquid chromatography) for absolute quantitative yield determination.     

Microtubule-organizing centres: a re-evaluation

The number, length, distribution and polarity of microtubules are largely controlled by microtubule-organizing centres, which nucleate and anchor microtubule minus ends in a process that requires gamma-tubulin. Here we discuss recent evidence indicating that gamma-tubulin-dependent formation of new microtubules is not restricted to conventional microtubule-organizing centres. These findings suggest that the spatio-temporal control of microtubule nucleation is more complex than previously thought, leading us to a re-evaluation of the concept of the microtubule-organizing center.     

The mechanisms of kin discrimination and the evolution of kin recognition in vertebrates: a critical re-evaluation

I re-examine the four most widely proposed mechanisms of kin discrimination among vertebrates and conclude that the current categorization of kin discrimination mechanisms has been counterproductive because it has a hindered a clear understanding of the basic mechanisms by which animals discriminate kin. I suggest that there likely is only one authentic mechanism of kin discrimination and that this mechanism is learning, particularly associative learning and habituation. Observed differences in the way animals discriminate between kin and non-kin are due only to the cues (e.g., individually-distinctive, family-distinctive, or self) that are used, and not to different mechanisms per se. I also consider whether kin discrimination is mediated by specially evolved kin recognition systems, defined as neural mechanisms that allow animals to directly classify conspecifics as either kin or non-kin. A preliminary analysis of vertebrate recognition systems suggests that specialized neural, endocrine, and developmental mechanisms specifically for recognizing kin have not evolved. Rather, kin discrimination results from an extension of other, non-specialized sensory and cognitive abilities of animals, and may be derived from other forms of social recognition, such as individual, group, or species recognition.