Morphine activates neuroinflammation in a manner parallel to endotoxin

Opioids create a neuroinflammatory response within the CNS, compromising opioid-induced analgesia and contributing to various unwanted actions. How this occurs is unknown but has been assumed to be via classic opioid receptors. Herein, we provide direct evidence that morphine creates neuroinflammation via the activation of an innate immune receptor and not via classic opioid receptors. We demonstrate that morphine binds to an accessory protein of Toll-like receptor 4 (TLR4), myeloid differentiation protein 2 (MD-2), thereby inducing TLR4 oligomerization and triggering proinflammation. Small-molecule inhibitors, RNA interference, and genetic knockout validate the TLR4/MD-2 complex as a feasible target for beneficially modifying morphine actions. Disrupting TLR4/MD-2 protein-protein association potentiated morphine analgesia in vivo and abolished morphine-induced proinflammation in vitro, the latter demonstrating that morphine-induced proinflammation only depends on TLR4, despite the presence of opioid receptors. These results provide an exciting, nonconventional avenue to improving the clinical efficacy of opioids.     

The neuronal connexin36 interacts with and is phosphorylated by CaMKII in a way similar to CaMKII interaction with glutamate receptors

Electrical synapses can undergo activity-dependent plasticity. The calcium/calmodulin-dependent kinase II (CaMKII) appears to play a critical role in this phenomenon, but the underlying mechanisms of how CaMKII affects the neuronal gap junction protein connexin36 (Cx36) are unknown. Here we demonstrate effective binding of ³⁵S-labeled CaMKII to 2 juxtamembrane cytoplasmic domains of Cx36 and in vitro phosphorylation of this protein by the kinase. Both domains reveal striking similarities with segments of the regulatory subunit of CaMKII, which include the pseudosubstrate and pseudotarget sites of the kinase. Similar to the NR2B subunit of the NMDA receptor both Cx36 binding sites exhibit phosphorylation-dependent interaction and autonomous activation of CaMKII. CaMKII and Cx36 were shown to be significantly colocalized in the inferior olive, a brainstem nucleus highly enriched in electrical synapses, indicating physical proximity of these proteins. In analogy to the current notion of NR2B interaction with CaMKII, we propose a model that provides a mechanistic framework for CaMKII and Cx36 interaction at electrical synapses.     

Activation of the bacterial thermosensor DesK involves a serine zipper dimerization motif that is modulated by bilayer thickness

DesK is a bacterial thermosensor protein involved in maintaining membrane fluidity in response to changes in environmental temperature. Most likely, the protein is activated by changes in membrane thickness, but the molecular mechanism of sensing and signaling is still poorly understood. Here we aimed to elucidate the mode of action of DesK by studying the so-called “minimal sensor DesK” (MS-DesK), in which sensing and signaling are captured in a single transmembrane segment. This simplified version of the sensor allows investigation of membrane thickness-dependent protein–lipid interactions simply by using synthetic peptides, corresponding to the membrane-spanning parts of functional and nonfunctional mutants of MS-DesK incorporated in lipid bilayers with varying thicknesses. The lipid-dependent behavior of the peptides was investigated by circular dichroism, tryptophan fluorescence, and molecular modeling. These experiments were complemented with in vivo functional studies on MS-DesK mutants. Based on the results, we constructed a model that suggests a new mechanism for sensing in which the protein is present as a dimer and responds to an increase in bilayer thickness by membrane incorporation of a C-terminal hydrophilic motif. This results in exposure of three serines on the same side of the transmembrane helices of MS-DesK, triggering a switching of the dimerization interface to allow the formation of a serine zipper. The final result is activation of the kinase state of MS-DesK.All organisms have to be able to rapidly adapt to a vast variety of external stimuli to survive. In bacteria, two-component signal transduction systems are some of the most abundant mechanisms for sensing and adapting to changes in the extracellular environment. These systems mediate responses in chemotaxis and phototaxis, and regulate feedback to changes in osmolarity, redox state, and temperature (1, 2). However, despite the evident importance of two-component systems for bacterial survival, the molecular mechanisms of signal transduction via these systems have barely begun to be untangled.The DesKR system is a two-component system first identified in the soil bacterium Bacillus subtilis (3). Together with other regulatory systems, it is involved in maintaining membrane fluidity when the environmental temperature changes. The DesKR system works as follows. The actual thermosensor—i.e., the protein that senses the temperature change—is DesK. This protein consists of five transmembrane helices and an intracellular catalytic domain (DesKC) and is believed to function as a dimer (2, 4). In response to decreased environmental temperature, DesKC phosphorylates the response regulator DesR, which in turn controls the expression levels of the effector enzyme, a desaturase. This desaturase is inserted into the membrane, where it can introduce double bonds into preexisting lipids, allowing the recovery of membrane fluidity at this lower temperature (3).In the present study, we focused on the first step of the signaling pathway, examining how the sensor is able to sense and transmit a temperature-dependent signal. This challenge was recently simplified by the discovery that both the sensing and the signal transduction properties of the membrane-spanning part of DesK can be captured into a single transmembrane segment by fusing the N-terminal part of the first transmembrane segment to the C-terminal part of the fifth transmembrane segment (5). The resulting protein is called the minimal sensor DesK (MS-DesK).Importantly, MS-DesK shows a temperature-dependent switch in activity comparable to the full-length DesK not only in vivo, but also when reconstituted in protein-free lipid bilayers made from bacterial lipids (5). Therefore, no other membrane proteins are involved in sensing or signal transduction. Furthermore, the activity of the catalytic domain DesKC itself, is not temperature-sensitive (5, 6), and thus it must be the transmembrane segment of MS-DesK that somehow reacts to changes in temperature, most likely by sensing corresponding changes in the physical properties of the lipids.Which properties of the membrane could be sensed by DesK and MS-DesK? On a decrease in environmental temperature, membrane lipids become more ordered, and consequently the membrane becomes thicker (7). Some evidence suggests that such changes in membrane thickness may be a key factor in the regulation of DesK sensing and signaling. First, an MS-DesK length mutant (4V) containing four extra valines in the C-terminal region of its transmembrane segment was found to be inactive and to remain locked in the phosphatase state on a decrease in temperature (5). Second, reconstitution studies showed increasing activity of both DesK and MS-DesK with increasing acyl chain length of the lipids in which the protein is reconstituted (5, 8). Third, increased incorporation of long-chain fatty acids into the membrane lipids was found to stimulate kinase activity of DesK in vivo, whereas increased levels of short-chain fatty acids result in loss of activity (9).How can membrane thickness regulate the activity of DesK? It has been shown that the N terminus of MS-DesK at the exoplasmic side of the membrane contains a motif that may render the protein sensitive to membrane thickness and interfacial hydration. This motif contains two hydrophilic amino acids, K10 and N12, that are essential for activity (5) and that presumably are located within the transmembrane region just below the lipid–water interface. Because their side chains can snorkel to the hydrophilic membrane–water interface, these amino acids were proposed to act as a buoy, stabilizing the position of the transmembrane segment. For this reason, this has been called the sunken-buoy (SB) motif (5). In addition to the SB motif, a charged linker region at the intracellular membrane–water interface was found to be important for activity (10). It has been proposed that both motifs act together as a molecular gauge that senses membrane thickness and thereby regulates the switching of activity of the intracellular catalytic domain of DesK (10). The molecular details of the mode of action of this molecular gauge have remained elusive, however.Because the activity of MS-DesK is most likely regulated by direct interactions of its single transmembrane segment with surrounding lipids, as discussed above, this system is ideally suited for studies on relatively simple model membranes. In such model systems, the biological complexity of the host membrane is reduced to allow for systematic studies. Here, to gain insight into the molecular mode of action of the sensor, we studied the behavior of peptides mimicking the membrane-spanning parts of a functional mutant and a nonfunctional mutant of MS-DesK in synthetic lipid bilayers by spectroscopic techniques and molecular modeling. Combined with in vivo functional studies on MS-DesK mutants and cross-linking experiments, our results lead to a new model of thermosensing in which changes in bilayer thickness trigger a switch between distinct dimerization interfaces within the membrane, resulting in activation of the sensor.     

Temporal activation of p53 by a specific MDM2 inhibitor is selectively toxic to tumors and leads to complete tumor growth inhibition

We have designed MI-219 as a potent, highly selective and orally active small-molecule inhibitor of the MDM2-p53 interaction. MI-219 binds to human MDM2 with a K(i) value of 5 nM and is 10,000-fold selective for MDM2 over MDMX. It disrupts the MDM2-p53 interaction and activates the p53 pathway in cells with wild-type p53, which leads to induction of cell cycle arrest in all cells and selective apoptosis in tumor cells. MI-219 stimulates rapid but transient p53 activation in established tumor xenograft tissues, resulting in inhibition of cell proliferation, induction of apoptosis, and complete tumor growth inhibition. MI-219 activates p53 in normal tissues with minimal p53 accumulation and is not toxic to animals. MI-219 warrants clinical investigation as a new agent for cancer treatment.