Conformational Dynamics and Structural Plasticity Play Critical Roles in the Ubiquitin Recognition of a UIM Domain

Ubiquitin-interacting motifs (UIMs) are an important class of protein domains that interact with ubiquitin or ubiquitin-like proteins. These approximately 20-residue-long domains are found in a variety of ubiquitin receptor proteins and serve as recognition modules towards intracellular targets, which may be individual ubiquitin subunits or polyubiquitin chains attached to a variety of proteins. Previous structural studies of interactions between UIMs and ubiquitin have shown that UIMs adopt an extended structure of a single α-helix, containing a hydrophobic surface with a conserved sequence pattern that interacts with key hydrophobic residues on ubiquitin. In light of this large body of structural studies, details regarding the presence and the roles of structural dynamics and plasticity are surprisingly lacking. In order to better understand the structural basis of ubiquitin-UIM recognition, we have characterized changes in the structure and dynamics of ubiquitin upon binding of a UIM domain from the yeast Vps27 protein. The solution structure of a ubiquitin-UIM fusion protein designed to study these interactions is reported here and found to consist of a well-defined ubiquitin core and a bipartite UIM helix. Moreover, we have studied the plasticity of the docking interface, as well as global changes in ubiquitin due to UIM binding at the picoseconds-to-nanoseconds and microseconds-to-milliseconds protein motions by nuclear magnetic resonance relaxation. Changes in generalized-order parameters of amide groups show a distinct trend towards increased structural rigidity at the UIM-ubiquitin interface relative to values determined in unbound ubiquitin. Analysis of ¹⁵N Carr-Purcell-Meiboom-Gill relaxation dispersion measurements suggests the presence of two types of motions: one directly related to the UIM-binding interface and the other induced to distal parts of the protein. This study demonstrates a case where localized interactions among protein domains have global effects on protein motions at timescales ranging from picoseconds to milliseconds.     

Impact of myocardial structure and function postinfarction on diastolic strain measurements: implications for assessment of myocardial viability

Venular control of arteriolar perfusion has been the focus of several investigations in recent years. This study investigated 1) whether endogenous adenosine helps control venule-dependent arteriolar dilation and 2) whether venular leukocyte adherence limits this response via an oxidant-dependent mechanism in which nitric oxide (NO) levels are decreased. Intravital microscopy was used to assess changes in arteriolar diameters and NO levels in rat mesentery. The average resting diameter of arterioles (27.5 +/- 1.0 microm) paired with venules with minimal leukocyte adherence (2.1 +/- 0.3 per 100-microm length) was significantly larger than that of unpaired arterioles (24.5 +/- 0.8 microm) and arterioles (23.3 +/- 1.3 microm) paired with venules with higher leukocyte adherence (9.0 +/- 0.5 per 100-microm length). Local superfusion of adenosine deaminase (ADA) induced significant decreases in diameter and perivascular NO concentration in arterioles closely paired to venules with minimal leukocyte adherence. However, ADA had little effect on arterioles closely paired to venules with high leukocyte adherence or on unpaired arterioles. To determine whether the attenuated response to ADA for the high-adherence group was oxidant dependent, the responses were also observed in arterioles treated with 10(-4) M Tempol. In the high-adherence group, Tempol fully restored NO levels to those of the low-adherence group; however, the ADA-induced constriction remained attenuated, suggesting a possible role for an oxidant-independent vasoconstrictor released from the inflamed venules. These findings suggest that adenosine- and venule-dependent dilation of paired arterioles may be mediated, in part, by NO and inhibited by venular leukocyte adherence.     

Metalloproteinase/Presenilin1 processing of ephrinB regulates EphB-induced Src phosphorylation and signaling

Bidirectional signaling triggered by interacting ephrinB receptors (EphB) and ephrinB ligands is crucial for development and function of the vascular and nervous systems. A signaling cascade triggered by this interaction involves activation of Src kinase and phosphorylation of ephrinB. The mechanism, however, by which EphB activates Src in the ephrinB-expressing cells is unknown. Here we show that EphB stimulates a metalloproteinase cleavage of ephrinB2, producing a carboxy-terminal fragment that is further processed by PS1/gamma-secretase to produce intracellular peptide ephrinB2/CTF2. This peptide binds Src and inhibits its association with inhibitory kinase Csk, allowing autophosphorylation of Src at residue tyr418. EphrinB2/CTF2-activated Src phosphorylates ephrinB2 and inhibits its processing by gamma-secretase. These data show that the PS1/gamma-secretase system controls Src activation and ephrinB phosphorylation by regulating production of Src activator ephrinB2/CTF2. Accordingly, gamma-secretase inhibitors prevented the EphB-induced sprouting of endothelial cells and the recruitment of Grb4 to ephrinB. PS1 FAD and gamma-secretase dominant-negative mutants inhibited the EphB-induced cleavage of ephrinB2 and Src autophosphorylation, raising the possibility that FAD mutants interfere with the functions of Src and ephrinB2 in the CNS.     

Inhibition of mRNA deadenylation by the nuclear cap binding complex (CBC)

Poly(A)-specific ribonuclease (PARN) is a cap-interacting and poly(A)-specific 3'-exoribonuclease. Here we have investigated how the cap binding complex (CBC) affects human PARN activity. We showed that CBC, via its 80-kDa subunit (CBP80), inhibited PARN, suggesting that CBC can regulate mRNA deadenylation. The CBC-mediated inhibition of PARN was cap-independent, and in keeping with this, the CBP80 subunit alone inhibited PARN. Our data suggested a new function for CBC, identified CBC as a potential regulator of PARN, and emphasized the importance of communication between the two extreme ends of the mRNA as a key strategy to regulate mRNA degradation. Based on our data, we have proposed a model for CBC-mediated regulation of PARN, which relies on an interaction between CBP80 and PARN. Association of CBC with PARN might have importance in the regulated recruitment of PARN to the nonsense-mediated decay pathway during the pioneer round of translation.     

Influence of sequential oligopeptide carriers on the bioactive structure of conjugated epitopes: comparative study of the conformation of a Herpes simplex virus glycoprotein gD-1 epitope in the free and conjugated form, and protein "built-in" crystal structure

Synthetic carriers play an important role in immunogen presentation, due to their ability of inducing improved and specific responses to conjugated epitopes. Their influence on the bioactive conformation of the epitope, though admittedly crucial for relevant in vitro and in vivo applications, is difficult to evaluate, given the usual lack of information on the complex conformational features determined by the nature of the carrier and the mode of ligation. Using the Herpes simplex virus glycoprotein D-1 epitope (Leu(9)-Lys-Nle-Ala-Asp-Pro-Asn-Arg-Phe-Arg-Gly-Lys-Asp-Leu(22)) as a model, we have performed a detailed conformational analysis on the free epitope peptide in solution and on three constructs in which the epitope was conjugated to sequential oligopeptide carriers {Ac-[Lys-Aib-Gly](4)-OH (SOC(4))} (through either a thioether or an amide bond; Ac: acetyl) and polytuftsin oligomers {H-[Thr-Lys-Pro-Lys-Gly](4)-NH(2) (T20)}, (through a thioether bond). The analysis of the epitope conformation in the parent protein, in carrier-conjugated and free form, suggests that the beta-turn structure of the -Asp(13)-Pro-Asn-Arg(16)- segment is highly conserved and independent of the epitope form. However, small conformational variations were observed at the C-terminal part of the epitope, depending on the nature of the carrier.     

Putrescine stimulates chemiosmotic ATP synthesis

Putrescine is a main polyamine found in animals, plants and microbes, but the molecular mechanism underlying its mode of action is still obscure. In vivo chlorophyll a fluorescence in tobacco leaf discs indicated that putrescine treatment affects the energization of the thylakoid membrane. Molecular dissection of the electron transport chain by biophysical and biochemical means provided new evidence that putrescine can play an important bioenergetic role acting as a cation and as a permeant natural buffer. We demonstrate that putrescine increases chemiosmotic ATP synthesis more than 70%. Also a regulation of the energy outcome by small changes in putrescine pool under the same photonic environment (i.e., photosynthetically active radiation) is shown. The proposed molecular mechanism has at least four conserved features: (i) presence of a membrane barrier, (ii) a proton-driven ATPase, (iii) a DeltapH and (iv) a pool of putrescine.     

Crystal structure of the soluble form of equinatoxin II, a pore-forming toxin from the sea anemone Actinia equina

BACKGROUND: Membrane pore-forming toxins have a remarkable property: they adopt a stable soluble form structure, which, when in contact with a membrane, undergoes a series of transformations, leading to an active, membrane-bound form. In contrast to bacterial toxins, no structure of a pore-forming toxin from an eukaryotic organism has been determined so far, an indication that structural studies of equinatoxin II (EqtII) may unravel a novel mechanism. RESULTS: The crystal structure of the soluble form of EqtII from the sea anemone Actinia equina has been determined at 1.9 A resolution. EqtII is shown to be a single-domain protein based on a 12 strand beta sandwich fold with a hydrophobic core and a pair of alpha helices, each of which is associated with the face of a beta sheet. CONCLUSIONS: The structure of the 30 N-terminal residues is the largest segment that can adopt a different structure without disrupting the fold of the beta sandwich core. This segment includes a three-turn alpha helix that lies on the surface of a beta sheet and ends in a stretch of three positively charged residues, Lys-30, Arg-31, and Lys-32. On the basis of gathered data, it is suggested that this segment forms the membrane pore, whereas the beta sandwich structure remains unaltered and attaches to a membrane as do other structurally related extrinsic membrane proteins or their domains. The use of a structural data site-directed mutagenesis study should reveal the residues involved in membrane pore formation.     

Endothelial Ca2+ wavelets and the induction of myoendothelial feedback

When arteries constrict to agonists, the endothelium inversely responds, attenuating the initial vasomotor response. The basis of this feedback mechanism remains uncertain, although past studies suggest a key role for myoendothelial communication in the signaling process. The present study examined whether second messenger flux through myoendothelial gap junctions initiates a negative-feedback response in hamster retractor muscle feed arteries. We specifically hypothesized that when agonists elicit depolarization and a rise in second messenger concentration, inositol trisphosphate (IP(3)) flux activates a discrete pool of IP(3) receptors (IP(3)Rs), elicits localized endothelial Ca(2+) transients, and activates downstream effectors to moderate constriction. With use of integrated experimental techniques, this study provided three sets of supporting observations. Beginning at the functional level, we showed that blocking intermediate-conductance Ca(2+)-activated K(+) channels (IK) and Ca(2+) mobilization from the endoplasmic reticulum (ER) enhanced the contractile/electrical responsiveness of feed arteries to phenylephrine. Next, structural analysis confirmed that endothelial projections make contact with the overlying smooth muscle. These projections retained membranous ER networks, and IP(3)Rs and IK channels localized in or near this structure. Finally, Ca(2+) imaging revealed that phenylephrine induced discrete endothelial Ca(2+) events through IP(3)R activation. These events were termed recruitable Ca(2+) wavelets on the basis of their spatiotemporal characteristics. From these findings, we conclude that IP(3) flux across myoendothelial gap junctions is sufficient to induce focal Ca(2+) release from IP(3)Rs and activate a discrete pool of IK channels within or near endothelial projections. The resulting hyperpolarization feeds back on smooth muscle to moderate agonist-induced depolarization and constriction.     

The canonical NF-κB pathway differentially protects normal and human tumor cells from ROS-induced DNA damage

DNA damage responses (DDR) invoke senescence or apoptosis depending on stimulus intensity and the degree of activation of the p53-p21(Cip1/Waf1) axis; but the functional impact of NF-κB signaling on these different outcomes in normal vs. human cancer cells remains poorly understood. We investigated the NF-κB-dependent effects and mechanism underlying reactive oxygen species (ROS)-mediated DDR outcomes of normal human lung fibroblasts (HDFs) and A549 human lung cancer epithelial cells. To activate DDR, ROS accumulation was induced by different doses of H(2)O(2). The effect of ROS induction caused a G2 or G2-M phase cell cycle arrest of both human cell types. However, ROS-mediated DDR eventually culminated in different end points with HDFs undergoing premature senescence and A549 cancer cells succumbing to apoptosis. NF-κB p65/RelA nuclear translocation and Ser536 phosphorylation were induced in response to H(2)O(2)-mediated ROS accumulation. Importantly, blocking the activities of canonical NF-κB subunits with an IκBα super-repressor or suppressing canonical NF-κB signaling by IKKβ knock-down accelerated HDF premature senescence by up-regulating the p53-p21(Cip1/Waf1) axis; but inhibiting the canonical NF-κB pathway exacerbated H(2)O(2)-induced A549 cell apoptosis. HDF premature aging occurred in conjunction with γ-H2AX chromatin deposition, senescence-associated heterochromatic foci and beta-galactosidase staining. p53 knock-down abrogated H(2)O(2)-induced premature senescence of vector control- and IκBαSR-expressing HDFs functionally linking canonical NF-κB-dependent control of p53 levels to ROS-induced HDF senescence. We conclude that IKKβ-driven canonical NF-κB signaling has different functional roles for the outcome of ROS responses in the contexts of normal vs. human tumor cells by respectively protecting them against DDR-dependent premature senescence and apoptosis.     

Mast cells and inflammation

Mast cells are well known for their role in allergic and anaphylactic reactions, as well as their involvement in acquired and innate immunity. Increasing evidence now implicates mast cells in inflammatory diseases where they are activated by non-allergic triggers, such as neuropeptides and cytokines, often exerting synergistic effects as in the case of IL-33 and neurotensin. Mast cells can also release pro-inflammatory mediators selectively without degranulation. In particular, IL-1 induces selective release of IL-6, while corticotropin-releasing hormone secreted under stress induces the release of vascular endothelial growth factor. Many inflammatory diseases involve mast cells in cross-talk with T cells, such as atopic dermatitis, psoriasis and multiple sclerosis, which all worsen by stress. How mast cell differential responses are regulated is still unresolved. Preliminary evidence suggests that mitochondrial function and dynamics control mast cell degranulation, but not selective release. Recent findings also indicate that mast cells have immunomodulatory properties. Understanding selective release of mediators could explain how mast cells participate in numerous diverse biologic processes, and how they exert both immunostimulatory and immunosuppressive actions. Unraveling selective mast cell secretion could also help develop unique mast cell inhibitors with novel therapeutic applications. This article is part of a Special Issue entitled: Mast cells in inflammation.