NAC (N-acetyl-L-cysteine) is commonly used to identify and test ROS (reactive oxygen species) inducers, and to inhibit ROS. In the present study, we identified inhibition of proteasome inhibitors as ...a novel activity of NAC. Both NAC and catalase, another known scavenger of ROS, similarly inhibited ROS levels and apoptosis associated with H₂O₂. However, only NAC, and not catalase or another ROS scavenger Trolox, was able to prevent effects linked to proteasome inhibition, such as protein stabilization, apoptosis and accumulation of ubiquitin conjugates. These observations suggest that NAC has a dual activity as an inhibitor of ROS and proteasome inhibitors. Recently, NAC was used as a ROS inhibitor to functionally characterize a novel anticancer compound, piperlongumine, leading to its description as a ROS inducer. In contrast, our own experiments showed that this compound depicts features of proteasome inhibitors including suppression of FOXM1 (Forkhead box protein M1), stabilization of cellular proteins, induction of ROS-independent apoptosis and enhanced accumulation of ubiquitin conjugates. In addition, NAC, but not catalase or Trolox, interfered with the activity of piperlongumine, further supporting that piperlongumine is a proteasome inhibitor. Most importantly, we showed that NAC, but not other ROS scavengers, directly binds to proteasome inhibitors. To our knowledge, NAC is the first known compound that directly interacts with and antagonizes the activity of proteasome inhibitors. Taken together, the findings of the present study suggest that, as a result of the dual nature of NAC, data interpretation might not be straightforward when NAC is utilized as an antioxidant to demonstrate ROS involvement in drug-induced apoptosis.
GTP-Dependent K-Ras Dimerization Muratcioglu, Serena; Chavan, Tanmay S.; Freed, Benjamin C. ...
Structure,
07/2015, Letnik:
23, Številka:
7
Journal Article
Recenzirano
Odprti dostop
Ras proteins recruit and activate effectors, including Raf, that transmit receptor-initiated signals. Monomeric Ras can bind Raf; however, activation of Raf requires its dimerization. It has been ...suspected that dimeric Ras may promote dimerization and activation of Raf. Here, we show that the GTP-bound catalytic domain of K-Ras4B, a highly oncogenic splice variant of the K-Ras isoform, forms stable homodimers. We observe two major dimer interfaces. The first, highly populated β-sheet dimer interface is at the Switch I and effector binding regions, overlapping the binding surfaces of Raf, PI3K, RalGDS, and additional effectors. This interface has to be inhibitory to such effectors. The second, helical interface also overlaps the binding sites of some effectors. This interface may promote activation of Raf. Our data reveal how Ras self-association can regulate effector binding and activity, and suggest that disruption of the helical dimer interface by drugs may abate Raf signaling in cancer.
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•The GTP-bound K-Ras4B catalytic domain can form stable homodimers•The K-Ras4B predicted β-sheet dimer interface overlaps the effectors' binding site•The K-Ras4B α3/α4 helical dimer interface may promote Raf dimerization•The two interfaces may illuminate K-Ras4B nanocluster formation
Muratcioglu et al. show that GTP-bound but not GDP-bound K-Ras4B catalytic domain can form stable homodimers. Modeling reveals two major dimer interfaces. The highly populated β-sheet interface is at the Switch I/effector binding regions. The second major dimerization mode involves helical interfaces at the C-terminal allosteric lobe of the G domain.
Is nucleotide exchange sufficient to activate K‐Ras4B? To signal, oncogenic rat sarcoma (Ras) anchors in the membrane and recruits effectors by exposing its effector lobe. With the use of NMR and ...molecular dynamics (MD) simulations, we observed that in solution, farnesylated guanosine 5'‐diphosphate (GDP)‐bound K‐Ras4B is predominantly autoinhibited by its hypervariable region (HVR), whereas the GTP‐bound state favors an activated, HVR‐released state. On the anionic membrane, the catalytic domain adopts multiple orientations, including parallel (~180°) and perpendicular (~90°) alignments of the allosteric helices, with respect to the membrane surface direction. In the autoinhibited state, the HVR is sandwiched between the effector lobe and the membrane; in the active state, with membrane‐anchored farnesyl and unrestrained HVR, the catalytic domain fluctuates reinlessly, exposing its effector‐binding site. Dimerization and clustering can reduce the fluctuations. This achieves preorganized, productive conformations. Notably, we also observe HVR‐autoinhibited K‐Ras4B‐GTP states, with GDP‐bound‐like orientations of the helices. Thus, we propose that the GDP/GTP exchange may not be sufficient for activation; instead, our results suggest that the GDP/GTP exchange, HVR sequestration, farnesyl insertion, and orientation/localization of the catalytic domain at the membrane conjointly determine the active or inactive state of K‐Ras4B. Importantly, K‐Ras4B‐GTP can exist in active and inactive states; on its own, GTP binding may not compel K‐Ras4B activation.—Jang, H., Banerjee, A., Chavan, T. S, Lu, S., Zhang, J., Gaponenko, V., Nussinov, R. The higher level of complexity of K‐Ras4B activation at the membrane. FASEB J. 30, 1643–1655 (2016). www.fasebj.org
Background: K-Ras4B modulates downstream signaling at different lipid microdomains.
Results: K-Ras4B farnesyl group spontaneously inserts into the disordered lipid microdomains, but phosphorylation ...prohibits the farnesyl membrane insertion.
Conclusion: The farnesyl may determine K-Ras4B function in different membrane microdomain environments.
Significance: Figuring out K-Ras4B localization at different membrane microdomains is important for a more complete understanding Ras-effector interactions mediating signaling pathways.
K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics simulations, we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, whereas the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion toward the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids but farnesyl membrane orientation. Phosphorylation of Ser-181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling.
Ras proteins are small GTPases that act as signal transducers between cell surface receptors and several intracellular signaling cascades. They contain highly homologous catalytic domains and ...flexible C-terminal hypervariable regions (HVRs) that differ across Ras isoforms. KRAS is among the most frequently mutated oncogenes in human tumors. Surprisingly, we found that the C-terminal HVR of K-Ras4B, thought to minimally impact the catalytic domain, directly interacts with the active site of the protein. The interaction is almost 100-fold tighter with the GDP-bound than the GTP-bound protein. HVR binding interferes with Ras-Raf interaction, modulates binding to phospholipids, and slightly slows down nucleotide exchange. The data indicate that contrary to previously suggested models of K-Ras4B signaling, HVR plays essential roles in regulation of signaling. High affinity binding of short peptide analogs of HVR to K-Ras active site suggests that targeting this surface with inhibitory synthetic molecules for the therapy of KRAS-dependent tumors is feasible.
Among coupled exchangers, CLCs uniquely catalyze the exchange of oppositely charged ions (Cl
for H
). Transport-cycle models to describe and explain this unusual mechanism have been proposed based on ...known CLC structures. While the proposed models harmonize with many experimental findings, gaps and inconsistencies in our understanding have remained. One limitation has been that global conformational change - which occurs in all conventional transporter mechanisms - has not been observed in any high-resolution structure. Here, we describe the 2.6 Å structure of a CLC mutant designed to mimic the fully H
-loaded transporter. This structure reveals a global conformational change to improve accessibility for the Cl
substrate from the extracellular side and new conformations for two key glutamate residues. Together with DEER measurements, MD simulations, and functional studies, this new structure provides evidence for a unified model of H
/Cl
transport that reconciles existing data on all CLC-type proteins.
NMR is commonly used to investigate macromolecular interactions. However, sensitivity problems hamper its use for studying such interactions at low physiologically relevant concentrations. At high ...concentrations, proteins or peptides tend to aggregate. In order to overcome this problem, we make use of reductive ¹³C-methylation to study protein interactions at low micromolar concentrations. Methyl groups in dimethyl lysines are degenerate with one ¹³CH₃ signal arising from two carbons and six protons, as compared to one carbon and three protons in aliphatic amino acids. The improved sensitivity allows us to study protein-protein or protein-peptide interactions at very low micromolar concentrations. We demonstrate the utility of this method by studying the interaction between the post-translationally lipidated hypervariable region of a human proto-oncogenic GTPase K-Ras and a calcium sensor protein calmodulin. Calmodulin specifically binds K-Ras and modulates its downstream signaling. This binding specificity is attributed to the unique lipidated hypervariable region of K-Ras. At low micromolar concentrations, the post-translationally modified hypervariable region of K-Ras aggregates and binds calmodulin in a non-specific manner, hence conventional NMR techniques cannot be used for studying this interaction, however, upon reductively methylating the lysines of calmodulin, we detected signals of the lipidated hypervariable region of K-Ras at physiologically relevant nanomolar concentrations. Thus, we utilize ¹³C-reductive methylation of lysines to enhance the sensitivity of conventional NMR methods for studying protein interactions at low concentrations.
CLC proteins are a ubiquitously expressed family of chloride-selective ion channels and transporters. A dearth of pharmacological tools for modulating CLC gating and ion conduction limits ...investigations aimed at understanding CLC structure/function and physiology. Herein, we describe the design, synthesis, and evaluation of a collection of N-arylated benzimidazole derivatives (BIMs), one of which (BIM1) shows unparalleled (>20-fold) selectivity for CLC-Ka over CLC-Kb, the two most closely related human CLC homologs. Computational docking to a CLC-Ka homology model has identified a BIM1 binding site on the extracellular face of the protein near the chloride permeation pathway in a region previously identified as a binding site for other less selective inhibitors. Results from site-directed mutagenesis experiments are consistent with predictions of this docking model. The residue at position 68 is 1 of only ∼20 extracellular residues that differ between CLC-Ka and CLC-Kb. Mutation of this residue in CLC-Ka and CLC-Kb (N68D and D68N, respectively) reverses the preference of BIM1 for CLC-Ka over CLC-Kb, thus showing the critical role of residue 68 in establishing BIM1 selectivity. Molecular docking studies together with results from structure–activity relationship studies with 19 BIM derivatives give insight into the increased selectivity of BIM1 compared with other inhibitors and identify strategies for further developing this class of compounds.
CLC secondary active transporters exchange Cl(-) for H(+). Crystal structures have suggested that the conformational change from occluded to outward-facing states is unusually simple, involving only ...the rotation of a conserved glutamate (Gluex) upon its protonation. Using (19)F NMR, we show that as H(+) is increased to protonate Gluex and enrich the outward-facing state, a residue ~20 Å away from Gluex, near the subunit interface, moves from buried to solvent-exposed. Consistent with functional relevance of this motion, constriction via inter-subunit cross-linking reduces transport. Molecular dynamics simulations indicate that the cross-link dampens extracellular gate-opening motions. In support of this model, mutations that decrease steric contact between Helix N (part of the extracellular gate) and Helix P (at the subunit interface) remove the inhibitory effect of the cross-link. Together, these results demonstrate the formation of a previously uncharacterized 'outward-facing open' state, and highlight the relevance of global structural changes in CLC function.
Significance α ₁-Adrenergic receptors are important for the regulation of vascular function and are targeted clinically for blood pressure control. Here, we provide evidence that α ₁A/B-adrenergic ...receptors (AR) form heteromeric complexes with chemokine (C-X-C motif) receptor 4 (CXCR4) on the cell surface of vascular smooth muscle cells. We show that disruption of α ₁A/B-AR:CXCR4 heteromeric complexes inhibits α ₁-AR–mediated functions in vascular smooth muscle cells and that treatment with CXCR4 agonists enhances the potency of the α ₁-AR agonist phenylephrine to increase blood pressure. These findings extend the current understanding of the molecular mechanisms regulating α ₁-AR and provide an example of G protein-coupled receptor heteromerization with important functional implications. Compounds targeting the α ₁A/B-AR:CXCR4 interaction could provide an alternative pharmacological approach to modulating blood pressure.
Recent evidence suggests that chemokine (C-X-C motif) receptor 4 (CXCR4) contributes to the regulation of blood pressure through interactions with α ₁-adrenergic receptors (ARs) in vascular smooth muscle. The underlying molecular mechanisms, however, are unknown. Using proximity ligation assays to visualize single-molecule interactions, we detected that α ₁A/B-ARs associate with CXCR4 on the cell surface of rat and human vascular smooth muscle cells (VSMC). Furthermore, α ₁A/B-AR could be coimmunoprecipitated with CXCR4 in a HeLa expression system and in human VSMC. A peptide derived from the second transmembrane helix of CXCR4 induced chemical shift changes in the NMR spectrum of CXCR4 in membranes, disturbed the association between α ₁A/B-AR and CXCR4, and inhibited Ca ²⁺ mobilization, myosin light chain (MLC) 2 phosphorylation, and contraction of VSMC upon α ₁-AR activation. CXCR4 silencing reduced α ₁A/B-AR:CXCR4 heteromeric complexes in VSMC and abolished phenylephrine-induced Ca ²⁺ fluxes and MLC2 phosphorylation. Treatment of rats with CXCR4 agonists (CXCL12, ubiquitin) reduced the EC ₅₀ of the phenylephrine-induced blood pressure response three- to fourfold. These observations suggest that disruption of the quaternary structure of α ₁A/B-AR:CXCR4 heteromeric complexes by targeting transmembrane helix 2 of CXCR4 and depletion of the heteromeric receptor complexes by CXCR4 knockdown inhibit α ₁-AR–mediated function in VSMC and that activation of CXCR4 enhances the potency of α ₁-AR agonists. Our findings extend the current understanding of the molecular mechanisms regulating α ₁-AR and provide an example of the importance of G protein-coupled receptor (GPCR) heteromerization for GPCR function. Compounds targeting the α ₁A/B-AR:CXCR4 interaction could provide an alternative pharmacological approach to modulate blood pressure.
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