Dynamics of NO interacting with soluble guanylate cyclase from 1 ps to 0.1 s and induced structural transitions

International audienc

Activation of MEK1 or MEK2 isoform is sufficient to fully transform intestinal epithelial cells and induce the formation of metastatic tumors

<p>Abstract</p> <p>Background</p> <p>The Ras-dependent ERK1/2 MAP kinase signaling pathway plays a central role in cell proliferation control and is frequently activated in human colorectal cancer. Small-molecule inhibitors of MEK1/MEK2 are therefore viewed as attractive drug candidates for the targeted therapy of this malignancy. However, the exact contribution of MEK1 and MEK2 to the pathogenesis of colorectal cancer remains to be established.</p> <p>Methods</p> <p>Wild type and constitutively active forms of MEK1 and MEK2 were ectopically expressed by retroviral gene transfer in the normal intestinal epithelial cell line IEC-6. We studied the impact of MEK1 and MEK2 activation on cellular morphology, cell proliferation, survival, migration, invasiveness, and tumorigenesis in mice. RNA interference was used to test the requirement for MEK1 and MEK2 function in maintaining the proliferation of human colorectal cancer cells.</p> <p>Results</p> <p>We found that expression of activated MEK1 or MEK2 is sufficient to morphologically transform intestinal epithelial cells, dysregulate cell proliferation and induce the formation of high-grade adenocarcinomas after orthotopic transplantation in mice. A large proportion of these intestinal tumors metastasize to the liver and lung. Mechanistically, activation of MEK1 or MEK2 up-regulates the expression of matrix metalloproteinases, promotes invasiveness and protects cells from undergoing anoikis. Importantly, we show that silencing of MEK2 expression completely suppresses the proliferation of human colon carcinoma cell lines, whereas inactivation of MEK1 has a much weaker effect.</p> <p>Conclusion</p> <p>MEK1 and MEK2 isoforms have similar transforming properties and are able to induce the formation of metastatic intestinal tumors in mice. Our results suggest that MEK2 plays a more important role than MEK1 in sustaining the proliferation of human colorectal cancer cells.</p

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Profil et tendances socio-économiques : îles de Montréal et Laval : document préparé pour le Regroupement des collèges du Montréal métropolitain /

Titre de la couv.(rel. à l'anglaise)Bibliogr.: f. 52-53Notes (part. bibliogr.) au bas des p

Understanding the NO-sensing mechanism at molecular level

International audienceWe present here how ultrafast time-resolved spectroscopy improves our understanding of a new class of proteins: Nitric Oxide sensors. Nitric oxide (NO) is a small, short-lived, and highly reactive gaseous molecule and it acts as a second messenger in several physiological systems. NO sensors are proteins which bind NO and are able to translate this binding into a signal for mammal cells as well as in bacteria. We have studied NO-sensors with the goal of understanding the activation and deactivation mechanism of the human NO-receptor, the enzyme guanylate cyclase (sGC), which is involved in communication between cells. Some bacterial sensors of NO (SONO) have structural homologies and common properties with sGC, but also have differences with sGC which make them valuable system to get structural and physiological information on sGC. To understand how NO-sensors interact with NO and control its reactivity, it is essential to probe dynamics and interactions when NO is present within protein core and what are the associated structural changes. For this purpose, we have used time-resolved absorption spectroscopy in the picoseconds (10(-12)s) time domain. NO can be photodissociated from heme by the pulse of femtosecond laser. Time-resolved transient absorption spectra on NO-sensors were recorded and NO-protein interacttion were recorded. In case of cytochrome c', we identified the formation of 5-coordinate (5c)-NO and 5c-His hemes from 4c-heme and demonstrate that proximal histidine precludes NO rebinding at the proximal site. In bacteria, the adaptation of SONO to temperature changes was not achieved by a simple temperature-dependent NO binding equilibrium, but by a change of the proportion between 5c-NO and 6c-NO species. This amplifies the response to temperature changes since a fast NO rebinding is the only property of a 5c-NO leading to 4c-heme after dissociation. Our results of NO dynamics provide a model for the regulation at molecular level in NO-sensing function

Contribution of Time-Resolved Absorption Spectroscopy to Study Biological Questions

International audienceIn this report, we illustrate through the study of two allosteric heme proteins the contribution of time-resolved absorption spectroscopy to the understanding of fundamental biological mechanisms. The first studied protein is the endogenous nitric oxide receptor (guanylate cyclase, sGC) whose activation and deactivation mechanisms are not yet fully resolved. We show that the rebinding of the proximal histidine occurs in similar to 100 picoseconds in sGC, which is the very first step of its deactivation following NO release. We also show that synergistic action of CO together with an allosteric activator induces the cleavage of the bond between heme iron and proximal histidine. The second one is the prototype of allosteric protein, the dioxygen transporter hemoglobin (Hb). In Hb, we show that the motion of the iron atom, central to the heme, moves in similar to 18 picoseconds after NO binding; this motion represents the very first step of the allosteric T -> R transition

Soluble Guanylate Cyclase Inhibitors Discovered among Natural Compounds

International audienceSoluble guanylate cyclase (sGC) is the human receptor of nitric oxide (NO) in numerous kinds of cells and produces the second messenger 3',5'-cyclic guanosine monophosphate (cGMP) upon NO binding to its heme. sGC is involved in many cell signaling pathways both under healthy conditions and under pathological conditions, such as angiogenesis associated with tumor growth. Addressing the selective inhibition of the NO/cGMP pathway is a strategy worthwhile to be investigated for slowing down tumoral angiogenesis or for curing vasoplegia. However, sGC inhibitors are lacking investigation. We have explored a chemical library of various natural compounds and have discovered inhibitors of sGC. The selected compounds were evaluated for their inhibition of purified sGC in vitro and sGC in endothelial cells. Six natural compounds, from various organisms, have IC50 in the range 0.2-1.5 μM for inhibiting the NO-activated synthesis of cGMP by sGC, and selected compounds exhibit a quantified antiangiogenic activity using an endothelial cell line. These sGC inhibitors can be used directly as tools to investigate angiogenesis and cell signaling or as templates for drug design

Quaternary Structure Controls Ligand Dynamics in Soluble Guanylate Cyclase

International audienceSoluble guanylate cyclase (sGC) is the mammalian endogenous nitric oxide (NO) receptor. The mechanisms of activation and deactivation of this heterodimeric enzyme are unknown. For deciphering them, functional domains can be overexpressed. We have probed the dynamics of the diatomic ligands NO and CO within the isolated heme domain β1(190) of human sGC by piconanosecond absorption spectroscopy. After photo-excitation of nitrosylated sGC, only NO geminate rebinding occurs in 7.5 ps. In β1(190), both photo-dissociation of 5c-NO and photo-oxidation occur, contrary to sGC, followed by NO rebinding (7 ps) and back-reduction (230 ps and 2 ns). In full-length sGC, CO geminate rebinding to the heme does not occur. In contrast, CO geminately rebinds to β1(190) with fast multiphasic process (35, 171, and 18 ns). We measured the bimolecular association rates kon = 0.075 ± 0.01 × 106 M−1*s−1 for sGC and 0.83 ± 0.1 × 106 M−1*s−1 for β1(190). These different dynamics reflect conformational changes and less proximal constraints in the isolated heme domain with respect to the dimeric native sGC. We concluded that the α-subunit and the β1(191-619) domain exert structural strains on the heme domain. These strains are likely involved in the transmission of the energy and relaxation toward the activated state after Fe2+-His bond breaking. This also reveals the heme domain plasticity modulated by the associated domains and subunit

Picosecond binding of the his ligand to four-coordinate heme in cytochrome c ': A one-way gate for releasing proximal NO

International audienceWe provide a direct demonstration of a "kinetic trap" mechanism in the proximal 5-coordinate heme-nitrosyl complex (5c-NO) of cytochrome c' from Alcaligenes xylosoxidans (AXCP) in which picosecond rebinding of the endogenous His ligand following heme-NO dissociation acts as a one-way gate for the release of proximal NO into solution. This demonstration is based upon picosecond transient absorption changes following NO photodissociation of the proximal 5c-NO AXCP complex. We have determined the absolute transient absorption spectrum of 4-coordinate ferrous heme to which NO rebinds with a time constant tNO = 7 ps (kNO = 1.4 × 1011 s-1) and shown that rebinding of the proximal histidine to the 4-coordinate heme takes place with a time constant tHis = 100 ± 10 ps (kHis = 1010 s-1) after the release of NO from the proximal heme pocket. This rapid His reattachment acts as a one-way gate for releasing proximal NO by precluding direct proximal NO rebinding once it has left the proximal heme pocket and requiring NO rebinding from solution to proceed via the distal heme face. Cop. 2013 American Chemical Society

Motion of proximal histidine and structural allosteric transition in soluble guanylate cyclase

International audienceWe investigated the changes of heme coordination in purified soluble guanylate cyclase (sGC) by time-resolved spectroscopy in a time range encompassing 11 orders of magnitude (from 1 ps to 0.2 s). After dissociation, NO either recombines geminately to the 4-coordinate (4c) heme (τG1 = 7.5 ps; 97 ± 1% of the population) or exits the heme pocket (3 ± 1%). The proximal His rebinds to the 4c heme with a 70-ps time constant. Then, NO is distributed in two approximately equal populations (1.5%). One geminately rebinds to the 5c heme (τG2 = 6.5 ns), whereas the other diffuses out to the solution, from where it rebinds bimolecularly (τ = 50 μs with [NO] = 200 μM) forming a 6c heme with a diffusion-limited rate constant of 2 × 10(8) M(-1)⋅s(-1). In both cases, the rebinding of NO induces the cleavage of the Fe-His bond that can be observed as an individual reaction step. Saliently, the time constant of bond cleavage differs depending on whether NO binds geminately or from solution (τ5C1 = 0.66 μs and τ5C2 = 10 ms, respectively). Because the same event occurs with rates separated by four orders of magnitude, this measurement implies that sGC is in different structural states in both cases, having different strain exerted on the Fe-His bond. We show here that this structural allosteric transition takes place in the range 1-50 μs. In this context, the detection of NO binding to the proximal side of sGC heme is discussed