Entropy-driven cAMP-dependent allosteric control of inhibitory interactions in exchange proteins directly activated by cAMP

Exchange proteins directly activated by cAMP (EPACs) are guanine nucleotide-exchange factors for the small GTPases Rap1 and Rap2 and represent a key receptor for the ubiquitous cAMP second messenger in eukaryotes. The cAMP-dependent activation of apoEPAC is typically rationalized in terms of a preexisting equilibrium between inactive and active states. Structural and mutagenesis analyses have shown that one of the critical determinants of the EPAC activation equilibrium is a cluster of salt bridges formed between the catalytic core and helices alpha1 and alpha2 at the N terminus of the cAMP binding domain and commonly referred to as ionic latch (IL). The IL stabilizes the inactive states in a closed topology in which access to the catalytic domain is sterically occluded by the regulatory moiety. However, it is currently not fully understood how the IL is allosterically controlled by cAMP. Chemical shift mapping studies consistently indicate that cAMP does not significantly perturb the structure of the IL spanning sites within the regulatory region, pointing to cAMP-dependent dynamic modulations as a key allosteric carrier of the cAMP-signal to the IL sites. Here, we have therefore investigated the dynamic profiles of the EPAC1 cAMP binding domain in its apo, cAMP-bound, and Rp-cAMPS phosphorothioate antagonist-bound forms using several 15N relaxation experiments. Based on the comparative analysis of dynamics in these three states, we have proposed a model of EPAC activation that incorporates the dynamic features allosterically modulated by cAMP and shows that cAMP binding weakens the IL by increasing its entropic penalty due to dynamic enhancements.     

Noradrenergic modulation of hypoglossal motoneuron excitability: developmental and putative state-dependent mechanisms

Hypoglossal (XII) motoneurons (MNs) contribute to diverse behaviors. Their innervation of the genioglossus muscle, a tongue protruder, plays a critical role in maintaining upper airway patency during breathing. Indeed, reduced activity in these motoneurons is implicated in sleep related disorders of breathing such as obstructive sleep apnea (OSA). The excitability of these MNs is modulated by multiple neurotransmitter systems. The focus of this review is on the modulation of XII MN excitability by norepinephrine (NE), which increases MN excitability through a variety of mechanisms. The level of noradrenergic drive, however, is very dynamic, varying on developmental, sleep-wake and even millisecond timescales relevant to transitions between behaviours. Here we review and provide new data on the maturation of the noradrenergic modulatory system, focusing on those elements specifically relevant to XII MN excitability including the: i) ontogeny of the noradrenergic cell group that provides the majority of the noradrenergic innervation to the XII nucleus, the Locus subcoeruleus (LsC); ii) time course over which the XII nucleus is innervated by noradrenergic nerve fibres, and; iii) ontogeny of XII MN sensitivity to NE. In the context of state-dependent changes in noradrenergic cell activity, we review mechanisms of NE action most relevant to its role in the muscle atonia of REM sleep. We conclude with a discussion of the hypothesis that the dynamics of MN modulation by NE extend to the spatial domain and recent data suggesting that noradrenergic modulation of the dendritic tree is not uniform but compartmentalized. Implications for information processing are discussed.     

A Novel Non-canonical Forkhead-associated (FHA) Domain-binding Interface Mediates the Interaction between Rad53 and Dbf4 Proteins

Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains are overrepresented in DNA damage and replication stress response proteins. They function primarily as phosphoepitope recognition modules but can also mediate non-canonical interactions. The latter are rare, and only a few have been studied at a molecular level. We have identified a crucial non-canonical interaction between the N-terminal FHA1 domain of the checkpoint effector kinase Rad53 and the BRCT domain of the regulatory subunit of the Dbf4-dependent kinase that is critical to suppress late origin firing and to stabilize stalled forks during replication stress. The Rad53-Dbf4 interaction is phosphorylation-independent and involves a novel non-canonical interface on the FHA1 domain. Mutations within this surface result in hypersensitivity to genotoxic stress. Importantly, this surface is not conserved in the FHA2 domain of Rad53, suggesting that the FHA domains of Rad53 gain specificity by engaging additional interaction interfaces beyond their phosphoepitope-binding site. In general, our results point to FHA domains functioning as complex logic gates rather than mere phosphoepitope-targeting modules.     

Increased frequency of drug-resistant bacteria and fecal coliforms in an Indiana Creek adjacent to farmland amended with treated sludge

Many studies indicate the presence of human pathogens and drug-resistant bacteria in treated sewage sludge. Since one of the main methods of treated sewage disposal is by application to agricultural land, the presence of these organisms is of concern to human health. The goal of this study was to determine whether the frequency of drug-resistant and indicator bacteria in Sugar Creek, which is used for recreational purposes, was influenced by proximity to a farmland routinely amended with treated sludge (site E). Surface water from 3 sites along Sugar Creek (site E, 1 upstream site (site C) and 1 downstream site (site K)) were tested for the presence of ampicillin-resistant (Amp(R)) bacteria, fecal and total coliforms over a period of 40 d. Site E consistently had higher frequencies of Amp(R) bacteria and fecal coliforms compared with the other 2 sites. All of the tested Amp(R) isolates were resistant to at least 1 other antibiotic. However, no isolate was resistant to more than 4 classes of antimicrobials. These results suggest that surface runoff from the farmland is strongly correlated with higher incidence of Amp(R) and fecal coliforms at site E.     

Human amnion as a novel cell delivery vehicle for chondrogenic mesenchymal stem cells

This study investigates the feasibility of processed human amnion (HAM) as a substrate for chondrogenic differentiation of mesenchymal stem cells (MSCs). HAM preparations processed by air drying (AD) and freeze drying (FD) underwent histological examination and MSC seeding in chondrogenic medium for 15 days. Monolayer cultures were used as control for chondrogenic differentiation and HAMs without cell seeding were used as negative control. Qualitative observations were made using scanning electron microscopy analysis and quantitative analyses were based on the sulfated glycosaminoglycans (GAG) assays performed on day 1 and day 15. Histological examination of HAM substrates before seeding revealed a smooth surface in AD substrates, while the FD substrates exhibited a porous surface. Cell attachment to AD and FD substrates on day 15 was qualitatively comparable. GAG were significantly highly expressed in cells seeded on FD HAM substrates. This study indicates that processed HAM is a potentially valuable material as a cell-carrier for MSC differentiation.     

The projection analysis of NMR chemical shifts reveals extended EPAC autoinhibition determinants

EPAC is a cAMP-dependent guanine nucleotide exchange factor that serves as a prototypical molecular switch for the regulation of essential cellular processes. Although EPAC activation by cAMP has been extensively investigated, the mechanism of EPAC autoinhibition is still not fully understood. The steric clash between the side chains of two conserved residues, L273 and F300 in EPAC1, has been previously shown to oppose the inactive-to-active conformational transition in the absence of cAMP. However, it has also been hypothesized that autoinhibition is assisted by entropic losses caused by quenching of dynamics that occurs if the inactive-to-active transition takes place in the absence of cAMP. Here, we test this hypothesis through the comparative NMR analysis of several EPAC1 mutants that target different allosteric sites of the cAMP-binding domain (CBD). Using what to our knowledge is a novel projection analysis of NMR chemical shifts to probe the effect of the mutations on the autoinhibition equilibrium of the CBD, we find that whenever the apo/active state is stabilized relative to the apo/inactive state, dynamics are consistently quenched in a conserved loop (β2-β3) and helix (α5) of the CBD. Overall, our results point to the presence of conserved and nondegenerate determinants of CBD autoinhibition that extends beyond the originally proposed L273/F300 residue pair, suggesting that complete activation necessitates the simultaneous suppression of multiple autoinhibitory mechanisms, which in turn confers added specificity for the cAMP allosteric effector.     

Dynamically Driven Ligand Selectivity in Cyclic Nucleotide Binding Domains

One of the mechanisms that minimize the aberrant cross-talk between cAMP- and cGMP-dependent signaling pathways relies on the selectivity of cAMP binding domains (CBDs). For instance, the CBDs of two critical eukaryotic cAMP receptors, i.e. protein kinase A (PKA) and the exchange protein activated by cAMP (EPAC), are both selectively activated by cAMP. However, the mechanisms underlying their cAMP versus cGMP selectivity are quite distinct. In PKA this selectivity is controlled mainly at the level of ligand affinity, whereas in EPAC it is mostly determined at the level of allostery. Currently, the molecular basis for these different selectivity mechanisms is not fully understood. We have therefore comparatively analyzed by NMR the cGMP-bound states of the essential CBDs of PKA and EPAC, revealing key differences between them. Specifically, cGMP binds PKA preserving the same syn base orientation as cAMP at the price of local steric clashes, which lead to a reduced affinity for cGMP. Unlike PKA, cGMP is recognized by EPAC in an anti conformation and generates several short and long range perturbations. Although these effects do not alter significantly the structure of the EPAC CBD investigated, remarkable differences in dynamics between the cAMP- and cGMP-bound states are detected for the ionic latch region. These observations suggest that one of the determinants of cGMP antagonism in EPAC is the modulation of the entropic control of inhibitory interactions and illustrate the pivotal role of allostery in determining signaling selectivity as a function of dynamic changes, even in the absence of significant affinity variations.In eukaryotes, protein kinase A (PKA)² and the exchange protein directly activated by cAMP (EPAC) are two major receptors for the cAMP second messenger (1–4). The activities of both PKA and EPAC are modulated in a cAMP-dependent manner through cAMP binding domains (CBDs) (1–4). In all isoforms of PKA, two tandem CBDs, denoted as CBD-A and CBD-B, are part of the regulatory subunit (R), in which they are preceded by an N-terminal dimerization docking module and a linker region (Fig. 1a) (1, 3). In the inactive state PKA exists as a tetrameric holo-enzyme complex, including two regulatory (R) subunits and two catalytic (C) subunits (1, 3). Binding of cAMP to the CBDs of the R subunits results in the release of the C subunits and in the activation of the kinase function (1, 3).Open in a separate windowFIGURE 1.Schematic representation of the domain organization in the regulatory subunit I-α of PKA (a) and in EPAC (b).The black circles indicate cAMP. a, D/D is the dimerization docking domain; the inhibitory site is shown in orange, and the two tandem cAMP binding domains, CBD-A and CBD-B, are highlighted in different shades of green. b, DEP, disheveled-egl-10-pleckstrin domain; REM, Ras exchange motif; RA, Ras-associated module, and the CDC25HD catalytic domain are represented in gray, green, orange, blue, and yellow, respectively. The black dashed line and the empty cAMP circle in EPAC2 indicate that this domain is not strictly necessary for the cAMP-dependent GEF activity. The module with question mark in EPAC1 denotes an unknown function for this domain. a and b, the CBD in light green shown below the full-length protein represents the construct used for the NMR studies. c, sequence alignment of the CBDs of bovine RIα- domain A and human EPAC1. Fully conserved residues are highlighted in green; cyan denotes conservation for the functional group only, and yellow indicates the residues present only in one of the CBDs. The secondary structure of apo-EPAC2_m (PDB ID 1O7F) is shown in red.Unlike PKA, EPAC is a single-chain protein that functions as a guanine nucleotide-exchange factor (GEF) for the small GTPase Rap1 and Rap2 (2, 4). The domain organization of EPAC includes an N-terminal regulatory region (RR) and a C-terminal catalytic region (CR) (Fig. 1b). There are two known homologous isoforms of EPAC, i.e. EPAC1 and EPAC2. One of the key differences between EPAC1 and EPAC2 is that in the former there is only a single CBD, whereas in the latter there are two noncontiguous CBDs, i.e. CBD-A and CBD-B. However, CBD-A has been shown not to be strictly necessary for the cAMP-dependent activation of EPAC (2, 4).Both PKA and EPAC are critical for the regulation of a wide range of cAMP-dependent physiological processes (1–4), and impaired activity of these cAMP sensors has been implicated in cardiovascular pathology, diabetes, and Alzheimer disease (1–4). Therefore, PKA and EPAC represent attractive therapeutic targets. However, the design and development of specific drug leads targeting the CBDs of either of these two eukaryotic protein systems require an in depth analysis of how PKA and EPAC selectively recognize and allosterically respond to diverse cNMPs. For instance, the cAMP and cGMP second messengers control distinct groups of essential signaling pathways (1–6). It is therefore critical to minimize the cross-talk between the cAMP- and cGMP-dependent cellular responses. Although in vivo the selective control of the cAMP- and/or cGMP-dependent signaling pathways is a complex process that depends on multiple factors, including the modulation of cNMP synthesis, degradation, and compartmentalization (5), one of the key mechanisms to reduce the cAMP/cGMP cross-talk relies on the ability of both PKA and EPAC CBDs to sense selectively cAMP as opposed to cGMP.Despite the fact that both PKA and EPAC CBDs are cAMP-selective sensors, these two signaling systems adopt different mechanisms to implement their cAMP-selective response. Specifically, in the PKA system cGMP is an agonist of cAMP, i.e. cGMP is able to activate PKA once it binds the R subunit. However, the affinity of cGMP for the PKA R subunit is significantly lower than that of cAMP, resulting in an activation constant that is 2 orders of magnitude higher than that of cAMP (i.e. K_a of 21 ± 2 nm for cAMP and of 4100 ± 20 nm for cGMP) (7, 8). Unlike PKA, EPAC preserves approximate micromolar affinities for both cAMP and cGMP, but in EPAC the latter cNMP is an antagonist of cAMP (9, 10), i.e. cGMP, like other N⁶-substituted cAMP analogs, binds effectively to the CBD of EPAC but fails to fully activate its GEF activity (9, 10).The molecular basis for the cGMP antagonism selectively observed in EPAC but not in PKA is currently not fully understood. An initial hypothesis to explain the antagonist function of cGMP in EPAC has been recently proposed based on the structure of the ternary (S_p)-cAMPS-EPAC2_m-Rap1 complex (11), which shows that the N⁶ of the (S_p)-cAMPS agonist forms an hydrogen-bond with the backbone carbonyl oxygen of Lys-450 located in the lid region (supplemental Table S1) (11). The disruption of this hydrogen bond by cGMP has been hypothesized to result in the inhibition of EPAC activation (11). However, a similar backbone hydrogen bond between the N⁶ of cAMP and the backbone carbonyl oxygen of Arg-632 has been observed also in the CBDs of the hyperpolarization-activated cyclic nucleotide-modulated channels (HCN) (supplemental Table S1), for which cGMP, like in PKA, is not an antagonist (12). This observation suggests that the elimination of the cAMP N⁶ hydrogen bond alone may not be sufficient to fully explain why cGMP is a cAMP antagonist with respect to the activation of EPAC. Furthermore, the previously proposed hypothesis based on the simple disruption by cGMP of the N⁶ hydrogen bond does not consider the possibility that cGMP may adopt an EPAC-bound conformation different from that of cAMP and/or that cGMP may affect also the inhibitory interactions between the ionic latch residues of the EPAC RR and the CDC25HD catalytic domain. These RR/CR salt bridges stabilize the EPAC system in an overall “closed” topology, whereby the RR sterically occludes access of Rap1 into the catalytic domain of EPAC (13). cAMP binding results in increased picosecond to nanosecond and millisecond to microsecond dynamics at the ionic latch region, which in turn leads to an increased entropic penalty for the inhibitory interactions mediated by the ionic latch (14). In other words, cAMP is able to weaken the inhibitory interactions between the regulatory and catalytic regions of EPAC by increasing the entropic cost associated with the formation of the cluster of ionic latch salt bridges between these two functional segments. This dynamically driven mechanism contributes to the observed cAMP-dependent shift toward active “open” conformations of EPAC, and we hypothesize that one of the effects of cGMP is to perturb the dynamic patterns of the EPAC CBD, thus altering the entropic control of the inhibitory interactions.To test our hypotheses on cGMP agonism/antagonism and to further understand the molecular mechanisms underlying the different signaling responses of PKA and EPAC to the two endogenous second messengers cAMP and cGMP, here we present a comparative NMR analysis of cGMP binding and allostery for the critical CBDs of both PKA and EPAC. These results were also compared with the data on the same domains in their apo- and cAMP-bound states (14–20). All these studies rely on the RIα-(119–244) and the related EPAC1_h-(149–318) constructs (Fig. 1), which have been previously validated as models of the essential CBDs of PKA and EPAC, respectively (14–20).Our comparative NMR analysis has revealed that the structure, dynamics, and allosteric activation pathways of the PKA CBD-A are not significantly altered when cAMP is replaced by cGMP. However, significant differences between these two cNMPs are found for the EPAC1_h CBD at the level of both ligand recognition and modulation of dynamic modes, leading to a mechanism in which cGMP shifts the activation equilibrium of EPAC toward the auto-inhibited state, thus accounting for its antagonistic function.     

Chondrocyte-alginate constructs with or without TGF-β1 produces superior extracellular matrix expression than monolayer cultures

Tissue engineering approaches often require expansion of cell numbers in vitro to accelerate tissue regenerative processes. Although several studies have used this technique for therapeutic purposes, a major concern involving the use of isolated chondrocyte culture is the reduction of extracellular matrix (ECM) protein expressed due to the transfer of cells from the normal physiological milieu to the artificial 2D environment provided by the cell culture flasks. To overcome this issue, the use of alginate hydrogel beads as a substrate in chondrocyte cultures has been suggested. However, the resultant characteristics of cells embedded in this bead is elusive. To elucidate this, a study using chondrocytes isolated from rabbit knee articular cartilage expanded in vitro as monolayer and chondrocyte-alginate constructs was conducted. Immunohistochemical evaluation and ECM distribution was examined with or without transforming growth factor (TGF-β1) supplement to determine the ability of cells to express major chondrogenic proteins in these environments. Histological examination followed by transmission electron microscopy and scanning electron microscopy was performed to determine the morphology and the ultrastructural characteristics of these cells. Results demonstrated a significant increase in glycosaminoglycan/mg protein levels in chondrocyte cultures grown in alginate construct than in monolayer cultures. In addition, an abundance of ECM protein distribution surrounding chondrocytes cultured in alginate hydrogel was observed. In conclusion, the current study demonstrates that the use of alginate hydrogel beads in chondrocyte cultures with or without TGF-β1 supplement provided superior ECM expression than monolayer cultures.