Protein kinase C (PKC)ζ-mediated Gαq stimulation of ERK5 protein pathway in cardiomyocytes and cardiac fibroblasts

Gq-coupled G protein-coupled receptors (GPCRs) mediate the actions of a variety of messengers that are key regulators of cardiovascular function. Enhanced Gα(q)-mediated signaling plays an important role in cardiac hypertrophy and in the transition to heart failure. We have recently described that Gα(q) acts as an adaptor protein that facilitates PKCζ-mediated activation of ERK5 in epithelial cells. Because the ERK5 cascade is known to be involved in cardiac hypertrophy, we have investigated the potential relevance of this pathway in cardiovascular Gq-dependent signaling using both cultured cardiac cell types and chronic administration of angiotensin II in mice. We find that PKCζ is required for the activation of the ERK5 pathway by Gq-coupled GPCR in neonatal and adult murine cardiomyocyte cultures and in cardiac fibroblasts. Stimulation of ERK5 by angiotensin II is blocked upon pharmacological inhibition or siRNA-mediated silencing of PKCζ in primary cultures of cardiac cells and in neonatal cardiomyocytes isolated from PKCζ-deficient mice. Moreover, upon chronic challenge with angiotensin II, these mice fail to promote the changes in the ERK5 pathway, in gene expression patterns, and in hypertrophic markers observed in wild-type animals. Taken together, our results show that PKCζ is essential for Gq-dependent ERK5 activation in cardiomyocytes and cardiac fibroblasts and indicate a key cardiac physiological role for the Gα(q)/PKCζ/ERK5 signaling axis.     

Thermodynamic and kinetic characterization of the association of triosephosphate isomerase: the role of diffusion

It is known that diffusion plays a central role in the folding of small monomeric proteins and in the rigid-body association of proteins, however, the role of diffusion in the association of the folding intermediates of oligomeric proteins has been scarcely explored. In this work, catalytic activity and fluorescence measurements were used to study the effect of viscosity in the unfolding and refolding of the homodimeric enzyme triosephosphate isomerase from Saccharamyces cerevisiae. Two transitions were found by equilibrium and kinetic experiments, suggesting a three-state model with a monomeric intermediate. Glycerol barely affects DeltaG(0)(fold) whereas DeltaG(0)(assoc) becomes more favourable in the presence of the cosolvent. From 0 to 60% (v/v) glycerol, the association rate constant showed a near unitary dependence on solvent viscosity. However, at higher glycerol concentrations deviations from Kramers theory were observed. The dissociation rate constant showed a viscosity effect much higher than one. This may be related to secondary effects such as short-range glycerol-induced repulsion between monomers. Nevertheless, after comparison under isostability conditions, a slope near one was also observed for the dissociation rate. These results strongly suggest that the bimolecular association producing the native dimer is limited by diffusional events of the polypeptide chains through the solvent.     

Influence of the germline sequence on the thermodynamic stability and fibrillogenicity of human lambda 6 light chains

Light chain-associated amyloidosis is a fatal disease characterized by the aggregation and pathologic deposition of monoclonal light chain-related fragments as amyloid fibrils in organs or tissues throughout the body. Notably, it has been observed that proteins encoded by the lambda variable light chain (V(L)) gene segment 6a are invariably associated with amyloid deposition; however, the contribution of the gene to this phenomenon has not been established. In this regard, we have determined the thermodynamic stability and kinetics of in vitro fibrillogenesis of a recombinant (r) V(L) protein, designated 6aJL2, which contains the predicted sequences encoded by the 6a and JL2 germline genes. Additionally, we studied a 6a mutant (6aJL2-Arg25Gly), that is present in approximately 25% of all amyloid-associated lambda6 light chains. Remarkably, the wild-type 6aJL2 protein was more stable than were all known amyloidogenic kappa and lambda light chains for which stability parameters are available; more importantly, it was even more so (and less fibrillogenic) than the only clinically proven nonamyloidogenic lambda6 protein, Jto. Conversely, the mutated 6aJL2-R25G molecule was considerably less stable and more fibrillogenic than was the native 6aJL2. Our data indicate that the propensity of lambda6 light chains to form amyloid can not be attributed to thermodynamic instability of the germline-encoded Vlambda6 domain, but rather, is dependent on sequence alterations that render such proteins amyloidogenic.     

Ca handling alterations and vascular dysfunction in diabetes

More than 65% of patients with diabetes mellitus die from cardiovascular disease or stroke. Hyperglycemia, due to either reduced insulin secretion or reduced insulin sensitivity, is the hallmark feature of diabetes mellitus. Vascular dysfunction is a distinctive phenotype found in both types of diabetes and could be responsible for the high incidence of stroke, heart attack, and organ damage in diabetic patients. In addition to well-documented endothelial dysfunction, Ca²⁺ handling alterations in vascular smooth muscle cells (VSMCs) play a key role in the development and progression of vascular complications in diabetes. VSMCs provide not only structural integrity to the vessels but also control myogenic arterial tone and systemic blood pressure through global and local Ca²⁺ signaling. The Ca²⁺ signalosome of VSMCs is integrated by an extensive number of Ca²⁺ handling proteins (i.e. channels, pumps, exchangers) and related signal transduction components, whose function is modulated by endothelial effectors. This review summarizes recent findings concerning alterations in endothelium and VSMC Ca²⁺ signaling proteins that may contribute to the vascular dysfunction found in the diabetic condition.     

PGE2 induces apoptosis of hepatic stellate cells and attenuates liver fibrosis in mice by downregulating miR-23a-5p and miR-28a-5p

MicroRNAs (miRNAs), small noncoding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key regulatory molecules in chronic liver diseases, whose end stage is hepatic fibrosis, a major global health burden. Pharmacological strategies for prevention or treatment of hepatic fibrosis are still limited, what makes it necessary to establish a better understanding of the molecular mechanisms underlying its pathogenesis. In this context, we have recently shown that cyclooxygenase-2 (COX-2) expression in hepatocytes restricts activation of hepatic stellate cells (HSCs), a pivotal event in the initiation and progression of hepatic fibrosis. Here, we evaluated the role of COX-2 in the regulation of a specific set of miRNAs on a mouse model of CCl₄ and bile duct ligation (BDL)-induced liver fibrosis. Our results provide evidence that COX-2 represses miR-23a-5p and miR-28-5p expression in HSC. The decrease of miR-23a-5p and miR-28-5p expression promotes protection against fibrosis by decreasing the levels of pro-fibrogenic markers α-SMA and COL1A1 and increasing apoptosis of HSC. Moreover, we demonstrate that serum levels of miR-28-5p are decreased in patients with chronic liver disease. These results suggest a protective effect exerted by COX-2-derived prostanoids in the process of hepatofibrogenesis.     

Unfolding of triosephosphate isomerase from Trypanosoma brucei: identification of intermediates and insight into the denaturation pathway using tryptophan mutants

The unfolding of triosephosphate isomerase (TIM) from Trypanosoma brucei (TbTIM) induced by guanidine hydrochloride (GdnHCl) was characterized. In contrast to other TIMs, where unfolding is a two or three state process, TbTIM showed two intermediates. The solvent exposure of different regions of the protein in the unfolding process was characterized spectroscopically with mutant proteins in which tryptophans (W) were changed to phenlylalanines (F). The midpoints of the transitions measured by circular dichroism, intrinsic fluorescence, and catalytic activity, as well as the increase in 1-aniline 8-naphthalene sulfonate fluorescence, show that the native state was destabilized in the W12F and W12F/W193F mutants, relative to the wild-type enzyme. Using the hydrodynamic profile for the unfolding of a monomeric TbTIM mutant (RMM0-1TIM) measured by size-exclusion chromatography as a standard, we determined the association state of these intermediates: D*, a partially expanded dimer, and M*, a partially expanded monomeric intermediate. High-molecular-weight aggregates were also detected. At concentrations over 2.0 M GdnHCl, the hydrodynamic properties of TbTIM and RMM0-1TIM are the same, suggesting that the dimeric intermediate dissociates and the unfolding proceeds through the denaturation of an expanded monomeric intermediate. The analysis of the denaturation process of the TbTIM mutants suggests a sequence for the gradual exposure of W residues: initially the expansion of the native dimer to form D* affects the environments of W12 and W159. The dissociation of D* to M* and further unfolding of M* to U induces the exposure of W170. The role of protein concentration in the formation of intermediates and aggregates is discussed considering the irreversibility of this unfolding process.     

X-ray structure of a truncated form of cytochrome f from chlamydomonas reinhardtii

A truncated form of cytochrome f from Chlamydomonas reinhardtii (an important eukaryotic model organism for photosynthetic electron transfer studies) has been crystallized (space group P2(1)2(1)2(1); three molecules/asymmetric unit) and its structure determined to 2.0 A resolution by molecular replacement using the coordinates of a truncated turnip cytochrome f as a model. The structure displays the same folding and detailed features as turnip cytochrome f, including (a) an unusual heme Fe ligation by the alpha-amino group of tyrosine 1, (b) a cluster of lysine residues (proposed docking site of plastocyanin), and (c) the presence of a chain of seven water molecules bound to conserved residues and extending between the heme pocket and K58 and K66 at the lysine cluster. For this array of waters, we propose a structural role. Two cytochrome f molecules are related by a noncrystallographic symmetry operator which is a distorted proper 2-fold rotation. This may represent the dimeric relation of the monomers in situ; however, the heme orientation suggested by this model is not consistent with previous EPR measurements on oriented membranes.     

NOD1 Activation Induces Cardiac Dysfunction and Modulates Cardiac Fibrosis and Cardiomyocyte Apoptosis

The innate immune system is responsible for the initial response of an organism to potentially harmful stressors, pathogens or tissue injury, and accordingly plays an essential role in the pathogenesis of many inflammatory processes, including some cardiovascular diseases. Toll like receptors (TLR) and nucleotide-binding oligomerization domain-like receptors (NLRs) are pattern recognition receptors that play an important role in the induction of innate immune and inflammatory responses. There is a line of evidence supporting that activation of TLRs contributes to the development and progression of cardiovascular diseases but less is known regarding the role of NLRs. Here we demonstrate the presence of the NLR member NOD1 (nucleotide-binding oligomerization domain containing 1) in the murine heart. Activation of NOD1 with the specific agonist C12-iEDAP, but not with the inactive analogue iE-Lys, induces a time- and dose-dependent cardiac dysfunction that occurs concomitantly with cardiac fibrosis and apoptosis. The administration of iEDAP promotes the activation of the NF-κB and TGF-β pathways and induces apoptosis in whole hearts. At the cellular level, both native cardiomyocytes and cardiac fibroblasts expressed NOD1. The NLR activation in cardiomyocytes was associated with NF-κB activation and induction of apoptosis. NOD1 stimulation in fibroblasts was linked to NF-κB activation and to increased expression of pro-fibrotic mediators. The down-regulation of NOD1 by specific siRNAs blunted the effect of iEDAP on the pro-fibrotic TGF-β pathway and cell apoptosis. In conclusion, our report uncovers a new pro-inflammatory target that is expressed in the heart, NOD1. The specific activation of this NLR induces cardiac dysfunction and modulates cardiac fibrosis and cardiomyocyte apoptosis, pathological processes involved in several cardiac diseases such as heart failure.     

Hydrophobic repacking of the dimer interface of triosephosphate isomerase by in silico design and directed evolution

Triosephosphate isomerase from Saccharomyces cerevisiae (wt-TIM) is an obligated homodimer. The interface of wt-TIM is formed by 34 residues. In the native dimer, each monomer buries nearly 2600 A(2) of accessible surface area (ASA), and 58.4% of the interface ASA is hydrophobic. We determined the thermodynamic and functional consequences of increasing the hydrophobic character of the wt-TIM interface. Mutations were restricted to a cluster of five nonconserved residues located far from the active site. Two different approaches, in silico design and directed evolution, were employed. In both methodologies, the obtained proteins were soluble, dimeric, and compact. In silico-designed proteins are very stable dimers that bind substrate with a wild-type-like K(m); albeit, they exhibited a very low k cat. Proteins obtained from directed evolution experiments show wild-type-like catalytic activity, while their stability is decreased. Hydrophobic replacements at the interface produced a remarkable shift in the dissociation step. For wt-TIM and for TIMs obtained by directed evolution, dissociation was observed in the first transition, with C(1/2) values ranging from 0.58 to 0.024 M GdnHCl, whereas for TIMs generated by in silico design, dissociation occurred in the last transition, with C(1/2) values ranging form 3.01 to 3.65 M GdnHCl. For the latter mutants, the stabilization of the interface changed the equilibrium transitions to a novel four-state process with two dimeric intermediates. The change in the intermediate nature suggests that the relative stabilities of different folding units are similar so that subtle alterations in their stability produce a total transformation of the folding pathway.