Remodeling Of Complexes Research Articles

RNA Helicase Proteins as Chaperones and Remodelers

Superfamily 2 helicase proteins are ubiquitous in RNA biology and have an extraordinarily broad set of functional roles. Central among these roles are the promotion of rearrangements of structured RNAs and the remodeling of ribonucleoprotein complexes (RNPs), allowing formation of native RNA structure or progression through a functional cycle of structures. Although all superfamily 2 helicases share a conserved helicase core, they are divided evolutionarily into several families, and it is principally proteins from three families, the DEAD-box, DEAH/RHA, and Ski2-like families, that function to manipulate structured RNAs and RNPs. Strikingly, there are emerging differences in the mechanisms of these proteins, both between families and within the largest family (DEAD-box), and these differences appear to be tuned to their RNA or RNP substrates and their specific roles. This review outlines basic mechanistic features of the three families and surveys individual proteins and the current understanding of their biological substrates and mechanisms.

Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed β2-Adrenergic Receptors

We present subclone sensitive cell phenotypic pharmacology of ligands at the β2-adrenergic receptor (β2-AR) stably expressed in HEK-293 cells. The parental cell line was transfected with green fluorescent protein (GFP)-tagged β2-AR. Four stable subclones were established and used to profile a library of sixty-nine AR ligands. Dynamic mass redistribution (DMR) profiling resulted in a pharmacological activity map suggesting that HEK293 endogenously expresses functional Gi-coupled α2-AR and Gs-coupled β2-AR, and the label-free cell phenotypic activity of AR ligands are subclone dependent. Pathway deconvolution revealed that the DMR of epinephrine is originated mostly from the remodeling of actin microfilaments and adhesion complexes, to less extent from the microtubule networks and receptor trafficking, and certain agonists displayed different efficacy towards the cAMP-Epac pathway. We demonstrate that receptor signaling and ligand pharmacology is sensitive to the receptor expression level, and the organization of the receptor and its signaling circuitry.

Chapter 1 - Fifty Years of Nuclear Pores and Nucleocytoplasmic Transport Studies: Multiple Tools Revealing Complex Rules

Nuclear pore complexes (NPCs) are multiprotein assemblies embedded within the nuclear envelope and involved in the control of the bidirectional transport of proteins and ribonucleoparticles between the nucleus and the cytoplasm. Since their discovery more than 50 years ago, NPCs and nucleocytoplasmic transport have been the focus of intense research. Here, we review how the use of a multiplicity of structural, biochemical, genetic, and cell biology approaches have permitted the deciphering of the main features of this macromolecular complex, its mode of assembly as well as the rules governing nucleocytoplasmic exchanges. We first present the current knowledge of the ultrastructure of NPCs, which reveals that they are modular and repetitive assemblies of subunits referred to as nucleoporins, associated into stable subcomplexes and composed of a limited set of protein domains, including phenylalanine-glycine (FG) repeats and membrane-interacting domains. The outcome of investigations on nucleocytoplasmic trafficking will then be detailed, showing how it involves a limited number of molecular factors and common mechanisms, namely (i) indirect association of cargos with nuclear pores through receptors in the donor compartment, (ii) progression within the channel through dynamic hydrophobic interactions with FG-Nups, and (iii) NTPase-driven remodeling of transport complexes in the target compartment. Finally, we also discuss the outcome of more recent studies, which indicate that NPCs and the transport machinery are dynamic and versatile devices, whose biogenesis is tightly coordinated with the cell cycle, and which carry nonconventional duties, in particular, in mitosis, gene expression, and genetic stability.

Partitioning and Remodeling of the Schizosaccharomyces japonicus Mitotic Nucleus Require Chromosome Tethers

During cellular proliferation, the mother nucleus divides into two daughters, each carrying a full complement of chromosomes and capable of driving the next cell cycle. Mitotic chromosome segregation must be coordinated with remodeling of other nuclear components, including the nuclear envelope (NE) and the non-membrane-bound nucleolus. Here, we show that in the fission yeast Schizosaccharomyces japonicus, which breaks and reforms the NE during mitosis, the evolutionary conserved LEM-domain protein Man1 promotes equal partitioning of the nuclear membrane, nucleolar remodeling, and efficient inheritance of the nuclear pore complexes (NPCs) by the daughter nuclei. Analyses of man1 mutants and results obtained using an artificial chromatin-nuclear pore tether suggest that Man1 may exert its function by linking the NPCs to segregating chromatin. By integrating the partitioning of the nuclear membrane and nucleolar material with chromosome segregation, cells build two nearly identical copies of the original nucleus.

Planar Cell Polarity Pathway Regulates Nephrin Endocytosis in Developing Podocytes

The noncanonical Wnt/planar cell polarity (PCP) pathway controls a variety of cell behaviors such as polarized protrusive cell activity, directional cell movement, and oriented cell division and is crucial for the normal development of many tissues. Mutations in the PCP genes cause malformation in multiple organs. Recently, the PCP pathway was shown to control endocytosis of PCP and non-PCP proteins necessary for cell shape remodeling and formation of specific junctional protein complexes. During formation of the renal glomerulus, the glomerular capillary becomes enveloped by highly specialized epithelial cells, podocytes, that display unique architecture and are connected via specialized cell-cell junctions (slit diaphragms) that restrict passage of protein into the urine; podocyte differentiation requires active remodeling of cytoskeleton and junctional protein complexes. We report here that in cultured human podocytes, activation of the PCP pathway significantly stimulates endocytosis of the core slit diaphragm protein, nephrin, via a clathrin/β-arrestin-dependent endocytic route. In contrast, depletion of the PCP protein Vangl2 leads to an increase of nephrin at the cell surface; loss of Vangl2 functions in Looptail mice results in disturbed glomerular maturation. We propose that the PCP pathway contributes to podocyte development by regulating nephrin turnover during junctional remodeling as the cells differentiate.

Nucleoid localization of Hsp40 Mdj1 is important for its function in maintenance of mitochondrial DNA

Faithful replication and propagation of mitochondrial DNA (mtDNA) is critical for cellular respiration. Molecular chaperones, ubiquitous proteins involved in protein folding and remodeling of protein complexes, have been implicated in mtDNA transactions. In particular, cells lacking Mdj1, an Hsp40 co-chaperone of Hsp70 in the mitochondrial matrix, do not maintain functional mtDNA. Here we report that the great majority of Mdj1 is associated with nucleoids, DNA-protein complexes that are the functional unit of mtDNA transactions. Underscoring the importance of Hsp70 chaperone activity in the maintenance of mtDNA, an Mdj1 variant having an alteration in the Hsp70-interacting J-domain does not maintain mtDNA. However, a J-domain containing fragment expressed at the level that Mdj1 is normally present is not competent to maintain mtDNA, suggesting a function of Mdj1 beyond that carried out by its J-domain. Nevertheless, loss of mtDNA function upon Mdj1 depletion is retarded when the J-domain, is overexpressed. Analysis of Mdj1 variants revealed a correlation between nucleoid association and DNA maintenance activity, suggesting that localization is functionally important. We found that Mdj1 has DNA binding activity and that variants retaining DNA-binding activity also retained nucleoid association. Together, our results are consistent with a model in which Mdj1, tethered to the nucleoid via DNA binding, thus driving a high local concentration of the Hsp70 machinery, is important for faithful DNA maintenance and propagation.

Automated Detection and Quantification of Granular Cell Compartments

Many cellular processes are organized in a compartmentalized and dynamic fashion to ensure effective adaptation to physiological changes. Thus, in response to stress and disease, cells initiate protective mechanisms to restore homeostasis. Among these mechanisms are the arrest of translation and remodeling of ribonucleoprotein complexes into granular compartments in the cytoplasm, known as stress granules (SGs). To date, the analysis of SGs has relied on the manual demarcation and measurement of the compartment, making quantitative studies time-consuming, while preventing the efficient use of high-throughput technology. We developed the first fully automated, computer-based procedures that measure the association of fluorescent molecules with granular compartments. Our methods quantify automatically multiple granule parameters and generate data at the level of single cells or individual SGs. These techniques detect simultaneously in an automated fashion proteins and RNAs located in SGs. The effectiveness of our protocols is demonstrated by studies that reveal several of the unique biological and structural characteristics of SGs. In particular, we show that the type of stress determines granule size and composition, as illustrated by the concentration of poly(A)-RNA and a specific SG marker protein. Furthermore, we took advantage of the computer-based and automated methods to design assays suitable for high-throughput screening.

Structure of an actin-related subcomplex of the SWI/SNF chromatin remodeler

The packaging of DNA into nucleosomal structures limits access for templated processes such as transcription and DNA repair. The repositioning or ejection of nucleosomes is therefore critically important for regulated events, including gene expression. This activity is provided by chromatin remodeling complexes, or remodelers, which are typically large, multisubunit complexes that use an ATPase subunit to translocate the DNA. Many remodelers contain pairs or multimers of actin-related proteins (ARPs) that contact the helicase-SANT-associated (HSA) domain within the catalytic ATPase subunit and are thought to regulate ATPase activity. Here, we determined the structure of a four-protein subcomplex within the SWI/SNF remodeler that comprises the Snf2 HSA domain, Arp7, Arp9, and repressor of Ty1 transposition, gene 102 (Rtt102). Surprisingly, unlike characterized actin-actin associations, the two ARPs pack like spoons and straddle the HSA domain, which forms a 92-Å-long helix. The ARP-HSA interactions are reminiscent of contacts between actin and many binding partners and are quite different from those in the Arp2/3 complex. Rtt102 wraps around one side of the complex in a highly extended conformation that contacts both ARPs and therefore stabilizes the complex, yet functions to reduce by ∼2.4-fold the remodeling and ATPase activity of complexes containing the Snf2 ATPase domain. Thus, our structure provides a foundation for developing models of remodeler function, including mechanisms of coupling between ARPs and the ATPase translocation activity.

Augmented generation of protein fragments during wakefulness as the molecular cause of sleep: a hypothesis.

Despite extensive understanding of sleep regulation, the molecular-level cause and function of sleep are unknown. I suggest that they originate in individual neurons and stem from increased production of protein fragments during wakefulness. These fragments are transient parts of protein complexes in which the fragments were generated. Neuronal Ca²⁺ fluxes are higher during wakefulness than during sleep. Subunits of transmembrane channels and other proteins are cleaved by Ca²⁺-activated calpains and by other nonprocessive proteases, including caspases and secretases. In the proposed concept, termed the fragment generation (FG) hypothesis, sleep is a state during which the production of fragments is decreased (owing to lower Ca²⁺ transients) while fragment-destroying pathways are upregulated. These changes facilitate the elimination of fragments and the remodeling of protein complexes in which the fragments resided. The FG hypothesis posits that a proteolytic cleavage, which produces two fragments, can have both deleterious effects and fitness-increasing functions. This (previously not considered) dichotomy can explain both the conservation of cleavage sites in proteins and the evolutionary persistence of sleep, because sleep would counteract deleterious aspects of protein fragments. The FG hypothesis leads to new explanations of sleep phenomena, including a longer sleep after sleep deprivation. Studies in the 1970s showed that ethanol-induced sleep in mice can be strikingly prolonged by intracerebroventricular injections of either Ca²⁺ alone or Ca²⁺ and its ionophore (Erickson et al., Science 1978;199:1219-1221; Harris, Pharmacol Biochem Behav 1979;10:527-534; Erickson et al., Pharmacol Biochem Behav 1980;12:651-656). These results, which were never interpreted in connection to protein fragments or the function of sleep, may be accounted for by the FG hypothesis about molecular causation of sleep.

Facio-Genital Dysplasia-5 Regulates Matrix Adhesion and Survival of Human Endothelial Cells

The function of the endothelial cell (EC)-enriched Rho family guanine nucleotide exchange factor, facio-genital dysplasia-5 (FGD5), is poorly understood. We sought to determine whether FGD5 regulates endothelial cytoskeletal reorganization and angiogenesis. We observed that FGD5 is expressed in primary human EC isolated from sites across the vasculature. Inhibition of FGD5 expression using RNA interference decreased the protein by ≈70%. In 3-dimensional vascular endothelial growth factor-stimulated angiogenesis in vitro, FGD5-deficient endothelial sprout protrusion was markedly blunted versus nonsilenced controls. FGD5 knockdown impaired adhesion to fibronectin and collagen IV and remodeling of matrix adhesion complexes. Similarly, monolayer electric impedance was decreased, and impedance increased at a slower rate after seeding FGD5-deficient cells versus controls, reflecting decreased EC spreading. Further, FGD5 plays a role in cell survival, because expression of cleaved caspase-3 was increased in FGD5-deficient EC after loss of cell-matrix contacts, and proapoptotic tumor necrosis factor-α stimulation elicited EC with subdiploid DNA content among FGD5-deficient EC. Mechanistically, the phosphatidylinositol 3-kinase/Akt pathway that regulates both adhesive and survival signal transduction pathways requires FGD5. Vascular endothelial growth factor-stimulated Akt phosphorylation and downstream forkhead box protein-O1 inactivation is inhibited by FGD5 loss. FGD5 regulates endothelial adhesion, survival, and angiogenesis by modulating phosphatidylinositol 3-kinase signaling.

Remodeling of mechanical junctions and of microtubule-associated proteins accompany cardiac connexin43 lateralization

Desmosomes and adherens junctions provide mechanical continuity between cardiac cells, whereas gap junctions allow for cell-cell electrical/metabolic coupling. These structures reside at the cardiac intercalated disc (ID). Also at the ID is the voltage-gated sodium channel (VGSC) complex. Functional interactions between desmosomes, gap junctions, and VGSC have been demonstrated. Separate studies show, under various conditions, reduced presence of gap junctions at the ID and redistribution of connexin43 (Cx43) to plaques oriented parallel to fiber direction (gap junction "lateralization"). To determine the mechanisms of Cx43 lateralization, and the fate of desmosomal and sodium channel molecules in the setting of Cx43 remodeling. Adult sheep were subjected to right ventricular pressure overload (pulmonary hypertension). Tissue was analyzed by quantitative confocal microscopy and by transmission electron microscopy. Ionic currents were measured using conventional patch clamp. Quantitative confocal microscopy demonstrated lateralization of immunoreactive junctional molecules. Desmosomes and gap junctions in lateral membranes were demonstrable by electron microscopy. Cx43/desmosomal remodeling was accompanied by lateralization of 2 microtubule-associated proteins relevant for Cx43 trafficking: EB1 and kinesin protein Kif5b. In contrast, molecules of the VGSC failed to reorganize in plaques discernable by confocal microscopy. Patch-clamp studies demonstrated change in amplitude and kinetics of sodium current and a small reduction in electrical coupling between cells. Cx43 lateralization is part of a complex remodeling that includes mechanical and gap junctions but may exclude components of the VGSC. We speculate that lateralization results from redirectionality of microtubule-mediated forward trafficking. Remodeling of junctional complexes may preserve electrical synchrony under conditions that disrupt ID integrity.

Does Lin28 Antagonize miRNA-Mediated Repression by Displacing miRISC from Target mRNAs?

Lin28 is a developmentally regulated RNA-binding protein that plays important roles in diverse physiological and pathological processes including oncogenesis and brain synaptic function. These pleiotropic roles of Lin28 are mechanistically linked both to its ability to directly stimulate translation of genes involved primarily in cell growth and metabolism and to its ability to block biogenesis of a subset of miRNAs including the let-7 family of miRNAs. In the case of direct stimulation of gene expression, Lin28 binds to targeted mRNAs through recognition of Lin28-responsive elements (LREs) within mRNAs and recruits RNA helicase A (RHA) to promote translation. RHA belongs to the DEAD-box protein family of RNA helicases, which generally catalyze ATP-dependent unwinding of RNA duplexes or remodeling of ribonucleoprotein complexes (RNPs). Since any given mRNA can potentially be inhibited by miRNAs bearing complementary sequences, we hypothesize that binding of Lin28 to LREs not only nucleates the binding of multiple Lin28 molecules to the same mRNA, but also leads to remodeling of RNPs through recruitment of RHA and causes release of inhibitory miRNA-induced silencing complexes bound to the mRNA. This mode of action may contribute to Lin28-mediated stimulation of translation in both tumor and neuronal cells.

Gle1 Is a Multifunctional DEAD-box Protein Regulator That Modulates Ded1 in Translation Initiation

DEAD-box protein (Dbp) family members are essential for gene expression; however, their precise roles and regulation are not fully defined. During messenger (m)RNA export, Gle1 bound to inositol hexakisphosphate (IP(6)) acts via Dbp5 to facilitate remodeling of mRNA-protein complexes. In contrast, here we define a novel Gle1 role in translation initiation through regulation of a different DEAD-box protein, the initiation factor Ded1. We find that Gle1 physically and genetically interacts with Ded1. Surprisingly, whereas Gle1 stimulates Dbp5, it inhibits Ded1 ATPase activity in vitro, and IP(6) does not affect this inhibition. Functionally, a gle1-4 mutant specifically suppresses initiation defects in a ded1-120 mutant, and ded1 and gle1 mutants have complementary perturbations in AUG start site recognition. Consistent with this role in initiation, Gle1 inhibits translation in vitro in competent extracts. These results indicate that Gle1 has a direct role in initiation and negatively regulates Ded1. Together, the differential regulation of two distinct DEAD-box proteins by a common factor (Gle1) establishes a new paradigm for controlling gene expression and coupling translation with mRNA export.

Remodeling of Nucleoprotein Complexes Is Independent of the Nucleotide State of a Mutant AAA+ Protein

DnaA protein, a member of the AAA+ (ATPase associated with various cellular activities) family, initiates DNA synthesis at the chromosomal origin of replication (oriC) and regulates the transcription of several genes, including its own. The assembly of DnaA complexes at chromosomal recognition sequences is affected by the tight binding of ATP or ADP by DnaA. DnaA with a point mutation in its membrane-binding amphipathic helix, DnaA(L366K), previously described for its ability to support growth in cells with altered phospholipid content, has biochemical characteristics similar to those of the wild-type protein. Yet DnaA(L366K) fails to initiate in vitro or in vivo replication from oriC. We found here, through in vitro dimethyl sulfate footprinting and gel mobility shift assays, that DnaA(L366K) in either nucleotide state was unable to assemble into productive prereplication complexes. In contrast, at the dnaA promoter, both the ATP and the ADP form of DnaA(L366K) generated active nucleoprotein complexes that efficiently repressed transcription in a manner similar to wild-type ATP-DnaA. Thus, it appears that unlike wild-type DnaA protein DnaA(L366K) can adopt architectures that are independent of its bound nucleotide, and instead the locus determines the functionality of the higher order DnaA(L366K)-DNA complexes.

Rab6, Rab8, and MICAL3 Cooperate in Controlling Docking and Fusion of Exocytotic Carriers

Rab6 is a conserved small GTPase that localizes to the Golgi apparatus and cytoplasmic vesicles and controls transport and fusion of secretory carriers [1]. Another Rab implicated in trafficking from the trans-Golgi to the plasma membrane is Rab8 [2-5]. Here we show that Rab8A stably associates with exocytotic vesicles in a Rab6-dependent manner. Rab8A function is not needed for budding or motility of exocytotic carriers but is required for their docking and fusion. These processes also depend on the Rab6-interacting cortical factor ELKS [1], suggesting that Rab8A and ELKS act in the same pathway. We show that Rab8A and ELKS can be linked by MICAL3, a member of the MICAL family of flavoprotein monooxygenases [6]. Expression of a MICAL3 mutant with an inactive monooxygenase domain resulted in a strong accumulation of secretory vesicles that were docked at the cell cortex but failed to fuse with the plasma membrane, an effect that correlated with the strongly reduced mobility of MICAL3. We propose that the monooxygenase activity of MICAL3 is required to regulate its own turnover and the concomitant remodeling of vesicle-docking protein complexes in which it is engaged. Taken together, the results of our study illustrate cooperation of two Rab proteins in constitutive exocytosis and implicates a redox enzyme in this process.

Glucocorticoid-induced p11 over-expression and chromatin remodeling: A novel molecular mechanism of traumatic stress?

While the actions of glucocorticoids on brain function have been comprehensively studied, understanding of the underlying genomic mechanisms is advancing slowly. Recently, it was found that p11 is associated with traumatic stress and depression, and glucocorticoids regulate expression of the p11 gene. The ligand-activated glucocorticoid receptor (GR) interacts with two glucocorticoid response elements (GREs) in the p11 promoter region to up-regulate the p11 gene. RU486, a glucocorticoid receptor antagonist, and mutation of GREs both block glucocorticoid-induced p11 over-expression, suggesting that glucocorticoid-induced p11 over-expression is mediated by GR and GREs. Thus, the p11 gene can be transcriptionally activated. There is evidence that this transcriptional activation is mediated by the remodeling of chromatin complexes in response to glucocorticoid receptor-regulated promotors. The regulation of eukaryotic gene expression by chromatin remodeling is complex and is essential for numerous cellular processes. The association of linker-histone, non-histone and heterochromatin-specific proteins plays a key role in the generation of higher-order chromatin structures. Understanding the chromatin remodeling involved in the glucocorticoid-mediated increase of p11 expression by stress may clarify stress-induced over-expression of p11 and also identify a new therapeutic target for post-traumatic disorder and depressive disorders, i.e., chromatin remodeling.

Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating

Heat shock cognate protein 70 (Hsc70) supports remodeling of protein complexes -- for example, disassembly of clathrin coats on endocytic coated vesicles. To understand how a simple ATP driven molecular clamp catalyzes a large-scale disassembly reaction, we have used single-particle fluorescence imaging to track the dynamics of Hsc70 and its clathrin substrate in real time. Hsc70 accumulates to a critical level, determined by kinetic modeling to be one Hsc70 for every two functional attachment sites; rapid, all-or-none uncoating then ensues. We propose that Hsc70 traps conformational distortions, seen previously by electron cryomicroscopy, in the vicinity of each occupied site and that accumulation of local strains destabilises the clathrin lattice. Capture of conformational fluctuations may be a general mechanism for chaperone-driven disassembly of protein complexes.

Conformational remodeling of femtomolar inhibitor-acetylcholinesterase complexes in the crystalline state.

The active center of acetylcholinesterase (AChE), a target site for competitive inhibitors, resides centrosymmetric to the subunit at the base of a deep, narrow gorge lined by aromatic residues. At the gorge entry, a peripheral site encompasses overlapping binding loci for noncompetitive inhibitors, which alter substrate access to the gorge. The click-chemistry inhibitor TZ2PA6 links the active center ligand, tacrine, to the peripheral site ligand, propidium, through a biorthogonal reaction of an acetylene and an azide that forms either a syn1 or an anti1 triazole. Compared with wild-type mouse AChE, a Tyr337Ala mutant displays full catalytic activity, albeit with 2-3 orders of magnitude higher affinities for the TZ2PA6 syn1 and anti1 regioisomers, reflected in low femtomolar K(d) values, diffusion-limited association, and dissociation half-times greater than 1 month and 1 week, respectively. Three structures of each of the co-crystallized syn1 and anti1 complexes of the Tyr337Ala mutant were solved at three distinct times of crystal maturation, consistent with or exceeding the half-lives of the complexes in solution, while crystalline complexes obtained from soaked Tyr337Ala crystals led to picturing "freshly formed" complexes. The structures, at 2.55-2.75 Å resolution, reveal a range of unprecedented conformations of the bound regioisomers, not observed in the wild-type AChE complexes, associated with concerted positional rearrangements of side chains in the enzyme gorge. Moreover, time-dependent conformational remodeling of the crystalline complexes appears to correlate with the dissociation half-times of the solution complexes. Hence, for the tight-binding TZ2PA6 inhibitors, the initial complexes kinetically driven in solution slowly form more stable complexes governed by thermodynamic equilibrium and observable in mature crystals.

Loss of PI3Kγ Enhances cAMP-Dependent MMP Remodeling of the Myocardial N-Cadherin Adhesion Complexes and Extracellular Matrix in Response to Early Biomechanical Stress

Mechanotransduction and the response to biomechanical stress is a fundamental response in heart disease. Loss of phosphoinositide 3-kinase (PI3K)γ, the isoform linked to G protein-coupled receptor signaling, results in increased myocardial contractility, but the response to pressure overload is controversial. To characterize molecular and cellular responses of the PI3Kγ knockout (KO) mice to biomechanical stress. In response to pressure overload, PI3KγKO mice deteriorated at an accelerated rate compared with wild-type mice despite increased basal myocardial contractility. These functional responses were associated with compromised phosphorylation of Akt and GSK-3α. In contrast, isolated single cardiomyocytes from banded PI3KγKO mice maintained their hypercontractility, suggesting compromised interaction with the extracellular matrix as the primary defect in the banded PI3KγKO mice. β-Adrenergic stimulation increased cAMP levels with increased phosphorylation of CREB, leading to increased expression of cAMP-responsive matrix metalloproteinases (MMPs), MMP2, MT1-MMP, and MMP13 in cardiomyocytes and cardiofibroblasts. Loss of PI3Kγ resulted in increased cAMP levels with increased expression of MMP2, MT1-MMP, and MMP13 and increased MMP2 activation and collagenase activity in response to biomechanical stress. Selective loss of N-cadherin from the adhesion complexes in the PI3KγKO mice resulted in reduced cell adhesion. The β-blocker propranolol prevented the upregulation of MMPs, whereas MMP inhibition prevented the adverse remodeling with both therapies, preventing the functional deterioration in banded PI3KγKO mice. In banded wild-type mice, long-term propranolol prevented the adverse remodeling and systolic dysfunction with preservation of the N-cadherin levels. The enhanced propensity to develop heart failure in the PI3KγKO mice is attributable to a cAMP-dependent upregulation of MMP expression and activity and disorganization of the N-cadherin/β-catenin cell adhesion complex. β-Blocker therapy prevents these changes thereby providing a novel mechanism of action for these drugs.

Loss of PI3Kγ Enhances cAMP-Dependent MMP Remodeling of N-Cadherin Adhesion Complexes and Extracellular Matrix in Response to Biomechanical Stress

Loss of PI3Kγ Enhances cAMP-Dependent MMP Remodeling of N-Cadherin Adhesion Complexes and Extracellular Matrix in Response to Biomechanical Stress