Characterisation of the Bax-nucleophosmin interaction: the importance of the Bax C-terminus

The molecular chaperone nucleophosmin has been identified as a novel Bax binding protein with this interaction proposed to be a key event in the activation and translocation of Bax in mitochondrial dysfunction and apoptotic cell death. Using a proximity assay, we have quantitatively defined the high affinity and saturable interaction between Bax and nucleophosmin indicative of a competitive and specific mechanism. Binding of full length Bax to nucleophosmin was only observed after conformational change was induced using non-ionic detergents (e.g., NP-40). The Bax-nucleophosmin interaction was inhibited by a Bax C-terminal antibody (IC₅₀ = 1 nM) but minimally affected by antibodies directed against either the N-terminus or α-helices 4 and 5. Bcl-2 and p53 inhibited the interaction between full length activated Bax and nucleophosmin. The proximity assay based on the Bax-nucleophosmin interaction was robust and reproducible (Z′ = 0.50) facilitating its use for screening a small chemical library. A low molecular weight non-peptide compound, 2-(5-methyl-2-phenyl-1,3-thiazol-4-yl)ethanohydrazide, partially inhibited the Bax-nucleophosmin interaction (IC₅₀ = 100 nM) and also attenuated UV-induced cell death of HEK293 cells. The present investigations demonstrate the importance of exposure of the C-terminus of Bax for its interaction with nucleophosmin. These protein–protein interaction assays provide a technical approach both for the study of Bax-interacting proteins and for the discovery of novel anti-apoptotic agents. L. Kerr and J. Sharkey have contributed equally to this work.     

Differential binding to dorsal and ventral cell surfaces of fibroblasts: effect on collagen phagocytosis

Matrix remodeling by phagocytic fibroblasts is essential for growth and development but the regulatory processes are undefined. We evaluated the impact of spreading on the binding step of collagen phagocytosis with a novel culture system that more closely replicates phagocytosis in vivo than previous models. 3T3 cells were plated on collagen-coated beads, thereby loading only ventral surfaces (adhesion with spreading), or were allowed to spread on collagen films and then loaded with beads on their dorsal surfaces (adhesion without spreading). Ventral surfaces bound three-fold more beads than dorsal surfaces which was accompanied by accelerated phagosomal maturation. Arp3 and cortactin, markers of the actin-associated spreading machinery, strongly accumulated around ventrally but not dorsally loaded beads, suggesting that spreading contributes to enhanced binding of ventral surfaces. Further, ventral surfaces exhibited two-fold more free alpha2beta1 integrins, the major collagen receptors. Notably, compared to cells spread on collagen substrates, spreading cells exhibited a three-fold higher alpha2beta1 mobile fraction which was correlated with limited engagement of ventral receptors by actin filaments. Thus integrin ligation by actin filaments regulates the mobility of collagen receptors which in turn mediates the enhanced binding of collagen beads on spreading surfaces.     

Bi-directional processing of DNA loops by mismatch repair-dependent and -independent pathways in human cells

Previous work has shown that small DNA loop heterologies are repaired not only through the mismatch repair (MMR) pathway but also via an MMR-independent pathway in human cells. However, how DNA loop repair is partitioned between these pathways and how the MMR-independent repair is processed are not clear. Using a novel construct that completely and specifically inhibits MMR in HeLa extracts, we demonstrate here that although MMR is capable of bi-directionally processing DNA loops of 2, 4, 5, 8, 10, or 12 nucleotides in length, the repair activity decreases with the increase of the loop size. Evidence is presented that the largest loop that the MMR system can process is 16 nucleotides. We also show that strand-specific MMR-independent loop repair occurs for all looped substrates tested and rigorously demonstrate that this repair is bi-directional. Analysis of repair intermediates generated by the MMR-independent pathway revealed that although the processing of looped substrates with a strand break 5' to the heterology occurred similarly to MMR (i.e. excision is conducted by exonucleases from the pre-existing strand break to the heterology), the processing of the heterology in substrates with a 3' strand break is consistent with the involvement of endonucleases.     

Modeling beta-adrenergic control of cardiac myocyte contractility in silico

The beta-adrenergic signaling pathway regulates cardiac myocyte contractility through a combination of feedforward and feedback mechanisms. We used systems analysis to investigate how the components and topology of this signaling network permit neurohormonal control of excitation-contraction coupling in the rat ventricular myocyte. A kinetic model integrating beta-adrenergic signaling with excitation-contraction coupling was formulated, and each subsystem was validated with independent biochemical and physiological measurements. Model analysis was used to investigate quantitatively the effects of specific molecular perturbations. 3-Fold overexpression of adenylyl cyclase in the model allowed an 85% higher rate of cyclic AMP synthesis than an equivalent overexpression of beta 1-adrenergic receptor, and manipulating the affinity of Gs alpha for adenylyl cyclase was a more potent regulator of cyclic AMP production. The model predicted that less than 40% of adenylyl cyclase molecules may be stimulated under maximal receptor activation, and an experimental protocol is suggested for validating this prediction. The model also predicted that the endogenous heat-stable protein kinase inhibitor may enhance basal cyclic AMP buffering by 68% and increasing the apparent Hill coefficient of protein kinase A activation from 1.0 to 2.0. Finally, phosphorylation of the L-type calcium channel and phospholamban were found sufficient to predict the dominant changes in myocyte contractility, including a 2.6x increase in systolic calcium (inotropy) and a 28% decrease in calcium half-relaxation time (lusitropy). By performing systems analysis, the consequences of molecular perturbations in the beta-adrenergic signaling network may be understood within the context of integrative cellular physiology.     

Inactivation of Mre11 does not affect VSG gene duplication mediated by homologous recombination in Trypanosoma brucei

We demonstrate, by gene deletion analysis, that Mre11 has a critical role in maintaining genomic integrity in Trypanosoma brucei. mre11(-/-) null mutant strains exhibited retarded growth but no delay or disruption of cell cycle progression. They showed also a weak hyporecombination phenotype and the accumulation of gross chromosomal rearrangements, which did not involve sequence translocation, telomere loss, or formation of new telomeres. The trypanosome mre11(-/-) strains were hypersensitive to phleomycin, a mutagen causing DNA double strand breaks (DSBs) but, in contrast to mre11(-/-) null mutants in other organisms and T. brucei rad51(-/-) null mutants, displayed no hypersensitivity to methyl methanesulfonate, which causes point mutations and DSBs. Mre11 therefore is important for the repair of chromosomal damage and DSBs in trypanosomes, although in this organism the intersection of repair pathways appears to differ from that in other organisms. Mre11 inactivation appears not to affect VSG gene switching during antigenic variation of a laboratory strain, which is perhaps surprising given the importance of homologous recombination during this process.     

Mechanical force regulation of myofibroblast differentiation in cardiac fibroblasts

The myocardium responds to chronic pressure or volume overload by activation and proliferation of cardiac fibroblasts and their differentiation into myofibroblasts. Because alpha-smooth muscle actin (SMA) expression is the classical marker for myofibroblast differentiation, we examined force-induced SMA expression and regulation by specific MAPK pathways. Rat cardiac fibroblasts were separated from myocytes and smooth muscle cells, cultured, and phenotyped by using the presence of SMA, vimentin, and ED-A fibronectin and the absence of desmin as myofibroblast markers. Static tensile forces (0.65 pN/microm2) were applied to fibroblasts via collagen-coated magnetite beads. In neonatal cardiac fibroblasts cultured for 1 day, immunostaining and Western and Northern blotting showed very low basal levels of SMA. After the application of force, there were 1.5- to 2-fold increases of SMA protein and mRNA within 4 h. Force-induced SMA expression was dependent on ERK phosphorylation and on intact actin filaments. In contrast to cells cultured for 1 day, cells grown for 3 days on rigid substrates showed prominent stress fibers and high basal levels of SMA, which were reduced by 32% within 4 h after force application. ERK was not activated by force, but p38 phosphorylation was required for force-induced inhibition of SMA expression. These results indicate that mechanical force-induced regulation of SMA content is dependent on myofibroblast differentiation and by selective activation of MAPKs.     

Glycated collagen cross-linking alters cardiac mechanics in volume-overload hypertrophy

Alteration of hemodynamic loading induces remodeling that includes changes in myocardial properties and extracellular matrix structure. We investigated the hypothesis that cardiac hypertrophy due to volume overload produces changes in myocardial diastolic mechanics and stiffness that are in part due to alterations in advanced glycation end-product (AGE) collagen cross-linking. Rats developed volume overload induced by arteriovenous fistula (AVF). To assess the dependence of AGE cross-linking on mechanics, we prevented AGE formation by administering the drug aminoguanidine (AG) to one group of AVF rats (AG+AVF). Volume overload did not modify collagen concentration. Right ventricular AGE cross-links were modestly elevated in AVF hearts but were significantly reduced by AG. AVF rats exhibited significantly increased septal AGE cross-links that were inhibited in the AG+AVF group. AVF-induced increases in left ventricular longitudinal stiffness and septal circumferential stiffness were prevented in AG+AVF hearts. Volume overload appears to regionally modify AGE collagen cross-linking and stiffness, and AG treatment prevented these increases, demonstrating that AGE cross-linking plays a role in mediating diastolic compliance in volume-overload hypertrophy.     

Smooth muscle actin determines mechanical force-induced p38 activation

The mitogen-activated protein kinase p38 is activated by mechanical force, but the cellular elements that mediate force-induced p38 phosphorylation are not defined. As alpha-smooth muscle actin (SMA) is an actin isoform associated with force generation in fibroblasts, we asked if SMA participates in the activation of p38 by force. Tensile forces (0.65 pn/mum(2)) generated by magnetic fields were applied to collagen-coated magnetite beads bound to Rat-2 cells. Immunoblotting showed that p38alpha was the predominant p38 isoform. Analysis of bead-associated proteins demonstrated that SMA enrichment of collagen receptor complexes required the alpha2beta1 integrin. SMA was present almost entirely as filaments. Swinholide depolymerized SMA filaments and blocked force-induced p38 phosphorylation and force-induced increases of SMA. Knockdown of SMA (70% reduction) using RNA interference did not affect beta-actin but inhibited force-induced p38 phosphorylation by 50%. Inhibition of Rho kinase blocked SMA filament assembly, force-induced increases of SMA, and force-induced p38 activation. Force application increased SMA content and enhanced the association of phosphorylated p38 with SMA filaments. Blockade of p38 phosphorylation by SB203586 abrogated force-induced increases of SMA. In cells transfected with SMA promoter-beta-galactosidase fusion constructs, co-transfection with constitutively active p38 or MKK6 increased SMA promoter activity by 2.5-3-fold. Dominant negative p38 blocked force-induced activation of the SMA promoter. In SMA negative cells, there was no force-induced p38 phosphorylation. We conclude that force-induced p38 phosphorylation is dependent on an SMA filament-dependent pathway that uses a feed-forward amplification loop to synergize force-induced SMA expression with p38 activation.     

Structural and functional roles of desmin in mouse skeletal muscle during passive deformation

Mechanical interactions between desmin and Z-disks, costameres, and nuclei were measured during passive deformation of single muscle cells. Image processing and continuum kinematics were used to quantify the structural connectivity among these structures. Analysis of both wild-type and desmin-null fibers revealed that the costamere protein talin colocalized with the Z-disk protein alpha-actinin, even at very high strains and stresses. These data indicate that desmin is not essential for mechanical coupling of the costamere complex and the sarcomere lattice. Within the sarcomere lattice, significant differences in myofibrillar connectivity were revealed between passively deformed wild-type and desmin-null fibers. Connectivity in wild-type fibers was significantly greater compared to desmin-null fibers, demonstrating a significant functional connection between myofibrils that requires desmin. Passive mechanical analysis revealed that desmin may be partially responsible for regulating fiber volume, and consequently, fiber mechanical properties. Kinematic analysis of alpha-actinin strain fields revealed that knockout fibers transmitted less shear strain compared to wild-type fibers and experienced a slight increase in fiber volume. Finally, linkage of desmin intermediate filaments to muscle nuclei was strongly suggested based on extensive loss of nuclei positioning in the absence of desmin during passive fiber loading.