Cardiac-specific deletion of the microtubule-binding protein CENP-F causes dilated cardiomyopathy.

CENP-F is a large multifunctional protein with demonstrated regulatory roles in cell proliferation, vesicular transport and cell shape through its association with the microtubule (MT) network. Until now, analysis of CENP-F has been limited to in vitro analysis. Here, using a Cre-loxP system, we report the in vivo disruption of CENP-F gene function in murine cardiomyocytes, a cell type displaying high levels of CENP-F expression. Loss of CENP-F function in developing myocytes leads to decreased cell division, blunting of trabeculation and an initially smaller, thin-walled heart. Still, embryos are born at predicted mendelian ratios on an outbred background. After birth, hearts lacking CENP-F display disruption of their intercalated discs and loss of MT integrity particularly at the costamere; these two structures are essential for cell coupling/electrical conduction and force transduction in the heart. Inhibition of myocyte proliferation and cell coupling as well as loss of MT maintenance is consistent with previous reports of generalized CENP-F function in isolated cells. One hundred percent of these animals develop progressive dilated cardiomyopathy with heart block and scarring, and there is a 20% mortality rate. Importantly, although it has long been postulated that the MT cytoskeleton plays a role in the development of heart disease, this study is the first to reveal a direct genetic link between disruption of this network and cardiomyopathy. Finally, this study has broad implications for development and disease because CENP-F loss of function affects a diverse array of cell-type-specific activities in other organs.

Murine CENP-F regulates centrosomal microtubule nucleation and interacts with Hook2 at the centrosome.

The microtubule (MT) network is essential in a broad spectrum of cellular functions. Many studies have linked CENP-F to MT-based activities as disruption of this protein leads to major changes in MT structure and function. Still, the basis of CENP-F regulation of the MT network remains elusive. Here, our studies reveal a novel and critical localization and role for CENP-F at the centrosome, the major MT organizing center (MTOC) of the cell. Using a yeast two-hybrid screen, we identify Hook2, a linker protein that is essential for regulation of the MT network at the centrosome, as a binding partner of CENP-F. With recently developed immunochemical reagents, we confirm this interaction and reveal the novel localization of CENP-F at the centrosome. Importantly, in this first report of CENP-F(-/-) cells, we demonstrate that ablation of CENP-F protein function eliminates MT repolymerization after standard nocodazole treatment. This inhibition of MT regrowth is centrosome specific because MT repolymerization is readily observed from the Golgi in CENP-F(-/-) cells. The centrosome-specific function of CENP-F in the regulation of MT growth is confirmed by expression of truncated CENP-F containing only the Hook2-binding domain. Furthermore, analysis of partially reconstituted MTOC asters in cells that escape complete repolymerization block shows that disruption of CENP-F function impacts MT nucleation and anchoring rather than promoting catastrophe. Our study reveals a major new localization and function of CENP-F at the centrosome that is likely to impact a broad array of MT-based actions in the cell.

Murine CENPF interacts with syntaxin 4 in the regulation of vesicular transport.

Syntaxin 4 is a component of the SNARE complex that regulates membrane docking and fusion. Using a yeast two-hybrid screen, we identify a novel interaction between syntaxin 4 and cytoplasmic murine CENPF, a protein previously demonstrated to associate with the microtubule network and SNAP-25. The binding domain for syntaxin 4 in CENPF was defined by yeast two-hybrid assay and co-immunoprecipitation. Confocal analyses in cell culture reveal a high degree of colocalization between endogenously expressed proteins in interphase cells. Additionally, the endogenous SNARE proteins can be isolated as a complex with CENPF in immunoprecipitation experiments. Further analyses demonstrate that murine CENPF and syntaxin 4 colocalize with components of plasma membrane recycling: SNAP-25 and VAMP2. Depletion of endogenous CENPF disrupts GLUT4 trafficking whereas expression of a dominant-negative form of CENPF inhibits cell coupling. Taken together, these studies demonstrate that CENPF provides a direct link between proteins of the SNARE system and the microtubule network and indicate a diverse role for murine CENPF in vesicular transport.

Oxidation of 5-thio-2-nitrobenzoic acid, by the biologically relevant oxidants peroxynitrite anion, hydrogen peroxide and hypochlorous acid.

The modification of protein and non-protein thiols by oxidants including hydrogen peroxide (H(2)O(2)), peroxynitrite anion (ONOO(-)) and hypochlorous acid (HOCl) is well documented. Using an aromatic thiol, 5-thio-2-nitrobenzoic acid, and biologically relevant oxidants, we have identified higher oxidation states of sulfur including the sulfonic acid derivative and the disulfide S-oxide, a thiosulfinate, by HPLC and mass spectrometry. The initial reaction of ONOO(-) with 5-thio-2-nitrobenzoic acid yielded a transient red intermediate, the sulfenate anion. The red intermediate was observed when ONOO(-) and H(2)O(2) were used to oxidize 5-thio-2-nitrobenzoic acid and it persisted for several seconds at pH 7. HOCl oxidized the disulfide, 5,5'dithiobis(2-nitrobenzoic acid) to the corresponding sulfonic acid and no additional products were detected. Using this system, we can directly compare the thiol-oxidizing abilities of several oxidants. Because 5-thio-2-nitrobenzoic acid is the product of the reaction of Ellman's reagent with protein thiols, a detailed study of its stability in biological matrices where oxidants may be generated is warranted.

Repair of peroxynitrite damage to tubulin by the thioredoxin reductase system.

Cumulative oxidative damage to proteins coupled with a decrease in repair has been implicated in the pathology of several neurodegenerative diseases. Herein we report that peroxynitrite-induced disulfides in porcine brain tubulin are repaired by the thioredoxin reductase system composed of rat liver thioredoxin reductase, human or Escherichia coli thioredoxin, and NADPH. Disulfide bonds between the alpha-tubulin and the beta-tubulin subunits were repaired by thioredoxin reductase as determined by Western blot under nonreducing conditions. Total disulfide repair by thioredoxin reductase was assessed using a sulfhydryl-specific labeling reagent, 5-iodoacetamido-fluorescein. Treatment of tubulin with 1.0 mM peroxynitrite anion decreased 5-iodoacetamido-fluorescein labeling by 48%; repair of peroxynitrite-damaged tubulin with thioredoxin reductase restored sulfhydryl labeling to control levels. Tubulin disulfide reduction by thioredoxin reductase restored tubulin polymerization activity that was lost after peroxynitrite was added. The extent of activity restored by thioredoxin reductase and by the nonspecific disulfide-reducing agent tris(2-carboxyethyl)phosphine hydrochloride was identical; however, activity was not restored to control levels. Tyrosine nitration of tubulin was detected at all concentrations of peroxynitrite tested; thus, tubulin nitration may be responsible for the fraction of activity that could not be restored. Thiol-disulfide exchange between tubulin and thioredoxin was detected by Western blot, thereby providing further support for our observations that optimal repair of tubulin disulfides required thioredoxin.

Modulation of the redox state of tubulin by the glutathione/glutaredoxin reductase system.

Alterations in the redox status of proteins have been implicated in the pathology of several neurodegenerative diseases. We report that peroxynitrite-induced disulfides in porcine brain tubulin are repaired by the glutaredoxin reductase system composed of glutathione reductase, human or Escherichia coli glutaredoxin, reduced glutathione, and NADPH. Reduction of disulfide bonds between the alpha- and beta-tubulin subunits by the glutathione reductase system was assessed by Western blot. Tubulin cysteine oxidation and reduction was quantitated by monitoring the incorporation of 5-iodoacetamido-fluorescein, a thiol-specific labeling reagent. Tubulin disulfide bond reduction by the glutaredoxin reductase system restored tubulin polymerization activity that was lost following peroxynitrite addition. In support of redox modulations of tubulin by glutathione, thiol-disulfide exchange between tubulin and oxidized glutathione was detected and quantitated by HPLC. In addition, glutathionylation of tubulin was detected by dot blot using an anti-GSH antibody.