An autonomous N-terminal transactivation domain in Fos protein plays a crucial role in transformation.

To date, three functional domains have been defined in c-Fos and v-Fos proteins and have been shown to play a role in transactivation: the leucine zipper mediating hetero-dimerization, the basic DNA contact site, and a C-terminally located transactivation domain (C-TA) harbouring the HOB1 and HOB2 motifs. While the bZip region, consisting of the leucine zipper and the DNA contact site, is indispensable for transformation, the C-TA domain is not required and is actually altered by internal deletions in the FBR-MuSV. We now show that the N-terminal regions of c-Fos and v-Fos contain a second transactivation domain (N-TA). A functionally crucial motif within the N-TA domain, termed NTM, was pinpointed to a approximately 25 amino acid stretch around positions 60-84 which is highly conserved in FosB. Analysis of LexA fusion proteins showed that the N-TA domains of both c-Fos and FosB function in an autonomous fashion in both fibroblasts and yeast. Most importantly, deletion of the NTM motif impairs the transforming properties of v-Fos. Apart from the bZip region, the N-TA domain is the only functional domain required for transformation by v-Fos, at least when its expression is driven by the strong FBR-MuSV-LTR promoter.     

p38 mitogen-activated protein kinase plays a key role in regulating MAPKAPK2 expression

One of three major families of the mitogen-activated kinases (MAPK), p38 as well as JNK, has been shown to transduce extracellular stress stimuli into cellular responses by phospho-relay cascades. Among p38 families, p38alpha is a widely characterized isoform and the biological phenomena are explained by its kinase activity regulating functions of its downstream substrates. However, its specific contributions to each phenomenon are yet not fully elucidated. For better understanding of the role of MAPKs, especially p38alpha, we utilized newly established mouse fibroblast cell lines originated from a p38alpha null mouse, namely, a parental cell line without p38alpha gene locus, knockout of p38alpha (KOP), Zeosin-resistant (ZKOP), revertant of p38alpha (RKOP), and Exip revertant (EKOP). EKOP is smaller in size but grows faster than the others. Although comparable amounts of ERK and JNK are expressed in each cell line, ERK is highly phosphorylated in EKOP even in normal culture conditions. Serum stimulation after serum starvation led to ERK phosphorylation in RKOP and ZKOP, but not in EKOP as much. On the contrary, relative phosphorylation level of JNK to total JNK in response to UV was low in RKOP. And its phosphorylation as well as total JNK is slightly lower in EKOP. RKOP is less sensitive to UV irradiation as judged by the survival rate. Stress response upon UV or sorbitol stimuli, leading to mitogen activate protein kinase activated kinase 2 (MAPKAPK2) phosphorylation, was only observed in RKOP. Further experiments reveal that MAPKAPK2 expression is largely suppressed in ZKOP and EKOP. Its expression was recovered by re-introduction of p38alpha. The loss of MAPKAPK2 expression accompanied by the defect of p38alpha is confirmed in an embryonic extract prepared from p38alpha null mice. These data demonstrate that p38 signal pathway is regulated not only by phosphorylation but also by modulation of the expression of its component. Together, we have established cell lines that can be used in analyzing the functions of MAPKs, especially p38alpha, and show that p38 is indispensable for MAPKAPK2 expression.     

PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization

PTX3 is a prototypic long pentraxin that plays a non-redundant role in innate immunity against selected pathogens and in female fertility. Here, we report that the infertility of Ptx3(-/-) mice is associated with severe abnormalities of the cumulus oophorus and failure of in vivo, but not in vitro, oocyte fertilization. PTX3 is produced by mouse cumulus cells during cumulus expansion and localizes in the matrix. PTX3 is expressed in the human cumulus oophorus as well. Cumuli from Ptx3(-/-) mice synthesize normal amounts of hyaluronan (HA), but are unable to organize it in a stable matrix. Exogenous PTX3 restores a normal cumulus phenotype. Incorporation in the matrix of inter-alpha-trypsin inhibitor is normal in Ptx3(-/-) cumuli. PTX3 does not interact directly with HA, but it binds the cumulus matrix hyaladherin tumor necrosis factor alpha-induced protein 6 (TNFAIP6, also known as TSG6) and thereby may form multimolecular complexes that can cross-link HA chains. Thus, PTX3 is a structural constituent of the cumulus oophorus extracellular matrix essential for female fertility.     

The extracellular microenvironment plays a key role in regulating the redox status of cell surface proteins in HIV-infected subjects

There is an overwhelming interest in the study of the redox status of the cell surface affecting redox signaling in the cells and also predicting the total redox status of the cells. Measuring the total surface thiols (cell surface molecule thiols, csm-SH) we have shown that the overall level of surface thiols is tightly controlled. In vitro, the total concentration of intracellular glutathione (iGSH) seems to play a regulatory role in determination of the amounts of reduced proteins on cells. In addition, short term exposure of the cell surface to glutathione disulfide (GSSG, oxidized GSH) seems to reduce the overall levels of csm-SH suggesting that the function of some cysteine containing proteins on the cell surface may be regulated by the amount of GSSG secreted from the cells or the GSSG available in the extracellular environment. Examination of peripheral blood mononuclear cells (PBMCs) from healthy or HIV-infected subjects failed to reveal a similar correlation between the intra- and extracellular thiol status of cells. Although there is a relatively wide variation between individuals in both csm-SH and iGSH there is no correlation between the iGSH and csm-SH levels measured for healthy and HIV-infected individuals. There are many reports suggesting different redox active proteins on the cell surface to be the key players in the total cell surface redox regulation. However, we suggest that the redox status of the cells is regulated through a complex and tightly regulated mechanism that needs further investigation. In the mean time, overall surface thiol measurements together with case specific protein determinations may offer the most informative approach. In this review, we discuss our own results as well as results from other laboratories to argue that the overall levels of surface thiols on the exofacial membrane are regulated primarily by redox status of the cell surface microenvironment.     

The Phe105 loop of Alix Bro1 domain plays a key role in HIV-1 release

Alix and cellular paralogs HD-PTP and Brox contain N-terminal Bro1 domains that bind ESCRT-III CHMP4. In contrast to HD-PTP and Brox, expression of the Bro1 domain of Alix alleviates HIV-1 release defects that result from interrupted access to ESCRT. In an attempt to elucidate this functional discrepancy, we solved the crystal structures of the Bro1 domains of HD-PTP and Brox. They revealed typical boomerang folds they share with the Bro1 Alix domain. However, they each contain unique structural features that may be relevant to their specific function(s). In particular, phenylalanine residue in position 105 (Phe105) of Alix belongs to a long loop that is unique to its Bro1 domain. Concurrently, mutation of Phe105 and surrounding residues at the tip of the loop compromise the function of Alix in HIV-1 budding without affecting its interactions with Gag or CHMP4. These studies identify a new functional determinant in the Bro1 domain of Alix.     

The forkhead-associated domain 2 (FHA2) in Arabidopsis plays a role in plant fertility by regulating stamen development

The forkhead-associated (FHA) domain is involved in protein–protein interaction by recognizing a phosphothreonine epitope on target proteins. In this study, we investigated in planta functions of the Arabidopsis FHA domain 2. AtFHA2 was mainly localized in the nucleus. Arabidopsis fha2 null mutants grew normally during the vegetative stage, but had severely reduced fertility during reproductive stage. The reduced fertility was mainly caused by defective stamen filament elongation, while female flower parts of the mutants were fertile. Additionally, the mutants had fewer stamens than the wild type and the vegetative organs of the mutants, such as cotyledons and leaves, had increased ploidy. These results suggest that AtFHA2 may play a role in a signaling pathway for the control of plant organ development.     

Induction of AGAMOUS gene expression plays a key role in ripening of tomato sepals in vitro

In vitro culture of VFNT Cherry tomato sepals (calyx) at 16–21 °C results in developmental changes that are similar to those that occur in fruit tissue [10]. Sepals become swollen, red, and succulent, produce ethylene, and have increased levels of polygalacturonase RNA. They also produce many flavor volatiles characteristic of ripe tomato fruit and undergo similar changes in sugar content [11]. We examined the expression of the tomato AGAMOUS gene, TAG1, in ripening, in vitro sepal cultures and other tissues from the plant and found that TAG1 RNA accumulates to higher levels than expected from data from other plants. Contrary to reports on the absence of AGAMOUS in sepals, TAG1 RNA levels in green sepals from greenhouse-grown plants is detectable, its concentration increasing with in vitro ripening to levels that were even higher than in red, ripe fruit. Sepals of fruit on transgenic tomato plants that expressed TAG1 ectopically were induced by low temperature to ripen in vivo, producing lycopene and undergoing cell wall softening as is characteristic of pericarpic tissue. We therefore propose that the induction of elevated TAG1 gene expression plays a key role in developmental changes that result in sepal ripening.     

Cross-talk between the paired domain and the homeodomain of Pax3: DNA binding by each domain causes a structural change in the other domain, supporting interdependence for DNA Binding

The Pax3 protein has two DNA binding domains, a Paired domain (PD) and a paired-type Homeo domain (HD). Although the PD and HD can bind to cognate DNA sequences when expressed individually, genetic and biochemical data indicate that the two domains are functionally interdependent in intact Pax3. The mechanistic basis of this functional interdependence is unknown and was studied by protease sensitivity. Pax3 was modified by the creation of Factor Xa cleavage sites at discrete locations in the PD, the HD, and in the linker segment joining the PD and the HD (Xa172, Xa189, and Xa216) in individual Pax3 mutants. The effect of Factor Xa insertions on protein stability and on DNA binding by the PD and the HD was measured using specific target site sequences. Independent insertions at position 100 in the linker separating the first from the second helix-turn-helix motif of the PD and at position 216 immediately upstream of the HD were found to be readily accessible to Factor Xa cleavage. The effect of DNA binding by the PD or the HD on accessibility of Factor Xa sites inserted in the same or in the other domain was monitored and quantitated for multiple mutants bearing different numbers of Xa sites at each position. In general, DNA binding reduced accessibility of all sites, suggesting a more compact and less solvent-exposed structure of DNA-bound versus DNA-free Pax3. Results of dose response and time course experiments were consistent and showed that DNA binding by the PD not only caused a local structural change in the PD but also caused a conformational change in the HD (P3OPT binding to Xa216 mutants); similarly, DNA binding by the HD also caused a conformational change in the PD (P2 binding to Xa100 mutants). These results provide a structural basis for the functional interdependence of the two DNA binding domains of Pax3.     

Rho plays a central role in regulating local cell-matrix mechanical interactions in 3D culture

The purpose of this study was to assess quantitatively the role of the small GTPase Rho on cell morphology, f-actin organization, and cell-induced matrix remodeling in 3D culture. Human corneal fibroblasts (HTK) were infected with adenoviruses that express green fluorescent protein (GFP) or GFP-N19Rho (dominant negative Rho). One day later cells were plated inside collagen matrices and allowed to spread for 24 h. Cells were fixed and stained for f-actin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) images were acquired using confocal microscopy. Fourier transform analysis was used to assess local collagen fibril alignment, and changes in cell morphology and collagen density were measured using MetaMorph. The decrease in matrix height was used as an indicator of global matrix contraction. HTK and HTK-GFP cells induced significant global matrix contraction; this was inhibited by N19Rho. HTK and HTK-GFP fibroblasts generally had a bipolar morphology and occasional intracellular stress fibers. Collagen fibrils were compacted and aligned parallel to stress fibers and pseudopodia. In contrast, HTK-GFPN19 cells were elongated, and had a more cortical f-actin distribution. Numerous small extensions were also observed along the cell body. In addition, both local collagen fibril density and alignment were significantly reduced. Rho plays a key role in regulating both the morphology and mechanical behavior of corneal fibroblasts in 3D culture. Overall, the data suggest that Rho-kinase dependent cell contractility contributes to global and local matrix remodeling, whereas Rho dependent activation of mDia and/or other downstream effectors regulates the structure and number of cell processes.     

The proline-rich domain of tau plays a role in interactions with actin

Background  

The microtubule-associated protein tau is able to interact with actin and serves as a cross-linker between the microtubule and actin networks. The microtubule-binding domain of tau is known to be involved in its interaction with actin. Here, we address the question of whether the other domains of tau also interact with actin.     

Mitochondrial oxidative stress plays a key role in aging and apoptosis

Harman first suggested in 1972 that mitochondria might be the biological clock in aging, noting that the rate of oxygen consumption should determine the rate of accumulation of mitochondrial damage produced by free radical reactions. Later in 1980 Miquel and coworkers proposed the mitochondrial theory of cell aging. Mitochondria from postmitotic cells use O2 at a high rate, hence releasing oxygen radicals that exceed the cellular antioxidant defences. The key role of mitochondria in cell aging has been outlined by the degeneration induced in cells microinjected with mitochondria isolated from fibroblasts of old rats, especially by the inverse relationship reported between the rate of mitochondrial production of hydroperoxide and the maximum life span of species. An important change in mitochondrial lipid composition is the age-related decrease found in cardiolipin content. The concurrent enhancement of lipid peroxidation and oxidative modification of proteins in mitochondria further increases mutations and oxidative damage to mitochondrial DNA (mtDNA) in the aging process. The respiratory enzymes containing the defective mtDNA-encoded protein subunits may increase the production of reactive oxygen species, which in turn would aggravate the oxidative damage to mitochondria. Moreover, superoxide radicals produced during mitochondrial respiration react with nitric oxide inside mitochondria to yield damaging peroxynitrite. Treatment with certain antioxidants, such as sulphur-containing antioxidants, vitamins C and E, or the Ginkgo biloba extract EGb 761, protects against the age-associated oxidative damage to mtDNA and the oxidation of mitochondrial glutathione. Moreover, the EGb 761 extract also prevents changes in mitochondrial morphology and function associated with aging of the brain and liver.