Helix 2 of the paired domain plays a key role in the regulation of DNA-binding by the Pax-3 homeodomain.

Pax3 contains two structurally independent DNA-binding domains, a paired domain (PD) and a homeodomain (HD). Biochemical and mutagenesis studies have shown that both domains are functionally interdependent. In particular, it has been shown that the PD can regulate the DNA-binding specificity and dimerization potential of the HD. To delineate Pax3 protein segments that are involved in the regulation of HD DNA-binding, a series of chimeric proteins were created in which the HD and linker region were gradually replaced with corresponding sequences from a heterologous HD protein, Phox. Characterization of chimeric proteins by electrophoretic mobility shift analysis (EMSA) suggests that a portion of the linker region contributes to the functional interaction between the PD and HD. In addition, stepwise removal of sequences from the Pax3 PD was used to define regions within this domain that are involved in the regulation of HD DNA-binding. EMSA of these proteins in the context of the chimeric Pax3/Phox backbone provided two key findings: (i) the C-terminal subdomain of the PD does not play a major role in the regulation of HD DNA-binding and (ii) the N-terminal subdomain and, in particular, the second alpha-helix are essential for modulation of HD DNA-binding. Significantly, deletion of helix 2 was found to be sufficient to uncouple regulation of HD DNA-binding by the PD.     

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.     

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.     

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.     

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.     

Pax8 plays a pivotal role in regulation of cardiomyocyte growth and senescence

Congenital heart disease (CHD) is a worldwide health problem, particularly in young populations. In spite of the advancement and progress in medical research and technology, the underlying causative factors and mechanisms of CHD still remain unclear. Bone morphogenetic protein receptor IA (ALK3) mediates the development of ventricular septal defect (VSD). We have recently found that paired box gene 8 (Pax8) may be the downstream molecule of ALK3. Paired box gene 8 plays an essential role in VSD, and apoptosis and proliferation imbalance leads to septal dysplasia. Recent studies have also disclosed that cellular senescence also participates in embryonic development. Whether programmed senescence exists in cardiac organogenesis has not ever been reported. We hypothesized that together with various biological processes, such as apoptosis, enhanced cellular senescence may occur actively in the development of Pax8 null mice murine hearts. In H9C2 myogenic cells, Pax8 overexpression can rescue caspase‐dependent apoptosis induced by ALK3 silencing. Senescent cells and senescence‐associated mediators in Pax8 knockout hearts increased compared with the wild‐type ones in an age‐dependent manner. These results suggest that Pax8 maybe the downstream molecule of ALK3, it mediates the murine heart development perhaps via cellular senescence, which may serve as a mechanism that compensates for the cell loss via apoptosis in heart development.     

Phosphoinositide signaling plays a key role in cytokinesis

To perform the vital functions of motility and division, cells must undergo dramatic shifts in cell polarity. Recent evidence suggests that polarized distributions of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, which are clearly important for regulating cell morphology during migration, also play an important role during the final event in cell division, which is cytokinesis. Thus, there is a critical interplay between the membrane phosphoinositides and the cytoskeletal cortex that regulates the complex series of cell shape changes that accompany these two processes.     

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.     

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.     

Pax3 and Pax7 expression during myoblast differentiation in vitro and fast and slow muscle regeneration in vivo

In this report, we focused on Pax3 and Pax7 expression in vitro during myoblast differentiation and in vivo during skeletal muscle regeneration. We showed that Pax3 and Pax7 were present in EDL (extensor digitorum longus) and Soleus muscle derived cells. These cells express in vitro a similar level of Pax3 mRNA, however, differ in the levels of mRNA encoding Pax7. Analysis of Pax3 and Pax7 proteins showed that Soleus and EDL satellite cells differ in the level of Pax3/7 proteins and also in the number of Pax3/7 positive cells. Moreover, Pax3/7 expression was restricted to undifferentiated cells, and both proteins were absent at further stages of myoblast differentiation, indicating that Pax3 and Pax7 are down-regulated during myoblast differentiation. However, we noted that the population of undifferentiated Pax3/7 positive cells was constantly present in both in vitro cultured satellite cells of EDL and Soleus. In contrast, there was no significant difference in Pax3 and Pax7 during in vivo differentiation accompanying regeneration of EDL and Soleus muscle. We demonstrated that Pax3 and Pax7, both in vitro and in vivo, participated in the differentiation and regeneration events of muscle and detected differences in the Pax7 expression pattern during in vitro differentiation of myoblasts isolated from fast and slow muscles.     

The COOH-terminal tail of the GAT-2 GABA transporter contains a novel motif that plays a role in basolateral targeting

The ability of polarized epithelia to perform vectorial transport depends on the asymmetrical distribution of transmembrane proteins among their plasma membrane domains. The establishment and maintenance of these polar distributions relies on molecular signals embedded in the proteins themselves and the interpretation of these signals by cellular sorting machinery. Using Madin-Darby canine kidney (MDCK) cells as an in vitro model of polarized epithelia, our laboratory has previously shown that the COOH-terminal cytoplasmic 22 amino acids of the GAT-2 isoform of the -amino butyric acid (GABA) transporter are necessary for its basolateral distribution. We demonstrate that the COOH-terminal tail of the transporter can function as an autonomous basolateral distribution signal, independently of the rest of the transporter. We find that the three-amino acid PDZ domain-interacting motif at the COOH-terminus of GAT-2 is not necessary for its basolateral distribution. Instead, the more proximal seven amino acids are necessary both for targeting and for steady-state distribution. Because this sequence resembles no other known basolateral sorting information, we conclude that these seven amino acids contain a novel basolateral targeting and distribution motif. GABA transporter; traffic; sorting signal; targeting signal