Viral Oncoproteins Discriminate between p53 and the p53 Homolog p73

p73 is a recently identified member of the p53 family. Previously it was shown that p73 can, when overproduced in p53-defective tumor cells, activate p53-responsive promoters and induce apoptosis. In this report we describe the generation of anti-p73 monoclonal antibodies and confirm that two previously described p73 isoforms are produced in mammalian cells. Furthermore, we show that these two isoforms can bind to canonical p53 DNA-binding sites in electrophoretic mobility shift assays. Despite the high degree of similarity between p53 and p73, we found that adenovirus E1B 55K, simian virus 40 T, and human papillomavirus E6 do not physically interact with p73. The observation that viral oncoproteins discriminate between p53 and p73 suggests that the functions of these two proteins may differ under physiological conditions. Furthermore, they suggest that inactivation of p73 may not be required for transformation.     

On the shoulders of giants: p63, p73 and the rise of p53

The discoveries of the p53 homologs, p63 and p73, have both fueled new insights and exposed enigmas in our understanding of the iconic p53 tumor suppressor. Although the pivotal role of p53 in cancer pathways remains unchallenged, because p63 and p73 are now implicated in stem cell identity, neurogenesis, natural immunity and homeostatic control. Despite their seemingly separate tasks, there are hints that the p53 family members both collaborate and interfere with one another. The question remains, therefore, as to whether these genes evolved to function independently or whether their familial ties still bind them in pathways of cell proliferation, death and tumorigenesis.     

Nuclear accumulation of the small GTPase Gsp1p depends on nucleoporins Nup133p,Rat2p/Nup120p,Nup85p,Nic96p,and the acetyl-CoA carboxylase Acc1p

The small GTPase Ran/Gsp1p plays an essential role in nuclear trafficking of macromolecules, as Ran/Gsp1p regulates many transport processes across the nuclear pore complex (NPC). To determine the role of nucleoporins in the generation of the nucleocytoplasmic Gsp1p concentration gradient, mutations in various nucleoporin genes were analyzed in the yeast Saccharomyces cerevisiae. We show that the nucleoporins Nup133p, Rat2p/Nup120p, Nup85p, Nic96p, and the enzyme acetyl-CoA carboxylase (MTR7) control the distribution and cellular concentration of Gsp1p. At the restrictive temperature the reporter protein GFP-Gsp1p, which is too large to diffuse across the nuclear envelope, fails to concentrate in nuclei of nup133delta, rat2-1, nup85delta, nic96deltaC, and mtr7-1 cells, demonstrating that GFP-Gsp1p nuclear import is deficient. In addition, the concentration of Gsp1p is severely reduced in mutants nup133Delta and mtr7-1 under these conditions. We have now identified the molecular mechanisms that contribute to the dissipation of the Gsp1p concentration gradient in these mutants. Loss of the Gsp1p gradient in nup133delta and rat2-1 can be explained by reduced binding of the Gsp1p nuclear carrier Ntf2p to NPCs. Likewise, nup85delta cells that mislocalize GFP-Gsp1p at the permissive as well as non-permissive temperature have a diminished association of Ntf2p-GFP with nuclear envelopes under both conditions. Moreover, under restrictive conditions Prp20p, the guanine nucleotide exchange factor for Gsp1p, mislocalizes to the cytoplasm in nup85delta, nic96deltaC, and mtr7-1 cells, thereby contributing to a collapse of the Gsp1p gradient. Taken together, components of the NPC subcomplex containing Rat2p/Nup120p, Nup133p, and Nup85p, in addition to proteins Nic96p and Mtr7p, are shown to be crucial for the formation of a nucleocytoplasmic Gsp1p gradient.     

Regulation of the formin for3p by cdc42p and bud6p

Formins are conserved actin nucleators responsible for the assembly of diverse actin structures. Many formins are controlled through an autoinhibitory mechanism involving the interaction of a C-terminal DAD sequence with an N-terminal DID sequence. Here, we show that the fission yeast formin for3p, which mediates actin cable assembly and polarized cell growth, is regulated by a similar autoinhibitory mechanism in vivo. Multiple sites govern for3p localization to cell tips. The localization and activity of for3p are inhibited by an intramolecular interaction of divergent DAD and DID-like sequences. A for3p DAD mutant expressed at endogenous levels produces more robust actin cables, which appear to have normal organization and dynamics. We identify cdc42p as the primary Rho GTPase involved in actin cable assembly and for3p regulation. Both cdc42p, which binds at the N terminus of for3p, and bud6p, which binds near the C-terminal DAD-like sequence, are needed for for3p localization and full activity, but a mutation in the for3p DAD restores for3p localization and other phenotypes of cdc42 and bud6 mutants. In particular, the for3p DAD mutation suppresses the bipolar growth (NETO) defect of bud6Delta cells. These findings suggest that cdc42p and bud6p activate for3p by relieving autoinhibition.     

Bet1p activates the v-SNARE Bos1p.

Bet1p is a type II membrane protein that is required for vesicular transport between the endoplasmic reticulum and Golgi complex in the yeast Saccharomyces cerevisiae. A domain of Bet1p, that shows potential to be involved in a coiled-coil interaction, is homologous to a region of the neuronal protein SNAP-25. Here, we used in vitro binding studies to demonstrate that Bet1p plays a role in potentiating soluble NSF attachment protein receptor (SNARE) interactions. Mutational analysis points to the coiled-coil region as necessary for Bet1p function, and circular dichroism experiments support this theory. In vitro binding studies were also used to demonstrate that a direct interaction between Bet1p and Bos1p is required for the efficient interaction of the vesicle SNARE with its SNARE target. Genetic studies suggest that the interactions of Bet1p with Bos1p are regulated by the small GTP-binding protein Ypt1p.     

p300/CBP/p53 interaction and regulation of the p53 response.

Substantial evidence points to a critical role for the p300/CREB binding protein (CBP) coactivators in p53 responses to DNA damage. p300/CBP and the associated protein P/CAF bind to and acetylate p53 during the DNA damage response, and are needed for full p53 transactivation as well as downstream p53 effects of growth arrest and/or apoptosis. Beyond this simplistic model, p300/CBP appear to be complex integrators of signals that regulate p53, and biochemically, the multipartite p53/p300/CBP interaction is equally complex. Through physical interaction with p53, p300/CBP can both positively and negatively regulate p53 transactivation, as well as p53 protein turnover depending on cellular context and environmental stimuli, such as DNA damage.     

From p63 to p53 across p73

Most genes are members of a family. It is generally believed that a gene family derives from an ancestral gene by duplication and divergence. The tumor suppressor p53 was a striking exception to this established rule. However, two new p53 homologs, p63 and p73, have recently been described [1, 2, 3, 4, 5 and 6]. At the sequence level, p63 and p73 are more similar to each other than each is to p53, suggesting the possibility that the ancestral gene is a gene resembling p63/p73, while p53 is phylogenetically younger [1 and 2].

The complexity of the family has also been enriched by the alternatively spliced forms of p63 and p73, which give rise to a complex network of proteins involved in the control of cell proliferation, apoptosis and development [1, 2, 4, 7, 8 and 9].

In this review we will mainly focus on similarities and differences as well as relationships among p63, p73 and p53.     

Predicted structure for the calcium-dependent membrane-binding proteins p35, p36, and p32

A new family of proteins (annexins) that bind to membranes at micromolar free Ca2+ has been recognized. Its members include an EGF-receptor kinase substrate (p35), a retroviral tyrosine kinase substrate (p36), the liver protein endonexin (p32) and an electric ray protein, calelectrin. Each protein contains four sequence repeats with a further 2-fold internal homology. Using the predicted secondary structure and pattern of conserved hydrophobic residues in each repeat, we have built a three-dimensional model that is largely isostructural with the known molecular conformation of bovine intestinal calcium-binding protein. The final (energy-refined) model had a core formed from the conserved hydrophobic residues. It differed from ICaBP principally in the length of the two Ca2+-binding loops with only one loop being able to bind. The model suggests a mechanism for interaction of these new Ca2+-binding proteins with phospholipid bilayers.     

Ugo1p links the Fzo1p and Mgm1p GTPases for mitochondrial fusion

In yeast, mitochondrial fusion requires Ugo1p and two GTPases, Fzo1p and Mgm1p. Ugo1p is anchored in the mitochondrial outer membrane with its N terminus facing the cytosol and C terminus in the intermembrane space. Fzo1p is also an outer membrane protein, whereas Mgm1p is located in the intermembrane space. Recent studies suggest that these three proteins form protein complexes that mediate mitochondrial fusion. Here, we show that the cytoplasmic domain of Ugo1p directly interacts with Fzo1p, whereas its intermembrane space domain binds to Mgm1p. We identified the Ugo1p-binding site in Fzo1p and demonstrated that Ugo1p-Fzo1p interaction is essential for the formation of mitochondrial shape, maintenance of mitochondrial DNA, and fusion of mitochondria. Although the GTPase domains of Fzo1p and Mgm1p regulate mitochondrial fusion, they were not required for association with Ugo1p. Furthermore, we found that Ugo1p bridges the interaction between Fzo1p and Mgm1p in mitochondria. Our data indicate that distinct regions of Ugo1p bind directly to Fzo1p and Mgm1p and thereby link these two GTPases during mitochondrial fusion.     

Vps51p links the VFT complex to the SNARE Tlg1p

Intracellular membrane fusion requires the complex coordination of SNARE, rab/ypt, and rab effector function. In the yeast Saccharomyces cerevisiae, fusion of endosome-derived vesicles with the late Golgi depends on a cascade of protein-protein interactions that results in the recruitment to Golgi membranes of a conserved docking complex, VFT. This complex binds to Ypt6-GTP, which is necessary for its localization to the Golgi, and also to the SNARE Tlg1p. We show here that the VFT complex contains a fourth, previously uncharacterized, subunit, Vps51p (Ykr020w). Yeast cells lacking VPS51 have defects in vacuole morphology and recycling of the SNARE Snc1p to the plasma membrane, but still assemble a core VFT complex consisting of Vps52p, Vps53p, and Vps54p that localizes properly to the Golgi. Binding to Ypt6-GTP is a property of Vps52p. In contrast, binding to Tlg1p is mediated by a short sequence at the N terminus of Vps51p. Recent evidence suggests that components of a number of rab/ypt effector complexes share a common, distantly related helical coiled-coil motif. We show that each VFT subunit requires this coiled-coil motif for assembly into the complex.     

Sgf1p, a New Component of the Sec34p/Sec35p Complex

Here we report the identification of SGF1 as a high-copy suppressor of the sec35–1 mutant. SGF1 encodes an essential hydrophilic protein of ∼ 100 kDa. Using the yeast two-hybrid system and coprecipitation studies, we demonstrate that Sgf1p is a new subunit of the multiprotein Sec34p/Sec35p complex. Reduced levels of Sgf1p lead to the accumulation of a variety of membranes as well as a kinetic block in endoplasmic reticulum to Golgi traffic. Immunofluorescence studies demonstrate that Sec34p is found throughout the Golgi, with a high concentration on early Golgi. Although an earlier study suggested that Sec34p (Grd20p) is not required for protein secretion, we show here that the sec34–2 and sec35–1 mutations lead to a pleiotropic block in the secretion of all proteins into the growth medium.     

Expression of p53, p63 and p73 in the orofacial region of human embryos

We studied p53, p63, p73 protein expression in the orofacial region of five human embryos aged 7-18 weeks of intrauterine development using a three-step immunohistochemical method. Expression of proteins in various locations was evaluated semiquantitatively. A decrease in p53, p63 and p73 proteins occurred in the 13-week-old material with the exception of the tooth germ where a drop in p73 appeared in the ninth week.     

Yrb1p interaction with the gsp1p C terminus blocks Mog1p stimulation of GTP release from Gsp1p

Mog1p, a multicopy suppressor of gsp1, the temperature-sensitive mutant of the Saccharomyces cerevisiae Ran homologue, binds to GTP-Gsp1p but not to GDP-Gsp1p. The function of Mog1p in the Ran cycle is as yet unknown. This study found that Mog1p releases a nucleotide from GTP-Gsp1p but not from GDP-Gsp1p. Yrb1p, the S. cerevisiae homologue of RanBP1, which is a strong inhibitor of RCC1-stimulated nucleotide release, also inhibited the Mog1p-stimulated nucleotide release from GTP-Gsp1p. At a concentration corresponding to the molar concentration of GTP-Gsp1p, Yrb1p completely inhibited the Mog1p-stimulated nucleotide release. Consistently, the Yrb1p.GTP-Gsp1p complex was more stable than the Mog1p.GTP-Gsp1p complex. Yrb1p did not inhibit the Mog1p-stimulated nucleotide release from GTP-Gsp1DeltaC. The Gsp1DeltaC protein lacks the final eight amino acids of the C terminus, and for this reason, the interaction between GTP-Gsp1DeltaC and Yrb1p was strongly reduced. On the other hand, Mog1p binds to GTP-Gsp1DeltaC more efficiently than to GTP-Gsp1p.     

Smut fungi (Basidiomycota p.p., Ascomycota p.p.) of the world. novelties, selected examples, trends

After defining the meaning of the term 'smut fungus', their current taxonomic classification is presented. There are to date, 1640 'true' species of smut fungi that are classified into 2 phyla, 2 subphyla, 4 classes, 8 orders, 24 families and 90 genera. Recent changes to the classification of the smut fungi have produced some surprises when compared to their traditional classification. The variability of the symptoms produced by the smut fungi on their host plants, and the great morphological diversity of the spores is illustrated by selected pictures. Trends and perspectives in the research of the taxonomy of smut fungi are discussed. On the occasion of the imminent world monograph of smut fungi the necessity for urgent measures ensuring their global conservation is stressed. If present trends of habitat destruction continue, caused in large part by the human population explosion, many of the estimated 4000 to 4500 species of smut fungi become extinct before they are even discovered.