The gene for the type II (p75) tumor necrosis factor receptor (TNF-RII) is localized on band 1p36.2–p36.3

Summary The gene encoding the type II (p75) tumor necrosis factor receptor (TNF-RII) has been localized on human chromosome 1, band 1p36.2 by nonradioactive in situ hybridization. The gene encoding the type I (p55) TNF-R, which is structurally homologous to the type II (p75) TNF-R, has been previously localized on chromosome 12 band 12p13. Thus, despite their probable common ancestry, the genes for the two TNF-Rs are localized on different chromosomes.     

Interaction of the Saccharomyces cerevisiae cortical actin patch protein Rvs167p with proteins involved in ER to Golgi vesicle trafficking

We have used affinity chromatography to identify two proteins that bind to the SH3 domain of the actin cytoskeleton protein Rvs167p: Gyp5p and Gyl1p. Gyp5p has been shown to be a GTPase activating protein (GAP) for Ypt1p, a Rab GTPase involved in ER to Golgi trafficking; Gyl1p is a protein that resembles Gyp5p and has recently been shown to colocalize with and belong to the same protein complex as Gyp5p. We show that Gyl1p and Gyp5p interact directly with each other, likely through their carboxy-terminal coiled-coil regions. In assays of GAP activity, Gyp5p had GAP activity toward Ypt1p and we found that this activity was stimulated by the addition of Gyl1p. Gyl1p had no GAP activity toward Ypt1p. Genetic experiments suggest a role for Gyp5p and Gyl1p in ER to Golgi trafficking, consistent with their biochemical role. Since Rvs167p has a previously characterized role in endocytosis and we have shown here that it interacts with proteins involved in Golgi vesicle trafficking, we suggest that Rvs167p may have a general role in vesicle trafficking.     

While p73 is essential,p63 is completely dispensable for the development of the central nervous system

The ancient p53 paralogs p63 and p73 regulate specific tissue formation, cell survival and cell death via their TA and ΔN isoforms. Targeted disruption of the p73 locus leads to severe defects in the development of the central nervous system (CNS), and p73 has recently been shown to be an essential regulator of neural stem cell maintenance and differentiation in both embryonal and adult neurogenesis. In contrast, global p63-/- mice lack skin and limbs. Moreover, p63 is detectable in embryonic cortex. It has previously been proposed to also play critical pro-death and pro-survival roles in neural precursors of the developing sympathetic and central nervous system, respectively, based on experimental overexpression and siRNA-mediated knockdown of p63. Here we perform an extensive analysis of the developing central nervous system in global p63-/- mice and their wildtype littermates. Brain and spinal cord of embryos and newborn mice were assessed in vivo for neuroanatomy, histology, apoptosis, proliferation, stemness and differentiation, and in vitro for self-renewal and maturation in neurosphere assays. None of these analyses revealed a detectable phenotype in p63-/- mice. Hence, despite the profound impact of p63 on the development of stratified epithelia and limbs, p63 is completely dispensable for proper development of the central nervous system. Thus, despite their strong homology, the non-overlapping tissue specificity of p63 and p73 functions appears more pronounced than previously anticipated.     

Expression of the Aplysia californica rho gene in Escherichia coli: purification and characterization of its encoded p21 product.

A new family of highly conserved genes, designated rho, has recently been isolated and characterized (P. Madaule and R. Axel, Cell 41:31-40, 1985). These genes have been found in Saccharomyces cerevisiae, Drosophila melanogaster, rats, and humans, and their 21,000-dalton products are highly homologous. The rho p21 protein shares 35% amino acid homology with the Harvey ras p21 protein and on this basis has been proposed to be a G protein. We expressed the Aplysia californica rho gene in Escherichia coli and purified its p21 protein to more than 90% purity. The availability of the rho protein in high quantities made it possible to establish its high affinity for guanine nucleotides. The rho p21 protein had nucleotide-binding properties similar to those of the ras p21 protein. However, a comparison of these proteins revealed some important differences regarding their specificities and affinities. Finally, the rho p21 protein had GTPase activity almost identical to that of a normal ras p21 protein, the rates being 0.106 and 0.105 mol/min per mol of p21, respectively. Thus, the results suggest that the degree of homology found between the ras and rho genes products most likely is related to the conservation of sequences relevant to their ability to bind and hydrolyze guanine nucleotides. The fact that the rho p21 protein binds and hydrolyzes GTP strongly suggests that it is a G protein with a potential regulatory function conserved in evolution.     

Multiple SNARE interactions of an SM protein: Sed5p/Sly1p binding is dispensable for transport

Sec1/Munc18 (SM) proteins are central to intracellular transport and neurotransmitter release but their exact role is still elusive. Several SM proteins, like the neuronal N-Sec1 and the yeast Sly1 protein, bind their cognate t-SNAREs with high affinity. This has been thought to be critical for their function. Here, we show that various mutant forms of Sly1p and the Golgi-localized syntaxin Sed5p, which abolish their high-affinity interaction, are fully functional in vivo, indicating that the tight interaction of the two molecules per se is not relevant for proper function. Mutant Sly1p unable to bind Sed5p is excluded from core SNARE complexes, also demonstrating that Sly1p function is not directly coupled to assembled SNARE complexes thought to execute membrane fusion. We also find that wild-type Sly1p and mutant Sly1p unable to bind Sed5p directly interact with selected ER-to-Golgi and intra-Golgi nonsyntaxin SNAREs. The newly identified, direct interactions of the SM protein with nonsytaxin SNAREs might provide a molecular mechanism by which SNAREs can be activated to engage in pairing and assemble into fusogenic SNARE complexes.     

Structural comparison of yeast snoRNP and spliceosomal protein Snu13p with its homologs

Snu13p is a bifunctional yeast protein involved in both messenger RNA splicing as well as ribosomal RNA maturation. Snu13p initiates assembly of ribonucleoprotein particles by interacting with a conserved RNA motif called kink turn. Unlike its archaeal homolog, L7Ae, Snu13p displays differential specificity for functionally distinct kink turns. Thus, the structures of Snu13p at different functional states, including those alone and bound with RNAs, are required to understand how the protein differentially interacts with kink turns. Although the structure of the human homolog of Snu13p bound with a spliceosomal RNA is known, there has not been a report of a structure of free Snu13p. This has hindered our ability to understand the structural basis for Snu13p's substrate specificity. We report a crystal structure of free Snu13p at 1.9A and a detailed structural comparison with its homologs. We show that free Snu13p has nearly an identical conformation as that of its human homolog bound with RNA. Interestingly, both eukaryotic proteins exhibit notable structural differences in their central beta-sheets as compared to their archaeal homolog, L7Ae. The observed structural differences offer a possible explanation to the observed difference in RNA specificity between Snu13p and L7Ae.     

The <Emphasis Type="Italic">p53</Emphasis> Gene Family: Control of Cell Proliferation and Developmental Programs

For a quarter of a century the gene p53 has attracted close attention of scientists who deal with problems of carcinogenesis and maintenance of genetic stability. Multicellular organisms on our planet owe their rich evolution in many respects to the ability of this gene to protect cells from oncogenic transformation and harmful changes in DNA. A relatively recent discovery of structural p53 homologs, the genes p63 and p73, which seem to have more ancient roots, has roused keen interest in their function. Do they carry out oncosuppressor functions in partnership with p53 or do they possess their own specific functions? This review analyzes data on p53, p63, and p73 functional activity at the levels of the molecule, cell, and whole organism with the accent on examination of specific p63/p73 targets indicating a unique role of these genes in control of developmental processes.     

Viral proteins expressed on the surface of murine leukemia cells.

Leukemic cells of AKR mice contain as constituents of their membranes the murine leukemia virus envelope protein gp70 and the precursor polyprotein of the viral internal (core) structural proteins. Both gp70 and the core polyprotein are represented on the cell surface as glycoproteins, as evidenced by incorporation of [3H]glucosamine into their structure and the binding of these proteins to lectins. The glycosylated core polyprotein exists in at least two serologically distinguishable forms: the 95,000-dalton polyprotein reacts with antisera prepared against the viral proteins p30, p12, and p10, whereas the 85,000-dalton polyprotein reacts with antisera prepared against the viral proteins p30 and p12, but not p10. Additional heterogeneity in these cell surface polyproteins has been observed wtih leukemias induced by exogenous leukemia viruses. Spontaneous leukemia cells of AKR mice invariably express gp70 and the core polyprotein on their cell surface; normal thymocytes of young AKR mice express gp70, but not the core polyprotein on their surface.     

Interrelations between p73 and p53: a model, neuroblastoma

Homologies in sequence and gene organization of p53 and their relatives, p73 and p63, suggest similar biological functions. However differences exist between the p53 family members. Indeed in human tumors p53 is often mutated while p63 and p73 are very rarely mutated. In addition, in contrast to p53 which is transcribed in a unique mRNA species spanning all gene exons, each homologue expresses two types of isoforms: some with transactivation domain (TAD) showing tumor suppressive properties, the others deprived of TAD, with oncogenic properties. If p53 responds to immediate genotoxic stress, its homologues participate to the cell homeostasis of specific tissues along their development and differentiation, neuronal tissue for p73, epithelial for p63. However a collaboration between the three p53 family members has been shown to occur in response to cell genotoxic damages. Neuroblastic tumors characterized by a large spectrum of neuronal differentiation constitute a good model to study relationship between p73 and p53 as well as the regulation of their respective expression.     

Probing the interaction between p53 and the bacterial protein azurin by single molecule force spectroscopy

p53 is a human tumour suppressor which regulates multiple cellular processes, including cell growth, genomic stability and cell death. Recent works have demonstrated the bacterial redox protein azurin to enter cancer cells and induce apoptosis through p53 stabilization, resulting in a tumour growth regression. Azurin has been shown to bind p53 although many details of the complex formed by these two proteins are still poorly characterized. Here, we get insight into the kinetics of this complex formation, by exploring the interaction between p53 and azurin in their environment by single molecule force spectroscopy. To this aim, azurin has been linked to the atomic force microscope tip, whereas p53 has been immobilized onto a gold substrate. Therefore, by performing force-distance cycles we have detected specific recognition events between p53 and azurin, displaying unbinding forces of around 70 pN for an applied loading rate of 3 nN s(-1). The specificity of these events has been assessed by the significant reduction of their frequency observed after blocking the p53 sample by an azurin solution. Moreover, by measuring the rupture force as a function of the loading rate we have determined the dissociation rate constant of this complex to be approximately 0.1 s(-1). Our findings are here discussed in connection with results obtained in bulk experiments, with the aim of clarifying some molecular details of the p53-azurin complex that may help designing new anticancer strategy.     

Exogenous Thyropin from p41 Invariant Chain Diminishes Cysteine Protease Activity and Affects IL-12 Secretion during Maturation of Human Dendritic Cells

Dendritic cells (DC) play a pivotal role as antigen presenting cells (APC) and their maturation is crucial for effectively eliciting an antigen-specific immune response. The p41 splice variant of MHC class II-associated chaperone, called invariant chain p41 Ii, contains an amino acid sequence, the p41 fragment, which is a thyropin-type inhibitor of proteolytic enzymes. The effects of exogenous p41 fragment and related thyropin inhibitors acting on human immune cells have not been reported yet. In this study we demonstrate that exogenous p41 fragment can enter the endocytic pathway of targeted human immature DC. Internalized p41 fragment has contributed to the total amount of the immunogold labelled p41 Ii-specific epitope, as quantified by transmission electron microscopy, in particular in late endocytic compartments with multivesicular morphology where antigen processing and binding to MHC II take place. In cell lysates of treated immature DC, diminished enzymatic activity of cysteine proteases has been confirmed. Internalized exogenous p41 fragment did not affect the perinuclear clustering of acidic cathepsin S-positive vesicles typical of mature DC. p41 fragment is shown to interfere with the nuclear translocation of NF-κB p65 subunit in LPS-stimulated DC. p41 fragment is also shown to reduce the secretion of interleukin-12 (IL-12/p70) during the subsequent maturation of treated DC. The inhibition of proteolytic activity of lysosomal cysteine proteases in immature DC and the diminished capability of DC to produce IL-12 upon their subsequent maturation support the immunomodulatory potential of the examined thyropin from p41 Ii.     

Pex19p binds Pex30p and Pex32p at regions required for their peroxisomal localization but separate from their peroxisomal targeting signals

The assembly of proteins in the peroxisomal membrane is a multistep process requiring their recognition in the cytosol, targeting to and insertion into the peroxisomal membrane, and stabilization within the lipid bilayer. The peroxin Pex19p has been proposed to be either the receptor that recognizes and targets newly synthesized peroxisomal membrane proteins (PMP) to the peroxisome or a chaperone required for stabilization of PMPs at the peroxisomal membrane. Differentiating between these two roles for Pex19p could be achieved by determining whether the peroxisomal targeting signal (PTS) and the region of Pex19p binding of a PMP are the same or different. We addressed the role for Pex19p in the assembly of two PMPs, Pex30p and Pex32p, of the yeast Saccharomyces cerevisiae. Pex30p and Pex32p control peroxisome size and number but are dispensable for peroxisome formation. Systematic truncations from the carboxyl terminus, together with in-frame deletions of specific regions, have identified PTSs essential for targeting Pex30p and Pex32p to peroxisomes. Both Pex30p and Pex32p interact with Pex19p in regions that do not overlap with their PTSs. However, Pex19p is required for localizing Pex30p and Pex32p to peroxisomes, because mutations that disrupt the interaction of Pex19p with Pex30p and Pex32p lead to their mislocalization to a compartment other than peroxisomes. Mutants of Pex30p and Pex32p that localize to peroxisomes but produce cells exhibiting the peroxisomal phenotypes of cells lacking these proteins demonstrate that the regions in these proteins that control peroxisomal targeting and cell biological activity are separable. Together, our data show that the interaction of Pex19p with Pex30p and Pex32p is required for their roles in peroxisome biogenesis and are consistent with a chaperone role for Pex19p in stabilizing or maintaining membrane proteins in peroxisomes.     

Oligomeric state and stoichiometry of p24 proteins in the early secretory pathway

The p24 proteins belong to a highly conserved family of membrane proteins that cycle in the early secretory pathway. They bind to the coat proteins of COPI and COPII vesicles, and are proposed to be involved in vesicle biogenesis, cargo uptake, and quality control, but their precise function is still under debate. Most p24 proteins form hetero-oligomers, essential for their correct localization and stability. Functional insights regarding the mechanisms of their steady state localization and the role of interaction with coat proteins has been hampered by a lack of data on their concentration and state of oligomerization within the endoplasmic reticulum, the intermediate compartment, and Golgi complex. We have determined for all mammalian p24 family members the size of the oligomers formed and their stoichiometric relation in each of these individual organelles. In contrast to earlier reports, we show that individual members exist as dimers and monomers and that the ratio between these two forms depends on both the organelle investigated and the p24 protein. We find unequal quantities, with p23 and p27 building up concentration gradients, ruling out a simple 1:1 stoichiometry. In addition, we show differential cycling of individual p24 members. These data point to a complex and dynamic system of altering dimerizations of the family members.     

Cloning and expression of the human p8, a nuclear protein with mitogenic activity.

We have previously identified a new rat mRNA, provisionally named p8, which showed a strong, but transient, induction in the pancreas in response to acute pancreatitis. We report here the cloning and sequencing of the human p8 cDNA. The human p8 polypeptide is 82 amino acids long and shows an overall identity of 74% with the rat counterpart. The complete structure of the p8 gene was also established. The p8 gene comprises three exons separated by two introns and the gene was mapped to chromosome 16. Analysis of the p8 primary structure suggested the presence of a bipartite motif of nuclear targeting, indicating that p8 may function within the nucleus. This presumption has been confirmed by transfection of COS-7 cells with the p8 cDNA followed by immunostaining with specific antibodies. p8 mRNA expression is induced in some, but not all, cells by serum starvation and ceramide. To analyze the p8 function, we stably transfected HeLa cells with a p8 expression plasmid, and measured their growth and their sensitivity to apoptosis. Response to TNF alpha and staurosporine-induced apoptosis was not modified by p8 expression. However, cells expressing p8 grew significantly more rapidly.     

p73 is expressed in human thymic epithelial cells.

The thymus is a heterogeneous immune organ in which immature T-cells develop and eventually specialize to make certain immune responses of their own. Among various types of stromal cells in the thymus, thymic epithelial cells (TECs) have a crucially important function for presenting self-antigens and secreting cytokines to thymocytes for their maturation into T-cells. In this study we show that the p73 gene, a homologue of the tumor suppressor gene p53, was expressed in the nucleus of the human TEC in vivo and in TEC lines in vitro. Because p73 has the capacity to be a transactivator like p53, it may contribute to T-cell development in the context of TEC biology as regulated in the cell cycle and apoptosis.     

Pex8p: an intraperoxisomal organizer of the peroxisomal import machinery

Peroxisomes transport folded and oligomeric proteins across their membrane. Two cytosolic import receptors, Pex5p and Pex7p, along with approximately 12 membrane-bound peroxins participate in this process. While interactions among individual peroxins have been described, their organization into functional units has remained elusive. We have purified and defined two core complexes of the peroxisomal import machinery: the docking complex comprising Pex14p and Pex17p, with the loosely associated Pex13p, and the RING finger complex containing Pex2p, Pex10p, and Pex12p. Association of both complexes into a larger import complex requires Pex8p, an intraperoxisomal protein. We conclude that Pex8p organizes the formation of the larger import complex from the trans side of the peroxisomal membrane and thus might enable functional communication between both sides of the membrane.