The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitro

The integration of viral DNA into the host cell chromosome is an essential feature of the retroviral life cycle. The integration reaction requires cis-acting sequences at the ends of linear viral DNA and a trans-acting product of the pol gene, the integration protein (IN). Previously, we demonstrated that avian sarcoma-leukosis virus (ASLV) IN is able to carry out the first step in the integration process in vitro: nicking of the ends of linear viral DNA. In this paper, using two independent assays, we demonstrate that IN, alone, is sufficient to carry out the second step: cleavage and joining to the target DNA. These results demonstrate that the retroviral IN protein is an integrase.     

Ral is both necessary and sufficient for the inhibition of myeloid differentiation mediated by Ras

Hyperactivation of Ras is one of the most common abnormalities in acute myeloid leukemia. In experimental models, Ras inhibits myeloid differentiation, which is characteristic of leukemia; however, the mechanism through which it disrupts hematopoiesis is poorly understood. In multipotent FDCP-mix cells, Ras inhibits terminal neutrophil differentiation, thereby indefinitely extending their proliferative potential. Ras also strongly promotes the sensitivity of these cells to granulocyte-macrophage colony-stimulating factor (GM-CSF). Using this model, we have dissected the signaling elements downstream of Ras to determine their relative contribution to the dysregulation of hematopoiesis. Cells expressing Ras mutants selectively activating Raf (Ras*T35S) or phosphatidylinositol 3-kinase (Ras*Y40C) did not significantly affect differentiation or proliferative capacity, whereas Ras*E37G (which selectively activates RalGEFs) perpetuated proliferation and blocked neutrophil development in a manner similar to that of Ras. Correspondingly, expression of constitutively active versions of these effectors confirmed the overriding importance of Ral guanine nucleotide exchange factors. Cells expressing Ras demonstrated hyperactivation of Ral, which itself was able to exactly mimic the phenotype of Ras, including hypersensitivity to GM-CSF. Conversely, dominant negative Ral promoted spontaneous neutrophil development. Ral, in turn, appears to influence differentiation through multiple effectors. These data show, for the first time, the importance of Ral in regulating differentiation and self-renewal in hematopoietic cells.     

The signal Peptide of a vacuolar protein is necessary and sufficient for the efficient secretion of a cytosolic protein

A cytosolic pea (Pisum sativum) seed albumin (ALB) and a chimeric protein (PHALB) consisting of the signal peptide and first three amino acids of phytohemagglutinin (PHA) and the amino acid sequence of ALB were expressed in parallel suspension cultures of tobacco (Nicotiana tabacum) cells and their intracellular fates examined. PHALB was efficiently secreted by the cells whereas ALB remained intracellular. These experiments show that the information contained in the signal peptide of a vacuolar protein is both necessary and sufficient for efficient secretion, and define secretion as a default or bulk-flow pathway. Entry into the secretory pathway was accompanied by glycosylation and the efficient conversion of the high mannose glycans into complex glycans indicating that transported glycoproteins do not need specific recognition domains for the modifying enzymes in the Golgi. Tunicamycin depressed the accumulation of the unglycosylated polypeptide in the culture medium much less than the accumulation of other glycoproteins. We interpret this as evidence that glycans on proteins that are not normally glycosylated do not have the same function of stabilizing and protecting the polypeptide as on natural glycoproteins.     

A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability.

The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.     

The C-terminal globular domain of the prion protein is necessary and sufficient for import into the endoplasmic reticulum

The mammalian prion protein (PrP) is composed of an unstructured flexible N-terminal region and a C-terminal globular domain. We examined the import of PrP into the endoplasmic reticulum (ER) of neuronal cells and show that information present in the C-terminal globular domain is required for ER import of the N terminus. N-terminal fragments of PrP, devoid of structural domains located in the C terminus, remained in the cytosol with an uncleaved signal peptide and were rapidly degraded by the proteasome. Conversely, the separate C-terminal domain of PrP, comprising the highly ordered helix 2-loop-helix 3 motif, was entirely imported into the ER. As a consequence, two PrP mutants linked to inherited prion disease in humans, PrP-W145Stop and PrP-Q160Stop, were partially retained in the cytosol. The cytosolic fraction was characterized by an uncleaved N-terminal signal peptide and was degraded by the proteasome. Our study identified a new regulatory element in the C-terminal globular domain of PrP necessary and sufficient to promote import of PrP into the ER.     

Repression of c-myc is necessary but not sufficient for terminal differentiation of B lymphocytes in vitro

The importance of c-myc as a target of the Blimp-1 repressor has been studied in BCL-1 cells, in which Blimp-1 is sufficient to trigger terminal B-cell differentiation. Our data show that Blimp-1-dependent repression of c-myc is required for BCL-1 differentiation, since constitutive expression of c-Myc blocked differentiation. Furthermore, ectopic expression of cyclin E mimicked the effects of c-Myc on both proliferation and differentiation, indicating that the ability of c-Myc to drive proliferation is responsible for blocking BCL-1 differentiation. However, inhibition of c-Myc by a dominant negative form was not sufficient to drive BCL-1 differentiation. Thus, during Blimp-1-dependent plasma cell differentiation, repression of c-myc is necessary but not sufficient, demonstrating the existence of additional Blimp-1 target genes.     

The lymphotoxin-beta receptor is necessary and sufficient for LIGHT-mediated apoptosis of tumor cells

LIGHT is a tumor necrosis factor (TNF) ligand superfamily member, which binds two known cellular receptors, lymphotoxin-beta receptor (LTbetaR) and the herpesvirus entry mediator (HveA). LIGHT is a homotrimer that activates proapoptotic and integrin-inducing pathways. Receptor binding residues via LIGHT were identified by introducing point mutations in the A' --> A" and D --> E loops of LIGHT, which altered binding to LTbetaR and HveA. One mutant of LIGHT exhibits selective binding to HveA and is inactive triggering cell death in HT29.14s cells or induction of ICAM-1 in fibroblasts. Studies with HveA- or LTbetaR-specific antibodies further indicated that HveA does not contribute, either cooperatively or by direct signaling, to the death pathway activated by LIGHT. LTbetaR, not HveA, recruits TNF receptor-associated factor-3 (TRAF3), and LIGHT-induced death is blocked by a dominant negative TRAF3 mutant. Together, these results indicate that TRAF3 recruitment propagates death signals initiated by LIGHT-LTbetaR interaction and implicates a distinct biological role for LIGHT-HveA system.     

Stat6 is necessary and sufficient for IL-4's role in Th2 differentiation and cell expansion

IL-4 plays a critical role in the differentiation of TCR-stimulated naive CD4 T cells to the Th2 phenotype. In response to IL-4, the IL-4R activates a set of phosphotyrosine binding domain-containing proteins, including insulin receptor substrate 1/2, Shc, and IL-4R interacting protein, as well as Stat6. Stat6 has been shown to be required for Th2 differentiation. To determine the roles of the phosphotyrosine binding adaptors in Th2 differentiation, we prepared a retrovirus containing a mutant of the human (h)IL-4R alpha-chain, Y497F, which is unable to recruit these adaptors. The mutant hIL-4Ralpha, as well as the wild-type (WT) hIL-4Ralpha, was introduced into naive CD4 T cells. Upon hIL-4 stimulation, Y497F worked as well as the WT hIL-4Ralpha in driving Th2 differentiation, as measured by Gata3 up-regulation and IL-4 production. Furthermore, IL-4-driven cell expansion was also normal in the cells infected with Y497F, although cells infected with Y497F were not capable of phosphorylating insulin receptor substrate 2. These results suggest that the signal pathway mediated by Y497 is dispensable for both IL-4-driven Th2 differentiation and cell expansion. Both WT and Y497F hIL-4Ralpha lose the ability to drive Th2 differentiation and cell expansion in Stat6-knockout CD4 T cells. A constitutively activated form of Stat6 introduced into CD4 T cells resulted in both Th2 differentiation and enhanced cell expansion. Thus, activated Stat6 is necessary and sufficient to mediate both IL-4-driven Th2 differentiation and cell expansion in CD4 T cells.     

Dimerization of the testis brain RNA-binding protein (translin) is mediated through its C-terminus and is required for DNA- and RNA-binding.

Testis brain-RNA-binding protein (TB-RBP) is a single-stranded DNA- and RNA-binding protein that is involved in chromosomal translocations, mRNA transport and translational regulation. Here we show from in vitro and in vivo protein binding studies that TB-RBP dimers are the minimum structural unit needed for DNA- and RNA-binding. Truncation studies demonstrate that the C-terminus of 55 amino acids of TB-RBP is essential, but not sufficient for DNA- or RNA-binding, and deletion of the leucine zipper motif in the C-terminus abolishes DNA- and RNA-binding. Changing cysteine 225 in the C-terminus to alanine does not significantly reduce DNA- or RNA-binding, but reduces the stability of the dimer. We conclude that the leucine zipper motif is required to maintain two molecules of TB-RBP as a dimer which is stabilized by a disulfide bond involving cysteine 225.     

Visinin-like protein (VILIP) is a neuron-specific calcium-dependent double-stranded RNA-binding protein.

Double-stranded RNA-binding proteins function in regulating the stability, translation, and localization of specific mRNAs. In this study, we have demonstrated that the neuron-specific, calcium-binding protein, visinin-like protein (VILIP) contains one double-stranded RNA-binding domain, a protein motif conserved among many double-stranded RNA-binding proteins. We showed that VILIP can specifically bind double-stranded RNA, and this interaction specifically requires the presence of calcium. Mobility shift studies indicated that VILIP binds double-stranded RNA as a single protein-RNA complex with an apparent equilibrium dissociation constant of 9.0 x 10(-6) M. To our knowledge, VILIP is the first double-stranded RNA-binding protein shown to be calcium-dependent. Furthermore, VILIP specifically binds the 3'-untranslated region of the neurotrophin receptor, trkB, an mRNA localized to hippocampal dendrites in an activity-dependent manner. Given that VILIP is also expressed in the hippocampus, these data suggest that VILIP may employ a novel, calcium-dependent mechanism to regulate its binding to important localized mRNAs in the central nervous system.     

A C-terminal fragment of an intron-encoded maturase is sufficient for promoting group I intron splicing

Group I introns often encode proteins that catalyze site-specific DNA hydrolysis. Some of these proteins have acquired the ability to promote splicing of their cognate intron, but whether these two activities reside in different regions of the protein remains obscure. A crystal structure of I-AniI, a dual function intron-encoded protein, has shown that the protein has two pseudo-symmetric domains of equal size. Each domain contacts its DNA substrate on either side of two cleavage sites. As a first step to identify the RNA binding surface, the N- and C-terminal domains of I-AniI were separately expressed and tested for promoting the splicing of the mitochondrial (mt) COB pre-RNA. The N-terminal protein showed no splicing activation or RNA binding, suggesting that this domain plays a minimal role in activity or is improperly folded. Remarkably, the 16-kDa C-terminal half facilitates intron splicing with a rate similar to that of the full-length protein. Both the C-terminal fragment and full-length proteins bind tightly to the COB intron. RNase footprinting shows that the C-terminal and full-length proteins bind to the same regions and induce the same conformational changes in the COB intron. Together, these results show that the C-terminal fragment of I-AniI is necessary and sufficient for maturase activity and suggests that I-AniI acquired splicing function by utilizing a relatively small protein surface that likely represents a novel RNA binding motif. This fragment of I-AniI represents the smallest group I intron splicing cofactor described to date.     

Helix 3 is necessary and sufficient for prion protein's anti-Bax function

To identify the structural elements of the prion protein (PrP) necessary for its protective function against Bcl-2 associated protein X (Bax), we performed structure–function analyses of the anti-Bax function of cytosolic PrP (CyPrP) in MCF-7 cells. Deletions of 1, 2, or 3 N-terminal Bcl-2 homology domain 2-like octapeptide repeats (BORs), but not deletion of all four BORs, abolish CyPrPs anti-Bax function. Deletion of α-helix 3 (PrP23–199) or further C-terminal deletions of α-helix 1 and 2, and β-strand 1 and 2 (PrP23–172, PrP23–160, PrP23–143, and PrP23–127) eliminates CyPrPs protection against Bax-mediated cell death. The substitution of helix 3 amino acid residues K204, V210, and E219 by proline inhibits the anti-Bax function of CyPrP. The substitution of K204, but not V210 and E219, by alanine residues also prevents CyPrPs anti-Bax function. Expression of PrPs helix 3 displays anti-Bax activity in MCF-7 cells and in human neurons. Together, these results indicate that although the BOR domain has an influence on PrPs anti-Bax function, the helix 3 is necessary and sufficient for the anti-Bax function of CyPrP. Identification of helix 3 as the structural element for the anti-Bax function thus provides a molecular target to modulate PrPs anti-Bax function in cancer and neurodegeneration.     

The translocation domain in trimeric autotransporter adhesins is necessary and sufficient for trimerization and autotransportation

Trimeric autotransporter adhesins (TAAs) comprise one of the secretion pathways of the type V secretion system. The mechanism of their translocation across the outer membrane remains unclear, but it most probably occurs by the formation of a hairpin inside the β-barrel translocation unit, leading to transportation of the passenger domain from the C terminus to the N terminus through the lumen of the β-barrel. We further investigated the phenomenon of autotransportation and the rules that govern it. We showed by coexpressing different Escherichia coli immunoglobulin-binding (Eib) proteins that highly similar TAAs could form stochastically mixed structures (heterotrimers). We further investigated this phenomenon by coexpressing two more distantly related TAAs, EibA and YadA. These, however, did not form heterotrimers; indeed, coexpression was lethal to the cells, leading to elimination of one or another of the genes. However, substituting in either protein the barrel of the other one so that the barrels were identical led to formation of heterotrimers as for Eibs. Our work shows that trimerization of the β-barrel, but not the passenger domain, is necessary and sufficient for TAA secretion while the passenger domain is not.     

Lactococcus lactis YfiA is necessary and sufficient for ribosome dimerization

Dimerization and inactivation of ribosomes in Escherichia coli is a two‐step process that involves the binding of ribosome modulation factor (RMF) and hibernation promotion factor (HPF). Lactococcus lactis MG1363 expresses a protein, YfiA^Ll, which associates with ribosomes in the stationary phase of growth and is responsible for dimerization of ribosomes. We show that full‐length YfiA^Ll is necessary and sufficient for ribosome dimerization in L. lactis but also functions heterologously in vitro with E. coli ribosomes. Deletion of the yfiA gene has no effect on the growth rate but diminishes the survival of L. lactis under energy‐starving conditions. The N‐terminal domain of YfiA^Ll is homologous to HPF from E. coli, whereas the C‐terminal domain has no counterpart in E. coli. By assembling ribosome dimers in vitro, we could dissect the roles of the N‐ and C‐terminal domains of YfiA^Ll. It is concluded that the dimerization and inactivation of ribosomes in L. lactis and E. coli differ in several cellular and molecular aspects. In addition, two‐dimensional maps of dimeric ribosomes from L. lactis obtained by single particle electron microscopy show a marked structural difference in monomer association in comparison to the ribosome dimers in E. coli.     

The gene 10 (UL49.5) product of equine herpesvirus 1 is necessary and sufficient for functional processing of glycoprotein M

The functional cooperation of equine herpesvirus 1 (EHV-1) glycoprotein M (gM) and the gene 10 (UL49.5) product was analyzed. Transient-transfection experiments using gM and UL49.5 expression plasmids as well as RK13 cell lines constitutively expressing UL49.5 (RK49.5) or gM (RKgM) demonstrated that the endo-beta-N-acetylglucosaminidase H (endo H)-resistant mature form of gM was detectable only after coexpression of the two proteins. Deletion of the EHV-1 UL49.5-homologous gene 10 in strain KyA resulted in a small-plaque phenotype and up to 190-fold-reduced virus titers. The growth defects of the mutant KyA Delta 49.5 virus, which were very similar to those of a gM-negative KyA virus, could be completely compensated for by growth of the mutant virus on RK49.5 cells or by repairing the deletion of gene 10 in the revertant virus KyA Delta 49.5R. Analysis of cells infected with the UL49.5-negative EHV-1 demonstrated that gM was not transported to the trans-Golgi network in the absence of the UL49.5 product. In contrast, gM was efficiently transported and processed to the endo H-resistant mature form in KyA Delta 49.5-infected RK49.5 cells. Furthermore, radioimmunoprecipitation experiments demonstrated that gM maturation was observed only if a 10,000-M(r) protein was coprecipitated with gM in KyA- or KyA Delta 49.5R-infected cells or virions. This protein was absent in cells infected with Ky Delta 49.5 or KyA Delta gM, suggesting that it was the EHV-1 UL49.5 product. Taken together, our results demonstrate that the expression of the EHV-1 UL49.5 product is necessary and sufficient for gM processing and that it is required for efficient virus replication.     

Polypyrimidine-tract binding protein (PTB) is necessary, but not sufficient, for efficient internal initiation of translation of human rhinovirus-2 RNA

Initiation of translation of the animal picornavirus RNAs is via a mechanism of direct internal ribosome entry, which requires a substantial segment of the viral 5'-untranslated region, generally known as the IRES (for "internal ribosome entry site"). Because, however, translation of the RNAs of members of the enterovirus, and more especially, the rhinovirus subgroups of the Picornaviridae is restricted in the reticulocyte lysate system, but is greatly stimulated by the addition of HeLa cell extracts, the implication is that, in these cases, internal initiation also requires cellular trans-acting factors that are more abundant in HeLa cell extracts than in rabbit reticulocytes. This was used as the basis of a functional assay for the purification of the HeLa cell factors required for translation dependent on the human rhinovirus-2 (HRV) IRES. There are two such HeLa cell factors separable by ion-exchange chromatography, each of which is individually active in the assay, although their combined effect is synergistic. One of these activities is shown to be polypyrimidine-tract binding protein (PTB) on the grounds that (1) the activity copurifies to homogeneity with PTB and (2) recombinant PTB expressed in Escherichia coli stimulates HRV IRES-dependent translation with a specific activity similar to that of the purified HeLa cell factor. Furthermore, it is shown that recombinant PTB also stimulates the translation of RNAs bearing the poliovirus type 1 (Mahoney) IRES.     

The carboxy-terminus of the hepatitis B virus X protein is necessary and sufficient for the activation of hypoxia-inducible factor-1alpha

Hepatitis B virus X protein (HBx) of the hepatitis B virus is strongly implicated in angiogenesis and metastasis during hepatocarcinogenesis. Previously, we reported that HBx enhances activity of hypoxia-inducible factor-1alpha (HIF-1alpha), a potent transactivator that induces angiogenic factors. Here, we delineate the structural region of HBx that potentiates HIF-1alpha. The carboxy-terminus of HBx increased the stability of HIF-1alpha protein, probably through inhibiting interaction with von Hippel-Lindau protein. Further, the carboxy-terminus of HBx enhanced the transactivation function of HIF-1alpha by enhancing its association with CREB binding protein (CBP). Finally, we demonstrated the physical association of HBx with the basic helix-loop-helix/PER-ARNT-SIM domain, the inhibitory domain, and the carboxy-terminal transactivation domain of HIF-1alpha in vivo.