Helix 6 of tBid is necessary but not sufficient for mitochondrial binding activity

The apoptosis effector Bid regulates cell death at the level of mitochondrial cytochrome c efflux. Bid consists of 8 -helices (designated H1 through H8, respectively) and is a soluble cytosolic protein in its native state. Proteolysis of the N-terminus (encompassing H1 and H2) of Bid yields activated tBid (truncated Bid), which translocates to the mitochondria and induces the efflux of cytochrome c. Here, we demonstrate that helix H6 of tBid is necessary, albeit not sufficient, for mitochondrial binding. In particular, a 33 amino acid long domain, which encompassed H6 and H7, behaved as the minimum domain in tBid that was sufficient for mitochondrial binding. Unexpectedly, the hydrophobic surface of these helices could be mutated without altering the binding activity of the domain, implying that the secondary structure of the helices may be the key determinant of binding. These experiments expand our mechanistic understanding of the apoptotic regulator, tBid.     

A Rep recognition sequence is necessary but not sufficient for nicking of DNA by adeno-associated virus type-2 Rep proteins

The strand-specific, site-specific endonuclease (nicking) activity of the Rep68 and Rep78 (Rep68/78) proteins of adeno-associated virus type 2 (AAV) is involved in AAV replication, and appears to be involved in AAV site-specific integration. Rep68/78 cuts within the inverted terminal repeats (ITRs) of the AAV genome and in the AAV preferred integration locus on human chromosome 19 (AAVS1). The known endonuclease cut sites are 11-16 bases away from the primary binding sites, known as Rep recognition sequences (RRSs). A linear, double-stranded segment of DNA, containing an RRS and a cut site, has previously been shown to function as a substrate for the Rep68/78 endonuclease activity. We show here that mutation of the Rep recognition sequence, within such a DNA segment derived from the AAV ITRs, eliminates the ability of this substrate to be cleaved detectably by Rep78. Rep78 nicks the RRS-containing site from AAVS1 about half as well as the linear ITR sequence. Eighteen other RRS-containing sequences found in the human genome, but outside AAVS1, are not cleaved by Rep78. These results may help to explain the specificity of AAV integration.     

Interaction with the Epstein-Barr virus helicase targets Zta to DNA replication compartments

Zta has a dual role in the Epstein-Barr virus (EBV) lytic cycle, acting as a key regulator of EBV lytic gene expression and also being essential for lytic viral DNA replication. Zta's replication function is mediated in part through interactions with the core viral replication proteins. We now show interaction between Zta and the helicase (BBLF4) and map the binding region to within amino acids (aa) 22 to 86 of the Zta activation domain. In immunofluorescence assays, green fluorescent protein (GFP)-tagged BBLF4 localized to the cytoplasm of transfected cells. Cotransfection of Zta resulted in translocation of BBLF4-GFP into the nucleus indicating interaction between these two proteins. However, Zta with a deletion of aa 24 to 86 was unable to mediate nuclear translocation of BBLF4-GFP. Results obtained with Zta variants carrying deletions across the aa 24 to 86 region indicated more than one contact site for BBLF4 within this domain, and this was reinforced by the behavior of the four-point mutant Zta (m22/26,74/75), which was severely impaired for BBLF4 interaction. Binding of BBLF4 to Zta was confirmed using GST affinity assays. In both cotransfection-replication assays and replication assays performed in EBV-positive P3HR1 cells, the Zta (m22/26,74/75) mutant was replication defective. In Zta-transfected D98-HR1 cells, replication compartments could be detected by immunofluorescence staining using anti-BMRF1 monoclonal antibody. Cells transfected with Zta variants that were defective for helicase binding still formed replication compartments, but Zta was excluded from these compartments. These experiments reveal a role for the Zta-helicase interaction in targeting Zta to sites of viral DNA replication.     

CENP-C is necessary but not sufficient to induce formation of a functional centromere.

CENP-C is an evolutionarily conserved centromeric protein. We have used the chicken DT40 cell line to test the idea that CENP-C is sufficient as well as necessary for the formation of a functional centromere. We have compared the effects of disrupting the localization of CENP-C with those of inducibly overexpressing the protein. Removing CENP-C from the centromere causes disassembly of the centromere protein complex and blocks cells at the metaphase-anaphase junction. Overexpressed CENP-C is associated with an increase in errors of chromosome segregation and inhibits the completion of mitosis. However, the excess CENP-C does not disrupt the native centromeres detectably and does not associate with another conserved centromere protein, ZW10. The distribution of the excess CENP-C changes during the cell cycle. In metaphase, the excess CENP-C coats the chromosome arms. At the metaphase-anaphase transition, the excess CENP-C clusters, and during interphase it is present in large bodies which form around pre-existing centromeres which are also clustered. These results indicate that CENP-C is necessary but not sufficient for the formation of a functional centromere and suggest that the structure of CENP-C may be regulated during the cell cycle.     

Deglycosylation is necessary but not sufficient for activation of proconcanavalin A.

Concanavalin A (ConA), one of the most studied plant lectins, is formed in jack bean (Canavalia ensiformis) seeds. ConA is synthesized as an inactive glycoprotein precursor proConA. Different processing events such as endoproteolytic cleavages, ligation of peptides and deglycosylation of the precursor are required to generate the different polypeptides constitutive of mature ConA. Among these events, deglycosylation of the prolectin appears as a key step in the lectin activation. The detection of deglycosylated proConA in immature jack bean seeds indicates that endoproteolytic cleavages are not prerequisite for its deglycosylation. Both the structure of the lectin precursor N-glycans Man8-9GlcNAc2 and the capacity of Endo H to cleave these oligosaccharide from native proConA in vitro favoured Endo H-type glycosidases as candidates for proConA deglycosylation in planta. Evidence for pH-dependent changes in the prolectin folding were obtained from analysis of the N-glycan accessibility and activation of the deglycosylated lectin precursor in acidic conditions. These data are consistent with the observation that both deglycosylation and acidification of the pH are the minimum requirements to convert the inactive precursor into an active lectin.     

Heparin binding is necessary, but not sufficient, for fibronectin aggregation. A fluorescence polarization study

Analysis of parameters governing heparin binding to fibronectin indicates that heparin binding is a necessary, but insufficient, condition for fibronectin cryoprecipitation. Heparin binding to fibronectin is a rapid, readily reversible event which can occur under several conditions which prohibit fibronectin cryoprecipitation. While cryoprecipitation of fibronectin is abolished at temperatures in excess of 10 degrees C, appreciable heparin binding to fibronectin does occur even at 40 degrees C. While increasing ionic strength and pH inhibit both heparin binding and cryoprecipitation of fibronectin, heparin binding can still occur at high ionic strengths and pH values which completely abolish cryoprecipitation. Scatchard analysis of fluorescent polarization data reveals a biphasic heparin binding curve with high and low affinity Kd values of 3.5 X 10(-8) and 10(-6) M, respectively. In contrast to heparin binding, fibronectin aggregation is a cooperative phenomenon. Fibronectin cryoprecipitation is greatly reduced at temperatures above 10 degrees C, at pH values above pH 10, and at ionic strengths above 0.3 M. Thus, heparin binding and protein aggregation are separate events which occur during fibronectin cryoprecipitation. Results obtained here via fluorescence polarization in conjunction with other physical measurements suggest that a decrease in flexibility of the fibronectin molecule is associated with the protein aggregation step of cryoprecipitation. The role of heparin in the mechanism of fibronectin cryoprecipitation is discussed.     

E2-c-Cbl recognition is necessary but not sufficient for ubiquitination activity

The E2 ubiquitin-conjugating enzymes UbcH7 and UbcH5B both show specific binding to the RING (really interesting new gene) domain of the E3 ubiquitin-protein ligase c-Cbl, but UbcH7 hardly supports ubiquitination of c-Cbl and substrate in a reconstituted system. Here, we found that neither structural changes nor subtle differences in the E2-E3 interaction surface are possible explanations for the functional specificity of UbcH5B and UbcH7 in their interaction with c-Cbl. The quick transfer of ubiquitin from the UbcH5B∼Ub thioester to c-Cbl or other ubiquitin acceptors suggests that UbcH5B might functionally be a relatively pliable E2 enzyme. In contrast, the UbcH7∼Ub thioester is too stable to transfer ubiquitin under our assay conditions, indicating that UbcH7 might be a more specific E2 enzyme. Our results imply that the interaction specificity between c-Cbl and E2 is required but not sufficient for transfer of ubiquitin to potential targets.     

The direct binding of the catalytic subunit of protein phosphatase 1 to the PKR protein kinase is necessary but not sufficient for inactivation and disruption of enzyme dimer formation

The PKR protein kinase is among the best-studied effectors of the host interferon (IFN)-induced antiviral and antiproliferative response system. In response to stress signals, including virus infection, the normally latent PKR becomes activated through autophosphorylation and dimerization and phosphorylates the eIF2alpha translation initiation factor subunit, leading to an inhibition of mRNA translation initiation. While numerous virally encoded or modulated proteins that bind and inhibit PKR during virus infection have been studied, little is known about the cellular proteins that counteract PKR activity in uninfected cells. Overexpression of PKR in yeast also leads to an inhibition of eIF2alpha-dependent protein synthesis, resulting in severe growth suppression. Screening of a human cDNA library for clones capable of counteracting the PKR-mediated growth defect in yeast led to the identification of the catalytic subunit (PP1(C)) of protein phosphatase 1alpha. PP1(C) reduced double-stranded RNA-mediated auto-activation of PKR and inhibited PKR transphosphorylation activities. A specific and direct interaction between PP1(C) and PKR was detected, with PP1(C) binding to the N-terminal regulatory region regardless of the double-stranded RNA-binding activity of PKR. Importantly, a consensus motif shared by many PP1(C)-interacting proteins was necessary for PKR binding to PP1(C). The PKR-interactive site was mapped to a C-terminal non-catalytic region that is conserved in the PP1(C)2 isoform. Indeed, co-expression of PP1(C) or PP1(C)2 inhibited PKR dimer formation in Escherichia coli. Interestingly, co-expression of a PP1(C) mutant lacking the catalytic domain, despite retaining its ability to bind PKR, did not prevent PKR dimerization. Our findings suggest that PP1(C) modulates PKR activity via protein dephosphorylation and subsequent disruption of PKR dimers.     

Interaction of leukocyte integrins with ligand is necessary but not sufficient for function

The leukocyte integrins (CD11/CD18 or beta 2-type integrins) are expressed exclusively on leukocytes and participate in many adhesion- dependent functions (Arnaout, M.A. 1990. Blood. 75:1037-1050; Springer, T. A. 1990. Nature. (Lond.) 346:425-434; Dustin, M. L., and T. S. Springer. 1991. Annu. Rev. Immunol. 9:27-66). The avidity of leukocyte integrin binding to their ligands or counter-receptors is dependent upon response to intracellular signals (Wright, S. D., and B. C. Meyer. 1986. J. Immunol. 136:1759-1764; Dustin, M. A., and T. S. Springer. 1989. Nature (Lond.). 341:619-624). We have investigated the effects of a novel mAb (mAb 24) which defines a leukocyte integrin alpha subunit epitope that is Mg(2+)-dependent and may be used as a "reporter" of the activation state of these receptors (Dransfield, I., and N. Hogg. 1989. EMBO (Eur. Mol. Biol. Organ) J. 8:3759-3765; Dransfield, I., A.-M. Buckle, and N. Hogg. 1990. Immunol. Rev. 114:29-44; Dransfield, I., C. Cabanas, A. Craig, and N. Hogg. 1992. J. Cell Biol.) Data is presented to show that this mAb inhibits monocyte-dependent, antigen-specific T cell proliferation and IL-2-activated natural killer cell assays which are both dependent on lymphocyte function-associated antigen-1 (LFA-1), and complement receptor type 3 (CR3)-mediated neutrophil chemotaxis to f-Met-Leu-Phe. This inhibitory effect is not caused by the prevention of receptor/ligand binding because LFA-1/ICAM-1, LFA-1/ICAM-2,3 and CR3/iC3b interactions are, under activating conditions, promoted rather than blocked by mAb 24. As it does not interfere with mitogen- stimulated T cell proliferation, it is unlikely that mAb 24 transduces a "negative" or antiproliferative signal to the T cells to which it is bound. Using a model system of transient activation of LFA-1, we have found that mAb 24 prevents "deadhesion" of receptor/ligand pairs, possibly locking leukocyte integrins in an "active" conformation. It is speculated that inhibition of leukocyte integrin function by this mAb reflects the necessity for dynamic leukocyte integrin/ligand interactions.     

The C-terminal region of ammodytoxins is important but not sufficient for neurotoxicity.

Ammodytoxins (Atxs) are presynaptically acting snake venom phospholipase A2 (PLA2) toxins the molecular mechanism of whose neurotoxicity is not completely understood. Two chimeric PLA2s were prepared by replacing the C-terminal part of a nontoxic venom PLA2, ammodytin I2, with that of AtxA(K108N). The chimeras were not toxic, but were able to bind strongly to an Atxs-specific neuronal receptor, R25. They also showed an increased affinity for calmodulin, a recently identified high-affinity binding protein for Atxs, whereas affinity for a neuronal M-type PLA2 receptor remained largely unchanged. The results show that the C-terminal region of Atxs, which is known to be involved in neurotoxicity, is critical for their interaction with specific binding proteins, but that some other part of the molecule also contributes to toxicity.