Interaction of FIE,a Polycomb protein,with pRb: a possible mechanism regulating endosperm development

Inactivation of the Arabidopsis protein FERTILIZATION INDEPENDENT ENDOSPERM (FIE) induces division of the central cell of the embryo sac, leading to endosperm development in the absence of fertilization. The mechanism whereby FIE regulates this process is unknown. We postulated that activation of central cell division in fie mutant plants might involve the retinoblastoma protein (pRb), a cell cycle regulatory element. Pull-down and surface plasmon resonance assays demonstrated that FIE interacts in-vitro with the pRb homologues from Arabidopsis (AtRb), maize (ZmRb) and human (HuRb). The interaction of FIE with ZmRB and HuRb in the yeast two-hybrid system supports the possibility that a FIE-pRb interaction may occur also in planta. Mutational analysis showed that this interaction does not occur via the LxCxE motif of the FIE protein nor via the pocket B domain of pRb. These results suggest that FIE may inhibit premature division of the central cell of the embryo sac, at least partly, through interaction with pRb, and suppression of pRb-regulated genes.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by R. G. Herrmann     

Identification and characterization of a novel human cDNA encoding a 21 kDa pRb-associated protein

The retinoblastoma protein (pRb) functions as a critical master regulator in cell cycle regulation, which is an important cell-regulatory process, through its interaction with various cellular proteins. Using the C-terminus of human pRb and the yeast two-hybrid system, a novel protein named RBP21 that contains 187 amino acid residues with a calculated molecular weight of 21 kDa was identified as a pRb-binding protein. Sequence analysis indicates that RBP21 shares homology with other retinoblastoma-binding proteins in the pRb-binding motif LxCxE at the C-terminal region. In vitro specific interaction between pRb and RBP21 was confirmed using in vitro translation products. When overexpressed in COS-7 cells, RBP21 could co-immunoprecipitate with pRb. This interaction requires the LxCxE motif of RBP21 and the entire pocket region of pRb. Each point mutation of the conserved amino acid residues in pRb-binding motif of RBP21 abolished its specific interaction with pRb. RH mapping result showed that this novel gene was mapped to chromosome region 15q21.1-21.3. Northern blot analysis suggested that RBP21 was widely expressed in various human tissues and cancer cell lines. When expressed in HeLa cells as a green fluorescent protein fusion, RBP21 was distributed throughout the cell.     

Understanding the targeting of the RB family proteins by viral oncoproteins to defeat their oncogenic machinery

The retinoblastoma (RB) family consists of three genes, RB1, RBL1, and RBL2, that code for the pRb, p107, and pRb2/p130 proteins, respectively. All these factors have pivotal roles in controlling fundamental cellular mechanisms such as cell cycle, differentiation and apoptosis. The founder and the most investigated RB family protein is pRb, which is considered to be the paradigm of tumor suppressors. However, p107 and pRb2/p130 clearly display a high degree of structural and functional homology with pRb. Interestingly, these factors were first identified as physical targets of the Adenovirus E1A oncoprotein. Indeed, RB family proteins are the most important and widely investigated targets of small DNA virus oncoproteins, such as Adenovirus E1A, human papillomavirus E7 and Simian virus 40 large T antigen. By interacting with pRb and with other RB family members, these oncoproteins neutralize their growth suppressive properties, thus stimulating proliferation of the infected cells, de‐differentiation, and resistance to apoptosis. All these acquired features strongly favor the rise and selection of immortalized and mutation‐prone cells, leading to a higher propensity in undergoing transformation. Our present work aims to illustrate and delve into these protein–protein interactions. Considering that these viral oncoproteins are dispensable for normal cellular functions, they can create “oncogene addiction” in the infected/transformed cells. This makes the possibility to dismantle these interactions extremely attractive, thus promoting the development of highly specific smart molecules capable of targeting only the infected/transformed cells that express these viral factors. J. Cell. Physiol. 228: 285–291, 2013. © 2012 Wiley Periodicals, Inc.     

Molluscum contagiosum virus interleukin-18 (IL-18) binding protein is secreted as a full-length form that binds cell surface glycosaminoglycans through the C-terminal tail and a furin-cleaved form with only the IL-18 binding domain

Some poxviruses and their mammalian hosts encode homologous proteins that bind interleukin-18 (IL-18) with high affinity and inhibit IL-18-mediated immune responses. MC54L, the IL-18 binding protein of the human poxvirus that causes molluscum contagiosum, is unique in having a C-terminal tail of nearly 100 amino acids that is dispensable for IL-18 binding. When recombinant MC54L was expressed and purified via a C-terminal six-histidine tag, a shorter fragment was detected in addition to the full-length protein. This C-terminal fragment resulted from the cleavage of MC54L by cellular furin, as it was greatly diminished when furin was specifically inhibited or when a furin-deficient cell line was used for expression. Furthermore, the N- and C-terminal fragments of MC54L were generated by cleavage of the recombinant protein with furin in vitro. The furin cleavage site was mapped within a 32-amino-acid segment that is C terminal to the IL-18 binding domain. Full-length MC54L, but not the N-terminal IL-18 binding fragment, bound to cells and to purified heparin and other glycosaminoglycans that are commonly found on the cell surface and in the extracellular matrix. MC54L bound to heparin with a nanomolar K(d) and could simultaneously bind to IL-18. Their different glycosaminoglycan and cell binding properties may allow the long and short forms of MC54L to inactivate IL-18 near the site of infection and at more distal locations, respectively.     

Vesicular stomatitis viruses expressing wild-type or mutant M proteins activate apoptosis through distinct pathways

Vesicular stomatitis virus (VSV) induces apoptosis by at least two mechanisms. The viral matrix (M) protein induces apoptosis via the mitochondrial pathway due to the inhibition of host gene expression. However, in some cell types, the inhibition of host gene expression by VSV expressing wild-type (wt) M protein delays VSV-induced apoptosis, indicating that another mechanism is involved. In support of this, the recombinant M51R-M (rM51R-M) virus, expressing a mutant M protein that is defective in its ability to inhibit host gene expression, induces apoptosis much more rapidly in L929 cells than do viruses expressing wt M protein. Here, we determine the caspase pathways by which the rM51R-M virus induces apoptosis. An analysis of caspase activity, using fluorometric caspase assays and Western blots, indicated that each of the main initiator caspases, caspase-8, caspase-9, and caspase-12, were activated during infection with the rM51R-M virus. The overexpression of Bcl-2, an inhibitor of the mitochondrial pathway, or MAGE-3, an inhibitor of caspase-12 activation, did not delay apoptosis induction in rM51R-M virus-infected L929 cells. However, an inhibitor of caspase-8 activity significantly delayed apoptosis induction. Furthermore, the inhibition of caspase-8 activity prevented the activation of caspase-9, suggesting that caspase-9 is activated by cross talk with caspase-8. These data indicate that VSV expressing the mutant M protein induces apoptosis via the death receptor apoptotic pathway, a mechanism distinct from that induced by VSV expressing the wt M protein.     

Disparity between levels of in vitro neutralization of vaccinia virus by antibody to the A27 protein and protection of mice against intranasal challenge

Immunization with recombinant proteins may provide a safer alternative to live vaccinia virus for prophylaxis of poxvirus infections. Although antibody protects against vaccinia virus infection, the mechanism is not understood and the selection of immunogens is daunting as there are dozens of surface proteins and two infectious forms known as the mature virion (MV) and the enveloped virion (EV). Our previous studies showed that mice immunized with soluble forms of EV membrane proteins A33 and B5 and MV membrane protein L1 or passively immunized with antibodies to these proteins survived an intranasal challenge with vaccinia virus. The present study compared MV protein A27, which has a role in virus attachment to glycosaminoglycans on the cell surface, to L1 with respect to immunogenicity and protection. Although mice developed similar levels of neutralizing antibody after immunizations with A27 or L1, A27-immunized mice exhibited more severe disease upon an intranasal challenge with vaccinia virus. In addition, mice immunized with A27 and A33 were not as well protected as mice receiving L1 and A33. Polyclonal rabbit anti-A27 and anti-L1 IgG had equivalent MV-neutralizing activities when measured by the prevention of infection of human or mouse cells or cells deficient in glycosaminoglycans or by adding antibody prior to or after virus adsorption. Nevertheless, the passive administration of antibody to A27 was poorly protective compared to the antibody to L1. These studies raise questions regarding the basis for antibody protection against poxvirus disease and highlight the importance of animal models for the early evaluation of vaccine candidates.     

Antitumor protein (AP) from a mushroom induced apoptosis to transformed human keratinocyte by controlling the status of prb, c-MYC, cyclin E-cdk2, and p21WAF1 in the G1/S transition

Antitumor protein (AP) from a mushroom, induced the morphological changes typical to apoptosis such as nuclear condensation, aneuploidity, and DNA fragmentation at concentrations as low as 5-20 ng/ml to cancer cells. Molecular alterations related to cell cycle. Molecular alterations related to cell cycle, especially G1/S transition were investigated with a human keratinocyte transformed with oncoproteins, E6 and E7 of human pappiloma virus(HPV)-16. AP didn't alter significantly and oncosuppressor p53 level, but induced hyperphosphorylation of pRb. Time-dependent change of G1 cyclins, cdk2 and cdk4 after addition of AP showed that expression level of cdk inhibitors, INK4 family, and p27KIP1 did not altered, while that of p21WAF1 was downregulated.     

Induction of apoptosis in human leukemia cells by the CDK1 inhibitor CGP74514A

We have examined the effects of the CDK1 inhibitor CGP74514A on cell cycle- and apoptosis-related events in human leukemia cells. An 18-hr exposure to 5 microM CGP74514A induced mitochondrial damage (i.e., loss of Delta psi(m)) and apoptosis in multiple human leukemia cell lines (e.g., U937, HL-60, KG-1, CCRF-CEM, Raji, and THP; range 30-95%). In U937 cells, CGP74514A- induced apoptosis (5 microM) became apparent within 4 hr and approached 100% by 24 hr. The pan- caspase inhibitor Boc-fmk and the caspase-8 inhibitor lETD-fmk opposed CGP74514A-induced caspase-9 activation and PARP degradation, but not cytochrome c or Smac/DIABLO release. CGP74514A-mediated apoptosis was substantially blocked by ectopic expression of full-length Bel- 2, a loop-deleted mutant Bcl-2, and Bcl-x(L). CGP74514A treatment (5 microM; 18 hr) resulted in increased p21(CIP1) expression, p27(KIP1) degradation, diminished E2F1 expression, and dephosphorylation of p34(CDC2). It also induced early (i.e., within 2 hr) inhibition of CDK1 activity and dephosphorylation of pRb, followed by pRb degradation, but did not block pRb phosphorylation at CDK2- and CDK4- specific sites. These findings indicate that the selective CDK1 inhibitor, CGP74514A, induces complex changes in cell cycle-related proteins in human leukemia cells accompanied by extensive mitochondrial damage, caspase activation, and apoptosis.     

The MC159 protein from the molluscum contagiosum poxvirus inhibits NF-κB activation by interacting with the IκB kinase complex

Molluscum contagiosum virus (MCV) causes persistent neoplasms in healthy and immunocompromised people. Its ability to persist likely is due to its arsenal of viral immunoevasion proteins. For example, the MCV MC159 protein inhibits TNF-R1-induced NF-κB activation and apoptosis. The MC159 protein is a viral FLIP and, as such, possesses two tandem death effector domains (DEDs). We show in this article that, in human embryonic kidney 293 T cells, the expression of wild-type MC159 or a mutant MC159 protein containing the first DED (MC159 A) inhibited TNF-induced NF-κB, or NF-κB activated by PMA or MyD88 overexpression, whereas a mutant protein lacking the first DED (MC159 B) did not. We hypothesized that the MC159 protein targeted the IκB kinase (IKK) complex to inhibit these diverse signaling events. Indeed, the MC159 protein, but not MC159 B, coimmunoprecipitated with IKKγ. MC159 coimmunoprecipitated with IKKγ when using mouse embryonic fibroblasts that lack either IKKα or IKKβ, suggesting that the MC159 protein interacted directly with IKKγ. MC159-IKKγ coimmunoprecipitations were detected during infection of cells with either MCV isolated from human lesions or with a recombinant MC159-expressing vaccinia virus. MC159 also interacts with TRAF2, a signaling molecule involved in NF-κB activation. However, mutational analysis of MC159 failed to reveal a correlation between MC159-TRAF2 interactions and MC159's inhibitory function. We propose that MC159-IKK interactions, but not MC159-TRAF2 interactions, are responsible for inhibiting NF-κB activation.     

Differential regulation of retinoblastoma protein by hormonal and antihormonal agents in T47D breast cancer cells

Phosphorylation of the tumor suppressor protein, retinoblastoma (pRb), regulates the progression of the cell cycle. Previous work from this laboratory had shown that estradiol (E(2)) regulates tumor suppressor proteins, p53 and retinoblastoma in breast cancer cells. In the present study, we have examined the phosphorylation of pRB in T47D breast cancer cells following treatments with R5020 and antiprogestins. In growth medium containing serum depleted of endogenous steroids by charcoal treatment, pRb appeared mainly in its hypophosphorylated form. Addition of 10 nM R5020 to the culture medium caused hyperphosphorylation of pRb within 24 h, but the hypophosphorylated form of pRb began to accumulate after 72 h. Upon prolonged R5020 treatment (72-96 h), pRb was detected exclusively in its hypophosphorylated form. While treatment of cells with R5020 caused a transient increase in the level of cyclin D1, E(2) addition caused a sustained increase in the level of cyclin D1 consistent with its role in stimulating pRb phosphorylation. Antagonists of both estrogen receptor (ER) and progesterone receptor (PR) blocked the E(2) and R5020-induced pRb phosphorylation, respectively. These results suggest that R5020 induces pRb phosphorylation via a transient increased expression of cyclin D1, whereas E(2) treatment results in sustained expression of cyclin D1 and increased pRb phosphorylation. Furthermore, R5020 effects on pRb phosphorylation appear PR-mediated as no cross-antagonism of pRb phosphorylation was observed: the R5020 effects were blocked by RU486 and ZK98299, but not by the pure ER antagonist, ICI 182, 780 (ICI).     

Human cytomegalovirus pp71 stimulates cell cycle progression by inducing the proteasome-dependent degradation of the retinoblastoma family of tumor suppressors

The oncoproteins of the DNA tumor viruses, adenovirus E1A, simian virus 40 T antigen, and papillomavirus E7, each interact with the retinoblastoma family of tumor suppressors, leading to cell cycle stimulation, apoptosis induction, and cellular transformation. These proteins utilize a conserved LXCXE motif, which is also found in cellular proteins, to target the retinoblastoma family. Here, we describe a herpesvirus protein that shares a subset of the properties of the DNA tumor virus oncoproteins but maintains important differences as well. The human cytomegalovirus pp71 protein employs an LXCXD motif to attack the retinoblastoma family members and induce DNA synthesis in quiescent cells. pp71 binds to and induces the degradation of the hypophosphorylated forms of the retinoblastoma protein and its family members p107 and p130 in a proteasome-dependent manner. However, pp71 does not induce apoptosis and fails to transform cells. Thus, the similarities and differences in comparison to E1A, T antigen, and E7 make pp71 an interesting new tool with which to further dissect the role of the retinoblastoma/E2F pathway in cellular growth control and carcinogenesis.     

Regulation of retinoblastoma protein functions by ectopic expression of human cyclins.

The retinoblastoma susceptibility gene (RB) product, the retinoblastoma protein (pRb), functions as a regulator of cell proliferation. Introduction of the RB gene into SAOS-2 osteosarcoma cells, which lack functional pRb, prevents cell cycle progression. Such growth-suppressive functions can be modulated by phosphorylation of pRb, which occurs via cell cycle-regulated kinases. We show that constitutively expressed cyclins A and E can overcome pRb-mediated suppression of proliferation. pRb becomes hyperphosphorylated in cells overexpressing these cyclins, and this phosphorylation is essential for cyclin A- and cyclin E-mediated rescue of pRb-blocked cells. This suggests that G1 and S phase cyclins can act as regulators of pRb function in the cell cycle by promoting pRb phosphorylation.     

Molecular determinants for the complex formation between the retinoblastoma protein and LXCXE sequences

The retinoblastoma tumor suppressor protein (pRb) is a key negative regulator of cell proliferation that is frequently disregulated in human cancer. Many viral oncoproteins (for example, HPV E7 and E1A) are known to bind to the pRb pocket domain via a LXCXE binding motif. There are also some 20 cellular proteins that contain a LXCXE motif and have been reported to associate with the pocket domain of pRb. Using NMR spectroscopy and isothermal calorimetry titration, we show that LXCXE peptides of viral oncoproteins bind strongly to the pocket domain of pRb. Additionally, we show that LXCXE-like peptides of HDAC1 bind to the same site on pRb with a weak (micromolar) and transient association. Systematic substitution of residues other than conserved Leu, Cys, and Glu show that the residues flanking the LXCXE are important for the binding, whereas positively charged amino acids in the XLXCXEXXX sequence significantly weaken the interaction.     

The High-Risk HPV16 E7 Oncoprotein Mediates Interaction between the Transcriptional Coactivator CBP and the Retinoblastoma Protein pRb

The oncoprotein E7 from human papillomavirus (HPV) strains that confer high cancer risk mediates cell transformation by deregulating host cellular processes and activating viral gene expression through recruitment of cellular proteins such as the retinoblastoma protein (pRb) and the cyclic-AMP response element binding binding protein (CBP) and its paralog p300. Here we show that the intrinsically disordered N-terminal region of E7 from high-risk HPV16 binds the TAZ2 domain of CBP with greater affinity than E7 from low-risk HPV6b. HPV E7 and the tumor suppressor p53 compete for binding to TAZ2. The TAZ2 binding site in E7 overlaps the LxCxE motif that is crucial for interaction with pRb. While TAZ2 and pRb compete for binding to a monomeric E7 polypeptide, the full-length E7 dimer mediates an interaction between TAZ2 and pRb by promoting formation of a ternary complex. Cell-based assays show that expression of full-length HPV16 E7 promotes increased pRb acetylation and that this response depends both on the presence of CBP/p300 and on the ability of E7 to form a dimer. These observations suggest a model for the oncogenic effect of high-risk HPV16 E7. The disordered region of one E7 molecule in the homodimer interacts with the pocket domain of pRb, while the same region of the other E7 molecule binds the TAZ2 domain of CBP/p300. Through its ability to dimerize, E7 recruits CBP/p300 and pRb into a ternary complex, bringing the histone acetyltransferase domain of CBP/p300 into proximity to pRb and promoting acetylation, leading to disruption of cell cycle control.