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// Martin C. Pearce 1 , John T. Gamble 1, 2 , Prasad R. Kopparapu 1 , Edmond F. O'Donnell 1 , Monica J. Mueller 1 , Hyo Sang Jang 1 , Julie A. Greenwood 2 , Arnold C. Satterthwait 3 , Robert L. Tanguay 4, 5, 6 , Xiao-Kun Zhang 3 and Siva Kumar Kolluri 1, 4, 5, 6 1 Cancer Research Laboratory, Department of Environmental & Molecular Toxicology, Oregon State University, Corvallis, Oregon 97331, USA 2 Department of Biochemistry & Biophysics, Oregon State University, Corvallis, Oregon 97331, USA 3 Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92031, USA 4 Department of Environmental & Molecular Toxicology, Environmental Health Sciences Center, Oregon State University, Corvallis, Oregon 97331, USA 5 Linus Pauling Institute, Oregon State University, Corvallis, OR 97331, USA 6 Center for Genome Research and Biocomputing, Oregon State University, Corvallis, OR 97331, USA Correspondence to: Siva Kumar Kolluri, email: siva.kolluri@oregonstate.edu Keywords: chemoresistance; Bcl-2; NuBCP-9; paclitaxel; lung cancer Received: February 19, 2018 Accepted: April 24, 2018 Published: May 25, 2018 ABSTRACT Resistance to chemotherapy is a major cause of treatment failure and poor overall survival in patients with lung cancer. Identification of molecular targets present in resistant cancer cells is essential for addressing therapeutic resistance and prolonging lung cancer patient survival. Members of the B-cell lymphoma 2 (Bcl-2) family of proteins are associated with chemotherapeutic resistance. In this study, we found that pro-survival protein Bcl-2 is upregulated in paclitaxel resistant cells, potentially contributing to chemotherapy resistance. To exploit the increase in Bcl-2 expression for targeting therapy resistance, we investigated the effects of a peptide derived from the nuclear receptor Nur77 that converts Bcl-2 from an anti-apoptotic protein to a pro-apoptotic protein. The Nur77 derived peptide preferentially induced apoptosis in paclitaxel-resistant cancer cells with high expression of Bcl-2. This peptide also induced apoptosis of multidrug resistant H69AR lung cancer cells that express Bcl-2 and inhibited their growth in 3D spheroids. The Nur77 peptide strongly suppressed the growth of paclitaxel-resistant lung cancer cells in a zebrafish xenograft tumor model. Taken together, our data supports a new strategy for treating lung cancers that acquire resistance to chemotherapy through overexpression of Bcl-2.

Host innate immune responses to DNA viruses involve members of the nucleotide-binding domain, leucine-rich repeat and pyrin domain containing protein (NLRP) family, which form “inflammasomes” that activate caspase-1, resulting in proteolytic activation of cytokines interleukin (IL)-1β and IL-18. We hypothesized that DNA viruses would target inflammasomes to overcome host defense. A Vaccinia virus (VACV) B-cell CLL/lymphoma 2 (Bcl-2) homolog, F1L, was demonstrated to bind and inhibit the NLR family member NLRP1 in vitro. Moreover, infection of macrophages in culture with virus lacking F1L (ΔF1L) caused increased caspase-1 activation and IL-1β secretion compared with wild-type virus. Virulence of ΔF1L virus was attenuated in vivo, causing altered febrile responses, increased proteolytic processing of caspase-1, and more rapid inflammation in lungs of infected mice without affecting cell death or virus replication. Furthermore, we found that a hexapeptide from F1L is necessary and sufficient for inhibiting the NLRP1 inflammasome in vitro, thus identifying a peptidyl motif required for binding and inhibiting NLRP1. The functional importance of this NLRP1-binding motif was further confirmed by studies of recombinant ΔF1L viruses reconstituted either with the wild-type F1L or a F1L mutant that fails to bind NLRP1. Cellular infection with wild-type F1L reconstituted virus-suppressed IL-1β production, whereas mutant F1L did not. In contrast, both wild-type and mutant versions of F1L equally suppressed apoptosis. In vivo, the NLR nonbinding F1L mutant virus exhibited an attenuated phenotype similar to ΔF1L virus, thus confirming the importance of F1L interactions with NLRP1 for viral pathogenicity in mice. Altogether, these findings reveal a unique viral mechanism for evading host innate immune responses.

The K7L gene product of the smallpox virus is a protease implicated in the maturation of viral proteins. K7L belongs to protease Clan CE, which includes distantly related cysteine proteases from eukaryotes, pathogenic bacteria, and viruses. Here, we describe its recombinant high level expression, biochemical mechanism, substrate preference, and regulation. Earlier studies inferred that the orthologous I7L vaccinia protease cleaves at an AG-X motif in six viral proteins. Our data for K7L suggest that the AG-X motif is necessary but not sufficient for optimal cleavage activity. Thus, K7L requires peptides extended into the P7 and P8 positions for efficient substrate cleavage. Catalytic activity of K7L is substantially enhanced by homodimerization, by the substrate protein P25K as well as by glycerol. RNA and DNA also enhance cleavage of the P25K protein but not of synthetic peptides, suggesting that nucleic acids augment the interaction of K7L with its protein substrate. Library-based peptide preference analyses enabled us to design an activity-based probe that covalently and selectively labels K7L in lysates of transfected and infected cells. Our study thus provides proof-of-concept for the design of inhibitors and probes that may contribute both to a better understanding of the role of K7L in the virus life cycle and the design of novel anti-virals.

The human Bcl-2 family includes six antiapoptotic members (Bcl-2, Bcl-B, Bcl-W, Bcl-X(L), Bfl-1, and Mcl-1) and many proapoptotic members, wherein a balance between the two determines cell life or death in many physiological and disease contexts. Elevated expression of various antiapoptotic Bcl-2 members is commonly observed in cancers, and chemical inhibitors of these proteins have been shown to promote apoptosis of malignant cells in culture, in animal models, and in human clinical trials. All six antiapoptotic members bind a helix from the proapoptotic family member Bim, thus quenching Bim's apoptotic signal. Here, we describe the use of a multiplex, high-throughput flow cytometry assay for the discovery of small molecule modulators that disrupt the interaction between the antiapoptotic members of the Bcl-2 family and Bim. The six antiapoptotic Bcl-2 family members were expressed as glutathione-S-transferase fusion proteins and bound individually to six glutathione bead sets, with each set having a different intensity of red fluorescence. A fluorescein-conjugated Bcl-2 homology region 3 (BH3) peptide from Bim was employed as a universal ligand. Flow cytometry measured the amount of green peptide bound to each bead set in a given well, with inhibitory compounds resulting in a decrease of green fluorescence on one or more bead set(s). Hits and cheminformatically selected analogs were retested in a dose-response series, resulting in three "active" compounds for Bcl-B. These three compounds were validated by fluorescence polarization and isothermal titration calorimetry. We discuss some of the lessons learned about screening a chemical library provided by the National Institutes of Health Small Molecule Repository (∼195,000 compounds) using high-throughput flow cytometry.

NLRP1 (NLR family, pyrin domain-containing 1) is a contributor to innate immunity involved in intracellular sensing of pathogens, as well as danger signals related to cell injury. NLRP1 is one of the core components of caspase-1-activating platforms termed “inflammasomes,” which are involved in proteolytic processing of interleukin-1β (IL-1β) and in cell death. We previously discovered that anti-apoptotic proteins Bcl-2 and Bcl-X L bind to and inhibit NLRP1 in cells. Using an in vitro reconstituted system employing purified recombinant proteins, we studied the mechanism by which Bcl-2 and Bcl-X L inhibit NLRP1. Bcl-2 and Bcl-X L inhibited caspase-1 activation induced by NLRP1 in a concentration-dependent manner, with K i ≈ 10 nM. Bcl-2 and Bcl-X L were also determined to inhibit ATP binding to NLRP1, which is required for oligomerization of NLRP1, and Bcl-X L was demonstrated to interfere with NLRP1 oligomerization. Deletion of the flexible loop regions of Bcl-2 and Bcl-X L , which are located between the first and second α-helices of these anti-apoptotic proteins and which were previously shown to be required for binding NLRP1, abrogated ability to inhibit caspase-1 activation, ATP binding and oligomerization of NLRP1. Conversely, synthetic peptides corresponding to the loop region of Bcl-2 were sufficient to potently inhibit NLRP1. These findings thus demonstrate that the loop domain is necessary and sufficient to inhibit NLRP1, providing insights into the mechanism by which anti-apoptotic proteins Bcl-2 and Bcl-X L inhibit NLRP1.

Histone lysine methyltransferases (HKMTs) are involved in regulation of chromatin structure, and, as such, are important for longterm gene activation and repression that is associated with cell memory and establishment of cell-type specific transcriptional programs. Most HKMTs contain a SET domain, which is responsible for their catalytic activity. RIZ1 is a transcription regulator and tumor suppressor that catalyzes methylation of lysine 9 of histone H3 and contains a rather distinct SET domain. Similar SET domains, sometimes refererred to as PR (PRDI-BF1 and RIZ1 homology) domains, are also found in other proteins including Blimp-1/PRDI-BF1, MDS1-EVI1 and Meisetz. We determined the solution structure of the PR domain from RIZ1 and characterized its interaction with S-adenosyl homocysteine (SAH) and a peptide from histone H3. Despite low sequence identity with canonical SET domains, the PR domain displays a typical SET fold including a pseudo-knot at the C-terminus. The N-flanking sequence of RIZ1 PR domain adopts a novel conformation and interacts closely with the SET fold. The C-flanking sequence contains an α-helix that exhibits higher mobility than the SET fold and points away from the protein face that harbors active site in other SET domains. Residues that interact with the methylation cofactor in SET domainsmore » are not conserved in RIZ1 or other PR domains, and the SET fold of RIZ1 does not bind SAH. However, the PR domain of RIZ1 interacts specifically with a synthetic peptide comprising residues 1-20 of histone H3.« less

Epstein-Barr virus is a human herpesvirus that causes infectious mononucleosis and lymphoproliferative malignancies. LMP1 (latent membrane protein-1), which is encoded by this virus and which is essential for transformation of B lymphocytes, acts as a constitutively active mimic of the tumor necrosis factor receptor (TNFR) CD40. LMP1 is an integral membrane protein containing six transmembrane segments and a cytoplasmic domain at the C terminus that binds to intracellular TNFR-associated factors (TRAFs). TRAFs are intracellular co-inducers of downstream signaling from CD40 and other TNFRs, and TRAF3 is required for activation of B lymphocytes by LMP1. Cytoplasmic C-terminal activation region 1 of LMP1 bears a motif (PQQAT) that conforms to the TRAF recognition motif PVQET in CD40. In this study, we report the crystal structure of this portion of LMP1 C-terminal activation region-1 (204PQQATDD210) bound in complex with TRAF3. The PQQAT motif is bound in the same binding crevice on TRAF3 where CD40 is bound, providing a molecular mechanism for LMP1 to act as a CD40 decoy for TRAF3. The LMP1 motif is presented in the TRAF3 crevice as a close structural mimic of the PVQET motif in CD40, and the intermolecular contacts are similar. However, the viral protein makes a unique contact: a hydrogen bond network formed between Asp210 in LMP1 and Tyr395 and Arg393 in TRAF3. This intermolecular contact is not made in the CD40-TRAF3 complex. The additional hydrogen bonds may stabilize the complex and strengthen the binding to permit LMP1 to compete with CD40 for binding to the TRAF3 crevice, influencing downstream signaling to B lymphocytes and contributing to dysregulated signaling by LMP1.

Humanin (HN) is a recently identified endogenous peptide that protects cells against cytotoxicity induced by various stimuli. Recently, we showed that HN binds to and inhibits Bax, a proapoptotic Bcl-2 family protein, suggesting a mechanism for HN action. In this study, we identified Bim, a Bcl-2 homology 3-only member of the Bcl-2/Bax family, as an additional HN target protein. Using in vitro protein binding, immunoprecipitation, and coimmunolocalization assays, we demonstrated that HN binds directly to the extra long isoform of Bim (BimEL) but not the long (BimL) or short (BimS) isoforms. HN also protects cells against apoptosis induced by BimEL but not BimL and BimS in gene transfection studies. In contrast, mutants of HN which failed to bind BimEL failed to protect from BimEL-induced cell death. Moreover, HN inhibited BimEL-induced release of SMAC and cytochrome c from mitochondria isolated from bax-/-cells, indicating that HN can suppress BimEL independently of its effect on Bax. Finally, we demonstrate that HN prevents BimEL-induced oligomerization of Bak using isolated mitochondria. Taken together, our results indicate that the inhibition of BimEL may contribute to the antiapoptotic properties of the HN peptide.

B cell-activating factor belonging to the TNF family receptor (BAFF-R), a member of the TNFR superfamily, plays a role in autoimmunity after ligation with BAFF ligand (also called TALL-1, BLyS, THANK, or zTNF4). BAFF/BAFF-R interactions are critical for B cell regulation, and signaling from this ligand-receptor complex results in NF-κB activation. Most TNFRs transmit signals intracellularly by recruitment of adaptor proteins called TNFR-associated factors (TRAFs). However, BAFF-R binds only one TRAF adaptor, TRAF3, and this interaction negatively regulates activation of NF-κB. In this study, we report the crystal structure of a 24-residue fragment of the cytoplasmic portion of BAFF-R bound in complex with TRAF3. The recognition motif 162PVPAT166 in BAFF-R is accommodated in the same binding crevice on TRAF3 that binds two related TNFRs, CD40 and LTβR, but is presented in a completely different structural framework. This region of BAFF-R assumes an open conformation with two extended strands opposed at right angles that each make contacts with TRAF3. The recognition motif is located in the N-terminal arm and intermolecular contacts mediate TRAF recognition. In the C-terminal arm, key stabilizing contacts are made, including critical hydrogen bonds with Gln379 in TRAF3 that define the molecular basis for selective binding of BAFF-R solely to this member of the TRAF family. A dynamic conformational adjustment of Tyr377 in TRAF3 occurs forming a new intermolecular contact with BAFF-R that stabilizes the complex. The structure of the complex provides a molecular explanation for binding affinities and selective protein interactions in TNFR-TRAF interactions.

Lymphotoxin-beta receptor (LTbetaR) and CD40 are members of the tumor necrosis factor family of signaling receptors that regulate cell survival or death through activation of NF-kappaB. These receptors transmit signals through downstream adaptor proteins called tumor necrosis factor receptor-associated factors (TRAFs). In this study, the crystal structure of a region of the cytoplasmic domain of LTbetaR bound to TRAF3 has revealed an unexpected new recognition motif, 388IPEEGD393, for TRAF3 binding. Although this motif is distinct in sequence and structure from the PVQET motif in CD40 and PIQCT in the regulator TRAF-associated NF-kappaB activator (TANK), recognition is mediated in the same binding crevice on the surface of TRAF3. The results reveal structurally adaptive "hot spots" in the TRAF3-binding crevice that promote molecular interactions driving specific signaling after contact with LTbetaR, CD40, or the downstream regulator TANK.

TRAFs (tumor necrosis factor receptor [TNFR]-associated factors) bind to the cytoplasmic portion of liganded TNFRs and stimulate activation of NF-κB or JNK pathways. A modulator of TRAF signaling, TANK, serves as either an enhancer or an inhibitor of TRAF-mediated signaling pathways. The crystal structure of a region of TANK bound to TRAF3 has been determined and compared to a similar CD40/TRAF3 complex. TANK and CD40 bind to the same crevice on TRAF3. The recognition motif PxQxT is presented in a boomerang-like structure in TANK that is markedly different from the hairpin loop that forms in CD40 upon binding to TRAF3. Critical TANK contact residues were confirmed by mutagenesis to be required for binding to TRAF3 or TRAF2. Binding affinity, measured by isothermal titration calorimetry and competition assays, demonstrated that TANK competes with CD40 for the TRAF binding site.

BAG-family proteins share a conserved protein interaction region, called the 'BAG domain', which binds and regulates Hsp70/Hsc70 molecular chaperones. This family of cochaperones functionally regulates signal transducing proteins and transcription factors important for cell stress responses, apoptosis, proliferation, cell migration and hormone action. Aberrant overexpression of the founding member of this family, BAG1, occurs in human cancers. In this study, a structure-based approach was used to identify interacting residues in a BAG1--Hsc70 complex. An Hsc70-binding fragment of BAG1 was shown by multidimensional NMR methods to consist of an antiparallel three-helix bundle. NMR chemical shift experiments marked surface residues on the second (alpha 2) and third (alpha 3) helices in the BAG domain that are involved in chaperone binding. Structural predictions were confirmed by site-directed mutagenesis of these residues, resulting in loss of binding of BAG1 to Hsc70 in vitro and in cells. Molecular docking of BAG1 to Hsc70 and mutagenesis of Hsc70 marked the molecular surface of the ATPase domain necessary for interaction with BAG1. The results provide a structural basis for understanding the mechanism by which BAG proteins link molecular chaperones and cell signaling pathways.

Background: The third hypervariable (V3) loop of HIV-1 gp120 has been termed the principal neutralizing determinant (PND) of the virus and is involved in many aspects of virus infectivity. The V3 loop is required for viral entry into the cell via membrane fusion and is believed to interact with cell surface chemokine receptors on T cells and macrophages. Sequence changes in V3 can affect chemokine receptor usage, and can, therefore, modulate which types of cells are infected. Antibodies raised against peptides with V3 sequences can neutralize laboratory-adapted strains of the virus and inhibit syncytia formation. Fab fragments of these neutralizing antibodies in complex with V3 loop peptides have been studied by X-ray crystallography to determine the conformation of the V3 loop. Results: We have determined three crystal structures of Fab 58.2, a broadly neutralizing antibody, in complex with one linear and two cyclic peptides the amino acid sequence of which comes from the MN isolate of the gp120 V3 loop. Although the peptide conformations are very similar for the linear and cyclic forms, they differ from that seen for the identical peptide bound to a different broadly neutralizing antibody, Fab 59.1, and for a similar peptide bound to the MN-specific Fab 50.1. The conformational difference in the peptide is localized around residues Gly-Pro-Gly-Arg, which are highly conserved in different HIV-1 isolates and are predicted to adopt a type II β turn. Conclusions: The V3 loop can adopt at least two different conformations for the highly conserved Gly-Pro-Gly-Arg sequence at the tip of the loop. Thus, the HIV-1 V3 loop has some inherent conformational flexibility that may relate to its biological function.

We have recently identified a series of compounds which efficiently inhibit Anthrax lethal factor (LF) metallo-protease. Here we present further structure activity relationship and CoMFA (Comparative Molecular Field Analysis) studies on newly derived inhibitors. The obtained 3D QSAR model was subsequently compared with the X-ray structure of the complex between LF and a representative compound. Our studies form the basis for the rational design of additional compounds with improved activity and selectivity.

Antigenic variation among different HIV-1 isolates has been a major problem in the development of an effective vaccine against AIDS. Peptide vaccines incorporating structural elements common to groups of viral isolates, such as the clade subtypes of HIV-1, hold promise; however, the design of such immunogens has been hampered by the lack of specific structural information on the viral proteins to be targeted. As part of a structure-based approach to this problem, we report the design and characterization of a conformationally restricted peptide analog (Aib142) of a highly conserved HIV-1 clade-B sequence from the third variable loop of the membrane glycoprotein gp120. The design strategy incorporates peptide conformational data derived from crystal structure analysis of an MN-isolate peptide (RP142) in complex with the Fab fragment (Fab59.1) of a broadly neutralizing antibody. The synthetic peptide (Aib142) replaces an alanine residue within the V3 loop epitope sequence GPGRAF by the conformationally restricted helicogenic α-aminoisobutyryl residue. As expected, the crystal structure of the Fab 59.1-Aib142 complex at 2.8 A resolution shows that the peptide interacts very similarly with the neutralizing antibody. Proton nuclear magnetic resonance (NMR) studies indicate that the free Aib142 peptide is indeed more ordered in solution with a conformational preference that corresponds to the X-ray structure of its Fab-bound form. Aib142 thus represents the first step in the design of conformationally constrained peptide analogs built to mimic biologically relevant structural forms of HIV-1 neutralization sites.

An antibody engineering strategy was employed to build high affinity ligands and antagonists of integrins alpha v beta 3 and alpha IIb beta 3. Previously, we inserted the integrin recognition motif, RGD, into the antigen binding site of a human antibody and selected the optimal flanking sequences from a phage-display library (Barbas, C. F., Languino, L. R., and Smith, J. W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10003-10007). The resulting antibody, Fab-9, blocked the function of integrin alpha v beta 3 but also bound to the ligand binding site of platelet integrin alpha IIb beta 3. In this report, the antibody engineering effort has been extended by 1) redesigning Fab-9 to achieve specificity for platelet integrin alpha IIb beta 3, 2) building non-RGD-containing antibodies that bind the ligand binding site of both beta 3-integrins, and 3) testing the hypothesis that peptides derived from complementarity determining regions (CDR) can be used to emulate the activity of the parent synthetic antibody. These goals were accomplished by subjecting the original antibody, Fab-9, to a "motif optimization" (MTF). A phage library was constructed in which the residues flanking the RGD motif in Fab-9 were maintained, but the RGDX sequence was randomized. This library was panned on purified alpha IIb beta 3 to identify high affinity binders. Four function-blocking antibodies lacking RGD, but with specificity for alpha IIb beta 3, were characterized. The antibody with the highest preference for alpha IIb beta 3, MTF-10, had an adhesion sequence of KGDN. This sequence is similar in primary structure to the active sequence within the disintegrin barbourin, which also antagonizes alpha IIb beta 3 (Scarborough, R. M., Rose, J. W., Hsu, M. A., Phillips, D. R., Fried, V. A., Campbell, A. M., Nannizzi, L., and Charo, I. F. (1991) J. Biol. Chem. 266, 9359-9362). MTF-10 had a 70-fold higher affinity for alpha IIb beta 3 than alpha v beta 3. Through our selection strategy, we also identified several antibodies that lack RGD but still blocked ligand binding to both integrins with high affinity. Therefore, the RGD sequence is not necessary for a high affinity interaction with the ligand binding site of beta 3-integrins. Further investigation showed that the activity of inhibitory antibodies could be emulated by synthetic peptides derived from the protein sequences of the antibody's HCDR3. CDR-derived peptides blocked ligand binding to integrins and maintained essentially the same specificity as the parent antibody.

X-ray quality crystals of an Fab fragment from a transmission-blocking monoclonal antibody 4B7 (MAb 4B7) against a sexual stage protein Pfs25 of Plasmodium falciparum were grown as uncomplexed and peptide-complexed forms. Initially, the intact immunoglobulin was crystallized because cleavage with pepsin or papain did not produce a homogeneous product. Further proteolytic trials with elastase produced a suitable Fab fragment from which crystals have been obtained, both for the free Fab and in complex with cyclic peptides and in the presence of linear peptides. While linear peptides bind to MAb 4B7, cyclic peptides modeled on a predicted beta-hairpin loop of the third EGF-like domain of Pfs25 bind better and readily co-crystallize with the Fab. The genes for the variable domain of the Fab have been cloned, sequenced and the primary amino-acid sequence for the complete Fab deduced. This work explores the use of glycerol as an additive and the modification of the peptide sequence outside the epitope for improving in the crystallization. Data sets have been collected from crystals of several Fab-peptide complexes and from uncomplexed Fab to resolutions ranging from 2.4 to 3.3 A. The packing arrangements of several crystal forms have been determined by molecular replacement, and refinement of their three-dimensional structures is in progress. The three-dimensional structure of this Fab complexed with the various peptides will aid in an understanding of the mode by which this antibody recognizes and prevents transmission of the malaria parasite.

Bcl-2 can be converted into a pro-apoptotic molecule by nuclear receptor Nur77. The development of Bcl-2 converters as anti-cancer therapeutics has been explored by us. We reported recently the identification of a Nur77-derived Bcl-2 converting peptide (NuBCP) and its enantiomer, which induce apoptosis of cancer cells in vitro and in animals. The apoptotic effect of NuBCP enantiomers and their activation of Bax are not inhibited but rather potentiated by Bcl-2. Using fluorescence polarization assays, we determined that NuBCP enantiomers bind both quantitatively and stoichiometrically to the Bcl-2 loop, which shares the characteristics of structurally adaptable regions with many cancer-associated signaling proteins. NuBCP-9 enantiomers act as molecular switches to dislodge the Bcl-2 BH4 motif exposing its BH3 motif. Mechanistically we demonstrated, using fluorescence quenching based liposome assays, that NuBCP-9-induced Bcl-2 conformational change not only neutralizes Bcl-2's inhibition of Bax-mediated membrane permeabilization but also exposes the Bcl-2's BH3 motif that in turn neutralizes Bcl-XL's inhibition of Bax like BH3 motif-derived peptides and compounds. Our results provide mechanistic insight into Bcl-2 conversion and identify a new direction for developing Bcl-2-based cancer therapeutics. (This work is in part supported by the grant GM062964 to J. Lin from the National Institute of Health.)

The natural product gambogic acid (GA) has been reported to have cytotoxic activity against tumor cells in culture and was identified as an active compound in a cell-based high-throughput screening assay for activators of caspases, proteases involved in apoptosis. Using the antiapoptotic Bcl-2 family protein, Bfl-1, as a target for screening of a library of natural products, we identified GA as a competitive inhibitor that displaced BH3 peptides from Bfl-1 in a fluorescence polarization assay. Analysis of competition for BH3 peptide binding revealed that GA inhibits all six human Bcl-2 family proteins to various extents, with Mcl-1 and Bcl-B the most potently inhibited [concentrations required for 50% inhibition (IC50)

The pro-apoptotic members of the Bcl-2 family include initiator proteins that contain only BH3 domains and downstream effector multi-BH domain-containing proteins, including Bax and Bak. In this report, we compared the ability of the six human anti-apoptotic Bcl-2 family members to suppress apoptosis induced by overexpression of Bax or Bak, correlating findings with protein interactions measured by three different methods: co-immunoprecipitation, glutathione S-transferase pulldown, and fluorescence polarization assays employing synthetic BH3 peptides from Bax and Bak. Bcl-B and Mcl-1 showed strong preferences for binding to and suppression of Bax and Bak, respectively. In contrast, the other anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-XL, Bcl-W, and Bfl-1) suppressed apoptosis induced by overexpression of either Bax or Bak, and they displayed an ability to bind both Bax and Bak by at least one of the three protein interaction methods. Interestingly, however, full-length Bax and Bak proteins and synthetic Bax and Bak BH3 peptides exhibited discernible differences in their interactions with some anti-apoptotic members of the Bcl-2 family, cautioning against reliance on a single method for detecting protein interactions of functional significance. Altogether, the findings reveal striking distinctions in the behaviors of Bcl-B and Mcl-1 relative to the other anti-apoptotic Bcl-2 family members, where Bcl-B and Mcl-1 display reciprocal abilities to bind and neutralize Bax and Bak.

Humanin is a short endogenous peptide, which can provide protection from cell death through its association with various receptors, including the pro-apoptotic Bcl-2 family proteins Bid, Bim, and Bax. By using NMR chemical shift mapping experiments, we demonstrate that the interaction between Humanin-derived peptides and Bid is specific, and we localize the binding site to a region on the surface of Bid, which includes residues from the conserved helical BH3 domain of the protein. The BH3 domain mediates the association of Bid with other Bcl-2 family members and is essential for the protein's cytotoxic activity. The data suggest that Humanin exerts its cytoprotective activity by engaging the Bid BH3 domain; this would hinder the association of Bid with other Bcl-2 family proteins, thereby mitigating its toxicity. The identification of a Humanin-specific binding site on the surface of Bid reinforces its importance as a direct modulator of programmed cell death, and suggests a strategy for the design of cytoprotective peptide inhibitors of Bid.

Defects in apoptosis mechanisms play important roles in malignancy and autoimmunity. Orphan nuclear receptor Nur77/TR3 has been demonstrated to bind antiapoptotic protein Bcl-2 and convert it from a cytoprotective to a cytodestructive protein, representing a phenotypic conversion mechanism. Of the 6 antiapoptotic human Bcl-2 family members, we found that Nur77/TR3 binds strongest to Bcl-B, showing selective reactivity with Bcl-B, Bcl-2, and Bfl-1 but not Bcl-XL, Mcl-1, or Bcl-W. Nur77 converts the phenotype of Bcl-B from antiapoptotic to proapoptotic. Bcl-B is prominently expressed in plasma cells and multiple myeloma. Endogenous Bcl-B associates with endogenous Nur77 in RPMI 8226 myeloma cells, where RNA interference experiments demonstrated dependence on Bcl-B for Nur77-induced apoptosis. Furthermore, a Nur77-mimicking peptide killed RPMI 8226 myeloma cells through a Bcl-B–dependent mechanism. Because Bcl-B is abundantly expressed in plasma cells and some myelomas, these findings raise the possibility of exploiting the Nur77/Bcl-B mechanism for apoptosis for eradication of autoimmune plasma cells or myeloma.

Recently, we discovered that Humanin (HN), a small endogenous peptide of 24 amino acids, binds to and inhibits the proapoptotic protein Bax. We show here that HN also interacts with the BH3-only Bcl-2/Bax family protein, Bid, as well as a truncated form of Bid (tBid) associated with protease-mediated activation of this proapoptotic protein. Synthetic HN peptide binds purified Bid and tBid in vitro and blocks tBid-induced release of cytochrome c and SMAC from isolated mitochondria, whereas mutant peptides that fail to bind Bid or tBid lack this activity. Moreover, HN peptide also retained protective activity on bax–/–mitochondria, indicating that HN can block tBid-induced release of apoptogenic proteins from these organelles in a Bax-independent manner. HN peptide inhibits tBid-induced oligomerization of Bax and Bak in mitochondrial membranes, as shown by experiments with chemical cross-linkers or gel filtration. Gene transfection experiments showed that HN (but not an inactive mutant of HN) also protects intact cells from apoptosis induced by overexpression of tBid. We conclude that Bid represents an additional cellular target of HN, and we propose that HN-mediated suppression of Bid contributes to the antiapoptotic activity of this endogenous peptide.

Bax (Bcl2-associated X protein) is an apoptosis-inducing protein that participates in cell death during normal development and in various diseases. Bax resides in an inactive state in the cytosol of many cells. In response to death stimuli, Bax protein undergoes conformational changes that expose membrane-targeting domains, resulting in its translocation to mitochondrial membranes, where Bax inserts and causes release of cytochrome c and other apoptogenic proteins. It is unknown what controls conversion of Bax from the inactive to active conformation. Here we show that Bax interacts with humanin (HN), an anti-apoptotic peptide of 24 amino acids encoded in mammalian genomes. HN prevents the translocation of Bax from cytosol to mitochondria. Conversely, reducing HN expression by small interfering RNAs sensitizes cells to Bax and increases Bax translocation to membranes. HN peptides also block Bax association with isolated mitochondria, and suppress cytochrome c release in vitro. Notably, the mitochondrial genome contains an identical open reading frame, and the mitochondrial version of HN can also bind and suppress Bax. We speculate therefore that HN arose from mitochondria and transferred to the nuclear genome, providing a mechanism for protecting these organelles from Bax.

BAG (Bcl-2-associated athanogene) proteins are molecular chaperone regulators that affect diverse cellular pathways. All members share a conserved motif, called the BAG domain (BD), which binds to Hsp70/Hsc70 family proteins and modulates their activity. We have determined the solution structure of BD from BAG4/SODD (silencer of death domains) by multidimensional nuclear magnetic resonance methods and compared it to the corresponding domain in BAG1 (Briknarova, K., Takayama, S., Brive, L., Havert, M. L., Knee, D. A., Velasco, J., Homma, S., Cabezas, E., Stuart, J., Hoyt, D. W., Satterthwait, A. C., Llinas, M., Reed, J. C., and Ely, K. R. (2001) Nat. Struct. Biol. 8, 349-352). The difference between BDs from these two BAG proteins is striking, and the structural comparison defines two subfamilies of mammalian BD-containing proteins. One subfamily includes the closely related BAG3, BAG4, and BAG5 proteins, and the other is represented by BAG1, which contains a structurally and evolutionarily distinct BD. BDs from both BAG1 and BAG4 are three-helix bundles; however, in BAG4, each helix in this bundle is three to four turns shorter than its counterpart in BAG1, which reduces the length of the domain by one-third. BAG4 BD thus represents a prototype of the minimal functional fragment that is capable of binding to Hsc70 and modulating its chaperone activity.

It was proposed to develop a structure-based approach for synthetic vaccine development designed to recapitulate the activities of potent neutralizing monoclonal antibodies (MAbs) using constrained peptides as immunogens. We focused on two antibodies, a MAb 58.2 that binds a sequential V3 epitope and IgG bl2 that binds a discontinuous epitope, both on HIV-1 gp120. We validated the structure-based approach with a V3 mimetic using MAb 58.2 and demonstrated the enormous effect that peptide conformation can have on affinity and immunogenicity. We were unable to mimic the more challenging discontinuous IgG bl2 epitope despite considerable effort. The enormous affinity enhancements we observed for the constrained V3 peptide and dearth of potent HIV-1 neutralizing antibodies, particularly those specific for sequential epitopes, led us to an important discovery. It suggested that new antibodies might be identified with constrained peptides that go undetected with peptides. It was found that several helix-stabilized gpl20 peptides detect new antibodies that go undetected with linear peptides implying a general phenomenon. Constrained peptides, tailored to mimic short, sequential regions of proposed neutralization sites might be used to widen the selection of template antibodies best suited for synthetic vaccine development.

The generation of neutralizing antibodies by peptide immunization is dependent on achieving conformational compatibility between antibodies and native protein. Consequently, approaches are needed for developing conformational mimics of protein neutralization sites. We replace putative main-chain hydrogen bonds (NH --O=CRNH) with a hydrazone link (N-N=CH-CH(2)CH(2)) and scan constrained peptides for fit with neutralizing monoclonal antibodies (MAbs). To explore this approach, a V3 MAb 58.2 that potently neutralizes T-cell lab-adapted HIV-1(MN) was used to identify a cyclic peptide, [JHIGPGR(Aib)F(D-Ala)GZ]G-NH(2) (loop 5), that binds with1000-fold higher affinity than the unconstrained peptide. NMR structural studies suggested that loop 5 stabilized beta-turns at GPGR and R(Aib)F(D-Ala) in aqueous solvent implying considerable conformational mimicry of a Fab 58.2 bound V3 peptide determined by X-ray crystallography [Stanfield, R. L. et al. (1999) Structure 142, 131-142]. Rabbit polyclonal antibodies (PAbs) generated to loop 5 but not to the corresponding uncyclized peptide bound the HIV-1(MN) envelope glycoprotein, gp120. When individual rabbit antisera were scanned with linear and cyclic peptides, further animal-to-animal differences in antibody populations were characterized. Loop 5 PAbs that most closely mimicked MAb 58.2 neutralized HIV-1(MN) with similar potency. These results demonstrate the remarkable effect that conformation can have on peptide affinity and immunogenicity and identify an approach that can be used to achieve these results. The implications for synthetic vaccine and HIV-1 vaccine research are discussed.

Our proposal was directed to the identification of synthetic peptide mimetics corresponding to neutralizing epitopes recognized by IgG1b12 and MAb 58.2. Herein, we report recent results on MAb 58.2 which provides important new information regarding methods and outcomes for synthetic vaccines as well as specific information about the role that conformation plays in neutralizing immune responses to the V3 epitope. MAb 58.2 displays broad reactivity against Clade B gp120s from primary isolates. It weakly neutralizes a cloned macrophage-tropic primary isolate, JR-CSF. We identified a constrained V-3 loop mimetic that binds >1,000 times better to MAb 58.2 than the corresponding linear peptide. Rabbit polyclonal antibodies to the constrained peptide but not the linear peptide bind rgp120 (MN). Furthermore, the polyclonal immune response to a constrained V3 peptide mimics the conformational preference and neutralizing activity of MAb 58.2. HIV-1 neutralization correlates with conformational preference and affinity for gp120. These results and others presented in the report establish beyond doubt the remarkable improvements in antigenicity and immunogenicity of constrained peptides compared to linear peptides.

Monoclonal antibody 4B7 is a neutralizing antibody that binds the protein Pfs25 in the sexual stages of the malaria parasite Plasmodium falciparum and completely blocks transmission of the parasite from human serum to the mosquito host. Here we report the identification of the epitope on Pfs25 recognized by 4B7 and the crystallization of the intact murine monoclonal antibody with peptides corresponding to that epitope. This study highlights the importance of ligands in the crystallization of proteins. In this case peptides have been used to modulate the solubility of the peptide–IgG complex and may have provided different or additional crystal contacts to create or enhance a crystalline reticulum. Multiple crystal forms characterize this crystallization and the various peptides, differing both in length and sequence, have been used to investigate how such changes affect nucleation and crystal growth.

A large number (33) of human Fab fragments reacting with HIV-1 surface glycoprotein gp120 have been generated by selection from a combinatorial IgG1 kappa library displayed on the surface of phage. The library was prepared from a long term asymptomatic HIV-seropositive donor. Analysis of the sequences from these Fabs shows the heavy chains can be placed in groups, many of which contain intraclonal variants, almost certainly corresponding to chains used in vivo. Further variants can be accessed via chain shuffling experiments in which a given light chain is recombined with a library of heavy chains. Heavy chain promiscuity, i.e. the ability of heavy chains to pair with different light chains with retention of antigen binding, is dependent on the particular heavy chain considered and probably excludes the identification of in vivo light chain partners. The antibodies examined here are primarily to the CD4 binding site on gp120 and broadly reflect the serum profile of the donor. The antibodies show evidence of extensive somatic modification indicative of an antigen-driven response. The heavy chain CDR3 regions of the antibodies show a remarkably conserved extended length. A number also show strong sequence conservation in CDR3 against a background of considerable diversity in the rest of the VH gene supporting a central role for this region in antigen recognition.

Nur77 (also known as TR3 and NGFI-B) is a nuclear orphan receptor that in response to various stimuli can translocate from the nucleus to the mitochondria, bind Bcl-2, and induce a conformational change in Bcl-2 that exposes its BH3 domain. This conformational change of Bcl-2 transforms this protein into a pro-apoptotic molecule that can induce cytochrome c release and apoptosis (Cell 116:527, 2004). Interestingly, in acute myeloid leukemia (AML) cell lines and blasts, Nur77 is absent. We have recently generated a nine amino acid peptide from the Nur77 protein (TR3) that is capable of inducing the same pro-apoptotic conformational change in Bcl-2 that Nur77 elicits (manuscript submitted). We tested the hypothesis that a cell-permeable version of this peptide can induce Bcl-2-dependent apoptosis in AML cells and AML stem cells. Primary AML samples (n=6) were incubated with 20 μM control or TR3 peptide for 24 hrs. Induction of apoptosis was measured as CD34+38−123+ PS/Annexin V+ by multiparametric flow cytometry. Our results demonstrate that the L-enantiomer form of TR3 indeed induces apoptosis in total AML cells (60%, p=0.001) and CD34+38−123+ stem cells (67%, p=0.001), as compared to control peptide. Bax expression did not affect the peptide’s ability to induce apoptosis, as wild-type HCT116 cells and HCT116 cells deficient in Bax showed similar levels of apoptosis after treatment with the TR3 peptide. However, Bcl-2 expression was shown to be critical since lentiviral shRNA ablation of Bcl-2 in KG1 cells completely prevented induction of apoptosis by TR3 peptides. We conclude that the TR3 peptide is a potent inducer of apoptosis that mimics the action of Nur77 on Bcl-2 at the mitochondrial level. These results suggest that the Nur77 peptide or compounds that mimic this peptide may have utility as a novel therapeutic agent against Bcl-2 expressing cancers and leukemias.

TR3 (Nur77) is an orphan member of the steroid/retinoid family of Nuclear Receptors (NRs) that translocates from nucleus to cytosol, where it binds Bcl-2 and converts its phenotype from anti-apoptotic to pro-apoptotic (Li, et al. Science289: 1159, 2000; Lin, et al CELL116: 527, 2004). We surveyed TR3 (Nur77) for interactions with all six human anti-apoptotic Bcl-2-family proteins: Bcl-2, Bcl-XL, Mcl-1, Bcl-W, Bfl-1, and Bcl-B. We observed that Bcl-2, Bfl-1, and Bcl-B interact with TR3 as determined by co-immunoprecipitation assays using lysates from transfected cells and by GST pull down assays using GST-Bcl-2-family fusion proteins. In contrast, Bcl-XL, Bcl-W, and Mcl-1 displayed little or no binding to TR3 (Nur77). Co-localization experiments using fluorescent protein tagging and confocal microscopy corroborated these findings, showing co-localization of a fragment of TR3 (Nur77) that accumulates in cytosol (rather than nucleus) [TR3 lacking the DNA-binding domain [DDBD]) with Bcl-2, Bfl-1, and Bcl-B on mitochondria and other intracellular organelles in intact cells, but not co-localization with Bcl-XL, Mcl-1 or Bcl-W. Co-expression of Bcl-2, Bfl-1, or Bcl-B with TR3DDBD by transfection resulted in robust apoptosis induction, while co-expression of Bcl-XL, Bcl-W, or Mcl-1 with TR3DDBD did not. In contrast to results obtained in TR3DDBD co-expression studies, expressing any of the anti-apoptotic Bcl-2-family proteins (Bcl-2, Bfl-1, Bcl-B, Bcl-XL, Mcl-1, Bcl-W) individually resulted in suppression of apoptosis induced by Staurosporine, illustrating the role of TR3 in converting the phenotypes of Bcl-2, Bfl-1 and Bcl-B from protector to killer. Because binding of TR3 (Nur77) to Bcl-B appeared to be strongest among the Bcl-2-family proteins, we focused on this Bcl-B for additional studies. (Ke, N. et al. J. Biol Chem276: 12481, 2001. Using monospecific antibodies, the endogenous Bcl-B protein was localized in human tissues, revealing predominant expression in plasma cells. Several myeloma cell lines also expressed Bcl-B protein, as determined by immunoblotting. Stimulating myeloma cell line RPMI8226 with ionomycin and phorbol ester TPA (agents that induce TR3 expression and accumulation in cytosol) resulted in association of endogenous TR3 with endogenous Bcl-B, as determined by co-immunoprecipitation experiments. Reducing endogenous Bcl-B levels using small interfering RNA (siRNA) reduced apoptosis induced by transfected TR3DDBD as well as apoptosis induced by a synthetic peptide that mimics TR3. We conclude that cytosolic TR3 (Nur77) interacts selectively with certain anti-apoptotic members of the Bcl-2-family (Bcl-2, Bfl-1, Bcl-B), converting them from protectors to killers. The ability of TR3 (Nur77) to convert Bcl-B suggests a possible novel strategy for triggering apoptosis of Bcl-B-expressing cells, which may be of utility for eradicating long-lived autoantibody-producing plasma cells or for killing malignant myeloma cells. (Supported by NIH GM60554 and SASS Foundation).

The preparation of a table or “key” for determining the several species of beetles of the rhynchophorus genus Calendra [*][1] in the pupa stage has been necessary in order to facilitate the study of the species involved in economic outbreaks. [1]: #fn-2

A B S T R A C T Optical microscopic studies of longitudinal sections of permineralized Rhynia axes from the lower (?) Devonian Rhynie Chert of Scotland have revealed the occurrence of numerous pores, 3-8 ,m in diam, distributed along lateral cell walls in the zone of tissue commonly regarded as phloem. In face view, these pores appear to be composed of circular subunits less than 1 Am in diam. A conductive function for the complex tissue of the Rhynia phloem zone seems evidenced by the occurrence of these pores and by the thin-walled, elongate, tapered IN A RECENT REVIEW ARTICLE, Chaloner (1970) presented a fresh approach toward deciphering major developments in the early evolution of the vascular flora. Abandoning the more traditional practice of attempting to identify the first appearance of the several systematic groups (the "first lycopod," "first fern," etc.), he sought to recognize the earliest securely dated occurrence of various morphological and anatomical features that together provide a composite picture of primitive tracheophytes (e.g., tracheids, stomata, cuticularized epidermis, spores with triradiate marks, etc.). Because of its rare histological preservation in plant fossils, phloem was omitted from Chaloner's analysis. In fact, as Esau (1969) noted in her recent comprehensive monograph, "phloem tissue is so poorly preserved in most fossils that

This corn billbug breeds, so far as known. chiefly below high-tide mark in salt reed grass (Spartina cynosuroides) in maritime marshes along the Atlantic Coast.

SummaryBcl-2 can be converted into a proapoptotic molecule by nuclear receptor Nur77. However, the development of Bcl-2 converters as anticancer therapeutics has not been explored. Here we report the identification of a Nur77-derived Bcl-2-converting peptide with 9 amino acids (NuBCP-9) and its enantiomer, which induce apoptosis of cancer cells in vitro and in animals. The apoptotic effect of NuBCPs and their activation of Bax are not inhibited but rather potentiated by Bcl-2. NuBCP-9 and its enantiomer bind to the Bcl-2 loop, which shares the characteristics of structurally adaptable regions with many cancer-associated and signaling proteins. NuBCP-9s act as molecular switches to dislodge the Bcl-2 BH4 domain, exposing its BH3 domain, which in turn blocks the activity of antiapoptotic Bcl-XL.

A new strategy is advanced for the conformational restriction of peptidyl immunogens. Our approach is to replace putative amide-amide hydrogen bonds with covalent hydrogen-bond mimics. Because on average every other amino acid in a protein engages in this bond, the syntheses of diversely shaped peptides can be contemplated. Synthetic methods for introducing a potential hydrogen-bond mimic into a peptide with α-helical potential is reported and the structural consequences are discussed. The replacement of the hydrogen bond with a chemical link will modify as well as shape the peptide. To explore the consequences of these changes, a potential synthetic vaccine for malaria, the repeating tetrapeptide Asn-Pro-Asn-Ala, was conformationally restricted. Antibodies to the shaped malarial peptide showed a strong cross reaction with Plasmodium falciparum sporozoites.

Under “Description of Pupae,” p. 147, insert the following description to precede C. inaequalis (Say). Calendra mormon (Chttn.) Rostrum with six seta-bearing tubercles only, basal pair on or not on prominences. Wrinkles sometimes present and almost identical about the bases of basal and second tubercles; sometimes smooth or less rough about basals than about second tubercles; meso- and metanotal setae lacking; setae of eighth tergite usually absent; prolateropronotal setae usually, and medio- and spiraculo-pronotal setae regularly, lacking.