A novel agent SL-401 induces anti-myeloma activity by targeting plasmacytoid dendritic cells, osteoclastogenesis and cancer stem-like cells.

Novel therapies for multiple myeloma (MM) can target mechanism(s) in the host-MM bone marrow (BM) microenvironment mediating MM progression and chemoresistance. Our studies showed increased numbers of tumor-promoting, immunosuppressive and drug-resistant plasmacytoid dendritic cells (pDCs) in the MM BM microenvironment. pDC-MM cell interactions upregulate interleukin-3 (IL-3), which stimulates both pDC survival and MM cell growth. Since IL-3 R is highly expressed on pDCs in the MM BM milieu, we here targeted pDCs using a novel IL-3 R-targeted therapeutic SL-401. In both in vitro and in vivo models of MM in its BM milieu, SL-401 decreases viability of pDCs, blocks pDC-induced MM cell growth, and synergistically enhances anti-MM activity of bortezomib and pomalidomide. Besides promoting pDC survival and MM cell growth, IL-3 also mediates progression of osteolytic bone disease in MM. Osteoclast (OCL) progenitor cells express IL-3 R, and we show that SL-401 abrogates monocyte-derived OCL formation and bone resorption. Finally, we show that SL-401 also decreases the viability of IL-3 R-expressing cancer stem-like cells in MM. Overall, our study provides the preclinical basis for clinical trials of SL-401 to block pDC-induced MM cell growth, inhibit osteoclastogenesis and target MM stem-like cell subpopulations to improve patient outcome in MM.

Conformational Sampling of Maltose-transporter Components in Cartesian Collective Variables is Governed by the Low-frequency Normal Modes.

We have studied large-scale conformational transitions in the maltose-binding protein, and the nucleotide binding domains of a maltose-transporter using enhanced conformational sampling in Cartesian collective variables (CVs) with temperature-accelerated molecular dynamics (TAMD), and C(α)-based elastic network normal mode analysis. Significantly, every functional displacement in the TAMD-generated pathways of each protein could be rationalized via a single low-frequency soft mode, while a combination of 2 to 3 low-frequency modes were found to describe the entire conformational change suggesting that collective functional movement in TAMD trajectories is facilitated by the intrinsically accessible low-frequency normal modes. By applying a harmonic potential to facilitate functional motion in TAMD simulations, we also provide a recipe to reproducibly generate structural transitions in both proteins, which can be used to characterize large-scale conformational changes in other biomolecules.

Inactivation of HAUSP in vivo modulates p53 function.

Hausp is a deubiquitinase that has been shown to regulate the p53-Mdm2 pathway. Cotransfection of p53 and Hausp stabilizes p53 through the removal of ubiquitin moieties from polyubiquitinated p53. Interestingly, knockout or RNA interference-mediated knockdown of Hausp in human cells also resulted in the stabilization of p53 due to the destabilization of Mdm2, suggesting a dynamic role of Hausp in p53 activation. To understand the physiological functions of Hausp, we generated hausp knockout mice. Hausp knockout mice die during early embryonic development between embryonic days E6.5 and E7.5. The hausp knockout embryos showed p53 activation, but no apparent increase in apoptosis. Embryonic lethality was caused by a dramatic reduction in proliferation and termination in development, in part due to p53 activation and/or abrogation of p53-independent functions. Although deletion of p53 did not completely rescue the embryonic lethality of the hausp knockout, embryonic development was extended in both hausp and p53 double knockout embryos. These data show that Hausp has a critical role in regulating the p53-Mdm2 pathway.

CHARMM: the biomolecular simulation program.

CHARMM (Chemistry at HARvard Molecular Mechanics) is a highly versatile and widely used molecular simulation program. It has been developed over the last three decades with a primary focus on molecules of biological interest, including proteins, peptides, lipids, nucleic acids, carbohydrates, and small molecule ligands, as they occur in solution, crystals, and membrane environments. For the study of such systems, the program provides a large suite of computational tools that include numerous conformational and path sampling methods, free energy estimators, molecular minimization, dynamics, and analysis techniques, and model-building capabilities. The CHARMM program is applicable to problems involving a much broader class of many-particle systems. Calculations with CHARMM can be performed using a number of different energy functions and models, from mixed quantum mechanical-molecular mechanical force fields, to all-atom classical potential energy functions with explicit solvent and various boundary conditions, to implicit solvent and membrane models. The program has been ported to numerous platforms in both serial and parallel architectures. This article provides an overview of the program as it exists today with an emphasis on developments since the publication of the original CHARMM article in 1983.

The p53--Mdm2--HAUSP complex is involved in p53 stabilization by HAUSP.

The ubiquitin-specific protease HAUSP is a critical component of the p53-Mdm2 pathway by acting as a specific deubiquitinase for both p53 and Mdm2. Recent structural studies have indicated that p53 and Mdm2 bind to the N-terminal TRAF-like domain of HAUSP in a mutually exclusive manner. To understand the mechanism of HAUSP-mediated effects, we have created a p53 mutant that lacks HAUSP binding based on the crystal structure analysis. Indeed, this mutant p53 protein can be degraded by Mdm2 but fails to interact with HAUSP both in vitro and in vivo. Surprisingly, however, we have found that direct interaction between HAUSP and p53 is not absolutely required for it to antagonize efficiently Mdm2-mediated ubiquitination of p53 and that HAUSP is capable of enzymatically functioning in trans on p53 by using Mdm2 as a bridge. Further, we show that a trimeric protein complex containing p53, Mdm2 and HAUSP can exist in vivo, despite mutually exclusive binding, with Mdm2 serving as a binding mediator for p53 and HAUSP. These findings reveal the complication of HAUSP-mediated effects in the p53-Mdm2 interplay. It also has important implications for the development of novel chemotherapeutic compounds aimed at blocking this protein-protein interaction.

ARF-BP1 as a potential therapeutic target.

In this review, we discuss the recent identification of ARF-BP1 (also known as Mule, UREB1, E3(histone), LASU1, and HectH9). ARF-BP1, a HECT domain-containing E3 ubiquitin ligase, interacts with ARF and p53. Its ubiquitin ligase activity is inhibited by ARF. Inactivation of ARF-BP1 stabilised p53 and induced apoptosis. Notably, inactivation of ARF-BP1 also caused cell growth repression in p53-null cells and breast cancer cells with mutant p53. Thus, ARF-BP1 emerges as a novel therapeutic target against cancer regardless of p53 status.

Effects of an S84E mutation of bovine growth hormone in transgenic mice.

The ability of mutant bovine growth hormones (bGH) to serve as either agonist or antagonist has been demonstrated in transgenic mice. We have prepared two transgenic strains of FVB/N mice, one expressing wild-type bGH and a second with a glutamic acid mutation at serine 84 in helix 2. Comparison of their phenotypes to those of nontransgenic littermates indicates that wild-type bGH induces a previously described phenotype for hyper-somatotrophic mice. In contrast, the replacement of the side chain hydroxyl at serine 84 with acetic acid produced a phenotype that expressed bGH at appreciable concentrations, but failed to elicit the phenotype observed with either an agonist or an antagonist of bGH. These results indicate that serine 84 is crucial for the activity of bGH despite this site being distal to the receptor binding surfaces.

Different elements of mini-helix 1 are required for human growth hormone or prolactin action via the prolactin receptor.

Human growth hormone (hGH) and prolactin (hPRL) have a low sequence homology, but both bind and activate hPRL receptors. hGH also binds hGH receptors. hGH has 22 and 20 kDa forms; residues 32-46 have been deleted by alternative RNA splicing to create the smaller form. hGH requires F44 for activity through the hPRL receptor, but not for activity through the hGH receptor. The deletion of F44 from hGH has the same effect as removal of residues 32-46 (approximately 200-fold loss in activity), indicating the importance of F44 in hGH when activating the hPRL receptor. In contrast, when the homologous F50 is deleted from hPRL little or no activity is lost, indicating that this highly conserved phenylalanine is not required for the action of hPRL. Deletion of residues 41-52 (a non-conserved sequence homologous to residues 32-46 of hGH) reduced the activity of hPRL by >14 000-fold. This region is essential for the biological activity of hPRL. As these two proteins have evolved from a common ancestor, they have retained the requirement for this region but need different structural elements to activate hPRL receptors. Such diversity represents an opportunity to fine-tune hormone activity.

Differential effects of zinc on functionally distinct human growth hormone mutations.

Human growth hormone (hGH) binds and activates lactogenic receptors by a sequential receptor dimerization mechanism. The affinity for the first lactogenic receptor is increased due to one zinc molecule linking hGH residues H18 and E174, located in helices 1 and 4, respectively, with two adjacent residues in the lactogenic receptor (D187 and H188). Two functionally unique groups of mutant hGHs have been identified. Addition of 25 microM zinc to lactogenic bioassays differentially affects mutant activities based on which group they belong to. One mutation (G120R) is located within the binding surface of hGH that interacts with the second lactogenic receptor. In the presence of endogenous zinc, G120R reduces the maximal activity of hGH without altering either the agonist or antagonist phases of the bell-shaped dose-response curve. Addition of zinc to this assay further reduces the activity of this protein. In contrast, mutations within a hydrophobic motif in hGH that functionally couples the two lactogenic receptor binding sites decrease the sensitivity (right-shift) of the agonist phase of the dose-response curve without similarly affecting the antagonist phase. The addition of zinc to these lactogenic assays increases the sensitivity (left-shifts) of the dose-response curves, largely negating the effect of these mutations. The effects of zinc differentiate between mutations within these two distinct functional motifs by limiting the pool of potential conformations that are available for binding within either of the receptor binding sites of this ligand.

Functional relationship of serine 90 phosphorylation and the surrounding putative salt bridge in bovine prolactin.

The biological activity of bovine prolactin (PRL) is reduced by in vivo phosphorylation of serine 90 (S90) that is located within a putative N+4 salt bridge (R89 and D93). We substituted hydrophobic, polar, or acidic residues for S90 and/or replaced members of the putative R89/D93 salt bridge to determine if a functional relationship between the putative salt bridge and the phosphorylation could be observed. At position 90 the bulk of the residue was the most important factor in modulating biological activity in either the rat Nb2 cell bioassay or PRL receptor binding. Charge played a smaller role. Replacement of either partner of the salt bridge reduced both biological and binding activities indicating the presence of a salt bridge at this position. The combination of replacing a salt bridge member and substituting glutamic acid at S90 produced greater than additive changes in our experimental endpoints, indicating a functional coupling between the salt bridge and phosphorylation site. We interpret the data to indicate that either in vivo phosphorylation or specific mutations that destabilize the salt bridge impairs biological activity.

Mutating aspartate in the calcium-binding site of alpha-lactalbumin: effects on the protein stability and cation binding.

The residue Asp87, which is in the calcium-binding loop of bovine alpha-lactalbumin (alpha-LA) and provides a side-chain carboxylate oxygen for ligand Ca(II) co-ordination, was substituted by either alanine or asparagine. The physical properties and calcium-binding affinities were monitored by intrinsic fluorescence and circular dichroism spectroscopy. D87A alpha-LA displayed a total loss of rigid tertiary structure, a dramatic loss in secondary structure and negligible calcium affinity [Anderson et al. (1997) Biochemistry, 36, 11648-11654]. On the contrary, D87N alpha-LA displayed native-like secondary structure with a somewhat de-stabilized tertiary structure. When the well-documented N-terminal methionine was enzymatically removed from D87N alpha-LA [Veprintsev et al. (1999) PROTEINS: Struct. Funct. Genet., 37, 65-72], the structure appeared to more closely resemble native alpha-LA. Remarkably, the thermal transition mid-temperature of apo-desMetD87N alpha-LA was approximately 31 degrees C versus native apo- alpha-LA (approximately 25 degrees C), probably due to negative charge 'compensation' in the calcium co-ordination site. On the other hand, the transition mid-temperature of Ca(II)-bound desMetD87N alpha-LA was approximately 57 degrees C versus native alpha-LA (approximately 66 degrees C), which was related to a decreased Ca(II) affinity (K = approximately 2.1 x 10(5) versus approximately 1.7 x 10(7)/M at 40 degrees C, respectively). These results reaffirm that alanine substitution in site specific mutagenesis is not always a prudent choice. Substitutions must be conservative with only minimal changes in functional groups and side-chain volume.

Ligand-protein database: linking protein-ligand complex structures to binding data.

In computational structure-based drug design, the scoring functions are the cornerstones to the success of design/discovery. Many approaches have been explored to improve their reliability and accuracy, leading to three families of scoring functions: force-field-based, knowledge-based, and empirical. The last family is the most widely used in association with docking algorithms because of its speed, even though such empirical scoring functions produce far too many false positives to be fully reliable. In this work, we describe a World Wide Web accessible database that gathers the structural information from known complexes of the PDB with experimental binding data. This database, the Ligand-Protein DataBase (LPDB), is designed to allow the selection of complexes based on various properties of receptors and ligands for the design and parametrization of new scoring functions or to assess and improve existing ones. Moreover, for each complex, a continuum of ligand positions ranging from the crystallographic position to points on the surface of the protein receptor allows an assessment of the energetic behavior of particular scoring functions.

Modeling of the metallo-beta-lactamase from B. fragilis: structural and dynamic effects of inhibitor binding.

The structure and dynamics of an inhibitor-bound complex of the metallo-beta-lactamase from Bacteroides fragilis are studied by using molecular dynamics. A search of the conformational space was performed to obtain three distinct models of the complex, which were then subjected to solvated molecular dynamics. A solvated molecular dynamics study of the apo protein was performed to serve as a baseline for comparison with the bound simulations. We find loop conformation changes due to binding as well as a decrease in flexibility of the protein as a whole and especially in the major loop of the beta-lactamase. We report the structural and dynamical features of the inhibitor-bound and apo models, as well as experimentally measurable quantities, which should be capable of distinguishing the two binding modes we have determined.

Accurate reconstruction of all-atom protein representations from side-chain-based low-resolution models.

A procedure for the reconstruction of all-atom protein structures from side-chain center-based low-resolution models is introduced and applied to a set of test proteins with high-resolution X-ray structures. The accuracy of the rebuilt all-atom models is measured by root mean square deviations to the corresponding X-ray structures and percentages of correct chi(1) and chi(2) side-chain dihedrals. The benefit of including C(alpha) positions in the low-resolution model is examined, and the effect of lattice-based models on the reconstruction accuracy is discussed. Programs and scripts implementing the reconstruction procedure are made available through the NIH research resource for Multiscale Modeling Tools in Structural Biology (http://mmtsb.scripps.edu).

Zinc binding in bovine alpha-lactalbumin: sequence homology may not be a predictor of subtle functional features.

alpha-Lactalbumin (alpha-LA), a calcium-binding protein, also possesses zinc-binding sites comprising a single strong site and several weaker secondary sites. The only site found by X-ray crystallography (Ren et. al., J. Biol. Chem. 1993;268:19292) was Glu 49 of human alpha-LA, but zinc binding had never been measured in solution for human alpha-LA. This residue was genetically substituted by Ala in bovine alpha-LA and the metal-binding properties of the resulting desMetE49A protein were compared with those for native alpha-LA by fluorescence methods. Surprisingly, desMetE49A alpha-LA and the native bovine protein had similar affinities for both Zn(2+) and Ca(2+). Genetic substitution of other possible candidates for Zn(2+) chelating residues, which included Glu 25, did not alter the affinity of bovine alpha-LA to Zn2+; however, substitution of Glu 1 by Met resulted in the disappearance of strong Zn(2+) binding. A proposed site involves Glu 1, Glu 7, Asp 11, and Asp 37, which would participate in strong Zn(2+) binding based on their propinquity to Glu 1. Human alpha-LA, which has a Lys at position 1 rather than Glu, binds zinc with a reduced affinity compared with native bovine alpha-LA, suggesting that the site identified from the X-ray structure did not correspond to strong zinc binding in solution.

The species specificity of growth hormone requires the cooperative interaction of two motifs.

Primate growth hormones (GH) activate both primate and non-primate somatotrophic receptors (GH receptors), but non-primate GHs do not activate primate GH receptors. Previous studies argued the interaction of Asp(171) of human GH and Arg(43) of the receptor produced an attractive ionic interaction. In non-primate GHs, His(170) replaces the homologous Asp(171), producing a repulsive interaction with Arg(43) of the primate receptor which was believed to reduce the attraction of non-primate GH for the human GH receptor, thus providing species specificity. In this report, H170D bovine GH had activity and affinity for human GH receptors approaching those of human GH. In contrast, replacing Asp(171) of human GH with His did not significantly reduce somatotrophic activity, indicating that species specificity is not wholly explained by this residue's interaction with Arg(43) of the receptor. Deletion of either Phe(44) (a residue present only in primate GHs) or residues 32-46 (20-kDa form of human GH) each only marginally reduced somatotrophic activities. But the combination of the D171H mutation with either DeltaPhe(44) or Delta32-46 in human GH reduced binding and activity in a greater than additive fashion, indicated a functional interaction between these distant structural features. In bovine GH addition of phenylalanine at position 44 increased the somatotrophic activity and receptor affinity in cells containing the human GH receptor. The combination of the H170D mutation and the addition of phenylalanine at position 44 created a bovine GH with activity indistinguishable from wild-type human GH. Based on evidence from both bovine and human GHs, the cooperative interaction of these two distant motifs determined the species specificity and indicated that structural plasticity was a critical feature necessary for the species specificity of somatotrophic activity.

Enhanced immunogenicity of a conformational epitope of human T-lymphotropic virus type 1 using a novel chimeric peptide.

The ability of a peptide vaccine derived from the human T-lymphotropic virus type 1 (HTLV-1) surface envelope glycoprotein protein (gp46) to mimic the native protein and elicit a protective immune response has been examined. This peptide construct, designated MVFMF2, comprises amino acids (aa) 175-218 of gp46 linked by a four residue turn (GPSL) to a promiscuous T-cell epitope from the measles virus fusion protein (MVF, aa 288-302). The peptide was structurally characterized by circular dichroism (CD) spectroscopy and was found to contain alpha-helical secondary structure. The immunogenicity of MVFMF2 in rabbits and mice was evaluated by direct ELISA and competitive ELISA using peptide constructs and the recombinant protein ACH-RE3 (aa 165-306). This peptide, when administered with adjuvant (N-acetyl-glucosamine-3yl-acetyl-L-alanyl-D-isoglutamine, nor-MDP) was immunogenic in an outbred population of both rabbits and mice. Furthermore, the peptide construct was encapsulated in biodegradable microspheres of poly(D,L-lactide-co-glycolide) to eliminate booster immunization and to examine adjuvant requirements. The data indicate that MVFMF2 shows enhanced immunogenicity when encapsulated in biodegradable microspheres. Inoculation of the encapsulated peptide produced a similar humoral response to that of the free peptide, but did not require the use of adjuvant. Elicited anti-rabbit and anti-mouse antibodies recognized whole viral preparations and the recombinant protein ACH-RE3 in ELISA assays. Additionally, inoculated rabbits exhibited enhanced reactivity to viral antigens by western blot compared to non-vaccinated controls. Although anti-rabbit and anti-mouse antibodies were capable of inhibiting syncytium formation at low dilutions, rabbits were not protected from cell-associated viral challenge. Future development of vaccines to HTLV-1 may need to incorporate the ability to elicit cell-mediated immune responses in order to protect against cell-associated viral infection.