Inhibition of the histone demethylase, KDM5B, directly induces re-expression of tumor suppressor protein HEXIM1 in cancer cells.

BACKGROUND: The tumor suppressor actions of hexamethylene bis-acetamide (HMBA)-inducible protein 1 (HEXIM1) in the breast, prostate, melanomas, and AML have been reported by our group and others. Increased HEXIM1 expression caused differentiation and inhibited proliferation and metastasis of cancer cells. Historically, HEXIM1 has been experimentally induced with the hybrid polar compound HMBA, but HMBA is a poor clinical candidate due to lack of a known target, poor pharmacological properties, and unfavorable ADMETox characteristics. Thus, HEXIM1 induction is an intriguing therapeutic approach to cancer treatment, but requires better chemical tools than HMBA. METHODS: We identified and verified KDM5B as a target of HEXIM1 inducers using a chemical proteomics approach, biotin-NeutrAvidin pull-down assays, surface plasmon resonance, and molecular docking. The regulation of HEXIM1 by KDM5B and KDM5B inhibitors was assessed using chromatin immunoprecipitation assays, RT-PCR, western blotting, and depletion of KDM5B with shRNAs. The regulation of breast cancer cell phenotype by KDM5B inhibitors was assessed using western blots, differentiation assays, proliferation assays, and a mouse model of breast cancer metastasis. The relative role of HEXIM1 in the action of KDM5B inhibitors was determined by depleting HEXIM1 using shRNAs followed by western blots, differentiation assays, and proliferation assays. RESULTS: We have identified a highly druggable target, KDM5B, which is inhibited by small molecule inducers of HEXIM1. RNAi knockdown of KDM5B induced HEXIM1 expression, thus validating the specific negative regulation of tumor suppressor HEXIM1 by the H3K4me3/2 demethylase KDM5B. Known inhibitors of KDM5B were also able to induce HEXIM1 expression, inhibit cell proliferation, induce differentiation, potentiate sensitivity to cancer chemotherapy, and inhibit breast tumor metastasis. CONCLUSION: HMBA and 4a1 induce HEXIM1 expression by inhibiting KDM5B. Upregulation of HEXIM1 expression levels plays a critical role in the inhibition of proliferation of breast cancer cells using KDM5B inhibitors. Based on the novel molecular scaffolds that we identified which more potently induced HEXIM1 expression and data in support that KDM5B is a target of these compounds, we have opened up new lead discovery and optimization directions.

Multiple glycosaminoglycan-binding epitopes of monocyte chemoattractant protein-3/CCL7 enable it to function as a non-oligomerizing chemokine.

The interaction of chemokines with glycosaminoglycans (GAGs) facilitates the formation of localized chemokine gradients that provide directional signals for migrating cells. In this study, we set out to understand the structural basis and impact of the differing oligomerization propensities of the chemokines monocyte chemoattractant protein (MCP)-1/CCL2 and MCP-3/CCL7 on their ability to bind GAGs. These chemokines provide a unique comparison set because CCL2 oligomerizes and oligomerization is required for its full in vivo activity, whereas CCL7 functions as a monomer. To identify the GAG-binding determinants of CCL7, an unbiased hydroxyl radical footprinting approach was employed, followed by a focused mutagenesis study. Compared with the size of the previously defined GAG-binding epitope of CCL2, CCL7 has a larger binding site, consisting of multiple epitopes distributed along its surface. Furthermore, surface plasmon resonance (SPR) studies indicate that CCL7 is able to bind GAGs with an affinity similar to CCL2 but higher than the non-oligomerizing variant, CCL2(P8A), suggesting that, in contrast to CCL2, the large cluster of GAG-binding residues in CCL7 renders oligomerization unnecessary for high affinity binding. However, the affinity of CCL7 is more sensitive than CCL2 to the density of heparan sulfate on the SPR surfaces; this is likely due to the inability of CCL7 to oligomerize because CCL2(P8A) also binds significantly less tightly to low than high density heparan sulfate surfaces compared with CCL2. Together, the data suggest that CCL7 and CCL2 are non-redundant chemokines and that GAG chain density may provide a mechanism for regulating the accumulation of chemokines on cell surfaces.

Circulating human CD4 and CD8 T cells do not have large intracellular pools of CCR5.

CC Chemokine Receptor 5 (CCR5) is an important mediator of chemotaxis and the primary coreceptor for HIV-1. A recent report by other researchers suggested that primary T cells harbor pools of intracellular CCR5. With the use of a series of complementary techniques to measure CCR5 expression (antibody labeling, Western blot, quantitative reverse transcription polymerase chain reaction), we established that intracellular pools of CCR5 do not exist and that the results obtained by the other researchers were false-positives that arose because of the generation of irrelevant binding sites for anti-CCR5 antibodies during fixation and permeabilization of cells.

Structural analysis of proinsulin hexamer assembly by hydroxyl radical footprinting and computational modeling.

Mutations in the insulin gene can impair proinsulin folding and cause diabetes mellitus. Although crystal structures of insulin dimers and hexamers are well established, proinsulin is refractory to crystallization. Although an NMR structure of an engineered proinsulin monomer has been reported, structures of the wild-type monomer and hexamer remain undetermined. We have utilized hydroxyl radical footprinting and molecular modeling to characterize these structures. Differences between the footprints of insulin and proinsulin, defining a "shadow" of the connecting (C) domain, were employed to refine the model. Our results demonstrate that in its monomeric form, (i) proinsulin contains a native-like insulin moiety and (ii) the C-domain footprint resides within an adjoining segment (residues B23-B29) that is accessible to modification in insulin but not proinsulin. Corresponding oxidation rates were observed within core insulin moieties of insulin and proinsulin hexamers, suggesting that the proinsulin hexamer retains an A/B structure similar to that of insulin. Further similarities in rates of oxidation between the respective C-domains of proinsulin monomers and hexamers suggest that this loop in each case flexibly projects from an outer surface. Although dimerization or hexamer assembly would not be impaired, an ensemble of predicted C-domain positions would block hexamer-hexamer stacking as visualized in classical crystal lattices. We anticipate that protein footprinting in combination with modeling, as illustrated here, will enable comparative studies of diabetes-associated mutant proinsulins and their aberrant modes of aggregation.

Integrated algorithms for high-throughput examination of covalently labeled biomolecules by structural mass spectrometry.

Mass spectrometry based structural proteomics approaches for probing protein structures are increasingly gaining in popularity. The potential for such studies is limited because of the lack of analytical techniques for the automated interpretation of resulting data. In this article, a suite of algorithms called ProtMapMS is developed, integrated, and implemented specifically for the comprehensive automatic analysis of mass spectrometry data obtained for protein structure studies using covalent labeling. The functions include data format conversion, mass spectrum interpretation, detection, and verification of all peptide species, confirmation of the modified peptide products, and quantification of the extent of peptide modification. The results thus obtained provide valuable data for use in combination with computational approaches for protein structure modeling. The structures of both monomeric and hexameric forms of insulin were investigated by oxidative protein footprinting followed by high-resolution mass spectrometry. The resultant data was analyzed both manually and using ProtMapMS without any manual intervention. The results obtained using the two methods were found to be in close agreement and overall were consistent with predictions from the crystallographic structure.

Modification of ß-Defensin-2 by Dicarbonyls Methylglyoxal and Glyoxal Inhibits Antibacterial and Chemotactic Function In Vitro.

BACKGROUND: Beta-defensins (hBDs) provide antimicrobial and chemotactic defense against bacterial, viral and fungal infections. Human ß-defensin-2 (hBD-2) acts against gram-negative bacteria and chemoattracts immature dendritic cells, thus regulating innate and adaptive immunity. Immunosuppression due to hyperglycemia underlies chronic infection in Type 2 diabetes. Hyperglycemia also elevates production of dicarbonyls methylgloxal (MGO) and glyoxal (GO). METHODS: The effect of dicarbonyl on defensin peptide structure was tested by exposing recombinant hBD-2 (rhBD-2) to MGO or GO with subsequent analysis by MALDI-TOF MS and LC/MS/MS. Antimicrobial function of untreated rhBD-2 vs. rhBD-2 exposed to dicarbonyl against strains of both gram-negative and gram-positive bacteria in culture was determined by radial diffusion assay. The effect of dicarbonyl on rhBD-2 chemotactic function was determined by chemotaxis assay in CEM-SS cells. RESULTS: MGO or GO in vitro irreversibly adducts to the rhBD-2 peptide, and significantly reduces antimicrobial and chemotactic functions. Adducts derive from two arginine residues, Arg22 and Arg23 near the C-terminus, and the N-terminal glycine (Gly1). We show by radial diffusion testing on gram-negative E. coli and P. aeruginosa, and gram-positive S. aureus, and a chemotaxis assay for CEM-SS cells, that antimicrobial activity and chemotactic function of rhBD-2 are significantly reduced by MGO. CONCLUSIONS: Dicarbonyl modification of cationic antimicrobial peptides represents a potential link between hyperglycemia and the clinical manifestation of increased susceptibility to infection, protracted wound healing, and chronic inflammation in undiagnosed and uncontrolled Type 2 diabetes.

Future directions of structural mass spectrometry using hydroxyl radical footprinting.

Hydroxyl radical protein footprinting coupled to mass spectrometry has been developed over the last decade and has matured to a powerful method for analyzing protein structure and dynamics. It has been successfully applied in the analysis of protein structure, protein folding, protein dynamics, and protein-protein and protein-DNA interactions. Using synchrotron radiolysis, exposure of proteins to a 'white' X-ray beam for milliseconds provides sufficient oxidative modification to surface amino acid side chains, which can be easily detected and quantified by mass spectrometry. Thus, conformational changes in proteins or protein complexes can be examined using a time-resolved approach, which would be a valuable method for the study of macromolecular dynamics. In this review, we describe a new application of hydroxyl radical protein footprinting to probe the time evolution of the calcium-dependent conformational changes of gelsolin on the millisecond timescale. The data suggest a cooperative transition as multiple sites in different molecular subdomains have similar rates of conformational change. These findings demonstrate that time-resolved protein footprinting is suitable for studies of protein dynamics that occur over periods ranging from milliseconds to seconds. In this review, we also show how the structural resolution and sensitivity of the technology can be improved as well. The hydroxyl radical varies in its reactivity to different side chains by over two orders of magnitude, thus oxidation of amino acid side chains of lower reactivity are more rarely observed in such experiments. Here we demonstrate that the selected reaction monitoring (SRM)-based method can be utilized for quantification of oxidized species, improving the signal-to-noise ratio. This expansion of the set of oxidized residues of lower reactivity will improve the overall structural resolution of the technique. This approach is also suggested as a basis for developing hypothesis-driven structural mass spectrometry experiments.

Ribosomal S6 kinase 2 directly phosphorylates the 5-hydroxytryptamine 2A (5-HT2A) serotonin receptor, thereby modulating 5-HT2A signaling.

The 5-hydroxytryptamine 2A (5-HT(2A)) receptor is a member of the G protein-coupled receptor superfamily (GPCR) and plays a key role in transducing a variety of cellular signals elicited by 5-hydroxytryptamine in both peripheral and central tissues. Despite its broad physiological importance, our current understanding of 5-HT(2A) receptor regulation is incomplete. We recently reported the novel finding that the multifunctional ERK effector ribosomal S6 kinase 2 (RSK2) physically interacts with the 5-HT(2A) receptor third intracellular (i3) loop and modulates receptor signaling (Sheffler, D. J., Kroeze, W. K., Garcia, B. G., Deutch, A. Y., Hufeisen, S. J., Leahy, P., Bruning, J. C., and Roth, B. L. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 4717-4722). We report here that RSK2 directly phosphorylates the 5-HT(2A) receptor i3 loop at the conserved residue Ser-314, thereby modulating 5-HT(2A) receptor signaling. Furthermore, these studies led to the discovery that RSK2 is required for epidermal growth factor-mediated heterologous desensitization of the 5-HT(2A) receptor. We arrived at these conclusions via multiple lines of evidence, including in vitro kinase experiments, tandem mass spectrometry, and site-directed mutagenesis. Our findings were further validated using phospho-specific Western blot analysis, metabolic labeling studies, and whole-cell signaling experiments. These results support a novel regulatory mechanism in which a downstream effector of the ERK/MAPK pathway directly interacts with, phosphorylates, and modulates signaling of the 5-HT(2A) serotonin receptor. To our knowledge, these findings are the first to demonstrate that a downstream member of the ERK/MAPK cascade phosphorylates a GPCR as well as mediates cross-talk between a growth factor and a GPCR.

Visualizing Arp2/3 complex activation mediated by binding of ATP and WASp using structural mass spectrometry.

Actin-related protein (Arp) 2/3 complex nucleates new branches in actin filaments playing a key role in controlling eukaryotic cell motility. This process is tightly regulated by activating factors: ATP and WASp-family proteins. However, the mechanism of activation remains largely hypothetical. We used radiolytic protein footprinting with mass spectrometry in solution to probe the effects of nucleotide- and WASp-binding on Arp2/3. These results represent two significant advances in such footprinting approaches. First, Arp2/3 is the most complex macromolecular assembly yet examined; second, only a few picomoles of Arp2/3 was required for individual experiments. In terms of structural biology of Arp 2/3, we find that ATP binding induces conformational changes within Arp2/3 complex in Arp3 (localized in peptide segments 5-18, 212-225, and 318-327) and Arp2 (within peptide segment 300-316). These data are consistent with nucleotide docking within the nucleotide clefts of the actin-related proteins promoting closure of the cleft of the Arp3 subunit. However, ATP binding does not induce conformational changes in the other Arp subunits. Arp2/3 complex binds to WASp within the C subdomain at residue Met 474 and within the A subdomain to Trp 500. Our data suggest a bivalent attachment of WASp to Arp3 (within peptides 162-191 and 318-329) and Arp2 (within peptides 66-80 and 87-97). WASp-dependent protections from oxidation within peptides 54-65 and 80-91 of Arp3 and in peptides 300-316 of Arp2 suggest domain rearrangements of Arp2 and Arp3 resulting in a closed conformational state consistent with an "actin-dimer" model for the active state.

Hydroxyl radical probe of the surface of lysozyme by synchrotron radiolysis and mass spectrometry.

A new approach is reported that combines synchrotron radiolysis and mass spectrometry to probe the surface of proteins. Hydroxyl radicals produced upon the radiolysis of protein solutions with synchrotron light for several milliseconds result in the reaction of amino acid side chains. This results in the formation of stable oxidation products where the level of oxidation at the reactive residues is influenced by the accessibility of their side chains to the bulk solvent. The aromatic and sulfur-containing residues have been found to react preferentially in accord with previous peptide studies. The sites of oxidation have been determined by tandem mass spectrometry. The rate of oxidation at these reactive markers has been measured for each of the proteolytic peptides as a function of exposure time based on the relative proportion of modified and unmodified peptide ions detected by mass spectrometry. Oxidation rates have been found to correlate closely with a theoretical measure of the accessibility of residue side chains to the bulk solvent in the native protein structure. The synchrotron-based approach is able to distinguish the relative accessibility of the tryptophan residue side chains of lysozyme at positions 62 and 123 from each other and all other tryptophan residues based on their rates of oxidation.

Visualizing the Ca2+-dependent activation of gelsolin by using synchrotron footprinting.

Radiolytic protein footprinting with a synchrotron source is used to reveal detailed structural changes that occur in the Ca(2+)-dependent activation of gelsolin. More than 80 discrete peptides segments within the structure, covering 95% of the sequence in the molecule, were examined by footprinting and mass spectrometry for their solvent accessibility as a function of Ca(2+) concentration in solution. Twenty-two of the peptides exhibited detectable oxidation; for seven the oxidation extent was seen to be Ca(2+) sensitive. Ca(2+)titration isotherms monitoring the oxidation within residues 49-72 (within subdomain S1), 121-135 (S1), 162-166 (S2), and 722-748 (S6) indicate a three-state activation process with a intermediate that was populated at a Ca(2+) concentration of 1-5 microM that is competent for capping and severing activity. A second structural transition with a midpoint of approximately 60-100 microM, where the accessibility of the above four peptides is further increased, is also observed. Tandem mass spectrometry showed that buried residues within the helical "latch" of S6 (including Pro-745) that contact an F-actin-binding site on S2 and buried F-actin-binding residues within S2 (including Phe-163) are unmasked in the submicromolar Ca(2+) transition. However, residues within S4 that are part of an extended beta-sheet with S6 (including Tyr-453) are revealed only in the subsequent transition at higher Ca(2+) concentrations; the disruption of this extended contact between S4 and S6 (and likely the analogous contact between S1 and S3) likely results in an extended structure permitting additional functions consistent with the fully activated gelsolin molecule.

Structural analysis of gelsolin using synchrotron protein footprinting.

Protein footprinting provides detailed structural information on protein structure in solution by directly identifying accessible and hydroxyl radical-reactive side chain residues. Radiolytic generation of hydroxyl radicals using millisecond pulses of a synchrotron "white" beam results in the formation of stable side chain oxidation products, which can be digested with proteases for mass spectrometry (MS) analysis. Liquid chromatography-coupled MS and tandem MS methods allow for the quantitation of the ratio of modified and unmodified peptides and identify the specific side chain probes that are oxidized, respectively. The ability to monitor the changes in accessibility of multiple side chain probes by monitoring increases or decreases in their oxidation rates as a function of ligand binding provides an efficient and powerful tool for analyzing protein structure and dynamics. In this study, we probe the detailed structural features of gelsolin in its "inactive" and Ca2+-activated state. Oxidation rate data for 81 peptides derived from the trypsin digestion of gelsolin are presented; 60 of these peptides were observed not to be oxidized, and 21 had detectable oxidation rates. We also report the Ca2+-dependent changes in oxidation for all 81 peptides. Fifty-nine remained unoxidized, five increased their oxidation rate, and two experienced protections. Tandem mass spectrometry was used to identify the specific side chain probes responsible for the Ca2+-insensitive and Ca2+-dependent responses. These data are consistent with crystallographic data for the inactive form of gelsolin in terms of the surface accessibility of reactive residues within the protein. The results demonstrate that radiolytic protein footprinting can provide detailed structural information on the conformational dynamics of ligand-induced structural changes, and the data provide a detailed model for gelsolin activation.