Lisa Tucker-Kellogg

De novo determination of peptide structure with solid-state magic-angle spinning NMR spectroscopy

The three-dimensional structure of the chemotactic peptide N-formyl-l-Met-l-Leu-l-Phe-OH was determined by using solid-state NMR (SSNMR). The set of SSNMR data consisted of 16 13C–15N distances and 18 torsion angle constraints (on 10 angles), recorded from uniformly 13C,15N- and 15N-labeled samples. The peptides structure was calculated by means of simulated annealing and a newly developed protocol that ensures that all of conformational space, consistent with the structural constraints, is searched completely. The result is a high-quality structure of a molecule that has thus far not been amenable to single-crystal diffraction studies. The extensions of the SSNMR techniques and computational methods to larger systems appear promising.

Engrailed (Gln50→Lys) homeodomain–DNA complex at 1.9 Å resolution: structural basis for enhanced affinity and altered specificity

BACKGROUND The homeodomain is one of the key DNA-binding motifs used in eukaryotic gene regulation, and homeodomain proteins play critical roles in development. The residue at position 50 of many homeodomains appears to determine the differential DNA-binding specificity, helping to distinguish among binding sites of the form TAATNN. However, the precise role(s) of residue 50 in the differential recognition of alternative sites has not been clear. None of the previously determined structures of homeodomain-DNA complexes has shown evidence for a stable hydrogen bond between residue 50 and a base, and there has been much discussion, based in part on NMR studies, about the potential importance of water-mediated contacts. This study was initiated to help clarify some of these issues. RESULTS The crystal structure of a complex containing the engrailed Gln50-->Lys variant (QK50) with its optimal binding site TAATCC (versus TAATTA for the wild-type protein) has been determined at 1.9 A resolution. The overall structure of the QK50 variant is very similar to that of the wild-type complex, but the sidechain of Lys50 projects directly into the major groove and makes several hydrogen bonds to the O6 and N7 atoms of the guanines at base pairs 5 and 6. Lys50 also makes an additional water-mediated contact with the guanine at base pair 5 and has an alternative conformation that allows a hydrogen bond with the O4 of the thymine at base pair 4. CONCLUSIONS The structural context provided by the folding and docking of the engrailed homeodomain allows Lys50 to make remarkably favorable contacts with the guanines at base pairs 5 and 6 of the binding site. Although many different residues occur at position 50 in different homeodomains, and although numerous position 50 variants have been constructed, the most striking examples of altered specificity usually involve introducing or removing a lysine sidechain from position 50. This high-resolution structure also confirms the critical role of Asn51 in homeodomain-DNA recognition and further clarifies the roles of water molecules near residues 50 and 51.

Cell-delivery therapeutics for liver regeneration☆

For acute, chronic, or hereditary diseases of the liver, cell transplantation therapies can stimulate liver regeneration or serve as a bridge until liver transplantation can be performed. Recently, fetal hepatocytes, stem cells, liver progenitor cells, or other primitive and proliferative cell types have been employed for cell transplantation therapies, in an effort to improve the survival, proliferation, and engraftment of the transplanted cells. Reviewing earlier studies, which achieved success by transplanting mature hepatocytes, we propose that there is a switch-like regulation of liver regeneration that changes state according to a stimulus threshold of extracellular influences such as cytokines, matrices and neighboring cells. Important determinants of a successful clinical outcome include sufficient quantities and functional levels of the transplanted cells (even for short periods to alter the environment), rather than just engraftment levels or survival durations of the exogenously transplanted cells. The relative importance of these determining factors will impact future choices of cell sources, delivery vehicles, and sites of cell transplantation to stimulate liver regeneration for patients with severe liver diseases.

The synergy in cytokine production through MyD88-TRIF pathways is co-ordinated with ERK phosphorylation in macrophages.

Although specific single Toll‐like receptor (TLR) ligands are known to drive the development of Th1 or Th2 immunity, the outcome of different combinations of TLR ligands on innate immunity is not well defined. Spatiotemporal dynamics are critical in determining the specificity of the immune response, but the mechanisms underlying combinatorial TLR stimulation remain unclear. Here, we tested pairwise combinations of TLR ligands separated by different time intervals for their effect on cytokine production in macrophages. We observed that stimulation via a combination of MyD88‐ and TRIF‐utilizing adaptors leads to a highly synergistic cytokine response. On a timescale of 4–24 h, macrophages pretreated with poly(I:C) (TLR3 ligand) are cross‐primed to a second stimulation with R848 (TLR7 ligand) and vice versa, and each condition exhibits different optimal time windows of synergistic response for each cytokine. We show that the synergy resulting from combinatorial stimuli (poly(I:C) and R848 is also regulated by the order and dosage of the TLR agonists. Secondary response genes, which depend on new protein synthesis for transcription, show greater synergy than primary response genes, and such enhancement is abolished when new protein synthesis is inhibited. Synergistic cytokine production appears concordant with sustained ERK phosphorylation, suggesting that the de novo factors act via inhibition of ERK dephosphorylation, for example, by the downregulation of dual specificity phosphatase 6. Taken together, our findings illustrate a checkpoint in the innate immune system, where the synchronization of timing of both MyD88 and TRIF pathways is required for a maximal cytokine response and potential memory effect in macrophages.

HGF regulates the activation of TGF-β1 in rat hepatocytes and hepatic stellate cells.

Hepatocyte growth factor (HGF) ameliorates experimental liver fibrosis through many mechanisms, including degradation of accumulated collagen and decreased expression of fibrotic genes. Investigating an upstream mechanism in which HGF could decrease many fibrotic effectors, we asked whether HGF regulates activation of the fibrotic cytokine transforming growth factor‐beta 1 (TGF‐β1). Specifically, we tested whether HGF decreases the levels of active TGF‐β1, and whether such decrease depends on the predominantly hepatocyte‐secreted protease plasmin, and whether it depends on the TGF‐β1 activator thrombospondin‐1 (TSP‐1). With hepatocyte monocultures, we found HGF‐induced hepatocyte proliferation did increase total levels of plasmin, while decreasing gene expression of fibrotic markers (PAI‐1, TGF‐β1, and TIMP‐2). With in vitro models of fibrotic liver (HSC‐T6 hepatic stellate cells, or co‐cultures of HSC‐T6 and hepatocytes), we found high levels of fibrosis‐associated proteins such as TSP‐1, active TGF‐β1, and Collagen I. HGF treatment on these fibrotic cultures stimulated plasmin levels; increased TSP‐1 protein cleavage; and decreased the levels of active TGF‐β1 and Collagen I. When plasmin was blocked by the inhibitor aprotinin, HGF could no longer decrease TGF‐β1 activation and Collagen I. Meanwhile, the TSP‐1‐specific peptide inhibitor, LSKL, reduced TGF‐β1 to the same level as in the HGF‐treated cultures; combining LSKL and HGF treatments caused no further decrease, suggesting that HGF affects the TSP‐1 dependent pathway of TGF‐β1 activation. Therefore, HGF can decrease TGF‐β1 activation and TGF‐β1‐dependent fibrotic markers, by stimulating hepatocytes to produce plasmin, and by antagonizing TSP‐1‐dependent activation of TGF‐β1. J. Cell. Physiol. 228: 393–401, 2013.

Delineation of Lipopolysaccharide (LPS)-binding Sites on Hemoglobin FROM IN SILICO PREDICTIONS TO BIOPHYSICAL CHARACTERIZATION

Background: Redox activity of hemoglobin (Hb) is augmented by lipopolysaccharide (LPS) to boost immune defense. Results: Computational analysis of Hb identified LPS-binding hot spots, which were further defined via peptide-based binding assays and confirmed by mutagenesis of Hb subunits. Conclusion: Regions of Hb that interact with LPS have been delineated. Significance: Knowledge of LPS-binding residues on Hb may be exploited for designing antimicrobial peptides. Hemoglobin (Hb) functions as a frontline defense molecule during infection by hemolytic microbes. Binding to LPS induces structural changes in cell-free Hb, which activates the redox activity of the protein for the generation of microbicidal free radicals. Although the interaction between Hb and LPS has implications for innate immune defense, the precise LPS-interaction sites on Hb remain unknown. Using surface plasmon resonance, we found that both the Hb α and β subunits possess high affinity LPS-binding sites, with KD in the nanomolar range. In silico analysis of Hb including phospho-group binding site prediction, structure-based sequence comparison, and docking to model the protein-ligand interactions showed that Hb possesses evolutionarily conserved surface cationic patches that could function as potential LPS-binding sites. Synthetic Hb peptides harboring predicted LPS-binding sites served to validate the computational predictions. Surface plasmon resonance analysis differentiated LPS-binding peptides from non-binders. Binding of the peptides to lipid A was further substantiated by a fluorescent probe displacement assay. The LPS-binding peptides effectively neutralized the endotoxicity of LPS in vitro. Additionally, peptide B59 spanning residues 59–95 of Hbβ attached to the surface of Gram-negative bacteria as shown by flow cytometry and visualized by immunogold-labeled scanning electron microscopy. Site-directed mutagenesis of the Hb subunits further confirmed the function of the predicted residues in binding to LPS. In summary, the integration of computational predictions and biophysical characterization has enabled delineation of multiple LPS-binding hot spots on the Hb molecule.

Membrane tension controls adhesion positioning at the leading edge of cells

Cell migration is dependent on adhesion dynamics and actin cytoskeleton remodeling at the leading edge. These events may be physically constrained by the plasma membrane. Here, we show that the mechanical signal produced by an increase in plasma membrane tension triggers the positioning of new rows of adhesions at the leading edge. During protrusion, as membrane tension increases, velocity slows, and the lamellipodium buckles upward in a myosin II–independent manner. The buckling occurs between the front of the lamellipodium, where nascent adhesions are positioned in rows, and the base of the lamellipodium, where a vinculin-dependent clutch couples actin to previously positioned adhesions. As membrane tension decreases, protrusion resumes and buckling disappears, until the next cycle. We propose that the mechanical signal of membrane tension exerts upstream control in mechanotransduction by periodically compressing and relaxing the lamellipodium, leading to the positioning of adhesions at the leading edge of cells.

Hepatic Stellate Cell–Targeted Delivery of Hepatocyte Growth Factor Transgene via Bile Duct Infusion Enhances Its Expression at Fibrotic Foci to Regress Dimethylnitrosamine-Induced Liver Fibrosis

Liver fibrosis generates fibrotic foci with abundant activated hepatic stellate cells and excessive collagen deposition juxtaposed with healthy regions. Targeted delivery of antifibrotic therapeutics to hepatic stellate cells (HSCs) might improve treatment outcomes and reduce adverse effects on healthy tissue. We delivered the hepatocyte growth factor (HGF) gene specifically to activated hepatic stellate cells in fibrotic liver using vitamin A-coupled liposomes by retrograde intrabiliary infusion to bypass capillarized hepatic sinusoids. The antifibrotic effects of DsRed2-HGF vector encapsulated within vitamin A-coupled liposomes were validated by decreases in fibrotic markers in vitro. Fibrotic cultures transfected with the targeted transgene showed a significant decrease in fibrotic markers such as transforming growth factor-β1. In rats, dimethylnitrosamine-induced liver fibrosis is manifested by an increase in collagen deposition and severe defenestration of sinusoidal endothelial cells. The HSC-targeted transgene, administered via retrograde intrabiliary infusion in fibrotic rats, successfully reduced liver fibrosis markers alpha-smooth muscle actin and collagen, accompanied by an increase in the expression of DsRed2-HGF near the fibrotic foci. Thus, targeted delivery of HGF gene to hepatic stellate cells increased the transgene expression at the fibrotic foci and strongly enhanced its antifibrotic effects.

FLIP: A flop for execution signals

Resistance to apoptosis is one of the established hallmarks of cancer cells. This is a function of an imbalance between the proteins that facilitate death execution and those that inhibit apoptosis or promote cell proliferation. The anti-apoptotic protein, FLICE inhibitory protein (FLIP), first identified as a viral protein, is over-expressed in a variety of human pathologies. Initial observations linked FLIP expression to inhibition of death receptor induced apoptosis, due to its structural homology to the cysteine protease, caspase-8. FLIP impedes full processing of pro-caspase-8 to its active form and its release to the cytosol, and by doing so blocks apoptotic signaling downstream of the membrane death initiating signaling complex (DISC). Recent observations have highlighted the complex regulation of this protein and its cross talk with diverse signaling networks and metabolic processes. As FLIP expression is directly associated with chemotherapy resistance, a better understanding of its genomic organization, gene transcription, as well as post-transcriptional regulation could yield novel targets with potential therapeutic implications against drug refractory cancers. In this short review, we provide a brief overview of the structural and functional biology of this somewhat complex protein with direct relevance to carcinogenesis.

Pani: a novel algorithm for fast discovery of putative target nodes in signaling networks

In biological network analysis, the goal of the target identification problem is to predict molecule to inhibit (or activate) to achieve optimum efficacy and safety for a disease treatment. A related problem is the target prioritization problem which predicts a subset of molecules in a given disease-related network which contains successful drug targets with highest probability. Sensitivity analysis prioritizes targets in a dynamic network model using principled criteria, but fails to penalize off-target effects, and does not scale for large networks. We describe Pani (Putative TArget Nodes PrIoritization), a novel method that prunes and ranks the possible target nodes by exploiting concentration-time profiles and network structure (topological) information. Pani and two sensitivity analysis methods were applied to three signaling networks, mapk-pi3k; myosin light chain (mlc) phosphorylation and sea urchin endomesoderm gene regulatory network which are implicated for example in ovarian cancer; atrial fibrillation and deformed embryos. Predicted targets were compared against the molecules known to be targeted by drugs in clinical use for the respective diseases. Pani is orders of magnitude faster and prioritizes the majority of known targets higher than both sensitivity methods. This highlights a potential disagreement between absolute mathematical sensitivity and our intuition of influence. We conclude that empirical, structural methods like Pani, which demand almost no run time, offer benefits not available from quantitative simulation and sensitivity analysis.