Identification of Salivary Metabolic Signatures Associated with Primary Sjögren's Disease.

Sjögren's disease (SjD) is the second most prevalent autoimmune disorder that involves chronic inflammation of exocrine glands. Correct diagnosis of primary SjD (pSjD) can span over many years since disease symptoms manifest only in advanced stages of salivary and lachrymal glandular destruction, and consensus diagnostic methods have critical sensitivity and selectivity limitations. Using nuclear magnetic resonance (NMR) spectroscopy, we determined the composition of metabolites in unstimulated saliva samples from 30 pSjD subjects and 30 participants who do not have Sjögren's disease (non-Sjögren's control group, NS-C). Thirty-four metabolites were quantified in each sample, and analysis was conducted on both non-normalized (concentration) and normalized metabolomics data from all study participants (ages 23-78) and on an age-restricted subset of the data (ages 30-70) while applying false discovery rate correction in determining data significance. The normalized data of saliva samples from all study participants, and of the age-restricted subset, indicated significant increases in the levels of glucose, glycerol, taurine, and lactate, as well as significant decreases in the levels of 5-aminopentanoate, acetate, butyrate and propionate, in subjects with pSjD compared to subjects in the NS-C group. Additionally, a significant increase in choline was found only in the age-restricted subset, and a significant decrease in fucose was found only in the whole study population in normalized data of saliva samples from the pSjD group compared to the NS-C group. Metabolite concentration data of saliva samples from all study participants, but not from the age-restricted subset, indicated significant increases in the levels of glucose, glycerol, taurine, and lactate in subjects with pSjD compared to controls. The study showed that NMR metabolomics can be implemented in defining salivary metabolic signatures that are associated with disease status, and can contribute to differential analysis between subjects with pSjD and those who are not affected with this disease, in the clinic.

Stapled ß-Hairpins Featuring 4-Mercaptoproline.

Peptides constrained by intramolecular cross-links, especially stapled α-helices, have emerged as versatile scaffolds for drug development. However, there are fewer examples of similarly constrained scaffolds for other secondary structures. Here, we used a novel computational strategy to identify an optimal staple for antiparallel ß-strands, and then we incorporated that staple within a ß-hairpin peptide. The hairpin uses 4-mercaptoproline as a novel staple component, which contributes to a unique, kinked structure. The stapled hairpins show a high degree of structure in aqueous solution, excellent resistance to degradation in cell lysates, and cytosolic penetration at micromolar concentrations. They also overlay with a unique subset of kinked hairpin motifs at protein-protein interaction interfaces. Thus, these scaffolds represent promising starting points for developing inhibitors of cellular protein-protein interactions.

Noninvasive Epidermal Metabolite Profiling.

A buffer placed in brief contact in the skin was assayed by 1H NMR spectroscopy. We found that this passive extraction of the skin surface yields abundant metabolites. Metabolites of the skin surface originate from a variety of sources, including the sweat gland, which produces lactate from the glucose received from its capillary bed. Little is known about how metabolites resident on and within the skin surface respond to a metabolic or hemodynamic perturbation. As a possible application of epidermal metabolite profiling, we asked whether metabolites extracted from the skin surface are indicative of heart failure. The levels of lactate and other molecules were significantly lower in patients in heart failure than in individuals who reported healthy heart function, possibly due to reduced blood flow to the sweat gland resulting in a lack of tissue perfusion. Most amino acids were unchanged in levels, except for glycine and serine that increased as a percentage of all amino acids. These results have the potential in the long term to help decide the extent to which a patient has heart failure for which objective measures are lacking. Moreover, the results suggest that epidermal metabolite profiling may be useful for other assessments of human health.

Involvement of a gut-retina axis in protection against dietary glycemia-induced age-related macular degeneration.

Age-related macular degeneration (AMD) is the major cause of blindness in developed nations. AMD is characterized by retinal pigmented epithelial (RPE) cell dysfunction and loss of photoreceptor cells. Epidemiologic studies indicate important contributions of dietary patterns to the risk for AMD, but the mechanisms relating diet to disease remain unclear. Here we investigate the effect on AMD of isocaloric diets that differ only in the type of dietary carbohydrate in a wild-type aged-mouse model. The consumption of a high-glycemia (HG) diet resulted in many AMD features (AMDf), including RPE hypopigmentation and atrophy, lipofuscin accumulation, and photoreceptor degeneration, whereas consumption of the lower-glycemia (LG) diet did not. Critically, switching from the HG to the LG diet late in life arrested or reversed AMDf. LG diets limited the accumulation of advanced glycation end products, long-chain polyunsaturated lipids, and their peroxidation end-products and increased C3-carnitine in retina, plasma, or urine. Untargeted metabolomics revealed microbial cometabolites, particularly serotonin, as protective against AMDf. Gut microbiota were responsive to diet, and we identified microbiota in the Clostridiales order as being associated with AMDf and the HG diet, whereas protection from AMDf was associated with the Bacteroidales order and the LG diet. Network analysis revealed a nexus of metabolites and microbiota that appear to act within a gut-retina axis to protect against diet- and age-induced AMDf. The findings indicate a functional interaction between dietary carbohydrates, the metabolome, including microbial cometabolites, and AMDf. Our studies suggest a simple dietary intervention that may be useful in patients to arrest AMD.

Amino Acid Biosynthetic Pathways Are Required for Klebsiella pneumoniae Growth in Immunocompromised Lungs and Are Druggable Targets during Infection.

The emergence of multidrug-resistant Klebsiella pneumoniae has rendered a large array of infections difficult to treat. In a high-throughput genetic screen of factors required for K. pneumoniae survival in the lung, amino acid biosynthesis genes were critical for infection in both immunosuppressed and wild-type (WT) mice. The limited pool of amino acids in the lung did not change during infection and was insufficient for K. pneumoniae to overcome attenuating mutations in aroA, hisA, leuA, leuB, serA, serB, trpE, and tyrA in WT and immunosuppressed mice. Deletion of aroA, which encodes 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase class I, resulted in the most severe attenuation. Treatment with the EPSP synthase-specific competitive inhibitor glyphosate decreased K. pneumoniae growth in the lungs. K. pneumoniae expressing two previously identified glyphosate-resistant mutations in EPSP synthase had significant colonization defects in lung infection. Selection and characterization of six spontaneously glyphosate-resistant mutants in K. pneumoniae yielded no mutations in aroA Strikingly, glyphosate treatment of mice lowered the bacterial burden of two of three spontaneous glyphosate-resistant mutants and further lowered the burden of the less-attenuated EPSP synthase catalytic mutant. Of 39 clinical isolate strains, 9 were resistant to glyphosate at levels comparable to those of selected resistant strains, and none appeared to be more highly resistant. These findings demonstrate amino acid biosynthetic pathways essential for K. pneumoniae infection are promising novel therapeutic targets.

Targeting Liver Fibrosis with a Cell-penetrating Protease-activated Receptor-2 (PAR2) Pepducin.

Chronic liver inflammation and fibrosis in nonalcoholic steatohepatitis can lead to cirrhosis and liver failure for which there are currently no approved treatments. Protease-activated receptor-2 (PAR2) is an emerging new target expressed on liver stellate cells and hepatocytes that regulates the response to liver injury and inflammation. Here, we identified a pepducin to block the deleterious actions of PAR2 in promoting liver fibrosis. Non-alcoholic fatty liver disease and early fibrosis were induced by the methionine-choline-deficient diet in mice. Fibrotic liver disease was induced by administering carbon tetrachloride for 8 weeks. Mice were treated with the pepducin PZ-235 either from onset of the experiment or after fibrosis was established. Hepatic fibrosis, collagen content, inflammatory cytokines, steatosis, triglycerides, and NAFLD activity score were assessed as primary outcome parameters depending on the model. The activity of the PAR2 pepducin on cultured stellate cell activation and hepatocyte reactive oxygen species production was evaluated. PZ-235 significantly suppressed liver fibrosis, collagen deposition, inflammatory cytokines, NAFLD activity score, steatosis, triglycerides, aspartate transaminase, alanine transaminase, and stellate cell proliferation by up to 50-100%. The PAR2 inhibitor afforded significant protective effects against hepatocellular necrosis and attenuated PAR2-mediated reactive oxygen species production in hepatocytes. PZ-235 was distributed to liver and other mouse tissues and was found to form a well structured α-helix that closely resembles the juxtamembrane helical region of the analogous TM6 and third intracellular region of the intact receptor that is critical for coupling to internal G proteins. The ability of PZ-235 to effectively suppress fibrosis, hepatocellular necrosis, reactive oxygen species production, steatosis, and inflammation indicates the potential for PAR2 pepducin inhibitors to be broadly efficacious in the treatment of liver fibrosis.

Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics.

G protein-coupled receptors (GPCRs) are remarkably versatile signaling systems that are activated by a large number of different agonists on the outside of the cell. However, the inside surface of the receptors that couple to G proteins has not yet been effectively modulated for activity or treatment of diseases. Pepducins are cell-penetrating lipopeptides that have enabled chemical and physical access to the intracellular face of GPCRs. The structure of a third intracellular (i3) loop agonist, pepducin, based on protease-activated receptor-1 (PAR1) was solved by NMR and found to closely resemble the i3 loop structure predicted for the intact receptor in the on-state. Mechanistic studies revealed that the pepducin directly interacts with the intracellular H8 helix region of PAR1 and allosterically activates the receptor through the adjacent (D/N)PXXYYY motif through a dimer-like mechanism. The i3 pepducin enhances PAR1/Gα subunit interactions and induces a conformational change in fluorescently labeled PAR1 in a very similar manner to that induced by thrombin. As pepducins can potentially be made to target any GPCR, these data provide insight into the identification of allosteric modulators to this major drug target class.

Structured cyclic peptides that bind the EH domain of EHD1.

EHD1 mediates long-loop recycling of many receptors by forming signaling complexes using its EH domain. We report the design and optimization of cyclic peptides as ligands for the EH domain of EHD1. We demonstrate that the improved affinity from cyclization allows fluorescence-based screening applications for EH domain inhibitors. The cyclic peptide is also unusually well-structured in aqueous solution, as demonstrated using nuclear magnetic resonance-based structural models. Because few EH domain inhibitors have been described, these more potent inhibitors will improve our understanding of the roles of EHD1 in the context of cancer invasion and metastasis.

Structural analysis of the N-terminal domain of subunit a of the yeast vacuolar ATPase (V-ATPase) using accessibility of single cysteine substitutions to chemical modification.

The vacuolar ATPase (V-ATPase) is a multisubunit complex that carries out ATP-driven proton transport. It is composed of a peripheral V1 domain that hydrolyzes ATP and an integral V0 domain that translocates protons. Subunit a is a 100-kDa integral membrane protein (part of V0) that possesses an N-terminal cytoplasmic domain and a C-terminal hydrophobic domain. Although the C-terminal domain functions in proton transport, the N-terminal domain is critical for intracellular targeting and regulation of V-ATPase assembly. Despite its importance, there is currently no high resolution structure for subunit a of the V-ATPase. Recently, the crystal structure of the N-terminal domain of the related subunit I from the archaebacterium Meiothermus ruber was reported. We have used homology modeling to construct a model of the N-terminal domain of Vph1p, one of two isoforms of subunit a expressed in yeast. To test this model, unique cysteine residues were introduced into a Cys-less form of Vph1p and their accessibility to modification by the sulfhydryl reagent 3-(N-maleimido-propionyl) biocytin (MPB) was determined. In addition, accessibility of introduced cysteine residues to MPB modification was compared in the V1V0 complex and the free V0 domain to identify residues protected from modification by the presence of V1. The results provide an experimental test of the proposed model and have identified regions of the N-terminal domain of subunit a that likely serve as interfacial contact sites with the peripheral V1 domain. The possible significance of these results for in vivo regulation of V-ATPase assembly is discussed.

A bicyclic peptide scaffold promotes phosphotyrosine mimicry and cellular uptake.

While peptides are promising as probes and therapeutics, targeting intracellular proteins will require greater understanding of highly structured, cell-internalized scaffolds. We recently reported BC1, an 11-residue bicyclic peptide that inhibits the Src homology 2 (SH2) domain of growth factor receptor-bound protein 2 (Grb2). In this work, we describe the unique structural and cell uptake properties of BC1 and similar cyclic and bicyclic scaffolds. These constrained scaffolds are taken up by mammalian cells despite their net neutral or negative charges, while unconstrained analogs are not. The mechanism of uptake is shown to be energy-dependent and endocytic, but distinct from that of Tat. The solution structure of BC1 was investigated by NMR and MD simulations, which revealed discrete water-binding sites on BC1 that reduce exposure of backbone amides to bulk water. This represents an original and potentially general strategy for promoting cell uptake.

Assessing the Performance of Peptide Force Fields for Modeling the Solution Structural Ensembles of Cyclic Peptides.

Molecular dynamics simulation is a powerful tool for characterizing the solution structural ensembles of cyclic peptides. However, the ability of simulation to recapitulate experimental results and make accurate predictions largely depends on the force fields used. In our work here, we evaluate the performance of seven state-of-the-art force fields in recapitulating the experimental NMR results in water of 12 benchmark cyclic peptides, consisting of 6 cyclic pentapeptides, 4 cyclic hexapeptides, and 2 cyclic heptapeptides. The results show that RSFF2+TIP3P, RSFF2C+TIP3P, and Amber14SB+TIP3P exhibit similar and the best performance, all recapitulating the NMR-derived structure information on 10 cyclic peptides. Amber19SB+OPC successfully recapitulates the NMR-derived structure information on 8 cyclic peptides. In contrast, OPLS-AA/M+TIP4P, Amber03+TIP3P, and Amber14SBonlysc+GB-neck2 could only recapitulate the NMR-derived structure information on 5 cyclic peptides, the majority of which are not well-structured.

Molecular remodeling mechanisms of the neural somatodendritic compartment.

Neuronal cells use the process of vesicle trafficking to manipulate the populations of neurotransmitter receptors and other membrane proteins. Long term potentiation (LTP) is a long-lived increase in synaptic strength between neurons and increases postsynaptic dendritic spine size and the concentration of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptor (AMPAR) located in the postsynaptic density. AMPAR is removed from the cell surface via clathrin-mediated endocytosis. While the adaptor protein 2 (AP2) complex of endocytosis seems to have the components needed to allow temporal and spatial regulations of internalization, many accessory proteins are involved, such as epidermal growth factor receptor phosphorylation substrate 15 (Eps15). A sequence of repeats in the Eps15 protein is known as the Eps15 homology (EH) domain. It has affinity for asparagine-proline-phenylalanine (NPF) sequences that are contained within vesicle trafficking proteins such as epsin, Rab11 family interacting protein 2 (Rab11-FIP2), and Numb. After endocytosis, a pool of AMPAR is stored in the endosomal recycling compartment that can be transported to the dendritic spine surface upon stimulation during LTP for lateral diffusion into the postsynaptic density. Rab11 and the Eps15 homologue EHD1 are involved in receptor recycling. EHD family members are also involved in transcytosis of the neuronal cell adhesion molecule neuron-glia cell adhesion molecule (NgCAM) from the somatodendritic compartment to the axon. Neurons have a unique morphology comprising many projections of membrane that is constructed in part by the effects of the Eps15 homologue, intersectin. Morphogenesis in the somatodendritic compartment is becoming better understood, but there is still much exciting territory to explore, especially regarding the roles of various EH domain-NPF interactions in endocytic and recycling processes.

Suppression of arterial thrombosis without affecting hemostatic parameters with a cell-penetrating PAR1 pepducin.

BACKGROUND: Thrombin-dependent platelet activation is heightened in the setting of percutaneous coronary intervention and may cause arterial thrombosis with consequent myocardial necrosis. Given the high incidence of adverse effects in patients with acute coronary syndromes, there remains an unmet need for the development of new therapeutics that target platelet activation without unduly affecting hemostasis. The thrombin receptor, PAR1, has recently emerged as a promising new target for therapeutic intervention in patients with acute coronary syndromes. METHODS AND RESULTS: We report the development of a first-in-class intracellular PAR1 inhibitor with optimized pharmacokinetic properties for use during percutaneous coronary intervention in patients with acute coronary syndromes. PZ-128 is a cell-penetrating pepducin inhibitor of PAR1 that targets the receptor-G-protein interface on the inside surface of platelets. The structure of PZ-128 closely resembles the predicted off-state of the corresponding juxtamembrane region of the third intracellular loop of PAR1. The onset of action of PZ-128 was rapid and suppressed PAR1 aggregation and arterial thrombosis in guinea pigs and baboons and strongly synergized with oral clopidogrel. There was full recovery of platelet function by 24 hours. Importantly, PZ-128 had no effect on bleeding or coagulation parameters in primates or in blood from patients undergoing percutaneous coronary intervention. CONCLUSIONS: Based on the efficacy data in nonhuman primates with no noted adverse effects on hemostasis, we anticipate that the rapid onset of platelet inhibition and reversible properties of PZ-128 are well suited to the acute interventional setting of percutaneous coronary intervention and may provide an alternative to long-acting small-molecule inhibitors of PAR1.

Cross-strand interactions of fluorinated amino acids in ß-hairpin constructs.

We describe herein the design, synthesis, and thermodynamic characterization of fluorinated ß-hairpin constructs. Introduction of hexafluoroleucine (Hfl) did not perturb ß-hairpin formation, as judged by (1)H NMR structures of four peptides determined to <1 Å backbone RMSDs, allowing direct comparison of thermodynamic stabilities of fluorinated peptides to their hydrocarbon counterparts. Judicious fluorination of peptides often results in increased thermal and chemical stability of the resultant folded structures. However, we found that when cross-strand residue partners were varied, the side-chain interaction energies followed the order Leu-Leu > Hfl-Leu > Hfl-Hfl. All peptides were more structured in 90% MeOH than in aqueous buffers. The peptides with Hfl-Leu or Hfl-Hfl cross-strand partners showed increased interaction energies in this solvent compared to those in water, in contrast to the insignificant effect on Leu-Leu. Our results inform the binding and assembly of peptides containing Hfl in the context of ß-sheet structures and may be useful in interpreting binding of fluorinated ligands and peptides to biological targets.

Design and characterization of an enhanced repressor of human papillomavirus E2 protein.

Papillomaviruses are causative agents of cervical and anogenital cancers. The viral E2 protein mediates viral DNA replication and transactivation of viral oncogenes and thus represents a specific target for therapeutic intervention. Short forms of E2, E2R, contain only the C-terminal dimerization domain, and repress the normal function of E2 due to formation of an inactive heterodimer. Using structure-guided design, we replaced conserved residues at the dimer interface to design a heterodimer with increased stability. One E2R mutant in which histidine was replaced by a glutamate residue showed preferential heterodimer formation in vitro, as well as an increase in plasticity at the interface, as a result of histidine-glutamate pair formation, as observed spectroscopically and in the crystal structure, determined to 2.2-Å resolution. In addition, the enhanced E2R showed greater repression of transcription from E2-responsive reporter plasmids in mammalian cell culture. Recent advances in protein delivery into the cell raise the possibility of using exogenously added proteins as therapeutic agents. More generally, this approach may be used to target the subunit interfaces of any multisubunit protein having a similar mechanism of action.

Membrane-binding properties of the Factor VIII C2 domain.

Factor VIII functions as a cofactor for Factor IXa in a membrane-bound enzyme complex. Membrane binding accelerates the activity of the Factor VIIIa-Factor IXa complex approx. 100000-fold, and the major phospholipid-binding motif of Factor VIII is thought to be on the C2 domain. In the present study, we prepared an fVIII-C2 (Factor VIII C2 domain) construct from Escherichia coli, and confirmed its structural integrity through binding of three distinct monoclonal antibodies. Solution-phase assays, performed with flow cytometry and FRET (fluorescence resonance energy transfer), revealed that fVIII-C2 membrane affinity was approx. 40-fold lower than intact Factor VIII. In contrast with the similarly structured C2 domain of lactadherin, fVIII-C2 membrane binding was inhibited by physiological NaCl. fVIII-C2 binding was also not specific for phosphatidylserine over other negatively charged phospholipids, whereas a Factor VIII construct lacking the C2 domain retained phosphatidyl-L-serine specificity. fVIII-C2 slightly enhanced the cleavage of Factor X by Factor IXa, but did not compete with Factor VIII for membrane-binding sites or inhibit the Factor Xase complex. Our results indicate that the C2 domain in isolation does not recapitulate the characteristic membrane binding of Factor VIII, emphasizing that its role is co-operative with other domains of the intact Factor VIII molecule.

Use of nuclear magnetic resonance spectroscopy in diagnosis of inborn errors of metabolism.

Nuclear Magnetic Resonance (NMR) spectroscopy has been applied in many fields of science and is increasingly being considered as a tool in the clinical setting. This review examines its application for diagnosis of inborn errors of metabolism (IEMs). IEMs, whether involving deficiency in the synthesis and degradation of metabolites, or in lipoprotein metabolism, affect nearly 3% of the global population. NMR is a preferred method for comprehensive evaluation of complex biofluids such as blood or urine, as it can provide a relatively unbiased overview of all compounds that are present and does not destroy or otherwise chemically alter the sample. While current newborn screening programs take advantage of other more sensitive methods, such as mass spectrometry, NMR has advantages especially for urine analysis with respect to ease of sample preparation and the reproducibility of results. NMR spectroscopy is particularly compatible with analysis of lipoproteins because it provides information about their size and density, not easily attained by other methods, that can help the clinician to better manage patients with dyslipidemia. We believe that NMR holds great potential for expanding clinical diagnosis in the future, in the field of IEMs and beyond.

A Warburg-like metabolic program coordinates Wnt, AMPK, and mTOR signaling pathways in epileptogenesis.

Epilepsy is a complex neurological condition characterized by repeated spontaneous seizures and can be induced by initiating seizures known as status epilepticus (SE). Elaborating the critical molecular mechanisms following SE are central to understanding the establishment of chronic seizures. Here, we identify a transient program of molecular and metabolic signaling in the early epileptogenic period, centered on day five following SE in the pre-clinical kainate or pilocarpine models of temporal lobe epilepsy. Our work now elaborates a new molecular mechanism centered around Wnt signaling and a growing network comprised of metabolic reprogramming and mTOR activation. Biochemical, metabolomic, confocal microscopy and mouse genetics experiments all demonstrate coordinated activation of Wnt signaling, predominantly in neurons, and the ensuing induction of an overall aerobic glycolysis (Warburg-like phenomenon) and an altered TCA cycle in early epileptogenesis. A centerpiece of the mechanism is the regulation of pyruvate dehydrogenase (PDH) through its kinase and Wnt target genes PDK4. Intriguingly, PDH is a central gene in certain genetic epilepsies, underscoring the relevance of our elaborated mechanisms. While sharing some features with cancers, the Warburg-like metabolism in early epileptogenesis is uniquely split between neurons and astrocytes to achieve an overall novel metabolic reprogramming. This split Warburg metabolic reprogramming triggers an inhibition of AMPK and subsequent activation of mTOR, which is a signature event of epileptogenesis. Interrogation of the mechanism with the metabolic inhibitor 2-deoxyglucose surprisingly demonstrated that Wnt signaling and the resulting metabolic reprogramming lies upstream of mTOR activation in epileptogenesis. To augment the pre-clinical pilocarpine and kainate models, aspects of the proposed mechanisms were also investigated and correlated in a genetic model of constitutive Wnt signaling (deletion of the transcriptional repressor and Wnt pathway inhibitor HBP1). The results from the HBP1-/- mice provide a genetic evidence that Wnt signaling may set the threshold of acquired seizure susceptibility with a similar molecular framework. Using biochemistry and genetics, this paper outlines a new molecular framework of early epileptogenesis and advances a potential molecular platform for refining therapeutic strategies in attenuating recurrent seizures.

Charge effects in the selection of NPF motifs by the EH domain of EHD1.

The Eps15 homology (EH) domain is found in proteins associated with endocytosis and vesicle trafficking. EH domains bind to their target proteins through an asparagine-proline-phenylalanine (NPF) motif. We have measured the interaction energetics of the EH domain from EHD1 with peptides derived from two of its binding partners: Rabenosyn-5 (Ac-GPSLNPFDEED-NH(2)) and Rab11-Fip2 (Ac-YESTNPFTAK-NH(2)). Heteronuclear single quantum coherence (HSQC) spectroscopy shows that both peptides bind in the canonical binding pocket of EHD1 EH and induce identical structural changes, yet the affinity of the negatively charged Ac-GPSLNPFDEED-NH(2) (K(a) = 8 x 10(5) M(-1)) is tighter by 2 orders of magnitude. The thermodynamic profiles (DeltaG, DeltaH, DeltaS) were measured for both peptides as a function of temperature. The enthalpies of binding are essentially identical, and the difference in affinity is a consequence of the difference in entropic cost. Ac-GPSLNPFDEED-NH(2) binding is salt-dependent, demonstrating an electrostatic component to the interaction, whereas Ac-YESTNPFTAK-NH(2) binding is independent of salt. Successive replacement of acidic residues in Ac-GPSLNPFDEED-NH(2) with neutral residues showed that all are important. Lysine side chains in EHD1 EH create a region of strong positive surface potential near the NPF binding pocket. Contributions by lysine epsilon-amino groups to complex formation with Ac-GPSLNPFDEED-NH(2) was shown using direct-observe (15)N NMR spectroscopy. These experiments have enabled us to define a new extended interaction motif for EHD proteins, N-P-F-[DE]-[DE]-[DE], which we have used to predict new interaction partners and hence broaden the range of cellular activities involving the EHD proteins.

Delineating cell behavior and metabolism of non-melanoma skin cancer in vitro.

Non-melanoma skin cancers - basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) - are the most frequent forms of malignant neoplasm in humans worldwide. The etiology of these carcinomas is multifactorial. In addition to the harmful effect of UV light, altered cross-talk between neoplastic epithelial cells and the supporting dermal fibroblasts contributes to the regulation of tumor cell behavior, growth and survival. Metabolic cooperation between these cell types allows them to adapt and react to changes in their surrounding microenvironment by modifying their cellular bioenergetics and biosynthesis. We characterized the growth, behavior, and metabolic activity of human BCC cells, E-cadherin-competent SCC cells and E-cadherin-suppressed SCC cells in the presence or absence of dermal fibroblasts. In mono-cultures and co-cultures, BCC and SCC cells demonstrated distinct morphology, growth and organizational patterns. These tumor cells also exhibited unique patterns of consumption and secretion profiles of glucose, lactate, acetate, glutamine, glutamate, and pyruvate. In comparison to mono-cultures, growth of fibroblasts with either BCC cells or SCC cells enriched the cell growth environment, allowed for metabolic cooperation between these two cell types, and resulted in alterations in the metabolic profiles of the co-cultures. These alterations were affected by the cancer cell type, culture confluence and the composition of the growth medium. Characterizing the bioenergetics of BCC and SCC cells in the context of tumor-stromal interactions is not only important for further understanding of tumor pathogenesis, but also can illuminate potential new targets for novel, metabolic-based therapies for non-melanoma skin cancers.