Native and engineered tropism of vectors derived from a rare species D adenovirus serotype 43.

Unique molecular properties of species D adenoviruses (Ads)-the most diverse yet underexplored group of Ads-have been used to develop improved gene vectors. The low seroprevalence in humans of adenovirus serotype 43 (Ad43), an otherwise unstudied species D Ad, identified this rare serotype as an attractive new human gene therapy vector platform. Thus, in this study we wished to assess biological properties of Ad43 essential to its vectorization.

Erythropoietin Stimulates Tumor Growth via EphB4.

While recombinant human erythropoietin (rhEpo) has been widely used to treat anemia in cancer patients, concerns about its adverse effects on patient survival have emerged. A lack of correlation between expression of the canonical EpoR and rhEpo's effects on cancer cells prompted us to consider the existence of an alternative Epo receptor. Here, we identified EphB4 as an Epo receptor that triggers downstream signaling via STAT3 and promotes rhEpo-induced tumor growth and progression.

Competition between Grb2 and Plcγ1 for FGFR2 regulates basal phospholipase activity and invasion.

FGFR2-expressing human cancer cells with low concentrations of the adaptor protein Grb2 show high prevalence for metastatic outcome. In nonstimulated cells, the SH3 domain (and not the SH2 domains) of Plcγ1 directly competes for a binding site at the very C terminus of FGFR2 with the C-terminal SH3 domain of Grb2. Reduction of Grb2 concentration permits Plcγ1 access to the receptor.

SLP-76 sterile α motif (SAM) and individual H5 α helix mediate oligomer formation for microclusters and T-cell activation.

Despite the importance of the immune adaptor SLP-76 in T-cell immunity, it has been unclear whether SLP-76 directly self-associates to form higher order oligomers for T-cell activation. In this study, we show that SLP-76 self-associates in response to T-cell receptor ligation as mediated by the N-terminal sterile α motif (SAM) domain. SLP-76 co-precipitated alternately tagged SLP-76 in response to anti-CD3 ligation.

Pharmacological inactivation of Skp2 SCF ubiquitin ligase restricts cancer stem cell traits and cancer progression.

Skp2 E3 ligase is overexpressed in numerous human cancers and plays a critical role in cell-cycle progression, senescence, metabolism, cancer progression, and metastasis. In the present study, we identified a specific Skp2 inhibitor using high-throughput in silico screening of large and diverse chemical libraries. This Skp2 inhibitor selectively suppresses Skp2 E3 ligase activity, but not activity of other SCF complexes.

Biophysical characterization of VEGF-aHt DNA aptamer interactions.

The binding of the well-studied DNA aptamer aHt (5'-ATACCAGTCTATTCAATTGGGCCCGTCCGTAT GGTGGGTGTGCTGGCCAG-3'), which has been demonstrated to recognize human vascular endothelial growth factor (VEGF₁₆₅) to recombinant VEGF was characterized using fluorescence anisotropy, isothermal titration calorimetry and analytical ultracentrifugation. The negatively-charged DNA aptamer is selective for VEGF and does not recognize positively-charged hen egg lysozyme, or bovine serum albumin. In contrast to the VEGF association of the previously-described aV DNA aptamer, where the binding is enthalpically driven and sequence-specific, the binding of the aHt aptamer to VEGF is entropically-driven and not abolished by scrambling of the sequence.

Solution structure of the tandem acyl carrier protein domains from a polyunsaturated fatty acid synthase reveals beads-on-a-string configuration.

The polyunsaturated fatty acid (PUFA) synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP) domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect) and in structural stabilization of the multidomain protein (synergistic effect). While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers.

Inhibition of basal FGF receptor signaling by dimeric Grb2.

Receptor tyrosine kinase activity is known to occur in the absence of extracellular stimuli. Importantly, this "background" level of receptor phosphorylation is insufficient to effect a downstream response, suggesting that strict controls are present and prohibit full activation. Here a mechanism is described in which control of FGFR2 activation is provided by the adaptor protein Grb2.

Catalytic protein modification with dirhodium metallopeptides: specificity in designed and natural systems.

In this study, we present advances in the use of rhodium(II) metallopeptides for protein modification. Site-specific, proximity-driven modification is enabled by the unique combination of peptide-based molecular recognition and a rhodium catalyst capable of modifying a wide range of amino-acid side chains. We explore catalysis based on coiled-coil recognition in detail, providing an understanding of the determinants of specificity and culminating in the demonstration of orthogonal modification of separate proteins in cell lysate.

Binding with nucleic acids or glycosaminoglycans converts soluble protein oligomers to amyloid.

Ample evidence suggests that almost all polypeptides can either adopt a native structure (folded or intrinsically disordered) or form misfolded amyloid fibrils. Soluble protein oligomers exist as an intermediate between these two states, and their cytotoxicity has been implicated in the pathology of multiple human diseases. However, the mechanism by which soluble protein oligomers develop into insoluble amyloid fibrils is not clear, and investigation of this important issue is hindered by the unavailability of stable protein oligomers.

Macromolecular crowding tunes folding landscape of parallel α/β protein, apoflavodoxin.

Proteins normally fold in crowded cellular environments. Here we use a set of Desulfovibrio desulfuricans apoflavodoxin variants to assess--with residue-specific resolution--how apoflavodoxin's folding landscape is tuned by macromolecular crowding. We find that, under crowded conditions, initial topological frustration is reduced, subsequent folding requires less ordering in the transition state, and β-strand 1 becomes more important in guiding the process.

Residue-specific analysis of frustration in the folding landscape of repeat beta/alpha protein apoflavodoxin.

Flavodoxin adopts the common repeat beta/alpha topology and folds in a complex kinetic reaction with intermediates. To better understand this reaction, we analyzed a set of Desulfovibrio desulfuricans apoflavodoxin variants with point mutations in most secondary structure elements by in vitro and in silico methods. By equilibrium unfolding experiments, we first revealed how different secondary structure elements contribute to overall protein resistance to heat and urea.

Pseudosymmetry, high copy number and twinning complicate the structure determination of Desulfovibrio desulfuricans (ATCC 29577) flavodoxin.

The crystal structure of oxidized flavodoxin from Desulfovibrio desulfuricans (ATCC 29577) was determined by molecular replacement in two crystal forms, P3(1)21 and P4(3), at 2.5 and 2.0 A resolution, respectively.

Macromolecular crowding modulates folding mechanism of alpha/beta protein apoflavodoxin.

Protein dynamics in cells may be different from those in dilute solutions in vitro, because the environment in cells is highly concentrated with other macromolecules. This volume exclusion because of macromolecular crowding is predicted to affect both equilibrium and kinetic processes involving protein conformational changes. To quantify macromolecular crowding effects on protein folding mechanisms, we investigated the folding energy landscape of an alpha/beta protein, apoflavodoxin, in the presence of inert macromolecular crowding agents, using in silico and in vitro approaches.

Effect of Hofmeister ions on protein thermal stability: roles of ion hydration and peptide groups?

We have systematically explored the Hofmeister effects of cations and anions (0.3-1.75 M range) for acidic Desulfovibrio desulfuricans apoflavodoxin (net charge -19, pH 7) and basic horse heart cytochrome c (net charge +17, pH 4.

Role of cations in stability of acidic protein Desulfovibrio desulfuricans apoflavodoxin.

Apoflavodoxin from the sulfate reducing bacteria Desulfovibrio desulfuricans is a small, acidic protein with a net charge of -19 at neutral pH. Here, we show that monovalent cations in biologically relevant amounts have dramatic effects on apoflavodoxin stability. The effect is largest for Gdm(+) and decreases as a function of increased cation charge density (Gdm(+)>NH(4)(+)K(+) approximately Cs(+) approximately Na(+)>Li(+)).

Molecular crowding enhances native structure and stability of alpha/beta protein flavodoxin.

To investigate the consequences of macromolecular crowding on the behavior of a globular protein, we performed a combined experimental and computational study on the 148-residue single-domain alpha/beta protein, Desulfovibrio desulfuricans apoflavodoxin. In vitro thermal unfolding experiments, as well as assessment of native and denatured structures, were probed by using far-UV CD in the presence of various amounts of Ficoll 70, an inert spherical crowding agent. Ficoll 70 has a concentration-dependent effect on the thermal stability of apoflavodoxin (DeltaT(m) of 20 degrees C at 400 mg/ml; pH 7).

Macromolecular crowding increases structural content of folded proteins.

Here we show that increased amount of secondary structure is acquired in the folded states of two structurally-different proteins (alpha-helical VlsE and alpha/beta flavodoxin) in the presence of macromolecular crowding agents. The structural content of flavodoxin and VlsE is enhanced by 33% and 70%, respectively, in 400 mg/ml Ficoll 70 (pH 7, 20 degrees C) and correlates with higher protein-thermal stability. In the same Ficoll range, there are only small effects on the unfolded-state structures of the proteins.