Daniel F. Lyons

A thermally targeted peptide inhibitor of symmetrical dimethylation inhibits cancer-cell proliferation.

Targeting splicing machinery components is an underdeveloped strategy for cancer therapy. Uridine-rich small nuclear ribonucleoproteins (UsnRNPs) are essential spliceosome components that recognize splice sites in newly transcribed RNA. The major spliceosomal snRNPs are comprised of UsnRNA bound by a ring of Sm proteins. The survival of motor neuron (SMN) complex provides specificity for binding of Sm proteins to UsnRNAs. Three of the seven proteins that comprise the Sm core possess post-translationally modified C-terminal symmetric dimethylarginine (sDMA) residues which promote binding of these proteins to SMN. Here we describe a peptide inhibitor of sDMA that is capable of interfering with SMN/SmB interaction. The inhibitory peptide was attached to elastin-like polypeptide, a thermally responsive macromolecular carrier, in order to increase its stability and allow enhancement of its cellular uptake by thermal targeting. The fusion polypeptide inhibited the interaction of SMN/SmB, inhibited proliferation, and induced apoptosis in HeLa cells.

Specific Soluble Oligomers of Amyloid-β Peptide Undergo Replication and Form Non-fibrillar Aggregates in Interfacial Environments

Background: Oligomers of amyloid-β peptides are implicated in the etiology of Alzheimer disease. Results: Specific “off-pathway” oligomers of Aβ42 show unique replication properties upon interacting with monomers. Conclusion: The results indicate that oligomers that are formed along pathways outside the fibril formation pathway may undergo replication. Significance: Mechanistic details of Aβ soluble oligomers will enable better understanding of Alzheimer disease pathology. Aggregates of amyloid-β (Aβ) peptides have been implicated in the etiology of Alzheimer disease. Among the different forms of Aβ aggregates, low molecular weight species ranging between ∼2- and 50-mers, also called “soluble oligomers,” have emerged as the species responsible for early synaptic dysfunction and neuronal loss. Emerging evidence suggests that the neurotoxic oligomers need not be formed along the obligatory nucleation-dependant fibril formation pathway. In our earlier work, we reported the isolation of one such “off-pathway” 12–18-mer species of Aβ42 generated from fatty acids called large fatty acid-derived oligomers (LFAOs) (Kumar, A., Bullard, R. L., Patel, P., Paslay, L. C., Singh, D., Bienkiewicz, E. A., Morgan, S. E., and Rangachari, V. (2011) PLoS One 6, e18759). Here, we report the physiochemical aspects of LFAO-monomer interactions as well as LFAO-LFAO associations in the presence of interfaces. We discovered that LFAOs are a replicating strain of oligomers that recruit Aβ42 monomers and quantitatively convert them into LFAO assemblies at the expense of fibrils, a mechanism similar to prion propagation. We also found that in the presence of hexane-buffer or chloroform-buffer interfaces LFAOs are able to associate with themselves to form larger but non-fibrillar aggregates. These results further support the hypothesis that low molecular weight oligomers can be generated via non-fibril formation pathways. Furthermore, the unique replicating property of off-pathway oligomers may hold profound significance for Alzheimer disease pathology.

Are fluorescence-detected sedimentation velocity data reliable?

Sedimentation velocity analytical ultracentrifugation is a classical biophysical technique that is commonly used to analyze the size, shape, and interactions of biological macromolecules in solution. Fluorescence detection provides enhanced sensitivity and selectivity relative to the standard absorption and refractrometric detectors, but data acquisition is more complex and can be subject to interference from several photophysical effects. Here, we describe methods to configure sedimentation velocity measurements using fluorescence detection and evaluate the performance of the fluorescence optical system. The fluorescence detector output is linear over a concentration range of at least 1 to 500nM fluorescein and Alexa Fluor 488. At high concentrations, deviations from linearity can be attributed to the inner filter effect. A duplex DNA labeled with Alexa Fluor 488 was used as a standard to compare sedimentation coefficients obtained using fluorescence and absorbance detectors. Within error, the sedimentation coefficients agree. Thus, the fluorescence detector is capable of providing precise and accurate sedimentation velocity results that are consistent with measurements performed using conventional absorption optics, provided the data are collected at appropriate sample concentrations and the optics are configured correctly.

Folding and Hydrodynamics of a DNA i-Motif from the c-MYC Promoter Determined by Fluorescent Cytidine Analogs

The four-stranded i-motif (iM) conformation of cytosine-rich DNA has importance to a wide variety of biochemical systems that range from their use in nanomaterials to potential roles in oncogene regulation. The iM structure is formed at slightly acidic pH, where hemiprotonation of cytosine results in a stable C-C(+) basepair. Here, we performed fundamental studies to examine iM formation from a C-rich strand from the promoter of the human c-MYC gene. We used a number of biophysical techniques to characterize both the hydrodynamic properties and folding kinetics of a folded iM. Our hydrodynamic studies using fluorescence anisotropy decay and analytical ultracentrifugation show that the iM structure has a compact size in solution and displays the rigidity of a double strand. By studying the rates of circular dichroism spectral changes and quenching of fluorescent cytidine analogs, we also established a mechanism for the folding of a random coil oligo into the iM. In the course of determining this folding pathway, we established that the fluorescent dC analogs tC° and PdC can be used to monitor individual residues of an iM structure and to determine the pKa of an iM. We established that the C-C(+) hydrogen bonding of certain bases initiates the folding of the iM structure. We also showed that substitutions in the loop regions of iMs give a distinctly different kinetic signature during folding compared with bases that are intercalated. Our data reveal that the iM passes through a distinct intermediate form between the unfolded and folded forms. Taken together, our results lay the foundation for using fluorescent dC analogs to follow structural changes during iM formation. Our technique may also be useful for examining folding and structural changes in more complex iMs.

Structural and Hydrodynamic Analysis of a Novel Drug Delivery Vector: ELP[V5G3A2-150]

The therapeutic potential of elastin-like polypeptide (ELP) conjugated to therapeutic compounds is currently being investigated as an approach to target drugs to solid tumors. ELPs are hydrophobic polymers that are soluble at low temperatures and cooperatively aggregate above a transition temperature (TT), allowing for thermal targeting of covalently attached drugs. They have been shown to cooperatively transition from a disordered structure to a repeating type II β-turn structure, forming a β-spiral above the TT. Here we present biophysical measurements of the structural, thermodynamic, and hydrodynamic properties of a specific ELP being investigated for drug delivery, ELP[V5G3A2-150]. We examine the biophysical properties below and above the TT to understand and predict the therapeutic potential of ELP-drug conjugates. We observed that below the TT, ELP[V5G3A2-150] is soluble, with an extended conformation consisting of both random coil and heterogeneous β structures. Sedimentation velocity experiments indicate that ELP[V5G3A2-150] undergoes weak self-association with increasing temperature, and above the TT the hydrophobic effect drives aggregation entropically. These experiments also reveal a previously unreported temperature-dependent critical concentration (Cc) that resembles a solubility constant. Labeling ELP[V5G3A2-150] with fluorescein lowers the TT by 3.5°C at 20 μM, whereas ELP[V5G3A2-150] dissolution in physiological media (fetal bovine serum) increases the TT by ∼2.2°C.

Biochemical evidence that human EB1 does not bind preferentially to the microtubule seam

EB1 is a highly conserved microtubule (MT) plus end tracking protein (+TIP) involved in regulating MT dynamics, but the mechanisms of its effects on MT polymerization remain undefined. Resolving this question requires understanding how EB1 interacts with MTs. Previous electron microscopy of the S. pombe EB1 homolog Mal3p suggested that Mal3p binds specifically to the MT seam, implying that EB1 family members promote MT polymerization by stabilizing the seam. However, more recent electron microscopy indicates that Mal3p binds everywhere except the seam. Neither set of experiments investigated the behavior of human EB1, or provided an explanation for why these studies arrived at different answers. To resolve these questions, we have used a combination of MT‐binding assays and theoretical modeling with MTBindingSim. Our results indicate that human EB1 binds to the lattice, consistent with the recent Mal3p results, and show that Mal3p‐binding assays that were previously interpreted as evidence for preferential seam binding are equally consistent with weak lattice binding. In addition, we used analytical ultracentrifugation to investigate the possibility that the EB1 monomer–dimer equilibrium might contribute to EB1 binding behavior, and determined that the EB1 dimerization dissociation constant is approximately 90 nM. We and others find that the cellular concentration of EB1 is on the order of 200 nM, suggesting that a portion of EB1 may be monomeric at physiological concentrations. These observations lead us to suggest that regulation of EB1 dimerization might play a role in controlling EB1 function.

Effect of basic cell-penetrating peptides on the structural, thermodynamic, and hydrodynamic properties of a novel drug delivery vector, ELP[V5G3A2-150].

Elastin-like polypeptides (ELPs) are large, nonpolar polypeptides under investigation as components of a novel drug delivery system. ELPs are soluble at low temperatures, but they desolvate and aggregate above a transition temperature (TT). This aggregation is being utilized for targeting systemically delivered ELP–drug conjugates to heated tumors. We previously examined the structural, thermodynamic, and hydrodynamic properties of ELP[V5G3A2-150] to understand its behavior as a therapeutic agent. In this study, we investigate the effect that adding basic cell-penetrating peptides (CPPs) to ELP[V5G3A2-150] has on the polypeptide’s solubility, structure, and aggregation properties. CPPs are known to enhance the uptake of ELP into cultured cells in vitro and into tumor tissue in vivo. Interestingly, the asymmetric addition of basic residues decreased the solubility of ELP[V5G3A2-150], although below the TT we still observed a low level of self-association that increased with temperature. The ΔH of the aggregation process correlates with solubility, suggesting that the basic CPPs stabilize the aggregated state. This is potentially beneficial as the decreased solubility will increase the fraction aggregated and enhance drug delivery efficacy at a heated tumor. Otherwise, the basic CPPs did not significantly alter the biophysical properties of ELP. All constructs were monomeric at low temperatures but self-associate with increasing temperature through an indefinite isodesmic association. This self-association was coupled to a structural transition to type II β-turns. All constructs reversibly aggregated in an endothermic reaction, consistent with a reaction driven by the release of water.

Techniques for Dissecting the Johnston-Ogston Effect

The development of the fluorescence detection system (Aviv-FDS) for the AUC allows a single fluorescently labeled species to be quantitatively characterized against a highly concentrated and heterogeneous background. During our use of the FDS to characterize ELP, a novel drug delivery vector (see Lyons et al., Biophys J 104:2009–2021, 2013), in serum, we encountered the Johnston-Ogston (J-O) effect. The J-O effect is a classical anomaly in sedimentation velocity theory and practice describing the nonideal sedimentation properties of a component as a function of high concentrations of other components. We examined the J-O effect using recent advances in AUC hardware, the AU-FDS (AVIV Biomedical), and data analysis methods, DCDT+ and SEDANAL global direct boundary fitting. We empirically quantified the self- and cross-sedimentation nonideality properties of ELP and the two most ubiquitous serum proteins, albumin (∼35–40 mg/ml) and γ-globulins (∼10–15 mg/ml). We have verified and measured the presence of cross-term hydrodynamic nonideality by running SV studies on a fluorescently labeled component (∼100 nM) in a titration experiment with high concentrations of unlabeled components. This has been accounted for through the introduction of a 3 × 3 nonideality matrix of Ks values into SEDANAL. ELP experiments with mixtures of albumin and γ-globulins were also performed in an attempt to recapitulate the J-O behavior of a serum solution. Clearly, other components or effects contribute to the serum J-O effect. Additional experiments with lipids, lipidated serum albumin, and PEG solutions are planned. These studies lay the groundwork for bringing quantitative hydrodynamic analyses into crowded environments and will allow measurement of hydrodynamic and equilibrium macromolecular properties in a physiological state.