Francisco N. Barrera

MicroRNA silencing for cancer therapy targeted to the tumour microenvironment

MicroRNAs are short non-coding RNAs expressed in different tissue and cell types that suppress the expression of target genes. As such, microRNAs are critical cogs in numerous biological processes, and dysregulated microRNA expression is correlated with many human diseases. Certain microRNAs, called oncomiRs, play a causal role in the onset and maintenance of cancer when overexpressed. Tumours that depend on these microRNAs are said to display oncomiR addiction. Some of the most effective anticancer therapies target oncogenes such as EGFR and HER2; similarly, inhibition of oncomiRs using antisense oligomers (that is, antimiRs) is an evolving therapeutic strategy. However, the in vivo efficacy of current antimiR technologies is hindered by physiological and cellular barriers to delivery into targeted cells. Here we introduce a novel antimiR delivery platform that targets the acidic tumour microenvironment, evades systemic clearance by the liver, and facilitates cell entry via a non-endocytic pathway. We find that the attachment of peptide nucleic acid antimiRs to a peptide with a low pH-induced transmembrane structure (pHLIP) produces a novel construct that could target the tumour microenvironment, transport antimiRs across plasma membranes under acidic conditions such as those found in solid tumours (pH approximately 6), and effectively inhibit the miR-155 oncomiR in a mouse model of lymphoma. This study introduces a new model for using antimiRs as anti-cancer drugs, which can have broad impacts on the field of targeted drug delivery.

The dimerization domain of the HIV-1 capsid protein binds a capsid protein-derived peptide: a biophysical characterization.

The type 1 HIV presents a conical capsid formed by ∼1500 units of the capsid protein, CA. Homodimer‐ization of CA via its C‐terminal domain, CA‐C, constitutes a key step in virion assembly. CA‐C dimerization is largely mediated by reciprocal interactions between residues of its second α‐helix. Here, we show that an N‐terminal‐acetylated and C‐terminal–amidated peptide, CAC1, comprising the sequence of the CA‐C dimerization helix plus three flanking residues at each side, is able to form a complex with the entire CA‐C domain. Thermal denaturation measurements followed by circular dichroism (CD), NMR, and size‐exclusion chromatography provided evidence of the interaction between CAC1 and CA‐C. The apparent dissociation constant of the heterocomplex formed by CA‐C and CAC1 was determined by several biophysical techniques, namely, fluorescence (using an anthraniloyl‐labeled peptide), affinity chromatography, and isothermal titration calorimetry. The three techniques yielded similar values for the apparent dissociation constant, in the order of 50 μM. This apparent dissociation constant was only five times higher than was the dissociation constant of both CA‐C and the intact capsid protein homodimers (10 μM).

The inactivating factor of glutamine synthetase, IF7, is a “natively unfolded” protein

Glutamine synthetase (GS) is the key enzyme responsible for the primary assimilation of ammonium in all living organisms, and it catalyses the synthesis of glutamine from glutamic acid, ATP, and ammonium. One of the recently discovered mechanisms of GS regulation involves protein‐protein interactions with a small 65‐residue‐long protein named IF7. Here, we study the structure and stability of IF7 and its binding properties to GS, by using several biophysical techniques (fluorescence, circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopies, and gel filtration chromatography) which provide complementary structural information. The findings show that IF7 has a small amount of residual secondary structure, but lacks a well defined tertiary structure, and is not compact. Thus, all of the studies indicate that IF7 is a “natively unfolded” protein. The binding of IF7 to GS, its natural binding partner, occurs with an apparent dissociation constant of KD = 0.3 ± 0.1 μM, as measured by fluorescence. We discuss the implications for the GS regulation mechanisms of IF7 being unfolded.

Protein Self-Assembly and Lipid Binding in the Folding of the Potassium Channel KcsA†

Moderate concentrations of the alcohol 2,2,2-trifluoroethanol (TFE) cause the coupled unfolding and dissociation into subunits of the homotetrameric potassium channel KcsA, in a process that is partially irreversible when the protein is solubilized in plain dodecyl beta-d-maltoside (DDM) micelles [Barrera et al. (2005) Biochemistry 44, 14344-52]. Here we report that the transition from the folded tetramer to the unfolded monomer becomes completely reversible when KcsA is solubilized in mixed micelles composed of the detergent DDM and the lipids DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and DOPG (1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]). This result suggests that lipids may act as effectors in the tetramerization of KcsA. The observed reversibility allowed the determination of the standard free energy of the folding reaction of KcsA: DeltaG = 30.5 +/- 3.1 kcal x mol-1. We also observed that, prior to the unfolding of the tetramer, the presence of lower TFE concentrations causes the disassembly of supramolecular clusters of KcsA into the individual tetrameric molecules. Within the limits of experimental resolution, this is also a reversible process, but unlike the tetramer to monomer transition from above, the level of clustering is not influenced by the presence of solubilized lipids. These observations suggest a distinct role of the lipids in the different in vitro assembly steps (folding/tetramerization and clustering) of KcsA.

Construction and genetic selection of small transmembrane proteins that activate the human erythropoietin receptor

This work describes a genetic approach to isolate small, artificial transmembrane (TM) proteins with biological activity. The bovine papillomavirus E5 protein is a dimeric, 44-amino acid TM protein that transforms cells by specifically binding and activating the platelet-derived growth factor β receptor (PDGFβR). We used the E5 protein as a scaffold to construct a retrovirus library expressing ∼500,000 unique 44-amino acid proteins with randomized TM domains. We screened this library to select small, dimeric TM proteins that were structurally unrelated to erythropoietin (EPO), but specifically activated the human EPO receptor (hEPOR). These proteins did not activate the murine EPOR or the PDGFβR. Genetic studies with one of these activators suggested that it interacted with the TM domain of the hEPOR. Furthermore, this TM activator supported erythroid differentiation of primary human hematopoietic progenitor cells in vitro in the absence of EPO. Thus, we have changed the specificity of a protein so that it no longer recognizes its natural target but, instead, modulates an entirely different protein. This represents a novel strategy to isolate small artificial proteins that affect diverse membrane proteins. We suggest the word “traptamer” for these transmembrane aptamers.

The transcriptional repressor RYBP is a natively unfolded protein which folds upon binding to DNA.

RYBP (Ring1A and YY1 binding protein) is a zinc finger protein with an essential role during embryonic development, which binds transcriptional factors, Polycomb products, and mediators of apoptosis, suggesting roles in, apparently, unrelated functions. To investigate mechanisms underlying its association with functionally diverse partners, we set out to study its structural properties using a number of biophysical (fluorescence, circular dichroism, Fourier transform infrared, and NMR spectroscopies) and hydrodynamic (analytical ultracentrifugation, DOSY-NMR, and gel filtration chromatography) techniques. We find RYBP to be a noncompact protein with little residual secondary structure, lacking a well-defined tertiary structure. These observations are also supported by theoretical calculations using neural networks and pairwise energy content, suggesting that RYBP is a natively unfolded protein. In addition, structural studies on its binding to the C-terminal region of the Polycomb protein Ring1B or to DNA show conformational changes in the complexed RYBP, consistent with the acquisition of a folded structure. The data provide a structural explanation for RYBP engagement in functionally unrelated pathways by means of its assembly into various macromolecular complexes as an unstructured protein with the ability to acquire a well-structured fold due to its association with different partners.

Aspartate Embedding Depth Affects pHLIP’s Insertion pKa

We have used the pHlow insertion peptide (pHLIP) family to study the role of aspartate embedding depth in pH-dependent transmembrane peptide insertion. pHLIP binds to the surface of a lipid bilayer as a largely unstructured monomer at neutral pH. When the pH is lowered, pHLIP inserts spontaneously across the membrane as a spanning α-helix. pHLIP insertion is reversible when the pH is adjusted back to a neutral value. One of the critical events facilitating pHLIP insertion is the protonation of aspartates in the spanning domain of the peptide: the negative side chains of these residues convert to uncharged, polar forms, facilitating insertion by altering the hydrophobicity of the spanning domain. To examine this protonation mechanism further, we created pHLIP sequence variants in which the two spanning aspartates (D14 and D25) were moved up or down in the sequence. We hypothesized that the aspartate depth in the inserted state would directly affect the proton affinity of the acidic side chains, altering the pKa of pH-dependent insertion. To this end, we also mutated the arginine at position 11 to determine whether arginine snorkeling modulates the insertion pKa by affecting the aspartate depth. Our results indicate that both types of mutations change the insertion pKa, supporting the idea that the aspartate depth is a participating parameter in determining the pH dependence. We also show that pHLIPs resistance to aggregation can be altered with our mutations, identifying a new criterion for improving the performance of pHLIP in vivo when targeting acidic disease tissues such as cancer and inflammation.

An extensive thermodynamic characterization of the dimerization domain of the HIV‐1 capsid protein

The type 1 human immunodeficiency virus presents a conical capsid formed by several hundred units of the capsid protein, CA. Homodimerization of CA occurs via its C‐terminal domain, CA‐C. This self‐association process, which is thought to be pH‐dependent, seems to constitute a key step in virus assembly. CA‐C isolated in solution is able to dimerize. An extensive thermodynamic characterization of the dimeric and monomeric species of CA‐C at different pHs has been carried out by using fluorescence, circular dichroism (CD), absorbance, nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR), and size‐exclusion chromatography (SEC). Thermal and chemical denaturation allowed the determination of the thermodynamic parameters describing the unfolding of both CA‐C species. Three reversible thermal transitions were observed, depending on the technique employed. The first one was protein concentration‐dependent; it was observed by FTIR and NMR, and consisted of a broad transition occurring between 290 and 315 K; this transition involves dimer dissociation. The second transition (Tm ∼ 325 K) was observed by ANS‐binding experiments, fluorescence anisotropy, and near‐UV CD; it involves partial unfolding of the monomeric species. Finally, absorbance, far‐UV CD, and NMR revealed a third transition occurring at Tm ∼ 333 K, which involves global unfolding of the monomeric species. Thus, dimer dissociation and monomer unfolding were not coupled. At low pH, CA‐C underwent a conformational transition, leading to a species displaying ANS binding, a low CD signal, a red‐shifted fluorescence spectrum, and a change in compactness. These features are characteristic of molten globule‐like conformations, and they resemble the properties of the second species observed in thermal unfolding.

Artificial Transmembrane Oncoproteins Smaller than the Bovine Papillomavirus E5 Protein Redefine Sequence Requirements for Activation of the Platelet-Derived Growth Factor β Receptor

ABSTRACT The bovine papillomavirus E5 protein (BPV E5) is a 44-amino-acid homodimeric transmembrane protein that binds directly to the transmembrane domain of the platelet-derived growth factor (PDGF) β receptor and induces ligand-independent receptor activation. Three specific features of BPV E5 are considered important for its ability to activate the PDGF β receptor and transform mouse fibroblasts: a pair of C-terminal cysteines, a transmembrane glutamine, and a juxtamembrane aspartic acid. By using a new genetic technique to screen libraries expressing artificial transmembrane proteins for activators of the PDGF β receptor, we isolated much smaller proteins, from 32 to 36 residues, that lack all three of these features yet still dimerize noncovalently, specifically activate the PDGF β receptor via its transmembrane domain, and transform cells efficiently. The primary amino acid sequence of BPV E5 is virtually unrecognizable in some of these proteins, which share as few as seven consecutive amino acids with the viral protein. Thus, small artificial proteins that bear little resemblance to a viral oncoprotein can nevertheless productively interact with the same cellular target. We speculate that similar cellular proteins may exist but have been overlooked due to their small size and hydrophobicity.

Effects of Conducting and Blocking Ions on the Structure and Stability of the Potassium Channel KcsA

This article reports on the interaction of conducting (K+) and blocking (Na+) monovalent metal ions with detergent-solubilized and lipid-reconstituted forms of the K+ channel KcsA. Monitoring of the protein intrinsic fluorescence reveals that the two ions bind competitively to KcsA with distinct affinities (dissociation constants for the KcsA·K+ and KcsA·Na+ complexes of ∼8 and 190 mm, respectively) and induce different conformations of the ion-bound protein. The differences in binding affinity as well as the higher K+ concentration bathing the intracellular mouth of the channel, through which the cations gain access to the protein binding sites, should favor that only KcsA·K+ complexes are formed under physiological-like conditions. Nevertheless, despite such prediction, it was also found that concentrations of Na+ well below its dissociation constant and even in the presence of higher K+ concentrations, cause a remarkable decrease in the protein thermal stability and facilitate thermal dissociation into subunits of the tetrameric KcsA, as concluded from the temperature dependence of the protein infrared spectra and from gel electrophoresis, respectively. These latter observations cannot be explained based on the occupancy of the binding sites from above and suggest that there must be additional ion binding sites, whose occupancy could not be detected by fluorescence and in which the affinity for Na+ must be higher or at least similar to that of K+. Moreover, cation binding as reported by means of fluorescence does not suffice to explain the large differences in free energy of stabilization involved in the formation of the KcsA·Na+ and KcsA·K+ complexes, which for the most part should arise from synergistic effects of the ion-mediated intersubunit interactions.