SUMMARY PARAGRAPH MicroRNAs (miRNAs) are short non-coding RNAs expressed in different tissue and cell types that suppress the expression of target genes. As such, miRNAs are critical cogs in numerous biological processes1,2, and dysregulated miRNA expression is correlated with many human diseases. Certain miRNAs, called oncomiRs, play a causal role in the onset and maintenance of cancer when overexpressed. Tumors that depend on these miRNAs are said to display oncomiR addiction3–5. Some of the most effective anticancer therapies target oncogenes like EGFR and HER2; similarly, inhibition of oncomiRs using antisense oligomers (i.e. antimiRs) is an evolving therapeutic strategy6,7. However, the in vivo efficacy of current antimiR technologies is hindered by physiological and cellular barriers to delivery into targeted cells8. Here we introduce a novel antimiR delivery platform that targets the acidic tumor microenvironment, evades systemic clearance by the liver, and facilitates cell entry via a non-endocytic pathway. We found that the attachment of peptide nucleic acid (PNA) antimiRs to a peptide with a low pH-induced transmembrane structure (pHLIP) produced a novel construct that could target the tumor microenvironment, transport antimiRs across plasma membranes under acidic conditions such as those found in solid tumors (pH ~6), and effectively inhibit the miR-155 oncomiR in a mouse model of lymphoma. This study introduces a new paradigm in the use of antimiRs as anti-cancer drugs, which can have broad impacts on the field of targeted drug delivery.
Cholesterol is an integral component of eukaryotic cell membranes and a key molecule in controlling membrane fluidity, organization, and other physicochemical parameters. It also plays a regulatory function in antibiotic drug resistance and the immune response of cells against viruses, by stabilizing the membrane against structural damage. While it is well understood that, structurally, cholesterol exhibits a densification effect on fluid lipid membranes, its effects on membrane bending rigidity are assumed to be nonuniversal; i.e., cholesterol stiffens saturated lipid membranes, but has no stiffening effect on membranes populated by unsaturated lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). This observation presents a clear challenge to structure–property relationships and to our understanding of cholesterol-mediated biological functions. Here, using a comprehensive approach—combining neutron spin-echo (NSE) spectroscopy, solid-state deuterium NMR (2H NMR) spectroscopy, and molecular dynamics (MD) simulations—we report that cholesterol locally increases the bending rigidity of DOPC membranes, similar to saturated membranes, by increasing the bilayer’s packing density. All three techniques, inherently sensitive to mesoscale bending fluctuations, show up to a threefold increase in effective bending rigidity with increasing cholesterol content approaching a mole fraction of 50%. Our observations are in good agreement with the known effects of cholesterol on the area-compressibility modulus and membrane structure, reaffirming membrane structure–property relationships. The current findings point to a scale-dependent manifestation of membrane properties, highlighting the need to reassess cholesterol’s role in controlling membrane bending rigidity over mesoscopic length and time scales of important biological functions, such as viral budding and lipid–protein interactions.
The practical application of nanoparticles (NPs) as chemotherapeutic drug delivery systems is often hampered by issues such as poor circulation stability and targeting inefficiency. Here, we have utilized a simple approach to prepare biocompatible and biodegradable pHresponsive hybrid NPs that overcome these issues. The NPs consist of a drug-loaded polylactic-co-glycolic acid (PLGA) core covalently 'wrapped' with a crosslinked bovine serum albumin (BSA) shell designed to minimize interactions with serum proteins and macrophages that inhibit target recognition. The shell is functionalized with the acidity-triggered rational membrane (ATRAM) peptide to facilitate internalization specifically into cancer cells within the acidic tumor microenvironment. Following uptake, the unique intracellular conditions of cancer cells degrade the NPs, thereby releasing the chemotherapeutic cargo. The drugloaded NPs showed potent anticancer activity in vitro and in vivo while exhibiting no toxicity to healthy tissue. Our results demonstrate that the ATRAM-BSA-PLGA NPs are a promising targeted cancer drug delivery platform.
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 K D ס 0.3 ± 0.1 M, as measured by fluorescence. We discuss the implications for the GS regulation mechanisms of IF7 being unfolded.Keywords: Inactivating factor; structure; glutamine synthetase; "natively unfolded" protein; intrinsic disorder A fundamental question in cellular physiology is how cells are able to recognize and respond to changes in their environment. To answer that question, it is necessary to identify not only the physiological signal, but also the protein(s) that senses and induces the responses to that signal. Different signal transduction pathways operate in bacteria to regulate gene expression. This regulation occurs at the transcriptional as well as the posttranscriptional levels. In some cases, a metabolite functions as the signal that triggers and alters gene expression by interacting directly with a transcription factor. However, protein-protein interaction mechanisms occur frequently in the regulatory systems.The response of bacteria to nitrogen limitation represents a paradigm of those regulatory systems (Merrick and Edwards 1995;Magasanik 1996). In bacteria, glutamine and 2-oxoglutarate are the most important molecules involved in nitrogen sensing. The intracellular concentration of these metabolites, and also the ratio between them, change depending on nitrogen nutritional conditions (Hu et al. 1999). Glutamine synthetase (GS), the enzyme that catalyses the synthesis of glutamine from glutamic acid, ATP, and ammonium, is then also subject to regulation depending on nitrogen availability. GS type I, the most common type found in prokaryotes, is a large protein with a molecular weight of about 600,000 Da. It consists of 12 identical subReprint requests to: José L. Neira, Instituto de Biología Molecular y Celular, Edificio Torregaitán, Universidad Miguel Hernández, Avda. del Ferrocarril s/n, 03202 Elche (Alicante), Spain; e-mail: jlneira@umh.es; fax: 34-96-665-8758. 4 These two authors contributed equally to this work. Abbreviations: ANS, 8-anilinonapthalene-1-sulfonic acid; CD, circular dichrois...
Different patterns of channel activity have been detected by patch clamping excised membrane patches from reconstituted giant liposomes containing purified KcsA, a potassium channel from prokaryotes. The more frequent pattern has a characteristic low channel opening probability and exhibits many other features reported for KcsA reconstituted into planar lipid bilayers, including a moderate voltage dependence, blockade by Na ؉ , and a strict dependence on acidic pH for channel opening. The predominant gating event in this low channel opening probability pattern corresponds to the positive coupling of two KcsA channels. However, other activity patterns have been detected as well, which are characterized by a high channel opening probability (HOP patterns), positive coupling of mostly five concerted channels, and profound changes in other KcsA features, including a different voltage dependence, channel opening at neutral pH, and lack of Na During the last decades, the use of high resolution electrophysiological techniques to study ion channels has provided a large amount of information on functional aspects of these important membrane proteins. Such a detailed information on channel function, however, has not been accompanied by structural knowledge until recently, when several structurally simpler homologues of mammalian ion channels found in extremophyle bacteria or Archaea and remarkably resistant to harsh experimental conditions, have been purified, crystallized and their structure solved at high resolution by x-ray diffraction methods (1-4). A K ϩ channel from the soil bacteria Streptomyces lividans named KcsA 4 (1), a homotetramer made up of identical 160-amino acid subunits, was the first of such structures to be solved (5, 6), and, although the x-ray structure corresponds to a closed channel conformation, it has contributed much to our current understanding of ion selectivity and permeation. Ironically, there was little or no functional information on KcsA by the time its structure was solved, and then several groups undertook the task of characterizing its single channel properties, which has been surrounded by controversy. For instance, Schrempf's group, discoverers of KcsA in S. lividans, reported a strong dependence of channel opening on acidic pH, multiple conductance states with opening probabilities near 0.5, and unusual permeabilities to Na ϩ , Li ϩ , Ca 2ϩ , or Mg 2ϩ , along with K ϩ (7-9). In contrast, Miller's group (10, 11) using purified KcsA reconstituted into planar lipid bilayers found a single conductance state with a much lower opening probability, as well as orthodox ion selectivity and other properties to validate KcsA as a bona fide K ϩ channel and as a faithful structural model for these molecules. The above discrepancies were never fully explained but, still, it became generally accepted that KcsA behaves as a moderately voltage-dependent, K ϩ -selective channel with a characteristic low opening probability and the peculiar property of opening only in response to very acidic pH condit...
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