Cell-penetrating peptides, PepFects, show no evidence of toxicity and immunogenicity in vitro and in vivo

Cell-penetrating peptide based vehicles have been developed for the delivery of different payloads into the cells in culture and in animals. However, several biological features, among which is the tendency to trigger innate immune response, limit the development of highly efficient peptide-based drug delivery vectors. This study aims to evaluate the influence of transportan 10 (TP10) and its chemically modified derivatives, PepFects (PFs), on the innate immune response of the host system. PFs have shown high efficiency in nucleic acid delivery in vitro and in vivo; hence, the estimation of their possible toxic side effects would be of particular interest. In this study, we analyzed cytotoxic and immunogenic response of PF3, PF4, and PF6 peptides in monocytic leukemia and peripheral blood mononuclear cell lines. In comparison with amphipathic PFs, TP10, TAT, stearyl-(RxR)(4) peptides, and the most widely used transfection reagents Lipofectamine 2000 and Lipofectamine RNAiMAX were also analyzed in this study. IL-1β, IL-18, and TNF-α cytokine release was detected using highly sensitive enzyme-linked immunosorbent assay (ELISA). Cell viability was detected by measuring the activity of cellular enzymes that reduce water-soluble tetrazolium salts to formazan dyes and apoptosis was evaluated by measuring the levels of caspase-1 and caspase-3/7 over untreated cells. All peptides were found to be nontoxic and nonimmunogenic in vitro at the concentrations of 10 μM and 5 μM, respectively, and at a dose of 5 mg/kg in vivo, suggesting that these CPPs exhibit a promising potential in the delivery of therapeutic molecules into the cell without risks of toxicity and inflammatory reactions.     

The genotype of early-transmitting HIV gp120s promotes α (4) β(7)-reactivity, revealing α (4) β(7) +/CD4+ T cells as key targets in mucosal transmission

Mucosal transmission of HIV is inefficient. The virus must breach physical barriers before it infects mucosal CD4⁺ T cells. Low-level viral replication occurs initially in mucosal CD4⁺ T cells, but within days high-level replication occurs in Peyer''s patches, the gut lamina propria and mesenteric lymph nodes. Understanding the early events in HIV transmission may provide valuable information relevant to the development of an HIV vaccine. The viral quasispecies in a donor contracts through a genetic bottleneck in the recipient, such that, in low-risk settings, infection is frequently established by a single founder virus. Early-transmitting viruses in subtypes A and C mucosal transmission tend to encode gp120s with reduced numbers of N-linked glycosylation sites at specific positions throughout the V1-V4 domains, relative to typical chronically replicating isolates in the donor quasispecies. The transmission advantage gained by the absence of these N-linked glycosylation sites is unknown. Using primary α₄β₇ ⁺/CD4⁺ T cells and a flow-cytometry based steady-state binding assay we show that the removal of transmission-associated N-linked glycosylation sites results in large increases in the specific reactivity of gp120 for integrin- α₄β₇. High-affinity for integrin α₄β₇, although not found in many gp120s, was observed in early-transmitting gp120s that we analyzed. Increased α₄β₇ affinity is mediated by sequences encoded in gp120 V1/V2. α₄β₇-reactivity was also influenced by N-linked glycosylation sites located in C3/V4. These results suggest that the genetic bottleneck that occurs after transmission may frequently involve a relative requirement for the productive infection of α₄β₇ ⁺/CD4⁺ T cells. Early-transmitting gp120s were further distinguished by their dependence on avidity-effects to interact with CD4, suggesting that these gp120s bear unusual structural features not present in many well-characterized gp120s derived from chronically replicating viruses. Understanding the structural features that characterize early-transmitting gp120s may aid in the design of an effective gp120-based subunit vaccine.     

Lipophilic derivatives of natural chlorins: Synthesis,mixed micelles with phospholipids,and uptake by cultured cells

The chemical synthesis of six lipophilic conjugates of chlorins was carried out, in which lipophilic fragment (either hexadecyl- or cholest-5-en-3β-yloxyethyl-) bound to 13¹-, 15²-, 17³-positions of macrocycle by formation of related carboxamides. Structure of synthesized conjugates was studied by spectral methods and molecular modeling. Lipophilic conjugates of chlorins, being mixed with egg yolk phosphatidyl choline, formed mixed micelles stable in aqueous media under physiological conditions. Mixed micelles of conjugates with phosphatidyl choline differing in stoichiometric compositions were prepared and characterized by absorption spectra, electron microscopy and laser scattering. These micelles were found to bind and internalized by human breast carcinoma MCF-7 cells. The presented data reveal that modification of macrocycle with lipophilic substituents, solubilization of obtained conjugates in aqueous medium as mixed micelles with phospholipids, and transfer of mixed micelles to cells is simple approach for targeting of chlorin derivatives, which apparently may be used in photodynamic therapy.     

Discovery of small molecule inhibitors of adenovirus by disrupting E3-19K/HLA-A2 interactions

The binding of the adenovirus (Ad) protein E3-19K with the human leukocyte antigen (HLA) plays an important role in Ad infections, which is the causative agent of a series of gastrointestinal, respiratory and ocular diseases. The objective of this research is to evaluate the essential interactions between E3-19K and HLA-A2 using the X-ray crystal structure of the E3-19K/HLA-A2 complex, and to identify small molecules that could potentially disrupt their binding. Computational methods, including molecular dynamic simulations, MM/GBSA calculations, and computational solvent mapping, were implemented to determine potential binding site(s) for small molecules. The previous experimentally determined hot spot residues, Q54 and E177 in HLA-A2, were also predicted to be the dominant residues for binding to E3-19K by our theoretical calculations. Several other residues were also found to play pivotal roles for the binding of E3-19K with HLA-A2. Residues adjacent to E177, including Q54 and several other residues theoretically predicted to be crucial in HLA-A2 were selected as a potential binding pocket to perform virtual screening with 1200 compounds from the Prestwick library. Seven hits were validated by surface plasmon resonance (SPR) as binders to HLA-A2 as a first step in identifying molecules that can perturb its association with the Ad E3-19K protein.     

Hypertrophic cardiomyopathy-causing Asp175asn and Glu180gly Tpm1 mutations shift tropomyosin strands further towards the open position during the ATPase cycle

Yeast calmodulin known to be ubiquitylated in vivo in a Ca²⁺ dependent manner has long remained an orphan substrate. Here we identify Saccharomyces cerevisiae Asr1p as an ubiquitin E3 ligase for yeast calmodulin, a protein involved in calcium signaling. A short region within Asr1p-C harboring two putative calmodulin-binding motifs is sufficient and necessary for interaction with calmodulin. The interaction is direct, occurs in vivo and depends on physiological concentrations of Ca²⁺. A minimal set of purified proteins including Asr1p E3 ligase was sufficient for in vitro ubiquitylation of calmodulin, a reaction that required a functional Asr1p Ring domain. We propose a role of the Asr1p E3 ligase activity in coping with stress.     

Evidence for a pathogenic role of BRCA1 L1705P and W1837X germ-line mutations

BRCA1 L1705P (c.5114T>C) has been classified in the NCBI SNP database as the variant with uncertain significance and is absent in major BRCA1 databases. BRCA1 W1837X (c.5511G>A) results in a loss of only last 27 residues of BRCA1 protein, thus its pathogenic role still requires a confirmation. This report describes two breast cancer (BC) patients carrying BRCA1 L1705P and W1837X germ-line mutations, respectively. Significant evidence for BC-predisposing impact of the mentioned mutations have been obtained: (1) both index cases presented with the triple-negative receptor status of BC disease; (2) complete segregation with BRCA1-related cancers was observed in the families of these patients; (3) somatic loss of the remaining (wild-type) BRCA1 allele was detected in tumor tissues of the affected women. The results of this study have to be taken into account while providing genetic counseling to cancer patients and while considering the use of BRCA1-specific therapeutic compounds for BC treatment.