Biology subjects

McMurry, J. L.

Publications and source records attributed to McMurry, J. L..

5 recordsLinked to original sources

bioRxiv2026

Utilization of Cell-penetrating Peptide Adaptors to Enhance Delivery of Variably Charged Protein Cargos

Cell-penetrating peptides (CPPs) can deliver biomacromolecular cargos into cells, potentially enabling a new mode of intracellular drug delivery. However, a major problem with CPP-mediated delivery is entrapment of CPPs within endosomes and covalent linkages ensure CPPs and cargos share a common fate. We previously developed a CPP-adaptor system based on reversible, calcium-dependent cargo binding that produces cargo release from adaptors as complexes dissociate following internalization and Ca2+ efflux from early endosomes. Having employed CPP-adaptors with an array of protein cargos of differing charges, it became apparent that positively charged cargos often appeared to dominate internalization and that association with the adaptor had little effect. To systematically address the effects of cargo charge and CPP function, we tested the ability of several adaptors to increase internalization of a set of adaptor binding GFP cargos having net charges of +9, +15, +20, +25 and +36. Intrinsic internalization of these cargos reproduced reported patterns showing that positive charge increases internalization. However, labeling these cargos with a chemical fluorophore revealed that GFP fluorescence grossly underestimated total internalization. Internalization was charge and concentration dependent with more positive cargos showing apparent saturation of internalization at 100-400 nM, well below the concentrations at which covalently linked CPP-cargos are dosed. We tested the ability of 5 adaptors to internalize these cargos. Our prototype adaptor, TAT-CaM, was completely ineffective with the +9 cargo, but internalized moderately charged cargos extremely efficiently at concentrations far below the {micro}M range. A derivative adaptor, TAT-LAH4-CaM, was highly effective with all cargos and produced similar maximal internalization at 100-400 nM. However, two adaptors specifically designed with increased positive charge inhibited internalization of the most positive cargos. One of these, GFP-CaM, based on the supercharged GFP with net charge of +36, did increase internalization of the least positive cargos, demonstrating an adaptor with high affinity for the cell surface can increase internalization of a neutral cargo at very low concentration. The common maximal level of intrinsic GFP cargo internalization correlated with surface loading of these cargos, suggesting a limit to the beneficial effects of increased plasma membrane association. However, TAT-CaM further increased internalization via an apparently distinct mechanism. In this limited study of the interaction of cargo charge and adaptor efficacy, we found diverse behaviors that hint at the power and flexibility possible with adaptor/cargo internalization.

cell biology↗

bioRxiv2021

A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos

Cell-penetrating peptides (CPPs) are capable of transporting molecules to which they are tethered across cellular membranes. Unsurprisingly, CPPs have attracted attention for their potential drug delivery applications, but several technical hurdles remain to be overcome. Chief among them is the so-called endosomal escape problem, i.e. the propensity of CPP-cargo molecules to be endocytosed but remain entrapped in endosomes rather than reaching the cytosol. Previously, a CPP fused to calmodulin that bound calmodulin binding site-containing cargos was shown to efficiently deliver cargos to the cytoplasm, effectively overcoming the endosomal escape problem. The CPP-adaptor, "TAT-CaM," evinces delivery at nM concentrations and more rapidly than we had previously been able to measure. To better understand the kinetics and mechanism of CPP-adaptor-mediated cargo delivery, a real-time cell penetrating assay was developed in which a flow chamber containing cultured cells was installed on the stage of a confocal microscope to allow for observation ab initio. Also examined in this study was an improved CPP-adaptor that utilizes naked mole rat (Heterocephalus glaber) calmodulin in place of human and results in superior internalization, likely due to its lesser net negative charge. Adaptor-cargo complexes were delivered into the flow chamber and fluorescence intensity in the midpoint of baby hamster kidney cells was measured as a function of time. Delivery of 400 nM cargo was observed within seven minutes and fluorescence continued to increase linearly as a function of time. Cargo-only control experiments showed that the minimal uptake which occurred independently of the CPP-adaptor resulted in punctate localization consistent with endosomal entrapment. A distance analysis was performed for cell-penetration experiments in which CPP-adaptor-delivered cargo showing wider dispersions throughout cells as compared to an analogous covalently-bound CPP-cargo. Small molecule endocytosis inhibitors did not have significant effects upon delivery. The real-time assay is an improvement upon static endpoint assays and should be informative in a broad array of applications.

cell biology↗

bioRxiv2020

Utilization of a cell-penetrating peptide-adaptor for delivery of HPV protein E2 into cervical cancer cells to arrest cell growth and promote cell death

Cervical cancer is the second leading cause of cancer deaths in women worldwide. Human papillomavirus (HPV) is the causative agent of nearly all forms of cervical cancer, which arises upon viral integration into the host genome and concurrent loss of regulatory gene E2. E2 protein regulates viral oncoproteins E6 and E7. Loss of E2 upon viral integration results in unregulated expression and activity of E6 and E7, which promotes carcinogenesis. Previous studies using gene-based delivery show that reintroduction of E2 into cervical cancer cell lines can reduce proliferative capacity and promote apoptosis. However, owing in part to limitations on transfection in vivo, E2 reintroduction has yet to achieve therapeutic usefulness. A promising new approach is protein-based delivery systems utilizing cell-penetrating peptides (CPPs). CPPs readily traverse the plasma membrane and are able to carry with them biomolecular cargos to which they are attached. Though more than two decades of research have been dedicated to their development for delivery of biomolecular therapeutics, the full potential of CPPs has yet to be realized as the field is hindered by the tendency of CPP-linked cargos to be trapped in endosomes as well as having significant off-target potential in vivo. Using a CPP-adaptor system that reversibly binds cargo thereby overcoming the endosomal entrapment that hampers other CPP methods, bioactive E2 protein was delivered into living cervical cancer cells, resulting in inhibition of cellular proliferation and promotion of cell death in a time- and dose-dependent manner. The results suggest that this nucleic acid- and virus-free delivery method could be harnessed to develop novel, effective protein therapeutics for treatment of cervical cancer.

cell biology↗

bioRxiv2019

Kinetic Analysis of Bacteriophage Sf6 Binding to Outer Membrane Protein A Using Whole Virions

For successful infection, viruses must recognize their respective host cells. A common mechanism of host recognition by viruses is to utilize a portion of the host cell as a receptor. Bacteriophage Sf6, which infects Shigella flexneri, uses lipopolysaccharide as a primary receptor and then requires interaction with a secondary receptor, a role that can be fulfilled by either outer membrane proteins (Omp) A or C. Our previous work showed that specific residues in the loops of OmpA mediate Sf6 infection. To better understand Sf6 interactions with OmpA loop variants, we determined the kinetics of these interactions through the use of biolayer interferometry, an optical biosensing technique that yields data similar to surface plasmon resonance. Here, we successfully tethered whole Sf6 virions, determined the binding constant of Sf6 to OmpA to be 36 nM. Additionally, we showed that Sf6 bound to five variant OmpAs and the resulting kinetic parameters varied only slightly. Based on these data, we propose a model in which Sf6: Omp receptor recognition is not solely based on kinetics, but likely also on the ability of an Omp to induce a conformational change that results in productive infection.

microbiology↗

bioRxiv2018

Control of eNOS and nNOS through regulation of obligatory conformational changes in the reductase catalytic cycle

Endothelial and neuronal nitric oxide synthases (eNOS, nNOS) are important signal generators in a number of processes including angiogenesis and neurotransmission. The homologous inducible isoform (iNOS) occupies a multitude of conformational states in a catalytic cycle, including subnanosecond input and output states and a distribution of open conformations with average lifetimes of ~4.3 ns. In this study, fluorescence lifetime spectroscopy was used to probe conformational states of purified eNOS and nNOS in the presence of chaotropes, calmodulin, NADP+ and NADPH. Two-domain FMN/oxygenase constructs of nNOS were also examined with respect to calmodulin effects. Optical biosensing was used to analyze calmodulin binding in the presence of NADP+ and NADPH. Calmodulin binding induced a shift of the population away from the input and to the open and output states of NOS. NADP+ shifted the population towards the input state. The oxygenase domain, lacking the input state, provided a measure of calmodulin-induced open-output transitions. A mechanism for regulation by calmodulin and an elucidation of the catalytic mechanism are suggested by a conformational lockdown model. Calmodulin speeds transitions between input and open and between open and output states, effectively reducing the conformational manifold, speeding catalysis. Conformational control of catalysis involves reorientation of the FMN binding domain, of which fluorescence lifetime is an indicator. The approach described herein is a new tool for biophysical and structural analysis of NOS enzymes, regulatory events and other homologous reductase-containing enzymes.\n\nA note to the readerThis manuscript has over the past several years been submitted to and rejected by several journals, usually on the basis of reviewer opinion that it was not an important enough result to merit inclusion in the journal. Owing to the passing of the first author and the loss of his expertise in fluorescence lifetime spectroscopy, it has become too onerous a task to continually revise the manuscript to suit the whims of reviewers who nevertheless still reject the work. We are thus simply releasing the final form of the manuscript to BioRxiv in the hopes that it finds a readership who will find, as we do, that the results are of value to the field.

biochemistry↗