Expression and subcellular localization of USH1C/harmonin in human retina provides insights into pathomechanisms and therapy.

Usher syndrome (USH) is the most common form of hereditary deaf-blindness in humans. USH is a complex genetic disorder, assigned to three clinical subtypes differing in onset, course and severity, with USH1 being the most severe. Rodent USH1 models do not reflect the ocular phenotype observed in human patients to date; hence, little is known about the pathophysiology of USH1 in the human eye. One of the USH1 genes, USH1C, exhibits extensive alternative splicing and encodes numerous harmonin protein isoforms that function as scaffolds for organizing the USH interactome. RNA-seq analysis of human retinae uncovered harmonin_a1 as the most abundant transcript of USH1C. Bulk RNA-seq analysis and immunoblotting showed abundant expression of harmonin in Müller glia cells (MGCs) and retinal neurons. Furthermore, harmonin was localized in the terminal endfeet and apical microvilli of MGCs, presynaptic region (pedicle) of cones and outer segments (OS) of rods as well as at adhesive junctions between MGCs and photoreceptor cells (PRCs) in the outer limiting membrane (OLM). Our data provide evidence for the interaction of harmonin with OLM molecules in PRCs and MGCs and rhodopsin in PRCs. Subcellular expression and colocalization of harmonin correlate with the clinical phenotype observed in USH1C patients. We also demonstrate that primary cilia defects in USH1C patient-derived fibroblasts could be reverted by the delivery of harmonin_a1 transcript isoform. Our studies thus provide novel insights into PRC cell biology, USH1C pathophysiology and development of gene therapy treatment(s).

Identification of a novel spirocyclic Nek2 inhibitor using high throughput virtual screening.

NIMA Related Kinase 2 (Nek2) kinase is an attractive target for the development of therapeutic agents for several types of highly invasive cancers. Despite this, no small molecule inhibitor has advanced to the late clinical stages thus far. In this work, we have identified a novel spirocyclic inhibitor (V8) of Nek2 kinase, utilizing a high-throughput virtual screening (HTVS) approach. Using recombinant Nek2 enzyme assays, we show that V8 can inhibit Nek2 kinase activity (IC50 = 2.4 ± 0.2 µM) by binding to the enzyme's ATP pocket. The inhibition is selective, reversible and is not time dependent. To understand the key chemotype features responsible for Nek2 inhibition, a detailed structure-activity relationships (SAR) was performed. Using molecular models of the energy-minimized structures of Nek2-inhibitory complexes, we identify key hydrogen-bonding interactions, including two from the hinge-binding region, likely responsible for the observed affinity. Finally, using cell-based studies, we show that V8 attenuates (a) pAkt/PI3 Kinase signaling in a dose-dependent manner, and (b) proliferative and migratory phenotypes of highly aggressive human MDA-MB-231 breast and A549 lung cancer cell lines. Thus, V8 is an important novel lead compound for the development of highly potent and selective Nek2 inhibitory agents.

Clickable, selective, and cell-permeable activity-based probe of human cathepsin B - Minimalistic approach for enhanced selectivity.

Human cathepsin B is a cysteine-dependent protease whose roles in both normal and diseased cellular states remain yet to be fully delineated. This is primarily due to overlapping substrate specificities and lack of unambiguously annotated physiological functions. In this work, a selective, cell-permeable, clickable and tagless small molecule cathepsin B probe, KDA-1, is developed and kinetically characterized. KDA-1 selectively targets active site Cys25 residue of cathepsin B for labeling and can detect active cellular cathepsin B in proteomes derived from live human MDA-MB-231 breast cancer cells and HEK293 cells. It is anticipated that KDA-1 probe will find suitable applications in functional proteomics involving human cathepsin B enzyme.

Cell penetrable, clickable and tagless activity-based probe of human cathepsin L.

Human cathepsin L is a ubiquitously expressed endopeptidase and is known to play critical roles in a wide variety of cellular signaling events. Its overexpression has been implicated in numerous human diseases, including highly invasive forms of cancer. Inhibition of cathepsin L is therefore considered a viable therapeutic strategy. Unfortunately, several redundant and even opposing roles of cathepsin L have recently emerged. Selective cathepsin L probes are therefore needed to dissect its function in context-specific manner before significant resources are directed into drug discovery efforts. Herein, the development of a clickable and tagless activity-based probe of cathepsin L is reported. The probe is highly efficient, active-site directed and activity-dependent, selective, cell penetrable, and non-toxic to human cells. Using zebrafish model, we demonstrate that the probe can inhibit cathepsin L function in vivo during the hatching process. It is anticipated that the probe will be a highly effective tool in dissecting cathepsin L biology at the proteome levels in both normal physiology and human diseases, thereby facilitating drug-discovery efforts targeting cathepsin L.

Ellagic acid promoted biomimetic synthesis of shape-controlled silver nanochains.

In this work, ellagic acid (EA), a naturally occurring plant polyphenol, was utilized for the biomimetic synthesis of silver (Ag) nanoparticles, which over a period of time formed extended branched nanochains of hexagonal-shaped silver nanoparticles. It was found that EA not only has the capability of reducing silver ions, resulting in the formation of Ag nanoparticles, due to its extended polyphenolic system, but also appears to recognize and affect the Ag nanocrystal growth on the (111) face, leading to the formation of hexagon-shaped Ag nanocrystals. Initially, various Ag nanocrystal shapes were observed; however, over a longer period of time, a majority of hexagonal-shaped nanocrystals were formed. Although the exact mechanism of formation of the nanocrystals is not known, it appears that EA attaches to the silver nuclei, leading to lower surface energy of the (111) face. Further, the nanocrystals fuse together, forming interfaces among the aggregates, and, with time, those interfaces become lesser, and the nanoparticles merge together and share the same single crystallographic orientation, which leads to the formation of long elongated chains of hexagonal nanoparticles. This biomimetic approach may be developed as a green synthetic method to prepare building blocks with tunable properties for the development of nanodevices. Further, we explored the antibacterial properties and found that the tandem of EA-Ag nanochains substantially enhanced the antibacterial properties of both gram-positive and gram-negative bacteria compared to silver nanoparticles or EA alone. Additionally, the materials were also utilized for imaging of mammalian NRK (normal rat kidney) cells.

pH dependent spontaneous growth of ellagic acid assemblies for targeting HeLa cells.

Herein, we have studied the self-assembly and the spontaneous growth of microassemblies of the plant polyphenol ellagic acid for HeLa cancer cell imaging and therapy. The growth of the assemblies was studied at varying pH over time. It was found that initially microspheres were formed which gradually transformed into microfibers via nucleation and polymerization process. The optimum growth of microfibers was found to be in the pH range of 6-8. We have shown that the microfibers successfully adhered to the HeLa cell membranes and inhibited their proliferation. This biological approach, using assemblies derived from plant polyphenols, may be used for direct cellular drug delivery and may potentially help develop a simple and economical method to create building blocks with desired properties for a new generation of sensors, bioimaging and drug delivery systems.

Development of self-assembled phytosterol based nanoassemblies as vehicles for enhanced uptake of doxorubicin to HeLa cells.

Nanoscale supramolecular systems have been increasingly gaining importance as drug release vehicles due to their ability to target tumor cells. In this work, we have developed a new class of nanoassemblies derived from the phytosterol 24-EpiBrassinolide (EpiB) for the development of nanocarriers for the anticancer drug Doxorubicin (DOX). EpiB is a biocompatible cholesterol mimic, and has inherent apoptotic properties toward cancer cells. Thus, by encapsulating DOX within a nanocarrier with innate anticancer ability we have developed a targeting system that can enhance the uptake and efficacy of DOX in tumor cells. The nanocarriers were formed by self-assembly of EpiB. The morphologies of assemblies formed were dependent upon the concentration of EpiB used. While at low concentrations, spherical nanoassemblies were formed, at higher concentration, lamellar aggregates with birefringence properties were observed. Our results indicated that the drug loaded nanocarriers showed diffusion controlled release of the drug, and demonstrated antiproliferative effects, cellular uptake and were apoptotic against HeLa cervical cancer cells. Furthermore, EpiB loaded DOX enhanced both apoptosis and uptake into the cell's nuclei. These supramolecular assemblies may have potential applications for enhancing efficacy of chemotherapeutic drugs through passive targeting.

Templated growth of calcium phosphate on tyrosine derived microtubules and their biocompatibility.

Microtubular structures were self-assembled in aqueous media from a newly synthesized bolaamphiphile, bis(N-alpha-amido-tyrosyl-tyrosyl-tyrosine)-1,5-pentane dicarboxylate. In order to increase the biocompatibility of the microtubules, they were functionalized with the peptide sequence GRGDSP. Further, calcium phosphate nanocrystals were grown on the microtubules. In some cases, collagen was added in order to mimic the components of natural bone tissue. The biomaterials obtained were characterized via transmission electron microscopy (TEM), atomic force microscopy (AFM), IR, and energy dispersive X-ray spectroscopy (EDX) analyses. The biocompatibility of the calcium phosphate-coated microtubules was studied by conducting in vitro cell-attachment, cell-proliferation and cytotoxicity studies using mouse embryonic fibroblast (MEF) cells. The studies revealed that the biomaterials were found to be non-toxic and biocompatible. The functionalized tubular assemblies coated with calcium phosphate nanocrystals mimic the nanoscale composition of natural bone and may potentially support bone in-growth and osseointegration when used in orthopaedic or dental applications.

Layer-by-layer assembly of peptide based bioorganic-inorganic hybrid scaffolds and their interactions with osteoblastic MC3T3-E1 cells.

In this work we have developed a new family of biocomposite scaffolds for bone tissue regeneration by utilizing self-assembled fluorenylmethyloxycarbonyl protected Valyl-cetylamide (FVC) nanoassemblies as templates. To tailor the assemblies for enhanced osteoblast attachment and proliferation, we incorporated (a) Type I collagen, (b) a hydroxyapatite binding peptide sequence (EDPHNEVDGDK) derived from dentin sialophosphoprotein and (c) the osteoinductive bone morphogenetic protein-4 (BMP-4) to the templates by layer-by-layer assembly. The assemblies were then incubated with hydroxyapatite nanocrystals blended with varying mass percentages of TiO2 nanoparticles and coated with alginate to form three dimensional scaffolds for potential applications in bone tissue regeneration. The morphology was examined by TEM and SEM and the binding interactions were probed by FITR spectroscopy. The scaffolds were found to be non-cytotoxic, adhered to mouse preosteoblast MC3T3-E1 cells and promoted osteogenic differentiation as indicated by the results obtained by alkaline phosphatase assay. Furthermore, they were found to be biodegradable and possessed inherent antibacterial capability. Thus, we have developed a new family of tissue-engineered biocomposite scaffolds with potential applications in bone regeneration.

Self-assembling peptide assemblies bound to ZnS nanoparticles and their interactions with mammalian cells.

Self-assembling peptide sequences (both synthetic and natural) have emerged as a new group of building blocks for diverse applications. In this work we investigated the formation of assemblies of three diverse peptide sequences derived from the crustacean cardioactive peptide CCAP (1-9), a cardioaccelerator and neuropeptide transmitter in crustaceans, atrial natriuretic hormone ANP (1-28), a powerful vasodilator secreted by heart muscle cells of mammals, as well as adamstsostatin peptide ADS (1-17), which functions as an inhibitor of angiogenesis. The formation of assemblies was found to be dependent upon the sequences as well as the pH in which the assemblies were grown. The secondary structural transformation of the peptides was studied by circular dichroism as well as FTIR spectroscopy. In order to render the sequences luminescent, we conjugated the assemblies with ZnS nanoparticles. Finally the interactions of the peptide bound ZnS nanoparticles with mammalian normal rat kidney cells were explored. In some cases the nanoconjugates were found to adhere not only to the cellular membranes but also extend into the cytoplasm. Thus, such nanocomposites may be utilized for cell penetration and have the potential to serve as coercive multifunctional vectors for bioimaging and cellular delivery.

Fabrication of collagen-elastin-bound peptide microtubes for mammalian cell attachment.

In this work we have designed self-assembled peptide-based microconstructs and examined their interactions with elastin and collagen for potential application as scaffolds for chondrocyte cell attachment. Being biological in nature, peptide-based nano- and microstructures have intrinsic molecular recognition properties which allow extensive chemical, conformational and functional diversity. We have synthesized a new peptide bolaamphiphile, bis(N-α-amido-val)-1,5-pentane dicarboxylate, and examined its self-assembly at varying pH values. The formation of high-density networks of nano- and microtubular structures was found to be in the range of pH 4-6. The formed microtubes were then covalently bound to varying concentrations of the extracellular matrix protein elastin, a versatile protein that allows for an extensive array of physical and chemical modifications to attune properties towards diverse necessities of biomedical applications. We found that binding to microtubes was concentration dependent. The morphological and chemical changes complementing the processes of self-assembly and binding to elastin were examined by electron microscopic and spectroscopic methods. Furthermore, we also incorporated the extracellular matrix protein type-I collagen, a critical constituent for designing biocompatible scaffolds, into the elastin functionalized micro-tubes. Since the main goal is to develop highly biocompatible protein functionalized microstructures that support cellular interactions, we examined the interactions of the microcomposites with chondrocyte cell line, in order to assess the biocompatibility and interaction between the microconstructs and the cells. The designed elastin and collagen-bound peptide microtubes may potentially serve as a new class of biomaterials by promoting cell growth and proliferation.

Fabrication of ellagic acid incorporated self-assembled peptide microtubes and their applications.

Ellagic acid (EA), a plant polyphenol known for its wide-range of health benefits was encapsulated within self-assembled threonine based peptide microtubes. The microtubes were assembled using the synthesized precursor bolaamphiphile bis(N-α-amido threonine)-1,5-pentane dicarboxylate. The self-assembly of the microstructures was probed at varying pH. In general, tubular formations were observed at a pH range of 4-6. The formed microtubes were then utilized for fabrication with EA. We probed the ability of the microtubes as drug release vehicles for EA as well as for antibacterial applications. It was found that the release of EA was both pH and concentration dependent. The biocompatibility as well as cytotoxicity of the EA-fabricated microtubes was examined in the presence of mammalian normal rat kidney (NRK) cells. Finally the antibacterial effects of the EA incorporated peptide microtubes was examined against Escherichia coli and Staphylococcus aureus.

Morphology controlled growth of chitosan-bound microtubes and a study of their biocompatibility and antibacterial activity.

Self-assembled peptide microtubes are fabricated with the biopolymer chitosan. The microtubes are covalently attached to chitosan and the morphology of the chitosan assembled on the surface of the microtubes can be tuned by altering the pH of the growth solution. Cytotoxicity studies in the presence of mouse embryonic fibroblasts indicate that the chitosan-bound microtubes are highly biocompatible and the cells are able to survive and proliferate at a similar rate to the control. Antibacterial studies in the presence of E. coli prove that the chitosan-bound microtubes are bactericidal. This simple method for the development of biocompatible microstructures will facilitate cell targeting, fabrication of efficient carrier devices, and the preparation of highly efficient antibacterial materials.

Characterization of myosin-II binding to Golgi stacks in vitro.

In addition to important roles near the actin-rich cell cortex, ample evidence indicates that multiple myosins are also involved in membrane movements in the endomembrane system. Nonmuscle myosin-II has been shown to have roles in anterograde and retrograde trafficking at the Golgi. Myosin-II is present on Golgi stacks isolated from intestinal epithelial cells and has been localized to the Golgi in several polarized and unpolarized cell lines. An understanding of roles of myosin-II in Golgi physiology will be facilitated by understanding the molecular arrangement of myosin-II at the Golgi. Salt-washing removes endogenous myosin-II from isolated Golgi and purified brush border myosin-II can bind in vitro. Brush border myosin-II binds to a tightly bound Golgi peripheral membrane protein with a K(1/2) of 75 nM and binding is saturated at 0.7 pmol myosin/microg Golgi. Binding studies using papain cleavage fragments of brush border myosin-II show that the 120-kDa rod domain, but not the head domain, of myosin heavy chain can bind directly to Golgi stacks. The 120-kDa domain does not bind to Golgi membranes when phosphorylated in vitro with casein kinase-II. These results suggest that phosphorylation in the rod domain may regulate the binding and/or release of myosin-II from the Golgi. These data support a model in which myosin-II is tethered to the Golgi membrane by its tail and actin filaments by its head. Thus, translocation along actin filaments may extend Golgi membrane tubules and/or vesicles away from the Golgi complex.

Myosin VIIa, harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle.

Deaf-blindness in three distinct genetic forms of Usher type I syndrome (USH1) is caused by defects in myosin VIIa, harmonin and cadherin 23. Despite being critical for hearing, the functions of these proteins in the inner ear remain elusive. Here we show that harmonin, a PDZ domain-containing protein, and cadherin 23 are both present in the growing stereocilia and that they bind to each other. Moreover, we demonstrate that harmonin b is an F-actin-bundling protein, which is thus likely to anchor cadherin 23 to the stereocilia microfilaments, thereby identifying a novel anchorage mode of the cadherins to the actin cytoskeleton. Moreover, harmonin b interacts directly with myosin VIIa, and is absent from the disorganized hair bundles of myosin VIIa mutant mice, suggesting that myosin VIIa conveys harmonin b along the actin core of the developing stereocilia. We propose that the shaping of the hair bundle relies on a functional unit composed of myosin VIIa, harmonin b and cadherin 23 that is essential to ensure the cohesion of the stereocilia.