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1.
PNAS Nexus ; 3(5): pgae192, 2024 May.
Article in English | MEDLINE | ID: mdl-38783894

ABSTRACT

Atrial fibrillation (AF), the most common cardiac arrhythmia, is strongly associated with several comorbidities including heart failure (HF). AF in general, and specifically in the context of HF, is progressive in nature and associated with poor clinical outcomes. Current therapies for AF are limited in number and efficacy and do not target the underlying causes of atrial remodeling such as inflammation or fibrosis. We previously identified the calcium-activated SK4 K+ channels, which are preferentially expressed in the atria relative to the ventricles in both rat and human hearts, as attractive druggable target for AF treatment. Here, we examined the ability of BA6b9, a novel allosteric inhibitor of SK4 channels that targets the specific calmodulin-PIP2 binding domain, to alter AF susceptibility and atrial remodeling in a systolic HF rat postmyocardial infarction (post-MI) model. Daily BA6b9 injection (20 mg/kg/day) for 3 weeks starting 1-week post-MI prolonged the atrial effective refractory period, reduced AF induction and duration, and dramatically prevented atrial structural remodeling. In the post-MI left atrium (LA), pronounced upregulation of the SK4 K+ channel was observed, with corresponding increases in collagen deposition, α-SMA levels, and NLRP3 inflammasome expression. Strikingly, BA6b9 treatment reversed these changes while also significantly reducing the lateralization of the atrial connexin Cx43 in the LA of post-MI rats. Our findings indicate that the blockade of SK4 K+ channels using BA6b9 not only favors rhythm control but also remarkably reduces atrial structural remodeling, a property that is highly desirable for novel AF therapies, particularly in patients with comorbid HF.

2.
J Med Chem ; 64(3): 1510-1523, 2021 02 11.
Article in English | MEDLINE | ID: mdl-33522230

ABSTRACT

Necrosis is the main mode of cell death, which leads to multiple clinical conditions affecting hundreds of millions of people worldwide. Its molecular mechanisms are poorly understood, hampering therapeutics development. Here, we identify key proteolytic activities essential for necrosis using various biochemical approaches, enzymatic assays, medicinal chemistry, and siRNA library screening. These findings provide strategies to treat and prevent necrosis, including known medicines used for other indications, siRNAs, and establish a platform for the design of new inhibitory molecules. Indeed, inhibitors of these pathways demonstrated protective activity in vitro and in vivo in animal models of traumatic brain injury, acute myocardial infarction, and drug-induced liver toxicity. Consequently, this study may pave the way for the development of novel therapies for the treatment, inhibition, or prevention of a large number of hitherto untreatable diseases.


Subject(s)
Necroptosis/drug effects , Necrosis/prevention & control , Pancreatic Elastase/antagonists & inhibitors , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Animals , Brain Injuries, Traumatic/drug therapy , Brain Injuries, Traumatic/pathology , Cell Death/drug effects , Chemical and Drug Induced Liver Injury/drug therapy , High-Throughput Screening Assays , Humans , Mice, Inbred BALB C , Mice, Inbred C57BL , Myocardial Infarction/drug therapy , Myocardial Infarction/pathology , RNA, Small Interfering , U937 Cells
3.
Gastroenterology ; 159(5): 1807-1823, 2020 11.
Article in English | MEDLINE | ID: mdl-32653496

ABSTRACT

BACKGROUND & AIMS: The intestinal barrier protects intestinal cells from microbes and antigens in the lumen-breaches can alter the composition of the intestinal microbiota, the enteric immune system, and metabolism. We performed a screen to identify molecules that disrupt and support the intestinal epithelial barrier and tested their effects in mice. METHODS: We performed an imaging-based, quantitative, high-throughput screen (using CaCo-2 and T84 cells incubated with lipopolysaccharide; tumor necrosis factor; histamine; receptor antagonists; and libraries of secreted proteins, microbial metabolites, and drugs) to identify molecules that altered epithelial tight junction (TJ) and focal adhesion morphology. We then tested the effects of TJ stabilizers on these changes. Molecules we found to disrupt or stabilize TJs were administered mice with dextran sodium sulfate-induced colitis or Citrobacter rodentium-induced intestinal inflammation. Colon tissues were collected and analyzed by histology, fluorescence microscopy, and RNA sequencing. RESULTS: The screen identified numerous compounds that disrupted or stabilized (after disruption) TJs and monolayers of epithelial cells. We associated distinct morphologic alterations with changes in barrier function, and identified a variety of cytokines, metabolites, and drugs (including inhibitors of actomyosin contractility) that prevent disruption of TJs and restore TJ integrity. One of these disruptors (putrescine) disrupted TJ integrity in ex vivo mouse colon tissues; administration to mice exacerbated colon inflammation, increased gut permeability, reduced colon transepithelial electrical resistance, increased pattern recognition receptor ligands in mesenteric lymph nodes, and decreased colon length and survival times. Putrescine also increased intestine levels and fecal shedding of viable C rodentium, increased bacterial attachment to the colonic epithelium, and increased levels of inflammatory cytokines in colon tissues. Colonic epithelial cells from mice given putrescine increased expression of genes that regulate metal binding, oxidative stress, and cytoskeletal organization and contractility. Co-administration of taurine with putrescine blocked disruption of TJs and the exacerbated inflammation. CONCLUSIONS: We identified molecules that disrupt and stabilize intestinal epithelial TJs and barrier function and affect development of colon inflammation in mice. These agents might be developed for treatment of barrier intestinal impairment-associated and inflammatory disorders in patients, or avoided to prevent inflammation.


Subject(s)
Colitis/drug therapy , Colon/drug effects , Enterobacteriaceae Infections/drug therapy , Epithelial Cells/drug effects , Gastrointestinal Agents/pharmacology , High-Throughput Screening Assays , Intestinal Absorption/drug effects , Intestinal Mucosa/drug effects , Tight Junctions/drug effects , Animals , Caco-2 Cells , Citrobacter rodentium/pathogenicity , Colitis/chemically induced , Colitis/metabolism , Colitis/microbiology , Colon/metabolism , Colon/microbiology , Colon/pathology , Dextran Sulfate , Disease Models, Animal , Enterobacteriaceae Infections/metabolism , Enterobacteriaceae Infections/microbiology , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Epithelial Cells/pathology , Gastrointestinal Microbiome , Host-Pathogen Interactions , Humans , Intestinal Mucosa/metabolism , Intestinal Mucosa/microbiology , Intestinal Mucosa/pathology , Male , Mice, Inbred C57BL , Permeability , Putrescine/pharmacology , Taurine/pharmacology , Tight Junctions/metabolism , Tight Junctions/microbiology , Tight Junctions/pathology
4.
ACS Appl Bio Mater ; 3(12): 8395-8401, 2020 Dec 21.
Article in English | MEDLINE | ID: mdl-35019611

ABSTRACT

The skin is a key site for drug administration because of its large surface area and noninvasive accessibility. However, the dermal architecture serves as an excellent barrier, protecting from external mechanical, chemical, microbial, and physical perturbations. Most drugs display poor permeability through this barrier, thus making dermal and subdermal delivery challenging. Cell-penetrating peptides (CPPs), a diverse group of relatively short cationic and amphipathic membrane-interacting peptides, are fast becoming an important class of drug carriers and could potentially be developed for the dermal delivery of active molecules. However, the mechanism of CPP transdermal delivery is not fully understood, and there is a genuine need for a minimal model to understand this important phenomenon. Here, we demonstrate the potent membrane interactions of a minimal four-amino-acid-long CPP as well as the significance of guanidinium patterning and cationic nature of this palindromic peptide on its bioactivity. Furthermore, we demonstrate the biocompatibility of this peptide as well as its rapid cellular uptake and endosomal distribution. Finally, by utilizing a porcine full-thickness skin model, we demonstrate the substantial independent dermal and sonophoresis-based transdermal penetration of this minimal model. These results provide a minimal model for CPPs which can be easily manipulated for further biophysical and biochemical evaluations as well as a potent functional CPP with excellent skin permeability, which can be utilized for a wide variety of cosmetic and medical applications.

5.
Angew Chem Int Ed Engl ; 57(38): 12444-12447, 2018 09 17.
Article in English | MEDLINE | ID: mdl-30088843

ABSTRACT

The formation of apoptosis-inducing amyloidal structures by metabolites has significantly extended the "amyloid hypothesis" to include non-proteinaceous, single metabolite building blocks. However, detection of metabolite assemblies is restricted compared to their larger protein-based counterparts owing to the hindrance of external labelling and limited immunohistochemical detection tools. Herein, we present the detection of the formation, dynamics, and cellular distribution of metabolite amyloid-like structures and provide mechanistic insights into the generation of supramolecular chromophores. Moreover, the intrinsic fluorescence properties allow the detection of metabolite assemblies in living cells without the use of external dyes. Altogether, this intrinsic fluorescence of metabolite assemblies further verifies their amyloidal nature, while providing an important tool for further investigation of their pathological role in inborn error of metabolism disorders.


Subject(s)
Amyloid/chemistry , Amyloid/metabolism , Cell Line, Tumor , Fluorescence , HEK293 Cells , Humans , Microscopy, Confocal
6.
Sci Rep ; 7(1): 544, 2017 03 28.
Article in English | MEDLINE | ID: mdl-28373710

ABSTRACT

The intercellular spreading of protein assemblies is a major factor in the progression of neurodegenerative disorders. The quantitative study and visualization of cell-to-cell propagation using tagged-proteins is challenging due to the steric effect of relatively large fluorescence tags and the risk of 'false positive' identification when analyzing these rare transmission events. Here, we established a cell culture model to characterize the cell-to-cell transmission of TAR DNA-binding protein and α-synuclein, involved in amyotrophic lateral sclerosis and Parkinson's disease, respectively, using the small nine amino acid influenza hemagglutinin tag. The novel use of single cell resolution imaging flow cytometry allowed the visualization and quantification of all individual transmission events. Cell-level analysis of these events indicated that the degree of transfer is lower than previously reported based on conventional flow cytometry. Furthermore, our analysis can exclude 'false positive' events of cellular overlap and extracellular debris attachment. The results were corroborated by high-resolution confocal microscopy mapping of protein localization.


Subject(s)
DNA-Binding Proteins/metabolism , Molecular Imaging , alpha-Synuclein/metabolism , Cell Line , Coculture Techniques , Flow Cytometry , Fluorescent Antibody Technique , Humans , Intracellular Space , Microscopy, Fluorescence , Molecular Imaging/methods , Protein Transport , Single-Cell Analysis
7.
Nat Cell Biol ; 13(12): 1457-65, 2011 Nov 13.
Article in English | MEDLINE | ID: mdl-22081092

ABSTRACT

Cell elongation and polarization are basic morphogenetic responses to extracellular matrix adhesion. We demonstrate here that human cultured fibroblasts readily polarize when plated on rigid, but not on compliant, substrates. On rigid surfaces, large and uniformly oriented focal adhesions are formed, whereas cells plated on compliant substrates form numerous small and radially oriented adhesions. Live-cell monitoring showed that focal adhesion alignment precedes the overall elongation of the cell, indicating that focal adhesion orientation may direct cell polarization. siRNA-mediated knockdown of 85 human protein tyrosine kinases (PTKs) induced distinct alterations in the cell polarization response, as well as diverse changes in cell traction force generation and focal adhesion formation. Remarkably, changes in rigidity-dependent traction force development, or focal adhesion mechanosensing, were consistently accompanied by abnormalities in the cell polarization response. We propose that the different stages of cell polarization are regulated by multiple, PTK-dependent molecular checkpoints that jointly control cell contractility and focal-adhesion-mediated mechanosensing.


Subject(s)
Cell Polarity/physiology , Extracellular Matrix/physiology , Fibroblasts/cytology , Fibroblasts/physiology , Focal Adhesions/physiology , Mechanotransduction, Cellular/physiology , Acrolein/analogs & derivatives , Acrolein/chemistry , Cells, Cultured , Dimethylpolysiloxanes/chemistry , Glass/chemistry , Humans , Nylons/chemistry , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Pyridines/chemistry , RNA, Small Interfering/pharmacology , Stress, Mechanical
8.
Mol Biol Cell ; 22(16): 2900-11, 2011 Aug 15.
Article in English | MEDLINE | ID: mdl-21680709

ABSTRACT

In mammalian cells, the Golgi apparatus is a ribbon-like, compact structure composed of multiple membrane stacks connected by tubular bridges. Microtubules are known to be important to Golgi integrity, but the role of the actin cytoskeleton in the maintenance of Golgi architecture remains unclear. Here we show that an increase in Rho activity, either by treatment of cells with lysophosphatidic acid or by expression of constitutively active mutants, resulted in pronounced fragmentation of the Golgi complex into ministacks. Golgi dispersion required the involvement of mDia1 formin, a downstream target of Rho and a potent activator of actin polymerization; moreover, constitutively active mDia1, in and of itself, was sufficient for Golgi dispersion. The dispersion process was accompanied by formation of dynamic F-actin patches in the Golgi area. Experiments with cytoskeletal inhibitors (e.g., latrunculin B, blebbistatin, and Taxol) revealed that actin polymerization, myosin-II-driven contractility, and microtubule-based intracellular movement were all involved in the process of Golgi dispersion induced by Rho-mDia1 activation. Live imaging of Golgi recovery revealed that fusion of the small Golgi stacks into larger compartments was repressed in cells with active mDia1. Furthermore, the formation of Rab6-positive transport vesicles derived from the Golgi complex was enhanced upon activation of the Rho-mDia1 pathway. Transient localization of mDia1 to Rab6-positive vesicles was detected in cells expressing active RhoA. Thus, the Rho-mDia1 pathway is involved in regulation of the Golgi structure, affecting remodeling of Golgi membranes.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Golgi Apparatus/metabolism , rhoA GTP-Binding Protein/metabolism , Actin Cytoskeleton/metabolism , Adaptor Proteins, Signal Transducing/genetics , Formins , Golgi Apparatus/drug effects , Golgi Apparatus/ultrastructure , HeLa Cells , Humans , Membrane Fusion , Myosin Type II/metabolism , Paclitaxel/pharmacology , Protein Multimerization , Protein Structure, Tertiary , Protein Transport , Signal Transduction , Time-Lapse Imaging , Tubulin Modulators/pharmacology , rab GTP-Binding Proteins/metabolism
9.
J Cell Sci ; 124(Pt 6): 910-20, 2011 Mar 15.
Article in English | MEDLINE | ID: mdl-21378309

ABSTRACT

The function of connexins, which form gap junctions, can be rapidly modulated by degradation, because they have half-lives of only a few hours. Autophagy is a degradation pathway that has been implicated in several diseases and can be induced by cellular stresses such as starvation. We investigated the involvement of autophagy in proteolysis of the wild-type connexins CX50 and CX43, and a cataract-associated connexin mutant, CX50P88S, which forms cytoplasmic accumulations. We observed that cytoplasmic connexins were partially (cup-shaped) or completely (ring-shaped) enclosed by structures containing the autophagy-related protein LC3. Intracellular connexins also colocalized with p62, a protein that might serve as a cargo receptor for autophagic degradation. Starvation induced a decrease in connexin levels that was blocked by treatment with chloroquine, a lysosomal protease inhibitor, or by knockdown of the autophagy-related protein Atg5. These results demonstrate that autophagy can regulate cellular levels of wild-type connexins and imply that the persistence of accumulations of CX50P88S results from insufficient degradation capacity of constitutive autophagy.


Subject(s)
Autophagy , Connexin 43/metabolism , Connexins/metabolism , Eye Proteins/metabolism , Animals , Connexin 43/genetics , Connexins/genetics , Eye Proteins/genetics , HeLa Cells , Humans , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Protein Transport
10.
Exp Eye Res ; 88(3): 600-9, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19073179

ABSTRACT

Mutant connexins have been linked to hereditary congenital cataracts. One such mutant causes a proline-to-serine substitution at position 88 in human connexin 50 (CX50P88S). In transfected cells, CX50P88S does not form gap junctions, but localizes in cytoplasmic multilamellar structures. We studied the dynamics of formation and the stability of these structures in HeLa cells stably transfected with CX50P88S containing a tetracysteine motif appended to its C-terminus (HeLa-CX50P88S(Cys)(4) cells). The tetracysteine motif binds the membrane-permeable biarsenical compounds, FlAsH and ReAsH, which become fluorescent upon binding allowing detection of CX50P88S(Cys)(4) by fluorescence microscopy or by transmission electron microscopy after the ReAsH-driven fluorescent photoconversion of diaminobenzidine. CX50P88S structures were long-lived. Pulse labeling of HeLa-CX50P88S(Cys)(4) cells with FlAsH followed by a chase and ReAsH labeling showed a differential distribution of the labels, with older CX50P88S surrounded by newly synthesized protein. Formation of CX50P88S accumulations was not affected by treatments that block ER-to-Golgi transport. Transmission electron microscopy and tomographic reconstruction revealed that CX50P88S accumulations corresponded to closely apposed circular or semicircular membrane stacks that were sometimes continuous with the rough endoplasmic reticulum. These results suggest that CX50P88S accumulations originate from the rough endoplasmic reticulum and that mutant protein is sequentially added resulting in long-lived cytoplasmic particles. The persistence of these particles in the lens may cause light scattering and the pulverulent cataracts observed in affected individuals.


Subject(s)
Cataract/genetics , Connexins/genetics , Cytoplasm/metabolism , Eye Proteins/genetics , Mutation , Apoptosis Regulatory Proteins/metabolism , Calcium-Binding Proteins/metabolism , Connexins/metabolism , Cytoplasm/ultrastructure , Electron Microscope Tomography , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/ultrastructure , Eye Proteins/metabolism , HeLa Cells , Humans , Microscopy, Electron
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