ABSTRACT
We examined the function of heparan-sulfate-modified proteoglycans (HSPGs) in pathways affecting Alzheimer disease (AD)-related cell pathology in human cell lines and mouse astrocytes. Mechanisms of HSPG influences on presenilin-dependent cell loss were evaluated in Drosophila using knockdown of the presenilin homolog, Psn, together with partial loss-of-function of sulfateless (sfl), a gene specifically affecting HS sulfation. HSPG modulation of autophagy, mitochondrial function, and lipid metabolism were shown to be conserved in human cell lines, Drosophila, and mouse astrocytes. RNA interference (RNAi) of Ndst1 reduced intracellular lipid levels in wild-type mouse astrocytes or those expressing humanized variants of APOE, APOE3, and APOE4. Neuron-directed knockdown of Psn in Drosophila produced apoptosis and cell loss in the brain, phenotypes suppressed by reductions in sfl expression. Abnormalities in mitochondria, liposomes, and autophagosome-derived structures in animals with Psn knockdown were also rescued by reduction of sfl. These findings support the direct involvement of HSPGs in AD pathogenesis.
ABSTRACT
Lipids are essential for tumours because of their structural, energetic, and signaling roles. While many cancer cells upregulate lipid synthesis, growing evidence suggests that tumours simultaneously intensify the uptake of circulating lipids carried by lipoproteins. Which mechanisms promote the uptake of extracellular lipids, and how this pool of lipids contributes to cancer progression, are poorly understood. Here, using functional genetic screens, we find that lipoprotein uptake confers resistance to lipid peroxidation and ferroptotic cell death. Lipoprotein supplementation robustly inhibits ferroptosis across numerous cancer types. Mechanistically, cancer cells take up lipoproteins through a pathway dependent on sulfated glycosaminoglycans (GAGs) linked to cell-surface proteoglycans. Tumour GAGs are a major determinant of the uptake of both low and high density lipoproteins. Impairment of glycosaminoglycan synthesis or acute degradation of surface GAGs decreases the uptake of lipoproteins, sensitizes cells to ferroptosis and reduces tumour growth in mice. We also find that human clear cell renal cell carcinomas, a distinctively lipid-rich tumour type, display elevated levels of lipoprotein-derived antioxidants and the GAG chondroitin sulfate than non-malignant human kidney. Altogether, our work identifies lipoprotein uptake as an essential anti-ferroptotic mechanism for cancer cells to overcome lipid oxidative stress in vivo, and reveals GAG biosynthesis as an unexpected mediator of this process.
ABSTRACT
Introduction: We describe a novel colopathy associated with pentosan polysulfate (PPS) use and measure the strength of the drug-disease association. Methods: Two-part investigation. In the cohort study of individuals with a history of prior long-term PPS use, case histories were obtained and gastrointestinal disease course was followed with review of endoscopy records and histopathology specimens. Findings were summarized with descriptive statistics. In the cross-sectional study of individuals with interstitial cystitis, drug exposure and medical histories were obtained for patients seen at a single clinical center. Strength of association between PPS use and diagnoses of inflammatory bowel disease (IBD) and/or irritable bowel syndrome (IBS) was measured with multivariate logistic regression. Results: In the cohort study of 13 participants, median PPS exposure was 2.04 kg (0.99-2.54). Eleven (84.6%) developed symptoms suggestive of IBD and/or IBS after initiation of PPS therapy. Of the 10 participants whose endoscopic and histopathologic findings we reviewed, six had abnormal-appearing colonic mucosa on endoscopy and all 10 had abnormal mucosal changes on histology. Clinical and histologic improvement was observed after PPS cessation. In the cross-sectional study of 219 subjects with interstitial cystitis, PPS use was a statistically significant predictor of both the IBD [adjusted odds ratio=3.3 (95% confidence interval, 1.2-8.8, p=0.02)] and the composite IBD+IBS [adjusted odds ratio=3.3 (95% confidence interval, 1.5-7.3, p=0.002)] outcomes. Discussion: We describe a strong association between PPS use and a clinical diagnosis of IBD and/or IBS. Histopathologic findings suggest a novel drug-associated colopathy, with some subjects requiring colectomy for dysplasia.
ABSTRACT
Heparan sulfate (HS) is a long, linear polysaccharide that is ubiquitously expressed in all animal cells and plays a key role in many cellular processes, including cell signaling and development. Dysregulation of HS assembly has been implicated in pathophysiological conditions, such as tumorigenesis and rare genetic disorders. HS biosynthesis occurs in a non-template-driven manner in the endoplasmic reticulum and Golgi through the activity of a large group of biosynthetic enzymes. While much is known about its biosynthesis, little is understood about the regulation of HS assembly across diverse tissue types and disease states. To address this gap in knowledge, we recently performed genome-wide CRISPR/Cas9 screens to identify novel regulatory factors of HS biosynthesis. From these screens, we identified the alpha globin transcription factor, TFCP2, as a top hit. To investigate the role of TFCP2 in HS assembly, we targeted TFCP2 expression in human melanoma cells using the CRISPR/Cas9 system. TFCP2 knockout cells exhibited decreased fibroblast growth factor binding to cell surface HS, alterations in HS composition, and slowed cell growth compared to wild-type cells. Additionally, RNA sequencing revealed that TFCP2 regulates the expression of multiple enzymes involved in HS assembly, including the secreted endosulfatase, SULF1. Pharmacological targeting of TFCP2 activity similarly reduced growth factor binding and increased SULF1 expression, and the knockdown of SULF1 expression in TFCP2 mutant cells restored melanoma cell growth. Overall, these studies identify TFCP2 as a novel transcriptional regulator of HS and highlight HS-protein interactions as a possible target to slow melanoma growth.
Subject(s)
Heparitin Sulfate , Melanoma , Animals , Humans , Heparitin Sulfate/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Gene Expression Regulation , Cell Proliferation , Melanoma/genetics , DNA-Binding Proteins/metabolismABSTRACT
Glycosaminoglycans (GAGs) are long, linear polysaccharides that are ubiquitously expressed on the cell surface and in the extracellular matrix of all animal cells. These complex carbohydrates are composed of alternating glucosamine and uronic acids that can be heterogeneously N- and O-sulfated. The arrangement and orientation of the sulfated sugar residues specify the location of distinct ligand binding sites on the cell surface, and their capacity to bind ligands impacts cell growth and development, the ability to form tissues and organs, and normal physiology. The heterogeneous nature of GAGs and their inherent structural diversity across different tissues, cell types, and disease states creates challenges to characterizing their structure and function. Here, we describe detailed methods to investigate GAG-protein interactions in vitro and evaluate the structural composition of two classes of sulfated GAGs, heparan sulfate and chondroitin/dermatan sulfate, using liquid chromatography, mass spectrometry, and radiolabeling techniques. Overall, these methods facilitate the evaluation of GAG structure and function to uncover the unique roles these molecules play in cell biology and human disease.
Subject(s)
Glycosaminoglycans , Heparitin Sulfate , Humans , Animals , Glycosaminoglycans/chemistry , Heparitin Sulfate/analysis , Heparitin Sulfate/chemistry , Chondroitin Sulfates/chemistry , Chromatography, Liquid , Uronic Acids , SulfatesABSTRACT
Glycosaminoglycans (GAGs) are long, linear polysaccharides that are ubiquitously expressed on the cell surface and in the extracellular matrix of all animal cells. These complex carbohydrates play important roles in many cellular processes and have been implicated in many disease states, including cancer, inflammation, and genetic disorders. GAGs are among the most complex molecules in biology with enormous information content and extensive structural and functional heterogeneity. GAG biosynthesis is a nontemplate-driven process facilitated by a large group of biosynthetic enzymes that have been extensively characterized over the past few decades. Interestingly, the expression of the enzymes and the consequent structure and function of the polysaccharide chains can vary temporally and spatially during development and under certain pathophysiological conditions, suggesting their assembly is tightly regulated in cells. Due to their many key roles in cell homeostasis and disease, there is much interest in targeting the assembly and function of GAGs as a therapeutic approach. Recent advances in genomics and GAG analytical techniques have pushed the field and generated new perspectives on the regulation of mammalian glycosylation. This review highlights the spatiotemporal diversity of GAGs and the mechanisms guiding their assembly and function in human biology and disease.
Subject(s)
Genomics , Glycosaminoglycans , Animals , Extracellular Matrix/metabolism , Glycosaminoglycans/chemistry , Glycosaminoglycans/metabolism , Glycosylation , Homeostasis , Humans , Mammals/metabolismABSTRACT
Heparan sulfate (HS) proteoglycans bind extracellular proteins that participate in cell signaling, attachment and endocytosis. These interactions depend on the arrangement of sulfated sugars in the HS chains generated by well-characterized biosynthetic enzymes; however, the regulation of these enzymes is largely unknown. We conducted genome-wide CRISPR-Cas9 screens with a small-molecule ligand that binds to HS. Screening of A375 melanoma cells uncovered additional genes and pathways impacting HS formation. The top hit was the epigenetic factor KDM2B, a histone demethylase. KDM2B inactivation suppressed multiple HS sulfotransferases and upregulated the sulfatase SULF1. These changes differentially affected the interaction of HS-binding proteins. KDM2B-deficient cells displayed decreased growth rates, which was rescued by SULF1 inactivation. In addition, KDM2B deficiency altered the expression of many extracellular matrix genes. Thus, KDM2B controls proliferation of A375 cells through the regulation of HS structure and serves as a master regulator of the extracellular matrix.
Subject(s)
F-Box Proteins/antagonists & inhibitors , Genome-Wide Association Study , Heparitin Sulfate/metabolism , Jumonji Domain-Containing Histone Demethylases/antagonists & inhibitors , Algorithms , CRISPR-Cas Systems , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Discovery , Extracellular Matrix/genetics , High-Throughput Screening Assays , Humans , Protein Binding/genetics , RNA-Seq , Sulfotransferases/antagonists & inhibitorsABSTRACT
We identify the prolyl-tRNA synthetase (PRS) inhibitor halofuginone 1 , a compound in clinical trials for anti-fibrotic and anti-inflammatory applications 2 , as a potent inhibitor of SARS-CoV-2 infection and replication. The interaction of SARS-CoV-2 spike protein with cell surface heparan sulfate (HS) promotes viral entry 3 . We find that halofuginone reduces HS biosynthesis, thereby reducing spike protein binding, SARS-CoV-2 pseudotyped virus, and authentic SARS-CoV-2 infection. Halofuginone also potently suppresses SARS-CoV-2 replication post-entry and is 1,000-fold more potent than Remdesivir 4 . Inhibition of HS biosynthesis and SARS-CoV-2 infection depends on specific inhibition of PRS, possibly due to translational suppression of proline-rich proteins. We find that pp1a and pp1ab polyproteins of SARS-CoV-2, as well as several HS proteoglycans, are proline-rich, which may make them particularly vulnerable to halofuginone's translational suppression. Halofuginone is orally bioavailable, has been evaluated in a phase I clinical trial in humans and distributes to SARS-CoV-2 target organs, including the lung, making it a near-term clinical trial candidate for the treatment of COVID-19.
ABSTRACT
Heparin is the most widely prescribed biopharmaceutical in production globally. Its potent anticoagulant activity and safety makes it the drug of choice for preventing deep vein thrombosis and pulmonary embolism. In 2008, adulterated material was introduced into the heparin supply chain, resulting in several hundred deaths and demonstrating the need for alternate sources of heparin. Heparin is a fractionated form of heparan sulfate derived from animal sources, predominantly from connective tissue mast cells in pig mucosa. While the enzymes involved in heparin biosynthesis are identical to those for heparan sulfate, the factors regulating these enzymes are not understood. Examination of the promoter regions of all genes involved in heparin/heparan sulfate assembly uncovered a transcription factor-binding motif for ZNF263, a C2H2 zinc finger protein. CRISPR-mediated targeting and siRNA knockdown of ZNF263 in mammalian cell lines and human primary cells led to dramatically increased expression levels of HS3ST1, a key enzyme involved in imparting anticoagulant activity to heparin, and HS3ST3A1, another glucosaminyl 3-O-sulfotransferase expressed in cells. Enhanced 3-O-sulfation increased binding to antithrombin, which enhanced Factor Xa inhibition, and binding of neuropilin-1. Analysis of transcriptomics data showed distinctively low expression of ZNF263 in mast cells compared with other (non-heparin-producing) immune cells. These findings demonstrate a novel regulatory factor in heparan sulfate modification that could further advance the possibility of bioengineering anticoagulant heparin in cultured cells.
Subject(s)
DNA-Binding Proteins/metabolism , Heparin/metabolism , Heparitin Sulfate/biosynthesis , Animals , Anticoagulants , Cell Line , Cells, Cultured , Chromatography, High Pressure Liquid , Gene Expression Regulation/genetics , HeLa Cells , Heparin/biosynthesis , Heparin/genetics , Heparitin Sulfate/genetics , Heparitin Sulfate/metabolism , Humans , Mast Cells/metabolism , Sulfotransferases/metabolism , Swine , Transcription FactorsABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Two-terminal memory elements, or memelements, capable of co-locating signal processing and memory via history-dependent reconfigurability at the nanoscale are vital for next-generation computing materials striving to match the brain's efficiency and flexible cognitive capabilities. While memory resistors, or memristors, have been widely reported, other types of memelements remain underexplored or undiscovered. Here we report the first example of a volatile, voltage-controlled memcapacitor in which capacitive memory arises from reversible and hysteretic geometrical changes in a lipid bilayer that mimics the composition and structure of biomembranes. We demonstrate that the nonlinear dynamics and memory are governed by two implicitly-coupled, voltage-dependent state variables-membrane radius and thickness. Further, our system is capable of tuneable signal processing and learning via synapse-like, short-term capacitive plasticity. These findings will accelerate the development of low-energy, biomolecular neuromorphic memelements, which, in turn, could also serve as models to study capacitive memory and signal processing in neuronal membranes.
Subject(s)
Cell Membrane/physiology , Electric Capacitance , Lipid Bilayers , Memory/physiology , Nonlinear Dynamics , Algorithms , Biomimetics/methods , Electrical Synapses/physiology , Learning/physiology , Models, Theoretical , Neuronal Plasticity/physiology , Neurons/cytology , Neurons/physiologyABSTRACT
The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the cognitive powers of the brain with comparable efficiency and density. To date, silicon-based three-terminal transistors and two-terminal memristors have been widely used in neuromorphic circuits, in large part due to their ability to co-locate information processing and memory. Yet these devices cannot achieve the interconnectivity and complexity of the brain because they are power-hungry, fail to mimic key synaptic functionalities, and suffer from high noise and high switching voltages. To overcome these limitations, we have developed and characterized a biomolecular memristor that mimics the composition, structure, and switching characteristics of biological synapses. Here, we describe the process of assembling and characterizing biomolecular memristors consisting of a 5 nm-thick lipid bilayer formed between lipid-functionalized water droplets in oil and doped with voltage-activated alamethicin peptides. While similar assembly protocols have been used to investigate biophysical properties of droplet-supported lipid membranes and membrane-bound ion channels, this article focuses on key modifications of the droplet interface bilayer method essential for achieving consistent memristor performance. Specifically, we describe the liposome preparation process and the incorporation of alamethicin peptides in lipid bilayer membranes, and the appropriate concentrations of each constituent as well as their impact on the overall response of the memristors. We also detail the characterization process of biomolecular memristors, including measurement and analysis of memristive current-voltage relationships obtained via cyclic voltammetry, as well as short-term plasticity and learning in response to step-wise voltage pulse trains.
Subject(s)
Lipid Bilayers , Synapses/physiology , Alamethicin , Biomimetics , Ion Channels , LiposomesABSTRACT
Solid-state neuromorphic systems based on transistors or memristors have yet to achieve the interconnectivity, performance, and energy efficiency of the brain due to excessive noise, undesirable material properties, and nonbiological switching mechanisms. Here we demonstrate that an alamethicin-doped, synthetic biomembrane exhibits memristive behavior, emulates key synaptic functions including paired-pulse facilitation and depression, and enables learning and computing. Unlike state-of-the-art devices, our two-terminal, biomolecular memristor features similar structure (biomembrane), switching mechanism (ion channels), and ionic transport modality as biological synapses while operating at considerably lower power. The reversible and volatile voltage-driven insertion of alamethicin peptides into an insulating lipid bilayer creates conductive pathways that exhibit pinched current-voltage hysteresis at potentials above their insertion threshold. Moreover, the synapse-like dynamic properties of the biomolecular memristor allow for simplified learning circuit implementations. Low-power memristive devices based on stimuli-responsive biomolecules represent a major advance toward implementation of full synaptic functionality in neuromorphic hardware.
ABSTRACT
Background: Tauopathies comprise a family of neurodegenerative disorders including Alzheimer's disease for which there is an urgent and unmet need for disease-modifying treatments. Tauopathies are characterized by pathological tau hyperphosphorylation, which has been shown to correlate tightly with disease progression and memory loss in patients suffering from Alzheimer's disease. We recently demonstrated an essential requirement for 3-O-sulfated heparan sulfate in pathological tau hyperphosphorylation in zebrafish, a prominent model organism for human drug discovery. Here, we investigated whether in vivo treatment with surfen or its derivatives oxalyl surfen and hemisurfen, small molecules with heparan sulfate antagonist properties, could mitigate tau hyperphosphorylation and neuronal deficits in a zebrafish model of tauopathies. Results: In vivo treatment of Tg[HuC::hTauP301L; DsRed] embryos for 2 days with surfen or oxalyl surfen significantly reduced the accumulation of the pThr181 tau phospho-epitope measured by ELISA by 30% and 51%, respectively. Western blot analysis also showed a significant decrease of pThr181 and pSer396/pSer404 in embryos treated with surfen or oxalyl surfen. Immunohistochemical analysis further confirmed that treatment with surfen or oxalyl surfen significantly decreased the AT8 tau epitope in spinal motoneurons. In addition, in vivo treatment of Tg[HuC::hTauP301L; DsRed] embryos with surfen or oxalyl surfen significantly rescued spinal motoneuron axon-branching defects and, as a likely consequence, the impaired stereotypical touch-evoked escape response. Importantly, treatment with hemisurfen, a surfen derivative devoid of heparan sulfate antagonist activity, does not affect tau hyperphosphorylation, nor neuronal or behavioural deficits in Tg[HuC::hTauP301L; DsRed] embryos. Conclusion: Our findings demonstrate for the first time that surfen, a well-tolerated molecule in clinical settings, and its derivative, oxalyl surfen, could mitigate or delay neuronal defects in tauopathies, including Alzheimer's disease.
ABSTRACT
Large-scale genetic screens using CRISPR/Cas9 technology have emerged as a major tool for functional genomics. With its increased popularity, experimental biologists frequently acquire large sequencing datasets for which they often do not have an easy analysis option. While a few bioinformatic tools have been developed for this purpose, their utility is still hindered either due to limited functionality or the requirement of bioinformatic expertise. To make sequencing data analysis of CRISPR/Cas9 screens more accessible to a wide range of scientists, we developed a Platform-independent Analysis of Pooled Screens using Python (PinAPL-Py), which is operated as an intuitive web-service. PinAPL-Py implements state-of-the-art tools and statistical models, assembled in a comprehensive workflow covering sequence quality control, automated sgRNA sequence extraction, alignment, sgRNA enrichment/depletion analysis and gene ranking. The workflow is set up to use a variety of popular sgRNA libraries as well as custom libraries that can be easily uploaded. Various analysis options are offered, suitable to analyze a large variety of CRISPR/Cas9 screening experiments. Analysis output includes ranked lists of sgRNAs and genes, and publication-ready plots. PinAPL-Py helps to advance genome-wide screening efforts by combining comprehensive functionality with user-friendly implementation. PinAPL-Py is freely accessible at http://pinapl-py.ucsd.edu with instructions and test datasets.
Subject(s)
CRISPR-Cas Systems/genetics , Computational Biology/instrumentation , Genetic Testing/instrumentation , Software , Genomics/instrumentation , InternetABSTRACT
Heparin and heparan sulfate glycosaminoglycans are long, linear polysaccharides that are made up of alternating dissacharide sequences of sulfated uronic acid and amino sugars. Unlike heparin, which is only found in mast cells, heparan sulfate is ubiquitously expressed on the cell surface and in the extracellular matrix of all animal cells. These negatively-charged glycans play essential roles in important cellular functions such as cell growth, adhesion, angiogenesis, and blood coagulation. These biomolecules are also involved in pathophysiological conditions such as pathogen infection and human disease. This review discusses past and current methods for targeting these complex biomolecules as a novel therapeutic strategy to treating disorders such as cancer, neurodegenerative diseases, and infection.
Subject(s)
Glycosaminoglycans/antagonists & inhibitors , Heparin/metabolism , Heparitin Sulfate/antagonists & inhibitors , Neoplasms/drug therapy , Small Molecule Libraries/pharmacology , Animals , Glycosaminoglycans/chemistry , Heparin/chemistry , Heparitin Sulfate/chemistry , Humans , Infections/drug therapy , Infections/metabolism , Neoplasms/metabolism , Neurodegenerative Diseases/drug therapy , Neurodegenerative Diseases/metabolism , Small Molecule Libraries/chemistryABSTRACT
Surfen, bis-2-methyl-4-amino-quinolyl-6-carbamide, was previously reported as a small molecule antagonist of heparan sulfate (HS), a key cell-surface glycosaminoglycan found on all mammalian cells. To generate structure-activity relationships, a series of rationally designed surfen analogs was synthesized, where its dimeric structure, exocyclic amines, and urea linker region were modified to probe the role of each moiety in recognizing HS. An in vitro assay monitoring inhibition of fibroblast growth factor 2 binding to wild-type CHO cells was utilized to quantify interactions with cell surface HS. The dimeric molecular structure of surfen and its aminoquinoline ring systems was essential for its interaction with HS, and certain dimeric analogs displayed higher inhibitory potency than surfen and were also shown to block downstream FGF signaling in mouse embryonic fibroblast cells. These molecules were also able to antagonize other HS-protein interactions including the binding of soluble RAGE to HS. Importantly, selected molecules were shown to neutralize heparin and other heparinoids, including the synthetic pentasaccharide fondaparinux, in a factor Xa chromogenic assay and in vivo in mice. These results suggest that small molecule antagonists of heparan sulfate and heparin can be of therapeutic potential for the treatment of disorders involving glycosaminoglycan-protein interactions.
ABSTRACT
Reactions of chlorine (Cl(2)) with 4-halo-1,1,2-trifluorobut-1-enes (1, 2, or 3) give open-ion intermediates A and E that are in equilibrium. The open-chloronium ions (E) rearrange to a five-membered-ring halonium ion during ionic chlorination of 3 when the number-4 halo-substituent is iodine. Three-membered-ring bromonium and iodonium ions from alkenes 1, 2, or 3 are rather symmetrical and similar in structure. Quantum chemical calculations show that five-membered-ring halonium ion intermediates are 11 to 27 kcal/mol more stable than the three-membered-ring halonium ions or the open-ions A and E. The five-membered-ring intermediates lead to rearranged products. Rearranged products increase as the number-4 halogen (Z) becomes more nucleophilic (Z: Cl < Br < I). Open chloronium ions from ionic chlorination of terminal fluorovinyl alkenes are compared to the open ions generated by protons to similar alkenes.